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WO2024094027A1 - Blocking sequence, kit thereof, and method for using same - Google Patents

Blocking sequence, kit thereof, and method for using same Download PDF

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Publication number
WO2024094027A1
WO2024094027A1 PCT/CN2023/128795 CN2023128795W WO2024094027A1 WO 2024094027 A1 WO2024094027 A1 WO 2024094027A1 CN 2023128795 W CN2023128795 W CN 2023128795W WO 2024094027 A1 WO2024094027 A1 WO 2024094027A1
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PCT/CN2023/128795
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French (fr)
Chinese (zh)
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唐劼
肖晶
李航
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深圳鸿生生物科技有限公司
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Publication of WO2024094027A1 publication Critical patent/WO2024094027A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the present invention relates to compositions for enhancing the delivery of active or therapeutic agents, such as therapeutic nucleic acids.
  • biological macromolecules such as proteins, DNA, siRNA, and in vitro transcribed messenger RNA (mRNA) can produce or act on specific protein functions to exert their efficacy, thereby achieving the purpose of disease prevention or treatment.
  • mRNA messenger RNA
  • These biological macromolecules are becoming an important method for preventing and treating a variety of diseases.
  • vaccines developed based on mRNA technology have shown great potential in combating the novel coronavirus (SARS-CoV-2) pandemic that broke out in 2019 with extremely high protection rates.
  • SARS-CoV-2 novel coronavirus
  • two COVID-19 mRNA vaccines have been approved by the US FDA for marketing worldwide.
  • mRNA technology also has great application potential in tumor immunotherapy, gene therapy, infectious disease prevention, gene editing, and genetic disease treatment.
  • mRNA technology has many advantages: it is manufactured in a cell-free manner, which enables rapid, economical and efficient production, and has a unique advantage in quickly responding to large-scale outbreaks of sudden infectious diseases; mRNA is almost never integrated into the genome, has high safety, and avoids the possibility of insertion mutations; in addition, a single mRNA vaccine can encode multiple antigens, enhance the immune response against adaptive pathogens, and can target multiple microorganisms or viral variants with a single formulation.
  • mRNA therapy involves administering specific mRNA to a subject in need of the therapy to produce proteins encoded by the mRNA in the patient. How to use a safe delivery system to efficiently and accurately deliver mRNA to target tissues and target cells and maximize its expression efficiency is the key to achieving successful clinical transformation of such drugs or vaccines.
  • mRNA is easily degraded by widely existing RNases, and its structural stability is a prerequisite for exerting biological effects; 2) mRNA is easily cleared after entering the body, has a short half-life in the body, and is difficult to reach specific target sites; 3) Due to its large relative molecular mass, strong hydrophilicity and electronegativity, mRNA itself is difficult to effectively cross various physiological barriers in the body, such as mucosal barriers, cell membranes and lysosomal barriers. Therefore, the effective targeted delivery of mRNA drugs/vaccines has always been challenging in medicine.
  • viral vectors Due to the relatively high in vivo transfection efficiency, early attempts to use viral vectors as a delivery method for gene delivery were made.
  • main problems of viral vectors such as the host's immune response to the viral vector itself, difficulty in repeated administration, possible activation of oncogenes that cause malignant tumors, and complications of inflammatory responses, have significantly hindered their development.
  • non-viral vectors have many advantages over viral vectors, such as high safety, low immunogenicity, reduced pathogenicity, reduced ability to insert mutations, and convenient large-scale preparation.
  • Nanoparticles developed based on nanobiomaterials are a typical type of non-viral vector.
  • Common non-viral vectors include lipid nanoparticles (LNP) and preparations based on cationic polymers, such as chitosan, polyethyleneimine, and dendrimers.
  • LNP lipid nanoparticles
  • preparations based on cationic polymers such as chitosan, polyethyleneimine, and dendrimers.
  • cationic polymers such as chitosan, polyethyleneimine, and dendrimers.
  • the LNP system is the most common, most advanced, and only mRNA drug delivery system that has successfully achieved clinical transformation.
  • LNP has developed into the "gold standard" delivery technology in the field of mRNA due to its many advantages, the system still has many defects: for example, the LNP system usually needs to be administered through injection routes such as intramuscular injection, subcutaneous injection or intravenous injection, and can only produce efficient gene transfection effects in tissues with large gaps such as the liver and spleen and in the intramuscular injection site (Pardi, N. et al. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes. J. Control. Release. 217, 345–351 (2015)). Studies have shown that after LNP preparations are administered via the respiratory route, they only mediate very low mRNA gene expression in mice (Zhang, N.-N.
  • mRNA drugs have great potential in the treatment of many lung diseases that have no clinical cure, such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), ⁇ 1-trypsin deficiency, asthma, pulmonary hypertension, primary ciliary dyskinesia, and dyskinesia) and idiopathic pulmonary fibrosis.
  • CF cystic fibrosis
  • COPD chronic obstructive pulmonary disease
  • ⁇ 1-trypsin deficiency asthma
  • pulmonary hypertension pulmonary hypertension
  • primary ciliary dyskinesia and dyskinesia
  • idiopathic pulmonary fibrosis idiopathic pulmonary fibrosis.
  • the special physiological structure of the lungs brings many advantages to its route of administration, and makes it an ideal expression site for mRNA drugs (Suberi, A., et al. Polymer nanoparticles deliver mRNA to the lung for mucosal vaccination. Sci Transl Med. 15,
  • Drugs delivered through the respiratory tract have simple administration methods, good patient compliance, are suitable for repeated administration regimens, and can also make the drug preparations evenly distributed in bronchial and alveolar epithelial tissues (Patel, AK et al. Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium. Adv. Mater. e1805116, (2019)).
  • Potential systemic side effects can be minimized, and problems such as cross infection caused by needle contamination can also be reduced.
  • the lung airway has a large absorption surface area and a rich capillary network, which is conducive to gene drug absorption and efficient transfection.
  • the lungs also have strong angiogenesis capabilities, which can mediate secretory proteins to enter the circulatory system to exert their effects. Therefore, the lungs are an ideal target for mRNA-based protein replacement therapy.
  • mRNA vaccines have great application prospects in inducing antigen-specific mucosal immunity in respiratory mucosal sites (e.g., nasal-associated lymphoid tissue NALT and bronchial-associated lymphoid tissue BALT). More than 90% of pathogens invade the human body through mucosal sites (e.g., respiratory tract, gastrointestinal tract, reproductive tract, etc.), including the new coronavirus (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS), influenza virus, respiratory syncytial virus, and Mycobacterium tuberculosis.
  • SARS-CoV-2 new coronavirus
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS Middle East respiratory syndrome coronavirus
  • influenza virus respiratory syncytial virus
  • Mycobacterium tuberculosis Mycobacterium tuberculosis.
  • vaccines administered by intramuscular injection can hardly induce mucosal immune responses, and thus cannot produce secretory IgA (sIgA) antibodies to protect the host from initial infection in the mucosal area.
  • the virus may still replicate in the respiratory mucosa and cause infection to others.
  • Studies have shown that the early specific humoral response in patients infected with SARS-CoV-2 is dominated by sIgA antibodies produced by mucosal immune responses (Sterlin, D. et al. IgA dominates the early neutralizing antibody response to SARS-CoV-2. Sci Transl Med. 13, eabd22234 (2021)).
  • the average neutralization potency of sIgA against the new coronavirus is more than seven times that of IgG, and it plays a dominant role in the virus neutralization process (Wang, Z. et al. Enhanced SARS-CoV-2 neutralization by dimeric IgA. Sci Transl Med.13,eabf1555(2021)). Therefore, efficient mucosal immune response is of great value to the efficacy of vaccines related to respiratory invasive pathogens, and may be the key to eradicating infection and transmission of related pathogens (Jeyanathan, M. et al. Immunological considerations for COVID-19 vaccine strategies. Nat. Rev. Immunol. 20, 615–632(2020)).
  • the present invention surprisingly found that adding amphiphilic block copolymers such as poloxamine or ) and or poloxamer or ) and other components, the obtained novel preparation can greatly improve the gene transfection efficiency of nucleic acid molecules such as mRNA mediated by such preparations in animals, especially when delivered through the mucosal part of the organism (such as respiratory delivery).
  • the preparation of the present invention is administered via the respiratory route, the gene transfection effect produced in the animal lung/respiratory mucosal tissue is significantly better than that of LNP preparations, poloxamine and poloxamer, thereby solving the problem of low delivery efficiency in the art.
  • the present invention provides a composition comprising a polymer-lipid, the composition comprising:
  • an active agent or therapeutic agent preferably the active agent or therapeutic agent comprises a nucleic acid
  • composition is formulated for delivery through a mucosal site of an organism, such as the respiratory tract, oral mucosa, gastrointestinal tract, ocular mucosa, ear mucosa, urethra, or reproductive tract, preferably the composition is formulated for delivery through the respiratory tract.
  • the nucleic acid comprises at least one selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) and micro RNA (miRNA), primary-miRNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), plasmid DNA (pDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), deoxyribozyme (DNAzyme), ribozyme (RNAzyme), nucleic acid aptamer (aptamer), clustered regularly interspaced short palindromic repeats (CRISPR)-related nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA, single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA).
  • the nucleic acid is a therapeutic nucleic acid. More preferably, the nucleic acid comprises mRNA.
  • the amphiphilic block copolymer accounts for 0.1%-98.0% weight percent of the composition, for example, 0.5%-95.0% weight percent, 1%-90.0% weight percent, 10%-80.0% weight percent, 20%-80.0% weight percent, 30%-80.0% weight percent, 40%-80.0% weight percent, 40%-70.0% weight percent or 50%-60.0% weight percent.
  • the cationic lipid comprises at least one selected from the group consisting of permanent cationic lipids, ionizable cationic lipids, cholesterol-derived cationic lipids and dendrimers or dendrons.
  • the cationic lipid comprises an ionizable cationic lipid.
  • the cationic lipid accounts for 23 mol%-83 mol% of the total lipids present in the composition, such as 30 mol%-80 mol%, 30 mol%-70 mol% or 40 mol%-60 mol%, or the cationic lipid accounts for about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol% or about 80 mol% of the total lipids present in the composition.
  • the non-cationic lipid comprises at least one selected from the group consisting of anionic lipids, zwitterionic lipids and neutral lipids, preferably, the non-cationic lipid comprises a neutral lipid.
  • the neutral lipid accounts for 19 mol%-75 mol% of the total lipids present in the composition.
  • the neutral lipid comprises:
  • a mixture of cholesterol or cholesterol-derived neutral lipids and phospholipids is provided.
  • the cholesterol comprises 14 mol%-70 mol% of the total lipids in the composition.
  • the phospholipids comprise about 5 mol% to about 30 mol% or 30 mol% to about 75 mol% of the total lipids in the composition.
  • composition of the present invention further comprises a lipid conjugate, wherein the lipid conjugate comprises at least one selected from the group consisting of: PEG-lipid conjugate, ATTA-lipid conjugate, polysarcosine-lipid conjugate, polypeptide/protein-lipid conjugate, cation-polymer-lipid conjugate (CPL) and derivatives thereof.
  • the lipid conjugate comprises a PEG-lipid conjugate.
  • the lipid conjugate comprises 0.1 mol%-10.0 mol% of the total lipids in the composition.
  • compositions of the present invention comprise:
  • an amphiphilic block copolymer a cationic lipid, a phospholipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 30.0 mol%-80.0 mol% of the total lipid present in the composition, the phospholipid accounts for 5.0 mol%-50.0 mol% of the total lipid, the cholesterol accounts for 14.0 mol%-64.0 mol% of the total lipid, the lipid conjugate accounts for 0.1 mol%-8.0 mol% of the total lipid, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight) of the composition;
  • a lipid conjugate such as a PEG-lipid conjugate
  • amphiphilic block copolymers cationic lipids, phospholipids, cholesterol, and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids account for 23.0 mol%-75.0 mol% of the total lipids present in the composition, the phospholipids account for 10.0 mol%-62.0 mol% of the total lipids, the cholesterol accounts for 14.0 mol%-46.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1%-95.0% by weight of the composition (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight);
  • an amphiphilic block copolymer a cholesterol-derived cationic lipid, a phospholipid, and a lipid conjugate such as a PEG-lipid conjugate, wherein the cholesterol-derived cationic lipid accounts for 29.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 19.0 mol%-70.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight). Compare);
  • an amphiphilic block copolymer a cationic lipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 25.0 mol%-80.0 mol% of the total lipids present in the composition, the cholesterol accounts for 15.0 mol%-50.0 mol% of the total lipids, the lipid conjugate accounts for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight) of the composition;
  • a lipid conjugate such as a PEG-lipid conjugate
  • amphiphilic block copolymers cationic lipids, phospholipids and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids account for 30.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 10.0 mol%-50.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1%-95.0% by weight of the composition (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight or 40%-70.0% by weight);
  • an amphiphilic block copolymer a cationic lipid, a phospholipid, and cholesterol
  • the cationic lipid accounts for 30.0 mol%-80.0 mol% of the total lipids present in the composition
  • the phospholipids account for 5.0 mol%-50.0 mol% of the total lipids
  • the cholesterol accounts for 15.0 mol%-50.0 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 0.1%-95.0% by weight (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight) of the composition; or
  • an amphiphilic block copolymer a cholesterol-derived cationic lipid and a phospholipid, wherein the cholesterol-derived cationic lipid accounts for 30.0 mol%-70.0 mol% of the total lipids present in the composition, the phospholipids account for 30.0 mol%-70.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% weight percent (e.g., 1%-90.0% weight percent, 10%-80.0% weight percent, 20%-80.0% weight percent, 30%-80.0% weight percent or 40%-70.0% weight percent) of the composition.
  • the cholesterol-derived cationic lipid accounts for 30.0 mol%-70.0 mol% of the total lipids present in the composition
  • the phospholipids account for 30.0 mol%-70.0 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 0.1%-95.0% weight percent (e.g., 1%-90.0% weight percent, 10%-
  • the amphiphilic block copolymer is a tetrafunctional amphiphilic block copolymer, wherein the tetrafunctional amphiphilic block copolymer comprises a block copolymer of four branches each comprising at least one hydrophilic block and at least one hydrophobic block, or the amphiphilic block copolymer is a linear amphiphilic block copolymer, wherein the linear amphiphilic block copolymer comprises a block copolymer of at least one hydrophilic block and at least one hydrophobic block.
  • the hydrophilic block is selected from polyoxyalkylenes, polyvinyl alcohol, polyvinyl pyrrolidone, poly(2-methyl-2-oxazoline) and sugars
  • the hydrophobic block is selected from polyoxyalkylenes, lipids, Fatty chains, alkylene polyesters, polyethylene glycol with benzyl polyether ends, and cholesterol
  • the hydrophilic block comprises polyethylene oxide units
  • the hydrophobic block comprises polypropylene oxide units.
  • the amphiphilic block copolymer comprises at least one selected from the group consisting of poloxamine or ), poloxamer or ), polyoxyethylene glycol dehydrated alcohol alkyl esters (polysorbates), polyvinyl pyrrolidone (PVP), polyethylene glycol ethers (BRIJ), polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, sorbitan and their derivatives.
  • the amphiphilic block copolymer comprises poloxamine or ), for example, the poloxamine is selected from poloxamine 304, poloxamine 701, poloxamine 704, poloxamine 901, poloxamine 904, poloxamine 908, poloxamine 1107, poloxamine 1301, poloxamine 1304, poloxamine 1307, poloxamine 90R4, poloxamine 150R1 or a combination thereof.
  • the amphiphilic block copolymer comprises poloxamer or ), for example, the poloxamer is selected from poloxamer 84, poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer Poloxamer 231, Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 304, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 402, Poloxa
  • the cationic lipid comprises at least one selected from the group consisting of DOTMA, DOSPA, DOTAP, ePC, DODAP, DODMA, DDAB, DSDMA, DODAC, DOAP, DMRIE, DOGS, DMOBA, HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4005, DLin-MC3-DMA, DLin-KC2-DMA, Acuitas ALC-0315, Acuitas A9, Acuitas Lipid 2,2, Moderna Lipid H (SM-102), Moderna Lipid 5, A2-Iso5-2DC18, BAME-O16B, 9A1P9, C12-200, cKK-E12, OF-Deg-Lin, 306Oi10, TT3, FTT5, Lipid319, 5A2-SC8, Genevant CL1, DLinDMA, DLenDMA, ClinDMA, CpLinDMA,
  • the cholesterol-derived cationic lipid comprises at least one selected from the group consisting of DC-Choi (N,N-dimethyl-N-ethylformamide cholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, N4-argininocholesterol carbonylamide (GL67), a cholesterol derivative coupled to a basic amino acid sequence, and imidazole cholesterol ester (ICE).
  • DC-Choi N,N-dimethyl-N-ethylformamide cholesterol
  • 1,4-bis(3-N-oleylamino-propyl)piperazine N4-argininocholesterol carbonylamide
  • GL67 N4-argininocholesterol carbonylamide
  • ICE imidazole cholesterol ester
  • the phospholipid comprises at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, dioleoylphosphatidylserine (DOPS), phosphatidylinositol, sphingomyelin, egg yolk sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphat
  • the cholesterol-derived neutral lipid comprises at least one selected from the group consisting of cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, BHEM-cholesterol, ⁇ -sitosterol, 20 ⁇ -hydroxycholesterol, cholesterol covalently linked to a polypeptide/protein, and derivatives thereof, preferably the cholesterol-derived neutral lipid comprises ⁇ -sitosterol.
  • the PEG-lipid conjugate comprises at least one selected from the following: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](DMPE-PEG2K), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000](DSPE-PEG2K), a conjugate of DSPE-PEG2K and mannose ( DSPE-PEG2K-Mannose), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000(DMG-PEG5K), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
  • compositions comprise an amphiphilic block copolymer and the following components:
  • composition comprises 40.0 mol% to 70.0 mol% of the total lipids, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE comprises 8.0 mol% to 39.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol comprises 20.0 mol% to 40.0 mol% of the total lipids, DMG-PEG
  • GL67, ICE, or HGT4002 DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE
  • DMG-PEG2K, DMG-PEG5K, or DSPE-PEG2K-Mannose wherein the GL67, ICE, or HGT4002 accounts for 40.0 mol%-80.0 mol% of the total lipids present in the composition
  • DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE accounts for 10.0 mol%-50.0 mol% of the total lipids
  • DMG-PEG2K, DMG-PEG5K, or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition
  • composition comprises the following components:
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein
  • the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition
  • DSPC, DPPC or DOPE account for 10.0 mol% of the total lipids
  • cholesterol or ⁇ -sitosterol account for 38.5 mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 1.5 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 89.9% by weight of the composition
  • an amphiphilic block copolymer (2) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.6 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 72.9% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 12.5 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 36.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.0% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 34.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 81.8% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 69.2% by weight of the composition;
  • mol% DSPC, DPPC or DOPE account for 14.1mol% of the total lipids
  • cholesterol or ⁇ -sitosterol account for 35.0mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 0.9mol% of the total lipids
  • amphiphilic block copolymer accounts for 43.3% by weight of the composition
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K
  • the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition
  • DSPC, DPPC or DOPE account for 14.3 mol% of the total lipids
  • cholesterol or ⁇ -sitosterol account for 35.0 mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 48.4% by weight of the composition
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K
  • the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition
  • DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids
  • cholesterol or ⁇ -sitosterol account for 35.0 mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 0.5 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 43.6% by weight of the composition
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 48.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.3% by weight of the composition;
  • an amphiphilic block copolymer (10) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 52.9% by weight of the composition;
  • Sterol accounts for 30.0 mol% of the total lipids
  • DMG-PEG2K or DSPE-PEG2K-Mannose accounts for 1.0 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 41.9% by weight of the composition
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.1 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.3% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 51.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 27.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.9% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.2% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 66.1% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the The amphiphilic block copolymer accounts for 74.5% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 79.6% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 83.0% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 88.2% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 43.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.3 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 33.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 39.1% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 32.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.3% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.1 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 28.4 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.9% by weight of the composition;
  • Amphiphilic block copolymers DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 34.7 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 40.1 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 24.3 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 32.5% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 29.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 50.1 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 28.0% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 35.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.6 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 46.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 35.9% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 55.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 16.9 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 27.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 44.6% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein
  • the DLin-MC3-DMA, ALC-0315 or SM-102 account for 65.0 mol% of the total lipids present in the composition
  • DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids
  • cholesterol or ⁇ -sitosterol account for 20.0 mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 21.0% by weight of the composition
  • an amphiphilic block copolymer GL67, ICE or HGT4002; DSPC, DPPC or DOPE; and DMG-PEG2K or DMG-PEG5K, wherein the GL67, ICE or HGT4002 accounts for 70.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE accounts for 28.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.1% by weight of the composition;
  • an amphiphilic block copolymer (4) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 61.3 mol% of the total lipids present in the composition, cholesterol or ⁇ -sitosterol account for 37.6 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.1 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition;
  • an amphiphilic block copolymer cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102 or C12-200; DOTAP, DODAP, DOTMA or DOSPA; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102 or C12-200 accounts for 30.0 mol% of the total lipids present in the composition, DOTAP, DODAP, DOTMA or DOSPA accounts for 39.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol accounts for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 57.7% by weight of the composition; or
  • An amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; and cholesterol or ⁇ -sitosterol, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 32.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 28.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.0% weight percent of the composition.
  • the molar ratio of nitrogen (amine) groups in the cationic lipids in the composition to phosphate groups in the nucleic acids is about 1.0 to about 30.0, about 4.0 to about 15.0, about 6.0 to about 8.0, or about 6.7 to about 7.6.
  • the ratio of lipid to nucleic acid (mass/mass ratio) in the composition is about 2 (2:1) to about 100 (100:1), about 5 (5:1) to about 60 (60:1), about 15 (15:1) to about 45(45:1), or about 20(20:1) to about 30(30:1).
  • the active agent or therapeutic agent further comprises a protein or polypeptide, and in some embodiments, the protein is a protein related to translation or transcription. In some embodiments, the protein is related to the CRISPR process. In some embodiments, the protein is a protein related to CRISPR. In some embodiments, the therapeutic agent is a protein or polypeptide. In some embodiments, the composition comprises both protein and nucleic acid. In some embodiments, the therapeutic agent is a small molecule.
  • the composition further comprises a targeting moiety to target the composition to a target organ, tissue or cell in a subject, preferably the targeting moiety comprises at least one selected from the following: glycosyl, lipid, nucleic acid aptamer, small molecule therapeutic agent, vitamin, polypeptide and protein such as antibody.
  • the composition further comprises an adjuvant, preferably the adjuvant comprises at least one selected from the following: CpG oligodeoxynucleotides, polyinosinic: polycytidylic acid, saponin extract (QS-21 extract), aluminum adjuvant, squalene, ⁇ -tocopherol, Tween, Span, lipopolysaccharide LPS, Pam 3 CSK 4 triacyl lipopeptide, cyclic adenosine diphosphate (c-di-AMP), 2′3′-cyclic guanosine monophosphate adenosine monophosphate (cGAMP), monophosphoryl-lipid A, MPL lipid, flagellin or immunomodulatory proteins such as IL-2, IL-12, GM-CSF, TSLP and nucleic acids encoding these immunomodulator proteins.
  • the adjuvant comprises at least one selected from the following: CpG oligodeoxynucleotides, polyinosinic: poly
  • the composition further comprises a transfection enhancer, preferably the transfection enhancer comprises at least one selected from the following: pulmonary surfactant protein, cell penetrating peptide, amphiphilic polypeptide, mucolytic enzyme, 1,2-propylene glycol, cellulose (such as carboxymethyl cellulose or hydroxypropyl cellulose), hyaluronate, alginate, pectin, polyethylene glycol, poloxamer, poloxamine, glucose, fructose, sucrose, trehalose, dextran, polyvinyl pyrrolidone, chitosan, polyvinyl alcohol, polyvinyl acetate, lectin, polylactic acid, polyhydroxybutyric acid, tromethamine, benzalkonium chloride, modified arginine, cetyl pyridinium chloride, L-lysine monohydrate, and polylactic acid-glycolic acid copolymer or salt solution.
  • pulmonary surfactant protein preferably the transfection enhancer comprises at least
  • the compositions are in the form of nanoparticles having an average size of about 1000 nm or less.
  • the nanoparticles have an average size of about 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 75 nm or less, or about 50 nm or less.
  • about 30% to about 100%, about 70% to about 100%, about 90% to about 100%, about 50% to about 90%, about 70% to about 90%, or about 80% to about 90% of the nanoparticles have the active or therapeutic agent encapsulated therein.
  • the composition is formulated in the form of a solution, dry powder, atomization or spray. In some embodiments, the composition is formulated for pulmonary and/or nasal administration by aerosolization, dry powder, inhalation, atomization or instillation.
  • the present invention provides a method for preparing a composition according to the first aspect of the present invention, the method comprising:
  • the method comprises:
  • the method comprises:
  • the method comprises:
  • the method further comprises the step of removing free lipid components and/or amphiphilic block copolymers, preferably by dialysis and/or tangential flow filtration. component and/or amphiphilic block copolymer.
  • the method further comprises the step of adding the amphiphilic block copolymer again after removing the free lipid component and/or the amphiphilic block copolymer.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the composition according to the first aspect of the present invention and a pharmaceutically acceptable carrier and/or excipient.
  • the present invention provides a method for delivering an active agent or a therapeutic agent to a target cell, the method comprising: contacting the cell with the composition according to the first aspect of the present invention under conditions sufficient to cause the active agent or the therapeutic agent to be taken up into the cell, preferably the cell is a mammalian cell.
  • the present invention provides a method for preventing and/or treating a disease or condition in a mammal, the method comprising administering a pharmaceutically effective amount of a composition according to the first aspect of the present invention or a pharmaceutical composition according to the third aspect of the present invention to a subject in need thereof, wherein the composition or pharmaceutical composition comprises an active agent or therapeutic agent for the disease or condition.
  • the disease or condition is selected from an immune system disease, a metabolic disease, a genetic disease, a cancer, a blood disease, a bacterial infection, or a viral infection.
  • the present invention provides a composition according to the first aspect of the present invention or a pharmaceutical composition according to the third aspect of the present invention for use in the preparation of a medicament for preventing and/or treating a disease in a subject, wherein the composition or pharmaceutical composition comprises an active agent or therapeutic agent for the disease or condition.
  • the disease or condition is selected from an immune system disease, a metabolic disease, a genetic disease, a cancer, a blood disease, a bacterial infection, or a viral infection.
  • Figure 1 Structural characterization of polymer-lipid composites (PoLixNano) and studies in in vitro models.
  • TEM Transmission electron microscopy
  • Figure 2 Study on the transfection efficiency of mRNA encoding firefly luciferase (Fluc-mRNA) in mice mediated by PoLixNano formulation and LNP control formulation via intratracheal spray (i.t.) administration for 6 hours.
  • Fluc-mRNA firefly luciferase
  • FIG. 3 Transfection efficiency study of PoLixNano formulations prepared with different types and concentrations of amphiphilic block copolymers in mice 6 h after intratracheal spray (i.t.) administration.
  • C Transfection efficiency study of PoLixNano formulations prepared with different types and concentrations of amphiphilic block copolymers in mice 6 h after intratracheal spray (i.t.) administration.
  • Figure 4 Study on the transfection efficiency of PoLixNano preparations prepared with different nitrogen-phosphorus ratios (N/P) and different lipid ratios in mice after 6 hours of intratracheal spray (it) administration.
  • Figure 5 Study on the transfection efficiency of PoLixNano formulations containing different types of lipids and lipid ratios in mice 6 hours after administration via intratracheal spray (i.t.).
  • A. Determination of Fluc expression levels in live mice and isolated lungs of PoLixNano formulations containing different types of cationic lipids (Dlin-MC3-DMA, ALC-0315, SM-102, C12-200 and HGT5000) and control LNP formulations (n 3)
  • FIG. 6 Transfection status of PoLixNano preparations prepared with different formulations in mice 6 hours after intranasal (i.n.) administration.
  • A. Quantitative determination of the Fluc bioluminescent signals mediated by PoLixNano preparations containing different concentrations of T704 (3 mg/mL-40 mg/mL), T904 (3 mg/mL), poloxamer 237 (10 mg/mL), poloxamer 338 (3 mg/mL) and different concentrations of poloxamer 124 (3 mg/mL and 20 mg/mL) and LNP control preparations in the isolated lungs of mice (n 3); B.
  • Figure 7 Study on the efficient transfection of Fluc-mRNA in mice mediated by the preparations (LNP+KG41, LNP+T904, LNP+10% sucrose, PoLixNano+KG41, PoLixNano+T904 and PoLixNano+10% sucrose) obtained by physical mixing of LNP preparations and PoLixNano preparations with different enhancers (KG41 polypeptide (KG41), T904 and sucrose solution with a final concentration of 10%) through different administration methods (i.t. or i.n.).
  • the preparations obtained by physical mixing of LNP preparations and PoLixNano preparations with different enhancers (KG41 polypeptide (KG41), T904 and sucrose solution with a final concentration of 10%
  • FIG. 8 Transfection efficacy of PoLixNano formulations containing unconventional lipid components and compositions in mice
  • CKK-E12: DOTAP: Chol: DMG-PEG2000 30:39:30:1
  • A. The Fluc expression level in the isolated lung of mice was determined by intratracheal spray (it) and nasal route (in) of PoLixNano preparations without DMG-PEG2000 lipid components and LNP control preparations in living mice and isolated lungs and livers (n 3);
  • FIG. 9 After the PoLixNano preparation was administered via nebulization, it mediated efficient Fluc-mRNA transfection in mice and studied the physicochemical properties of the nanoparticles.
  • A Schematic diagram of the mouse nebulization device and research process;
  • TEM Transmission electron microscopy
  • Figure 10 A study on the efficient mediating Fluc-mRNA transfection in mice of PoLixNano preparations containing different types of amphiphilic block copolymers, different molar ratios of lipid compositions and different dosages after administration via nebulization.
  • A. Quantitative determination of the Fluc bioluminescent signal in the isolated lungs of PoLixNano preparations containing different molar ratios of DMG-PEG2000 prepared with poloxamer 188 (left) or poloxamine 704 (right) and LNP control preparations 6 hours after administration via nebulization (n 3);
  • Fluc bioluminescent signal in the isolated lungs of PoLixNano preparations containing different molar ratios of lipid compositions prepared with poloxamer 188 (left) or poloxamine 704 (right) and LNP control preparations 6 hours after administration via nebulization (n 3);
  • FIG. 11 Study on the distribution, uptake and transfection efficiency of PoLixNano preparations in mice.
  • A. After 6 hours of administration of DIR-labeled PoLixNano preparations via intratracheal spray (i.t.), nasal route (i.n.), and nebulization, in vivo imaging technology (IVIS) was used to detect the distribution of PoLixNano preparations in living mice (left) and in vitro organs (right). The figure shows representative results; B.
  • IVIS in vivo imaging technology
  • FIG. 12 Transfection of PoLixNano preparations prepared with different formulations in mice after administration via different routes (i.t., i.n. and nebulization).
  • Exemplary results of the transfection mediated by the PoLixNano formulation and the LNP control formulation in living mice and isolated organs 6 hours after administration via the nasal route i.n.
  • G Representative results of the transfection of the PoLixNano formulation and the LNP control formulation in living mice and isolated organs 6 hours after administration via nebulization inhalation route (nebulization);
  • FIG. 13 Safety study of PoLixNano preparation in mice.
  • A. Six hours after administration of PoLixNano preparation via different routes (intratracheal spray, intranasal spray, and nebulization), the lungs of mice Representative H&E staining results of tissue sections, using control samples of PBS solution and LNP preparation inoculated by it route as negative control and positive control, respectively, scale bar 50 ⁇ m;
  • Figure 14 Fluc-mRNA transfection mediated by PoLixNano formulation and LNP control formulation in C57black 6 (C57BL/6) mice and Sprague-Dawley (SD) rats after administration via different routes (i.t., i.n. and nebulization).
  • A. Exemplary results of transfection induced by the above-mentioned preparations in living C57BL/6 mice and in ex vivo lungs, livers and spleens 6 h after administration by intratracheal spray (i.t.), nasal (i.n.) and nebulization (nebulization) routes (top) and quantitative results of Fluc bioluminescent signals in ex vivo lungs (bottom) (n 3);
  • B Exemplary results of transfection induced by the above-mentioned preparations in living C57BL/6 mice and in ex vivo lungs, livers and spleens 6 h after administration by intratracheal spray (i.t.), nasal (i.n.) and
  • Figure 15 Antigen-specific humoral immune response induced in mice after intratracheal spray (it) inoculation of PoLixNano preparation (RBD-mRNA/PoLixNano) loaded with mRNA encoding the SARS-CoV-2 RBD protein (RBD-mRNA).
  • PBS PBS buffer
  • LNP preparation LNP (it)
  • PoLixNano preparation and LNP preparation LNP (im)
  • FIG. 16 RBD-mRNA/PoLixNano formulation induced efficient and long-lasting adaptive cellular immune response in mice after intratracheal spray (i.t.) inoculation.
  • Figure 17 Challenge study of lethal SARS-CoV-2 ancestral strain and omicron mutant strain against mice immunized with PoLixNano preparations inoculated via intratracheal spray (i.t.).
  • A. Survival rate, weight change curve of mice immunized with PBS, LNP (i.m.), LNP (i.t.) and PoLixNano preparations after challenge with SARS-CoV-2 ancestral strain, and SARS-CoV-2 RNA load in lung tissue (Lungs) or nasal turbinates (Nasal turbinates) of the above mice 3 days after challenge (n 8). Samples of mice that were not challenged and did not undergo any intervention were used as controls (Control); B.
  • FIG. 18 PoLixNano preparation mediated Fluc-mRNA transfection in mice and mediated RBD-mRNA to produce efficient adaptive immune response after nasal administration (in).
  • the kinetic curve of protein expression in live mice and isolated lungs after administration of the preparation in vivo (n 3);
  • the ELISA method was used to detect the RBD antigen-specific IgG antibody titer in serum samples collected from mice immunized with PBS buffer (PBS), LNP control preparation (LNP) and PoLixNano preparation intranasally at 7 days, 14 days, 21 days, 28 days, 35 days, 42 days and 49 days after the first immunization, and at the same time, the BALF of mice 28 days after the first immunization was detected.
  • PBS PBS buffer
  • LNP LNP control preparation
  • PoLixNano preparation intranasally at 7 days, 14 days, 21 days, 28 days, 35 days, 42 days and 49 days after the first immunization, and at the same time, the BALF
  • Figure 19 Study on the transfection of Fluc-mRNA in mice and the induction of adaptive immune response mediated by PoLixNano preparations after intramuscular injection (i.m.).
  • A. Representative images of Fluc bioluminescence in live mice and different ex vivo organs (liver, spleen) and quantitative analysis of related signals (right) of PoLixNano preparations loaded with Fluc-mRNA and LNP control preparations after intramuscular injection (i.m.) (n 3);
  • the term “substantially” refers to the qualitative condition of exhibiting a target feature or characteristic in full or close to full extent or degree.
  • biological and chemical phenomena rarely, if ever, complete and/or proceed to complete or reach or avoid an absolute result. Therefore, the term “substantially” is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • the term “effective” means sufficient to achieve a desired, expected, or intended result.
  • “effective amount,” “therapeutically effective amount,” or “pharmaceutically effective amount” means an amount of the compound that, when administered to a subject or patient for treating a disease, is sufficient to achieve such treatment for the disease.
  • control sample is a sample subjected to the same conditions as a test sample except for the test article.
  • control subject is a subject having the same form of disease as a treated subject and whose age is about the same as that of the treated subject.
  • in vitro refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than in a multicellular organism.
  • in vivo refers to events that occur within multicellular organisms such as humans and non-human animals.
  • the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).
  • gene product refers to the product of a gene such as an RNA transcript, protein, or polypeptide.
  • lipid refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. They are generally divided into at least Three categories: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derivative lipids” such as steroids.
  • Lipid granule refers to a lipid formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a target site.
  • an active agent or therapeutic agent such as a nucleic acid (e.g., mRNA)
  • the active agent or therapeutic agent can be encapsulated in a lipid, thereby protecting the agent from enzymatic degradation.
  • Nucleic acid-lipid granules and methods for preparing them are disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and 20070042031, the disclosures of which are incorporated herein by reference for all purposes.
  • PoLixNano refers to a stable polymer-lipid composition or polymer-lipid particles or polymer-lipid compositions of nucleic acid loads or polymer-lipid particles of nucleic acid loads.
  • PoLixNano represents a composition made of amphiphilic block copolymers and lipids (e.g., cationic lipids, non-cationic lipids, and lipid conjugates to prevent particle aggregation), wherein the nucleic acid (e.g., mRNA, gRNA, siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, or plasmid, including plasmids transcribed by it interfering RNA) is completely or partially encapsulated in lipids.
  • nucleic acid e.g., mRNA, gRNA, siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA,
  • PoLixNano is a term for referring to nucleic acid-polymer-lipid compositions comprising nucleic acids (e.g., mRNA) encapsulated in polymer-lipid compositions.
  • PoLixNano typically contains amphiphilic block copolymers, cationic lipids, non-cationic lipids, and lipid conjugates (e.g., PEG-lipid conjugates).
  • nucleic acids when present in the polymer-lipid particles and/or lipid particles of the present invention, are resistant to degradation by nucleases in aqueous solutions.
  • PoLixNano is very effective for mucosal applications because they can efficiently penetrate the mucus (mucus penetration) present in large quantities in mucosal tissue sites, thereby safely delivering the loaded therapeutic agent (e.g., mRNA) to the target target cells (e.g., respiratory epithelial cells, dendritic cells, etc.), ultimately mediating the expression of transfected genes or silencing of target gene expression in these mucosal-related sites mediated by the therapeutic agent.
  • the loaded therapeutic agent e.g., mRNA
  • target target cells e.g., respiratory epithelial cells, dendritic cells, etc.
  • PoLixNano can also mediate the accumulation of therapeutic agents (e.g., mRNA) in distant sites (e.g., sites on the body separated from the site of administration), and it can mediate the expression of transfected genes or silencing of target gene expression at these distant sites.
  • therapeutic agents e.g., mRNA
  • the polymer-lipid composition particles of the invention typically have a median diameter of about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 80 nm to about 100 nm, and are substantially non-toxic.
  • the term "delivery” encompasses local delivery and systemic delivery.
  • the delivery of mRNA encompasses situations where the mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and retained in the target tissue (also referred to as “local distribution” or “local delivery”), and situations where the mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and secreted into the patient's circulatory system (e.g., serum), and is distributed throughout the body and absorbed by other tissues (also referred to as “systemic distribution” or “systemic delivery”).
  • the patient's circulatory system e.g., serum
  • Distal site refers to a separate site in the body that is not limited to adjacent capillary beds but includes sites that are widely distributed throughout the organism.
  • the term "encapsulation” or grammatical equivalents refers to the process of confining individual mRNA molecules within polymer-lipid nanoparticles.
  • encapsulated can refer to providing a fully encapsulated, partially encapsulated, or both active or therapeutic agents, such as lipid particles of nucleic acids (e.g., mRNA).
  • nucleic acids e.g., mRNA
  • nucleic acids are fully encapsulated in lipid particles (e.g., to form PoLixNano or other nucleic acid-nano particles).
  • cationic lipid refers to any of a number of lipid species carrying a net positive charge at a selected pH value, such as a physiological pH value (e.g., pH is about 7.0). It has been unexpectedly found that cationic lipids comprising alkyl chains with multiple unsaturated sites, for example, at least 2 or 3 unsaturated sites, are particularly effective for forming lipid particles with increased membrane fluidity. Many cationic lipids and related analogs that are also effective for the present invention have been described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Patent Nos.
  • the cationic lipid comprises a protonatable tertiary amine (e.g., pH titrable) head group, a C 18 alkyl chain, an ether bond between the head group and the alkyl chain, and 0 to 3 double bonds.
  • lipids include, for example, DSDMA, DLinDMA, DLenDMA, and DODMA.
  • amphiphilic lipid refers in part to any suitable material, wherein the hydrophobic portion of the lipid material is oriented into the hydrophobic phase, while the hydrophilic portion is oriented into the aqueous phase.
  • the hydrophilic nature comes from the presence of polar or charged groups such as sugars, phosphates, carboxyls, sulfates, aminos, sulfhydryls, nitros, hydroxyls and other similar groups.
  • Hydrophobicity can be imparted by the inclusion of non-polar groups, including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbons and such groups substituted by one or more aromatic, alicyclic or heterocyclic groups.
  • Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino lipids and sphingolipids.
  • phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, Phosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and dilinoleoylphosphatidylcholine.
  • amphipathic lipids Other compounds lacking phosphorus, such as sphingolipids, sphingolipid families, diacylglycerols and beta-acyloxy acids are also included in the group known as amphipathic lipids.
  • amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.
  • neutral lipid refers to any of a number of lipid species that exist as uncharged or neutral zwitterionic forms at a selected pH.
  • lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.
  • non-cationic lipid refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.
  • anionic lipid refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.
  • phosphatidylglycerol cardiolipin
  • diacylphosphatidylserine diacylphosphatidic acid
  • N-dodecanoylphosphatidylethanolamine N-succinylphosphatidylethanolamine
  • N-glutarylphosphatidylethanolamine N-glutarylphosphatidylethanol
  • lipid conjugate refers to a bound lipid that inhibits aggregation of lipid particles.
  • the lipid conjugate includes, but is not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), poly (ethylene glycol) -lipid conjugates (PEG-lipid conjugates), such as PEG coupled to a dialkoxypropyl group, PEG coupled to diacylglycerol, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, PEG coupled to ceramide (see, e.g., U.S. Patent No.
  • PEG can be directly conjugated to a lipid or can be connected to a lipid via a linker moiety.
  • Any linker moiety suitable for connecting PEG and a lipid can be used, such as a linker moiety without an ester and a linker moiety containing an ester. In a preferred embodiment, a linker moiety without an ester is used.
  • N/P ratio refers to the molar ratio of the positively charged molecular units in the cationic lipids in the polymer-lipid composition relative to the negatively charged molecular units in the mRNA encapsulated in the polymer-lipid composition. Therefore, the N/P ratio is usually calculated as the ratio of the molar number of amine groups in the cationic lipids in the polymer-lipid composition relative to the molar number of phosphate groups in the mRNA encapsulated in the polymer-lipid composition.
  • hydrophobic lipid refers to a compound having a non-polar group, including, but not limited to, long chain saturated and unsaturated aliphatic groups and such groups optionally substituted by one or more aromatic, alicyclic or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerols, dialkylglycerols, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.
  • the term "fusogenic” refers to the ability of polymer lipid particles, such as PoLixNano, to fuse with a cell membrane.
  • the membrane may be a plasma membrane or a membrane surrounding a cell organelle, such as an endosome, a lysosome, a cell nucleus, or the like.
  • aqueous solution refers to a composition comprising, in whole or in part, water.
  • organic lipid solution refers to a composition comprising in whole or in part an organic solvent with lipids.
  • “Isomers” of a first compound are individual compounds wherein each molecule contains the same constituent atoms as the first compound, but wherein the three-dimensional configuration of those atoms is different.
  • the term "patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof.
  • the patient or subject is a primate.
  • Non-limiting examples of human subjects are adults, adolescents, infants, and fetuses.
  • pharmaceutically acceptable refers to compounds, materials, compositions and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs and/or body fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable salt” refers to a salt of a compound of the present disclosure that is pharmaceutically acceptable as defined above and that has the desired pharmacological activity.
  • Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid , cinnamic acid,
  • Pharmaceutically acceptable salts also include those in which the acidic protons present are capable of The base addition salt that can be formed when reacting with an inorganic base or an organic base.
  • Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide.
  • Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. It should be recognized that the specific anion or cation that forms a part of any salt of the present disclosure is not critical, as long as the salt is pharmacologically acceptable as a whole.
  • Other examples of pharmaceutically acceptable salts and their preparation methods and methods of use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (PH Stahl & C.G. Wermuth, Verlag Helvetica Chimica Acta, 2002).
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition or vehicle involved in carrying or transporting a medicament, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
  • excipient includes, but is not limited to, any and all solvents, dispersion media or other liquid vehicles, dispersing or suspending agents, diluents, granulating and/or dispersing agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, binders, lubricants or coloring agents, sweeteners or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmotic pressure regulators, pH regulators, buffers, chelating agents, cold protection agents and/or plasticizers for a particular dosage form.
  • Prevention includes: (1) inhibiting the onset of a disease in a subject or patient who may be at risk for and/or susceptible to the disease but does not yet experience or display any or all of the symptoms or signs of the disease; and/or (2) slowing the onset of symptoms or signs of a disease in a subject or patient who may be at risk for and/or susceptible to the disease but does not yet experience or display any or all of the symptoms or signs of the disease.
  • Treating includes (1) inhibiting the disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., arresting further development of the symptoms and/or signs), (2) ameliorating the disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., reversing the symptoms and/or signs), and/or (3) achieving any measurable reduction in the disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease.
  • a “repeat unit” is the simplest structural entity of some material, e.g., a framework and/or a polymer, whether organic, inorganic, or metallo-organic.
  • the repeat units are linked together one after another along the chain, like the beads of a necklace.
  • the repeating unit is -CH2CH2- .
  • the subscript "n” indicates the degree of polymerization, that is, the number of repeating units linked together. When the value of "n" is undefined or in the absence of "n", it simply indicates the repetition of the formula within the brackets and the polymeric nature of the material.
  • repeating unit also applies where the connectivity between repeating units extends three-dimensionally, such as in metal organic frameworks, modified polymers, thermosetting polymers, etc.
  • repeating units may also be described as branching units, inner layers, or generations.
  • end-capping groups may also be described as surface groups.
  • the present invention encompasses the unexpected discovery that the addition of amphiphilic block copolymers such as poloxamine or styrene to LNP formulations (e.g., four-component LNP formulations consisting of ionizable/cationic lipids, neutral lipids, cholesterol, and PEG-lipids; three-component LNP formulations consisting of ionizable/cationic lipids, neutral lipids, and cholesterol; three-component LNP formulations comprising cholesterol-derivatized cationic lipids, neutral lipids, and PEG lipids; two-component LNP formulations comprising cholesterol-derivatized cationic lipids and neutral lipids) can enhance the performance of the LNP formulations.
  • LNP formulations e.g., four-component LNP formulations consisting of ionizable/cationic lipids, neutral lipids, cholesterol, and PEG-lipids
  • three-component LNP formulations consisting of ionizable/
  • the obtained novel composite preparation (referred to herein as a composition comprising polymer-lipid or PoLixNano) can greatly improve the gene transfection efficiency of nucleic acid molecules (such as mRNA) mediated by such preparations in animals (especially in cells related to respiratory tissues) after administration via the mucosal site of an organism, especially the respiratory tract (tracheal spray IT or nasal drops IN or atomized inhalation), so that the nucleic acid molecules can efficiently produce functional proteins in animal respiratory tract-related tissues and non-lung cells/tissues, thereby achieving the purpose of disease prevention or treatment.
  • nucleic acid molecules such as mRNA
  • Polymer-lipid preparations are particularly suitable for treating or preventing diseases related to respiratory tissue via non-invasive routes of administration (e.g., tracheal spray it, nasal drops in, and atomized inhalation, etc.).
  • non-invasive routes of administration e.g., tracheal spray it, nasal drops in, and atomized inhalation, etc.
  • the gene transfection effect produced in the lung/respiratory mucosal tissue of animals is significantly better than that of classic LNP preparations, poloxamine and poloxamer preparations.
  • mRNA delivered via the respiratory route is expected to make breakthroughs in the fields of mucosal vaccines (e.g., new coronavirus vaccines, influenza virus vaccines, tuberculosis vaccines, etc.), lung gene therapy (e.g., cystic fibrosis, ⁇ 1-trypsin deficiency, etc.), and refractory lung diseases (e.g., lung cancer, pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, etc.), solving the problem that there is currently no ideal delivery system in these fields.
  • mucosal vaccines e.g., new coronavirus vaccines, influenza virus vaccines, tuberculosis vaccines, etc.
  • lung gene therapy e.g., cystic fibrosis, ⁇ 1-trypsin deficiency, etc.
  • refractory lung diseases e.g., lung cancer, pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, etc.
  • polymer-lipid nanoformulations of specific formulations delivered via the respiratory route can be translocated after pulmonary delivery, that is, partially or completely moved from the site of administration (lungs) to the systemic blood supply by active or passive means, and then deposited in different non-lung cells or tissues, for example, the liver and/or spleen.
  • This transport of polymer-lipid compositions containing mRNA encoding therapeutic proteins constitutes non-invasive systemic delivery of active ingredients (i.e., mRNA) outside the lungs, resulting in the production of functional proteins in non-lung cells or tissues accessible to the whole body.
  • mRNA active ingredients
  • the main transfection organs of LNP preparations or poloxamine or poloxamer preparations administered under the same conditions are limited to the lungs.
  • the present invention provides a composition comprising a polymer-lipid, the composition comprising:
  • an active agent or therapeutic agent preferably the active agent or therapeutic agent comprises a nucleic acid
  • composition is formulated for delivery through a mucosal site of an organism, such as the respiratory tract, oral mucosa, gastrointestinal tract, ocular mucosa, ear mucosa, urethra, or reproductive tract, preferably the composition is formulated for delivery through the respiratory tract.
  • composition according to the present invention may preferably comprise ribonucleic acid (RNA), such as single-stranded RNA, more preferably messenger RNA (mRNA), such as in vitro transcribed mRNA.
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • the nucleic acid comprises at least one selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) and micro RNA (miRNA), primary-miRNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), plasmid DNA (pDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), deoxyribozyme (DNAzyme), ribozyme (RNAzyme), nucleic acid aptamer (aptamer), clustered regularly interspaced short palindromic repeats (CRISPR)-related nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA, single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA).
  • the composition comprises a first nucleic acid and a second nucleic acid.
  • the first nucleic acid is an mRNA.
  • the second nucleic acid is a single guide RNA.
  • the first nucleic acid is a messenger RNA (mRNA) and a single guide RNA (sgRNA).
  • RNA is a single-stranded RNA.
  • single-stranded RNA means a single continuous ribonucleotide chain, which is distinguished from RNA of a double-stranded molecule formed by hybridization of two or more separated chains.
  • single-stranded RNA does not exclude that the single-stranded molecule itself forms a double-stranded structure, such as a secondary (e.g., ring and stem-loop) structure or a tertiary structure.
  • RNA encompasses RNAs that encode amino acid sequences as well as RNAs that do not encode amino acid sequences.
  • RNA can be prepared by synthetic chemistry and enzymatic methods known to those of ordinary skill in the art, or by using recombinant technology, or can be isolated from natural sources, or by a combination thereof.
  • mRNA Messenger RNA
  • mRNA is a copolymer constructed from phosphate nucleoside building blocks, mainly adenosine, cytidine, uridine and guanosine, which acts as an intermediate carrier to introduce genetic information from DNA in the cell nucleus into the cytoplasm, where it is translated into protein. Therefore, mRNA is suitable as a substitute for gene expression.
  • mRNA should be understood to mean any polyribonucleotide molecule, if it enters a cell, is suitable for protein or its fragmentary expression, or can be translated into protein or its fragment.
  • Term " protein" covers any kind of amino acid sequence in this article, i.e. two or more amino acid whose chains connected by peptide bonds separately, and also comprises peptide and fusion protein.
  • mRNA contains a ribonucleotide sequence, which encodes a protein or a fragment thereof of a function needed or beneficial in or near a cell.
  • mRNA may contain a sequence of a complete protein or its functional variant.
  • a ribonucleotide sequence may encode a protein or its functional fragment acting as a factor, an inducer, a regulator, a stimulant or an enzyme, wherein such a protein is a protein necessary for its function to compensate for an obstacle (particularly a metabolic disorder) or to initiate a process in the body (such as the formation of new blood vessels, tissues, etc.) or to induce the immune system to produce an adaptive immune response.
  • a functional variant means the following fragment: it can assume the function of a protein in a cell, the function of the protein is needed in a cell, or the form of the absence or defect of the protein is pathogenic.
  • mRNA synthesis includes the addition of a "cap” on the 5' end and a “tail” on the 3' end.
  • the presence of the cap is important for providing resistance to nucleases present in most eukaryotic cells.
  • the presence of the "tail” serves to protect the mRNA from exonuclease degradation.
  • the mRNA includes a 5' cap structure.
  • the mRNA includes a 5' and/or 3' untranslated region.
  • the 5' untranslated region includes one or more elements that affect the stability or translation of the mRNA.
  • the 3' untranslated region includes one or more polyadenylation signals, protein binding sites that affect the localization stability of the mRNA in the cell, or one or more miRNA binding sites.
  • the term mRNA used herein encompasses modified mRNA, i.e., the mRNA can be a modified mRNA.
  • the present invention can be used to formulate and encapsulate unmodified mRNA or mRNA containing one or more modifications that generally enhance stability.
  • the modification is selected from modified nucleotides, modified sugar-phosphate backbones, and 5' and/or 3' untranslated regions.
  • the modification of mRNA may include modification of nucleotides of RNA.
  • the modified mRNA according to the present invention may include, for example, backbone modifications, sugar modifications, phosphate modifications, or base modifications.
  • mRNA may be synthesized from naturally occurring nucleotides and/or nucleotide analogs (modified nucleotides), the naturally occurring nucleotides and/or nucleotide analogs including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)) and modified nucleotide analogs or derivatives of purines and pyrimidines, the preparation of such analogs being known to those skilled in the art, for example, from U.S.
  • RNA preferably mRNA
  • all uridine nucleotides and cytidine nucleotides can be modified in the same form, or a mixture of modified nucleotides can be used for each.
  • the modified nucleotides can have natural or non-naturally occurring modifications. Mixtures of various modified nucleotides can be used.
  • one type of nucleotide uses at least two different modifications, wherein the modified nucleotide of one type has a functional group through which other groups can be connected. Nucleotides with different functional groups can also be used to provide binding sites for connecting different groups.
  • the RNA, preferably mRNA, according to the invention is characterized in that the modified uridine is selected from the group consisting of 2-thiouridine, 5-methyluridine, pseudouridine ( ⁇ ), 5-methyluridine 5'-triphosphate (m5U), N-1-methyl-pseudouridine (N1m ⁇ ), N-1-methyl-pseudouridine-triphosphate, 2-thiouridine 5'-triphosphate (S2U), 5-iodouridine 5'-triphosphate (I5U), 4-thiouridine 5'-triphosphate (S4U), 5-bromouridine 5'-triphosphate (Br5U), 2'-methyl-2'-deoxyuridine 5'-triphosphate (U2'm), 2'-amino-2'-deoxyuridine 5'-triphosphate ( U2'NH2 ), 2'-azido-2'-deoxyuridine 5'-triphosphate ( U2'N3 ), and 2'-fluoro-2'
  • the RNA, preferably mRNA, according to the invention is characterized in that the modified cytidine is selected from the group consisting of 5-methylcytidine, 5-hydroxymethylcytidine, 5-methoxycytidine, 3-methylcytidine, 2-thio-cytidine, 2'-methyl-2'-deoxycytidine 5'-triphosphate (C2'm), 2'-amino-2'-deoxycytidine 5'-triphosphate (C2'NH 2 ), 2'-fluoro-2'-deoxycytidine 5'-triphosphate (C2'F), 5-iodocytidine 5'-triphosphate (I5C), 5-bromocytidine 5'-triphosphate (Br5C), 5-methylcytidine 5'-triphosphate (m5C), 2-thiocytidine 5'-triphosphate (S2C) and 2'-azido-2'-deoxycytidine 5'-triphosphat
  • the RNA, preferably mRNA, according to the invention is characterized in that the modified adenosine is selected from the group consisting of N6-methyladenosine 5'-triphosphate (m6A), N1-methyladenosine 5'-triphosphate (m1A), 2'-O-methyladenosine 5'-triphosphate (A2'm), 2'-amino-2'-deoxyadenosine 5'-triphosphate (A2'NH 2 ), 2'-azido-2'-deoxyadenosine 5'-triphosphate (A2'N 3 ) and 2'-fluoro-2'-deoxyadenosine 5'-triphosphate (A2'F).
  • m6A N6-methyladenosine 5'-triphosphate
  • m1A N1-methyladenosine 5'-triphosphate
  • A2'm 2'-O-methyladenosine 5'-triphosphate
  • the RNA, preferably mRNA, according to the invention is characterized in that the modified guanosine is selected from N1-methylguanosine 5-triphosphate (m1G), 2'-O-methylguanosine 5'-triphosphate (G2'm), 2-amino-2-deoxyguanosine 5'-triphosphate (G2'NH 2 ), 2'-azido-2'-deoxyguanosine 5'-triphosphate (G2'N 3 ), 2'-fluoro-2'-deoxyguanosine 5'-triphosphate (G2'F).
  • m1G N1-methylguanosine 5-triphosphate
  • G2'm 2-amino-2-deoxyguanosine 5'-triphosphate
  • G2'N 3 2-azido-2'-deoxyguanosine 5'-triphosphate
  • G2'F 2'-fluoro-2'-deoxyguanosine 5'-triphosphate
  • the invention provides oligonucleotides comprising nucleosides connected.
  • nucleosides can be linked together using any nucleoside bond.
  • the two main categories of nucleoside linking groups are limited by the presence or absence of a phosphorus atom. Compared with natural phosphodiester bonds, modified bonds can be used to change (usually increase) the nuclease resistance of oligonucleotides.
  • nucleoside bonds with chiral atoms can be prepared as racemic mixtures or separate enantiomers. Representative chiral bonds include, but are not limited to, alkyl phosphonates and thiophosphates. The preparation methods of phosphorus-containing and non-phosphorus-containing nucleoside bonds are well known to those skilled in the art.
  • U.S. patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Patents 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118, 802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958, 013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,
  • the mRNA is an mRNA containing a combination of modified and unmodified nucleotides.
  • it is an mRNA containing a combination of modified and unmodified nucleotides as described in WO2011/012316.
  • the mRNA described therein shows improved stability and reduced immunogenicity.
  • mRNA contains labeled nucleic acid (preferably, nucleotides and/or ribonucleotides), such as, for example, isotope- and/or fluorescently labeled nucleotides.
  • labeled mRNA molecules play an important role in, for example, studying the intracellular conformation of RNA and DNA molecules and the distribution in animals and or cells.
  • mRNA can be synthesized according to any of a variety of known methods.
  • mRNA according to the present invention can be synthesized via in vitro transcription (IVT).
  • modified RNA preferably mRNA molecules can be chemically synthesized, for example, by conventional chemical synthesis on an automated nucleotide sequence synthesizer using a solid support and standard techniques, or by chemical synthesis of the corresponding DNA sequence and subsequent transcription thereof in vitro or in vivo.
  • mRNA provided by an in vitro transcription reaction is desirable, other sources of mRNA are contemplated within the scope of the present invention, including mRNA produced from bacteria, fungi, plants, and/or animals.
  • the mRNA can be combined with a target binding site, a targeting sequence and/or with a microRNA binding site to allow the activity of the desired mRNA only in the relevant cells.
  • the RNA can be combined with a microRNA or shRNA downstream of the 3' polyA tail.
  • in vitro synthesized mRNA can be purified prior to formulation and encapsulation to remove undesirable impurities (including various enzymes and other reagents used in the mRNA synthesis process).
  • the present invention can be used to prepare and encapsulate mRNAs of various lengths.
  • the present invention can be used to prepare and encapsulate in vitro synthesized mRNAs having a length equal to or greater than about 0.1 kb, 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb.
  • the present invention can be used to prepare and encapsulate in vitro synthesized mRNAs having a length of about 0.1-2 kb, about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb.
  • the present invention can achieve the effect of disease treatment, disease prevention and disease diagnosis through the interaction of ribonucleic acid with cell molecules and organelles.
  • This interaction can activate the innate immune system alone, such as certain CpG oligonucleotides and sequences designed to interact specifically with toll-like receptors and other extracellular or intracellular receptors.
  • ribonucleic acid preferably mRNA
  • the intake or introduction of ribonucleic acid (preferably mRNA) in the cell can be intended to cause nucleotide sequences, such as the expression of genes included in ribonucleic acid (preferably mRNA); it can be intended to be used for the expression of endogenous genes caused by the presence of introduced exogenous nucleic acid in the cell, silencing or knocking down; or it can be intended for the modification of endogenous nucleic acid sequences, such as the repair, excision, insertion or exchange of the whole stretches of the selected base or endogenous nucleic acid sequence; or it can be intended for the presence and interaction of introduced exogenous ribonucleic acid (preferably mRNA) in the cell and actually interfere with any cellular process.
  • nucleotide sequences such as the expression of genes included in ribonucleic acid (preferably mRNA)
  • endogenous genes caused by the presence of introduced exogenous nucleic acid in the cell, sile
  • the overexpression of the exogenous ribonucleic acid (preferred mRNA) introduced can be intended to compensate or complement endogenous gene expression, particularly in the case of endogenous gene defect or silence, causing gene expression product to be absent, insufficient or defective or malfunctioning, multiple metabolic and hereditary diseases, such as cystic fibrosis, hemophilia or muscular dystrophy, etc. are often such situations.
  • the overexpression of the exogenous ribonucleic acid (preferred mRNA) introduced can also be intended to make expression product interact with any endogenous cell process or interfere with any endogenous cell process, such as the regulation, signal transduction and other cell processes of gene expression.
  • the overexpression of the exogenous ribonucleic acid (preferred mRNA) introduced can also be intended to cause immune response in the background of the organism in which the cell of transfection or transduction resides or resides.
  • Example is the genetic modification of antigen presenting cells (such as dendritic cells) so that it presents antigens for vaccination purposes.
  • Other examples are the overexpression of cytokines in tumors, so as to cause tumor-specific immune response.
  • overexpression of introduced exogenous ribonucleic acid (preferably mRNA) can also be aimed at generating transiently genetically modified cells in vivo or ex vivo for cell therapy, such as modified T cells or precursors or stem cells or other cells for regenerative medicine.
  • the present invention can also realize the downregulation, silencing or knocking down of endogenous gene expression for therapeutic purposes by RNA interference (RNAi), using ribozymes, antisense oligonucleotides, tRNA, long double-stranded RNA.
  • RNA interference RNA interference
  • the downregulation, silencing or knocking down of endogenous gene expression of endogenous or pre-existing gene expression can be used to treat acquired, hereditary or spontaneous diseases, including viral infection and cancer.
  • nucleic acid can be introduced into cells as a preventive measure to practice, to prevent, for example, viral infection or tumor formation.
  • gene repair, base or sequence changes can be realized at the genome level and mRNA level (including exon skipping).
  • Base or sequence changes can be realized, for example, by RNA-guided site-specific DNA cutting, by utilizing trans-splicing, trans-splicing ribozymes, chimeric repair bodies (chimeraplasts), the shear and paste mechanism of RNA trans-splicing mediated by spliceosomes, or by utilizing class II or re-targeted introns, or by utilizing viral-mediated insertion mutagenesis or utilizing the targeted genome insertion using prokaryotic, eukaryotic or viral integrase systems.
  • RNA preferably mRNA therapeutic methods.
  • the disorder caused by a single gene mutation such as cystic fibrosis, hemophilia and multiple other diseases can be dominant or recessive.
  • polygenic disorders are caused by two or more genes, and the manifestation of the corresponding disease is often variable and related to environmental factors.
  • the example of polygenic disorder is hypertension, elevated cholesterol levels, cancer, neurodegenerative diseases, mental illness, etc.
  • the therapeutic RNA, preferably mRNA, representing one or more of these genes can be beneficial to those patients.
  • genetic disorders are not necessarily transmitted from parental genes, and may also be caused by new mutations.
  • the therapeutic RNA, preferably mRNA, representing the correct gene sequence can be beneficial to patients.
  • the coding sequence of the RNA, preferably mRNA, of the present invention can be transcribed and translated into a partial or full-length protein comprising a level of cellular activity equal to or greater than that of the native protein.
  • a genetic disease may be involved, for example, one that affects the lungs, such as SPB (surfactant protein B) deficiency, ABCA3 deficiency, cystic fibrosis (CF), primary ciliary dyskinesia, asthma, chronic obstructive pulmonary disease (COPD), and alpha 1-antitrypsin deficiency, or one that affects plasma proteins, such as congenital hemochromatosis (hepcidin Deficiency of the immune system, such as hemophilia a and b, and complement deficiencies (such as protein C deficiency), immunodeficiencies such as SCID (caused by mutations in various genes such as RAG1, RAG2, JAK3, IL7R, CD45, CD3 ⁇ , CD3 ⁇ ) or deficiency, due to a lack of adenosine deaminase, such as (ADA-SCID), septic granulomatosis (caused by mutations in the gp-91-pho
  • SPB
  • RNA preferably mRNA
  • Other diseases for which the RNA, preferably mRNA, of the present invention may exert a therapeutic effect include, for example, SMN1-related spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-related galactosemia; SLC3A1-related disorders, including cystinuria; COL4A5-related disorders, including Alport syndrome; galactocerebrosidase deficiency; X-linked leukoreflex atrophy and adrenoleukodystrophy; Friedreich's ataxia; Perelman-Merzbacher disease; TSC1 and TSC2-related tuberous sclerosis; Sanfilip B syndrome MPS IIIB; CTNS-related cystinosis; FMR1-related disorders, including fragile X syndrome, fragile X-linked tremor/ataxia syndrome, and fragile X premature ovarian failure syndrome; Prader-Willi syndrome; hereditary hemorrhagic tel
  • a protein such as an enzyme
  • RNA preferably mRNA
  • Transcript replacement therapy/enzyme replacement therapy does not affect the underlying genetic defect, but increases the concentration of the enzyme that the patient lacks.
  • GAA acid alpha-glucosidase
  • EPO erythropoietin
  • somatotropin somatotropin, hGH
  • cystic fibrosis transmembrane conductance regulator CFTR
  • growth factors such as GM-SCF, G-CSF, MPS, protein C, hepcidin, ABCA3 and surfactant protein B.
  • RNA according to the invention contains a coding sequence for cystic fibrosis transmembrane conductance regulator (CFTR), ⁇ 1-antitrypsin, Dynein axonemal intermediate chain 1 (DnaI1), surfactant protein B (SP-B) or erythropoietin.
  • proteins that can be encoded by the inventive RNA, preferably mRNA, according to the invention are growth factors, such as the human growth hormone hGH, BMP-2 or angiogenic factors.
  • the RNA may contain a ribonucleotide sequence encoding a full-length antibody or nanobody (e.g., both heavy and light chains), which may be used in therapeutic settings, such as to confer immunity to a subject.
  • a full-length antibody or nanobody e.g., both heavy and light chains
  • a full-length antibody or nanobody e.g., both heavy and light chains
  • the RNA can encode a functional monoclonal or polyclonal antibody that can be used to target and/or inactivate a biological target (e.g., a stimulatory cytokine such as tumor necrosis factor).
  • a biological target e.g., a stimulatory cytokine such as tumor necrosis factor
  • the RNA, preferably mRNA sequence can encode a functional anti-nephrotic factor antibody, for example, for the treatment of type II membranoproliferative glomerulonephritis or acute hemolytic uremic syndrome, or alternatively can encode an anti-vascular endothelial growth factor (VEGF) antibody for the treatment of VEGF-mediated diseases such as cancer.
  • VEGF vascular endothelial growth factor
  • RNA may contain a ribonucleotide sequence encoding a polypeptide or protein that can be used for genome editing technology.
  • a variety of genome editing systems using different polypeptides or proteins are known in the art, i.e., for example, CRISPR-Cas systems, large-range nucleases (homing nucleases, meganucleases), zinc finger nucleases (ZFNs), and nucleases (TALENs) based on transcription activator-like effectors.
  • CRISPR-Cas systems large-range nucleases (homing nucleases, meganucleases), zinc finger nucleases (ZFNs), and nucleases (TALENs) based on transcription activator-like effectors.
  • ZFNs zinc finger nucleases
  • TALENs nucleases
  • RNA may contain a ribonucleotide sequence encoding a polypeptide or protein of the Cas (CRISPR-associated protein) protein family, preferably Cas9 (CRISPR-associated protein 9).
  • Cas Cas9
  • Proteins of the Cas protein family, preferably Cas9 may be used in CRISPR/Cas9-based methods and/or CRISPR/Cas9 genome editing techniques. Nat. Biotechnol., 2014, 32(4): 347-355 reviews CRISPR-Cas systems for genome editing, regulation and targeting.
  • the RNA may contain a ribonucleotide sequence encoding a transcription activator-like effector nuclease (TALEN).
  • the RNA preferably the mRNA, may contain a ribonucleotide sequence encoding a zinc finger nuclease (ZFN).
  • the RNA preferably the mRNA, may contain a ribonucleotide sequence encoding a meganuclease.
  • RNA contains the ribonucleotide sequence that is not expressed into protein or polypeptide.
  • RNA should not be merely interpreted as meaning any such polynucleotide molecule: if it is introduced into a cell, it can be translated into polypeptide/protein or its fragment.
  • RNA contains the ribonucleotide sequence that is only transcribed into (functional) RNA, wherein the RNA is the final product (and therefore, does not need to be translated).
  • RNA contains the ribonucleotide sequence, which preferably provides the genetic information of the siRNA sequence or another desired ribonucleotide sequence.
  • the present invention provides a method for preparing a therapeutic composition comprising a polymer-lipid as described herein for delivering a specific antigen and/or a nucleic acid encoding a specific antigen, wherein the antigen may be an antigen from bacteria, virus, fungus or cancer cell.
  • antigen refers to a peptide or nucleotide-based biological material (natural, recombinant or synthetic) that stimulates a protective immune response in an animal.
  • Antigens suitable for the present invention can be amino acid sequences such as peptides or proteins, or nucleic acid sequences such as genomic DNA, cDNA, mRNA, saRNA, circRNA, tRNA, rRNA, small interfering RNA (iRNA) hybridization sequences, or modified or unmodified synthetic or semisynthetic oligonucleotide sequences.
  • Antigens suitable for the present invention may be obtained from an organism selected from the group consisting of bacteria, viruses, parasites, rickettsiae, protozoa and cancer cells.
  • polymer-lipid compositions of the invention can be used to treat or prevent a number of diseases and disorders, such as:
  • Retroviridae e.g., human immunodeficiency virus, including HIV-1
  • Flaviviridae e.g., dengue virus, encephalitis virus, yellow fever virus
  • Coronaviridae e.g., coronavirus
  • Rhabdoviridae e.g., vesicular stomatitis virus, rabies virus
  • Filoviridae e.g., Ebola virus
  • Paramyxoviridae e.g., parainfluenza virus, mumps virus
  • virus measles virus, respiratory syncytial virus
  • Orthomyxoviridae e.g., influenza virus
  • Reoviridae e.g., reovirus, orbivirus, and rotavirus
  • Binaviridae Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Herpesviridae (herpes simplex virus (HSV
  • the antigens selected for the polymer-lipid composition are derived from those antigens present in naturally occurring viruses (or expressed/induced during infection) (or designed with reference to those antigens).
  • H. pylori Helicobacter pylori, Legionella pneumophilia, Mycobacterium (e.g., M. tuberculosis, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Streptococcus agalactiae) (Group B Streptococcus), Streptococcus viridans, Streptococcus pneumoniae, Klebsiella spp. (including K.
  • Mycobacterium e.g., M. tuberculosis, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes
  • the antigens selected for the polymer-lipid composition are derived from those antigens present in naturally occurring bacteria (or expressed/induced during infection) (or artificially designed with reference to those antigens).
  • Cryptococcus neoformans Cryptococcus neoformans
  • Histoplasma capsulatum Coccidioides immitis
  • Blastomyces dermatitidis Chlamydia trachomatis
  • Candida albicans in which the antigen selected for the vaccine is derived from an existing Those antigens that exist in naturally occurring fungi (or are expressed/induced during infection) (or designed with reference to those antigens).
  • Plasmodium spp. including Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax
  • Toxoplasma spp. including T. gondii and T. cruzii
  • Cancers of the blood and lymphatic system including Hodgkin's disease, leukemia, lymphoma, multiple myeloma, and Diseases), melanoma (including melanoma of the eye), adenoma, sarcoma, cancer of solid tissue, melanoma, lung cancer, thyroid cancer, salivary gland cancer, leg cancer, tongue cancer, lip cancer, bile duct cancer, pelvic cancer, mediastinal cancer, urethral cancer, Kaposi's sarcoma (e.g., when associated with AIDS); skin cancer (including malignant melanoma), digestive tract cancer (including head and neck cancer, esophageal cancer, stomach cancer, pancreatic cancer, liver cancer, colon and rectal cancer, anal cancer), reproductive and urinary system cancer (including kidney cancer, bladder cancer, testicular cancer, prostate cancer), female cancer (including breast cancer, cervical cancer, ovarian cancer, gynecological cancer and chorio
  • helminths e.g. Schistosoma spp.
  • the antigen useful in the present invention is mRNA.
  • the antigen is naturally combined with the polymer-lipid composition of the present invention in an immunogenic effective amount.
  • Immunogenic effective amount means that when a polymer-lipid composition of the present invention and an antigen (e.g., mRNA) are administered to an individual, the antigen contains protective components at a concentration sufficient to protect the animal from the target disease.
  • an immunogenic effective amount of an antigen an amount of 0.01-100 ⁇ g can be mentioned.
  • the present invention provides a method for preparing a composition as described herein comprising delivering an immunomodulator and/or an mRNA encoding an immunomodulator.
  • the immunomodulator includes, but is not limited to, for example, interleukin 2 (IL-2), interleukin 12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 23 (IL-23), C-C domain chemokine ligand 28 (CCL28), interleukin 36 ⁇ (IL-36 ⁇ ), constitutively active variants of interferon gene (STING) protein stimulators, etc.
  • IL-2 interleukin 2
  • IL-12 interleukin 12
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • IL-23 interleukin 23
  • CCL28 C-C domain chemokine ligand 28
  • IL-36 ⁇ constitutively active variants of interferon gene (STING) protein stimulators, etc.
  • particles for use in the context of the present invention may comprise a single type of RNA, but may alternatively comprise a combination of two or more types of RNA, e.g. two or more types of RNA in a single particle.
  • the present invention may be in the form of particles containing one or more types of RNA, or in the form of a mixture of particles containing different types of RNA.
  • the polymer-lipid composition may further comprise one or more proteins.
  • proteins may include enzymes such as nucleases.
  • the compositions described herein may include one or more CRISPR-related proteins (e.g., CRISPR enzymes), including Cas proteins. These enzymes are known; for example, the amino acid sequence of Streptococcus pyogenes (S. pyogenes) Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.
  • the protein in the compositions described herein can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae (S. pneumonia)).
  • the CRISPR enzyme can direct the cleavage of one or both strands at the location of the target sequence, such as within the target sequence and/or within the complement of the target sequence.
  • the CRISPR enzyme can be mutated relative to the corresponding wild-type enzyme so that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence.
  • an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaving a single strand).
  • the Cas9 nickase can be used in combination with a guide sequence (e.g., two guide sequences) that target the sense and antisense strands of a DNA target, respectively. This combination allows both strands to be nicked and used to induce NHEJ or HDR.
  • the present disclosure provides compounds containing one or more therapeutic proteins.
  • Therapeutic proteins that can be included in the composition include a wide range of molecules such as cytokines, chemokines, interleukins, interferons, growth factors, coagulation factors, anticoagulants, blood factors, bone morphogenic proteins, immunoglobulins, and enzymes.
  • EPO erythropoietin
  • G-CSF granulocyte colony stimulating factor
  • ⁇ -galactosidase A ⁇ -L-iduronidase
  • rhASB N-acetylgalactosamine-4-sulfatase
  • TPA tissue-type plasminogen activator
  • IF interferon
  • IF interferon
  • rHGH human growth hormone
  • BHI human insulin
  • FSH follicle-stimulating hormone
  • amphiphilic block copolymers can be used to practice the present invention.
  • the amphiphilic block copolymers are also referred to as "surfactants” or “nonionic surfactants” or “nonionic amphiphilic block copolymers” or “block copolymers” or “amphiphilic polymers” or “polymers”.
  • block copolymer refers to a polymer comprising at least two groups or blocks of polymerized monomer units.
  • Block refers to a motif obtained by polymerization of monomers, which may be repeated within the polymer.
  • a block copolymer must contain blocks of at least two different types of polymerized monomers.
  • amphiphilic block copolymer refers to a block copolymer comprising at least one hydrophilic block and at least one hydrophobic block.
  • the blocks are nonionic, ie, do not comprise ion-forming moieties.
  • the amphiphilic block copolymer is a tetrafunctional amphiphilic block copolymer, wherein the tetrafunctional amphiphilic block copolymer comprises four branched block copolymers each comprising at least one hydrophilic block and at least one hydrophobic block, or the amphiphilic block copolymer is a linear amphiphilic block copolymer, wherein the linear amphiphilic block copolymer comprises a block copolymer of at least one hydrophilic block and at least one hydrophobic block.
  • the hydrophilic block is selected from polyoxyalkylenes, polyvinyl alcohol, polyvinyl pyrrolidone, poly(2-methyl-2-oxazoline) and sugars, and/or the hydrophobic block is selected from polyoxyalkylenes, fatty chains, alkylene polyesters, polyethylene glycol with benzyl polyether ends and cholesterol.
  • the hydrophilic block comprises polyethylene oxide units and the hydrophobic block comprises polypropylene oxide units.
  • the amphiphilic block copolymer comprises at least one selected from the group consisting of poloxamine or ), poloxamer or ), polyoxyethylene glycol dehydrated alcohol alkyl esters (polysorbates), polyvinyl pyrrolidone (PVP), polyethylene glycol ethers (BRIJ), polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, sorbitan and their derivatives.
  • the amphiphilic block copolymer comprises 0.1%-98.0% by weight of the composition, e.g., 0.5%-95.0% by weight, 5.0%-85.0% by weight, 10.0%-80.0% by weight, 20.0%-75.0% by weight, 30.0%-70.0% by weight, or 40.0%-60.0% by weight.
  • the poloxamines suitable for the present invention are also known as tetrafunctional nonionic amphiphilic block copolymers.
  • the tetrafunctional nonionic amphiphilic block copolymer of the present invention preferably comprises a hydrophilic block and a hydrophobic block in a hydrophilic block/hydrophobic block ratio of 0.5 to 1.5, preferably 0.8 to 1.3, more preferably 0.8 to 1.2.
  • the tetrafunctional nonionic amphiphilic tetrafunctional block copolymer useful in the present invention can be a (A-B)n-C branched block copolymer, wherein A represents a hydrophilic block, B represents a hydrophobic block, C represents a connecting portion, and n is 4 and represents the number of (A-B) groups connected to C.
  • the hydrophilic block A is a polyethylene oxide block (PEO)
  • the hydrophobic block B is a polypropylene oxide block (PPO).
  • the linking moiety C may be an alkylenediamine moiety, preferably an ethylenediamine moiety.
  • the tetrafunctional nonionic amphiphilic block copolymer useful in the present invention may be of the following structure:
  • i has a value of about 2 to about 225, particularly about 10 to about 100, more particularly about 10 to about 60, and j has a value of 2 to about 185, particularly about 10 to about 50, particularly about 10 to about 20, more particularly equal to or greater than 13,
  • R* is an alkylene group having 2 to 6 carbons, a cycloalkylene group having 5 to 8 carbons or a phenylene group, and is preferably an ethylene group
  • R1 and R2 either (a) both are hydrogen, or (b) one is hydrogen and the other is methyl,
  • R3 and R4 either (a) both are hydrogen, or (b) one is hydrogen and the other is methyl, and
  • R3 and R4 are both hydrogen, then one of R5 and R6 is hydrogen and the other is methyl, or if one of R3 and R4 is methyl, then R5 and R6 are both hydrogen.
  • nonionic amphiphilic tetrafunctional block copolymer useful in the present invention may be of the following structure:
  • the value of i is from about 2 to about 225, particularly from about 10 to about 100, more particularly from about 10 to about 60, and
  • the value of j is about 2 to about 185, particularly about 10 to about 50, particularly about 10 to about 20, and more particularly equal to or greater than 13,
  • R1 , R2 pair wherein for each R1 , R2 pair, one shall be hydrogen and the other shall be methyl.
  • i may be about 5 to about 125, particularly about 10 to about 100, more particularly about 10 to about 60, and j may be about 5 to about 50, particularly about 10 to about 25, particularly about 10 to about 20, more particularly equal to or greater than 13.
  • nonionic amphiphilic tetrafunctional block copolymers described herein have the structure of the following formula:
  • the value of i is from about 2 to about 225, particularly from about 10 to about 100, more particularly from about 10 to about 60, and
  • the value of j is about 2 to about 185, particularly about 10 to about 50, particularly about 10 to about 20, and more particularly is equal to or greater than 13,
  • R1 , R2 pair wherein for each R1 , R2 pair, one shall be hydrogen and the other shall be methyl.
  • i may be about 5 to about 125, particularly about 10 to about 100, more particularly about 10 to about 60, and j may be about 5 to about 50, particularly about 10 to about 25, particularly about 10 to about 20, more particularly equal to or greater than 13.
  • the molecular weight of the nonionic amphiphilic tetrafunctional block copolymer of the present invention may be 1,000 to 35,000, particularly 4,500 to 30,000, and more particularly 5,000 to 25,000.
  • the nonionic amphiphilic tetrafunctional block copolymer can be more specifically mentioned as having a molecular weight of 1650 g/mol and a PEO/PPO ratio of 15:16 (e.g., Poloxamine 304); or having a molecular weight of 5500 g/mol and a PEO/PPO ratio of 50:56 (e.g., Poloxamine
  • the nonionic amphiphilic tetrafunctional block copolymer of the present invention may comprise, preferably consists of, an ethylene oxide unit content of about 40%, particularly about 45%, particularly about 45% to about 80%, particularly about 45-70%, more particularly about 45-60%, more preferably about 50%.
  • tetrafunctional nonionic amphiphilic block copolymers of the present invention are generally known by the trade names "poloxamines" or Commercially available.
  • poloxamines suitable for use in the present invention may be found in Surfactant Systems, Eds. Attwood and Florence, Chapman and Hall, London 1983, p 356-361; The Condensed Encyclopaedia of Surfactants, Ed. Ash and Ash, Edward Arnold, London, 1989; Non-ionic Surfactants, pp. 300-371, Ed. Nace, Dekker, New York, 1996; Santon, Am. Perfumer Cosmet. 72(4): 54-58 (1958); (Dekker, N.Y., 1967); or US6,353,055.
  • a suitable poloxamine is poloxamine 304. In some embodiments, a suitable poloxamine is poloxamine 701. In some embodiments, a suitable poloxamine is poloxamine 704. In some embodiments, a suitable poloxamine is poloxamine 901. In some embodiments, a suitable poloxamine is poloxamine 904. In some embodiments, a suitable poloxamine is poloxamine 908. In some embodiments, a suitable poloxamine is poloxamine 1107. In some embodiments, a suitable poloxamine is poloxamine 1301. In some embodiments, a suitable poloxamine is poloxamine 1304. In some embodiments, a suitable poloxamine is poloxamine 1307. In some embodiments, a suitable poloxamine is poloxamine 90R4. In some embodiments, the suitable poloxamine is poloxamine 150R1. In some embodiments, the suitable poloxamine is a combination thereof.
  • amphiphilic block copolymers of the present invention can improve their targeting characteristics by chemical structure modification, such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification.
  • chemical structure modification such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification.
  • the glycosylated amphiphilic block copolymer comprises at least one terminal block conjugated to a glycosyl moiety, preferably a terminal hydrophilic block, and more preferably at least 25%, especially at least 50%, especially at least 75%, and more especially at least 100% of the terminal blocks of the glycosylated amphiphilic block copolymer are conjugated to the glycosyl moiety.
  • the glycosyl moiety can be conjugated to the block copolymer of the present invention by a covalent bond formed between a functional group of the glycosyl moiety and a functional group of the block copolymer.
  • the covalent bond can be formed by a reaction between two functional groups modified to be reactive, and the glycosyl moiety can be directly conjugated to the block copolymer. Alternatively, the glycosyl moiety can be conjugated to the block copolymer through a spacer.
  • Poloxamer (Poloxamer or )
  • suitable amphiphilic polymers are poloxamer.
  • suitable poloxamer has the following structure (PEO-PPO-PEO, i.e., polyethylene oxide-polypropylene oxide-polyethylene oxide structure):
  • a is an integer between 10 and 150
  • b is an integer between 20 and 60.
  • a is about 12 and b is about 20, or a is about 80 and b is about 27, or a is about 64 and b is about 37, or a is about 141 and b is about 44, or a is about 101 and b is about 56.
  • poloxamers suitable for the present invention have from about 10 to about 150 ethylene oxide units. In some embodiments, poloxamers have from about 10 to about 100 ethylene oxide units.
  • "reverse-poloxamer” i.e., poloxamer having a polypropylene oxide-polyethylene oxide-polypropylene oxide structure
  • a suitable "reverse-poloxamer” has the following structure (PPO-PEO-PPO):
  • a is an integer between 10 and 150, and b is an integer between 20 and 60.
  • reverse-poloxamers suitable for the present invention have from about 10 to about 150 ethylene oxide units. In some embodiments, “reverse-poloxamers” have from about 10 to about 100 ethylene oxide units.
  • a suitable poloxamer is poloxamer 84. In some embodiments, a suitable poloxamer is poloxamer 101. In some embodiments, a suitable poloxamer is poloxamer 105. In some embodiments, a suitable poloxamer is poloxamer 108. In some embodiments, a suitable poloxamer is poloxamer 122. In some embodiments, a suitable poloxamer is poloxamer 123. In some embodiments, a suitable poloxamer is poloxamer 124. In some embodiments, a suitable poloxamer is poloxamer 181. In some embodiments, a suitable poloxamer is poloxamer 182.
  • a suitable poloxamer is poloxamer 183. In some embodiments, a suitable poloxamer is poloxamer 184. In some embodiments, a suitable poloxamer is poloxamer 185. In some embodiments, a suitable poloxamer is poloxamer 188. In some embodiments, a suitable poloxamer is poloxamer 212. In some embodiments, a suitable poloxamer is poloxamer 215. In some embodiments, a suitable poloxamer is poloxamer 217. In some embodiments, a suitable poloxamer is poloxamer 231. In some embodiments, a suitable poloxamer is poloxamer 234.
  • a suitable poloxamer is poloxamer 235. In some embodiments, a suitable poloxamer is poloxamer 237. In some embodiments, a suitable poloxamer is poloxamer 238. In some embodiments, a suitable poloxamer is poloxamer 282. In some embodiments, a suitable poloxamer is poloxamer 284. In some embodiments, a suitable poloxamer is poloxamer 288. In one In some embodiments, a suitable poloxamer is poloxamer 304. In some embodiments, a suitable poloxamer is poloxamer 331. In some embodiments, a suitable poloxamer is poloxamer 333.
  • a suitable poloxamer is poloxamer 334. In some embodiments, a suitable poloxamer is poloxamer 335. In some embodiments, a suitable poloxamer is poloxamer 338. In some embodiments, a suitable poloxamer is poloxamer 401. In some embodiments, a suitable poloxamer is poloxamer 402. In some embodiments, a suitable poloxamer is poloxamer 403. In some embodiments, a suitable poloxamer is poloxamer 407. In some embodiments, suitable poloxamers are combinations thereof.
  • suitable poloxamers have an average molecular weight of about 4,000 g/mol to about 20,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 1,000 g/mol to about 50,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 1,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 2,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 3,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 4,000 g/mol.
  • suitable poloxamers have an average molecular weight of about 5,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 6,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 7,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 8,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 9,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 10,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 20,000 g/mol.
  • suitable poloxamers have an average molecular weight of about 30,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 40,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 50,000 g/mol.
  • the amphiphilic block copolymers of the present invention can improve their targeting characteristics by chemical structure modification, such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification.
  • chemical structure modification such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification.
  • glycosylation modification as an example, the glycosylated amphiphilic block copolymer comprises at least one terminal block conjugated to a glycosyl portion, preferably a terminal hydrophilic block, and more preferably at least 25%, especially at least 50%, of the glycosylated amphiphilic block copolymer.
  • the glycosyl moiety may be conjugated to the block copolymer of the invention via a covalent bond formed between one functional group of the glycosyl moiety and one functional group of the block copolymer.
  • the covalent bond may be formed by a reaction between two functional groups which are themselves modified to be reactive, and the glycosyl moiety may be conjugated directly to the block copolymer.
  • the glycosyl moiety may be conjugated to the block copolymer via a spacer.
  • the amphiphilic block copolymer is polyvinylpyrrolidone (PVP), such as PVP having a molecular weight of 3 kDa, 10 kDa, or 29 kDa.
  • PVP polyvinylpyrrolidone
  • the amphiphilic block copolymer is polyethylene glycol ether (BRIJ), polysorbate, sorbitan and their derivatives. In some embodiments, the amphiphilic polymer is a polysorbate, such as PS 20.
  • amphiphilic block copolymer is a polyethylene glycol ether.
  • a suitable polyethylene glycol ether is a compound of formula (S-1):
  • R 1BRIJ is C alkyl.
  • the polyethylene glycol ether is a compound of formula (S-1a):
  • S is an integer between 1 and 100.
  • R 1BRIJ is C alkenyl.
  • a suitable polyethylene glycol ether is a compound of formula (S-1b):
  • S is an integer between 1 and 100.
  • amphiphilic block copolymers of the present invention can improve their targeted delivery characteristics by chemical structure modification, such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification.
  • chemical structure modification such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification.
  • the glycosylated amphiphilic block copolymer comprises at least one terminal block conjugated to a glycosyl portion, preferably a terminal hydrophilic block, and more preferably at least 25%, especially at least 50%, especially at least 75%, and more especially at least 100% of the terminal blocks of the glycosylated amphiphilic block copolymer are conjugated to the glycosyl portion.
  • the glycosyl portion can be conjugated to the block copolymer of the present invention by a covalent bond formed between a functional group of the glycosyl portion and a functional group of the block copolymer.
  • the covalent bond can be formed by a reaction between two functional groups that are modified to be reactive, and the glycosyl portion can be directly conjugated to the block copolymer.
  • the glycosyl moiety can be conjugated to the block copolymer via a spacer.
  • cationic lipid refers to any of a number of lipid and lipidoid species having a net positive charge at a selected pH, such as physiological pH.
  • the cationic lipid of the present invention comprises at least one selected from the group consisting of permanent cationic lipids, ionizable cationic lipids, cholesterol-derived cationic lipids, and dendritic polymers or dendrons.
  • Such lipids include, but are not limited to, DOTMA, DOSPA, DOTAP, ePC, DODAP, DODMA, DDAB, DSDMA, DODAC, DOAP, DMRIE, DOGS, DMOBA, HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4005, DLin-MC3-DMA, DLin-KC2-DMA, Acuitas ALC-0315, Acuitas A9, Acuitas Lipid 2,2, Moderna Lipid H (SM-102), Moderna Lipid 5, A2-Iso5-2DC18, BAME-016B, 9A1P9, C12-200, cKK-E12, OF-Deg-Lin, 306Oi10, TT3, FTT5, Lipid319, 5A2-SC8, Genevan CL1, DLinDMA, DLenDMA, ClinDMA, CpLinDMA, imidazole cholesterol ester (ICE), DC-Choi (N
  • cationic lipid preparations are available and can be used in the present invention. These include, for example, (commercially available cationic liposomes comprising DOTMA and DOPE from GIBCO/BRL, Grand Island, New York, USA); (commercially available cationic liposomes comprising DOSPA and DOPE from GIBCO/BRL); and (Commercially available cationic liposomes containing DOGS are from Promega Corp., Madison, Wisconsin, USA).
  • the cationic lipids included in the compositions and methods of the present invention can be defined as compounds having the following structure or salts thereof:
  • R1 and R2 are independently selected and are H or C1 - C3 alkyl
  • R3 and R4 are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3 and R4 includes at least 2 sites of unsaturation.
  • R 3 and R 4 are the same, that is, R 3 and R 4 are linoleyl (C 18 ) and the like. In some other instances, R 3 and R 4 are different, that is, R 3 is tetradectrienyl (C 14 ) and R 4 is linoleyl (C 18 ).
  • the cationic lipid of Formula I is symmetrical, that is, R 3 and R 4 are the same. In another preferred embodiment, R 3 and R 4 both include at least 2 unsaturated sites.
  • R 3 and R 4 are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In a preferred embodiment, R 3 and R 4 are linoleyl. In some embodiments, R3 and R4 include at least 3 sites of unsaturation and are independently selected from, for example, dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.
  • the cationic lipid of Formula I is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or 1,2-dilinolenoyloxy-N,N-dimethylaminopropane (DLenDMA).
  • DLinDMA 1,2-dilinoleyloxy-N,N-dimethylaminopropane
  • DLenDMA 1,2-dilinolenoyloxy-N,N-dimethylaminopropane
  • a cationic lipid having the following structure (or a salt thereof) can be effectively used in the present invention.
  • R1 and R2 are the same or different and are independently optionally substituted C12 - C24 alkyl, optionally substituted C12 - C24 alkenyl, optionally substituted C12 - C24 alkynyl, or optionally substituted C12 - C24 acyl;
  • R3 and R4 are the same or different and are independently optionally substituted C1 - C6 alkyl, optionally substituted C1 - C6 alkenyl, or optionally substituted C1 - C6 alkynyl, or R3 and R4 may combine to form an optionally substituted heterocyclic ring of 4-6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen;
  • R 5 is absent or is hydrogen or C 1 -C 6 alkyl to provide a quaternary amine
  • n, n, and p are the same or different and are independently 0 or 1, provided that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are the same or different and are independently 0, S, or NH.
  • the cationic lipid having the above structure is 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino- [1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-di
  • compositions and methods of the present invention include permanent cationic lipids comprising quaternary ammonium ions.
  • permanent cationic lipids have the following general formula:
  • R1 and R2 are each independently C8 - C24 alkyl, C8 - C24 alkenyl, or a substituted form of either group;
  • R 3 , R 3 ′ and R 3 ′′ are each independently alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) ;
  • X ⁇ is a monovalent anion
  • the cationic lipids of the compositions and methods of the present invention are further defined as compounds having the following structure or salts thereof:
  • R1 and R2 are each independently C8 - C24 alkyl, C8 - C24 alkenyl, or a substituted form of either group;
  • R 3 , R 3 ′ and R 3 ′′ are each independently alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) ;
  • R4 is alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) ;
  • X ⁇ is a monovalent anion
  • the cationic lipids of the compositions and methods of the present invention are further defined as compounds having the following structure or salts thereof:
  • R1 and R2 are independently selected and are H or C1 - C3 alkyl
  • R3 and R4 are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3 and R4 includes at least 2 sites of unsaturation.
  • R 3 and R 4 are all the same, that is, R 3 and R 4 are all linoleyl (C 18 ), etc. In some other examples, R 3 and R 4 are different, that is, R 3 is tetradecatrienyl (C 14 ) and R 4 is linoleyl (C 18 ).
  • the cationic lipid of the present invention is symmetrical, that is, R 3 and R 4 are all the same. In another preferred embodiment, R 3 and R 4 both include at least 2 unsaturated sites.
  • R 3 and R 4 are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl and eicosadienyl. In a preferred embodiment, R 3 and R 4 are all linoleyl. In some embodiments, R 3 and R 4 include at least 3 unsaturated sites and are independently selected from, for example, dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linoleyl and eicosadienyl.
  • the cationic lipids of the compositions and methods of the present invention are further defined as compounds having the following structure or salts thereof:
  • R 4 and R 4 ' are each independently C 6 -C 24 alkyl, C 6 -C 24 alkenyl, or a substituted form of either group;
  • R 4 ′′ is alkyl (C ⁇ 24) , alkenyl (C ⁇ 24) , or a substituted form of either group;
  • R 4 ′′′ is C 1 -C 8 alkyl, C 2 -C 8 alkenyl, or a substituted form of either group;
  • X2 is a monovalent anion.
  • the permanent cationic lipid of the compositions and methods of the present invention is further defined as a compound having the following structure or a salt thereof:
  • Y 1 , Y 2 or Y 3 are each independently X 1 C(O)R 1 or X 2 N + R 3 R 4 R 5 ; provided that at least one of Y 1 , Y 2 and Y 3 is X 2 N + R 3 R 4 R 5 ;
  • R1 is C1 - C24 alkyl, C1 - C24 substituted alkyl, C1 - C24 alkenyl, C1 - C24 substituted alkenyl;
  • X1 is O or NR a , wherein R a is hydrogen, C 1 ⁇ C 4 alkyl or C 1 ⁇ C 4 substituted alkyl;
  • X2 is C1 - C6 alkanediyl or C1 - C6 substituted alkanediyl;
  • R 3 , R 4 and R 5 are each independently C 1 -C 24 alkyl, C 1 -C 24 substituted alkyl, C 1 -C 24 alkenyl, C 1 -C 24 substituted alkenyl;
  • A1 is an anion having a charge equal to the number of X2N + R3R4R5 groups in the compound.
  • Suitable cationic lipids for use in the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO 2010/144740 and U.S. Patent 8,058,069, which are incorporated herein by reference.
  • compositions and methods of the present invention include ionizable cationic lipids as described in International Patent Publication WO2013/149140, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid of one of the following formulas:
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, optionally substituted different saturated or unsaturated C 1 -C 20 alkyl groups, and optionally substituted different saturated or unsaturated C 6 -C 20 acyl groups; wherein L 1 and L 2 are each independently selected from the group consisting of hydrogen, optionally substituted C 1 -C 30 alkyl groups, optionally substituted different unsaturated C 1 -C 30 alkenyl groups, and optionally substituted C 1 -C 30 alkynyl groups; wherein m and o are each independently selected from the group consisting of: zero and any positive integer (eg, where m is three); and where n is zero or any positive integer (eg, where n is one).
  • the polymer-lipid composition provided herein comprises a cationic lipid of the formula:
  • p is an integer between 1 and 9, inclusive;
  • R 2 is independently hydrogen or optionally substituted C 1-6 alkyl
  • R 6 and R 7 are independently a group of formula (i), (ii) or (iii);
  • R' is independently hydrogen or optionally substituted alkyl
  • X is O, S or NR x , wherein RX is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;
  • Y is O, S or NR Y , wherein RY is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;
  • R is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group attached to an oxygen atom, a sulfur protecting group attached to a sulfur atom, or a nitrogen protecting group attached to a nitrogen atom.
  • protecting group; and RL is optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted heteroC1-50 alkyl, optionally substituted heteroC2-50 alkenyl, optionally substituted heteroC2-50 alkynyl, or a polymer.
  • compositions and methods of the invention include a cationic lipid having the formula:
  • compositions and methods of the present invention include cationic lipids as described in PCT Application Publication No. WO2020097384A1, the disclosure of which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid having the formula:
  • each R 1 and R 2 are independently H or C 1 -C 6 aliphatic; each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or an arylene group; each L 1 is independently an ester, a thioester, a disulfide bond or an anhydride group; each L 2 is independently C 2 -C 10 aliphatic; each X 1 is independently H or OH; and each R 3 is independently C 6 -C 20 aliphatic.
  • compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO2017/075531, which is incorporated herein by reference.
  • compositions and methods of the present invention include cationic lipids having the formula:
  • Suitable cationic lipids for use in the compositions and methods of the invention include cholesterol-based cationic lipids as described in International Patent Publications WO2018/089790 and WO2022/032154, which are incorporated herein by reference.
  • compositions and methods of the invention include a compound having the following structure: BL 1 -S,
  • B is a basic functional group, wherein the protonated form has a pKa of no more than about 8.0;
  • L 1 is an optionally substituted linking group, which is a C 1 -C 20 alkylene group or a 2 to 20-membered heteroalkylene group;
  • B is an optionally substituted 5-membered or 6-membered nitrogen-containing heteroaryl.
  • B is a group selected from pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl, each of which is optionally substituted.
  • L1 comprises a moiety that is an ester, amide, carbamate, carbonate or urea group, and L1 does not comprise a substituent having the structure -N(R') 2 , or a positively charged form thereof, wherein each R' is independently hydrogen or an optionally substituted C1 - C20 alkyl group.
  • S is a zoosterol, or an oxidized or reduced form thereof;
  • S is a phytosterol, or an oxidized or reduced form thereof;
  • S is a synthetic sterol, or an oxidized or reduced form thereof.
  • S is a sterol selected from the group consisting of cholesterol, an oxidized form of cholesterol, a reduced form of cholesterol, an alkyl lithochlate, stigmasterol, stigmasterol, campesterol, ergosterol and sitosterol.
  • compositions and methods of the invention include a compound having the structure
  • n is 0 or 1, preferably 0,
  • R 1 is a group –(CH 2 ) q -NH 2 or a group –(CH 2 ) r -NH-(CH 2 ) s -NH 2 ,
  • R 2 is a group –(CH 2 ) t -NH 2 or a group –(CH 2 ) u -NH-(CH 2 ) w -NH 2 ,
  • t, u and w are independently integers from 2 to 6,
  • R3 is a straight chain alkanediyl group having 1 to 4 carbon atoms
  • cationic lipids based on cholesterol can be used as a replacement of cationic lipids as described herein or in addition to cationic lipids as described herein.
  • Suitable cationic lipids based on sterols are cationic lipids based on sterols containing dialkylamino, imidazoles, basic amino acid sequences and guanidine.
  • compositions comprising one or more cationic lipids based on sterols comprising imidazoles, such as imidazole cholesterol ester or "ICE" lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopentadien[a]phenanthrene-3-yl 3-(1H-imidazole-4-yl) propionate.
  • imidazoles such as imidazole cholesterol ester or "ICE" lipid
  • imidazoles such as imidazole cholesterol ester or "ICE" lipid
  • the cholesterol-derived cationic lipid comprises at least one selected from the following: DC-Choi (N,N-dimethyl-N-ethylformamide cholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Patent No. 5,744,335), N4-arginine cholesterol carbonylamide (GL67), cholesterol derivatives coupled to basic amino acid sequences, imidazole cholesterol ester (ICE) and their derivatives.
  • DC-Choi N,N-dimethyl-N-ethylformamide cholesterol
  • 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23,
  • compositions and methods of the present invention include cleavable cationic lipids as described in International Patent Publication WO2012/170889, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid having the formula:
  • R 1 is selected from the group consisting of imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino) and pyridyl; wherein R 2 is selected from the group consisting of one of the following two general formulae:
  • R 3 and R 4 are each independently selected from the group consisting of: optionally substituted different saturated or unsaturated C 6 _C 20 alkyl groups and optionally substituted different saturated or unsaturated C 6 _C 20 acyl groups; and wherein n is zero or any positive integer (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more).
  • compositions and methods of the present invention include degradable cationic lipids, as described in PCT Application Publication No. WO2019222424A1, which is incorporated herein by reference.
  • the compositions and methods of the present invention include cationic lipids, which are any of the general formulas described in WO2019222424A1 or structures (1a)-(21a) and (1b)-(21b) and (22)-(237).
  • the compositions and methods of the present invention include cationic lipids having a structure according to the formula,
  • RX is independently -H, -L1-R1 or -L5A-L5B-B'; L1 , L2 and L3 are each independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S- or -C(O)NRL-; each L4A and L5A are independently -C(O)-, -C(O)O- or -C(O)NRL-; each L4B and L5B are independently C1 -C20 alkylene; C2 - C20 alkenylene; or C2 -C20 alkynylene; each B and B' is NR4R5 or a 5-10 membered nitrogen-containing heteroaryl group; each R1, R2 and R3 are independently C6 - C30 alkyl, C6-C30 alkenyl or C6- C30 alkynyl; each R4 and R5 are independently C6- C30
  • each RL is independently hydrogen, C 1 -C 20 alkyl, C 2 -C 20 alkenyl or C 2 -C 20 alkynyl.
  • the cationic lipid in the compositions and methods of the present invention is a dendritic polymer or dendron as described in International Patent Publication No. WO2020051220A1, which is incorporated herein by reference.
  • the cationic lipid compositions and methods of the present invention comprise one or more lipid-modified spermine derivatives having the following general formula:
  • X 1 is -(CH 2 ) n - or a carbonyl group, wherein n is 1, 2 or 3;
  • X 2 is selected from -(CH 2 )-, an ester group, an amide group, oxygen or sulfur;
  • R1 and R2 are independently selected from C6 - C18 alkyl, C6 - C18 alkyl containing olefinic bonds or lipophilic cholesterol molecules;
  • X1 and X2 are both -( CH2 )-, and R1 and R2 are independently selected from C10 - C18 alkyl groups.
  • X 1 is -(CH 2 ) 2 -
  • X 2 is oxygen
  • R 1 and R 2 are the same C 12 -C 18 alkyl group or a C 6 -C 18 alkyl group containing an olefinic bond.
  • X 1 is a carbonyl group
  • X 2 is -(CH 2 )-
  • R 1 and R 2 are the same C 12 -C 18 alkyl group or a C 6 -C 18 alkyl group containing an olefinic bond.
  • X 1 is -(CH 2 ) 2 -
  • X 2 is an ester group
  • R 1 and R 2 are the same C 12 -C 18 alkyl group or a C 6 -C 18 alkyl group containing an olefinic bond.
  • X 1 is -(CH 2 )-
  • X 2 is an amide group
  • R 1 and R 2 are the same C 12 -C 18 alkyl group or a C 6 -C 18 alkyl group containing an olefinic bond.
  • the lipid-modified spermine derivative of the invention is composed of spermine and oleyl alcohol coupled by different chemical bonds, wherein spermine is used as a head group with positive charge, and oleyl alcohol is linked to two tertiary amine groups in the middle.
  • Patent application publication number CN104876831 teaches some non-limiting examples of lipid-modified spermine derivatives that can be used in the present disclosure, and the patent application publication number CN104876831 is incorporated herein by reference.
  • compositions and methods of the invention include the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA”) (Feigner et al., Proc. Natl. Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, which is incorporated herein by reference).
  • DOTMA cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • cationic lipids suitable for use in the compositions and methods of the present invention include, for example, 5-carboxysperminylglycine dioctadecylamide (“DOGS”); 2,3-dioleyloxy-N-[2-(spermine-carboxyamide)ethyl]-N,N-dimethyl-1-propylammonium (“DOSPA”) (Behr et al., Proc. Natl. Acad. Sci. 86, 6982 (1989), U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761); 1,2-dioleoyl-3-dimethylammonium-propane (“DODAP”); 1,2-dioleoyl-3-trimethylammonium-propane (“DOTAP”).
  • DOGS 5-carboxysperminylglycine dioctadecylamide
  • DOSPA 2,3-dioleyloxy-N-[2-(spermine
  • Additional exemplary cationic lipids suitable for use in the compositions and methods of the present invention include: 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane (“DSDMA”); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (“DODMA”); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (“DLinDMA”); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (“DLenDMA”); N-dioleyl-N,N-dimethylammonium chloride (“DODAC”); N,N-distearoyl- N,N-dimethylammonium bromide (“DDAB”); N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (“DMRIE”); 3-di
  • the one or more cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.
  • one or more cationic lipids suitable for use in the compositions and methods of the invention include 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("XTC"); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadecane-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (“ALNY-100”) and/or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-di-undecyl-4,7,10,13-tetraazahexadecane-1,16-diamide (“NC98-5").
  • XTC 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-diox
  • Suitable cationic lipids for the compositions and methods of the present invention include compounds of the following formula and pharmaceutically acceptable salts thereof:
  • RCOO is selected from the list comprising: myristoyl, alpha-D-tocopheryl succinyl, linoleoyl, and oleoyl; and X is selected from the list comprising:
  • Suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publications WO2010/053572, WO2013/063468, WO2015/184256, WO2015/199952, WO2015/095340, WO2016/118725, WO2016/205691, WO2016/004202, WO2017/004143, WO2017/117528, WO2017/049245, WO2017/173054, which are incorporated herein by reference.
  • Suitable cationic lipids for use in the compositions and methods of the present invention include those described in J. McClellan, M.C. King, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference.
  • the cationic lipid comprises at least one selected from the group consisting of permanent cationic lipids, ionizable cationic lipids, cholesterol-derived cationic lipids, and dendrimers or dendrons. In preferred embodiments, the cationic lipid comprises an ionizable cationic lipid.
  • the cationic lipid may contain one or more asymmetrically substituted carbon or nitrogen atoms, and may be separated in an optically active or racemic form.
  • Cationic lipids may exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and each diastereomer. In some embodiments, a single diastereomer is obtained.
  • the chiral center of the cationic lipid of the present invention may have an S or R configuration.
  • one or more of the cationic lipids may exist as structural isomers.
  • the compound has the same chemical formula, but is different from the connectivity of the nitrogen atom of the core.
  • structural isomers may present a fully reacted primary amine, and then present a mixture of reacted secondary amines.
  • Chemical formula for representing cationic lipid of the present invention will usually only show one of several possible different tautomers.For example, many types of keto groups are known to exist in equilibrium with the corresponding enol groups. Similarly, many types of imino groups exist in equilibrium with the enamine groups. Whether for a given formula, which tautomer is described, and whether which tautomer is the most common, all tautomers of a given chemical formula are meant.
  • the atoms constituting the cationic lipids of the present invention are intended to include all isotopic forms of such atoms.
  • Isotopes used herein include those atoms with the same atomic number but with different mass numbers.
  • the isotopes of hydrogen include tritium and deuterium
  • the isotopes of carbon include 13 C and 14 C.
  • the cationic lipid comprises about 23 mol% to about 83 mol% of the total lipids present in the composition.
  • the cationic lipid accounts for about 25 mol% to about 80 mol%, e.g., about 30 mol% to about 80 mol%, about 35 mol% to about 80 mol%, about 40 mol% to about 80 mol%, about 45 mol% to about 80 mol%, about 50 mol% to about 80 mol%, about 55 mol% to about 80 mol%, about 60 mol% to about 80 mol%, about 65 mol% to about 80 mol%, about 70 mol% to about 80 mol%, or about 75 mol% to about 80 mol% of the total lipids present in the composition.
  • the cationic lipid accounts for about 25 mol% to about 70 mol%, e.g., about 30 mol% to about 70 mol%, about 35 mol% to about 70 mol%, about 40 mol% to about 70 mol%, about 45 mol% to about 70 mol%, about 50 mol% to about 70 mol%, about 55 mol% to about 70 mol%, about 60 mol% to about 70 mol%, or about 65 mol% to about 70 mol% of the total lipids present in the composition.
  • the cationic lipid accounts for about 25 mol% to about 60 mol%, such as about 30 mol% to about 60 mol%, about 35 mol% to about 60 mol%, about 40 mol% to about 60 mol%, about 45 mol% to about 60 mol%, about 50 mol% to about 60 mol%, or about 55 mol% to about 60 mol% of the total lipids present in the composition.
  • the cationic lipid accounts for about 25 mol% to about 50 mol%, such as about 30 mol% to about 50 mol%, about 35 mol% to about 50 mol%, or about 50 mol% of the total lipids present in the composition. About 40 mol% to about 50 mol% or about 45 mol% to about 50 mol%.
  • the cationic lipid comprises about 23 mol%, 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, or about 83 mol% of the total lipids present in the composition.
  • the proportions of the components of the composition may vary and the delivery efficiency of a particular formulation may be measured using, for example, endosomal release parameter (ERP) assays and/or in vivo gene transfection efficiency (such as in the case of nucleic acid loading) assays, etc.
  • ERP endosomal release parameter
  • in vivo gene transfection efficiency such as in the case of nucleic acid loading
  • the non-cationic lipid comprises at least one selected from the group consisting of anionic lipids, zwitterionic lipids and neutral lipids, preferably, the non-cationic lipid comprises a neutral lipid.
  • the phrase "non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid.
  • anionic lipid refers to any of a variety of lipid species that carry a net negative charge at a selected pH such as a physiological pH.
  • the neutral lipids account for 19 mol%-75 mol% of the total lipids present in the composition, such as 25 mol%-70 mol%, 30 mol%-70 mol%, 35 mol%-70 mol%, 40 mol%-70 mol%, 45 mol%-70 mol%, 50 mol%-65 mol% or 55 mol%-60 mol%.
  • the neutral lipids account for 20 mol%-65 mol% of the total lipids present in the composition, e.g., 25 mol%-65 mol%, 30 mol%-65 mol%, 35 mol%-65 mol%, 40 mol%-65 mol%, 45 mol%-65 mol%, 50 mol%-65 mol% or 55 mol%-65 mol%.
  • the neutral lipids account for 20 mol%-60 mol% of the total lipids present in the composition, e.g., 25 mol%-60 mol%, 30 mol%-60 mol%, 35 mol%-60 mol%, 40 mol%-60 mol%, 45 mol%-60 mol%, 50 mol%-60 mol% or 55 mol%-60 mol%.
  • the neutral lipids account for 20 mol%-55 mol%, e.g., 25 mol%-55 mol%, 30 mol%-55 mol%, 35 mol%-55 mol%, 40 mol%-55 mol%, 45 mol%-55 mol%, 50 mol%-55 mol%, or 20 mol%-50 mol%, e.g., 25 mol%-50 mol%, 30 mol%-50 mol%, 35 mol%-50 mol%, 40 mol%-50 mol% or 45 mol%-50 mol%, of the total lipids present in the composition.
  • the neutral lipids account for 20% of the total lipids present in the composition.
  • mol%-45mol% for example 25mol%-45mol%, 30mol%-45mol%, 35mol%-45mol% or 40mol%-45mol%, or 20mol%-40mol%, for example 25mol%-40mol%, 30mol%-40mol% or 35mol%-40mol%.
  • the neutral lipid comprises:
  • a mixture of cholesterol or cholesterol-derived neutral lipids and phospholipids is provided.
  • the cholesterol accounts for 14 mol%-70 mol% of the total lipids in the composition, for example, 20 mol%-70 mol%, 25 mol%-70 mol%, 30 mol%-70 mol%, 35 mol%-70 mol%, 40 mol%-70 mol%, 15 mol%-60 mol%, 20 mol%-60 mol%, 25 mol%-60 mol%, 30 mol%-60 mol%, 35 mol%-60 mol%, 40 mol%-60 mol%, 15 mol%-50 mol%, 20 mol%-50 mol%, 25 mol%-50 mol%, 30 mol%-50 mol%, 35 mol%-50 mol%, 15 mol%-40 mol%, 20 mol%-40 mol%, 25 mol%-40 mol%, 30 mol%-40 mol% or 35 mol%-40 mol%.
  • the phospholipids account for about 5 mol% to about 75 mol% of the total lipids in the composition, such as 5 mol% to 70 mol%, 8 mol% to 65 mol%, 10 mol% to 60 mol%, 10 mol% to 50 mol%, 10 mol% to 40 mol%, 10 mol% to 30 mol%, 10 mol% to 20 mol%, 15 mol% to 55 mol%, 15 mol% to 50 mol%, 15 mol% to 45 mol%, 10 mol% to 50 mol%, 10 ...
  • %-40mol% 15mol%-35mol%, 15mol%-30mol%, 15mol%-25mol%, 20mol%-45mol%, 20mol%-45mol%, 20mol%-40mol%, 20mol%-35mol%, 20mol%-30mol%, 20mol%-25mol%, 25mol%-40mol%, 25mol%-35mol% or 25mol%-30mol%.
  • the phospholipid comprises at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, dioleoylphosphatidylserine (DOPS), phosphatidylinositol, sphingomyelin, egg yolk sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylphosphatidylcholine (DPSC), dioleoylphosphatidylethanolamine (DOPE), dioleo
  • DPPC dioleoylphosphatidylglycerol
  • DOPG dipalmitoylphosphatidylglycerol
  • POPC palmitoyloleoyl-phosphatidylcholine
  • POPE palmitoyloleoyl-phosphatidylethanolamine
  • POPG palmitoyloleoyl-phosphatidylglycerol
  • DGTS 1,2-Dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)-homoserine
  • MGDG monogalactosyldiacylglycerol
  • DGDG diacetyldiacylglycerol
  • SQLDG sulfaquinolinediacylglycerol
  • 16-O-monomethyl PE 16-O-dimethyl PE, 18-1-trans PE and their derivatives.
  • compositions and methods of the invention include a non-cationic lipid having the formula:
  • R 1 and R 2 are each independently C 8 -C 24 alkyl, C 8 -C 24 alkenyl or a substituted form of either group;
  • R 3 , R 3 ′ and R 3 ′′ are each independently alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) ;
  • X ⁇ is a monovalent anion.
  • R 1 is C 8 -C 24 alkenyl or substituted C 8 -C 24 alkenyl.
  • R 2 is C 8 -C 24 alkenyl or substituted C 8 -C 24 alkenyl.
  • R 1 is C 8 -C 24 alkyl or substituted C 8 -C 24 alkyl.
  • R 2 is C 8 -C 24 alkyl or substituted C 8 -C 24 alkyl.
  • both R 1 and R 2 are the same.
  • R 3 , R 3 ′, and R 3 ′′ are each the same. In some embodiments, R 3 , R 3 ′, and R 3 ′′ are each methyl. In some embodiments, X - is a halide anion such as bromide or chloride.
  • compositions and methods of the invention include a phosphoglyceride or a salt thereof of the formula:
  • R4 is a straight chain alkyl group having 10 to 24 carbon atoms or a straight chain alkyl group having 1 to 3 double bonds and Straight-chain alkenyl groups of 10 to 24 carbon atoms;
  • R5 is a straight chain alkyl group having 10 to 24 carbon atoms or a straight chain alkenyl group having 1 to 3 double bonds and 10 to 24 carbon atoms;
  • compositions and methods of the invention include an anionic lipid of the formula:
  • R1 and R2 are each independently alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of either group;
  • R3 is hydrogen, alkyl (C ⁇ 6) or substituted alkyl (C ⁇ 6) or -Y1 - R4 , wherein: Y1 is alkanediyl (C ⁇ 6) or substituted alkanediyl (C ⁇ 6) ; and R4 is acyloxy (C ⁇ 8-24) or substituted acyloxy (C ⁇ 8-24) .
  • non-cationic lipids suitable for use in the present invention include phosphorus-free lipids, such as stearylamide, dodecylamine, hexadecylamine, acetylapalmitate, glyceryl ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic acid polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethoxylated fatty acid amides, distearyl dimethyl bromide, ceramides, sphingomyelin and their derivatives, etc.
  • phosphorus-free lipids such as stearylamide, dodecylamine, hexadecylamine, acetylapalmitate, glyceryl ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic acid
  • the cholesterol-derived neutral lipid comprises at least one selected from the group consisting of cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, BHEM-cholesterol, ⁇ -sitosterol, 20 ⁇ -hydroxycholesterol, polypeptide/protein covalently modified cholesterol, and derivatives thereof, wherein the synthesis of cholesteryl-2'-hydroxyethyl ether is described in U.S. Pat. No. 8,058,069, the disclosure of which is incorporated herein by reference for all purposes.
  • the cholesterol-derived lipid comprises ⁇ -sitosterol.
  • compositions and methods of the present invention include a steroid having a ring structure comprising three fused cyclohexyl rings and a fused cyclopentyl ring, as shown in the following formula:
  • the steroid derivative comprises a moiety having one or more non-alkyl substitutions.
  • the steroid or steroid derivative is a sterol, wherein the formula is further defined as:
  • the steroid or steroid derivative of the compositions and methods of the present invention is cholestane or a cholestane derivative.
  • the ring structure is further defined by the formula:
  • cholestane derivatives include non-alkyl substitutions of one or more of the above ring systems.
  • the cholestane or cholestane derivative is cholestene or a cholestene derivative or a sterol or a sterol derivative.
  • the cholestane or cholestane derivative is cholestene and a sterol or a derivative thereof.
  • the composition further comprises a lipid conjugate, wherein the lipid conjugate comprises at least one selected from the group consisting of a poly(ethylene glycol)-lipid conjugate (PEG-lipid conjugate or PEG-lipid), an ATTA-lipid conjugate, a polysarcosine-lipid conjugate, a polypeptide/protein-lipid conjugate, and a cation-polymer-lipid conjugate (CPL), preferably, the lipid conjugate comprises a PEG-lipid conjugate.
  • PEG-lipid conjugate or PEG-lipid poly(ethylene glycol)-lipid conjugate
  • ATTA-lipid conjugate ATTA-lipid conjugate
  • a polysarcosine-lipid conjugate a polypeptide/protein-lipid conjugate
  • CPL cation-polymer-lipid conjugate
  • the lipid conjugate is a PEG-lipid.
  • PEG-lipids include, but are not limited to, PEG coupled to a dialkoxypropyl group (PEG-DAA) as described in, for example, PCT Publication No. WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in, for example, U.S. Patent Publication Nos. 20030077829 and 2005008689, PEG coupled to a phospholipid such as phosphatidylethanolamine (PEG-PE), PEG coupled to ceramide as described in, for example, U.S. Patent No. 5,885,613, PEG conjugated to cholesterol or its derivatives, and mixtures thereof.
  • PEG-lipids include, but are not limited to, PEG coupled to a dialkoxypropyl group (PEG-DAA) as described in, for example, PCT Publication No. WO 05/026372, PEG coupled to diacylglycerol (PEG
  • Additional PEG-lipids include, but are not limited to, PEG-C-DOMG, DMG-PEG2000 ((R)-2,3-bis(myristoyloxy)propyl-1-(methoxy poly(ethylene glycol)2000)carbamate, DMG-PEG2K), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; 2-[(polyethylene glycol)-2000]-N,N-diethylacetamide), and mixtures thereof.
  • the PEG-lipid described herein can be synthesized as described in International Patent PCT/US2016/000129.
  • the PEG-lipid that can be used in the present invention can be a PEGylated lipid described in International Patent WO2012099755, the disclosures of which are incorporated herein by reference in their entirety for all purposes.
  • PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups.
  • PEG is classified by its molecular weight; for example, PEG2000 (PEG2K) has an average molecular weight of about 2,000 Daltons, and PEG5000 (PEG5K) has an average molecular weight of about 5,000 Daltons.
  • PEG is commercially available from Sigma-Aldrich Chemical Co.
  • 6,774,180 and 7,053,150 are also effective for preparing the PEG-lipid conjugates of the present invention.
  • mPEG (20 KDa) amine e.g., 6,774,180 and 7,053,150
  • mPEG (20 KDa) amine e.g., 6,774,180 and 7,053,150
  • mPEG (20 KDa) amine e.g., 6,774,180 and 7,053,150
  • mPEG (20 KDa) amine e.g., mPEG (20 KDa) amine
  • MePEG-CH 2 COOH monomethoxypolyethylene glycol-acetic acid
  • the PEG moiety of the PEG-lipid conjugate described herein can include an average molecular weight in the range of about 550 Daltons to about 10,000 Daltons. In certain instances, the PEG moiety has an average molecular weight of about 750 Daltons to about 5,000 Daltons (e.g., about 1,000 Daltons to about 5,000 Daltons, about 1,500 Daltons to about 3,000 Daltons, about 750 Daltons to about 3,000 Daltons, about 750 Daltons to about 2,000 Daltons, etc.). In a preferred embodiment, the PEG moiety has an average molecular weight of about 5,000 Daltons or about 2,000 Daltons or about 750 Daltons.
  • PEG can be optionally substituted with alkyl, alkoxy, acyl or aryl groups.
  • PEG can be directly conjugated to the lipid or can be connected to the lipid through a linker moiety.
  • Any linker moiety suitable for coupling PEG to the lipid can be used including, for example, non-ester-containing linker moieties and ester-containing linker moieties.
  • the linker moiety is a non-ester-containing linker moiety.
  • non-ester-containing linker moiety refers to a linker moiety that does not contain a carboxylate bond (-OC(O)-).
  • Suitable non-ester-containing linkers are preferably non-ester-containing linkers.
  • Moieties include, but are not limited to, amine (-C(O)NH-), amino (-NR-), carboxyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O ) CCH2CH2C ( O)-), succinamido (-NHC(O) CH2CH2C (O)NH-), and combinations thereof (such as linkers comprising both carbamate linker moieties and amine linker moieties).
  • a carbamate linker is used to couple the PEG and lipid.
  • an ester-containing linker moiety is used to couple the PEG and lipid.
  • Suitable ester-containing linker moieties include, for example, carbonate (—OC(O)O—), succinyl, phosphate (—O—(O)POH—O—), sulfonate, and combinations thereof.
  • Phosphatidylethanolamines with various acyl chain groups of different chain lengths and degrees of saturation can be conjugated with PEG to form lipid conjugates.
  • Such phosphatidylethanolamines are commercially available, or can be separated or synthesized using conventional techniques known to those skilled in the art.
  • Phosphatidyl-ethanolamines containing saturated or unsaturated fatty acids with a preferred carbon chain length in the range of C10 - C20 Phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used.
  • Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE) and distearoyl-phosphatidylethanolamine (DSPE).
  • DMPE dimyristoyl-phosphatidylethanolamine
  • DPPE dipalmitoyl-phosphatidylethanolamine
  • DOPE dioleoyl-phosphatidylethanolamine
  • DSPE distearoyl-phosphatidylethanolamine
  • the PEG-lipid conjugate of the compositions and methods of the present invention comprises a PEGylated phosphoglyceride of the formula:
  • p is an integer from 5 to 200, preferably from 10 to 170, and most preferably from 10 to 140;
  • R6 is a straight chain alkyl group having 10 to 20 carbon atoms or a straight chain alkenyl group having 1 to 3 double bonds and 10 to 20 carbon atoms;
  • R7 is a straight chain alkyl group having 10 to 20 carbon atoms or a straight chain alkenyl group having 1 to 3 double bonds and 10 to 20 carbon atoms;
  • the compositions and methods of the invention has the formula:
  • R 12 and R 13 are each independently alkyl (C ⁇ 24) , alkenyl (C ⁇ 24) , or a substituted form of any of these groups; Re is hydrogen, alkyl (C ⁇ 8) , or substituted alkyl (C ⁇ 8) ; and x is 1-250. In some embodiments, Re is alkyl (C ⁇ 8) such as methyl. R 12 and R 13 are each independently alkyl (C ⁇ 4-20) . In some embodiments, x is 5-250. In one embodiment, x is 5-125 or x is 100-250. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.
  • the lipid conjugate of the compositions and methods of the invention has the formula:
  • n1 is an integer between 1-100, and n2 and n3 are each independently selected from an integer between 1-29.
  • n1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70 , 75, 80, 85, 90, 95 or 100 or any range derivable therein.
  • n1 is about 30 to about 50.
  • n2 is 5-23.
  • n2 is 11 to about 17.
  • n3 is 5-23.
  • n3 is 11 to about 17.
  • R is a member selected from the group consisting of hydrogen, alkyl, and acyl
  • R1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R1 and the nitrogen to which they are attached form an azide moiety
  • R2 is a member selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, and an amino acid side chain
  • R3 is a member selected from the group consisting of hydrogen, halogen, hydroxyl, alkoxy, thiol, hydrazine, amino, and NR4R5 , wherein R4 and R5 are independently hydrogen or alkyl
  • n is 4-80
  • m is 2-6
  • p is 1-4
  • q is 0 or 1.
  • diacylglycerol refers to a compound having two fatty acyl chains, R1 and R2 , each independently having 2-30 carbons, which are bound to the 1- and 2-positions of glycerol via ester bonds.
  • the acyl groups may be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauryl ( C12 ), myristyl ( C14 ), palmityl ( C16 ), stearyl ( C18 ), and eicosyl ( C20 ).
  • R1 and R2 are the same, i.e., both R1 and R2 are myristyl (i.e., dimyristyl), both R1 and R2 are stearyl (i.e., distearyl), etc.
  • Diacylglycerol has the following general formula:
  • dialkoxypropyl refers to a compound having two alkyl chains, R1 and R2 , each independently having 2-30 carbons.
  • the alkyl group may be saturated or have varying degrees of unsaturation.
  • Dialkoxypropyl has the following general formula:
  • the PEG-lipid is a PEG-DAA conjugate having the following general formula:
  • R 1 and R 2 are independently selected and are long chain alkyl groups having from about 10 to about 22 carbon atoms; PEG is polyethylene glycol; and L is a non-ester-containing linker moiety or an ester-containing linker moiety as described above.
  • the long chain alkyl group may be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, lauryl (C 12 ), myristyl (C 14 ), palmityl (C 16 ), stearyl (C 18 ), and eicosyl (C 20 ).
  • R 1 and R 2 are the same, i.e., both R 1 and R 2 are myristyl (i.e., dimyristyl), both R 1 and R 2 are stearyl (i.e., distearyl), etc.
  • PEG has an average molecular weight in the range of about 550 daltons to about 10,000 daltons. In some instances, PEG has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons, etc.). In a preferred embodiment, PEG has an average molecular weight of about 2,000 daltons or about 750 daltons. PEG can be optionally substituted with an alkyl, alkoxy, acyl or aryl group. In certain embodiments, the terminal hydroxyl group is substituted with an ethoxy or methyl group.
  • L is a non-ester-containing linker moiety.
  • Suitable non-ester-containing linkers include, but are not limited to, amine linker moieties, amino linker moieties, carboxyl linker moieties, carbamate linker moieties, urea linker moieties, ether linker moieties, disulfide linker moieties, succinyl linker moieties, and combinations thereof.
  • the non-ester-containing linker moiety is a carbamate
  • the non-ester-containing linker moiety is an amine linker moiety (i.e., a PEG-A-DAA conjugate).
  • the non-ester-containing linker moiety is a succinyl linker moiety (i.e., a PEG-S-DAA conjugate).
  • the PEG-lipid conjugate is selected from:
  • the PEG-DAA conjugates are synthesized using standard techniques and reagents known to those skilled in the art. It should be recognized that the PEG-DAA conjugates will contain a variety of amide, amine, ether, thio, carbamate, and urea bonds. Those skilled in the art should recognize that methods and reagents for forming these bonds are well known and readily available. See, for example, March, ADVANCED ORGANIC CHEMISTRY (Wiley 1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); and Furniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th Edition (Longman 1989).
  • the PEG-DAA conjugate is a dilauryloxypropyl (C 12 )-PEG conjugate, a dimyristyloxypropyl (C 14 )-PEG conjugate, a dipalmityloxypropyl (C 16 )-PEG conjugate, or a distearyloxypropyl (C 18 )-PEG conjugate.
  • C 12 dilauryloxypropyl
  • C 14 dimyristyloxypropyl
  • C 16 dipalmityloxypropyl
  • C 18 distearyloxypropyl
  • hydrophilic polymers may be Instead of PEG.
  • suitable polymers that can be used instead of PEG include, but are not limited to, polyvinyl pyrrolidone, polymethyl oxazoline, polyethyl oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethyl acrylamide, polylactic acid, polyglycolic acid, poloxamer, poloxamine, derivatized cellulose such as hydroxymethyl cellulose or carboxyethyl cellulose, polysarcosine-lipid conjugates, and conjugates of polysarcosine with lipid-like substances.
  • polysarcosine-lipid conjugate or the conjugate of polysarcosine with lipid-like substances can be selected from the following group: polysarcosine-diacylglycerol conjugate, polysarcosine-dialkoxypropyl conjugate, polysarcosine-phospholipid conjugate, polysarcosine-ceramide conjugate, and mixtures thereof.
  • Suitable polysarcosine-lipid conjugates or conjugates of polysarcosine with lipid-like substances for use in the present invention, and methods for preparing and using polysarcosine-lipid conjugates or conjugates of polysarcosine with lipid-like substances are disclosed in, for example, U.S. Patent No.
  • suitable polysarcosine-lipid conjugates for use in the compositions and methods of the present invention include polysarcosine lipids as described in International Patent Publication WO2020070040, which is incorporated herein by reference.
  • the polymer-lipid composition (e.g., PoLixNano) of the present invention may also include a cation-polymer-lipid conjugate (CPL) (see, e.g., Chen et al., Bioconj. Chem., 11: 433-437 (2000)).
  • CPL cation-polymer-lipid conjugate
  • Suitable PoLixNano-CPLs for use in the present invention, and methods for preparing and using PoLixNano-CPLs are disclosed in, e.g., U.S. Pat. No. 6,852,334 and PCT Publication No. WO 00/62813, the disclosures of which are incorporated herein by reference in their entirety for all purposes.
  • the polycationic moiety may have a connected ligand, such as a targeting ligand or a chelating moiety for complexing calcium.
  • a connected ligand such as a targeting ligand or a chelating moiety for complexing calcium.
  • the cationic moiety maintains a positive charge.
  • the connected ligand has a positive charge.
  • Suitable ligands include, but are not limited to, compounds or devices with reactive functional groups and include lipids, amphipathic lipids, carrier compounds, bioaffinity compounds, biomaterials, biopolymers, biomedical devices, compounds that can be analyzed and detected, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immunostimulants, radioactive markers, fluorophores, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, other targeting moieties, or toxins.
  • the present invention also contemplates the use of PEG-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-l-[succinyl(methoxypolyethylene glycol)-2000] (C8PEG-2000 ceramide).
  • PEG-CER derivatized ceramides
  • Contemplated PEG-modified lipids include But not limited to a polyethylene glycol chain of up to 2 kDa, up to 3 kDa, up to 4 kDa, or up to 5 kDa in length, covalently attached to a lipid having an alkyl chain of C 6 -C 20 in length.
  • the PEG-modified or PEGylated lipid is PEGylated cholesterol. In some embodiments, the PEG-modified or PEGylated lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG (DMPE-PEG), wherein the PEG portion contains 10 to 140 repeating units, more preferably 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG2000 (DMPE-PEG2K).
  • DMPE-PEG2K 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG2000
  • the addition of such components can prevent complex aggregation, and can also provide a method for increasing the circulation life and increasing the delivery of compositions comprising polymer-lipids to target tissues (Klibanov et al.
  • Suitable PEG-modified lipids for the compositions and methods of the present invention include PEG lipids as described in International Patent Publication WO 2020061295 and U.S. Patent Publication US 20220016029, which are incorporated herein by reference.
  • the lipid conjugates of the present invention can improve their targeting characteristics by chemical structural modification, such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification.
  • chemical structural modification such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification.
  • the glycosylated PEG lipid comprises at least one terminal block conjugated to a glycosyl moiety, preferably a terminal hydrophilic block, and the glycosyl moiety can be conjugated to the PEG lipid of the present invention by a covalent bond formed between a functional group of the glycosyl moiety and a functional group of the PEG lipid.
  • the covalent bond can be formed by the reaction between two functional groups that are modified to be reactive, and the glycosyl moiety can be directly conjugated to the PEG lipid.
  • the glycosyl moiety can be conjugated to the PEG lipid through a spacer.
  • the lipid conjugates of the invention may have a ligand attached.
  • Suitable ligands include, but are not limited to, compounds or devices having reactive functional groups and include lipids, carrier compounds, bioaffinity compounds, biomaterials, biopolymers, biomedical devices, analytically detectable compounds, therapeutically active compounds, enzymes, immunostimulants, radioactive labels, fluorophores, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, toxins or other targeting moieties.
  • the lipid conjugate accounts for 0.1 mol%-10.0 mol% of the total lipids in the composition, e.g., 0.1 mol%-10.0 mol%, 1 mol%-10.0 mol%, 2 mol%-10.0 mol%, 3mol%-10.0mol%, 5mol%-10.0mol%, 0.1mol%-5mol%, 1mol%-5mol%, 2mol%-5mol% or 3mol%-5.0mol%.
  • compositions comprising polymer-lipids described herein contain less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1% of the total lipids by mole or weight. In some embodiments, the compositions comprising polymer-lipids described herein contain 0.4% or less of PEG-modified lipids or PEG, 0.3% or less of PEG-modified lipids or PEG, 0.2% or less of PEG-modified lipids or PEG, or 0.1% or less of PEG-modified lipids or PEG by mole or weight of total lipids.
  • compositions comprising polymer-lipids described herein contain 0.01% or less of PEG-modified lipids or PEG by mole or weight of total lipids. In some embodiments, the compositions of polymer-lipids described herein do not include lipid conjugates (e.g., PEG-lipids) or PEG.
  • the concentration of the lipid conjugate may vary, depending on the rate at which the lipid conjugate and polymer-lipid composition used form fusions.
  • the rate at which the lipid conjugate is exchanged from the polymer-lipid composition can be controlled, and the rate at which the nucleic acid/polymer-lipid composition forms a fusion.
  • the rate at which the nucleic acid/polymer-lipid composition forms a fusion can be varied, for example, by changing the lipid conjugate concentration, by changing the PEG molecular weight, or by changing the chain length and degree of saturation of the acyl chain groups on the phosphatidylethanolamine or ceramide.
  • variables include, for example, pH, temperature, ionic strength, etc., which can be used to change and/or control the rate at which the nucleic acid/polymer-lipid composition forms a fusion.
  • Other methods that can be used to control the rate at which the nucleic acid/polymer-lipid composition forms a fusion will be clear to those skilled in the art when reading the disclosure of the present invention.
  • the PEG-lipid conjugate comprises at least one selected from the group consisting of DMG-PEG2K, DMPE-PEG2K, DSPE-PEG2K, DSPE-PEG2K-Mannose, DMG-PEG5K, DMPE-PEG5K, DSPE-PEG5K-Mannose and DSPE-PEG5K.
  • compositions of the present invention comprise:
  • amphiphilic block copolymers cationic lipids, phospholipids, cholesterol and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipid accounts for 30.0% of the total lipids present in the composition mol%-80.0mol%, phospholipids account for 5.0mol%-50.0mol% of the total lipids, cholesterol accounts for 14.0mol%-64.0mol% of the total lipids, lipid conjugates account for 0.1mol%-8.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% weight percent of the composition;
  • an amphiphilic block copolymer a cationic lipid, a phospholipid, cholesterol, and a lipid conjugate such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 23.0 mol%-75.0 mol% of the total lipid present in the composition, the phospholipid accounts for 10.0 mol%-62.0 mol% of the total lipid, the cholesterol accounts for 14.0 mol%-46.0 mol% of the total lipid, the lipid conjugate accounts for 0.1 mol%-8.0 mol% of the total lipid, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition;
  • amphiphilic block copolymers cholesterol-derived cationic lipids, phospholipids, and lipid conjugates such as PEG-lipid conjugates, wherein the cholesterol-derived cationic lipids account for 29.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 19.0 mol%-70.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1%-95.0% by weight of the composition;
  • an amphiphilic block copolymer a cationic lipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 25.0 mol%-80.0 mol% of the total lipids present in the composition, cholesterol accounts for 15.0 mol%-50.0 mol% of the total lipids, the lipid conjugate accounts for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition;
  • amphiphilic block copolymers cationic lipids, phospholipids and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids account for 30.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 10.0 mol%-50.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1%-95.0% by weight of the composition;
  • an amphiphilic block copolymer a cationic lipid, a phospholipid, and cholesterol
  • the cationic lipid accounts for 30.0 mol%-80.0 mol% of the total lipids present in the composition
  • the phospholipids account for 5.0 mol%-50.0 mol% of the total lipids
  • the cholesterol accounts for 15.0 mol%-50.0 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition
  • an amphiphilic block copolymer a cholesterol-derived cationic lipid and a phospholipid, wherein the cholesterol-derived cationic lipid accounts for 30.0 mol%-70.0 mol% of the total lipids present in the composition, the phospholipids account for 30.0 mol%-70.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition.
  • the composition comprises an amphiphilic block copolymer and the following components:
  • composition comprises 40.0 mol% to 70.0 mol% of the total lipids, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE comprises 8.0 mol% to 39.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol comprises 20.0 mol% to 40.0 mol% of the total lipids, DMG-PEG
  • DOTAP DODAP
  • DOTMA DOSPA
  • DSPC DPPC
  • DOPS SOPE
  • DOPG DOPG
  • DSPE ESM or DOPE
  • cholesterol or ⁇ -sitosterol DOTAP, DODAP, DOTMA or DOSPA
  • DSPC DPPC
  • DOPS SOPE
  • DOPG DOPG
  • DSPE ESM or DOPE
  • cholesterol or ⁇ -sitosterol accounts for 15.0mol%-50.0mol% of the total lipids
  • DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1mol%-5.0mol% of the total lipids
  • amphiphilic block copolymer accounts for 30.0%-90.
  • GL67, ICE, or HGT4002 DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE
  • DMG-PEG2K, DMG-PEG5K, or DSPE-PEG2K-Mannose wherein the GL67, ICE, or HGT4002 accounts for 40.0 mol%-80.0 mol% of the total lipids present in the composition
  • DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE accounts for 10.0 mol%-50.0 mol% of the total lipids
  • DMG-PEG2K, DMG-PEG5K, or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition
  • compositions comprise the following components:
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 89.9% by weight of the composition;
  • an amphiphilic block copolymer (2) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.6 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 72.9% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 12.5 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 36.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.0% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 34.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 81.8% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 69.2% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.3% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K
  • the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition
  • DSPC, DPPC or DOPE account for 14.3 mol% of the total lipids
  • cholesterol or ⁇ -sitosterol account for 35.0 mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 48.4% by weight of the composition
  • Amphiphilic block copolymers DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.6% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 48.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.3% by weight of the composition;
  • an amphiphilic block copolymer (10) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 52.9% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.1 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.3% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein
  • the DLin-MC3-DMA, ALC-0315 or SM-102 account for 51.6 mol% of the total lipids present in the composition
  • DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids
  • cholesterol or ⁇ -sitosterol account for 27.5 mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids
  • the amphiphilic block copolymer accounts for 42.9% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.2% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 66.1% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 74.5% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 79.6% by weight of the composition;
  • an amphiphilic block copolymer (18) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 accounts for 46.0% of the total lipids present in the composition.
  • mol% DSPC, DPPC or DOPE account for 23.0mol% of the total lipids
  • cholesterol or ⁇ -sitosterol account for 30.0mol% of the total lipids
  • DMG-PEG2K or DMG-PEG5K account for 1.0mol% of the total lipids
  • amphiphilic block copolymer accounts for 83.0% by weight of the composition
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 88.2% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 43.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.3 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 33.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 39.1% by weight of the composition;
  • 30.0 mol% of lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.0 mol% of total lipids, and the amphiphilic block copolymer accounts for 66.0% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 32.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.3% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.1 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 28.4 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.9% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 34.7 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 40.1 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 24.3 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 32.5% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 29.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 50.1 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the The amphiphilic block copolymer accounts for 28.0% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 35.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.6 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 46.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 35.9% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 55.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 16.9 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 27.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 44.6% by weight of the composition;
  • an amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 65.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 21.0% by weight of the composition;
  • an amphiphilic block copolymer GL67, ICE or HGT4002; DSPC, DPPC or DOPE; and DMG-PEG2K or DMG-PEG5K, wherein the GL67, ICE or HGT4002 accounts for 70.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE accounts for 28.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.1% by weight of the composition;
  • Amphiphilic block copolymers DLin-MC3-DMA, ALC-0315 or SM-102; cholesterol Or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 61.3 mol% of the total lipids present in the composition, cholesterol or ⁇ -sitosterol account for 37.6 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.1 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition;
  • an amphiphilic block copolymer cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102 or C12-200; DOTAP, DODAP, DOTMA or DOSPA; cholesterol or ⁇ -sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102 or C12-200 accounts for 30.0 mol% of the total lipids present in the composition, DOTAP, DODAP, DOTMA or DOSPA accounts for 39.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol accounts for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 57.7% by weight of the composition; or
  • An amphiphilic block copolymer DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; and cholesterol or ⁇ -sitosterol, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 32.0 mol% of the total lipids, cholesterol or ⁇ -sitosterol account for 28.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.0% weight percent of the composition.
  • the molar ratio of nitrogen (amine) groups in the cationic lipid to phosphate groups of the nucleic acid (N/P ratio) in the composition is about 0.5 to about 40, about 1 to about 30, about 2 to about 25, about 3 to about 20, about 4 to about 15, about 5 to about 10, about 6 to about 8, about 2 to about 12, about 4 to about 8, about 5 to about 8, about 6 to about 7.8, about 6.7 to about 7.6 or about 6.8 to about 7.5.
  • the ratio of lipid to nucleic acid in the composition is about 1 (1:1) to about 100 (100:1), about 5 (5:1) to about 90 (90:1), about 1 (1:1) to about 50 (50:1), about 5 (5:1) to about 45 (45:1), about 10 (10:1) to about 40 (40:1), about 15 (15:1) to about 35 (35:1), about 20 (20:1) to about 30 (30:1), about 1 (1:1) to about 25 (25:1), about 5 (5:1) to about 30 (30:1), about 5 (5:1) to about 20 (20:1), about 5 (5:1) to about 15 (15:1) or about 5 (5:1) to about 10 (10:1).
  • a mixture of non-identical polymer-lipid compositions formed from at least a first and a second separate entity is included.
  • Each polymer-lipid composition comprises an mRNA and one or more cationic lipids, wherein the first polymer-lipid composition comprises a first cationic lipid and the second polymer-lipid composition comprises a second cationic lipid; wherein the first cationic lipid and the second cationic lipid are not the same; and wherein expression of a protein or peptide encoded by the mRNA after administration of the pharmaceutical composition to a subject
  • the expression of the mRNA encoding protein or peptide is at least about two-fold greater than that of an otherwise identical amount administered with the first lipid nanoparticle but without the second lipid nanoparticle.
  • the first polymer-lipid composition and the second polymer-lipid composition comprise one or more non-cationic lipids.
  • the first non-cationic lipid and the second non-cationic lipid are not the same; and wherein the expression of a protein or peptide encoded by an mRNA after administering the pharmaceutical composition to a subject exceeds the expression of an otherwise identical amount of mRNA-encoded protein or peptide administered with the first polymer-lipid composition but not with the second polymer-lipid composition by at least about two times.
  • the first polymer-lipid composition and the second polymer-lipid composition comprise one or more phospholipids. wherein the first phospholipid and the second phospholipid are not identical; and wherein the expression of a protein or peptide encoded by the mRNA after administration of the pharmaceutical composition to a subject exceeds the expression of an otherwise identical amount of mRNA-encoded protein or peptide administered with the first polymer-lipid composition but without the second polymer-lipid composition by at least about two times.
  • first polymer-lipid composition and the second polymer-lipid composition comprise one or more lipid conjugates. wherein the first lipid conjugate and the second lipid conjugate are not the same; and wherein the expression of a protein or peptide encoded by the mRNA after administration of the pharmaceutical composition to a subject exceeds the expression of an otherwise identical amount of mRNA-encoded protein or peptide administered with the first polymer-lipid composition but without the second polymer-lipid composition by at least about two times.
  • first polymer-lipid composition and the second polymer-lipid composition comprise one or more amphiphilic block copolymers.
  • first amphiphilic block copolymer and the second amphiphilic block copolymer are not the same; and wherein the expression of a protein or peptide encoded by mRNA after administering the pharmaceutical composition to a subject exceeds the expression of an otherwise identical amount of mRNA encoded protein or peptide administered with the first polymer-lipid composition but not with the second polymer-lipid composition by at least about two times.
  • the ratio of the first polymer-lipid composition to the second polymer-lipid composition in the pharmaceutical composition is about 1:1 or about 2:1 or about 3:1 or about 4:1.
  • cationic lipids (or non-lipid cationic agents) that can be distributed on the outer surface of polymer-lipid composition nanoparticles can be additionally added to the polymer-lipid composition.
  • the additionally added cationic lipid can be a sterolamine, such as GL67, ICE, etc.
  • the additional non-lipid cationic agent may be tromethamine, benzalkonium chloride, modified arginine, cetylpyridinium chloride, L-lysine monohydrate, etc.
  • the contact of the polymer-lipid composition with the cationic lipid (or non-lipid cationic agent) comprises dissolving the cationic lipid (or non-lipid cationic agent) in a non-ionic excipient.
  • the non-ionic excipient is selected from polyethylene glycol (15)-hydroxystearate ( HS 15), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K), polyoxyethylene sorbitan monooleate (Tween-80), sorbitan monooleate (Span-85), polyoxyethylene fatty acid esters (such as Myrij 52), polyoxyethylene fatty alcohol ethers (such as Brij 35) and ⁇ -tocopherol polyethylene glycol succinate (TPGS).
  • the contacting of the polymer-lipid composition with the cationic lipid (or non-lipid cationic agent) comprises dissolving the cationic lipid (or non-lipid cationic agent) in a buffer solution, such as PBS and Tris buffer.
  • Suitable mixtures of polymer-lipid compositions for use in the compositions and methods of the present invention include synergistically enhanced nucleic acid delivery mixed formulations as described in International Patent Publications WO 2014144196, WO 2022032154 and U.S. Patent US 10,130,649, which are incorporated herein by reference.
  • the active agent or therapeutic agent further comprises a protein or polypeptide, in some embodiments, the protein is a protein related to translation or transcription. In some embodiments, the protein is related to the CRISPR process. In some embodiments, the protein is a CRISPR-related protein.
  • the protein is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a, Cas13a, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, a homologue thereof, or a modified form thereof.
  • the protein is Cas9.
  • the protein or polypeptide is present in a molar ratio of about 1:1 to about 1:20 to the nucleic acid. In some embodiments, the molar ratio is about 1:1 to about 1:10. In some embodiments, the molar ratio is about 1:3 to about 1:8.
  • the therapeutic agent is a protein or polypeptide.
  • the composition comprises both a protein and a nucleic acid.
  • the composition comprises a Cas9 protein and a single guide nucleic acid.
  • the composition comprises a Cas9 protein, a single guide nucleic acid, and a donor DNA.
  • the therapeutic agent is a small molecule such as a small molecule selected from the group consisting of: anticancer anti-inflammatory drugs, anti-fungal drugs, psychiatric drugs such as analgesics, consciousness-altering agents such as anesthetics or hypnotics, nonsteroidal anti-inflammatory drugs (NSAIDS), anthelmintics, anti-acne agents, anti-angina agents, anti-arrhythmic agents, anti-asthma drugs, anti-bacterial agents, anti-benign prostatic hypertrophy agents, anticoagulants, antidepressants, anti-diabetic agents, antiemetics, anti-epileptic drugs, anti-gout drugs, anti-hypertensive agents, anti-inflammatory agents, anti-malarials, anti-migraine drugs, anti-muscarinics, anti-neoplastic agents, anti-obesity agents, anti-osteoporosis agents, anti-Parkinson's syndrome agents, anti- prolifer, anti- pro
  • the composition further comprises a targeting moiety to target the composition to a target organ, tissue or cell in a subject, preferably the targeting moiety comprises at least one selected from the following: a glycosyl, a lipid, a nucleic acid aptamer, a small molecule therapeutic agent, a vitamin, a polypeptide and a protein such as an antibody.
  • a targeting moiety comprises at least one selected from the following: a glycosyl, a lipid, a nucleic acid aptamer, a small molecule therapeutic agent, a vitamin, a polypeptide and a protein such as an antibody.
  • the targeting moiety is preferably selected from the following group: an epithelial cell ligand, in particular a respiratory epithelial cell ligand, a gastrointestinal epithelial cell ligand, a reproductive epithelial cell ligand or a microfolding cell ligand; an immune cell ligand, in particular a dendritic cell ligand, a T cell ligand, a B cell ligand or a macrophage ligand; an endothelial cell ligand, in particular a lung endothelial cell ligand or a liver endothelial cell ligand; a tumor cell ligand, in particular a melanoma, a lung or liver tissue-associated tumor cell ligand; and/or a skin cell ligand, in particular a dermal fibroblast ligand or a keratinocyte ligand.
  • an epithelial cell ligand in particular a respiratory epithelial cell ligand, a gastrointestinal epit
  • the composition further comprises an adjuvant, preferably the adjuvant comprises at least one selected from the following: CpG oligodeoxynucleotides, polyinosinic: polycytidylic acid, saponin extract (QS-21 extract), aluminum adjuvant, squalene, ⁇ -tocopherol, Tween, Span, lipopolysaccharide LPS, Pam 3 CSK 4 triacyl lipopeptide, cyclic adenosine diphosphate (c-di-AMP), 2′3′-cyclic guanosine monophosphate adenosine monophosphate (cGAMP), monophosphoryl-lipid A, MPL lipid, flagellin or immunomodulatory proteins such as IL-2, IL-12, GM-CSF, TSLP and nucleic acids encoding these immunomodulator proteins.
  • the adjuvant comprises at least one selected from the following: CpG oligodeoxynucleotides, polyinosinic: poly
  • Suitable adjuvants may include, but are not limited to, inorganic salts (e.g., AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH(SO 4 ) 2 , silica, aluminum, aluminum hydroxide, Ca 3 (PO 4 ) 2 , kaolin or carbon), polynucleotides with or without immune stimulating complexes (ISCOMs) (e.g., CpG oligonucleotides, such as Chuang, TH et al., (2002) J. Leuk. Biol. 71(3):538-44; Ahmad Nejad, P. et al., (2003) J. Leuk. Biol. 71(3):538-44). (2002) Eur.
  • inorganic salts e.g., AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH(SO 4 ) 2 , silica, aluminum, aluminum hydroxide, Ca 3 (PO 4 ) 2
  • the adjuvant is another RNA.
  • a suitable adjuvant is aluminum phosphate.
  • a suitable adjuvant is aluminum hydroxide.
  • a suitable adjuvant is a combination of aluminum phosphate and aluminum hydroxide.
  • the composition further comprises a transfection enhancer, preferably the transfection enhancer comprises at least one selected from the group consisting of a pulmonary surfactant protein, a cell-penetrating peptide, an amphiphilic polypeptide, a mucolytic enzyme, 1,2-propylene glycol, a cellulose (such as carboxymethyl cellulose or hydroxypropyl cellulose), a hyaluronate, alginate, pectin, polyethylene glycol, a poloxamer, a poloxamine, glucose, fructose, sucrose, trehalose, dextran, polyvinyl pyrrolidone, chitosan, polyvinyl alcohol, polyvinyl acetate, agglutinin, polylactic acid, polyhydroxybutyric acid, tromethamine, benzalkonium chloride, modified arginine, cetylpyridinium chloride, L-lysine
  • the compositions are in the form of nanoparticles having an average size of about 1000 nm or less.
  • the nanoparticles have an average size of about 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 75 nm or less, or about 50 nm or less.
  • about 30% to about 100%, about 70% to about 100%, about 90% to about 100%, about 50% to about 90%, about 70% to about 90%, or about 80% to about 90% of the nanoparticles have the active or therapeutic agent encapsulated therein.
  • the composition comprising polymer-lipid of the present invention
  • the composition is formulated as a solution, dry powder, atomization or spray.
  • the composition is formulated to be administered to the lung and/or nose by aerosolization, dry powder, inhalation, atomization or instillation.
  • the present invention particularly provides mRNA-polymer-lipid compositions prepared using the methods of the present invention as described herein.
  • the method for encapsulating mRNA as described herein includes a step of mixing a lipid solution with an mRNA solution in the presence of an amphiphilic polymer (e.g., poloxamine and/or poloxamer) to form polymer-lipid nanoparticles that encapsulate mRNA.
  • an amphiphilic polymer is present in the mRNA solution before mixing.
  • the amphiphilic polymer is present in the lipid solution before mixing.
  • an amphiphilic polymer is added during the mixing of the mRNA solution and the lipid solution.
  • an amphiphilic polymer is added after the mixing of the mRNA solution and the lipid solution.
  • the lipid solution in a suitable polymer-lipid comprises a cationic lipid and a non-cationic lipid (also referred to as a helper lipid).
  • a suitable lipid solution comprises a cationic lipid, a non-cationic lipid and a PEG-modified lipid or PEG.
  • a suitable lipid solution comprises a cationic lipid, a non-cationic lipid, a cholesterol-based lipid and a PEG-modified lipid or PEG.
  • Various lipids can be dissolved in a suitable solvent with the required respective amount and/or ratio to prepare a lipid solution for the methods described herein. A variety of methods can be used to prepare a suitable lipid solution. Exemplary methods are described in US 2016/0038432, US 2018/0153822 and US 2018/0125989, which are incorporated herein by reference.
  • the lipid solution in a suitable polymer-lipid comprises one or more cationic lipids, non-cationic lipids, cholesterol and/or PEG-modified lipids.
  • the polymer-lipid can comprise at least one of the following cationic lipids: DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, A2-Iso5-2DC18, BAME-016B, 9A1P9, OF-Deg-Lin, 306Oi10, TT3, FTT5, C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, GL67, HGT5000 or HGT5001, etc.
  • the polymer-lipid may include at least one of the following non-cationic lipids: DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (2-dioleoyl-sn-glycero-3-phosphoethanolamine).
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DOPE 1,2-dioleyl-sn-glycero-3-phosphoethanolamine
  • the polymer-lipid may include cholesterol and/or ⁇ -sitosterol, etc.
  • the polymer-lipid includes PEG-modified/modified lipids, and the PEG-modified lipids may include (but not exclusively) lipids covalently linked to alkyl chains having a length of C 6 -C 20 , and PEG chains up to 5 kDa.
  • the polymer-lipid comprises DMG-PEG2K, DMPE-PEG2K, DSPE-PEG2K, DSPE-PEG2K-Mannose, DMG-PEG5K, DMPE-PEG5K, DSPE-PEG5K, DSPE-PEG5K-Mannose, etc.
  • the polymer-lipid comprises one of the following lipid formulations: DLin-MC3-DMA, DSPC, cholesterol, DMG-PEG2K; DLin-MC3-DMA, DPPC, cholesterol, DMG-PEG2K; DLin-MC3-DMA, DOPE, cholesterol, DMG-PEG2K; DLin-MC3-DMA, DSPC, cholesterol, DMG-PEG5K; DLin-MC3-DMA, DPPC, cholesterol, DMG-PEG5K; DLin-MC3-DMA, DOPE, cholesterol, DMG-PEG5K; ALC-0315, DSPC, cholesterol, DMG-PEG2K; ALC-0315, DPPC, cholesterol, DMG-PEG2K; ALC-0 315, DOPE, cholesterol, DMG-PEG2K; ALC-0315, DSPC, cholesterol, DMG-PEG5K; ALC-0315, DPPC, cholesterol, DMG-PEG
  • the method comprises:
  • the method comprises:
  • the method comprises:
  • the method comprises:
  • the method further comprises the step of removing free lipid components and/or amphiphilic block copolymers, preferably by dialysis and/or tangential flow filtration.
  • the method further comprises the step of adding the amphiphilic block copolymer again after removing the free lipid component and/or the amphiphilic block copolymer.
  • mRNA solution or lipid solution, or both can be heated to a predetermined temperature higher than ambient temperature before mixing. In some embodiments, mRNA solution and lipid solution are heated to a predetermined temperature respectively before mixing. In some embodiments, mRNA solution and lipid solution are mixed at ambient temperature, and then heated to a predetermined temperature after mixing. In some embodiments, lipid solution is heated to a predetermined temperature and mixed with mRNA solution at ambient temperature. In some embodiments, mRNA solution is heated to a predetermined temperature and mixed with lipid solution at ambient temperature. In some embodiments, compared with other identical methods without heating steps, a heating step is included in the method process (before, during or after formation) to provide a higher encapsulation rate of mRNA.
  • the encapsulation of mRNA in polymer-lipid can be further enhanced by heating a formulation solution comprising mRNA-polymer-lipid and some free mRNA not encapsulated in polymer-lipid forming solution to a predetermined temperature as described herein.
  • the mRNA solution is heated to a predetermined temperature by adding an mRNA stock solution at ambient temperature to a buffer solution that is heated to reach the desired predetermined temperature.
  • the term "ambient temperature” refers to the temperature in a room, or the temperature surrounding an object of interest without heating or cooling.
  • the ambient temperature maintained by one or more solutions is or is less than about 35°C, 30°C, 25°C, 20°C, or 16°C.
  • the ambient temperature maintained by one or more solutions is in the range of about 15-35°C, about 15-30°C, about 15-25°C, about 15-20°C, about 20-35°C, about 25-35°C, about 30-35°C, about 20-30°C, about 25-30°C, or about 20-25°C.
  • the ambient temperature maintained by one or more solutions is 20-25°C.
  • the predetermined temperature above ambient temperature is typically greater than about 25°C.
  • the predetermined temperature suitable for the present invention is or is greater than about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C.
  • the predetermined temperature suitable for the present invention is in the range of about 25-70°C, about 30-70°C, about 35-70°C, about 40-70°C, about 45-70°C, about 50-70°C, or about 60-70°C.
  • the predetermined temperature suitable for the present invention is about 65°C.
  • a pump is used to mix the mRNA solution and the lipid solution. Since the encapsulation procedure with such mixing can be carried out on various scales, different types of pumps can be used to adapt to the required scale. However, it is generally desirable to use a pulseless flow pump. As used herein, a pulseless flow pump refers to any pump that can establish a continuous flow rate at a stable flow rate. The type of suitable pump may include, but is not limited to, a gear pump and a centrifugal pump.
  • the mRNA solution and lipid solution can be mixed at various flow rates.
  • the mRNA solution can be mixed at a flow rate greater than the flow rate of the lipid solution.
  • the mRNA storage solution can be mixed at a flow rate greater than at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 times greater than the flow rate of the lipid solution.
  • the suitable flow rate for mixing can be determined based on the scale.
  • the lipid solution is mixed at a flow rate within the range of about 1-4 ml/min, 2-6 ml/min, 3-8 ml/min, 5-10 ml/min, 10-20 ml/min, 15-30 ml/min, 25-75 ml/min, 20-50 ml/min, 25-75 ml/min, 30-90 ml/min, 40-100 ml/min, 50-110 ml/min, 75-200 ml/min, 200-350 ml/min, 350-500 ml/min, 500-650 ml/min, 650-850 ml/min, or 850-1000 ml/min.
  • the mRNA solution is mixed at a flow rate in the range of about 1000-2000 ml/min, 2000-3000 ml/min, 3000-4000 ml/min, or 4000-5000 ml/min.
  • the present polymer-lipid composition can be prepared using nanoprecipitation, which can be performed by the following unit operation, wherein the polymer-lipid composition is mixed by kinetics, and then matured and serially diluted from its individual lipid component self-assembly.
  • the unit operation generally includes a continuous online combination of three liquid streams and an online maturation step: an aqueous buffer is mixed with a lipid stock solution, matured via a controlled residence time, and the nanoparticles are diluted.
  • the nanoprecipitation itself occurs in a mixer suitable for scale, which is designed to allow the continuous, high-energy combination of an aqueous solution and a lipid stock solution dissolved in ethanol.
  • the aqueous solution and the lipid stock solution are all continuously flowed into the mixer at the same time.
  • the ethanol content of the lipid dissolution is kept to decrease suddenly, and the lipids are all precipitated from each other.
  • the nanoparticles are self-assembled in the mixing chamber.
  • the present invention provides a polymer-lipid composition produced by a continuous mixing method, for example, a process comprising the steps of providing an aqueous solution (which may include an amphiphilic block polymer) containing a nucleic acid such as mRNA in a first reservoir, providing an organic lipid solution (which may include an amphiphilic block polymer) in a second reservoir, and mixing the aqueous solution and the organic lipid solution so that the organic lipid solution mixes with the aqueous solution to substantially immediately produce a polymer-lipid composition encapsulating the nucleic acid (e.g., mRNA).
  • aqueous solution which may include an amphiphilic block polymer
  • organic lipid solution which may include an amphiphilic block polymer
  • the present invention provides polymer-lipid compositions produced by direct dilution method, and the direct dilution method comprises forming lipid solution and immediately and directly introducing lipid solution into the collection container containing controlled amount of dilution buffer.
  • the collection container comprises one or more components configured to stir the collection container contents to promote dilution.
  • the amount of dilution buffer present in the collection container is substantially equal to the volume of lipid solution introduced therein.
  • the liposome solution in 45% ethanol will advantageously produce smaller particles when introduced into the collection container containing equal volume of dilution buffer.
  • the present invention provides a polymer-lipid composition produced by a direct dilution method, wherein a third reservoir containing a dilution buffer is fluidically connected to the second mixing zone.
  • ethanol-drop precipitation has become the industry standard for preparing nucleic acid lipid nanoparticles. Precipitation reactions are favored for their continuity, scalability, and ease of use.
  • Such methods typically use high-energy mixers (e.g., T-junctions, confined impinging jets, microfluidic mixers, vortex mixers) to introduce lipids (in ethanol) into a suitable reverse phase solvent (i.e., water) in a controlled manner, thereby driving liquid supersaturation and spontaneous precipitation into lipid particles.
  • the vortex mixer used is described in U.S.
  • the microfluidic mixer used may employ the mixer described in PCT patent WO/2014/172045, which is incorporated herein by reference in its entirety.
  • the mixing step is performed using a T-junction, a confined impinging jet, a microfluidic mixer, or a vortex mixer.
  • the loading step is performed using a T-junction, a confined impinging jet, a microfluidic mixer, or a vortex mixer.
  • the present invention provides PoLixNano-CPLs (polymer-lipid compositions containing cation-polymer-lipid conjugates CPL) and/or PoLixNano-sterolamines (e.g., polymer-lipid compositions containing sterolamine GL67) prepared by "standard” techniques and "post-insertion” techniques, wherein the "post-insertion” techniques, i.e., CPL or GL67 is inserted into, e.g., a preformed polymer-lipid composition (PoLixNano), and the "standard” techniques, wherein CPL or GL67 is included in the lipid mixture during, e.g., the PoLixNano formation step.
  • PoLixNano-CPLs polymer-lipid compositions containing cation-polymer-lipid conjugates CPL
  • PoLixNano-sterolamines e.g., polymer-lipid compositions containing sterolamine GL67
  • Methods for preparing PoLixNano-CPLs Methods for preparing PoLixNano-GL67 are described in, for example, U.S. Patent Nos. 5,705,385; 6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent Publication No. 20020072121; and PCT Publication No. WO 00/62813, the disclosures of which are incorporated herein by reference in their entirety for all purposes.
  • Methods for preparing PoLixNano-GL67 are described in PCT Publication No. WO 2022/032154, the disclosures of which are incorporated herein by reference in their entirety for all purposes.
  • the polymer-lipid compositions of the present invention can be sized by any method useful for adjusting the size of liposomes. Size adjustment can be performed to obtain a desired size range and a relatively narrow particle size distribution.
  • nucleic acids in a polymer-lipid composition are pre-concentrated, for example, as described in U.S. Patent Application No. 09/744,103, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
  • the method provided by the present invention also includes adding a transfection enhancer to achieve enhanced nucleic acid transfection effect.
  • suitable transfection enhancers include pulmonary surfactant proteins, cell-penetrating peptides, amphipathic polypeptides, mucolytic enzymes, 1,2-propylene glycol, cellulose (such as carboxymethyl cellulose or hydroxypropyl cellulose), hyaluronate, alginate, pectin, polyethylene glycol, poloxamer, poloxamine, glucose, fructose, sucrose, trehalose, dextran, sucrose, trehalose, mannose, polyvinyl pyrrolidone, chitosan, polyvinyl alcohol, polyvinyl acetate, lectin, polylactic acid, polyhydroxybutyric acid, tromethamine, benzalkonium chloride, modified arginine, cetyl pyridinium chloride, L-lysine monohydrate, and polylactic acid-glycoli
  • the method may further include adding a non-lipid polycation, which is used to achieve lipid transfection of cells using the composition of the present invention.
  • suitable non-lipid polycations include hexadimethrine bromide (trade name from Sigma-Aldrich Chemical Co.) or other hexadimethrine salts.
  • suitable polycations include, but are not limited to, for example, poly-L-ornithine, poly-L-arginine, poly-D-arginine, poly-L-lysine, poly-D-lysine, polyallylamine, chitosan, and polyethyleneimine and pharmaceutically acceptable salts thereof. These polycations are preferably added after the particles have been formed.
  • the inventive method as herein described comprises the step of removing lipid and/or amphipathic polymer (for example poloxamine and/or poloxamer) that do not form particles.
  • free lipid and/or amphipathic polymer can be removed by buffer exchange techniques such as dialysis.
  • polymer-lipid formation solution is exchanged in the solution constituting the product formulation solution.
  • the mixture of the polymer-lipid nanoparticle formed can be dialyzed in one or more formulation solutions to remove free lipid and/or amphipathic polymer present during the formation of the polymer-lipid nanoparticle.
  • the solution comprising polymer-lipid nanoparticles can be replaced from polymer-lipid forming solution to formulation solution by any of a variety of buffer exchange techniques known in the art.
  • the step of replacing the polymer-lipid forming solution into formulation solution is accompanied by the purification and/or concentration of polymer-lipid.
  • Various methods can be used to realize the replacement of solution and the purification of polymer-lipid or the concentration of polymer-lipid in solution. For example, in some embodiments, this solution replacement is realized by diafiltration.
  • the solution is replaced, and tangential flow filtration (TFF, also referred to as cross-flow filtration) is used to purify polymer-lipid.
  • TMF tangential flow filtration
  • TFF non-desired permeate passes through the filter, and the desired retentate (polymer-lipid nanoparticles and free mRNA) is passed along the filter and collected downstream.
  • desired retentate polymer-lipid nanoparticles and free mRNA
  • Exemplary TFF purification methods are described in US2016/0040154 and US2015/0376220, which are incorporated herein by reference.
  • the residual amount of the amphiphilic polymer remains in the formulation after removal.
  • residual amount means the remaining amount after substantially all substances (amphiphilic polymers such as poloxamine and/or poloxamer described herein) are removed in the composition.
  • the residual amount can be detected qualitatively or quantitatively using known techniques. The residual amount may not be detected using known techniques.
  • excess mRNA is also removed along with the amphiphilic polymer (eg, poloxamine and/or poloxamer) present during mRNA-polymer-lipid formation.
  • amphiphilic polymer eg, poloxamine and/or poloxamer
  • a suitable formulation solution comprises only residual citrate. In some embodiments, a suitable formulation solution comprises only residual non-aqueous solvents such as ethanol. In some embodiments, a suitable formulation solution contains less than about 10 mM citrate. In some embodiments, a suitable formulation solution contains less than about 25% non-aqueous solvents such as ethanol. In some embodiments, a suitable formulation solution does not require any further lyophilization prior to lyophilization.
  • the formulation solution does not require any further downstream processing (e.g., buffer exchange and/or further purification steps and/or additional excipients) prior to administration to a sterile fill in a vial, syringe, or other container. In some embodiments, the formulation solution does not require any further downstream processing prior to administration to a subject.
  • further downstream processing e.g., buffer exchange and/or further purification steps and/or additional excipients
  • a suitable formulation solution after freeze-thawing, can improve or enhance the mRNA encapsulation capacity of the polymer-lipid composition after heating.
  • a suitable formulation solution is 10% sucrose and can be freeze-stable.
  • the polymer-lipid composition in the suitable formulation solution after heating can be stably frozen (e.g., retaining enhanced encapsulation) in about 1%, about 3%, about 5%, about 7%, about 9%, about 10%, about 13%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% sucrose solution.
  • the suitable formulation solution may not require any downstream purification or processing and can be stably stored in frozen form.
  • suitable formulation solutions may include buffers or salts.
  • Exemplary buffers may include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), calcium chloride, ammonium sulfate, magnesium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate.
  • Exemplary salts may include sodium chloride, magnesium chloride, lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, sodium malonate, sodium nitrate, sodium sulfate, and potassium chloride.
  • suitable formulation solutions may include a combination of cations and anions contained in the above-mentioned buffer salts.
  • a suitable formulation solution comprises one or more protective agents, and each of the one or more protective agents is independently a polyol (e.g., a diol or triol, such as propylene glycol (i.e., 1,2-propylene glycol)), 1,3-propylene glycol, glycerol, (+/-)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a non-detergent sulfobetaine (e.g., NDSB-201 (3-(1-pyridyl)-1-propanesulfonate), an osmotic agent (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000,
  • suitable formulation solutions are aqueous solutions comprising pharmaceutically acceptable excipients (including but not limited to cryoprotectants).
  • suitable formulation solutions are aqueous solutions comprising pharmaceutically acceptable excipients, and the excipients include but are not limited to sugars, such as one or more of trehalose, sucrose, mannose, lactose and mannitol.
  • suitable formulation solutions include trehalose.
  • suitable formulation solutions include sucrose.
  • suitable formulation solutions include mannose.
  • suitable formulation solutions include lactose.
  • suitable formulation solutions include mannitol.
  • a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume sugars, such as trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume trehalose. In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume sucrose. In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume mannose. In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume lactose.
  • a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume mannitol.
  • a suitable formulation solution is an aqueous solution containing about 10% weight/volume sugars, such as trehalose, sucrose, mannose, lactose, and mannitol.
  • a suitable formulation solution is an aqueous solution containing about 10% weight/volume trehalose.
  • a suitable formulation solution is an aqueous solution containing about 10% weight/volume sucrose.
  • a suitable formulation solution is an aqueous solution comprising about 10% weight/volume mannose.
  • a suitable formulation solution is an aqueous solution comprising about 10% weight/volume lactose.
  • a suitable formulation solution is an aqueous solution comprising about 10% weight/volume mannitol.
  • suitable formulation solutions have a pH between pH 4.5 and pH 7.5. In some embodiments, suitable formulation solutions have a pH between pH 5.0 and pH 7.0. In some embodiments, suitable formulation solutions have a pH between pH 5.5 and pH 7.0. In some embodiments, suitable formulation solutions have a pH higher than pH 4.5. In some embodiments, suitable formulation solutions have a pH higher than pH 5.0. In some embodiments, suitable formulation solutions have a pH higher than pH 5.5. In some embodiments, suitable formulation solutions have a pH higher than pH 6.0. In some embodiments, suitable formulation solutions have a pH higher than pH 6.5.
  • the present invention also provides the polymer-lipid composition in the form of a kit.
  • the kit may include containers divided into various components (e.g., various lipids, polymer components of active agents or therapeutic agents such as nucleic acids and particles) for accommodating the polymer-lipid composition.
  • the kit also includes an endosomal membrane destabilizer (e.g., calcium ions).
  • the kit typically includes the polymer-lipid composition of the present invention preferably in a dehydrated form and instructions for their rehydration and administration.
  • polymer-lipid compositions of the present invention can be customized to preferentially target special target tissues, organs or tumors.
  • the preferential targeting of polymer-lipid compositions can be carried out by controlling the particle components themselves. For example, as described in Example 11 and Example 16, it has been found that (PoLixNano+T904) physical mixed formulations can be used for preferentially targeting liver and spleen, and polymer-lipid composition formulations can be used for preferentially targeting lungs.
  • a targeting moiety may be desirable to attach to the surface of the polymer-lipid composition to further enhance the targeting of the particle.
  • Methods for attaching a targeting moiety e.g., a glycosyl, polypeptide, antibody, protein, etc.
  • a lipid or amphiphilic polymer such as those used in the particles of the present invention
  • the polymer-lipid composition of the present invention is effective for introducing an active agent or therapeutic agent (e.g., mRNA) into a cell. Therefore, the present invention also provides a method for introducing an active agent or therapeutic agent such as a nucleic acid (e.g., mRNA) into a cell. The method is performed in vitro or in vivo by the following steps: first forming a particle as described above, then contacting the particle with a cell to be sufficient to cause an active agent or therapeutic agent to be introduced into a cell for a period of time.
  • Polymer-lipid compositions of the present invention can be adsorbed on almost any cell type mixed or contacted therewith. Once adsorbed, the particle can be endocytosed by a part of the cell, exchange lipids with the cell membrane, or merge with the cell. Transfer or combination of the active agent or therapeutic agent (e.g., nucleic acid) part of the particle can occur by any of these approaches. Specifically, when fusion occurs, the particle membrane is integrated into the cell membrane and the content of the particle is combined with the intracellular fluid.
  • the active agent or therapeutic agent e.g., nucleic acid
  • the polymer-lipid composition of the present invention can be administered alone or in a mixture with a pharmaceutical carrier (e.g., saline or phosphate buffered saline PBS), which is selected according to the route of administration and standard pharmaceutical practice.
  • a pharmaceutical carrier e.g., saline or phosphate buffered saline PBS
  • standard buffered saline e.g., 135-150mM NaCl
  • suitable carriers include, for example, water (nuclease-free water), buffered water, 0.4% saline, 0.3% glycine, etc., including glycoproteins such as albumin, lipoproteins, globulins, etc. for increasing stability.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • the pharmaceutically acceptable carrier is generally added after the particles are formed.
  • the particles can be diluted into a pharmaceutically acceptable carrier such as standard buffered saline.
  • the concentration of particles in the pharmaceutical preparation can vary widely, usually at or at least about 0.1-30% by weight, to about 10-90% by weight at most, and is selected mainly by fluid volume, viscosity, etc. according to the specific mode of administration selected.
  • the concentration can be increased to reduce the fluid load associated with the treatment. This may be particularly desirable in patients with congestive heart failure or severe hypertension associated with atherosclerosis.
  • the particles made of irritating lipids can be diluted to low concentrations, thereby alleviating inflammation at the administration site.
  • the pharmaceutical composition of the present invention can be sterilized by conventional, well-known sterilization techniques.
  • the aqueous solution can be packaged for standby use or filtered and lyophilized under aseptic conditions, and the lyophilized preparation is combined with a sterile aqueous solution before administration.
  • the composition can contain pharmaceutical auxiliary substances as needed to approach physiological conditions, such as pH adjustment and buffers, tension regulators, etc.
  • the particle suspension can include a lipid-protectant that protects lipids from free radicals and lipid-peroxidation damage during storage. Lipophilic free radical quenchers, such as alpha tocopherol and water-soluble ion-specific chelators, such as ferrioxamine are suitable.
  • therapeutic agents such as nucleic acids (e.g., mRNA) can be delivered to any cell grown in culture, regardless of plant or animal origin, vertebrate or invertebrate, and any tissue or type.
  • the cell is an animal cell, more preferably a mammalian cell, and most preferably a human cell.
  • the contact between cells and the polymer-lipid composition of the present invention occurs in a biocompatible culture medium when performed in vitro.
  • the particle concentration varies widely, depending on the specific application, but is generally between about 1 ⁇ mol and about 10 mmol.
  • Treatment of cells with the polymer-lipid composition is generally performed at physiological temperature (about 37° C.) for about 1-48 hours, preferably for a period of about 2-6 hours.
  • the polymer-lipid composition suspension is added to seeded cells at a cell density of about 10 3 to about 10 6 cells/ml, more preferably about 1x10 5 cells/ml, at a confluency of 60-80%.
  • the concentration of the suspension added to the cells is preferably about 0.01-0.4 ⁇ g/ml, more preferably about 0.2 ⁇ g/ml.
  • the delivery efficiency of the polymer-lipid compositions of the present invention can be optimized using the endosomal release parameter (ERP) assay.
  • ERP endosomal release parameter
  • administration can be by any means known in the art, for example, by injection, nasal drops, inhalation (e.g., atomization or dry powder formulations, inhaled intranasally or intratracheally), oral administration, transdermal administration, genital tract administration, or rectal administration.
  • Administration can be achieved by single or divided doses.
  • the pharmaceutical composition can be administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly.
  • the pharmaceutical composition is administered intravenously or intraperitoneally by push injection (see, e.g., U.S. Patent No. 5,286,634).
  • Intracellular nucleic acid delivery has also been discussed in Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino et al., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. Drug Carrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993).
  • Other methods of administering lipid-based therapeutics are described, for example, in U.S. Pat. Nos. 3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578.
  • polymer-lipid compositions of the present invention can be administered by direct injection at the disease site or by injection at a site remote from the disease site (see, e.g., Culver, HUMAN GENE THERAPY, Mary Ann Liebert, Inc., Publishers, New York. Pages 70-71 (1994)).
  • Culver HUMAN GENE THERAPY
  • Mary Ann Liebert, Inc. Publishers, New York. Pages 70-71 (1994)
  • the disclosures of the above references are incorporated herein by reference in their entirety for all purposes.
  • compositions of the present invention may be in liquid form or solid form.
  • the compositions are in liquid form and can be delivered to the lungs via aerosolization, typically using various commercially available aerosol devices.
  • the compositions are in solid form and are suitable for inhalation to administer the compositions to the respiratory tract.
  • compositions can be delivered to the respiratory tract by suitable methods such as intranasal instillation, intratracheal instillation, and intratracheal injection.
  • suitable methods such as intranasal instillation, intratracheal instillation, and intratracheal injection.
  • the composition or nanoparticles are delivered intranasally, intrabronchially, or pulmonary.
  • a nebulizer or inhaler is used to deliver the composition or nanoparticles.
  • the composition is delivered to the lungs by a nebulized inhalation route.
  • Inhalation can be performed through the nose and/or mouth of an individual.
  • Administration can be performed by actively inhaling the composition or by administering the composition to an individual via a respirator.
  • Exemplary devices for delivering the composition to the respiratory tract include, but are not limited to, dry powder inhalers (DPI), pressurized metered dose inhalers (pMDI), nebulizers, and electric aerosol devices.
  • DPI dry powder inhalers
  • pMDI pressurized metered dose inhalers
  • nebulizers nebulizers
  • electric aerosol devices electric aerosol devices.
  • compositions of the present invention can be made into aerosol preparations (i.e., they can be "nebulized") alone or in combination with other suitable ingredients for administration by inhalation (e.g., intranasal or intratracheal) (see, Brigham et al., Am. J. Sci., 298: 278 (1989); and Patel et al., Advanced Materials, 31(8): e1805116 (2019)).
  • Aerosol preparations can be placed in a propellant that allows pressurization, such as dichlorodifluoromethane, propane, nitrogen, etc., or can be generated by a nebulizer.
  • composition of the present invention can be made into sprays individually or in combination with other suitable ingredients, so as to be used by nasal inhalation.
  • the pharmaceutical composition can be delivered by intranasal spray, inhalation, and/or other aerosol delivery vehicles.
  • the method for directly delivering nucleic acid compositions to the lungs by nasal aerosol spray has been described in, for example, U.S. Patent Nos. 5,756,353 and 5,804,212.
  • drug delivery using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (U.S. Patent No. 5,725,871) is also well known in the pharmaceutical field.
  • transmucosal drug delivery using a polytetrafluoroethylene carrier matrix is described in U.S. Patent No. 5,780,045. The disclosure of the above patent is incorporated herein by reference as a whole for all purposes.
  • Formulations suitable for parenteral administration such as, for example, by intraarticular (in a joint), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes include aqueous and nonaqueous, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives.
  • the composition is preferably administered, for example, by intravenous infusion, oral, topical, intraperitoneal, intravesical, or intrathecal administration.
  • Systemic delivery for in vivo therapy e.g., delivery of therapeutic nucleic acids through the body's circulatory system
  • To distant target cells such as PCT Publication Nos. WO 05/007196, WO 05/121348, WO 05/120152, and WO 04/002453 describe the use of nucleic acid-lipid particles to achieve such purposes, and the disclosure thereof is incorporated herein by reference for all purposes.
  • the present invention also provides a fully encapsulated polymer-lipid composition that protects nucleic acids from being degraded by nucleases in serum, is non-immunogenic, is very small in size, and is suitable for repeated administration.
  • polymer-lipid compositions are formulated using suitable pharmaceutical carriers.
  • suitable pharmaceutical carriers can be used in the compositions and methods of the present invention, and a variety of aqueous carriers can be used, for example, water, buffered water, physiological saline, 0.3% glycine, etc., and glycoproteins for enhancing stability, such as albumin, lipoprotein, globulin, etc., can also be used.
  • aqueous carriers for example, water, buffered water, physiological saline, 0.3% glycine, etc.
  • glycoproteins for enhancing stability such as albumin, lipoprotein, globulin, etc.
  • These compositions can be sterilized by conventional liposome sterilization techniques such as filtration.
  • compositions can contain pharmaceutical auxiliary substances as required to approach physiological conditions, such as pH adjustment and buffers, tension regulators, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • pharmaceutical auxiliary substances such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • the polymer-lipid composition disclosed herein can be delivered to an individual via oral administration.
  • the particles can be combined with excipients and in the form of ingestible tablets, buccal tablets, troche, capsules, pills, lozenges, elixirs, mouthwashes, suspensions, oral sprays, syrups, wafers, etc. (see, e.g., U.S. Patent Nos. 5,641,515, 5,580,579, and 5,792,451, the disclosure of which is incorporated herein by reference for all purposes).
  • These oral dosage forms may also include the following: adhesives, gelatin; excipients, lubricants, and/or flavorings.
  • the unit dosage form When the unit dosage form is a capsule, it may contain a lipid carrier in addition to the above-mentioned substances. A variety of other substances may exist as a coating or additionally modify the appearance of the dosage unit. Of course, any substance used in the preparation of any unit dose should be pharmaceutically pure and substantially nontoxic in the amount used.
  • these oral formulations can contain at least about 0.1% polymer-lipid composition or more, although the percentage of polymer-lipid composition can certainly be different, and can be conveniently between about 1% or 2% to about 60% or 70% or more of the total formulation weight or volume.
  • the amount of particles in each therapeutically effective composition that can be prepared is such that a suitable dose should be obtained in any given unit dose of the compound.
  • the technician in the field of preparing the pharmaceutical preparation should consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and as such, many dosages and treatment regimens may be desirable.
  • Formulations suitable for oral administration may consist of: (a) a lipid solution, such as a suspension in dilute (a) a therapeutic agent such as a nucleic acid (e.g., mRNA) in an effective amount of a packaged therapeutic agent such as a nucleic acid (e.g., mRNA) in a dispensing agent such as water, saline, or PEG400; (b) a capsule, sachet, or tablet, each containing a predetermined amount of a therapeutic agent such as a nucleic acid (e.g., mRNA), such as a liquid, solid, granular, or gelatin; (c) a suspension in an appropriate liquid; and (d) a suitable emulsion.
  • a therapeutic agent such as a nucleic acid (e.g., mRNA) in an effective amount of a packaged therapeutic agent such as a nucleic acid (e.g., mRNA) in a dispensing agent such as water, saline,
  • Tablet forms may include lactose, sucrose, mannose, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and one or more of pharmaceutically compatible carriers.
  • Pastille forms can include a therapeutic agent such as a nucleic acid (e.g., mRNA) in a flavoring agent, such as sucrose, as well as pastilles containing the therapeutic agent in an inert matrix such as gelatin and glycerin or sucrose and acacia emulsions, gelatin, and similar pastilles containing in addition to the therapeutic agent carriers known in the art.
  • a therapeutic agent such as a nucleic acid (e.g., mRNA) in a flavoring agent, such as sucrose, as well as pastilles containing the therapeutic agent in an inert matrix such as gelatin and glycerin or sucrose and acacia emulsions, gelatin, and similar pastilles containing in addition to the therapeutic agent carriers known in the art.
  • the polymer-lipid composition can be incorporated into a wide range of topical dosage forms.
  • a suspension of the polymer-lipid composition containing nucleic acids can be formulated and applied as a gel, oil, emulsion, topical cream, suppository, paste, ointment, lotion, foam, mousse, etc.
  • the method of the present invention can be implemented in a variety of hosts.
  • Preferred hosts include mammalian species, such as primates (e.g., humans and chimpanzees and other non-human primates), canines, felines, equines, bovines, sheep, goats, rodents (e.g., rats and mice), lagomorphs, and pigs.
  • the amount of formulation administered will depend on the ratio of therapeutic agent (e.g., nucleic acid) to polymer-lipid component, the specific therapeutic agent (e.g., nucleic acid) used, the disease or functional disorder to be treated, the age, weight and condition of the patient, and the judgment of the clinician, but will generally be about 0.01 to about 50 mg/kg body weight, preferably about 0.1 to about 5 mg/kg body weight, or about 10 8 -10 10 particles/administration (e.g., nebulized inhalation).
  • therapeutic agent e.g., nucleic acid
  • polymer-lipid component e.g., polymer-lipid component
  • specific therapeutic agent e.g., nucleic acid
  • the pharmaceutical compositions described herein are formulated for administration by inhalation, orally, intraadipose, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesically, intravitreally, topically, mucosally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, topically, buccally, transdermally, vaginally, in the form of creams, via catheter, via lavage, via continuous infusion, via infusion, via inhalation
  • the pharmaceutical composition is formulated for intravenous or intra-arterial injection.
  • compositions and methods of the present invention are used to treat a wide variety of cell types in vivo and in vitro.
  • Suitable cells include, for example, alveolar cells (including type I alveolar epithelial cells and type II alveolar epithelial cells), bronchial epithelial cells, goblet cells, ciliated cells, club cells, undifferentiated basal cells, pulmonary ionocytes, microfold cells (M cells), dendritic cells (DC cells), macrophages, T cells, B cells, NK cells, neutrophils, eosinophils, basophils, monocytes, hematopoietic precursor (stem) cells, fibroblasts, keratinocytes, hepatocytes, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, resting lymphocytes, terminally differentiated cells, slow or non-circulating primary cells, parenchymal cells, lymphoid cells, epithelial cells, osteocytes, etc.
  • active agents or therapeutic agents such as RNA (e.g., mRNA) are delivered to bronchial epithelial cells, ciliated cells, pulmonary ionocytes, microfold cells (M cells), dendritic cells (DC cells), T cells.
  • RNA e.g., mRNA
  • M cells microfold cells
  • DC cells dendritic cells
  • active agents or therapeutic agents such as RNA (e.g., mRNA) are delivered to cancer cells such as, for example, lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, bile duct cancer cells, small intestine cancer cells, gastric cancer cells, esophageal cancer cells, gallbladder cancer cells, liver cancer cells, pancreatic cancer cells, appendix cancer cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, renal cancer cells, central nervous system cancer cells, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteogenic sarcoma tumor cells, and blood cancer cells.
  • cancer cells such as, for example, lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, bile duct cancer cells, small intestine cancer cells, gastric cancer cells, esophageal cancer cells, gallbladder cancer cells, liver cancer cells, pancreatic cancer cells,
  • RNA e.g., mRNA
  • the methods and compositions can be used with a wide variety of vertebrates, including mammals, such as, for example, canines, felines, equines, bovines, sheep, goats, rodents (such as mice, rats and guinea pigs), lagomorphs, pigs, and primates (e.g., monkeys, chimpanzees and humans) cells.
  • mammals such as, for example, canines, felines, equines, bovines, sheep, goats, rodents (such as mice, rats and guinea pigs), lagomorphs, pigs, and primates (e.g., monkeys, chimpanzees and humans) cells.
  • tissue culture of cells may be required, this is well known in the art.
  • Freshney Culture of Animal Cells, a Manual of Basic Technique, 3rd ed., Wiley-Liss, New York (1994), Kuchler et al., Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977), and references cited therein provide a general description of cell culture.
  • General Guidance Cultured cell systems are most often in the form of cell monolayers, although cell suspensions are also used.
  • polymer-lipid composition of the present invention can be detected in a subject at about 1, 2, 3, 4, 5, 6, 7, 8 or more hours. In other embodiments, polymer-lipid composition of the present invention can be detected in a subject at about 8, 12, 24, 48, 60, 72 or 96 hours after applying particles, or about 6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 25 or 28 days.
  • the presence of polymer-lipid composition particles can be detected in cells, tissues or other biological samples from a subject.
  • Polymer-lipid composition particles can, for example, by directly detecting particles, detecting therapeutic nucleic acids such as RNA (e.g., mRNA) sequences, detecting the target protein or polypeptide sequence encoded therein, or a combination thereof.
  • RNA e.g., mRNA
  • the polymer-lipid composition particles of the present invention can be detected using any known method in the art.
  • a marker can be coupled directly or indirectly to a component of the polymer-lipid composition particle using methods known in the art.
  • a wide variety of markers can be used, and the selection of the marker depends on the required sensitivity, the ease of conjugation with the polymer-lipid composition particle component, stability requirements, and available instruments and processing regulations.
  • Suitable labels include, but are not limited to, spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives, such as fluorescein isothiocyanate (FITC) and Oregon Green TM ; rhodamine and derivatives, such as Texas Red, tetramethylrhodamine isothiocyanate (TRITC), etc., digoxigenin, biotin, phycoerythrin, AMCA, CyDyes TM , etc.; radioactive labels such as 3 H, 125 I, 35 S, 14 C, 32 P, 33 P, etc.; enzymes such as horseradish peroxidase, alkaline phosphatase, etc.; spectral colorimetric labels such as colloidal gold or colored glass or plastic beads such as polystyrene, polypropylene, latex, etc.
  • the labels can be detected by any means known in the art.
  • Nucleic acids are detected and quantified herein by any of a number of means known to those skilled in the art. Detection of nucleic acids can be performed by known methods such as Southern analysis, Northern analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Other analytical biochemical methods such as spectrophotometry, X-ray photography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and high diffusion chromatography can also be used.
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • the present disclosure provides a method for preventing and/or treating a disease or condition in a subject, the method comprising administering a pharmaceutically effective amount of a composition or pharmaceutical composition according to the present invention to a subject in need thereof in vivo, wherein the composition or pharmaceutical composition comprises an activating agent or therapeutic agent for the disease or condition.
  • the disease or condition is selected from an immune system disease, a metabolic disease, a genetic disease, a cancer, a blood disease, a bacterial infection, or a viral infection.
  • the present invention provides the use of a composition or pharmaceutical composition according to the present invention in the preparation of a medicament for preventing and/or treating a disease in a subject, wherein the composition or pharmaceutical composition comprises the activating agent or therapeutic agent for the disease or condition.
  • the disease or condition is selected from immune system diseases, metabolic diseases, hereditary diseases, cancer, blood diseases, bacterial infections or viral infections.
  • the compositions are used for delivery to target organs and/or target tissues and/or target cells for treating human subjects.
  • the compositions described herein are used for delivery to the lungs or lung cells of a subject.
  • the compositions described herein are used for delivery to the liver or liver cells of a subject.
  • the compositions described herein are used for delivery to the spleen or spleen cells of a subject.
  • the compositions described herein are used for delivery to the muscles or muscle cells of a subject.
  • the present invention provides a method for delivering a composition described herein comprising an mRNA encoding a protein or polypeptide, the therapeutic composition being used to treat a lung disease.
  • the present invention can be used in a method for producing an mRNA encoding the cystic fibrosis transmembrane conductance regulator CFTR (CFTR mRNA).
  • the CFTR mRNA is delivered to the lungs of a subject in need of a therapeutic composition for treating cystic fibrosis.
  • the present invention can be used in a method for producing an mRNA encoding ⁇ 1-trypsin (A1AT mRNA).
  • the A1AT mRNA is delivered to the lungs and or liver of a subject in need of a therapeutic composition for ⁇ 1-trypsin deficiency.
  • the present invention can be used in a method for producing mRNA encoding the medium and heavy chains of axonal dynein (DNAI1 mRNA and DNAH5 mRNA).
  • DNAI1 mRNA or DNAH5 mRNA is delivered to the lungs of a subject in need of a therapeutic composition for treating primary ciliary dyskinesia.
  • the present invention provides a method for delivering a composition as described herein comprising mRNA encoding a protein or polypeptide, wherein the therapeutic composition is used to treat liver disease or metabolic disease.
  • Such proteins and polypeptides may include those associated with urea cycle disorders, lysosomal storage disorders, glycogen storage disorders, amino acid metabolism disorders, lipid metabolism or fibrosis disorders, methylmalonic acidemia, or any other metabolic disorder for which delivery or treatment of the liver or hepatocytes with enriched full-length mRNA provides a therapeutic benefit.
  • the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a protein associated with a urea cycle disorder. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding an ornithine transcarbamylase (OTC) protein.
  • OTC ornithine transcarbamylase
  • the invention provides a method for delivering a composition described herein comprising mRNA encoding a protein associated with a lysosomal storage disorder. In certain embodiments, the invention provides a method for delivering a composition described herein comprising mRNA encoding an alpha galactosidase protein.
  • the invention provides a method for delivering a composition as described herein comprising mRNA encoding a protein associated with glycogen storage disease. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising mRNA encoding an acid alpha-glucosidase protein.
  • the invention provides a method for delivering a composition as described herein comprising mRNA encoding a protein related to amino acid metabolism. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising mRNA encoding phenylalanine hydroxylase.
  • the present invention provides a method for delivering a composition as described herein comprising an mRNA encoding a protein associated with lipid metabolism or fibrotic diseases. In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising an mRNA encoding an mTOR inhibitor.
  • the present invention provides a method for delivering a composition described herein comprising an mRNA encoding a protein associated with methylmalonic acidemia.
  • the present invention provides a method for delivering a composition described herein comprising an mRNA encoding a methylmalonyl-CoA mutant enzyme protein.
  • the therapeutic composition is used to deliver to or treat the liver or hepatocytes of a subject.
  • the invention provides a method for delivering a composition comprising a protein encoding ATP7B.
  • the present invention provides a method for delivering a composition as described herein comprising mRNA encoding porphobilinogen deaminase.
  • the present invention provides a method for delivering a composition as described herein comprising mRNA encoding human hemochromatosis (HFE) protein.
  • HFE human hemochromatosis
  • the therapeutic composition is used to deliver to or treat the cardiovascular system or cardiovascular cells of a subject.
  • the invention provides a method for delivering a composition described herein comprising mRNA encoding vascular endothelial growth factor A protein.
  • the therapeutic composition is used to deliver to or treat a subject's muscle or muscle cells.
  • the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a dystrophin.
  • the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a protein or polypeptide, the therapeutic composition being used to deliver to or treat a subject's myocardium or myocardial cells.
  • the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a protein or polypeptide that modulates one or both of a potassium channel and a sodium channel in a muscle tissue or muscle cell.
  • the therapeutic composition is used to deliver to or treat the nervous system or nervous system cells of a subject.
  • the invention provides a method for delivering a composition described herein comprising an mRNA encoding a survival motor neuron 1 protein.
  • the therapeutic composition is for delivery to or treatment of the blood or bone marrow, or blood cells or bone marrow cells, of a subject.
  • the invention provides a method for delivering a composition described herein comprising mRNA encoding beta globin.
  • the therapeutic composition is used to deliver to or treat a kidney or renal cell of a subject.
  • the invention provides a method for delivering a composition described herein comprising mRNA encoding type IV collagen alpha 5 chain (COL4A5) protein.
  • the therapeutic composition is used to deliver to or treat an eye or ocular cell of a subject.
  • the invention provides a method for delivering a composition described herein comprising an mRNA encoding an ATP-binding cassette subfamily A member 4 (ABCA4) protein.
  • the invention provides a method for delivering a composition described herein comprising an mRNA encoding a retinoschizin protein.
  • the therapeutic composition is used to deliver a vaccine to a subject or a subject's cell or to treat with a vaccine.
  • the invention provides a method for delivering a composition described herein comprising mRNA encoding an antigen from an infectious source such as a virus.
  • the invention provides a method for preparing a composition described herein comprising delivering mRNA encoding a novel coronavirus (SARS-CoV-2) antigen.
  • SARS-CoV-2 novel coronavirus
  • the present invention provides mRNA for containing an antigen from an infectious source such as bacteria.
  • the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Mycobacterium tuberculosis antigen.
  • the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Pseudomonas aeruginosa antigen.
  • the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Klebsiella pneumoniae antigen.
  • the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Staphylococcus aureus antigen. In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Baumannii antigen. In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Baumannii antigen. In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Legionella pneumophila antigen.
  • the present invention provides a method for delivering a therapeutic composition encoding a cancer-associated antigen of a subject or an mRNA of an antigen identified from a subject's cancer cells. In certain embodiments, the present invention provides a method for preparing a composition described herein comprising delivering an mRNA encoding an antigen determined from a subject's own cancer cells, i.e., providing a personalized cancer vaccine. In certain embodiments, the present invention provides a method for preparing a composition described herein comprising delivering an mRNA encoding an antigen expressed from a mutant KRAS gene.
  • the present invention provides a method for delivering a composition described herein comprising mRNA encoding an antibody.
  • the antibody may be a bispecific antibody.
  • the antibody may be part of a fusion protein.
  • the two independent mRNA-loaded polymer-lipid compositions (mRNA-PoLixNano) in the method comprise mRNA encoding an antibody light chain and a heavy chain.
  • the mRNA-PoLixNano composition of the present invention may include a combination of different PoLixNano containing different lipid compositions and mRNA encapsulating an antibody light chain or heavy chain.
  • the present invention provides a method for delivering a composition described herein comprising mRNA encoding an antibody against OX40. In certain embodiments, the present invention provides a method for delivering a composition comprising mRNA encoding an antibody against VEGF. In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising mRNA encoding an antibody to tissue necrosis factor alpha. In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising mRNA encoding an antibody to CD3. In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising mRNA encoding an antibody to CD19.
  • the present invention provides a method for delivering a composition described herein comprising an mRNA encoding an immunomodulator. In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding interleukin 2 (IL-2). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding interleukin 12 (IL-12). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding granulocyte-macrophage colony stimulating factor (GM-CSF).
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • the present invention provides a method for delivering a composition described herein comprising an mRNA encoding interleukin 23 (IL-23). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding C-C domain chemokine ligand 28 (CCL28). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding interleukin 36 ⁇ (IL-36 ⁇ ). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding thymic stromal lymphopoietin or a derivative thereof. In certain embodiments, the invention provides a method for delivering a composition described herein comprising mRNA encoding one or more constitutively active variants of the stimulator of interferon genes (STING) protein.
  • STING interferon genes
  • the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a nuclease. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a DNA endonuclease protein (such as a Cas 9 protein) guided by RNA. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a meganuclease protein. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a transcription activator-like effector nuclease protein. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a zinc finger nuclease protein.
  • the formulations described herein can be used to deliver any mRNA, in particular, in vitro transcribed mRNA (IVT-mRNA) encoding a specific antigen or therapeutic protein.
  • IVT-mRNA in vitro transcribed mRNA
  • the formulations described herein can also be used to deliver any modified or unmodified mRNA, or mRNA with a naturally occurring sequence or codon optimization.
  • the mRNA delivered by the formulations described herein can (but not exclusively) encode firefly luciferase (Fluc) and the new coronavirus SARS-CoV-2 spike protein receptor binding domain RBD antigen.
  • the mRNA encoding firefly luciferase (Fluc-mRNA) and encoding the RBD antigen (RBD-mRNA) of the spike protein of the novel coronavirus SARS-CoV-2 described herein can be synthesized by constructing a recombinant DNA plasmid comprising a T7 promoter, 5'UTR, ORF (Fluc or RBD), 3'UTR, and a 3' poly (A) tail of about 100 nucleotides in length; preparing a linearized DNA template by enzyme digestion or PCR amplification; performing in vitro transcription with the linearized DNA template, and then adding a 5' cap structure (Cap 1) to prepare Fluc-mRNA and RBD-mRNA. After purification, the IVT-mRNA band size and purity are determined by gel electrophoresis.
  • aqueous buffer 50 mM citrate buffer, pH 4.0
  • Fluc-mRNA or RBD-mRNA
  • 12.68 mg/mL citric acid and 8.77 mg/mL sodium citrate were mixed in a volume ratio of 1:1, and the pH of the mixture was about 4.0.
  • Fluc-mRNA (or RBD-mRNA) was diluted to 0.17 mg/mL with the above buffer.
  • the lipid-dissolved ethanol solution was fully mixed with the citrate buffer containing mRNA using a microfluidic mixing device (NanoFac series, Sichuan Shuogen Technology Co., Ltd.) (the volume ratio of aqueous phase to organic phase was 3:1) at a total flow rate of 12 mL/min.
  • a microfluidic mixing device NaFac series, Sichuan Shuogen Technology Co., Ltd.
  • the volume ratio of aqueous phase to organic phase was 3:1
  • the solution was mixed, it was dialyzed with 1 ⁇ PBS (pH 7.4) at 4°C (dialysis bag molecular weight cutoff MWCO: 8000-14000) for 20 hours, filtered with a 0.22 ⁇ m filter membrane, and stored at 2-8°C.
  • the final concentration of Fluc-mRNA and RBD-mRNA encapsulated in the prepared LNP preparation was about 0.12 mg/mL.
  • Preparation of aqueous phase Prepare 50 mM citrate buffer by the method described in "Formulation Example #1", use this buffer to prepare Fluc-mRNA (or RBD-mRNA) and T904 solutions into 0.34 mg/mL and 8 mg/mL stock solutions, respectively, then mix the above Fluc-mRNA (or RBD-mRNA) stock solution and T904 stock solution in a volume ratio of 1:1 and set aside.
  • Preparation of organic phase 50 mg/mL of Dlin-MC3-DMA in ethanol, 50 mg/mL of DSPC in ethanol, 50 mg/mL of cholesterol in ethanol and 50 mg/mL of DMG-PEG2K in ethanol were mixed in a molar ratio of 50:14:35:1, and diluted with ethanol to a total lipid concentration of 15.30 mg/mL.
  • the aqueous solution and the organic solution were fully mixed using a microfluidic mixing device (NanoFac series, Sichuan Shuogeng Technology Co., Ltd.) (the volume ratio of aqueous phase to organic phase was 3:1, and the total flow rate was 12 mL/min).
  • the mixed solution was collected, the collected solution was dialyzed (dialysis bag MWCO: 8000-14000) for 20 h in a 4°C environment using 1 ⁇ PBS (pH 7.4), filtered with a 0.22 ⁇ m filter membrane, and stored at 2-8°C.
  • the final concentration of Fluc-mRNA (or RBD-mRNA) encapsulated in the prepared PoLixNano preparation was approximately 0.10 mg/mL.
  • T904 was diluted to 75 mg/mL with 1 ⁇ PBS solution, filtered through a 0.22 ⁇ m filter membrane, and set aside.
  • the LNP solution prepared and purified by the method described in "Formulation Example #1" was mixed with the above T904 solution at a volume ratio of 4:1. After standing at room temperature for 20 minutes, it was filtered with a 0.22 ⁇ m filter membrane and stored at 2-8°C. The final concentration of Fluc-mRNA (or RBD-mRNA) encapsulated in the prepared LNP+T904 formulation was about 0.10 mg/mL.
  • T904 was diluted to 15 mg/mL with 1 ⁇ PBS, filtered through a 0.22 ⁇ m filter membrane, and set aside.
  • the PoLixNano solution prepared and purified in the above "Formulation Example #2" and the above T904 were mixed thoroughly at a volume ratio of 4:1. After incubation at room temperature for 20 minutes, the mixture was filtered through a 0.22 ⁇ m filter membrane and stored at 2-8°C. The final concentration of Fluc-mRNA (or RBD-mRNA) encapsulated in the prepared PoLixNano+T904 formulation was about 0.08 mg/mL.
  • PoLixNano solution prepared and purified in the above "Formulation Example #2” and 50% sucrose solution were mixed thoroughly at a volume ratio of 4:1. After incubation at room temperature for 20 minutes, the mixture was filtered through a 0.22 ⁇ m filter membrane and stored at 2-8°C. The final concentration of Fluc-mRNA encapsulated in the prepared PoLixNano + 10% sucrose formulation was about 0.08 mg/mL.
  • PoLixNano formulation prepared from three-component lipids consisting of ionizable/cationic lipids, neutral lipids, and cholesterol (i.e., PoLixNano without a PEG-lipid component):
  • Preparation of aqueous phase Use 50 mM citrate buffer to dilute Fluc-mRNA (or RBD-mRNA) and Poloxamer 407 solutions to 0.34 mg/mL and 13.33 mg/mL solutions, respectively, and then mix the Fluc-mRNA (or RBD-mRNA) solution and Poloxamer 407 solution in a volume ratio of 1:1 for standby use.
  • Preparation of organic phase 50 mg/mL of Dlin-MC3-DMA in ethanol, 50 mg/mL of DOPE in ethanol, and 50 mg/mL of cholesterol in ethanol were mixed at a molar ratio of 40:32:28 and diluted with ethanol to a total lipid concentration of 18.96 mg/mL.
  • the aqueous phase solution and the organic phase solution were fully mixed using a microfluidic mixing device (NanoFac series, Sichuan Shuogeng Technology Co., Ltd.) (the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min).
  • a microfluidic mixing device NanoFac series, Sichuan Shuogeng Technology Co., Ltd.
  • the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min.
  • the mixed solution was collected, it was dialyzed (dialysis bag MWCO: 8000-14000) for 20 h in a 4°C environment using 1 ⁇ PBS (pH 7.4), filtered with a 0.22 ⁇ m filter membrane, and stored at 2-8°C.
  • PoLixNano formulation prepared from three-component lipids consisting of cholesterol-derived cationic lipids, neutral lipids, and PEG-lipids:
  • Fluc-mRNA (or RBD-mRNA) and T904 solutions were diluted to 0.34 mg/mL and 8 mg/mL solutions, respectively, and then the Fluc-mRNA (or RBD-mRNA) solution and the T904 solution were mixed evenly at a volume ratio of 1:1 for standby use.
  • the aqueous phase solution and the organic phase solution were fully mixed using a microfluidic mixing device (NanoFac series, Sichuan Shuogeng Technology Co., Ltd.) (the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min).
  • a microfluidic mixing device NanoFac series, Sichuan Shuogeng Technology Co., Ltd.
  • the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min.
  • the mixed solution was collected, it was dialyzed (dialysis bag MWCO: 8000-14000) for 20 h in a 4°C environment using 1 ⁇ PBS (pH 7.4), filtered with a 0.22 ⁇ m filter membrane, and stored at 2-8°C.
  • PoLixNano formulation prepared from three-component lipids consisting of ionizable/cationic lipids, cholesterol, and PEG-lipids (i.e., PoLixNano without a phospholipid component):
  • Preparation of aqueous phase Use 50 mM citrate buffer to dilute Fluc-mRNA (or RBD-mRNA) and T904 solution to 0.34 mg/mL and 8 mg/mL solution, respectively, and then mix the Fluc-mRNA (or RBD-mRNA) solution and T904 solution in a volume ratio of 1:1 for standby use.
  • Preparation of organic phase 50 mg/mL ethanol solution of CKK-E12, 50 mg/mL ethanol solution of DOTAP, 50 mg/mL ethanol solution of cholesterol and 50 mg/mL ethanol solution of DMG-PEG2K were mixed at a molar ratio of 30:39:30:1 and diluted with ethanol to a total lipid concentration of 5.86 mg/mL.
  • the aqueous phase solution and the organic phase solution were fully mixed using a microfluidic mixing device (NanoFac series, Sichuan Shuogeng Technology Co., Ltd.) (the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min).
  • a microfluidic mixing device NanoFac series, Sichuan Shuogeng Technology Co., Ltd.
  • the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min.
  • the mixed solution was collected, it was dialyzed (dialysis bag MWCO: 8000-14000) for 20 h in a 4°C environment using 1 ⁇ PBS (pH 7.4), filtered with a 0.22 ⁇ m filter membrane, and stored at 2-8°C.
  • the particle size of the LNP and PoLixNano preparations was determined by a laser particle size analyzer. PDI, potential information and encapsulation efficiency. Encapsulation of nucleic acids was determined using the Quant-iT RiboGreen RNA assay. The RiboGreen assay was essentially as described in the article published by Heyes et al., Journal of Controlled Released, 2005, 107:276-287. The above physicochemical characterization results and the lipid components and amphiphilic block copolymer compositions of the formulations are summarized in Table 1.
  • This example illustrates exemplary methods of administering relevant formulations loaded with mRNA and methods for analyzing proteins expressed by the delivered mRNA in target tissues in vivo.
  • mice were housed in a specific pathogen-free (SPF) animal room with a 12-hour/12-hour light/dark cycle and free access to food and water. All experimental animals had at least 7 days of acclimatization time before the start of the experiment.
  • SPF pathogen-free
  • a high-pressure MicroSprayer needle (PennCentury, USA) was used to spray the mRNA dose of 2.5 ⁇ g mRNA/50 ⁇ L of PoLixNano or LNP formulation into the trachea of a single mouse. Unless otherwise specified, this dose was used in all experiments of the same type.
  • mice were administered PoLixNano or LNP formulations at an mRNA dose of 2.0 ⁇ g mRNA/40 ⁇ L.
  • the solution was applied as a drop to the nostril of a single animal during isoflurane (Isothesia) inhalation anesthesia, and the complex formulation was naturally inhaled. Unless otherwise specified, all experiments of the same type used this dose.
  • the PoLixNano formulation or LNP formulation with a dose of 25 ⁇ g mRNA/mouse was placed in a nebulizer system (Aerogen, Dangan, Ireland) with a nebulization rate of 30 ⁇ L/10 s and a nebulization time of 15 min. Unless otherwise specified, this dose was used in all experiments of the same type.
  • the skin surface of the outer thigh muscle of Balb/c mice was disinfected with 75% alcohol cotton balls, and the muscle was quickly pierced with a syringe to inject PoLixNano or LNP preparations with a mRNA dose of 5 ⁇ g mRNA/100 ⁇ L. Unless otherwise specified, all experiments of the same type used this dose.
  • IVIS in vivo imaging device In order to detect the expression of Luciferase reporter gene in mice administered with Fluc-mRNA preparation, IVIS in vivo imaging device was used for investigation. First, anesthesia was performed using an isoflurane anesthesia device at 1-3% (usually 2.5%). D-luciferin solution (3 mg D-luciferin dissolved in 100 ⁇ L PBS) was administered to each animal via intraperitoneal injection (i.p.) at a dose of 150 mg/kg. Allow the luciferin to distribute for 10 minutes. The animal was placed in an isoflurane chamber until anesthetized. The anesthetized animal was placed in the dorsal recumbency of the IVIS imaging chamber with the head placed in the ventilator.
  • Bioluminescence imaging was performed using the IVIS in vivo imaging system (Perkin Elmer, USA).
  • the instrument settings were as follows: the camera height was at D level, F/Stop was f1, the pixel combination (binning) was 8 ⁇ 8, and the exposure time was automatic.
  • the animals were killed by cervical dislocation, and the lungs, liver, or spleen were quickly removed, and the isolated organs were imaged again for bioluminescence.
  • the images were analyzed by measuring the radiance in the set region of interest (ROI) using Living Image software (PerkinElmer, USA). The values are shown as total radiance [p/s].
  • PoLixNano formulations containing different concentrations of T904 and T704 were prepared using the method described in the above "Formulation Example #2" using different lipid ratios.
  • PolixNano nanoparticles formed a multi-chamber structure ( Figure 1A). Studies have shown that the chamber structure in this type of nanoparticle can increase the transfection efficiency of mRNA (Cheng MHY, Cullis PR, et al. Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency. Adv Mater. 2023, 35 (31): e2303370).
  • the PoLixNano preparation containing 6 mg/mL T704 can be efficiently taken up by human bronchial epithelial cells 16HBE, dendritic cells DC2.4 and mouse bone marrow-derived dendritic cells (BMDC) cells, effectively express Fluc-mRNA, and is non-toxic to cells (Figure 1B and C).
  • the PoLixNano preparation has a stronger ability to penetrate the mucus layer ( Figure 1D).
  • the PoLixNano preparation can more effectively stimulate the differentiation and maturation of BMDC cells and better mediate subsequent adaptive immune responses (Figure 1E).
  • Example 6 Gene transfection effects of different preparations 6 hours after intratracheal spray (i.t.) administration
  • LNPs were prepared using the method described in Formulation Example #1, and PoLixNano formulations containing 0.025 mg/mL T904, 3 mg/mL T904, 0.025 mg/mL T704, and 6 mg/mL T704 were prepared using the method described in Formulation Example #2.
  • T904-mRNA preparation (or T704-mRNA preparation): Mix 4 ⁇ Tyrode buffer and 0.24 mg/mL Fluc-mRNA in a volume ratio of 1:1, and then mix the mixture with 6 mg/mL T904 solution (or T704 solution) in a volume ratio of 1:1. Incubate at room temperature for 20 minutes to obtain T904-mRNA preparation (or T704-mRNA preparation) with a Fluc-mRNA dose of 3 ⁇ g/50 ⁇ L.
  • Fluc-mRNA was diluted to 3 ⁇ g/50 ⁇ L with PBS.
  • Example 7 Intratracheal spraying of PoLixNano formulations containing different types of amphiphilic polymers (i.t.) Transfection effect 6h after administration
  • PoLixNano formulations containing different types of amphiphilic polymers as shown in FIG3 were prepared using the method described in the above “Formulation Example #2”.
  • the concentration of the amphiphilic polymer in FIG3 is the final concentration in the PoLixNano particles.
  • the results are shown in Figure 3.
  • the PoLixNano preparations containing different concentrations of T904 and T704 can significantly increase the luciferase level produced by Fluc-mRNA in living mice and lungs, especially the PoLixNano preparations containing 40 mg/mL T904 and 6 mg/mL T704 significantly improved the transfection efficiency of mRNA in the lungs ( Figure 3A and B).
  • the intratracheal spray (i.t.) administration of PoLixNano preparations prepared by replacing T704 or T904 with Tween 80, other Poloxamers or Poloxamines can still efficiently transfect Fluc-mRNA in the lungs ( Figure 3C and D).
  • Example 8 PoLixNano preparations prepared with different N/P ratios and lipid molar ratios were administered intratracheally Transfection effect 6 hours after intravenous (i.t.) administration
  • PoLixNano formulations Using the method described in the above "Formulation Example #2", keeping the molar ratio of each lipid component unchanged, PoLixNano formulations with N/P ratios of 2, 4, 5, 6, 7, 8, 9, 10, 12 and 15 were prepared. Using the method described in the above “Formulation Example #2", adjusting N/P to 8, PoLixNano formulations containing lipids in different molar ratios were prepared, wherein the molar ratio of DMG-PEG2000 was between 0.1 and 5, the molar ratio of DSPC was between 8 and 50.1, the molar ratio of Dlin-MC3-DMA was between 25 and 70, and the molar ratio of cholesterol was between 20.2 and 46.5.
  • PoLixNano preparations prepared with different lipid molar ratios had different gene transfection abilities in living mice and lungs and were significantly higher than the LNP control group, especially PoLixNano preparations (DMG-PEG2000 molar ratio between 0.3 and 1, DSPC molar ratio between 10 and 30.1, Dlin-MC3-DMA molar ratio between 35 and 65, and cholesterol molar ratio between 21 and 46.5) all had the potential for efficient transfection of Fluc-mRNA ( Figures 4B and C).
  • Example 9 PoLixNano formulations prepared with different types of lipids were administered via intratracheal spray (i.t.) Transfection effect after 6 hours
  • the results are shown in Figure 5.
  • the PoLixNano preparations prepared with different lipids can significantly increase the luciferase level produced by Fluc-mRNA in living mice and lungs.
  • the PoLixNano preparation groups prepared with Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 the PoLixNano prepared by replacing lipids with different structures (except SM-102) has a comparable level of luciferase produced by Fluc-mRNA in the lungs, and the PoLixNano prepared by SM-102 significantly improves the transfection efficiency of Fluc-mRNA in the lungs, which indicates that the PoLixNano preparations prepared by replacing lipids with different structures have the potential to achieve comparable or even more efficient mRNA transfection through respiratory administration.
  • Example 10 Preparation of Poloxamers, Poloxamines and Lipids Transfection effect of PoLixNano formulation 6 hours after i.n. administration
  • PoLixNano formulations Using the method described in the above "Formulation Example #5", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 49:20.1:30:0.9, and N/P was 8. PoLixNano + 10% sucrose formulations containing different types of poloxamers, poloxamines and lipids were prepared as shown in Figure 6. The concentrations of poloxamers and poloxamines were Final concentration within PoLixNano particles.
  • the PoLixNano preparations could efficiently transfect Fluc-mRNA in the lungs, especially the PoLixNano preparations (DMG-PEG2000 molar ratio between 0.5 and 2, DSPC molar ratio between 8 and 20.1, Dlin-MC3-DMA molar ratio between 47.9 and 56.4, and cholesterol molar ratio between 30 and 34.7 ( Figure 6B)).
  • Example 11 Physically mixed poloxamers, poloxamines, polypeptides and sucrose to obtain The preparation can further promote gene transfection of Fluc-mRNA in the lungs
  • LNP was prepared by the method described in "Formulation Example #1"; the corresponding LNP physical mixed preparation in Figure 7 was prepared by the method described in "Formulation Example #3".
  • LNP formulations The LNP control formulations without Poloxamer and Poloxamine were prepared by the methods described in the above "Formulation Example #6", “Formulation Example #7” and “Formulation Example #8", respectively.
  • the LNP formulation lacking the DSPC component was prepared by adjusting the lipid components and molar ratio to 8 N/P using the method described in the above "Formulation Example #1".
  • Preparation of PoLixNano preparations The methods described in the above-mentioned "Preparation Example #6", “Preparation Example #7” and “Preparation Example #8” were respectively used to adjust the lipid components and their molar ratios of the method described in "Preparation Example #2” (lacking DSPC component group), with N/P being 8, to prepare the corresponding PoLixNano preparations.
  • PoLixNano (GL67) and PoLixNano (CKK-E12) preparations were able to significantly increase the level of luciferase produced by Fluc-mRNA in the lungs after 6 hours of intratracheal spray (i.t.) administration ( Figures 8A and B).
  • PoLixNano preparations without DMG-PEG2000 or DSPC effectively increased the transfection efficiency of Fluc-mRNA in the liver, lungs and spleen after 6 hours of intratracheal spray (i.t.) administration.
  • PoLixNano preparations without DMG-PEG2000 or DSPC significantly increased the luciferase produced by Fluc-mRNA in the lungs via the nasal route (i.n.) ( Figures 8C and D).
  • Example 13 Analysis of the physicochemical properties of LNP and PoLixNano preparations before and after atomization and the Transfection effects of Poloxamers, Poloxamines and lipid-based PoLixNano formulations after 6 hours
  • the PoLixNano preparation or LNP control preparation was atomized using the atomization device shown in FIG9A.
  • the morphology of the LNP nanoparticles and the PoLixNano nanoparticles before and after atomization was observed using a transmission electron microscope (TEM).
  • TEM transmission electron microscope
  • the LNP preparation nanoparticles before atomization were uniform in size and intact in structure, while the LNP preparation nanoparticles after atomization were aggregated, broken and deformed, showing irregular morphology (FIG9B).
  • poloxamer 188 When poloxamer 188 was added to the above components at a final concentration of 10 mg/mL, the particle size and structural shape of the prepared PolixNano preparation particles did not change significantly before and after atomization (Table 2 and FIG9B), indicating that poloxamer 188 can protect the PolixNano nanoparticles during the atomization process, reduce the impact of the mechanical force of the atomization device on the nanoparticles, ensure the integrity of their morphological structure, and thus ensure the transfection efficiency of the mRNA loaded in the PolixNano nanoparticles.
  • Example 14 Administration of PoLixNano formulations containing different lipid ratios and doses by aerosol inhalation Transfection effect after h
  • Preparation of LNP preparation Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA (or SM-102), DSPC, cholesterol and DMG-PEG2000 was adjusted to 48.5:25:25:1.5, and N/P was 8.
  • Preparation of PoLixNano preparation Using the method described in the above-mentioned "Preparation Example #2", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted, wherein the molar ratio of DMG-PEG2000 was between 0.5 and 3, the molar ratio of DSPC was between 14 and 40, the molar ratio of Dlin-MC3-DMA was between 39 and 63, and the molar ratio of cholesterol was between 17.5 and 35.
  • the level of luciferase produced by Fluc-mRNA in the 50 ⁇ g/mouse group was higher than that in the 25 ⁇ g/mouse group and lower than that in the 150 ⁇ g/mouse group, indicating that the expression of Fluc-mRNA in the PoLixNano preparation administered by nebulized inhalation was dose-dependent (Figure 10C).
  • bronchial epithelial cells CD326 +
  • DC cells CD11c +
  • endothelial cells CD31 +
  • type I alveolar cells Podoplanin +
  • macrophages F4/80 +
  • the expression trend of luciferase produced by Fluc-mRNA and FLuc-circRNA at different times of administration of PoLixNano preparations by intratracheal spray was further analyzed. The results showed that luciferase with comparable expression levels was detected in the lungs 6h, 12h and 24h after administration, but the luciferase signal detected in the liver and spleen 24h after administration was significantly lower than that at 12h.
  • the PoLixNano preparation encapsulating Fluc-circRNA was administered by intratracheal spray (i.t.). The expression level of FLuc-circRNA was the highest at 6h after administration, and the expression level of Fluc-circRNA gradually decreased over time. A trace amount of luciferase signal could still be detected on the ninth day of administration.
  • PoLixNano preparation can mediate efficient transfection of mRNA and circRNA in mice and achieve sustained expression to a certain extent via intratracheal spray (i.t.) administration.
  • Preparation of LNP preparations Using the method described in the above-mentioned "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1 (intratracheal spray (i.t.)), 49:20.1:30:0.9 (nasal route (i.n.)) or 46:23:30:1 (atomized inhalation), and N/P was 8.
  • Preparation of PoLixNano preparations Use the methods described in the above "Preparation Example #2” and “Preparation Example #4" to prepare PoLixNano preparations containing different Poloxamer, Poloxamine or Tween 80 corresponding to the LNP preparations.
  • luciferase signals were detected in the lungs, liver and spleen of the LNP preparation group; luciferase signals were specifically detected in the lung tissue of the PoLixNano preparation containing 3mg/mL 188 or 3mg/mL T304, and the PoLixNano preparation containing 3mg/mL 338 was first prepared and then physically mixed with 1mg/mL 338.
  • Preparation of LNP preparation Using the method described in the above "Preparation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 49:20.1:30:0.9, and N/P was 8.
  • the expression levels of IL-4, IL-6, IL-17 and TNF- ⁇ in the PoLixNano preparation group containing 3 mg/mL T904 were comparable to those in the PBS group (i.t.), and were significantly lower than those in the LNP preparation group (i.t.) ( Figure 13C).
  • the liver and kidney function indicators in the serum of the PoLixNano preparation group containing 3 mg/mL T904 after i.t. administration were normal, and no specific IgG antibodies induced by the T904 component and lipid component were detected in the serum.
  • PoLixNano formulation can promote C57black 6 after 6 hours of administration via the respiratory route Gene transfection in the lungs of C57BL/6 mice and Sprague-Dawley (SD) rats
  • PoLixNano formulations Using the method described in the above "Formulation Example #2", prepare the corresponding PoLixNano formulations containing 3 mg/mL T904 (intratracheal spray (i.t.) or nasal route (i.n.)) and 10 mg/mL 188 (nebulized inhalation) of the LNP formulation.
  • Example 19 PoLixNano formulation encapsulated RBD-mRNA vaccine via intratracheal spray route (i.t.) Induces a long-lasting and highly effective immune response in mice after vaccination
  • the mRNA encoding the RBD antigen of the SARS-CoV-2 spike protein (RBD-mRNA) was used as a vaccine model, and the immune response mediated by the RBD-mRNA/PoLixNano preparation in mice was investigated via intratracheal spray (i.t.).
  • LNP formulation Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1, and N/P was 8. LNP formulation encapsulating RBD-mRNA vaccine formulation (hereinafter referred to as "LNP”) was prepared.
  • PoLixNano preparation Using the method described in the above-mentioned "Preparation Example #2", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1, N/P was 8, and the PoLixNano preparation containing T904 at a final concentration of 3.0 mg/mL was prepared to encapsulate RBD-mRNA vaccine preparation (hereinafter referred to as "PoLixNano").
  • the immunization dose was 2.5 ⁇ g RBD-mRNA/mouse.
  • the first vaccination time was counted as "day 0", and the same vaccination route, the same preparation and the same dose were used for booster immunization on day 21.
  • Serum, bronchoalveolar lavage fluid (BALF), nasal lavage fluid (NLF), pulmonary lymphocytes and splenocytes were collected from mice 28 days after the first immunization and the humoral immune response, mucosal immune response and adaptive immune response were detected.
  • mice inoculated with LNP preparations in different ways under the same conditions were used as controls, and mouse samples inoculated with PBS solution under the same conditions (hereinafter referred to as "PBS" group) were used as negative controls.
  • PBS mouse samples inoculated with PBS solution under the same conditions
  • the immunization dose was 3 ⁇ g RBD-mRNA/mouse, and the first vaccination time was counted as "Day 0", and booster immunization was performed on Day 21 using the same vaccination route, the same preparation and the same dose.
  • Serum was collected from mice 14 days, 28 days and 280 days after the first immunization, and bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF) were collected from mice on Day 28 to detect the ability of PoLixNano preparation to induce humoral immune response and mucosal immune response in mice, and mediastinal lymph node cells and splenocytes were collected from mice.
  • BALF bronchoalveolar lavage fluid
  • NVF nasal lavage fluid
  • the IgG antibody titer of RBD antigen in the mouse serum samples collected 14 days, 28 days and 280 days after the first immunization was determined by enzyme linked immunosorbent assay (ELISA).
  • ELISA enzyme linked immunosorbent assay
  • ELISA was used to measure the IgG antibody titer of RBD antigen in the bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF) samples collected from mice 28 days after the first immunization.
  • the IgG antibody titer produced in the BALF and NLF of mice induced by intratracheal spray (it) was significantly higher than that of the LNP-it group.
  • the results of Figure 15B show that the sIgA antibody titer of RBD antigen in the BALF and NLF samples collected 28 days and 280 days after the first immunization was measured by ELISA.
  • the sIgA antibody titer produced in the BALF and NLF of mice induced by intratracheal spray (it) PoLixNano was significantly higher than that of the LNP-it group.
  • the serum gradient dilution samples and BALF gradient dilution samples collected 28 days after the first immunization were mixed with pseudovirus and added to hACE-293T cells for infection for 24 hours, and the pseudovirus neutralization titer was calculated by detecting the relative luciferase unit (RLU).
  • the neutralizing antibody titers in the serum and BALF of mice in the PoLixNano preparation group were significantly higher than those in the LNP preparation group using different vaccination methods, indicating that the PoLixNano preparation group can significantly improve the level of neutralizing antibodies.
  • the enzyme-linked immunospot technique (ELISpot) was used to detect the production of IgG and sIgA antibody-secreting cells (ASC) in the mediastinal lymph node cells (Mediastinal lymphocytes) and spleen cells (Splenocytes) of mice 28 days after immunization.
  • the IgG antibody produced by PoLixNano in the mediastinal lymph node cells and spleen cells was significantly higher than that in the LNP preparation group using different vaccination methods.
  • Example 20 PoLixNano formulation encapsulated RBD-mRNA vaccine via intratracheal spray route (i.t.) inoculation induces cellular immune response, innate trained immune response and long-term adaptation in mice sexual Immune Response
  • the mRNA encoding the RBD antigen of the spike protein of the new coronavirus (RBD-mRNA) was used as a vaccine model to investigate the immune response mediated by the RBD-mRNA/PoLixNano preparation in mice via intratracheal spray (i.t.).
  • the preparation methods of the LNP preparation and PoLixNano preparation, as well as the immunization dose and method, were the same as those in Example 19.
  • BALF samples were collected from mice 28 days, 35 days, and 280 days after the first immunization. Pulmonary lymphocytes and spleen cells were also collected from mice.
  • the enzyme-linked immunospot technique was used to analyze the ability of the splenocytes and pulmonary lymphocytes of mice to produce cytokines interferon- ⁇ (IFN- ⁇ ), interleukin-4 (IL-4), and interleukin-17 (IL-17) 28 days after the first immunization.
  • IFN- ⁇ interferon- ⁇
  • IL-4 interleukin-4
  • IL-17 interleukin-17
  • LNP preparation groups with different inoculation methods induced the production of CD4+IFN- ⁇ + cells and CD8+IFN- ⁇ + cells Similar to the PBS group.
  • the above results show that compared with the LNP preparation, the PoLixNano preparation can significantly increase the IFN- ⁇ secretion level of lung CD4+T cells and CD8+T cells, and the PoLixNano preparation can improve the immune response of CD4+ and CD8+T cells.
  • the results of Figure 16C show that the tissue-resident memory T cell (Trm) subpopulation, effector memory T cell (Tem) subpopulation, and central memory T cell (Tcm) subpopulation in mice 28 days and 280 days after the first immunization were analyzed by flow cytometry.
  • Inoculation of PoLixNano preparations via intratracheal spray (it) can induce mice to produce more MHC+IM cells.
  • intratracheal spray (it) mRNA/PoLixNano formulation can induce a higher level of innate trained immune response in mice.
  • Example 21 RBD-mRNA vaccine encapsulated by PoLixNano formulation via intratracheal spray route Evaluation of protection effect of challenge after (i.t.) vaccination
  • the mRNA encoding the RBD antigen of the spike protein of the new coronavirus (RBD-mRNA) was used as a vaccine model to investigate the immune response mediated by the RBD-mRNA/PoLixNano preparation in mice via intratracheal spray (i.t.).
  • the preparation methods of the LNP preparation and PoLixNano preparation, as well as the immunization dose and method, were the same as those in Example 19.
  • the mRNA PoLixNano vaccine can completely protect balb/c mice from challenge with lethal SARS-CoV-2 strains after immunization with the intratracheal spray route (IT).
  • Mice were immunized by intratracheal spray route (IT) on days 0 and 21. 28 days after the initial immunization, the mice were challenged with a lethal dose of the original strain of SARS-CoV-2 and the Omicron variant strain by nasal drops, and specific tissues were collected at designated time points after the challenge to detect viral load and lung pathology.
  • the "gold standard" LNP preparation is difficult to mediate the mRNA vaccine to produce an efficient antigen-specific immune response in the respiratory mucosa, and the mRNA/LNP vaccine inoculated by the respiratory route is extremely limited in its ability to induce antigen-specific humoral immune responses and cellular immunity.
  • the PoLixNano preparation administered by the respiratory route can induce the mRNA vaccine to produce a strong, balanced and lasting multiple antigen-specific immune response, including mucosal immune response, humoral immune response and cellular immune response.
  • the mRNA/PoLixNano vaccine inoculated by the respiratory route is expected to achieve the ideal effect of providing strong immune protection to the vaccine recipients and blocking the spread of pathogens.
  • Example 22 RBD-mRNA vaccine encapsulated in PoLixNano formulation was administered via nasal route (i.n.) Post-immune response evaluation
  • LNP formulation Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1, and N/P was 8. The vaccine formulation of LNP formulation encapsulating RBD-mRNA was prepared.
  • the immunization dose was 2.0 ⁇ g Fluc-mRNA/mouse, and the expression of the reporter gene in mice was investigated by IVIS live imaging technology at 6h, 12h, 24h, 48h and 72h.
  • the reporter gene was highly expressed in mice at 12h and 24h, but no longer expressed after 72h (Figure 18A).
  • the immunization dose was 2.0 ⁇ g RBD-mRNA/mouse, and the first vaccination time was counted as "Day 0". On the 21st day, booster immunization was performed using the same vaccination route, the same preparation and the same dose.
  • the ELISA method was used to determine the RBD antigen-specific IgG in the serum of mice vaccinated with PoLixNano preparations on days 14, 21, 28, 35, 42 and 49. After booster immunization, the antibody level was significantly increased, and the LNP preparations injected via nasal injection did not induce the body to produce IgG ( Figure 18B).
  • the levels of sIgA antibodies produced in the BALF, NLF and saliva of mice vaccinated with PoLixNano preparations were significantly higher than those of the LNP group.
  • the enzyme-linked immunospot technique (ELISpot) was further used to analyze the production of cytokine interferon- ⁇ (IFN- ⁇ ) by the lung lymphocytes (Splenocytes) of mice.
  • IFN- ⁇ cytokine interferon- ⁇
  • Splenocytes lung lymphocytes
  • IL-4 Interleukin-4
  • IL-17 interleukin-17
  • the mouse lung lymphocytes obtained on the 28th day were stimulated by the RBD overlapping peptide library (covering the overlapping peptide library of the new crown RBD protein, a single polypeptide length of 14 amino acids, and adjacent polypeptides overlapped 9 amino acids) to secrete IFN- ⁇ , IL-4, and IL-17.
  • the ability is far superior to the control group of the LNP preparation inoculated via the nasal route.
  • Figure 18D shows that the resident memory T cells (Trm) and effector memory T cells (Tem) subpopulations of the lung tissue of immunized mice (inoculated on days 0 and 21) were analyzed by flow cytometry.
  • Preparation of LNP preparations Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:10:38.5:1.5, and N/P was 6; using the methods described in the above "Formulation Example #6", “Formulation Example #7” and “Formulation Example #8", LNP control preparations without Poloxamer and Poloxamine were prepared respectively. Using the method described in the above “Formulation Example #1”, the lipid components and molar ratios were adjusted, and N/P was 8 to prepare LNP preparations lacking DMG-PEG2000 components as shown in Figure 8. Vaccine preparations encapsulating mRNA in LNP preparations were prepared.
  • the inoculation dose was 2.5 ⁇ g Fluc-mRNA/mouse.
  • IVIS In vivo imaging technology
  • mice 6 hours after administration Mouse samples of LNP preparations inoculated in the same way under the same conditions were used as controls.
  • the intramuscular administration route (im) Administration of PoLixNano can induce higher luciferase expression in living mice and isolated organs ( Figure 19A).
  • DMG-PEG2000 is missing in the lipid component
  • administration of PoLixNano via intramuscular administration (im) can induce higher expression of luciferase in living mice and isolated organs ( Figure 19B).
  • the ELISA method was used to determine the RBD antigen-specific IgG antibody titer in the serum samples of mice administered with PoLixNano preparations via intramuscular administration (im) 14 days, 21 days, 28 days, and 35 days after the first immunization.
  • the antigen-specific humoral immune response ability induced by the LNP preparation (LNP-im) via intramuscular injection was significantly improved.
  • the ELISpot method was further used to analyze the ability of mouse spleen cells (Splenocytes) to produce cytokines, interleukin-4 (IL-4) and interleukin-17 (IL-17).
  • the PoLixNano preparation was administered by intramuscular injection (im).
  • the number of cells secreting IL-4 and IL-17 in mouse spleen cells obtained after stimulation with the RBD overlapping peptide library was significantly higher than that in the LNP control preparation group administered by intramuscular injection.

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Abstract

The present invention relates to a composition for enhancing the delivery of an active agent or a therapeutic agent, such as a therapeutic nucleic acid. The composition comprises: (A) the active agent or the therapeutic agent, wherein preferably, the active agent or the therapeutic agent comprises a nucleic acid; (B) an amphiphilic block copolymer; (C) a cationic lipid; and (D) a non-cationic lipid. The present invention further provides a method for preparing, delivering and using the composition, and use of the composition for treating and/or preventing related diseases or conditions.

Description

用于增强核酸递送的组合物Compositions for enhancing nucleic acid delivery 发明领域Field of the Invention
本发明涉及用于增强活性剂或治疗剂，例如治疗性核酸，的递送的组合物。The present invention relates to compositions for enhancing the delivery of active or therapeutic agents, such as therapeutic nucleic acids.
背景技术Background technique
诸如蛋白质、DNA、siRNA、以及体外转录mRNA(in vitro transcribed messenger RNA，以下简称mRNA)等生物大分子在成功导入生物体细胞后，可通过产生或作用于特定蛋白功能发挥药效，从而达到疾病预防或治疗目的。这些生物大分子正在成为防治多种疾病的重要方法。其中，基于mRNA技术研发的疫苗以极高的保护率在对抗2019年爆发的新型冠状病毒(SARS-CoV-2)大流行方面显示出巨大潜力。目前，全球已有2款新冠肺炎(COVID-19)mRNA疫苗获得美国FDA的批准上市。除新冠mRNA疫苗外，mRNA技术在肿瘤免疫治疗、基因治疗、传染病预防、基因编辑、遗传病治疗等领域同样具有极大的应用潜力。After being successfully introduced into biological cells, biological macromolecules such as proteins, DNA, siRNA, and in vitro transcribed messenger RNA (mRNA) can produce or act on specific protein functions to exert their efficacy, thereby achieving the purpose of disease prevention or treatment. These biological macromolecules are becoming an important method for preventing and treating a variety of diseases. Among them, vaccines developed based on mRNA technology have shown great potential in combating the novel coronavirus (SARS-CoV-2) pandemic that broke out in 2019 with extremely high protection rates. Currently, two COVID-19 mRNA vaccines have been approved by the US FDA for marketing worldwide. In addition to the COVID-19 mRNA vaccine, mRNA technology also has great application potential in tumor immunotherapy, gene therapy, infectious disease prevention, gene editing, and genetic disease treatment.
mRNA技术具有诸多优点：其采用无细胞的方式制造，从而实现快速、经济、高效的生产，在快速应对大规模突发性传染疾病爆发方面具有得天独厚的优势；mRNA几乎不会整合到基因组中，安全性高，避免了出现插入突变的可能性；此外，单个mRNA疫苗可以编码多种抗原，增强针对适应性病原体的免疫反应，并能够以单一配方针对多种微生物或病毒变体。mRNA technology has many advantages: it is manufactured in a cell-free manner, which enables rapid, economical and efficient production, and has a unique advantage in quickly responding to large-scale outbreaks of sudden infectious diseases; mRNA is almost never integrated into the genome, has high safety, and avoids the possibility of insertion mutations; in addition, a single mRNA vaccine can encode multiple antigens, enhance the immune response against adaptive pathogens, and can target multiple microorganisms or viral variants with a single formulation.
mRNA疗法涉及向需要所述疗法的受试者施用特定的mRNA，以在患者体内产生由所述mRNA编码的蛋白质。如何使用安全的递送系统将mRNA高效而精准地递送至目标组织及靶细胞、最大程度提升其表达效率，是实现这类药物或疫苗成功临床转化的关键。mRNA在体内的成功表达是一个系统性级联过程，主要难点在于：1)mRNA极易被广泛存在的RNA酶降解，其结构稳定性是发挥生物学效应的前提；2)mRNA进入体内后易被清除，体内半衰期短，难以到达特定靶部位；3)由于相对分子质量较大、亲水性和电负性较强，mRNA自身难以有效跨越体内的各种生理屏障，如黏膜屏障、细胞膜及溶酶体屏障等。因此mRNA药物/疫苗的有效靶向递送在医学上一直是具有挑战性的。 mRNA therapy involves administering specific mRNA to a subject in need of the therapy to produce proteins encoded by the mRNA in the patient. How to use a safe delivery system to efficiently and accurately deliver mRNA to target tissues and target cells and maximize its expression efficiency is the key to achieving successful clinical transformation of such drugs or vaccines. The successful expression of mRNA in vivo is a systemic cascade process, and the main difficulties are: 1) mRNA is easily degraded by widely existing RNases, and its structural stability is a prerequisite for exerting biological effects; 2) mRNA is easily cleared after entering the body, has a short half-life in the body, and is difficult to reach specific target sites; 3) Due to its large relative molecular mass, strong hydrophilicity and electronegativity, mRNA itself is difficult to effectively cross various physiological barriers in the body, such as mucosal barriers, cell membranes and lysosomal barriers. Therefore, the effective targeted delivery of mRNA drugs/vaccines has always been challenging in medicine.
目前常用的基因药物体内递送系统分为病毒载体和非病毒载体两类。由于体内转染效率相对较高，早期尝试使用病毒载体作为基因递送的递送方法，然而，病毒载体的主要问题，如宿主对病毒载体本身产生的免疫应答、难以重复给药、可能激活引起恶性肿瘤的致癌基因以及炎症反应的并发症等，显著阻碍了其发展。相比之下，非病毒载体与病毒载体相比具有安全性高、免疫原性低、致病性降低、插入突变能力降低、大规模制备方便等诸多优点。基于纳米生物材料发展而来的纳米粒是一类典型的非病毒载体。常见的非病毒载体包括脂质纳米颗粒(lipid nanoparticle,LNP)以及基于阳离子多聚物的制剂，如壳聚糖、聚乙烯亚胺、树枝状大分子等。其中LNP体系是迄今最常见、最先进也是唯一成功实现临床转化的mRNA药物递送系统。Currently, the commonly used in vivo delivery systems for gene drugs are divided into two categories: viral vectors and non-viral vectors. Due to the relatively high in vivo transfection efficiency, early attempts to use viral vectors as a delivery method for gene delivery were made. However, the main problems of viral vectors, such as the host's immune response to the viral vector itself, difficulty in repeated administration, possible activation of oncogenes that cause malignant tumors, and complications of inflammatory responses, have significantly hindered their development. In contrast, non-viral vectors have many advantages over viral vectors, such as high safety, low immunogenicity, reduced pathogenicity, reduced ability to insert mutations, and convenient large-scale preparation. Nanoparticles developed based on nanobiomaterials are a typical type of non-viral vector. Common non-viral vectors include lipid nanoparticles (LNP) and preparations based on cationic polymers, such as chitosan, polyethyleneimine, and dendrimers. Among them, the LNP system is the most common, most advanced, and only mRNA drug delivery system that has successfully achieved clinical transformation.
尽管LNP凭借诸多优势已发展成为目前mRNA领域的“金标准”递送技术，然而该体系仍然存在诸多缺陷：例如，LNP体系通常需要通过肌肉注射、皮下注射或静脉注射等注射途径给药，只能在肝脏和脾脏等空隙较大的组织以及肌肉注射部位产生高效的基因转染效果(Pardi,N.et al.Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes.J.Control.Release.217,345–351(2015))。已有研究表明，LNP制剂经呼吸道途径给药后，在小鼠体内仅介导极低的mRNA基因表达(Zhang,N.-N.et al.A thermostable mRNA vaccine against COVID-19.Cell.182,1271-1283(2020))。其次，LNP制剂有过敏反应较为严重的问题，在高剂量给药方案中存在风险(Ndeupen,S.et al.The mRNA-LNP platform's lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory.iScience.24,103479(2021)；Landesman-Milo,D.&Peer,D.Toxicity profiling of several common RNAi-based nanomedicines:a comparative study.Drug Deliv.Transl.Res.4,96–103(2014)；)。在mRNA递送领域，大多数基于LNP体系的成功应用进展仍局限于肝脏组织的递送(Samaridou,E.et al.Lipid nanoparticles for nucleic acid delivery:Current perspectives.Adv Drug Deliv Rev.154-155,37–63(2020))，这极大地限制了mRNA/LNP制剂在其他靶器官疾病领域的广泛应用，例如肺部、鼻部、胃肠道、生殖道等黏膜部位。Although LNP has developed into the "gold standard" delivery technology in the field of mRNA due to its many advantages, the system still has many defects: for example, the LNP system usually needs to be administered through injection routes such as intramuscular injection, subcutaneous injection or intravenous injection, and can only produce efficient gene transfection effects in tissues with large gaps such as the liver and spleen and in the intramuscular injection site (Pardi, N. et al. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes. J. Control. Release. 217, 345–351 (2015)). Studies have shown that after LNP preparations are administered via the respiratory route, they only mediate very low mRNA gene expression in mice (Zhang, N.-N. et al. A thermostable mRNA vaccine against COVID-19. Cell. 182, 1271-1283 (2020)). Secondly, LNP preparations have the problem of severe allergic reactions and pose risks in high-dose administration regimens (Ndeupen, S. et al. The mRNA-LNP platform's lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory. iScience. 24, 103479 (2021); Landesman-Milo, D. & Peer, D. Toxicity profiling of several common RNAi-based nanomedicines: a comparative study. Drug Deliv. Transl. Res. 4, 96–103 (2014);). In the field of mRNA delivery, most of the successful application progress based on LNP systems is still limited to delivery to liver tissue (Samaridou, E. et al. Lipid nanoparticles for nuclear acid delivery: Current perspectives. Adv Drug Deliv Rev. 154-155, 37–63 (2020)), which greatly limits the wide application of mRNA/LNP preparations in other target organ disease fields, such as the lungs, nose, gastrointestinal tract, reproductive tract and other mucosal sites.
以肺部为例，mRNA药物在许多临床上暂无治愈方案的肺部疾病治疗方面极具应用潜力，例如囊性纤维化(CF)，慢性阻塞性肺病(COPD)、α1-胰蛋白酶缺乏症、哮喘、肺动脉高压、原发性纤毛运动障碍(Primary cliliary  dyskinesia)以及特发性肺纤维化等。肺部的特殊生理结构为其给药途径带来了诸多优势，并使其成为mRNA药物的理想表达场所(Suberi,A.,et al.Polymer nanoparticles deliver mRNA to the lung for mucosal vaccination.Sci Transl Med.15,eabq0603(2023))。经呼吸道递送的药物，给药方式简单，患者顺应性好，适用于多次重复给药方案，还可以使药物制剂在支气管和肺泡上皮组织中均匀分布(Patel,A.K.et al.Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium.Adv.Mater.e1805116，(2019))。相较于注射给药方式，能将潜在的全身性副作用降至最低，还可降低针头污染导致的交叉感染等问题。肺气道吸收表面积大，毛细血管网丰富，有利于基因药物吸收及高效转染。并且肺部具有很强的血管生成能力，能够介导分泌蛋白进入到循环系统发挥作用。因此，肺部是基于mRNA药物蛋白质替代疗法的理想靶点。Taking the lungs as an example, mRNA drugs have great potential in the treatment of many lung diseases that have no clinical cure, such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), α1-trypsin deficiency, asthma, pulmonary hypertension, primary ciliary dyskinesia, and dyskinesia) and idiopathic pulmonary fibrosis. The special physiological structure of the lungs brings many advantages to its route of administration, and makes it an ideal expression site for mRNA drugs (Suberi, A., et al. Polymer nanoparticles deliver mRNA to the lung for mucosal vaccination. Sci Transl Med. 15, eabq0603 (2023)). Drugs delivered through the respiratory tract have simple administration methods, good patient compliance, are suitable for repeated administration regimens, and can also make the drug preparations evenly distributed in bronchial and alveolar epithelial tissues (Patel, AK et al. Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium. Adv. Mater. e1805116, (2019)). Compared with the injection administration method, potential systemic side effects can be minimized, and problems such as cross infection caused by needle contamination can also be reduced. The lung airway has a large absorption surface area and a rich capillary network, which is conducive to gene drug absorption and efficient transfection. The lungs also have strong angiogenesis capabilities, which can mediate secretory proteins to enter the circulatory system to exert their effects. Therefore, the lungs are an ideal target for mRNA-based protein replacement therapy.
其次，mRNA疫苗在呼吸道黏膜部位(例如，鼻相关淋巴组织NALT以及支气管相关淋巴样组织BALT)诱导抗原特异性黏膜免疫方面具有巨大的应用前景。90％以上病原体均由人体的黏膜部位(例如呼吸道，胃肠道，生殖道等)入侵，包含新型冠状病毒(SARS-CoV-2)、重症急性呼吸综合征冠状病毒(SARS-CoV)中东呼吸综合征冠状病毒(MERS)、流感病毒、呼吸道合胞病毒、肺结核杆菌在内的多种烈性致病病原体主要通过呼吸道黏膜部位感染宿主(Lavelle,E.C.&Ward,R.W.Mucosal vaccines-fortifying the frontiers.Nat.Rev.Immunol.22,236-250(2022))，这意味着在诱导阶段和效应阶段，病原体与宿主免疫系统的相互作用首先发生在呼吸道黏膜。以新冠疫苗为例，绝大多数上市的品种均采用肌肉注射方式接种，该途径只能诱导以血清IgG抗体为主的系统免疫应答，产生的IgG中和抗体很难到达和存在于呼吸道黏膜表面，因此难以清除该部位残留的病毒。不仅如此，肌肉注射途径接种的疫苗几乎不能诱导黏膜免疫应答，进而无法产生分泌型IgA(sIgA)抗体保护宿主免受黏膜部位的初始感染，病毒仍然可能在呼吸道黏膜复制并造成他人感染。有研究表明，感染SARS-CoV-2的病人体内早期特异性体液反应以黏膜免疫应答产生的sIgA抗体为主(Sterlin,D.et al.IgA dominates the early neutralizing antibody response to SARS-CoV-2.Sci Transl Med.13,eabd22234(2021))。并且sIgA对新冠病毒的平均中和效力是IgG的七倍以上，在病毒中和过程中占据主导地位(Wang,Z.et al.Enhanced SARS-CoV-2neutralization by dimeric IgA.Sci Transl  Med.13,eabf1555(2021))。因此，高效的黏膜免疫应答对于呼吸道入侵病原体相关疫苗的功效具有重要价值，或许是根除相关病原体感染与传播的关键(Jeyanathan,M.et al.Immunological considerations for COVID-19 vaccine strategies.Nat.Rev.Immunol.20,615–632(2020))。Secondly, mRNA vaccines have great application prospects in inducing antigen-specific mucosal immunity in respiratory mucosal sites (e.g., nasal-associated lymphoid tissue NALT and bronchial-associated lymphoid tissue BALT). More than 90% of pathogens invade the human body through mucosal sites (e.g., respiratory tract, gastrointestinal tract, reproductive tract, etc.), including the new coronavirus (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS), influenza virus, respiratory syncytial virus, and Mycobacterium tuberculosis. A variety of virulent pathogens mainly infect the host through the respiratory mucosal sites (Lavelle, EC & Ward, RW Mucosal vaccines-fortifying the frontiers. Nat. Rev. Immunol. 22, 236-250 (2022)), which means that in the induction stage and effect stage, the interaction between the pathogen and the host immune system first occurs in the respiratory mucosa. Taking the COVID-19 vaccine as an example, most of the products on the market are administered by intramuscular injection, which can only induce a systemic immune response dominated by serum IgG antibodies. The IgG neutralizing antibodies produced are difficult to reach and exist on the surface of the respiratory mucosa, making it difficult to remove the virus remaining in this area. Not only that, vaccines administered by intramuscular injection can hardly induce mucosal immune responses, and thus cannot produce secretory IgA (sIgA) antibodies to protect the host from initial infection in the mucosal area. The virus may still replicate in the respiratory mucosa and cause infection to others. Studies have shown that the early specific humoral response in patients infected with SARS-CoV-2 is dominated by sIgA antibodies produced by mucosal immune responses (Sterlin, D. et al. IgA dominates the early neutralizing antibody response to SARS-CoV-2. Sci Transl Med. 13, eabd22234 (2021)). Moreover, the average neutralization potency of sIgA against the new coronavirus is more than seven times that of IgG, and it plays a dominant role in the virus neutralization process (Wang, Z. et al. Enhanced SARS-CoV-2 neutralization by dimeric IgA. Sci Transl Med.13,eabf1555(2021)). Therefore, efficient mucosal immune response is of great value to the efficacy of vaccines related to respiratory invasive pathogens, and may be the key to eradicating infection and transmission of related pathogens (Jeyanathan, M. et al. Immunological considerations for COVID-19 vaccine strategies. Nat. Rev. Immunol. 20, 615–632(2020)).
尽管经呼吸道途径给予mRNA药物/疫苗具有诸多优势，但如何实现mRNA在呼吸道内的高效递送是全球范围内尚未攻克的难题。由于呼吸道黏膜组织微环境复杂，存在大量的酶类物质，抗原分子极易被降解。呼吸道系统经过长时间进化产生了强大的生理屏障(如支气管结构屏障、黏液屏障、免疫屏障及细胞膜屏障等)以及能够高效清除外源性异物的生理机制(如纤毛清除作用)，mRNA分子很难到达效应靶点，这对mRNA的递送技术提出了极高要求。如前所述，即使如LNP这类“金标准”递送系统，仍难以在呼吸道内介导令人满意的mRNA基因转染。因此迫切需要研发能够在呼吸道等黏膜部位高效递送mRNA分子的新型递送体系。Although there are many advantages to administering mRNA drugs/vaccines through the respiratory route, how to achieve efficient delivery of mRNA in the respiratory tract is a problem that has not yet been overcome worldwide. Due to the complex microenvironment of the respiratory mucosal tissue and the presence of a large number of enzyme substances, antigen molecules are easily degraded. After a long period of evolution, the respiratory system has developed strong physiological barriers (such as bronchial structural barriers, mucus barriers, immune barriers, and cell membrane barriers, etc.) and physiological mechanisms that can efficiently remove exogenous foreign bodies (such as ciliary clearance). It is difficult for mRNA molecules to reach the effector target, which places extremely high demands on mRNA delivery technology. As mentioned earlier, even "gold standard" delivery systems such as LNP are still difficult to mediate satisfactory mRNA gene transfection in the respiratory tract. Therefore, there is an urgent need to develop a new delivery system that can efficiently deliver mRNA molecules in mucosal sites such as the respiratory tract.
发明概述SUMMARY OF THE INVENTION
本发明出人意料地发现，向LNP制剂添加两亲性嵌段共聚物例如洛沙姆(poloxamine或)和或泊洛沙胺(poloxamer或)等组分后，所获得的新型制剂能够大幅提升此类制剂介导核酸类分子例如mRNA在动物体内的基因转染效率，尤其是当通过生物体黏膜部位递送(例如呼吸道递送)时。当本发明的制剂经呼吸道途径给药时，在动物肺部/呼吸道黏膜组织产生的基因转染效果显著优于LNP制剂、poloxamine以及poloxamer等制剂，从而解决了本领域中递送效率低的问题。The present invention surprisingly found that adding amphiphilic block copolymers such as poloxamine or ) and or poloxamer or ) and other components, the obtained novel preparation can greatly improve the gene transfection efficiency of nucleic acid molecules such as mRNA mediated by such preparations in animals, especially when delivered through the mucosal part of the organism (such as respiratory delivery). When the preparation of the present invention is administered via the respiratory route, the gene transfection effect produced in the animal lung/respiratory mucosal tissue is significantly better than that of LNP preparations, poloxamine and poloxamer, thereby solving the problem of low delivery efficiency in the art.
相应地，在第一方面，本发明提供了一种包含聚合物-脂质的组合物，所述组合物包含：Accordingly, in a first aspect, the present invention provides a composition comprising a polymer-lipid, the composition comprising:
(A)活性剂或治疗剂，优选所述活性剂或治疗剂包含核酸；(A) an active agent or therapeutic agent, preferably the active agent or therapeutic agent comprises a nucleic acid;
(B)两亲性嵌段共聚物；(B) amphiphilic block copolymers;
(C)阳离子脂质；和(C) a cationic lipid; and
(D)非阳离子脂质，(D) non-cationic lipids,
其中所述组合物配制为用于通过生物体黏膜部位(Mucosa)递送，例如呼吸道递送、口腔黏膜递送、胃肠道递送、眼部黏膜递送、耳部黏膜递送、尿道递送、或生殖道递送，优选所述组合物配制为用于通过呼吸道递送。 Wherein the composition is formulated for delivery through a mucosal site of an organism, such as the respiratory tract, oral mucosa, gastrointestinal tract, ocular mucosa, ear mucosa, urethra, or reproductive tract, preferably the composition is formulated for delivery through the respiratory tract.
在一些实施方案中，所述核酸包含选自以下的至少一种：信使RNA(mRNA)、自我扩增RNA(saRNA)、环状RNA(circRNA)、小干扰RNA(siRNA)、短发夹RNA(shRNA)和微小RNA(miRNA)、初级‐miRNA、反义寡核苷酸(ASO)、转运RNA(tRNA)、质粒DNA(pDNA)、单链DNA(ssDNA)、双链DNA(dsDNA)、脱氧核酶(DNAzyme)、核酶(RNAzyme)、核酸适配体(aptamer)、成簇的规律间隔的短回文重复序列(CRISPR)相关的核酸、单指导RNA(sgRNA)、CRISPR‐RNA(crRNA)、反式活化crRNA(tracrRNA)、指导RNA、单链RNA(ssRNA)和双链RNA(dsRNA)。In some embodiments, the nucleic acid comprises at least one selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) and micro RNA (miRNA), primary-miRNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), plasmid DNA (pDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), deoxyribozyme (DNAzyme), ribozyme (RNAzyme), nucleic acid aptamer (aptamer), clustered regularly interspaced short palindromic repeats (CRISPR)-related nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA, single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA).
在一些实施方案中，所述核酸是治疗性核酸。更优选地，所述核酸包含mRNA。In some embodiments, the nucleic acid is a therapeutic nucleic acid. More preferably, the nucleic acid comprises mRNA.
在本发明的组合物一些实施方案中，所述两亲性嵌段共聚物占所述组合物的0.1％-98.0％重量百分比，例如0.5％-95.0％重量百分比、1％-90.0％重量百分比、10％-80.0％重量百分比、20％-80.0％重量百分比、30％-80.0％重量百分比、40％-80.0％重量百分比、40％-70.0％重量百分比或50％-60.0％重量百分比。In some embodiments of the composition of the present invention, the amphiphilic block copolymer accounts for 0.1%-98.0% weight percent of the composition, for example, 0.5%-95.0% weight percent, 1%-90.0% weight percent, 10%-80.0% weight percent, 20%-80.0% weight percent, 30%-80.0% weight percent, 40%-80.0% weight percent, 40%-70.0% weight percent or 50%-60.0% weight percent.
在本发明的组合物的一些实施方案中，所述阳离子脂质包含选自以下的至少一种：永久阳离子脂质、可电离的阳离子脂质、胆固醇衍生的阳离子脂质和树枝状聚合物或树枝块(dendron)，优选地，所述阳离子脂质包含可电离的阳离子脂质。In some embodiments of the composition of the present invention, the cationic lipid comprises at least one selected from the group consisting of permanent cationic lipids, ionizable cationic lipids, cholesterol-derived cationic lipids and dendrimers or dendrons. Preferably, the cationic lipid comprises an ionizable cationic lipid.
在一些实施方案中，所述阳离子脂质占所述组合物中存在的总脂质的23mol％-83mol％，例如30mol％-80mol％、30mol％-70mol％或40mol％-60mol％、或所述阳离子脂质占所述组合物中存在的总脂质的约25mol％、约30mol％、约35mol％、约40mol％、约45mol％、约50mol％、约55mol％、约60mol％、约65mol％、约70mol％、约75mol％或约80mol％。In some embodiments, the cationic lipid accounts for 23 mol%-83 mol% of the total lipids present in the composition, such as 30 mol%-80 mol%, 30 mol%-70 mol% or 40 mol%-60 mol%, or the cationic lipid accounts for about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol% or about 80 mol% of the total lipids present in the composition.
在本发明的组合物一些实施方案中，所述非阳离子脂质包含选自以下的至少一种：阴离子脂质、两性离子脂质和中性脂质，优选地，所述非阳离子脂质包含中性脂质。在一些实施方案中，所述中性脂质占所述组合物中存在的总脂质的19mol％-75mol％。In some embodiments of the composition of the present invention, the non-cationic lipid comprises at least one selected from the group consisting of anionic lipids, zwitterionic lipids and neutral lipids, preferably, the non-cationic lipid comprises a neutral lipid. In some embodiments, the neutral lipid accounts for 19 mol%-75 mol% of the total lipids present in the composition.
在一些实施方案中，所述中性脂质包含：In some embodiments, the neutral lipid comprises:
胆固醇或胆固醇衍生的中性脂质；Cholesterol or cholesterol-derived neutral lipids;
磷脂；或phospholipids; or
胆固醇或胆固醇衍生的中性脂质和磷脂的混合物。 A mixture of cholesterol or cholesterol-derived neutral lipids and phospholipids.
在一些实施方案中，所述胆固醇占所述组合物中的总脂质的14mol％-70mol％。In some embodiments, the cholesterol comprises 14 mol%-70 mol% of the total lipids in the composition.
在一些实施方案中，所述磷脂占所述组合物中的总脂质的约5mol％-约30mol％或30mol％-约75mol％。In some embodiments, the phospholipids comprise about 5 mol% to about 30 mol% or 30 mol% to about 75 mol% of the total lipids in the composition.
在本发明的组合物的一些实施方案中，其还包含脂质缀合物，所述脂质缀合物包含选自以下的至少一种：PEG-脂质缀合物、ATTA-脂质缀合物、聚肌氨酸‐脂质缀合物、多肽/蛋白-脂质缀合物、阳离子-聚合物-脂质缀合物(CPL)以及它们的衍生物。优选地，所述脂质缀合物包含PEG-脂质缀合物。In some embodiments of the composition of the present invention, it further comprises a lipid conjugate, wherein the lipid conjugate comprises at least one selected from the group consisting of: PEG-lipid conjugate, ATTA-lipid conjugate, polysarcosine-lipid conjugate, polypeptide/protein-lipid conjugate, cation-polymer-lipid conjugate (CPL) and derivatives thereof. Preferably, the lipid conjugate comprises a PEG-lipid conjugate.
在一些实施方案中，所述脂质缀合物占所述组合物中的总脂质的0.1mol％-10.0mol％。In some embodiments, the lipid conjugate comprises 0.1 mol%-10.0 mol% of the total lipids in the composition.
在本发明的组合物的一些实施方案中，所述组合物包含：In some embodiments of the compositions of the present invention, the compositions comprise:
(1)两亲性嵌段共聚物、阳离子脂质、磷脂、胆固醇和脂质缀合物例如PEG-脂质缀合物，其中所述阳离子脂质占所述组合物中存在的总脂质的30.0mol％-80.0mol％，磷脂占总脂质的5.0mol％-50.0mol％，胆固醇占总脂质的14.0mol％-64.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比(例如1％-90.0％重量百分比、10％-80.0％重量百分比、20％-80.0％重量百分比、30％-80.0％重量百分比或40％-70.0％重量百分比)；(1) an amphiphilic block copolymer, a cationic lipid, a phospholipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 30.0 mol%-80.0 mol% of the total lipid present in the composition, the phospholipid accounts for 5.0 mol%-50.0 mol% of the total lipid, the cholesterol accounts for 14.0 mol%-64.0 mol% of the total lipid, the lipid conjugate accounts for 0.1 mol%-8.0 mol% of the total lipid, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight) of the composition;
(2)两亲性嵌段共聚物、阳离子脂质、磷脂、胆固醇和脂质缀合物例如PEG-脂质缀合物，其中所述阳离子脂质占所述组合物中存在的总脂质的23.0mol％-75.0mol％，磷脂占总脂质的10.0mol％-62.0mol％，胆固醇占总脂质的14.0mol％-46.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比(例如1％-90.0％重量百分比、10％-80.0％重量百分比、20％-80.0％重量百分比、30％-80.0％重量百分比或40％-70.0％重量百分比)；(2) amphiphilic block copolymers, cationic lipids, phospholipids, cholesterol, and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids account for 23.0 mol%-75.0 mol% of the total lipids present in the composition, the phospholipids account for 10.0 mol%-62.0 mol% of the total lipids, the cholesterol accounts for 14.0 mol%-46.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1%-95.0% by weight of the composition (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight);
(3)两亲性嵌段共聚物、胆固醇衍生的阳离子脂质、磷脂和脂质缀合物例如PEG-脂质缀合物，其中所述胆固醇衍生的阳离子脂质占所述组合物中存在的总脂质的29.0mol％-80.0mol％，磷脂占总脂质的19.0mol％-70.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比(例如1％-90.0％重量百分比、10％-80.0％重量百分比、20％-80.0％重量百分比、30％-80.0％重量百分比或40％-70.0％重量百分 比)；(3) an amphiphilic block copolymer, a cholesterol-derived cationic lipid, a phospholipid, and a lipid conjugate such as a PEG-lipid conjugate, wherein the cholesterol-derived cationic lipid accounts for 29.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 19.0 mol%-70.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight). Compare);
(4)两亲性嵌段共聚物、阳离子脂质、胆固醇和脂质缀合物例如PEG-脂质缀合物，其中所述阳离子脂质占所述组合物中存在的总脂质的25.0mol％-80.0mol％，胆固醇占总脂质的15.0mol％-50.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比(例如1％-90.0％重量百分比、10％-80.0％重量百分比、20％-80.0％重量百分比、30％-80.0％重量百分比或40％-70.0％重量百分比)；(4) an amphiphilic block copolymer, a cationic lipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 25.0 mol%-80.0 mol% of the total lipids present in the composition, the cholesterol accounts for 15.0 mol%-50.0 mol% of the total lipids, the lipid conjugate accounts for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight) of the composition;
(5)两亲性嵌段共聚物、阳离子脂质、磷脂和脂质缀合物例如PEG-脂质缀合物，其中所述阳离子脂质占所述组合物中存在的总脂质的30.0mol％-80.0mol％，磷脂占总脂质的10.0mol％-50.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比(例如1％-90.0％重量百分比、10％-80.0％重量百分比、20％-80.0％重量百分比、30％-80.0％重量百分比或40％-70.0％重量百分比)；(5) amphiphilic block copolymers, cationic lipids, phospholipids and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids account for 30.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 10.0 mol%-50.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1%-95.0% by weight of the composition (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight or 40%-70.0% by weight);
(6)两亲性嵌段共聚物、阳离子脂质、磷脂和胆固醇，其中所述阳离子脂质占所述组合物中存在的总脂质的30.0mol％-80.0mol％，磷脂占总脂质的5.0mol％-50.0mol％，胆固醇占总脂质的15.0mol％-50.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比(例如1％-90.0％重量百分比、10％-80.0％重量百分比、20％-80.0％重量百分比、30％-80.0％重量百分比或40％-70.0％重量百分比)；或(6) an amphiphilic block copolymer, a cationic lipid, a phospholipid, and cholesterol, wherein the cationic lipid accounts for 30.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 5.0 mol%-50.0 mol% of the total lipids, the cholesterol accounts for 15.0 mol%-50.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight (e.g., 1%-90.0% by weight, 10%-80.0% by weight, 20%-80.0% by weight, 30%-80.0% by weight, or 40%-70.0% by weight) of the composition; or
(7)两亲性嵌段共聚物、胆固醇衍生的阳离子脂质和磷脂，其中所述胆固醇衍生的阳离子脂质占所述组合物中存在的总脂质的30.0mol％-70.0mol％,磷脂占总脂质的30.0mol％-70.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比(例如1％-90.0％重量百分比、10％-80.0％重量百分比、20％-80.0％重量百分比、30％-80.0％重量百分比或40％-70.0％重量百分比)。(7) an amphiphilic block copolymer, a cholesterol-derived cationic lipid and a phospholipid, wherein the cholesterol-derived cationic lipid accounts for 30.0 mol%-70.0 mol% of the total lipids present in the composition, the phospholipids account for 30.0 mol%-70.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% weight percent (e.g., 1%-90.0% weight percent, 10%-80.0% weight percent, 20%-80.0% weight percent, 30%-80.0% weight percent or 40%-70.0% weight percent) of the composition.
在本发明的组合物的一些实施方案中，所述两亲性嵌段共聚物为四官能的两亲性嵌段共聚物，其中所述四官能的两亲性嵌段共聚物包含各自包含至少一个亲水性嵌段和至少一个疏水性嵌段的四个支链的嵌段共聚物，或所述两亲性嵌段共聚物为线性的两亲性嵌段共聚物，其中所述线性的两亲性嵌段共聚物包含至少一个亲水性嵌段和至少一个疏水性嵌段的嵌段共聚物。In some embodiments of the composition of the present invention, the amphiphilic block copolymer is a tetrafunctional amphiphilic block copolymer, wherein the tetrafunctional amphiphilic block copolymer comprises a block copolymer of four branches each comprising at least one hydrophilic block and at least one hydrophobic block, or the amphiphilic block copolymer is a linear amphiphilic block copolymer, wherein the linear amphiphilic block copolymer comprises a block copolymer of at least one hydrophilic block and at least one hydrophobic block.
在一些实施方案中，所述亲水性嵌段选自聚氧化烯、聚乙烯醇、聚乙烯吡咯烷酮、聚(2-甲基-2-噁唑啉)和糖，和/或所述疏水性嵌段选自聚氧化烯、脂 肪链、亚烷基聚酯、具有苄基聚醚端部的聚乙二醇和胆固醇，优选地，所述亲水性嵌段包含聚氧化乙烯单元，且所述疏水性嵌段包含聚氧化丙烯单元。In some embodiments, the hydrophilic block is selected from polyoxyalkylenes, polyvinyl alcohol, polyvinyl pyrrolidone, poly(2-methyl-2-oxazoline) and sugars, and/or the hydrophobic block is selected from polyoxyalkylenes, lipids, Fatty chains, alkylene polyesters, polyethylene glycol with benzyl polyether ends, and cholesterol, preferably, the hydrophilic block comprises polyethylene oxide units and the hydrophobic block comprises polypropylene oxide units.
在一些实施方案中，所述两亲性嵌段共聚物包含选自以下的至少一种：泊洛沙胺(poloxamine或)、泊洛沙姆(poloxamer或)、聚氧乙二醇脱水醇烷基酯(聚山梨醇酯)、聚乙烯吡咯烷酮(PVP)、聚乙二醇醚(BRIJ)、聚氧乙烯脂肪酸酯、聚氧乙烯脂肪醇醚、脱水山梨糖醇以及它们的衍生物。In some embodiments, the amphiphilic block copolymer comprises at least one selected from the group consisting of poloxamine or ), poloxamer or ), polyoxyethylene glycol dehydrated alcohol alkyl esters (polysorbates), polyvinyl pyrrolidone (PVP), polyethylene glycol ethers (BRIJ), polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, sorbitan and their derivatives.
在一些实施方案中，所述两亲性嵌段共聚物包含泊洛沙胺(poloxamine或)，例如所述泊洛沙胺选自泊洛沙胺304、泊洛沙胺701、泊洛沙胺704、泊洛沙胺901、泊洛沙胺904、泊洛沙胺908、泊洛沙胺1107、泊洛沙胺1301、泊洛沙胺1304、泊洛沙胺1307、泊洛沙胺90R4、泊洛沙胺150R1或其组合。In some embodiments, the amphiphilic block copolymer comprises poloxamine or ), for example, the poloxamine is selected from poloxamine 304, poloxamine 701, poloxamine 704, poloxamine 901, poloxamine 904, poloxamine 908, poloxamine 1107, poloxamine 1301, poloxamine 1304, poloxamine 1307, poloxamine 90R4, poloxamine 150R1 or a combination thereof.
在一些实施方案中，所述两亲性嵌段共聚物包含泊洛沙姆(poloxamer或)，例如所述泊洛沙姆选自泊洛沙姆84、泊洛沙姆101、泊洛沙姆105、泊洛沙姆108、泊洛沙姆122、泊洛沙姆123、泊洛沙姆124、泊洛沙姆181、泊洛沙姆182、泊洛沙姆183、泊洛沙姆184、泊洛沙姆185、泊洛沙姆188、泊洛沙姆212、泊洛沙姆215、泊洛沙姆217、泊洛沙姆231、泊洛沙姆234、泊洛沙姆235、泊洛沙姆237、泊洛沙姆238、泊洛沙姆282、泊洛沙姆284、泊洛沙姆288、泊洛沙姆304、泊洛沙姆331、泊洛沙姆333、泊洛沙姆334、泊洛沙姆335、泊洛沙姆338、泊洛沙姆401、泊洛沙姆402、泊洛沙姆403、泊洛沙姆407或其组合。In some embodiments, the amphiphilic block copolymer comprises poloxamer or ), for example, the poloxamer is selected from poloxamer 84, poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer Poloxamer 231, Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 304, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer 407, or a combination thereof.
在本发明的组合物的一些实施方案中，所述阳离子脂质包含选自以下的至少一种：DOTMA、DOSPA、DOTAP、ePC、DODAP、DODMA、DDAB、DSDMA、DODAC、DOAP、DMRIE、DOGS、DMOBA、HGT5000、HGT5001、HGT5002、HGT4001、HGT4002、HGT4003、HGT4005、DLin-MC3-DMA、DLin-KC2-DMA、Acuitas ALC-0315、Acuitas A9、Acuitas Lipid 2,2、Moderna Lipid H(SM-102)、Moderna Lipid 5、A2-Iso5-2DC18、BAME-O16B、9A1P9、C12-200、cKK-E12、OF-Deg-Lin、306Oi10、TT3、FTT5、Lipid319、5A2-SC8、Genevant CL1、DLinDMA、DLenDMA、ClinDMA、CpLinDMA、咪唑胆固醇酯(ICE)、RE-1、RE-2、RE-3、GL-67、5A2-SC8、Acuitas A9、Arcturus Lipid2,2(8,8)4C CH3、OF-02、A18-Iso5-2DC18、BAME-O16B、A6、98N12-5、L319、L343、304O13、306O138、306O12B、306-O12B、LP01、G0-C14、7C1、Cephalin、 Dlin-EG-DMA、DLinAP、DLin-MPZ、DLin-C-DAP、DLin-2-DMAP、Dlin-S-DMA、DLinDAP、DLin-MA、DLin-DAC、DLin-K-DMA、DLin-K-MPZ、DLin-K-DMA、DLin-K6-C4-DMA、DLin-K-C4-DMA、DLin-K-C3-DMA、CpLinDMA、DOcarbDAP、DLincarbDAP、C12-(2-3-2)、Genevant Lipid CL1、XTC、ALNY-100、NC98-5以及它们的衍生物。In some embodiments of the compositions of the invention, the cationic lipid comprises at least one selected from the group consisting of DOTMA, DOSPA, DOTAP, ePC, DODAP, DODMA, DDAB, DSDMA, DODAC, DOAP, DMRIE, DOGS, DMOBA, HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4005, DLin-MC3-DMA, DLin-KC2-DMA, Acuitas ALC-0315, Acuitas A9, Acuitas Lipid 2,2, Moderna Lipid H (SM-102), Moderna Lipid 5, A2-Iso5-2DC18, BAME-O16B, 9A1P9, C12-200, cKK-E12, OF-Deg-Lin, 306Oi10, TT3, FTT5, Lipid319, 5A2-SC8, Genevant CL1, DLinDMA, DLenDMA, ClinDMA, CpLinDMA, imidazole cholesteryl ester (ICE), RE-1, RE-2, RE-3, GL-67, 5A2-SC8, Acuitas A9, Arcturus Lipid2,2(8,8)4C CH3, OF-02, A18-Iso5-2DC18, BAME-O16B, A6, 98N12-5, L319, L343, 304O13, 306O138, 306O12B, 306-O12B, LP01, G0-C14, 7C1, Cephalin, Dlin-EG-DMA, DLinAP, DLin-MPZ, DLin-C-DAP, DLin-2-DMAP, Dlin-S-DMA, DLinDAP, DLin-MA, DLin-DAC, DLin-K-DMA, DLin-K-MPZ, DLin-K-DMA, DLin-K6-C4-DMA, DLin-K-C4-DMA, DLin-K-C3-DMA, CpLinDMA, DOcarbDAP, DLincarbDAP, C12-(2-3-2), Genevan Lipid CL1, XTC, ALNY-100, NC98-5 and their derivatives.
在本发明的组合物的一些实施方案中，所述胆固醇衍生的阳离子脂质包含选自以下的至少一种：DC‐Choi(N,N‐二甲基‐N‐乙基甲酰胺胆固醇)、1,4-双(3‐N‐油基氨基‐丙基)哌嗪、N4-精氨胆固醇羰酰氨(GL67)、与碱性氨基酸序列偶联的胆固醇衍生物和咪唑胆固醇酯(ICE)。In some embodiments of the compositions of the present invention, the cholesterol-derived cationic lipid comprises at least one selected from the group consisting of DC-Choi (N,N-dimethyl-N-ethylformamide cholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, N4-argininocholesterol carbonylamide (GL67), a cholesterol derivative coupled to a basic amino acid sequence, and imidazole cholesterol ester (ICE).
在本发明的组合物的一些实施方案中，所述磷脂包含选自以下的至少一种：卵磷脂、磷脂酰乙醇胺、溶血卵磷脂、溶血磷脂酰乙醇胺、磷脂酰丝氨酸、二油酰基磷脂酰丝氨酸(DOPS)、磷脂酰肌醇、鞘磷脂、蛋黄鞘磷脂(ESM)、脑磷脂、心磷脂、磷脂酸、脑苷脂、联十六烷基磷酸酯、二硬脂酰磷脂酰胆碱(DSPC)、二油酰磷脂酰乙醇胺(DOPE)、二油酰磷脂酰胆碱(DOPC)、二棕榈酰磷脂酰胆碱(DPPC)、二油酰磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、棕榈酰油酰-磷脂酰胆碱(POPC)、棕榈酰油酰-磷脂酰乙醇胺(POPE)、棕榈酰油酰-磷脂酰甘油(POPG)、二油酰磷脂酰乙醇胺4-(N-马来酰亚胺基甲基)-环己烷-1-甲酸酯(DOPE-mal)、二棕榈酰磷脂酰乙醇胺(DPPE)、二肉豆蔻酰-磷脂酰乙醇胺(DMPE)、二硬脂酰磷脂酰乙醇胺(DSPE)、单甲基-磷脂酰乙醇胺、二甲基-磷脂酰乙醇胺、二反油酰-磷脂酰乙醇胺(DEPE)、硬脂酰油酰-磷脂酰乙醇胺(SOPE)、溶血磷脂酰胆碱、蛋黄磷脂酰胆碱(EPC)、二亚油酰磷脂酰胆碱、1,2-二棕榈酰-sn-甘油-3-O-4'-(N，N，N-三甲基)-高丝氨酸(DGTS)、单半乳糖二酰甘油(MGDG)、二乙酰二酰基甘油(DGDG)、磺胺喹啉二酰基甘油(SQDG)、1-棕榈酰-2-顺式-9,10-亚甲基己基-癸酰基-sn-甘油-3-磷酸胆碱(Cyclo PC)、16‐O‐单甲基PE、16‐O‐二甲基PE、18‐1‐反式PE以及它们的衍生物。In some embodiments of the composition of the present invention, the phospholipid comprises at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, dioleoylphosphatidylserine (DOPS), phosphatidylinositol, sphingomyelin, egg yolk sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), Dipalmitoylphosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, egg yolk phosphatidylcholine (EPC), dilinoleoylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-O -4'-(N,N,N-trimethyl)-homoserine (DGTS), monogalactosyldiacylglycerol (MGDG), diacetyldiacylglycerol (DGDG), sulfaquinolinediacylglycerol (SQDG), 1-palmitoyl-2-cis-9,10-methylenehexyl-decanoyl-sn-glycero-3-phosphocholine (Cyclo PC), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE and their derivatives.
在本发明的组合物的一些实施方案中，所述胆固醇衍生的中性脂质包含选自以下的至少一种：胆甾烷醇、胆甾烷酮、胆甾烯酮、粪甾醇、胆固醇基-2'-羟基乙醚、胆固醇基-4'-羟基丁醚、BHEM-胆固醇、β-谷甾醇、20α-羟基胆固醇、与多肽/蛋白共价连接的胆固醇以及它们的衍生物，优选所述胆固醇衍生的中性脂质包含β-谷甾醇。 In some embodiments of the composition of the present invention, the cholesterol-derived neutral lipid comprises at least one selected from the group consisting of cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, BHEM-cholesterol, β-sitosterol, 20α-hydroxycholesterol, cholesterol covalently linked to a polypeptide/protein, and derivatives thereof, preferably the cholesterol-derived neutral lipid comprises β-sitosterol.
在本发明的组合物的一些实施方案中，所述PEG-脂质缀合物包含选自以下的至少一种：1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000(DMG-PEG2K)、1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](DMPE-PEG2K)、1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000](DSPE-PEG2K)、DSPE-PEG2K与甘露糖的偶联物(DSPE-PEG2K-Mannose)、1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000(DMG-PEG5K)、1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](DMPE-PEG5K)、1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000](DSPE-PEG5K)和DSPE-PEG5K与甘露糖的偶联物(DSPE-PEG5K-Mannose)。In some embodiments of the composition of the present invention, the PEG-lipid conjugate comprises at least one selected from the following: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](DMPE-PEG2K), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000](DSPE-PEG2K), a conjugate of DSPE-PEG2K and mannose ( DSPE-PEG2K-Mannose), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000(DMG-PEG5K), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](DMPE-PEG5K), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000](DSPE-PEG5K) and the conjugate of DSPE-PEG5K and mannose (DSPE-PEG5K-Mannose).
在本发明的包含聚合物-脂质的组合物的一些实施方案中，所述组合物包含两亲性嵌段共聚物和以下组分：In some embodiments of the polymer-lipid-containing compositions of the invention, the compositions comprise an amphiphilic block copolymer and the following components:
(1)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的40.0mol％-70.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的8.0mol％-39.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的15.0％-90.0％重量百分比；(1) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 The composition comprises 40.0 mol% to 70.0 mol% of the total lipids, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE comprises 8.0 mol% to 39.0 mol% of the total lipids, cholesterol or β-sitosterol comprises 20.0 mol% to 40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose comprises 0.1 mol% to 5.0 mol% of the total lipids, and the amphiphilic block copolymer comprises 15.0% to 90.0% by weight of the composition;
(2)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的30mol％-60mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的10.0mol％-49.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的20.0％-90.0％重量百分比； (2) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 1 accounts for 30 mol%-60 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 10.0 mol%-49.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 20.0%-90.0% weight percentage of the composition;
(3)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的24.0mol％-40.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的30.0mol％-64.0mol％，胆固醇或β-谷甾醇占总脂质的15.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的20.0％-90.0％重量百分比；(3) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 accounts for 24.0mol%-40.0mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE account for 30.0mol%-64.0mol% of the total lipids, cholesterol or β-sitosterol account for 15.0mol%-40.0mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1mol%-5.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 20.0%-90.0% weight percent of the composition;
(4)DOTAP、DODAP、DOTMA或DOSPA；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DOTAP、DODAP、DOTMA或DOSPA占所述组合物中存在的总脂质的23.0mol％-60mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的14.0mol％-60.0mol％，胆固醇或β-谷甾醇占总脂质的15.0mol％-50.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的30.0％-90.0％重量百分比；(4) DOTAP, DODAP, DOTMA or DOSPA; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DOTAP, DODAP, DOTMA or DOSPA accounts for 23.0 mol%-60 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 14.0 mol%-60.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 15.0 mol%-50.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition;
(5)GL67、ICE、或HGT4002；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述GL67、ICE、或HGT4002占所述组合物中存在的总脂质的40.0mol％-80.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的10.0mol％-50.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的30.0％-90.0％重量百分比；(5) GL67, ICE, or HGT4002; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE; and DMG-PEG2K, DMG-PEG5K, or DSPE-PEG2K-Mannose, wherein the GL67, ICE, or HGT4002 accounts for 40.0 mol%-80.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE accounts for 10.0 mol%-50.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K, or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition;
(6)cKK-E12、DLin-MC3-DMA、ALC-0315、SM-102、C12-200、DOTAP、DODAP、DOTMA、DOSPA、HGT5000或HGT5001；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述cKK-E12、DLin-MC3-DMA、ALC-0315、SM-102、C12-200、DOTAP、DODAP、DOTMA、DOSPA、HGT5000或HGT5001占所述组合物中存在的总脂质的45.0mol％-75.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-45.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述 两亲性嵌段共聚物占所述组合物的30.0％-90.0％重量百分比；(6) cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, C12-200, DOTAP, DODAP, DOTMA, DOSPA, HGT5000 or HGT5001; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, C12-200, DOTAP, DODAP, DOTMA, DOSPA, HGT5000 or HGT5001 02, C12-200, DOTAP, DODAP, DOTMA, DOSPA, HGT5000 or HGT5001 account for 45.0mol%-75.0mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 20.0mol%-45.0mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1mol%-5.0mol% of the total lipids, and the The amphiphilic block copolymer accounts for 30.0% to 90.0% by weight of the composition;
(7)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的30.0mol％-65.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的40.0％-90.0％重量百分比；(7) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 account for 30.0 mol%-65.0 mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 20.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% by weight of the composition;
(8)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的35.0mol％-70.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的10.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的40.0％-90.0％重量百分比；(8) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT50 00 or HGT5001 accounts for 35.0mol%-70.0mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 10.0mol%-40.0mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1mol%-5.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% weight percentage of the composition;
(9)GL67、ICE、或HGT4002；和DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE，其中所述GL67、ICE、或HGT4002占所述组合物中存在的总脂质的40.0mol％-80.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的10.0mol％-50.0mol％，且所述两亲性嵌段共聚物占所述组合物的40.0％-90.0％重量百分比；或(9) GL67, ICE, or HGT4002; and DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE, wherein the GL67, ICE, or HGT4002 accounts for 40.0 mol%-80.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE accounts for 10.0 mol%-50.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% weight percent of the composition; or
(10)DLin-MC3-DMA、ALC-0315、SM-102、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；和胆固醇或β-谷甾醇，其中所述DLin-MC3-DMA、ALC-0315、SM-102、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的30.0mol％-60.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的20.0mol％-45.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-45.0mol％，且所述两亲性嵌段共聚物占所述组合物的30.0％-90.0％重量百分比。(10) DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and cholesterol or β-sitosterol, wherein the DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, HGT5000 or HGT5001 accounts for 30.0 mol%-60.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 20.0 mol%-45.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol%-45.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% weight percent of the composition.
在本发明的组合物的一些实施方案中，所述组合物包含以下组分：In some embodiments of the composition of the present invention, the composition comprises the following components:
(1)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所 述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的10.0mol％，胆固醇或β-谷甾醇占总脂质的38.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.5mol％，且所述两亲性嵌段共聚物占所述组合物的89.9％重量百分比；(1) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein The DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.0 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 89.9% by weight of the composition;
(2)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的10.6mol％，胆固醇或β-谷甾醇占总脂质的38.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的72.9％重量百分比；(2) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.6 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 72.9% by weight of the composition;
(3)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的12.5mol％，胆固醇或β-谷甾醇占总脂质的36.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.5mol％，且所述两亲性嵌段共聚物占所述组合物的43.0％重量百分比；(3) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 12.5 mol% of the total lipids, cholesterol or β-sitosterol account for 36.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.0% by weight of the composition;
(4)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.5mol％，胆固醇或β-谷甾醇占总脂质的34.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.5mol％，且所述两亲性嵌段共聚物占所述组合物的81.8％重量百分比；(4) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or β-sitosterol account for 34.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 81.8% by weight of the composition;
(5)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.0mol％，胆固醇或β-谷甾醇占总脂质的35.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的69.2％重量百分比；(5) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.0 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 69.2% by weight of the composition;
(6)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0 mol％，DSPC、DPPC或DOPE占总脂质的14.1mol％，胆固醇或β-谷甾醇占总脂质的35.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的43.3％重量百分比；(6) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 accounts for 50.0% of the total lipids present in the composition. mol%, DSPC, DPPC or DOPE account for 14.1mol% of the total lipids, cholesterol or β-sitosterol account for 35.0mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.3% by weight of the composition;
(7)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.3mol％，胆固醇或β-谷甾醇占总脂质的35.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.7mol％，且所述两亲性嵌段共聚物占所述组合物的48.4％重量百分比；(7) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.3 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.4% by weight of the composition;
(8)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.5mol％，胆固醇或β-谷甾醇占总脂质的35.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.5mol％，且所述两亲性嵌段共聚物占所述组合物的43.6％重量百分比；(8) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.6% by weight of the composition;
(9)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的48.5mol％，DSPC、DPPC或DOPE占总脂质的17.0mol％，胆固醇或β-谷甾醇占总脂质的32.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的42.3％重量百分比；(9) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 48.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.3% by weight of the composition;
(10)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.5mol％，DSPC、DPPC或DOPE占总脂质的20.0mol％，胆固醇或β-谷甾醇占总脂质的32.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的52.9％重量百分比；(10) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 52.9% by weight of the composition;
(11)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的49.0mol％，DSPC、DPPC或DOPE占总脂质的20.0mol％，胆固醇或β-谷 甾醇占总脂质的30.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的41.9％重量百分比；(11) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, and cholesterol or β-sitosterol account for 20.0 mol% of the total lipids. Sterol accounts for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose accounts for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition;
(12)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的49.0mol％，DSPC、DPPC或DOPE占总脂质的20.1mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的40.3％重量百分比；(12) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.1 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.3% by weight of the composition;
(13)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的51.6mol％，DSPC、DPPC或DOPE占总脂质的20.0mol％，胆固醇或β-谷甾醇占总脂质的27.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的42.9％重量百分比；(13) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 51.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 27.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.9% by weight of the composition;
(14)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的50.2％重量百分比；(14) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.2% by weight of the composition;
(15)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的66.1％重量百分比；(15) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 66.1% by weight of the composition;
(16)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述 两亲性嵌段共聚物占所述组合物的74.5％重量百分比；(16) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the The amphiphilic block copolymer accounts for 74.5% by weight of the composition;
(17)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的79.6％重量百分比；(17) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 79.6% by weight of the composition;
(18)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的83.0％重量百分比；(18) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 83.0% by weight of the composition;
(19)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的88.2％重量百分比；(19) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 88.2% by weight of the composition;
(20)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的43.0mol％，DSPC、DPPC或DOPE占总脂质的23.3mol％，胆固醇或β-谷甾醇占总脂质的33.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.7mol％，且所述两亲性嵌段共聚物占所述组合物的39.1％重量百分比；(20) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 43.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.3 mol% of the total lipids, cholesterol or β-sitosterol account for 33.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 39.1% by weight of the composition;
(21)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的44.0mol％，DSPC、DPPC或DOPE占总脂质的25.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的63.6％重量百分比； (21) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 44.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 63.6% by weight of the composition;
(22)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的39.5mol％，DSPC、DPPC或DOPE占总脂质的25.3mol％，胆固醇或β-谷甾醇占总脂质的34.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.7mol％，且所述两亲性嵌段共聚物占所述组合物的37.2％重量百分比；(22) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.3 mol% of the total lipids, cholesterol or β-sitosterol account for 34.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 37.2% by weight of the composition;
(23)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的41.0mol％，DSPC、DPPC或DOPE占总脂质的28.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的66.0％重量百分比；(23) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 41.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 66.0% by weight of the composition;
(24)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的39.0mol％，DSPC、DPPC或DOPE占总脂质的28.0mol％，胆固醇或β-谷甾醇占总脂质的32.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的36.3％重量百分比；(24) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.3% by weight of the composition;
(25)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的39.0mol％，DSPC、DPPC或DOPE占总脂质的30.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的62.9％重量百分比；(25) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 62.9% by weight of the composition;
(26)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的40.6mol％，DSPC、DPPC或DOPE占总脂质的30.1mol％，胆固醇或β-谷甾醇占总脂质的28.4mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的36.9％重量百分比；(26) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.1 mol% of the total lipids, cholesterol or β-sitosterol account for 28.4 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.9% by weight of the composition;
(27)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、 DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的34.7mol％，DSPC、DPPC或DOPE占总脂质的40.1mol％，胆固醇或β-谷甾醇占总脂质的24.3mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的32.5％重量百分比；(27) Amphiphilic block copolymers; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 34.7 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 40.1 mol% of the total lipids, cholesterol or β-sitosterol account for 24.3 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 32.5% by weight of the composition;
(28)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的29.0mol％，DSPC、DPPC或DOPE占总脂质的50.1mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的28.0％重量百分比；(28) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 29.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 50.1 mol% of the total lipids, cholesterol or β-sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 28.0% by weight of the composition;
(29)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的35.0mol％，DSPC、DPPC或DOPE占总脂质的17.6mol％，胆固醇或β-谷甾醇占总脂质的46.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的35.9％重量百分比；(29) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 35.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.6 mol% of the total lipids, cholesterol or β-sitosterol account for 46.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 35.9% by weight of the composition;
(30)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的55.0mol％，DSPC、DPPC或DOPE占总脂质的16.9mol％，胆固醇或β-谷甾醇占总脂质的27.2mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的44.6％重量百分比；(30) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 55.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 16.9 mol% of the total lipids, cholesterol or β-sitosterol account for 27.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 44.6% by weight of the composition;
(31)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的60.0mol％，DSPC、DPPC或DOPE占总脂质的14.9mol％，胆固醇或β-谷甾醇占总脂质的24.2mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的46.5％重量百分比；(31) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 60.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.9 mol% of the total lipids, cholesterol or β-sitosterol account for 24.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 46.5% by weight of the composition;
(32)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所 述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的65.0mol％，DSPC、DPPC或DOPE占总脂质的14.1mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的21.0％重量百分比；(32) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein The DLin-MC3-DMA, ALC-0315 or SM-102 account for 65.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or β-sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 21.0% by weight of the composition;
(33)两亲性嵌段共聚物；GL67、ICE或HGT4002；DSPC、DPPC或DOPE；和DMG-PEG2K或DMG-PEG5K，其中所述GL67、ICE或HGT4002占所述组合物中存在的总脂质的70.0mol％，DSPC、DPPC或DOPE占总脂质的28.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.5mol％，且所述两亲性嵌段共聚物占所述组合物的48.1％重量百分比；(33) an amphiphilic block copolymer; GL67, ICE or HGT4002; DSPC, DPPC or DOPE; and DMG-PEG2K or DMG-PEG5K, wherein the GL67, ICE or HGT4002 accounts for 70.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE accounts for 28.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.1% by weight of the composition;
(34)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的61.3mol％，胆固醇或β-谷甾醇占总脂质的37.6mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.1mol％，且所述两亲性嵌段共聚物占所述组合物的41.9％重量百分比；(34) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 61.3 mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 37.6 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.1 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition;
(35)两亲性嵌段共聚物；cKK-E12、DLin-MC3-DMA、ALC-0315、SM-102或C12-200；DOTAP、DODAP、DOTMA或DOSPA；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述cKK-E12、DLin-MC3-DMA、ALC-0315、SM-102或C12-200占所述组合物中存在的总脂质的30.0mol％，DOTAP、DODAP、DOTMA或DOSPA占总脂质的39.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的57.7％重量百分比；或(35) an amphiphilic block copolymer; cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102 or C12-200; DOTAP, DODAP, DOTMA or DOSPA; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102 or C12-200 accounts for 30.0 mol% of the total lipids present in the composition, DOTAP, DODAP, DOTMA or DOSPA accounts for 39.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 57.7% by weight of the composition; or
(36)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；和胆固醇或β-谷甾醇，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的40.0mol％，DSPC、DPPC或DOPE占总脂质的32.0mol％，胆固醇或β-谷甾醇占总脂质的28.0mol％，且所述两亲性嵌段共聚物占所述组合物的50.0％重量百分比。(36) An amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; and cholesterol or β-sitosterol, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 32.0 mol% of the total lipids, cholesterol or β-sitosterol account for 28.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.0% weight percent of the composition.
在本发明的组合物的一些实施方案中，所述组合物中的阳离子脂质中的氮(胺)基团与核酸的磷酸酯基团的摩尔比(N/P比)为约1.0-约30.0，约4.0-约15.0，约6.0-约8.0或约6.7至约7.6。In some embodiments of the compositions of the invention, the molar ratio of nitrogen (amine) groups in the cationic lipids in the composition to phosphate groups in the nucleic acids (N/P ratio) is about 1.0 to about 30.0, about 4.0 to about 15.0, about 6.0 to about 8.0, or about 6.7 to about 7.6.
在本发明的组合物的一些实施方案中，所述组合物中的脂质与核酸的比(质量/质量比)为约2(2:1)-约100(100:1)，约5(5:1)-约60(60:1)，约15(15:1)-约 45(45:1)，或约20(20:1)-约30(30:1)。In some embodiments of the compositions of the invention, the ratio of lipid to nucleic acid (mass/mass ratio) in the composition is about 2 (2:1) to about 100 (100:1), about 5 (5:1) to about 60 (60:1), about 15 (15:1) to about 45(45:1), or about 20(20:1) to about 30(30:1).
在本发明的组合物的一些实施方案中，所述活性剂或治疗剂进一步包含蛋白质或多肽，在一些实施方案中，所述蛋白是与翻译或转录有关的蛋白。在一些实施方案中，所述蛋白与CRISPR过程有关。在一些实施方案中，所述蛋白是CRISPR有关的蛋白。在一些实施方案中，所述治疗剂是蛋白或多肽。在一些实施方案中，所述组合物包含蛋白和核酸两者。在一些实施方案中，所述治疗剂是小分子。在本发明的组合物的一些实施方案中，所述组合物进一步包含靶向部分以将所述组合物靶向至受试者中的目标器官、组织或细胞，优选所述靶向部分包含选自以下中的至少一种：糖基、脂质、核酸适配体、小分子治疗剂、维生素、多肽和蛋白质例如抗体。In some embodiments of the composition of the present invention, the active agent or therapeutic agent further comprises a protein or polypeptide, and in some embodiments, the protein is a protein related to translation or transcription. In some embodiments, the protein is related to the CRISPR process. In some embodiments, the protein is a protein related to CRISPR. In some embodiments, the therapeutic agent is a protein or polypeptide. In some embodiments, the composition comprises both protein and nucleic acid. In some embodiments, the therapeutic agent is a small molecule. In some embodiments of the composition of the present invention, the composition further comprises a targeting moiety to target the composition to a target organ, tissue or cell in a subject, preferably the targeting moiety comprises at least one selected from the following: glycosyl, lipid, nucleic acid aptamer, small molecule therapeutic agent, vitamin, polypeptide and protein such as antibody.
在本发明的组合物的一些实施方案中，所述组合物还包含佐剂，优选所述佐剂包含选自以下的至少一种：CpG寡脱氧核苷酸、聚肌苷：聚胞苷酸、皂角提取物(QS-21提取物)、铝佐剂、角鲨烯、α-生育酚、吐温(Tween)、司盘(Span)、脂多糖LPS、Pam3CSK4三酰脂肽、环腺苷二磷酸(c-di-AMP)、2′3′-环鸟苷一磷酸腺苷一磷酸(cGAMP)、单磷酰-脂质A、MPL脂质、鞭毛蛋白或免疫调节蛋白如IL-2、IL-12、GM-CSF、TSLP和编码这些免疫调节剂蛋白的核酸。In some embodiments of the composition of the present invention, the composition further comprises an adjuvant, preferably the adjuvant comprises at least one selected from the following: CpG oligodeoxynucleotides, polyinosinic: polycytidylic acid, saponin extract (QS-21 extract), aluminum adjuvant, squalene, α-tocopherol, Tween, Span, lipopolysaccharide LPS, Pam 3 CSK 4 triacyl lipopeptide, cyclic adenosine diphosphate (c-di-AMP), 2′3′-cyclic guanosine monophosphate adenosine monophosphate (cGAMP), monophosphoryl-lipid A, MPL lipid, flagellin or immunomodulatory proteins such as IL-2, IL-12, GM-CSF, TSLP and nucleic acids encoding these immunomodulator proteins.
在本发明的组合物的一些实施方案中，所述组合物还包含转染增效剂，优选所述转染增效剂包含选自以下中的至少一种：肺部表面活性蛋白、细胞穿膜肽、两亲性多肽、黏液溶解酶、1,2-丙二醇、纤维素(如羧甲基纤维素或羟丙基纤维素)、透明质酸盐、海藻酸盐、果胶、聚乙二醇、泊洛沙姆、泊洛沙胺、葡萄糖、果糖、蔗糖、海藻糖、葡聚糖、聚乙烯吡咯烷酮、壳聚糖、聚乙烯醇、聚乙酸乙烯酯、凝集素、聚乳酸、聚羟基丁酸、氨丁三醇、氯化苄烷铵、经修饰精氨酸、氯化鲸蜡基吡啶鎓、L-赖氨酸单水合物，以及聚乳酸-羟基乙酸共聚物或盐溶液。In some embodiments of the composition of the present invention, the composition further comprises a transfection enhancer, preferably the transfection enhancer comprises at least one selected from the following: pulmonary surfactant protein, cell penetrating peptide, amphiphilic polypeptide, mucolytic enzyme, 1,2-propylene glycol, cellulose (such as carboxymethyl cellulose or hydroxypropyl cellulose), hyaluronate, alginate, pectin, polyethylene glycol, poloxamer, poloxamine, glucose, fructose, sucrose, trehalose, dextran, polyvinyl pyrrolidone, chitosan, polyvinyl alcohol, polyvinyl acetate, lectin, polylactic acid, polyhydroxybutyric acid, tromethamine, benzalkonium chloride, modified arginine, cetyl pyridinium chloride, L-lysine monohydrate, and polylactic acid-glycolic acid copolymer or salt solution.
在本发明的组合物的一些实施方案中，所述组合物为纳米颗粒的形式，所述纳米颗粒具有约1000nm或更小的平均尺寸。在一些实施方案中，所述纳米颗粒具有约500nm或更小、400nm或更小、300nm或更小、200nm或更小、150nm或更小、125nm或更小、100nm或更小、75nm或更小、或约50nm或更小的平均尺寸。In some embodiments of the compositions of the invention, the compositions are in the form of nanoparticles having an average size of about 1000 nm or less. In some embodiments, the nanoparticles have an average size of about 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 75 nm or less, or about 50 nm or less.
在一些实施方案中，约30％-约100％、约70％-约100％、约90％-约100％、 约50％-约90％、约70％-约90％或约80％-约90％的所述纳米颗粒具有包封在其中的活性剂或治疗剂。In some embodiments, about 30% to about 100%, about 70% to about 100%, about 90% to about 100%, About 50% to about 90%, about 70% to about 90%, or about 80% to about 90% of the nanoparticles have the active or therapeutic agent encapsulated therein.
在本发明的组合物的一些实施方案中，所述组合物配制为溶液、干粉、雾化或喷雾的形式。在一些实施方案中，所述组合物配制为通过气溶胶化、干粉、吸入、雾化或滴注进行所述肺部和/或鼻部施用。In some embodiments of the composition of the present invention, the composition is formulated in the form of a solution, dry powder, atomization or spray. In some embodiments, the composition is formulated for pulmonary and/or nasal administration by aerosolization, dry powder, inhalation, atomization or instillation.
在第二方面，本发明提供了制备根据本发明的第一方面的组合物的方法，所述方法包括：In a second aspect, the present invention provides a method for preparing a composition according to the first aspect of the present invention, the method comprising:
(A)在两亲性嵌段共聚物的存在下将包含所述活性剂或治疗剂的溶液和包含所述脂质的溶液混合以形成所述组合物；或(A) mixing a solution comprising the active agent or therapeutic agent and a solution comprising the lipid in the presence of an amphiphilic block copolymer to form the composition; or
(B)将包含所述活性剂或治疗剂的溶液和包含所述脂质的溶液混合以形成包封所述活性剂或治疗剂的脂质纳米颗粒，随后将所述两亲性嵌段共聚物与所述脂质纳米颗粒溶液混合以形成所述组合物。(B) mixing a solution containing the active agent or therapeutic agent and a solution containing the lipid to form lipid nanoparticles encapsulating the active agent or therapeutic agent, and then mixing the amphiphilic block copolymer with the lipid nanoparticle solution to form the composition.
在一些实施方案中，所述方法包括：In some embodiments, the method comprises:
1)将所述两亲性嵌段共聚物加入到包含所述活性剂或治疗剂的溶液中和/或包含所述脂质的溶液中，和1) adding the amphiphilic block copolymer to a solution comprising the active agent or therapeutic agent and/or a solution comprising the lipid, and
2)将所述包含活性剂或治疗剂的溶液和所述包含脂质的溶液混合，2) mixing the solution containing the active agent or therapeutic agent and the solution containing the lipid,
从而形成所述组合物。Thereby forming the composition.
在另一些实施方案中，所述方法包括：In other embodiments, the method comprises:
1)在包含所述脂质的溶液中，使得所述脂质预形成无活性剂或治疗剂的脂质纳米颗粒；和1) in a solution comprising the lipid, allowing the lipid to pre-form into lipid nanoparticles without an active agent or therapeutic agent; and
2)将包含所述活性剂或治疗剂和两亲性嵌段共聚物的溶液与所述脂质纳米颗粒溶液混合，2) mixing a solution comprising the active agent or therapeutic agent and the amphiphilic block copolymer with the lipid nanoparticle solution,
从而形成所述组合物。Thereby forming the composition.
在另一些实施方案中，所述方法包括：In other embodiments, the method comprises:
1)将所述脂质和两亲性嵌段共聚物预形成无活性剂或治疗剂的聚合物-脂质纳米颗粒；和1) preforming the lipid and the amphiphilic block copolymer into polymer-lipid nanoparticles without active agent or therapeutic agent; and
2)将包含所述活性剂或治疗剂的溶液与所述聚合物-脂质纳米颗粒溶液混合，2) mixing a solution containing the active agent or therapeutic agent with the polymer-lipid nanoparticle solution,
从而形成所述组合物。Thereby forming the composition.
在一些实施方案中，所述方法进一步包括去除游离的脂质组分和/或两亲性嵌段共聚物的步骤，优选通过透析和/或切向流过滤去除所述游离的脂 质组分和/或两亲性嵌段共聚物。In some embodiments, the method further comprises the step of removing free lipid components and/or amphiphilic block copolymers, preferably by dialysis and/or tangential flow filtration. component and/or amphiphilic block copolymer.
在一些实施方案中，所述方法进一步包括在去除游离的脂质组分和/或两亲性嵌段共聚物之后，再次加入两亲性嵌段共聚物的步骤。In some embodiments, the method further comprises the step of adding the amphiphilic block copolymer again after removing the free lipid component and/or the amphiphilic block copolymer.
在第三方面，本发明提供了一种药物组合物，其包含根据本发明第一方面所述的组合物和药学上可接受的载体和/或赋形剂。In a third aspect, the present invention provides a pharmaceutical composition comprising the composition according to the first aspect of the present invention and a pharmaceutically acceptable carrier and/or excipient.
在第四方面，本发明提供了一种向靶细胞递送活性剂或治疗剂的方法，所述方法包括：在足以造成所述活性剂或治疗剂被摄入所述细胞中的情况下，使所述细胞与根据本发明第一方面所述的组合物接触，优选所述细胞是哺乳动物细胞。In a fourth aspect, the present invention provides a method for delivering an active agent or a therapeutic agent to a target cell, the method comprising: contacting the cell with the composition according to the first aspect of the present invention under conditions sufficient to cause the active agent or the therapeutic agent to be taken up into the cell, preferably the cell is a mammalian cell.
在第五方面，本发明提供了一种用于预防和/或治疗哺乳动物的疾病或病症的方法，所述方法包括给有此需要的受试者于体内施用药学有效量的根据本发明第一方面所述的组合物或根据本发明第三方面所述的药物组合物的步骤，其中所述组合物或药物组合物包含所述针对疾病或病症的活性剂或治疗剂。在一些实施方案中，所述疾病或病症选自免疫系统疾病、代谢性疾病、遗传性疾病、癌症、血液疾病、细菌感染或病毒感染。In a fifth aspect, the present invention provides a method for preventing and/or treating a disease or condition in a mammal, the method comprising administering a pharmaceutically effective amount of a composition according to the first aspect of the present invention or a pharmaceutical composition according to the third aspect of the present invention to a subject in need thereof, wherein the composition or pharmaceutical composition comprises an active agent or therapeutic agent for the disease or condition. In some embodiments, the disease or condition is selected from an immune system disease, a metabolic disease, a genetic disease, a cancer, a blood disease, a bacterial infection, or a viral infection.
在第六方面，本发明提供了根据本发明第一方面所述的组合物或根据本发明第三方面所述的药物组合物在制备用于预防和/或治疗受试者中的疾病的药物中的用途，其中所述组合物或药物组合物包含所述针对疾病或病症的活性剂或治疗剂。在一些实施方案中，所述疾病或病症选自免疫系统疾病、代谢性疾病、遗传性疾病、癌症、血液疾病、细菌感染或病毒感染。In a sixth aspect, the present invention provides a composition according to the first aspect of the present invention or a pharmaceutical composition according to the third aspect of the present invention for use in the preparation of a medicament for preventing and/or treating a disease in a subject, wherein the composition or pharmaceutical composition comprises an active agent or therapeutic agent for the disease or condition. In some embodiments, the disease or condition is selected from an immune system disease, a metabolic disease, a genetic disease, a cancer, a blood disease, a bacterial infection, or a viral infection.
从下述详细描述将会明白本公开内容的其它目的、特征和优点。但是，应当理解，尽管详细描述和具体实施例指示了本公开内容的某些实施方案，但是它们仅以说明的方式给出，因为本领域技术人员从该详细描述会明白在本公开内容的精神和范围内的各种变化和修改。Other objects, features and advantages of the present disclosure will be apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate certain embodiments of the present disclosure, they are given by way of illustration only, because those skilled in the art will appreciate various changes and modifications within the spirit and scope of the present disclosure from the detailed description.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1：聚合物-脂质组合物(PoLixNano)结构表征及在体外模型中的研究。Figure 1: Structural characterization of polymer-lipid composites (PoLixNano) and studies in in vitro models.
A.透射电镜(TEM)拍摄不同脂质比例制备的LNP纳米粒以及不同配方制备的PoLixNano纳米粒结构。标尺＝100nm；B.PoLixNano制剂在体外培养的16HBE细胞，DC2.4细胞及BMDC细胞中的摄取、转染效率(n＝6)。C.不同制剂在上述体外培养细胞模型中的毒性考察，PoLixNano制剂， Lipofecatmine2000(Lipo2000)及brPEI分别与16HBE细胞，DC2.4细胞及BMDC细胞共孵育4h后，对细胞存活率进行分析(n＝6)。D.PoLixNano制剂在Transwell小室中穿透Calu-3单层细胞膜，成功转染基底层培养的DC2.4细胞的情况，验证PoLixNano制剂穿越粘液屏障以及单层细胞膜的效率(n＝3)；E.BMDC细胞分化与成熟实验，将BMDC细胞与裸mRNA，LNP制剂及PoLixNano制剂共孵育24h后，通过流式细胞术检测BMDC细胞特异性标记物CD40+、CD80+、CD86+与MHCII+的表达情况(n＝3)。A. Transmission electron microscopy (TEM) images of LNP nanoparticles prepared with different lipid ratios and PoLixNano nanoparticles prepared with different formulas. Scale bar = 100nm; B. Uptake and transfection efficiency of PoLixNano preparations in 16HBE cells, DC2.4 cells and BMDC cells cultured in vitro (n = 6). C. Toxicity study of different preparations in the above in vitro cultured cell models, PoLixNano preparations, After lipofecatmine2000 (Lipo2000) and brPEI were co-incubated with 16HBE cells, DC2.4 cells and BMDC cells for 4 hours, the cell survival rate was analyzed (n=6). D. The PoLixNano preparation penetrated the Calu-3 monolayer cell membrane in the Transwell chamber and successfully transfected the DC2.4 cells cultured in the basal layer, verifying the efficiency of the PoLixNano preparation in crossing the mucus barrier and the monolayer cell membrane (n=3); E. BMDC cell differentiation and maturation experiment, after BMDC cells were co-incubated with naked mRNA, LNP preparations and PoLixNano preparations for 24 hours, the expression of BMDC cell-specific markers CD40+, CD80+, CD86+ and MHCII+ was detected by flow cytometry (n=3).
图2：PoLixNano制剂与LNP对照制剂经气管内喷雾途径(i.t.)给药6h后介导编码萤火虫荧光素酶的mRNA(Fluc-mRNA)在小鼠体内转染效率的研究。A.泊洛沙胺904制剂(T904)、泊洛沙胺704制剂(T704)、T904配制的PoLixNano制剂(PoLixNano(T904))、T704配制的PoLixNano制剂(PoLixNano(T704))以及LNP对照制剂(LNP)在活体小鼠中的萤火虫荧光素酶(Fluc)转染信号测定，分别以裸Fluc-mRNA(naked-mRNA)和PBS给药组作为对照组；B.定量测定A中所述制剂在小鼠离体肺脏中介导的Fluc生物发光信号情况(n＝3)；C.使用痕迹量T904(0.025mg/mL)、T704(0.025mg/mL)制备的PoLixNano制剂与LNP对照制剂在活体小鼠和离体肺脏中的示例性转染情况(左)以及Fluc生物发光信号定量结果(右)(n＝3)。Figure 2: Study on the transfection efficiency of mRNA encoding firefly luciferase (Fluc-mRNA) in mice mediated by PoLixNano formulation and LNP control formulation via intratracheal spray (i.t.) administration for 6 hours. A. Determination of firefly luciferase (Fluc) transfection signal of poloxamine 904 preparation (T904), poloxamine 704 preparation (T704), PoLixNano preparation prepared with T904 (PoLixNano (T904)), PoLixNano preparation prepared with T704 (PoLixNano (T704)) and LNP control preparation (LNP) in living mice, with naked Fluc-mRNA (naked-mRNA) and PBS administration groups as control groups, respectively; B. Quantitative determination of Fluc bioluminescent signal mediated by the preparations described in A in isolated mouse lungs (n=3); C. Exemplary transfection conditions (left) of PoLixNano preparations prepared with trace amounts of T904 (0.025 mg/mL) and T704 (0.025 mg/mL) and LNP control preparations in living mice and isolated lungs, as well as Fluc bioluminescent signal quantification results (right) (n=3).
图3：采用不同类型和浓度的两亲性嵌段共聚物制备的PoLixNano制剂经气管内喷雾途径(i.t.)给药6h后在小鼠体内的转染效率研究。A.含有不同浓度T904的PoLixNano制剂和LNP对照制剂在活体小鼠以及离体肺脏中Fluc表达水平测定(n＝3)；B.含有不同浓度T704的PoLixNano制剂和LNP对照制剂在活体小鼠和离体肺脏中Fluc表达水平测定(n＝3)；C.含有不同浓度T90R4、T304、T901以及Tween 80的PoLixNano制剂和对照LNP制剂在活体小鼠及离体肺脏中Fluc表达水平测定(n＝3)；D.含有不同浓度泊洛沙姆407、泊洛沙姆338、泊洛沙姆124、泊洛沙姆237、泊洛沙姆188、泊洛沙姆P105的PoLixNano制剂和对照LNP制剂在活体小鼠及离体肺脏中Fluc表达水平测定(n＝3)。Figure 3: Transfection efficiency study of PoLixNano formulations prepared with different types and concentrations of amphiphilic block copolymers in mice 6 h after intratracheal spray (i.t.) administration. A. Determination of Fluc expression levels in living mice and isolated lungs of PoLixNano preparations containing different concentrations of T904 and LNP control preparations (n=3); B. Determination of Fluc expression levels in living mice and isolated lungs of PoLixNano preparations containing different concentrations of T704 and LNP control preparations (n=3); C. Determination of Fluc expression levels in living mice and isolated lungs of PoLixNano preparations containing different concentrations of T90R4, T304, T901 and Tween 80 and control LNP preparations (n=3); D. Determination of Fluc expression levels in living mice and isolated lungs of PoLixNano preparations containing different concentrations of poloxamer 407, poloxamer 338, poloxamer 124, poloxamer 237, poloxamer 188 and poloxamer P105 and control LNP preparations (n=3).
图4：不同氮磷比(N/P)和不同脂质比例配制的PoLixNano制剂经气管内喷雾途径(i.t.)给药6h后在小鼠体内转染效率研究。A.不同氮磷比的PoLixNano制剂与对照LNP制剂在活体小鼠以及离体肺脏中Fluc表达水平测定(n＝3)；B-D：由不同摩尔比的脂质组分(Dlin-MC3-DMA：DSPC：Chol：DMG-PEG2000)配制的PoLixNano制剂与对照LNP制剂在活体小鼠以及离体 肺脏中Fluc表达水平测定(n＝3)。Figure 4: Study on the transfection efficiency of PoLixNano preparations prepared with different nitrogen-phosphorus ratios (N/P) and different lipid ratios in mice after 6 hours of intratracheal spray (it) administration. A. Determination of Fluc expression levels in the lungs of living mice and in vitro PoLixNano preparations with different nitrogen-phosphorus ratios and control LNP preparations (n=3); BD: Determination of Fluc expression levels in the lungs of living mice and in vitro PoLixNano preparations with different molar ratios of lipid components (Dlin-MC3-DMA: DSPC: Chol: DMG-PEG2000) and control LNP preparations (n=3); The expression level of Fluc in lung was determined (n=3).
图5：含有不同类型脂质及脂质比例的PoLixNano制剂经气管内喷雾途径(i.t.)给药6h后在小鼠体内转染效率研究。A.包含不同类型阳离子脂质(Dlin-MC3-DMA、ALC-0315、SM-102、C12-200和HGT5000)的PoLixNano制剂与对照LNP制剂在活体小鼠及离体肺脏中Fluc表达水平测定(n＝3)；B.包含不同类型磷脂(DSPC、DPPC、DOPE和ESM)的PoLixNano制剂与对照LNP制剂在活体小鼠以及离体肺脏中Fluc表达水平测定(n＝3)；C.包含不同类型胆固醇(胆固醇、β-谷甾醇、β-谷甾醇：胆固醇＝1:1)的PoLixNano制剂与对照LNP制剂在活体小鼠以及离体肺脏中Fluc表达水平测定(n＝3)；D.包含不同类型PEG脂质(DMG-PEG2000、DSPE-PEG-Mannose及DMG-PEG5000)和不同摩尔比DMG-PEG5000的PoLixNano制剂与对照LNP制剂在活体小鼠以及离体肺脏中Fluc表达水平测定(n＝3)。Figure 5: Study on the transfection efficiency of PoLixNano formulations containing different types of lipids and lipid ratios in mice 6 hours after administration via intratracheal spray (i.t.). A. Determination of Fluc expression levels in live mice and isolated lungs of PoLixNano formulations containing different types of cationic lipids (Dlin-MC3-DMA, ALC-0315, SM-102, C12-200 and HGT5000) and control LNP formulations (n=3); B. Determination of Fluc expression levels in live mice and isolated lungs of PoLixNano formulations containing different types of phospholipids (DSPC, DPPC, DOPE and ESM) and control LNP formulations (n=3); C. Determination of Fluc expression levels in live mice and isolated lungs of PoLixNano formulations containing different types of phospholipids (DSPC, DPPC, DOPE and ESM) and control LNP formulations (n=3); A. Determination of the Fluc expression level in the lungs of living mice and in vitro by PoLixNano preparations containing PEG lipids (cholesterol, β-sitosterol, β-sitosterol: cholesterol = 1:1) and control LNP preparations (n = 3); D. Determination of the Fluc expression level in the lungs of living mice and in vitro by PoLixNano preparations containing different types of PEG lipids (DMG-PEG2000, DSPE-PEG-Mannose and DMG-PEG5000) and different molar ratios of DMG-PEG5000 and control LNP preparations (n = 3).
图6：采用经鼻途径(i.n.)给药后6h，不同配方制备的PoLixNano制剂在小鼠体内的转染情况。A.定量测定含不同浓度T704(3mg/mL-40mg/mL)，T904(3mg/mL)，泊洛沙姆237(10mg/mL)，泊洛沙姆338(3mg/mL)及不同浓度泊洛沙姆124(3mg/mL及20mg/mL)的PoLixNano制剂及LNP对照制剂在小鼠离体肺脏中介导的Fluc生物发光信号情况(n＝3)；B.定量测定不同摩尔比的Dlin-MC3-DMA：DSPC：Chol：DMG-PEG2000配制的PoLixNano制剂及LNP对照制剂在小鼠离体肺脏中介导的Fluc生物发光信号情况(n＝3)；C.定量测定包含不同类型阳离子脂质、不同类型中性脂质、不同类型PEG脂质和不同摩尔比DMG-PEG5000的PoLixNano制剂及LNP对照制剂在小鼠离体肺脏中介导的Fluc生物发光信号情况(n＝3)。Figure 6: Transfection status of PoLixNano preparations prepared with different formulations in mice 6 hours after intranasal (i.n.) administration. A. Quantitative determination of the Fluc bioluminescent signals mediated by PoLixNano preparations containing different concentrations of T704 (3 mg/mL-40 mg/mL), T904 (3 mg/mL), poloxamer 237 (10 mg/mL), poloxamer 338 (3 mg/mL) and different concentrations of poloxamer 124 (3 mg/mL and 20 mg/mL) and LNP control preparations in the isolated lungs of mice (n=3); B. Quantitative determination of the Fluc bioluminescent signals mediated by PoLixNano preparations prepared with different molar ratios of Dlin-MC3-DMA:DSPC:Chol:DMG-PEG2000 and LNP control preparations in the isolated lungs of mice (n=3); C. Quantitative determination of the Fluc bioluminescent signals mediated by PoLixNano preparations containing different types of cationic lipids, different types of neutral lipids, different types of PEG lipids and different molar ratios of DMG-PEG5000 and LNP control preparations in the isolated lungs of mice (n=3).
图7：LNP制剂和PoLixNano制剂与不同增效剂(KG41多肽(KG41)、T904以及终浓度为10％的蔗糖溶液(10％sucrose))物理混合后所获得的制剂(LNP+KG41、LNP+T904、LNP+10％sucrose、PoLixNano+KG41、PoLixNano+T904以及PoLixNano+10％sucrose)经不同给药方式(i.t.或i.n.)在小鼠体内介导Fluc-mRNA高效转染的研究。A.定量测定经气管内喷雾途径(i.t.)将上述制剂给药6h后在活体小鼠及离体器官(肺、肝及脾)中Fluc生物发光信号的情况(n＝3)；B.定量测定经鼻途径(i.n.)将上述制剂给药6h后在活体小鼠及离体器官(肺、肝及脾)中Fluc生物发光信号的情况(n＝3)。Figure 7: Study on the efficient transfection of Fluc-mRNA in mice mediated by the preparations (LNP+KG41, LNP+T904, LNP+10% sucrose, PoLixNano+KG41, PoLixNano+T904 and PoLixNano+10% sucrose) obtained by physical mixing of LNP preparations and PoLixNano preparations with different enhancers (KG41 polypeptide (KG41), T904 and sucrose solution with a final concentration of 10%) through different administration methods (i.t. or i.n.). A. Quantitative determination of the Fluc bioluminescent signal in living mice and isolated organs (lung, liver and spleen) 6 hours after the above preparations were administered via the intratracheal spray route (i.t.) (n=3); B. Quantitative determination of the Fluc bioluminescent signal in living mice and isolated organs (lung, liver and spleen) 6 hours after the above preparations were administered via the nasal route (i.n.) (n=3).
图8：含有非常规脂质组分以及组成的PoLixNano制剂在小鼠体内转染效 率研究。A.同时含有CKK-E12和DOTAP两种阳离子脂质的PoLixNano制剂(CKK-E12：DOTAP：Chol：DMG-PEG2000＝30:39:30:1)以及LNP对照制剂经气管内喷雾途径(i.t.)在活体小鼠以及离体肺脏中Fluc表达水平测定(n＝3)；B.含有GL67阳离子脂质：DOPE：DMG-PEG2000＝70:28.5:1.5的PoLixNano制剂以及LNP对照制剂经气管内喷雾途径(i.t.)在小鼠离体肺脏中Fluc表达水平测定(n＝3)；C.不含有DMG-PEG2000脂质组分的PoLixNano制剂与LNP对照制剂经气管内喷雾途径(i.t.)和经鼻途径(i.n.)在活体小鼠及离体肺脏和肝脏中Fluc表达水平测定(n＝3)；D.不含有磷脂组分的PoLixNano制剂与LNP对照制剂经气管内喷雾途径(i.t.)和经鼻途径(i.n.)在活体小鼠以及离体肺脏和肝脏中Fluc表达水平测定(n＝3)。Figure 8: Transfection efficacy of PoLixNano formulations containing unconventional lipid components and compositions in mice A. The expression level of Fluc in live mice and isolated lungs was measured by intratracheal spray (it) of PoLixNano formulations containing both CKK-E12 and DOTAP cationic lipids (CKK-E12: DOTAP: Chol: DMG-PEG2000 = 30:39:30:1) and LNP control formulations (n = 3); B. The expression level of Fluc in live mice and isolated lungs was measured by intratracheal spray (it) of PoLixNano formulations containing GL67 cationic lipids: DOPE: DMG-PEG2000 = 70:28.5:1.5 and LNP control formulations (n = 3). A. The Fluc expression level in the isolated lung of mice was determined by intratracheal spray (it) and nasal route (in) of PoLixNano preparations without DMG-PEG2000 lipid components and LNP control preparations in living mice and isolated lungs and livers (n=3); D. The Fluc expression level in the isolated lungs and liver of PoLixNano preparations without phospholipid components was determined by intratracheal spray (it) and nasal route (in) of LNP control preparations (n=3).
图9：PoLixNano制剂经雾化吸入(nebulization)途径给药后，在小鼠体内介导Fluc-mRNA高效转染以及纳米粒理化性质的研究。A.小鼠雾化装置及研究流程示意图；B.透射电镜(TEM)拍摄LNP纳米粒(上，标尺＝200nm)和PoLixNano纳米粒(下，标尺＝100nm)雾化前后纳米粒形态对比；C.定量测定含不同类型两亲性嵌段共聚物(泊洛沙胺704、泊洛沙姆188、泊洛沙姆237、泊洛沙姆338、泊洛沙姆124、泊洛沙姆P105及泊洛沙姆407)的PoLixNano制剂与LNP对照制剂经nebulization给药6h后在离体肺脏中Fluc生物发光信号情况(n＝3)；D.定量测定包含不同类型阳离子脂质和不同类型中性脂质的PoLixNano制剂及LNP对照制剂经nebulization给药6h后在小鼠离体肺脏中介导的Fluc生物发光信号情况(n＝3)。Figure 9: After the PoLixNano preparation was administered via nebulization, it mediated efficient Fluc-mRNA transfection in mice and studied the physicochemical properties of the nanoparticles. A. Schematic diagram of the mouse nebulization device and research process; B. Transmission electron microscopy (TEM) photography of LNP nanoparticles (top, scale bar = 200 nm) and PoLixNano nanoparticles (bottom, scale bar = 100 nm) before and after nebulization to compare the morphology of nanoparticles; C. Quantitative determination of the Fluc bioluminescent signals in the isolated lungs of PoLixNano preparations containing different types of amphiphilic block copolymers (poloxamine 704, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 124, poloxamer P105 and poloxamer 407) and LNP control preparations 6 hours after nebulization administration (n = 3); D. Quantitative determination of the Fluc bioluminescent signals mediated by PoLixNano preparations containing different types of cationic lipids and different types of neutral lipids and LNP control preparations in the isolated lungs of mice 6 hours after nebulization administration (n = 3).
图10：包含不同类型两亲性嵌段共聚物、不同摩尔比脂质组成和不同给药剂量的PoLixNano制剂经雾化吸入途径(nebulization)给药后在小鼠体内高效介导Fluc-mRNA转染的研究。A.定量测定采用泊洛沙姆188(左)或泊洛沙胺704(右)配制的包含不同摩尔比DMG-PEG2000的PoLixNano制剂及LNP对照制剂经雾化吸入途径(nebulization)给药6h后在离体肺脏中Fluc生物发光信号情况(n＝3)；B.定量测定采用泊洛沙姆188(左)或泊洛沙胺704(右)配制的包含不同摩尔比脂质组成的PoLixNano制剂及LNP对照制剂经雾化吸入途径(nebulization)给药6h后在离体肺脏中Fluc生物发光信号情况(n＝3)；C.考察不同Fluc-mRNA给药剂量(25μg/mouse、50μg/mouse以及150μg/mouse)的PoLixNano制剂经雾化吸入途径(nebulization)给药24h后在活体小鼠及离体肺脏的Fluc生物发光信号情况(n＝3)。 Figure 10: A study on the efficient mediating Fluc-mRNA transfection in mice of PoLixNano preparations containing different types of amphiphilic block copolymers, different molar ratios of lipid compositions and different dosages after administration via nebulization. A. Quantitative determination of the Fluc bioluminescent signal in the isolated lungs of PoLixNano preparations containing different molar ratios of DMG-PEG2000 prepared with poloxamer 188 (left) or poloxamine 704 (right) and LNP control preparations 6 hours after administration via nebulization (n=3); B. Quantitative determination of the Fluc bioluminescent signal in the isolated lungs of PoLixNano preparations containing different molar ratios of lipid compositions prepared with poloxamer 188 (left) or poloxamine 704 (right) and LNP control preparations 6 hours after administration via nebulization (n=3); C. Investigate the Fluc bioluminescent signal in the living mice and isolated lungs of PoLixNano preparations with different Fluc-mRNA dosages (25μg/mouse, 50μg/mouse and 150μg/mouse) 24 hours after administration via nebulization (n=3).
图11：PoLixNano制剂在小鼠体内的分布、摄取及转染效率研究。A.DIR标记的PoLixNano制剂经气管内喷雾途径(i.t.)，经鼻途径(i.n.)，雾化途径(nebulization)给药6h后，采用活体成像技术(IVIS)检测PoLixNano制剂在活体小鼠体内(左)以及离体器官(右)中的分布，图示为代表性结果；B.RT-qPCR测定PoLixNano制剂所负载的IVT-mRNA(编码新冠病毒刺突蛋白受体结合域RBD)经气管内喷雾途径(i.t.)给药6h后在小鼠不同器官的内的含量(n＝3)；C.流式细胞术检测经气管内喷雾(i.t.)途径给药的DID标记PoLixNano制剂及DID标记LNP制剂在肺部DC细胞(CD11c+)，内皮细胞(CD31+)，上皮细胞(CD326+)、I型肺泡细胞(Podoplanin+)以及巨噬细胞(F4/80+)中的摄取情况(n＝3)；D.PoLixNano制剂经气管内喷雾途径(i.t.)接种后在活体小鼠(Mice)及不同器官(Lung、Liver及Spleen)内的蛋白表达量随时间变化的动力学曲线(n＝5)；E.负载编码Fluc的环状RNA(circRNA)的PoLixNano制剂经气管内喷雾途径(i.t.)给药后，在活体小鼠(Mice)中的蛋白表达量随时间变化的动力学曲线(n＝3)。Figure 11: Study on the distribution, uptake and transfection efficiency of PoLixNano preparations in mice. A. After 6 hours of administration of DIR-labeled PoLixNano preparations via intratracheal spray (i.t.), nasal route (i.n.), and nebulization, in vivo imaging technology (IVIS) was used to detect the distribution of PoLixNano preparations in living mice (left) and in vitro organs (right). The figure shows representative results; B. RT-qPCR was used to determine the content of IVT-mRNA (encoding the receptor binding domain RBD of the spike protein of the new coronavirus) loaded by PoLixNano preparations in different organs of mice 6 hours after administration via intratracheal spray (i.t.) (n=3); C. Flow cytometry was used to detect the DID-labeled PoLixNano preparations and DID-labeled L Uptake of NP preparations in lung DC cells (CD11c+), endothelial cells (CD31+), epithelial cells (CD326+), type I alveolar cells (Podoplanin+) and macrophages (F4/80+) (n=3); D. Kinetic curves of protein expression of PoLixNano preparations in living mice (Mice) and different organs (Lung, Liver and Spleen) over time after inoculation via intratracheal spray (i.t.) (n=5); E. Kinetic curves of protein expression of PoLixNano preparations loaded with circular RNA (circRNA) encoding Fluc in living mice (Mice) over time after administration via intratracheal spray (i.t.) (n=3).
图12：不同配方制备的PoLixNano制剂经不同途径(i.t.、i.n.以及nebulization)给药后在小鼠体内的转染情况。A.含泊洛沙姆188(3mg/mL)，T304(3mg/mL)及泊洛沙姆338(3mg/mL)的PoLixNano制剂及LNP对照制剂经气管内喷雾途径(i.t.)给药6h后在活体小鼠及离体器官中的转染情况示例性结果；B.定量测定A中所述制剂在小鼠离体肺脏中介导的Fluc生物发光信号情况(n＝3)；C.含T904(3mg/mL度)，T704(6mg/mL)，407(3mg/mL)，Tween80(3mg/mL)的PoLixNano制剂及LNP对照制剂经气管内喷雾途径(i.t.)给药6h后在活体小鼠及离体器官中介导的转染情况示例性结果；D.定量测定C中所述制剂在不同离体器官中介导的Fluc生物发光信号情况(n＝3)；E.PoLixNano制剂与LNP对照制剂经鼻途径(i.n.)给药6h后在活体小鼠及离体器官中介导的转染情况示例性结果；F.定量测定E中所述制剂在不同离体器官中介导的Fluc生物发光信号情况(n＝3)。G.PoLixNano制剂与LNP对照制剂经雾化吸入途径(nebulization)给药6h后在活体小鼠及离体器官中转染情况代表性结果；H.定量测定G中所述制剂在小鼠离体肺脏中介导的Fluc生物发光信号情况(n＝3)。Figure 12: Transfection of PoLixNano preparations prepared with different formulations in mice after administration via different routes (i.t., i.n. and nebulization). A. Exemplary results of transfection of PoLixNano preparations containing poloxamer 188 (3 mg/mL), T304 (3 mg/mL) and poloxamer 338 (3 mg/mL) and LNP control preparations in living mice and isolated organs 6 hours after administration via intratracheal spray route (i.t.); B. Quantitative determination of Fluc bioluminescent signal mediated by the preparations described in A in isolated mouse lungs (n=3); C. Exemplary results of transfection of PoLixNano preparations containing T904 (3 mg/mL), T704 (6 mg/mL), 407 (3 mg/mL), Tween80 (3 mg/mL) and LNP control preparations in living mice and isolated organs 6 hours after administration via intratracheal spray route (i.t.); ) in living mice and isolated organs 6 hours after administration of the PoLixNano formulation and the LNP control formulation via intratracheal spray route (i.t.); D. Quantitative determination of the Fluc bioluminescent signal mediated by the formulation in C in different isolated organs (n=3); E. Exemplary results of the transfection mediated by the PoLixNano formulation and the LNP control formulation in living mice and isolated organs 6 hours after administration via the nasal route (i.n.); F. Quantitative determination of the Fluc bioluminescent signal mediated by the formulation in E in different isolated organs (n=3). G. Representative results of the transfection of the PoLixNano formulation and the LNP control formulation in living mice and isolated organs 6 hours after administration via nebulization inhalation route (nebulization); H. Quantitative determination of the Fluc bioluminescent signal mediated by the formulation in G in isolated mouse lungs (n=3).
图13：PoLixNano制剂在小鼠体内的安全性考察。A.PoLixNano制剂经不同途径(气管内喷雾i.t.，经鼻i.n.以及雾化nebulization)给药6h后，小鼠肺部 组织切片代表性H&E染色结果，分别采用i.t.途径接种PBS溶液与LNP制剂的对照样品作为阴性对照以及阳性对照，标尺＝50μm；B.采用不同浓度的泊洛沙姆237和泊洛沙姆338制备的PoLixNano制剂经i.t.途径给药6h后，小鼠肺部组织切片示例性H&E染色结果，分别采用经i.t.途径接种PBS溶液以及LNP制剂的对照样品作为阴性对照及阳性对照。标尺＝50μm；C.经i.t.途径接种PBS溶液、PoLixNano制剂及LNP制剂48h后，小鼠肺组织研磨液中IL-4、IL-6、IL-17及TNF-α等炎症因子的表达情况(n＝3)；D.经i.t.途径接种PoLixNano制剂后，在不同时间点收集血清检测小鼠体内的各项生化指标的含量(n＝3)；E.在第7、第14和第21天收集血清，采用ELISA方法检测PoLixNano纳米粒中的聚合物组分(T904)以及脂质组分(empty-LNP)在小鼠体内的免疫原性(n＝3)。Figure 13: Safety study of PoLixNano preparation in mice. A. Six hours after administration of PoLixNano preparation via different routes (intratracheal spray, intranasal spray, and nebulization), the lungs of mice Representative H&E staining results of tissue sections, using control samples of PBS solution and LNP preparation inoculated by it route as negative control and positive control, respectively, scale bar = 50μm; B. Exemplary H&E staining results of mouse lung tissue sections 6 hours after it administration of PoLixNano preparations prepared with different concentrations of poloxamer 237 and poloxamer 338, using control samples of PBS solution and LNP preparation inoculated by it route as negative control and positive control, respectively. Scale bar = 50 μm; C. Expression of inflammatory factors such as IL-4, IL-6, IL-17 and TNF-α in the abrasive fluid of mouse lung tissue 48 h after inoculation with PBS solution, PoLixNano preparation and LNP preparation via the it route (n = 3); D. After inoculation with PoLixNano preparation via the it route, serum was collected at different time points to detect the content of various biochemical indicators in mice (n = 3); E. Serum was collected on the 7th, 14th and 21st days, and the immunogenicity of the polymer component (T904) and lipid component (empty-LNP) in PoLixNano nanoparticles in mice was detected by ELISA method (n = 3).
图14：PoLixNano制剂与LNP对照制剂经不同途径(i.t.、i.n.与nebulization)给药后，在C57black 6(C57BL/6)小鼠和Sprague-Dawley(SD)大鼠体内介导Fluc-mRNA转染的情况。A.上述制剂通过气管内喷雾途径(i.t.)、经鼻途径(i.n.)和雾化途径(nebulization)给药后6h在C57BL/6活体小鼠与离体肺脏、肝脏和脾脏中介导的转染情况示例性结果(上)以及离体肺脏Fluc生物发光信号定量结果(下)(n＝3)；B.上述制剂经i.t.途径(20μg Fluc-mRNA/rat)、i.n.途径(给药量同i.t.途径)和nebulization途径(45μg Fluc-mRNA/rat)给药6h后在活体SD大鼠以及离体肺脏、肝脏、脾脏中介导的Fluc生物发光信号测定情况。Figure 14: Fluc-mRNA transfection mediated by PoLixNano formulation and LNP control formulation in C57black 6 (C57BL/6) mice and Sprague-Dawley (SD) rats after administration via different routes (i.t., i.n. and nebulization). A. Exemplary results of transfection induced by the above-mentioned preparations in living C57BL/6 mice and in ex vivo lungs, livers and spleens 6 h after administration by intratracheal spray (i.t.), nasal (i.n.) and nebulization (nebulization) routes (top) and quantitative results of Fluc bioluminescent signals in ex vivo lungs (bottom) (n=3); B. Determination of Fluc bioluminescent signals induced by the above-mentioned preparations in living SD rats and in ex vivo lungs, livers and spleens 6 h after administration by i.t. route (20 μg Fluc-mRNA/rat), i.n. route (dosage amount is the same as that of i.t. route) and nebulization route (45 μg Fluc-mRNA/rat).
图15：经气管内喷雾途径(i.t.)接种负载编码新冠病毒RBD蛋白mRNA(RBD-mRNA)的PoLixNano制剂(RBD-mRNA/PoLixNano)后诱导小鼠产生的抗原特异性体液免疫应答情况。A.采用ELISA方法检测经i.t.途径接种的PBS缓冲液(PBS)、LNP制剂(LNP(i.t.))、PoLixNano制剂以及通过肌注接种途径(i.m.)的LNP制剂(LNP(i.m.))在首次免疫后14天，28天及280天采集的小鼠血清样品中的RBD抗原特异性IgG抗体滴度(n＝5)，同时检测初免后第28天小鼠肺泡灌洗液(BALF)及鼻腔灌洗液(NLF)样品中的RBD抗原特异性IgG抗体滴度；B.ELISA法检测上述PBS、LNP(i.t.)、LNP(i.m.)以及PoLixNano组于首次免疫后28天及280天所采集的小鼠BALF，NLF样品中的RBD抗原特异性分泌型sIgA抗体滴度(n＝5)；C.假病毒中和实验验证初免后28天上述制剂免疫小鼠血清及BALF样品对新冠病毒原始毒株(ancestral strain)及奥密克戎突变株(omicron)的中和效价(n＝8)；D.通过ELIspot方法检测上述制 剂初免后28天小鼠的脾脏(Spleens)及纵膈淋巴结(MLN)样品中IgG-和IgA-抗体分泌细胞(ASC)的含量(n＝3)；E.采用流式细胞术检测上述样品初免后第10天小鼠的MLN样品中Tfh及GCB阳性细胞的比例(n＝3)。Figure 15: Antigen-specific humoral immune response induced in mice after intratracheal spray (it) inoculation of PoLixNano preparation (RBD-mRNA/PoLixNano) loaded with mRNA encoding the SARS-CoV-2 RBD protein (RBD-mRNA). A. The ELISA method was used to detect the RBD antigen-specific IgG antibody titer in the mouse serum samples collected 14 days, 28 days and 280 days after the first immunization of PBS buffer (PBS), LNP preparation (LNP (it)), PoLixNano preparation and LNP preparation (LNP (im)) inoculated by it route (n=5), and the RBD antigen-specific IgG antibody titer in the mouse bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF) samples on the 28th day after the first immunization was detected; B. The ELISA method was used to detect the RBD antigen-specific secretory sIgA antibody titer in the mouse BALF and NLF samples collected 28 days and 280 days after the first immunization of the above-mentioned PBS, LNP (it), LNP (im) and PoLixNano groups (n=5); C. Pseudovirus neutralization experiments verified the serum and BALF samples of mice immunized with the above-mentioned preparations 28 days after the first immunization against the original strain of the new coronavirus (ancestral strain) and the mutant strain of omicron (n=8); D. Detection of the neutralization titer of the above strains by ELIspot method E. The content of IgG- and IgA-antibody secreting cells (ASC) in the spleen (Spleens) and mediastinal lymph node (MLN) samples of mice 28 days after the initial immunization (n=3); E. Flow cytometry was used to detect the proportion of Tfh and GCB positive cells in the MLN samples of mice 10 days after the initial immunization of the above samples (n=3).
图16：经气管内喷雾途径(i.t.)接种RBD-mRNA/PoLixNano制剂后诱导小鼠产生了高效且持久的适应性细胞免疫应答。A.分别收集PBS、LNP(i.t.)、LNP(i.m.)以及PoLixNano制剂在初免后28天的脾细胞及肺淋巴细胞样品，使用RBD重叠肽库刺激24h后，采用ELISpot技术检测细胞因子IFN-γ，IL-4以及IL-17的产生情况(n＝5)；B.采用流式细胞术，检测经RBD重叠肽库刺激6h后的免疫小鼠肺淋巴细胞样品分泌IFN-γ的情况(n＝5)；C.分别收集初免后28天及280天小鼠的肺淋巴细胞，采用流式细胞术检测样品中所含组织驻留记忆T细胞(Trm)、效应记忆T细胞(Tem)及中央型记忆T细胞(Tcm)的比例(n＝5)；D.PoLixNano制剂诱导小鼠产生训练固有免疫应答的情况。收集LNP(i.t.)、LNP(i.m.)以及PoLixNano制剂初免后11天小鼠BALF样品，采用流式细胞术检测MHC II+肺泡巨噬细胞(AM)或MHC II+肺间质巨噬细胞(IM)的比率(n＝5)。Figure 16: RBD-mRNA/PoLixNano formulation induced efficient and long-lasting adaptive cellular immune response in mice after intratracheal spray (i.t.) inoculation. A. Splenocytes and lung lymphocytes samples of PBS, LNP (i.t.), LNP (i.m.) and PoLixNano formulations were collected 28 days after primary immunization, and the production of cytokines IFN-γ, IL-4 and IL-17 was detected by ELISpot technology after 24 hours of stimulation with RBD overlapping peptide library (n=5); B. Flow cytometry was used to detect the secretion of IFN-γ in lung lymphocyte samples of immunized mice after 6 hours of stimulation with RBD overlapping peptide library (n=5); C. Lung lymphocytes of mice were collected 28 days and 280 days after primary immunization, and flow cytometry was used to detect the proportion of tissue-resident memory T cells (Trm), effector memory T cells (Tem) and central memory T cells (Tcm) in the samples (n=5); D. The situation of PoLixNano formulation inducing trained innate immune response in mice. BALF samples of mice were collected 11 days after primary immunization with LNP (i.t.), LNP (i.m.) and PoLixNano preparations, and the ratio of MHC II+ alveolar macrophages (AM) or MHC II+ interstitial macrophages (IM) was detected by flow cytometry (n=5).
图17：致死型新冠病毒原始株(ancestral strain)以及奥密克戎突变株(omicron)针对经气管内喷雾途径(i.t.)接种的PoLixNano制剂免疫小鼠的攻毒挑战研究。A.经PBS、LNP(i.m.)、LNP(i.t.)与PoLixNano制剂免疫后的小鼠在新冠病毒ancestral strain攻毒后的存活率、体重变化曲线以及攻毒后3天上述小鼠的肺组织(Lungs)或鼻甲(Nasal turbinates)中的新冠病毒RNA载量(n＝8)。未攻毒且未经任何干预的小鼠样品作为对照(Control)；B.经PBS、LNP(i.m.)、LNP(i.t.)及PoLixNano制剂免疫后的小鼠在新冠病毒omicron突变株攻毒后的存活率、体重变化曲线以及攻毒后3天上述小鼠的肺组织(Lungs)以及鼻甲(Nasal turbinates)中的新冠病毒RNA载量(n＝8)。未攻毒且未经任何干预的小鼠样品作为对照(Control)；C.新冠病毒ancestral strain攻毒3天后，不同制剂免疫小鼠肺组织的代表性H&E染色切片(上)以及代表性新冠病毒N蛋白免疫组化切片(下)，标尺＝200μm(n＝8)。D.omicron突变株攻毒3天后，不同制剂免疫小鼠肺组织的代表性H&E染色切片(上)及代表性新冠病毒N蛋白免疫组化切片(下)，标尺＝200μm(n＝8)。Figure 17: Challenge study of lethal SARS-CoV-2 ancestral strain and omicron mutant strain against mice immunized with PoLixNano preparations inoculated via intratracheal spray (i.t.). A. Survival rate, weight change curve of mice immunized with PBS, LNP (i.m.), LNP (i.t.) and PoLixNano preparations after challenge with SARS-CoV-2 ancestral strain, and SARS-CoV-2 RNA load in lung tissue (Lungs) or nasal turbinates (Nasal turbinates) of the above mice 3 days after challenge (n=8). Samples of mice that were not challenged and did not undergo any intervention were used as controls (Control); B. Survival rate, weight change curve of mice immunized with PBS, LNP (i.m.), LNP (i.t.) and PoLixNano preparations after challenge with SARS-CoV-2 omicron mutant strain, and SARS-CoV-2 RNA load in lung tissue (Lungs) and nasal turbinates (Nasal turbinates) of the above mice 3 days after challenge (n=8). Samples of mice that were not challenged and did not receive any intervention were used as controls (Control); C. Representative H&E staining sections of lung tissues of mice immunized with different preparations 3 days after challenge with the ancestral strain of the new coronavirus (upper) and representative immunohistochemical sections of the new coronavirus N protein (lower), scale bar = 200μm (n = 8). D. Representative H&E staining sections of lung tissues of mice immunized with different preparations 3 days after challenge with the omicron mutant strain (upper) and representative immunohistochemical sections of the new coronavirus N protein (lower), scale bar = 200μm (n = 8).
图18：PoLixNano制剂经鼻途径(i.n.)给药后介导Fluc-mRNA在小鼠体内的转染以及介导RBD-mRNA产生高效适应性免疫应答的研究。A.PoLixNano 制剂i.n.给药后在活体小鼠及离体肺脏内的蛋白表达量随时间变化的动力学曲线(n＝3)；B.采用ELISA方法检测经鼻接种的PBS缓冲液(PBS)、LNP对照制剂(LNP)与PoLixNano制剂免疫小鼠在首次免疫后7天，14天，21天，28天，35天，42天以及49天采集的血清样品(serum)中的RBD抗原特异性IgG抗体滴度，同时检测初免后28天，小鼠BALF、NLF及唾液(Saliva)样品中的RBD抗原特异性分泌型sIgA抗体水平(n＝5)；C.ELISpot法检测上述制剂免疫小鼠在初免后28天的肺淋巴细胞样品中IFN-γ、IL-4以及IL-17等细胞因子的产生情况(n＝5)；D.收集初免后28天小鼠的肺淋巴细胞，采用流式细胞术检测样品中所含组织驻留记忆T细胞(Trm)以及效应记忆T细胞(Tem)的比例(n＝5)Figure 18: PoLixNano preparation mediated Fluc-mRNA transfection in mice and mediated RBD-mRNA to produce efficient adaptive immune response after nasal administration (in). The kinetic curve of protein expression in live mice and isolated lungs after administration of the preparation in vivo (n=3); B. The ELISA method was used to detect the RBD antigen-specific IgG antibody titer in serum samples collected from mice immunized with PBS buffer (PBS), LNP control preparation (LNP) and PoLixNano preparation intranasally at 7 days, 14 days, 21 days, 28 days, 35 days, 42 days and 49 days after the first immunization, and at the same time, the BALF of mice 28 days after the first immunization was detected. The levels of RBD antigen-specific secretory sIgA antibodies in NLF and saliva samples (n=5); C. ELISpot method was used to detect the production of cytokines such as IFN-γ, IL-4 and IL-17 in lung lymphocyte samples of mice immunized with the above preparations 28 days after primary immunization (n=5); D. Lung lymphocytes of mice were collected 28 days after primary immunization, and the proportion of tissue-resident memory T cells (Trm) and effector memory T cells (Tem) in the samples were detected by flow cytometry (n=5)
图19：PoLixNano制剂经肌肉注射途径(i.m.)给药后介导Fluc-mRNA在小鼠体内的转染以及诱导适应性免疫应答的研究。A.负载Fluc-mRNA的PoLixNano制剂与LNP对照制剂经肌肉注射途径(i.m.)给药后在活体小鼠及不同离体器官(肝、脾)中Fluc生物发光代表性图片(左)及相关信号定量测定分析(右)(n＝3)；B.不含有DMG-PEG2000脂质组分的PoLixNano制剂与LNP对照制剂经肌肉注射途径(i.m.)给药后在活体小鼠及不同离体器官(肝、脾)中Fluc生物发光代表性图片(左)以及相关Fluc信号定量测定分析结果(右)(n＝3)；C.经肌肉注射途径(i.m.)两次接种(第0天及第21天)RBD-mRNA/PoLixNano制剂与LNP对照制剂后，采用ELISA法检测首次免疫后14天、21天、28天以及35天采集的小鼠血清样品中RBD特异性IgG抗体滴度(n＝3)；D.通过ELIspot方法检测C中所述制剂初免后28天小鼠脾淋巴细胞经RBD重叠肽库刺激后产生的细胞因子IL-4和IL-17斑点数量(n＝3)。Figure 19: Study on the transfection of Fluc-mRNA in mice and the induction of adaptive immune response mediated by PoLixNano preparations after intramuscular injection (i.m.). A. Representative images of Fluc bioluminescence in live mice and different ex vivo organs (liver, spleen) and quantitative analysis of related signals (right) of PoLixNano preparations loaded with Fluc-mRNA and LNP control preparations after intramuscular injection (i.m.) (n=3); B. Representative images of Fluc bioluminescence in live mice and different ex vivo organs (liver, spleen) and quantitative analysis of related Fluc signals after intramuscular injection (i.m.) of PoLixNano preparations without DMG-PEG2000 lipid components and LNP control preparations Measurement and analysis results (right) (n=3); C. After two intramuscular injections (i.m.) (on day 0 and day 21) of the RBD-mRNA/PoLixNano preparation and the LNP control preparation, the ELISA method was used to detect the RBD-specific IgG antibody titer in the mouse serum samples collected 14 days, 21 days, 28 days and 35 days after the first immunization (n=3); D. The ELIspot method was used to detect the number of cytokine IL-4 and IL-17 spots produced by mouse spleen lymphocytes after stimulation with the RBD overlapping peptide library 28 days after the initial immunization with the preparation described in C (n=3).
本文附图中所有统计数据均以平均值±标准差(mean±SD)表示。通过GraphPad Prism 8软件进行统计学分析，并采用双尾t检验分析，ns表示无显著性差异，*p＜0.05，**p＜0.01，***p＜0.001。All statistical data in the figures in this article are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism 8 software and two-tailed t-test analysis. ns indicates no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001.
发明详述DETAILED DESCRIPTION OF THE INVENTION
定义definition
当在权利要求书和/或说明书中结合术语“包含”使用时，词语“一个/种”或“一个/种”的使用可以表示“一个/种”，但它也与“一个/种或多个/种”、“至少一个/种”以及“一个/种或多于一个/种”的含义一致。 When used in conjunction with the term "comprising" in the claims and/or the specification, the use of the word "a" or "an" may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".
贯穿本文，术语“约”用于表示，值包括用于测定所述值的装置、方法的误差的固有变异、或研究受试者之间存在的变异。Throughout this document, the term "about" is used to indicate that a value includes the inherent variation for the device or method employed to determine the value, or the variation that exists between study subjects.
如在本文中使用的，术语“基本上”是指表现出全部或接近全部范围或程度的目标特征或特性的定性条件。生命科学领域的普通技术人员将理解，生物学和化学现象很少(如果曾经有)完成和/或继续完成或达到或避免绝对结果。因此，在本文中使用术语“基本上”来捕获许多生物学和化学现象中固有的潜在完整性缺失。As used herein, the term "substantially" refers to the qualitative condition of exhibiting a target feature or characteristic in full or close to full extent or degree. One of ordinary skill in the life sciences will appreciate that biological and chemical phenomena rarely, if ever, complete and/or proceed to complete or reach or avoid an absolute result. Therefore, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
术语“包含”、“具有”和“包括”是开放式系动词。这些动词中的一个或多个的任何形式或时态，诸如“包含”、“具有”、“包括”也是开放式的。例如，“包含”、“具有”或“包括”一个或多个步骤的任何方法并不限于仅具有那一个或多个步骤，并且还涵盖其它未列出的步骤。The terms "comprising," "having," and "including" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprising," "having," "including," is also open-ended. For example, any method that "comprising," "having," or "including" one or more steps is not limited to having only those one or more steps, and also encompasses other unlisted steps.
当在本说明书和/或权利要求书中使用时，术语“有效的”意指足以实现期望的、预期的或想要的结果。当在用化合物治疗患者或受试者的背景下使用时，“有效量”、“治疗有效量”或“药学有效量”意指所述化合物在对受试者或患者施用以治疗疾病时足以实现对所述疾病的这类治疗的量。As used in this specification and/or claims, the term "effective" means sufficient to achieve a desired, expected, or intended result. When used in the context of treating a patient or subject with a compound, "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" means an amount of the compound that, when administered to a subject or patient for treating a disease, is sufficient to achieve such treatment for the disease.
如本文所用，术语“改善”、“增加”或“减少”或语法等同项表示相对于基线测量值，诸如在本文所述治疗开始之前同一个体的测量值，或在没有本文所述治疗的情况下对照样品或受试者(或多个对照样品或受试者)的测量值的值。“对照样品”是除测试物品之外经受与测试样品相同条件的样品。“对照受试者”是患有与所治受试者相同形式的疾病，其年龄与所治受试者大约相同的受试者。As used herein, the terms "improve," "increase," or "decrease," or grammatical equivalents, refer to values relative to a baseline measurement, such as a measurement of the same individual before the start of a treatment described herein, or a measurement of a control sample or subject (or multiple control samples or subjects) in the absence of a treatment described herein. A "control sample" is a sample subjected to the same conditions as a test sample except for the test article. A "control subject" is a subject having the same form of disease as a treated subject and whose age is about the same as that of the treated subject.
如本文所用，术语“体外”是指在人工环境中，例如在试管或反应容器中，在细胞培养物中等，而不是在多细胞生物体内发生的事件。As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than in a multicellular organism.
如本文所用，术语“体内”是指在多细胞生物体诸如人和非人动物内发生的事件。在基于细胞的系统的上下文中，该术语可用于指在活细胞内发生的事件(与例如体外系统相反)。As used herein, the term "in vivo" refers to events that occur within multicellular organisms such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).
术语“基因产物”用于本文中时，指基因的产物诸如RNA转录物、蛋白质或多肽。The term "gene product" as used herein refers to the product of a gene such as an RNA transcript, protein, or polypeptide.
术语“脂质”是指一组有机化合物，其包括但不仅限于脂肪酸的酯，并且特征是在水中不溶，但是在许多有机溶剂中是可溶的。通常将它们分成至少 三类：(1)“简单脂质”，其包括脂肪和油以及蜡；(2)“化合物脂质”，其包括磷脂和糖脂；和(3)“衍生的脂质”诸如类固醇。The term "lipid" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. They are generally divided into at least Three categories: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derivative lipids" such as steroids.
“脂质颗粒”、“脂质纳米粒”或“LNP”用于本文中时，指可以用于递送活性剂或治疗剂，诸如核酸(例如，mRNA)，至目标靶部位的脂质制剂。在本发明的典型由阳离子脂质、非-阳离子脂质、和防止颗粒聚集的脂质缀合物形成的脂质颗粒中，所述活性剂或治疗剂可以包封在脂质中，由此保护试剂免受酶降解。核酸-脂质颗粒及其制备方法公开在，例如，美国专利公开号20040142025和20070042031，通过引用将其公开内容整体引入本文中用于全部目的。"Lipid granule", "lipid nanoparticle" or "LNP" as used herein refers to a lipid formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a target site. In the lipid granules typically formed by cationic lipids, non-cationic lipids and lipid conjugates that prevent particle aggregation of the present invention, the active agent or therapeutic agent can be encapsulated in a lipid, thereby protecting the agent from enzymatic degradation. Nucleic acid-lipid granules and methods for preparing them are disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and 20070042031, the disclosures of which are incorporated herein by reference for all purposes.
用于本文中时，术语“PoLixNano”指稳定的聚合物-脂质组合物或聚合物-脂质颗粒或负载核酸的聚合物-脂质组合物或负载核酸的聚合物-脂质颗粒。PoLixNano代表由两亲性嵌段共聚物以及脂质(例如，阳离子脂质，非-阳离子脂质，和防止颗粒聚集的脂质缀合物)制成的组合物，其中所述核酸(例如，mRNA,gRNA,siRNA,aiRNA,miRNA,ssDNA,dsDNA,ssRNA,短发夹RNA(shRNA)，dsRNA,或质粒，包括由其转录干扰RNA的质粒)完全或部分包封在脂质中。用于本文中时，术语“PoLixNano”是用于指代包含包封在聚合物-脂质组合物中的核酸(例如，mRNA)的核酸-聚合物-脂质组合物的术语。PoLixNano典型地含有两亲性嵌段共聚物，阳离子脂质，非-阳离子脂质，和脂质缀合物(例如，PEG-脂质缀合物)。另外，核酸，在存在于本发明的聚合物-脂质颗粒和/或脂质颗粒中时，在水溶液中对核酸酶的降解具有抗性。PoLixNano非常有效用于黏膜部位应用，因为它们可以高效穿透黏膜组织部位大量存在的黏液(mucus penetration)，从而将所负载的治疗剂(例如，mRNA)安全递送至目标靶细胞(例如，呼吸道上皮细胞，树突状细胞等)内，最终介导治疗剂在这些黏膜相关部位介导转染基因的表达或靶基因表达的沉默。与此同时，通过使用特定制剂配方，PoLixNano还可以介导治疗剂(例如，mRNA)积聚在远处部位(例如，身体上与施药部位分开的部位)，并且它可以在这些远处部位处介导转染基因的表达或靶基因表达的沉默。When used herein, the term "PoLixNano" refers to a stable polymer-lipid composition or polymer-lipid particles or polymer-lipid compositions of nucleic acid loads or polymer-lipid particles of nucleic acid loads. PoLixNano represents a composition made of amphiphilic block copolymers and lipids (e.g., cationic lipids, non-cationic lipids, and lipid conjugates to prevent particle aggregation), wherein the nucleic acid (e.g., mRNA, gRNA, siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, or plasmid, including plasmids transcribed by it interfering RNA) is completely or partially encapsulated in lipids. When used herein, the term "PoLixNano" is a term for referring to nucleic acid-polymer-lipid compositions comprising nucleic acids (e.g., mRNA) encapsulated in polymer-lipid compositions. PoLixNano typically contains amphiphilic block copolymers, cationic lipids, non-cationic lipids, and lipid conjugates (e.g., PEG-lipid conjugates). In addition, nucleic acids, when present in the polymer-lipid particles and/or lipid particles of the present invention, are resistant to degradation by nucleases in aqueous solutions. PoLixNano is very effective for mucosal applications because they can efficiently penetrate the mucus (mucus penetration) present in large quantities in mucosal tissue sites, thereby safely delivering the loaded therapeutic agent (e.g., mRNA) to the target target cells (e.g., respiratory epithelial cells, dendritic cells, etc.), ultimately mediating the expression of transfected genes or silencing of target gene expression in these mucosal-related sites mediated by the therapeutic agent. At the same time, by using a specific formulation formula, PoLixNano can also mediate the accumulation of therapeutic agents (e.g., mRNA) in distant sites (e.g., sites on the body separated from the site of administration), and it can mediate the expression of transfected genes or silencing of target gene expression at these distant sites.
本发明的聚合物-脂质组合物颗粒(例如，PoLixNano)典型地具有中位直径约40nm-约150nm，约50nm-约150nm，约60nm-约130nm，约70nm-约110nm，或约80nm-约100nm，且基本无毒。 The polymer-lipid composition particles of the invention (e.g., PoLixNano) typically have a median diameter of about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 80 nm to about 100 nm, and are substantially non-toxic.
如本文所用，术语“递送”涵盖局部递送和全身递送。例如，mRNA的递送涵盖其中将mRNA递送至靶组织并且编码的蛋白质或肽在靶组织内表达且保留的情况(也称为“局部分布”或“局部递送”)，以及其中将mRNA递送至靶组织并且编码的蛋白质或肽表达且分泌到患者的循环系统(例如血清)内，并且全身分布且被其他组织吸收的情况(也称为“全身分布”或“全身递送”)。As used herein, the term "delivery" encompasses local delivery and systemic delivery. For example, the delivery of mRNA encompasses situations where the mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and retained in the target tissue (also referred to as "local distribution" or "local delivery"), and situations where the mRNA is delivered to a target tissue and the encoded protein or peptide is expressed and secreted into the patient's circulatory system (e.g., serum), and is distributed throughout the body and absorbed by other tissues (also referred to as "systemic distribution" or "systemic delivery").
“远处部位”用于本文中时，指身体上分隔的部位，其不局限于邻近的毛细管床，而是包括遍及生物体广泛分布的部位。"Distal site" as used herein refers to a separate site in the body that is not limited to adjacent capillary beds but includes sites that are widely distributed throughout the organism.
如本文所用，术语“包封”或语法等同形式是指将单独的mRNA分子限制在聚合物-脂质纳米颗粒内的过程。As used herein, the term "encapsulation" or grammatical equivalents refers to the process of confining individual mRNA molecules within polymer-lipid nanoparticles.
用于本文中时，“包封的”可以指提供完全包封、部分包封、或二者的活性剂或治疗剂，诸如核酸(例如，mRNA)的脂质颗粒。在优选的实施方案中，核酸完全包封在脂质颗粒中(例如，以形成PoLixNano或其他核酸-纳米颗粒)。When used herein, "encapsulated" can refer to providing a fully encapsulated, partially encapsulated, or both active or therapeutic agents, such as lipid particles of nucleic acids (e.g., mRNA). In preferred embodiments, nucleic acids are fully encapsulated in lipid particles (e.g., to form PoLixNano or other nucleic acid-nano particles).
术语“阳离子脂质”指在选定的pH值，诸如生理pH值(例如，pH为约7.0)下携带净正电荷的许多脂质物种中的任何一种。已经意外地发现，包含具有多个不饱和位点，例如，至少2或3个不饱和位点的烷基链的阳离子脂质特别有效用于形成具有增加的膜流动性的脂质颗粒。也有效用于本发明的许多阳离子脂质和相关类似物已经记述在美国专利公开号20060083780和20060240554；美国专利号5,208,036；5,264,618；5,279,833；5,283,185；5,753,613；和5,785,992；和PCT公开号WO 96/10390中，通过引用将其公开内容整体引入本文中用于全部目的。阳离子脂质的非限制性实例详细记述在本文中。在一些情形中，阳离子脂质包含可质子化叔胺(例如，可pH滴定的)头基，C18烷基链，所述头基和烷基链之间的醚键，和0至3个双键。这样的脂质包括，例如，DSDMA,DLinDMA,DLenDMA，和DODMA。The term "cationic lipid" refers to any of a number of lipid species carrying a net positive charge at a selected pH value, such as a physiological pH value (e.g., pH is about 7.0). It has been unexpectedly found that cationic lipids comprising alkyl chains with multiple unsaturated sites, for example, at least 2 or 3 unsaturated sites, are particularly effective for forming lipid particles with increased membrane fluidity. Many cationic lipids and related analogs that are also effective for the present invention have been described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Patent Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96/10390, the disclosure of which is incorporated herein by reference for all purposes. Non-limiting examples of cationic lipids are described in detail herein. In some cases, the cationic lipid comprises a protonatable tertiary amine (e.g., pH titrable) head group, a C 18 alkyl chain, an ether bond between the head group and the alkyl chain, and 0 to 3 double bonds. Such lipids include, for example, DSDMA, DLinDMA, DLenDMA, and DODMA.
术语“两亲性脂质”部分地指任何适合的材料，其中脂质材料的疏水部分定向到疏水相中，而亲水部分定向到水相。亲水性质来自极性或带电基团诸如糖类、磷酸根、羧基、硫酸根、氨基、巯基、硝基、羟基和其它类似基团的存在。疏水性可以通过包含非极性基团而赋予，所述基团包括，但不限于，长链饱和和不饱和脂族烃基和由一个或多个芳族、脂环族或杂环基团取代的这样的基团。两亲性化合物的实例包括，但不限于，磷脂、氨基脂和鞘脂类。The term "amphiphilic lipid" refers in part to any suitable material, wherein the hydrophobic portion of the lipid material is oriented into the hydrophobic phase, while the hydrophilic portion is oriented into the aqueous phase. The hydrophilic nature comes from the presence of polar or charged groups such as sugars, phosphates, carboxyls, sulfates, aminos, sulfhydryls, nitros, hydroxyls and other similar groups. Hydrophobicity can be imparted by the inclusion of non-polar groups, including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbons and such groups substituted by one or more aromatic, alicyclic or heterocyclic groups. Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino lipids and sphingolipids.
磷脂的代表性实例包括，但不限于，磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酸、棕榈酰油酰磷脂酰胆碱、溶血磷脂酰胆碱、溶 血磷脂酰乙醇胺、二棕榈酰磷脂酰胆碱、二油酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱和二亚油酰磷脂酰胆碱。缺乏磷的其它化合物，诸如鞘脂、鞘糖脂家族、二酰甘油和β-酰氧酸也包括在被称为两亲性脂质的组中。另外，上述的两亲性脂质可与其它脂质混和，所述其他脂质包括甘油三酯类和固醇类。Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, Phosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus, such as sphingolipids, sphingolipid families, diacylglycerols and beta-acyloxy acids are also included in the group known as amphipathic lipids. In addition, the above-mentioned amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.
术语“中性脂质”指在选定的pH值下以不带电荷或中性两性离子形式存在的许多脂质物种中的任何一种。在生理pH值下，这样的脂质包括，例如，二酰磷脂酰胆碱、二酰磷脂酰乙醇胺、神经酰胺、鞘磷脂、脑磷脂、胆固醇、脑苷脂和二酰甘油。The term "neutral lipid" refers to any of a number of lipid species that exist as uncharged or neutral zwitterionic forms at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.
术语“非阳离子脂质”指任何两亲性脂质以及任何其他中性脂质或阴离子脂质。The term "non-cationic lipid" refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.
术语“阴离子脂质”指在生理pH值下带负电荷的任何脂质。这些脂质包括，但不限于，磷脂酰甘油、心磷脂、二酰磷脂酰丝氨酸、二酰磷脂酸、N-十二烷酰磷脂酰乙醇胺、N-琥珀酰磷脂酰乙醇胺、N-戊二酰磷脂酰乙醇胺、赖氨酰磷脂酰甘油、棕榈酰油酰磷脂酰甘油(POPG)，和其它与中性脂质连接的阴离子修饰基团。The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.
术语“脂质缀合物”指抑制脂质颗粒聚集的结合脂质。所述脂质缀合物包括，但不仅限于，聚酰胺低聚物(例如，ATTA-脂质缀合物)，聚(乙二醇)-脂质缀合物(PEG-脂质缀合物)，诸如与二烷氧基丙基偶联的PEG，与二酰甘油偶联的PEG，与胆固醇偶联的PEG，与磷脂酰乙醇胺偶联的PEG，与神经酰胺偶联的PEG(参见，例如，美国专利号5,885,613,以及国际专利PCT/US2016/000129，通过引用将其公开内容整体引入本文中用于全部目的)，聚肌氨酸‐脂质缀合物，阳离子PEG脂质，及其混合物。PEG可以与脂质直接缀合或可以通过连接体结构部分与脂质相连。可以使用适合于连接PEG和脂质的任何连接体结构部分，例如不含酯的连接体结构部分和含酯的连接体结构部分。在优选的实施方案中，使用不含酯的连接体结构部分。The term "lipid conjugate" refers to a bound lipid that inhibits aggregation of lipid particles. The lipid conjugate includes, but is not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), poly (ethylene glycol) -lipid conjugates (PEG-lipid conjugates), such as PEG coupled to a dialkoxypropyl group, PEG coupled to diacylglycerol, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, PEG coupled to ceramide (see, e.g., U.S. Patent No. 5,885,613, and International Patent PCT/US2016/000129, the disclosure of which is incorporated herein by reference for all purposes), polysarcosine-lipid conjugates, cationic PEG lipids, and mixtures thereof. PEG can be directly conjugated to a lipid or can be connected to a lipid via a linker moiety. Any linker moiety suitable for connecting PEG and a lipid can be used, such as a linker moiety without an ester and a linker moiety containing an ester. In a preferred embodiment, a linker moiety without an ester is used.
如本文所用，术语“N/P比”是指聚合物-脂质组合物中的阳离子脂质中的带正电的分子单元相对于包封于聚合物-脂质组合物内的mRNA中的带负电的分子单元的摩尔比。因此，N/P比通常以聚合物-脂质组合物中的阳离子脂质中的胺基团的摩尔数相对于包封于聚合物-脂质组合物内的mRNA中的磷酸基团的摩尔数之比计算。 As used herein, the term "N/P ratio" refers to the molar ratio of the positively charged molecular units in the cationic lipids in the polymer-lipid composition relative to the negatively charged molecular units in the mRNA encapsulated in the polymer-lipid composition. Therefore, the N/P ratio is usually calculated as the ratio of the molar number of amine groups in the cationic lipids in the polymer-lipid composition relative to the molar number of phosphate groups in the mRNA encapsulated in the polymer-lipid composition.
术语“疏水性脂质”指具有非极性基团的化合物，所述基团包括，但不限于，长链饱和和不饱和脂族坯基团和任选由一个或多个芳族、脂环族或杂环基团取代的这样的基团。合适的实例包括，但不仅限于，二酰甘油，二烷基甘油，N-N-二烷基氨基,1,2-二酰氧基-3-氨基丙烷，和1,2-二烷基-3-氨基丙烷。The term "hydrophobic lipid" refers to a compound having a non-polar group, including, but not limited to, long chain saturated and unsaturated aliphatic groups and such groups optionally substituted by one or more aromatic, alicyclic or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerols, dialkylglycerols, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.
术语“融合的”指聚合物脂质颗粒，诸如PoLixNano与细胞膜融合的能力。所述膜可以是质膜或围绕细胞器的膜，所述细胞器例如，内体、溶酶体、细胞核等。The term "fusogenic" refers to the ability of polymer lipid particles, such as PoLixNano, to fuse with a cell membrane. The membrane may be a plasma membrane or a membrane surrounding a cell organelle, such as an endosome, a lysosome, a cell nucleus, or the like.
用于本文中时，术语“水溶液”指全部或部分地包含水的组合物。As used herein, the term "aqueous solution" refers to a composition comprising, in whole or in part, water.
用于本文中时，术语“有机脂质溶液”指全部或部分地包含具有脂质的有机溶剂的组合物。As used herein, the term "organic lipid solution" refers to a composition comprising in whole or in part an organic solvent with lipids.
第一化合物的“异构体”是这样的单独化合物：其中每个分子含有与所述第一化合物相同的组分原子，但其中那些原子的三维构型不同。"Isomers" of a first compound are individual compounds wherein each molecule contains the same constituent atoms as the first compound, but wherein the three-dimensional configuration of those atoms is different.
本文中使用的术语“患者”或“受试者”表示活的哺乳动物生物体，诸如人、猴、牛、绵羊、山羊、狗、猫、小鼠、大鼠、豚鼠或其转基因物种。在某些实施方案中，所述患者或受试者是灵长类动物。人类受试者的非限制性例子是成人、青少年、婴儿和胎儿。As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects are adults, adolescents, infants, and fetuses.
本文中普遍使用的“药学上可接受的”表示这样的化合物、材料、组合物和/或剂型：其在合理的医学判断范围内适合用于与人类和动物的组织、器官和/或体液接触，而没有过度的毒性、刺激、变应性应答或其它问题或并发症，与合理的收益/风险比相称。As used generally herein, "pharmaceutically acceptable" refers to compounds, materials, compositions and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs and/or body fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
“药学上可接受的盐”是指如上文所定义的药学上可接受的并且具有期望的药理学活性的本公开内容的化合物的盐。这样的盐包括与以下酸形成的酸加成盐：无机酸诸如盐酸、氢溴酸、硫酸、硝酸、磷酸等；或有机酸诸如1,2‐乙烷二磺酸、2‐羟基乙磺酸、2‐萘磺酸、3‐苯基丙酸、4,4′‐亚甲基双(3‐羟基‐2‐烯‐1‐甲酸)、4‐甲基二环[2.2.2]辛‐2‐烯‐1‐甲酸、乙酸、脂族单和二羧酸、脂族硫酸、芳族硫酸、苯磺酸、苯甲酸、樟脑磺酸、碳酸、肉桂酸、柠檬酸、环戊烷丙酸、乙磺酸、富马酸、葡萄庚糖酸、葡糖酸、谷氨酸、羟乙酸、庚酸、己酸、羟基萘甲酸、乳酸、月桂基硫酸、马来酸、苹果酸、丙二酸、扁桃酸、甲磺酸、粘康酸、邻‐(4‐羟基苯甲酰基)苯甲酸、草酸、对氯苯磺酸、苯基‐取代的链烷酸、丙酸、对甲苯磺酸、丙酮酸、水杨酸、硬脂酸、琥珀酸、酒石酸、叔丁基乙酸、三甲基乙酸等。药学上可接受的盐还包括在存在的酸性质子能 够与无机碱或有机碱反应时可以形成的碱加成盐。可接受的无机碱包括氢氧化钠、碳酸钠、氢氧化钾、氢氧化铝和氢氧化钙。可接受的有机碱包括乙醇胺、二乙醇胺、三乙醇胺、氨丁三醇、N‐甲基葡糖胺等。应当认识到，形成本公开内容的任何盐的一部分的特定阴离子或阳离子不是至关重要的，只要所述盐作为整体是药理学上可接受的即可。药学上可接受的盐的其它例子和它们的制备方法与使用方法呈现于Handbook of Pharmaceutical Salts:Properties,and Use(P.H.Stahl&C.G.Wermuth编，Verlag Helvetica Chimica Acta,2002)。"Pharmaceutically acceptable salt" refers to a salt of a compound of the present disclosure that is pharmaceutically acceptable as defined above and that has the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid , cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, caproic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfate, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, trimethylacetic acid, etc. Pharmaceutically acceptable salts also include those in which the acidic protons present are capable of The base addition salt that can be formed when reacting with an inorganic base or an organic base. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. It should be recognized that the specific anion or cation that forms a part of any salt of the present disclosure is not critical, as long as the salt is pharmacologically acceptable as a whole. Other examples of pharmaceutically acceptable salts and their preparation methods and methods of use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (PH Stahl & C.G. Wermuth, Verlag Helvetica Chimica Acta, 2002).
本文中使用的术语“药学上可接受的载体”是指参与运载或运输药剂的药学上可接受的材料、组合物或媒介物，诸如液体或固体填充剂、稀释剂、赋形剂、溶剂或包囊材料。本文中使用的术语“赋形剂”包括但不限于所要特定剂型的任何及所有溶剂、分散介质或其他液体媒介、分散或助悬剂、稀释剂、造粒剂及/或分散剂、表面活性剂、等渗剂、增稠剂或乳化剂、防腐剂、黏合剂、润滑剂或著色剂、甜味剂到或调味剂、稳定剂、抗氧化剂、抗微生物剂或抗真菌剂、渗透压调节剂、pH调节剂、缓冲剂、螯合剂、冷保保护剂及/或增塑剂。用于调配药物制剂组成物的各种赋形剂及用于制备借该组成物的技术为此项技术中所已知(参见Remington:The Science and Practice f Pharmacy,第21版AR.Gennaro(Lippincott,Williams&Wilkins,Baltimore,MD,2006；该文以全文引用的方式并入本文中)。The term "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition or vehicle involved in carrying or transporting a medicament, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. The term "excipient" as used herein includes, but is not limited to, any and all solvents, dispersion media or other liquid vehicles, dispersing or suspending agents, diluents, granulating and/or dispersing agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, binders, lubricants or coloring agents, sweeteners or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmotic pressure regulators, pH regulators, buffers, chelating agents, cold protection agents and/or plasticizers for a particular dosage form. Various excipients used to formulate pharmaceutical preparation compositions and techniques for preparing such compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st edition, AR. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; which is incorporated herein by reference in its entirety).
“预防”包括：(1)抑制受试者或患者的疾病发作，所述受试者或患者可能有患所述疾病的风险和/或易患所述疾病，但尚未经历或表现出所述疾病的任何或所有病状或征状；和/或(2)减慢受试者或患者中的疾病的病状或征状发作，所述受试者或患者可能有患所述疾病的风险和/或易患所述疾病，但尚未经历或表现出所述疾病的任何或所有病状或征状。"Prevention" includes: (1) inhibiting the onset of a disease in a subject or patient who may be at risk for and/or susceptible to the disease but does not yet experience or display any or all of the symptoms or signs of the disease; and/or (2) slowing the onset of symptoms or signs of a disease in a subject or patient who may be at risk for and/or susceptible to the disease but does not yet experience or display any or all of the symptoms or signs of the disease.
“治疗”包括(1)抑制正经历或表现出疾病的病状或征状的受试者或患者中的所述疾病(例如，阻止所述病状和/或征状的进一步发展)，(2)改善正经历或表现出疾病的病状或征状的受试者或患者中的所述疾病(例如，逆转所述病状和/或征状)，和/或(3)实现正经历或表现出疾病的病状或征状的受试者或患者中的所述疾病的任何可测量的减轻。"Treating" includes (1) inhibiting the disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., arresting further development of the symptoms and/or signs), (2) ameliorating the disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease (e.g., reversing the symptoms and/or signs), and/or (3) achieving any measurable reduction in the disease in a subject or patient who is experiencing or exhibiting symptoms or signs of the disease.
“重复单元”是某些材料的最简单的结构实体，例如，框架和/或聚合物，无论是有机的、无机的还是金属有机的。在聚合物链的情况下，重复单元沿着链依次连接在一起，就像项链的珠子一样。例如，在聚乙烯‐[‐CH2CH2‐]n‐中， 重复单元是‐CH2CH2‐。下标“n”表示聚合度，也就是说，连接在一起的重复单元的数量。当“n”的值未定义时或在“n”不存在的情况下，它只是指示括号内该式的重复以及材料的聚合性质。重复单元的概念同样适用于重复单元之间的连接性三维地延伸的场合，诸如在金属有机框架、改性聚合物、热固性聚合物等中。在树枝状聚合物的上下文中，重复单元也可以被描述为分支单元、内层或代。类似地，封端基团也可以被描述为表面基团。A "repeat unit" is the simplest structural entity of some material, e.g., a framework and/or a polymer, whether organic, inorganic, or metallo-organic. In the case of a polymer chain, the repeat units are linked together one after another along the chain, like the beads of a necklace. For example, in polyethylene -[-CH 2 CH 2 -] n -, The repeating unit is -CH2CH2- . The subscript "n" indicates the degree of polymerization, that is, the number of repeating units linked together. When the value of "n" is undefined or in the absence of "n", it simply indicates the repetition of the formula within the brackets and the polymeric nature of the material. The concept of repeating unit also applies where the connectivity between repeating units extends three-dimensionally, such as in metal organic frameworks, modified polymers, thermosetting polymers, etc. In the context of dendrimers, repeating units may also be described as branching units, inner layers, or generations. Similarly, end-capping groups may also be described as surface groups.
以上定义替代在通过引用并入本文的任何参考文献中的任何冲突的定义。但是，对某些术语加以定义的事实不应当被认为表示未被定义的任何术语是不确定的。相反，所使用的所有术语均被认为以使得普通技术人员可以明白范围并实施本公开内容的方式描述本公开内容。The above definitions supersede any conflicting definitions in any references incorporated herein by reference. However, the fact that certain terms are defined should not be taken to mean that any term not defined is undefined. Instead, all terms used are considered to describe the present disclosure in a manner that allows a person of ordinary skill to understand the scope and practice the present disclosure.
本发明涵盖以下意外发现，即向LNP制剂(例如：由可电离/阳离子脂质、中性脂质、胆固醇以及PEG-脂质组成的四组分LNP制剂；由可电离/阳离子脂质、中性脂质以及胆固醇组成的三组分LNP制剂；包含胆固醇衍生的阳离子脂质、中性脂质以及PEG脂质的三组分LNP制剂；包含胆固醇衍生的阳离子脂质以及中性脂质的两组分LNP制剂)中添加两亲性嵌段共聚物例如洛沙姆(poloxamine或)和/或泊洛沙胺(poloxamer或)组分后，所获得的新型复合制剂(本文中称为包含聚合物-脂质的组合物或PoLixNano)经生物体黏膜部位途径特别是呼吸道途径(气管喷雾i.t.或滴鼻i.n.或雾化吸入)给药后，能够大幅度提升这类制剂介导核酸分子(如mRNA)在动物体内(尤其是呼吸道组织相关细胞内)的基因转染效率，使核酸分子能够在动物呼吸道相关组织以及非肺细胞/组织高效产生功能性蛋白质，从而实现疾病预防或治疗的目的。The present invention encompasses the unexpected discovery that the addition of amphiphilic block copolymers such as poloxamine or styrene to LNP formulations (e.g., four-component LNP formulations consisting of ionizable/cationic lipids, neutral lipids, cholesterol, and PEG-lipids; three-component LNP formulations consisting of ionizable/cationic lipids, neutral lipids, and cholesterol; three-component LNP formulations comprising cholesterol-derivatized cationic lipids, neutral lipids, and PEG lipids; two-component LNP formulations comprising cholesterol-derivatized cationic lipids and neutral lipids) can enhance the performance of the LNP formulations. ) and/or poloxamer or ) components, the obtained novel composite preparation (referred to herein as a composition comprising polymer-lipid or PoLixNano) can greatly improve the gene transfection efficiency of nucleic acid molecules (such as mRNA) mediated by such preparations in animals (especially in cells related to respiratory tissues) after administration via the mucosal site of an organism, especially the respiratory tract (tracheal spray IT or nasal drops IN or atomized inhalation), so that the nucleic acid molecules can efficiently produce functional proteins in animal respiratory tract-related tissues and non-lung cells/tissues, thereby achieving the purpose of disease prevention or treatment.
聚合物-脂质制剂特别适用于经由非侵入给药途径(例如气管喷雾i.t.、滴鼻i.n.、以及雾化吸入等)治疗或预防呼吸道组织相关的疾病。装载mRNA的聚合物-脂质组合物经呼吸道途径给药后，在动物肺部/呼吸道黏膜组织产生的基因转染效果显著优于经典的LNP制剂、poloxamine以及poloxamer制剂。经由呼吸道途径递送的mRNA有望在黏膜型疫苗(例如新冠病毒疫苗、流感病毒疫苗、肺结核疫苗等)、肺部基因治疗(如囊性纤维化、α1-胰蛋白酶缺乏症等)、肺部难治疾病(如肺癌、肺纤维化、哮喘、慢性阻塞性肺病等)领域中取得突破，解决这些领域中目前尚无理想递送系统的难题。 Polymer-lipid preparations are particularly suitable for treating or preventing diseases related to respiratory tissue via non-invasive routes of administration (e.g., tracheal spray it, nasal drops in, and atomized inhalation, etc.). After the polymer-lipid composition loaded with mRNA is administered via the respiratory route, the gene transfection effect produced in the lung/respiratory mucosal tissue of animals is significantly better than that of classic LNP preparations, poloxamine and poloxamer preparations. mRNA delivered via the respiratory route is expected to make breakthroughs in the fields of mucosal vaccines (e.g., new coronavirus vaccines, influenza virus vaccines, tuberculosis vaccines, etc.), lung gene therapy (e.g., cystic fibrosis, α1-trypsin deficiency, etc.), and refractory lung diseases (e.g., lung cancer, pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, etc.), solving the problem that there is currently no ideal delivery system in these fields.
其次，经由呼吸道途径递送特定配方的聚合物-脂质纳米制剂，能够在肺部递送之后易位，即，通过主动或被动的方式从给药部位(肺部)部分或完整地移动至全身血液供给，并随后沉积于不同非肺细胞或组织，例如，肝脏或/和脾脏中。包含编码治疗性蛋白(如囊性纤维跨膜电导调节因子CFTR，抗α1-胰蛋白酶等)或特定抗原(如肿瘤特异性抗原，新冠病毒刺突蛋白等)的mRNA的聚合物-脂质组合物的此转运构成活性成分(即mRNA)在肺部以外的非侵入性全身递送，导致为全身可接近的非肺细胞或组织产生功能性蛋白质。而在相同条件下施用的LNP制剂或poloxamine或poloxamer制剂，其主要转染器官仅限于肺部。Secondly, polymer-lipid nanoformulations of specific formulations delivered via the respiratory route can be translocated after pulmonary delivery, that is, partially or completely moved from the site of administration (lungs) to the systemic blood supply by active or passive means, and then deposited in different non-lung cells or tissues, for example, the liver and/or spleen. This transport of polymer-lipid compositions containing mRNA encoding therapeutic proteins (such as cystic fibrosis transmembrane conductance regulator CFTR, anti-α1-trypsin, etc.) or specific antigens (such as tumor-specific antigens, new coronavirus spike proteins, etc.) constitutes non-invasive systemic delivery of active ingredients (i.e., mRNA) outside the lungs, resulting in the production of functional proteins in non-lung cells or tissues accessible to the whole body. However, the main transfection organs of LNP preparations or poloxamine or poloxamer preparations administered under the same conditions are limited to the lungs.
相应地，在一方面，本发明提供了一种包含聚合物-脂质的组合物，所述组合物包含：Accordingly, in one aspect, the present invention provides a composition comprising a polymer-lipid, the composition comprising:
(A)活性剂或治疗剂，优选所述活性剂或治疗剂包含核酸；(A) an active agent or therapeutic agent, preferably the active agent or therapeutic agent comprises a nucleic acid;
(B)两亲性嵌段共聚物；(B) amphiphilic block copolymers;
(C)阳离子脂质；和(C) a cationic lipid; and
(D)非阳离子脂质，(D) non-cationic lipids,
其中所述组合物配制为用于通过生物体黏膜部位(Mucosa)递送，例如呼吸道递送、口腔黏膜递送、胃肠道递送、眼部黏膜递送、耳部黏膜递送、尿道递送、或生殖道递送，优选所述组合物配制为用于通过呼吸道递送。Wherein the composition is formulated for delivery through a mucosal site of an organism, such as the respiratory tract, oral mucosa, gastrointestinal tract, ocular mucosa, ear mucosa, urethra, or reproductive tract, preferably the composition is formulated for delivery through the respiratory tract.
核酸Nucleic Acids
根据本发明的组合物优选可以包含核糖核酸(RNA)，例如单链RNA，更优选是信使RNA(mRNA)，例如体外转录mRNA。The composition according to the present invention may preferably comprise ribonucleic acid (RNA), such as single-stranded RNA, more preferably messenger RNA (mRNA), such as in vitro transcribed mRNA.
在一些实施方案中，所述核酸包含选自以下的至少一种：信使RNA(mRNA)、自我扩增RNA(saRNA)、环状RNA(circRNA)、小干扰RNA(siRNA)、短发夹RNA(shRNA)和微小RNA(miRNA)、初级‐miRNA、反义寡核苷酸(ASO)、转运RNA(tRNA)、质粒DNA(pDNA)、单链DNA(ssDNA)、双链DNA(dsDNA)、脱氧核酶(DNAzyme)、核酶(RNAzyme)、核酸适配体(aptamer)、成簇的规律间隔的短回文重复序列(CRISPR)相关的核酸、单指导RNA(sgRNA)、CRISPR‐RNA(crRNA)、反式活化crRNA(tracrRNA)、指导RNA、单链RNA(ssRNA)和双链RNA(dsRNA)。在一些实施方案中，所述核酸是治疗性核酸。更优选的，所述核酸包含mRNA。 In some embodiments, the nucleic acid comprises at least one selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) and micro RNA (miRNA), primary-miRNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), plasmid DNA (pDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), deoxyribozyme (DNAzyme), ribozyme (RNAzyme), nucleic acid aptamer (aptamer), clustered regularly interspaced short palindromic repeats (CRISPR)-related nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA, single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA). In some embodiments, the nucleic acid is a therapeutic nucleic acid. More preferably, the nucleic acid comprises mRNA.
在一些实施方案中，所述组合物包含第一核酸和第二核酸。在一些实施方案中，所述第一核酸是mRNA。在一些实施方案中，所述第二核酸是单指导RNA。在一些实施方案中，所述第一核酸是信使RNA(mRNA)和单指导RNA(sgRNA)。In some embodiments, the composition comprises a first nucleic acid and a second nucleic acid. In some embodiments, the first nucleic acid is an mRNA. In some embodiments, the second nucleic acid is a single guide RNA. In some embodiments, the first nucleic acid is a messenger RNA (mRNA) and a single guide RNA (sgRNA).
原则上任何类型的RNA都可以在本发明的背景下采用。在优选的实施方案中，RNA是单链RNA。术语“单链RNA”意为单条连续的核糖核苷酸链，其区分于由两条或更多条分开的链杂交形成的双链分子的RNA。术语“单链RNA”不排除单链分子本身形成双链结构，如二级(例如环和茎环)结构或三级结构。In principle, any type of RNA can be used in the context of the present invention. In a preferred embodiment, the RNA is a single-stranded RNA. The term "single-stranded RNA" means a single continuous ribonucleotide chain, which is distinguished from RNA of a double-stranded molecule formed by hybridization of two or more separated chains. The term "single-stranded RNA" does not exclude that the single-stranded molecule itself forms a double-stranded structure, such as a secondary (e.g., ring and stem-loop) structure or a tertiary structure.
术语“RNA”涵盖编码氨基酸序列的RNA以及不编码氨基酸序列的RNA。RNA可以通过本领域普通技术人员已知的合成化学和酶促方法制备，或者通过使用重组技术来制备，或者可以从天然来源分离，或者通过其组合来制备。The term "RNA" encompasses RNAs that encode amino acid sequences as well as RNAs that do not encode amino acid sequences. RNA can be prepared by synthetic chemistry and enzymatic methods known to those of ordinary skill in the art, or by using recombinant technology, or can be isolated from natural sources, or by a combination thereof.
信使RNA(mRNA)是由磷酸核苷构造单元构造的共聚物，主要以腺苷、胞苷、尿苷和鸟苷为核苷，其作为中间载体将遗传信息从细胞核中的DNA引入细胞质中，在细胞质中翻译成蛋白质。因此，mRNA适合作为基因表达的替代物。Messenger RNA (mRNA) is a copolymer constructed from phosphate nucleoside building blocks, mainly adenosine, cytidine, uridine and guanosine, which acts as an intermediate carrier to introduce genetic information from DNA in the cell nucleus into the cytoplasm, where it is translated into protein. Therefore, mRNA is suitable as a substitute for gene expression.
在本发明的上下文中，mRNA应被理解为意指任意的多核糖核苷酸分子，如果其进入细胞，则适合蛋白质或其片段的表达，或者能够翻译成蛋白质或其片段。术语“蛋白质”在本文中涵盖任意种类的氨基酸序列，即两个或更多个各自通过肽键连接的氨基酸的链，并且还包括肽和融合蛋白。In the context of the present invention, mRNA should be understood to mean any polyribonucleotide molecule, if it enters a cell, is suitable for protein or its fragmentary expression, or can be translated into protein or its fragment.Term " protein " covers any kind of amino acid sequence in this article, i.e. two or more amino acid whose chains connected by peptide bonds separately, and also comprises peptide and fusion protein.
mRNA含有核糖核苷酸序列，该核糖核苷酸序列编码在细胞中或细胞附近需要的或有益的功能的蛋白质或其片段。mRNA可以含有完整蛋白质或其功能性变体的序列。进一步，核糖核苷酸序列可以编码充当因子、诱导物、调节剂、刺激剂或酶的蛋白质或其功能性片段，其中此类蛋白质是其功能对于弥补障碍(特别是代谢障碍)或者启动体内过程(如新血管、组织等的形成)或诱导免疫系统产生适应性免疫应答所必需的蛋白质。本文中，功能性变体意指以下片段：其在细胞中可以承担蛋白质的功能，该蛋白质的功能在细胞中是需要的，或者该蛋白质的缺失或缺陷的形式是致病性的。mRNA contains a ribonucleotide sequence, which encodes a protein or a fragment thereof of a function needed or beneficial in or near a cell. mRNA may contain a sequence of a complete protein or its functional variant. Further, a ribonucleotide sequence may encode a protein or its functional fragment acting as a factor, an inducer, a regulator, a stimulant or an enzyme, wherein such a protein is a protein necessary for its function to compensate for an obstacle (particularly a metabolic disorder) or to initiate a process in the body (such as the formation of new blood vessels, tissues, etc.) or to induce the immune system to produce an adaptive immune response. Herein, a functional variant means the following fragment: it can assume the function of a protein in a cell, the function of the protein is needed in a cell, or the form of the absence or defect of the protein is pathogenic.
通常，mRNA合成包括在5’末端上添加“帽”和在3’末端上添加“尾”。帽的存在对于提供对存在于大多数真核细胞中的核酸酶的抗性是重要的。“尾巴”的存在用于保护mRNA免受核酸外切酶降解。因此，在一些实施方案中， mRNA包括5’帽结构。在一些实施方案中，mRNA包括5’和/或3’非翻译区。在一些实施方案中，5’非翻译区包括一个或多个影响mRNA的稳定性或翻译的元件。在一些实施方案中，3’非翻译区包括一个或多个聚腺苷酸化信号，影响mRNA在细胞中定位稳定性的蛋白质结合位点，或一个或多个miRNA结合位点。如上所述，除非在具体上下文中另外指出，否则本文所用的术语mRNA涵盖修饰的mRNA，即mRNA可以是修饰的mRNA。Typically, mRNA synthesis includes the addition of a "cap" on the 5' end and a "tail" on the 3' end. The presence of the cap is important for providing resistance to nucleases present in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from exonuclease degradation. Thus, in some embodiments, The mRNA includes a 5' cap structure. In some embodiments, the mRNA includes a 5' and/or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect the stability or translation of the mRNA. In some embodiments, the 3' untranslated region includes one or more polyadenylation signals, protein binding sites that affect the localization stability of the mRNA in the cell, or one or more miRNA binding sites. As described above, unless otherwise indicated in the specific context, the term mRNA used herein encompasses modified mRNA, i.e., the mRNA can be a modified mRNA.
本发明可以用于调配和包封未经修饰的mRNA或者含有通常增强稳定性的一个或多个修饰的mRNA。在一些实施方案中，修饰选自经修饰的核苷酸、经修饰的糖磷酸骨架以及5’和/或3’非翻译区。The present invention can be used to formulate and encapsulate unmodified mRNA or mRNA containing one or more modifications that generally enhance stability. In some embodiments, the modification is selected from modified nucleotides, modified sugar-phosphate backbones, and 5' and/or 3' untranslated regions.
在一些实施方案中，mRNA的修饰可以包括RNA的核苷酸的修饰。根据本发明的经修饰的mRNA可以包括例如主链修饰、糖修饰、磷酸基修饰或碱基修饰。在一些实施方案中，mRNA可以从天然存在的核苷酸和/或核苷酸类似物(经修饰的核苷酸)合成，所述天然存在的核苷酸和/或核苷酸类似物包括但不限于嘌呤(腺嘌呤(A)、鸟嘌呤(G))或嘧啶(胸腺嘧啶(T)、胞嘧啶(C)、尿嘧啶(U))以及嘌呤和嘧啶的经修饰的核苷酸类似物或衍生物，此类类似物的制备是本领域的技术人员已知的，例如来自美国专利号4,373,071、美国专利号4,401,796、美国专利号4,415,732、美国专利号4,458,066、美国专利号4,500,707、美国专利号4,668,777、美国专利号4,973,679、美国专利号5,047,524、美国专利号5,132,418、美国专利号5,153,319、美国专利号5,262,530和5,700,642，这些专利的全部公开内容以引用的方式包括于本文中。In some embodiments, the modification of mRNA may include modification of nucleotides of RNA. The modified mRNA according to the present invention may include, for example, backbone modifications, sugar modifications, phosphate modifications, or base modifications. In some embodiments, mRNA may be synthesized from naturally occurring nucleotides and/or nucleotide analogs (modified nucleotides), the naturally occurring nucleotides and/or nucleotide analogs including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)) and modified nucleotide analogs or derivatives of purines and pyrimidines, the preparation of such analogs being known to those skilled in the art, for example, from U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642, the entire disclosures of which are incorporated herein by reference.
对于根据本发明的RNA优选mRNA，所有尿苷核苷酸和胞苷核苷酸都可以各自以相同的形式修饰，或者修饰的核苷酸的混合物可以用于每一种。修饰的核苷酸可以具有天然或非天然存在的修饰。可以使用各种修饰的核苷酸的混合物。For RNA, preferably mRNA, according to the present invention, all uridine nucleotides and cytidine nucleotides can be modified in the same form, or a mixture of modified nucleotides can be used for each. The modified nucleotides can have natural or non-naturally occurring modifications. Mixtures of various modified nucleotides can be used.
在本发明的一个实施方案中，一种类型的核苷酸使用至少两种不同的修饰，其中一种类型的修饰的核苷酸具有官能团，通过该官能团可以连接其它基团。也可以使用具有不同官能团的核苷酸，以提供用于连接不同基团的结合位点。In one embodiment of the invention, one type of nucleotide uses at least two different modifications, wherein the modified nucleotide of one type has a functional group through which other groups can be connected. Nucleotides with different functional groups can also be used to provide binding sites for connecting different groups.
在优选的实施方案中，根据本发明的RNA优选mRNA的特征在于，修饰的尿苷选自2-硫代尿苷、5-甲基尿苷、假尿苷(ψ)、5-甲基尿苷5’-三磷酸(m5U)、N-1‐甲基‐假尿苷(N1mΨ)、N-1‐甲基‐假尿苷-三磷酸、2-硫代尿苷5’-三磷酸 (S2U)、5-碘尿苷5’-三磷酸(I5U)、4-硫代尿苷5’-三磷酸(S4U)、5-溴尿苷5’-三磷酸(Br5U)、2’-甲基-2’-脱氧尿苷5’-三磷酸(U2’m)、2’-氨基-2’-脱氧尿苷5’-三磷酸(U2’NH2)、2’-叠氮基-2’-脱氧尿苷5’-三磷酸(U2’N3)和2’-氟-2’-脱氧尿苷5’-三磷酸(U2’F)。In a preferred embodiment, the RNA, preferably mRNA, according to the invention is characterized in that the modified uridine is selected from the group consisting of 2-thiouridine, 5-methyluridine, pseudouridine (ψ), 5-methyluridine 5'-triphosphate (m5U), N-1-methyl-pseudouridine (N1mΨ), N-1-methyl-pseudouridine-triphosphate, 2-thiouridine 5'-triphosphate (S2U), 5-iodouridine 5'-triphosphate (I5U), 4-thiouridine 5'-triphosphate (S4U), 5-bromouridine 5'-triphosphate (Br5U), 2'-methyl-2'-deoxyuridine 5'-triphosphate (U2'm), 2'-amino-2'-deoxyuridine 5'-triphosphate ( U2'NH2 ), 2'-azido-2'-deoxyuridine 5'-triphosphate ( U2'N3 ), and 2'-fluoro-2'-deoxyuridine 5'-triphosphate (U2'F).
在另一优选的实施方案中，根据本发明的RNA，优选mRNA的特征在于，修饰的胞苷选自5-甲基胞苷、5-羟甲基胞苷、5-甲氧基胞苷、3-甲基胞苷、2-硫-胞苷、2’-甲基-2’-脱氧胞苷5’-三磷酸(C2’m)、2’-氨基-2’-脱氧胞苷5’-三磷酸(C2’NH2)、2’-氟-2’-脱氧胞苷5’-三磷酸(C2’F)、5-碘胞苷5’-三磷酸(I5C)、5-溴胞苷5’-三磷酸(Br5C)、5-甲基胞苷5’-三磷酸(m5C)、2-硫胞苷5’-三磷酸(S2C)和2’-叠氮基-2’-脱氧胞苷5’-三磷酸(C2’N3)。In another preferred embodiment, the RNA, preferably mRNA, according to the invention is characterized in that the modified cytidine is selected from the group consisting of 5-methylcytidine, 5-hydroxymethylcytidine, 5-methoxycytidine, 3-methylcytidine, 2-thio-cytidine, 2'-methyl-2'-deoxycytidine 5'-triphosphate (C2'm), 2'-amino-2'-deoxycytidine 5'-triphosphate (C2'NH 2 ), 2'-fluoro-2'-deoxycytidine 5'-triphosphate (C2'F), 5-iodocytidine 5'-triphosphate (I5C), 5-bromocytidine 5'-triphosphate (Br5C), 5-methylcytidine 5'-triphosphate (m5C), 2-thiocytidine 5'-triphosphate (S2C) and 2'-azido-2'-deoxycytidine 5'-triphosphate (C2'N 3 ).
在另一优选的实施方案中，根据本发明的RNA，优选mRNA的特征在于，修饰的腺苷选自N6-甲基腺苷5’-三磷酸(m6A)、N1-甲基腺苷5’-三磷酸(m1A)、2’-O-甲基腺苷5’-三磷酸(A2’m)、2’-氨基-2’-脱氧腺苷5’-三磷酸(A2’NH2)、2’-叠氮基-2’-脱氧腺苷5’-三磷酸(A2’N3)以及2’-氟-2’-脱氧腺苷5’-三磷酸(A2’F)。In another preferred embodiment, the RNA, preferably mRNA, according to the invention is characterized in that the modified adenosine is selected from the group consisting of N6-methyladenosine 5'-triphosphate (m6A), N1-methyladenosine 5'-triphosphate (m1A), 2'-O-methyladenosine 5'-triphosphate (A2'm), 2'-amino-2'-deoxyadenosine 5'-triphosphate (A2'NH 2 ), 2'-azido-2'-deoxyadenosine 5'-triphosphate (A2'N 3 ) and 2'-fluoro-2'-deoxyadenosine 5'-triphosphate (A2'F).
在另一优选的实施方案中，根据本发明的RNA，优选mRNA的特征在于，修饰的鸟苷选自N1-甲基鸟苷5-三磷酸(m1G)、2’-O-甲基鸟苷5’-三磷酸(G2’m)、2-氨基-2-脱氧鸟苷5’-三磷酸(G2’NH2)、2’-叠氮基-2’-脱氧鸟苷5’-三磷酸(G2’N3)、2’-氟-2’-脱氧鸟苷5’-三磷酸(G2’F)。In another preferred embodiment, the RNA, preferably mRNA, according to the invention is characterized in that the modified guanosine is selected from N1-methylguanosine 5-triphosphate (m1G), 2'-O-methylguanosine 5'-triphosphate (G2'm), 2-amino-2-deoxyguanosine 5'-triphosphate (G2'NH 2 ), 2'-azido-2'-deoxyguanosine 5'-triphosphate (G2'N 3 ), 2'-fluoro-2'-deoxyguanosine 5'-triphosphate (G2'F).
教导某些上述经修饰的核苷碱基以及其它经修饰的核苷碱基的制备的代表性美国专利包括、但不限于美国专利3,687,808；4,845,205；5,130,302；5,134,066；5,175,273；5,367,066；5,432,272；5,457,187；5,459,255；5,484,908；5,502,177；5,525,711；5,552,540；5,587,469；5,594,121；5,596,091；5,614,617；5,645,985；5,681,941；5,750,692；5,763,588；5,830,653和6,005,096，它们中的每一篇通过引用整体并入本文。Representative U.S. patents that teach the preparation of some of the above-mentioned modified nucleoside bases, as well as other modified nucleoside bases, include, but are not limited to, U.S. Patents 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, each of which is incorporated herein by reference in its entirety.
在一些实施方案中，本发明提供了包含连接的核苷的寡核苷酸。在这样的实施方案中，核苷可以使用任何核苷间键连接在一起。核苷间连接基团的两个主要类别由磷原子的存在或不存在限定。与天然磷酸二酯键相比，经修饰的键可以用于改变(通常增加)寡核苷酸的核酸酶抗性。在一些实施方案中，具有手性原子的核苷间键可以制备为外消旋混合物或单独的对映异构体。代表性的手性键包括、但不限于烷基膦酸酯和硫代磷酸酯。含磷的和不含磷的核苷间键的制备方法是本领域技术人员众所周知的。 In some embodiments, the invention provides oligonucleotides comprising nucleosides connected. In such embodiments, nucleosides can be linked together using any nucleoside bond. The two main categories of nucleoside linking groups are limited by the presence or absence of a phosphorus atom. Compared with natural phosphodiester bonds, modified bonds can be used to change (usually increase) the nuclease resistance of oligonucleotides. In some embodiments, nucleoside bonds with chiral atoms can be prepared as racemic mixtures or separate enantiomers. Representative chiral bonds include, but are not limited to, alkyl phosphonates and thiophosphates. The preparation methods of phosphorus-containing and non-phosphorus-containing nucleoside bonds are well known to those skilled in the art.
教导这样的寡核苷酸缀合物的制备的代表性美国专利包括、但不限于美国专利4,828,979；4,948,882；5,218,105；5,525,465；5,541,313；5,545,730；5,552,538；5,578,717；5,580,731；5,580,731；5,591,584；5,109,124；5,118,802；5,138,045；5,414,077；5,486,603；5,512,439；5,578,718；5,608,046；4,587,044；4,605,735；4,667,025；4,762,779；4,789,737；4,824,941；4,835,263；4,876,335；4,904,582；4,958,013；5,082,830；5,112,963；5,214,136；5,082,830；5,112,963；5,214,136；5,245,022；5,254,469；5,258,506；5,262,536；5,272,250；5,292,873；5,317,098；5,371,241；5,391,723；5,416,203；5,451,463；5,510,475；5,512,667；5,514,785；5,565,552；5,567,810；5,574,142；5,585,481；5,587,371；5,595,726；5,597,696；5,599,923；5,599,928和5,688,941,它们中的每一篇通过引用并入本文。Representative U.S. patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Patents 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118, 802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958, 013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416, and 5,688,941, each of which is incorporated herein by reference.
在优选的实施方案中，mRNA是含有修饰的和未修饰的核苷酸的组合的mRNA。优选地，它是含有如WO2011/012316中所描述的修饰的和未修饰的核苷酸的组合的mRNA。其中描述的mRNA显示出提高的稳定性和减弱的免疫原性。In a preferred embodiment, the mRNA is an mRNA containing a combination of modified and unmodified nucleotides. Preferably, it is an mRNA containing a combination of modified and unmodified nucleotides as described in WO2011/012316. The mRNA described therein shows improved stability and reduced immunogenicity.
在另一实施方案中，mRNA含有标记的核酸(优选地，核苷酸和/或核糖核苷酸)，如，例如同位素和/或荧光标记的核苷酸。标记的mRNA分子在例如研究RNA和DNA分子的胞内构象以及在动物体内和或细胞中的分布情况起重要作用。In another embodiment, mRNA contains labeled nucleic acid (preferably, nucleotides and/or ribonucleotides), such as, for example, isotope- and/or fluorescently labeled nucleotides. Labeled mRNA molecules play an important role in, for example, studying the intracellular conformation of RNA and DNA molecules and the distribution in animals and or cells.
可以根据多种已知方法中的任一种合成mRNA。例如，可以经由体外转录(IVT)来合成根据本发明的mRNA。mRNA can be synthesized according to any of a variety of known methods. For example, mRNA according to the present invention can be synthesized via in vitro transcription (IVT).
此外，修饰的RNA，优选mRNA分子可以化学合成，例如，通过使用固相支持物和标准技术在自动化核苷酸序列合成仪上进行常规化学合成，或者通过化学合成相应的DNA序列，随后在体外或体内对其进行转录。Furthermore, modified RNA, preferably mRNA molecules can be chemically synthesized, for example, by conventional chemical synthesis on an automated nucleotide sequence synthesizer using a solid support and standard techniques, or by chemical synthesis of the corresponding DNA sequence and subsequent transcription thereof in vitro or in vivo.
虽然在一些实施方案中体外转录反应所提供的mRNA是所期望的，但是在本发明的范围内设想了mRNA的其他来源，包括从细菌、真菌、植物和/或动物产生的mRNA。While in some embodiments mRNA provided by an in vitro transcription reaction is desirable, other sources of mRNA are contemplated within the scope of the present invention, including mRNA produced from bacteria, fungi, plants, and/or animals.
在另一优选的实施方案中，mRNA可以与目标结合位点、靶向序列和/或与微小RNA结合位点组合，以便仅在相关细胞中允许期望的mRNA的活性。在进一步优选的实施方案中，RNA可以与3’polyA尾下游的微小RNA或shRNA组合。 In another preferred embodiment, the mRNA can be combined with a target binding site, a targeting sequence and/or with a microRNA binding site to allow the activity of the desired mRNA only in the relevant cells. In a further preferred embodiment, the RNA can be combined with a microRNA or shRNA downstream of the 3' polyA tail.
在一些实施方案中，可以在调配和包封之前纯化体外合成的mRNA，以除去不期望的杂质(包括在mRNA合成过程中使用的各种酶和其他试剂)。In some embodiments, in vitro synthesized mRNA can be purified prior to formulation and encapsulation to remove undesirable impurities (including various enzymes and other reagents used in the mRNA synthesis process).
本发明可以用于调配和包封各种长度的mRNA。在一些实施方案中，本发明可以用于调配和包封长度等于或大于约0.1kb、0.5kb、1kb、1.5kb、2kb、2.5kb、3kb、3.5kb、4kb、4.5kb、5kb、6kb、7kb、8kb、9kb、10kb、11kb、12kb、13kb、14kb、15kb或20kb的体外合成的mRNA。在一些实施方案中，本发明可以用于调配和包封长度在约0.1‐2kb、约1‐20kb、约1‐15kb、约1‐10kb、约5‐20kb、约5‐15kb、约5‐12kb、约5‐10kb、约8‐20kb或约8‐15kb范围内的体外合成的mRNA。The present invention can be used to prepare and encapsulate mRNAs of various lengths. In some embodiments, the present invention can be used to prepare and encapsulate in vitro synthesized mRNAs having a length equal to or greater than about 0.1 kb, 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb. In some embodiments, the present invention can be used to prepare and encapsulate in vitro synthesized mRNAs having a length of about 0.1-2 kb, about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb.
总体上，本发明可以通过核糖核酸与细胞分子和细胞器的相互作用来实现疾病治疗治疗、疾病预防和疾病诊断的效果。这种相互作用可以单独激活先天免疫系统，例如对某些CpG寡核苷酸和设计为与toll样受体和其它胞外或胞内受体特异性相互作用的序列而言即属于这类情况。此外，细胞中核糖核酸(优选mRNA)的摄入或引入可以旨在导致核苷酸序列，如核糖核酸(优选mRNA)中包含的基因的表达；可以旨在用于由于引入的外源核酸在细胞内的存在而导致的内源基因表达下调、沉默或敲低；或者可以旨在用于内源核酸序列的修饰，如所选择的碱基或内源核酸序列的整段序列(whole stretches)的修复、切除、插入或交换；或者可以旨在用于由于引入的外源核糖核酸(优选mRNA)在细胞内的存在和相互作用而实际上干扰任何细胞过程。引入的外源核糖核酸(优选mRNA)的过表达可以旨在补偿或互补内源基因表达，特别是在内源基因缺陷或沉默的情况下，导致基因表达产物不存在、不足或有缺陷或功能异常，多种代谢性和遗传性疾病，如囊性纤维化、血友病或肌肉营养不良等常常是这样的情况。引入的外源核糖核酸(优选mRNA)的过表达也可以旨在使表达产物与任意内源细胞过程相互作用或干扰任意内源细胞过程，如基因表达的调控、信号转导和其它细胞过程。引入的外源核糖核酸(优选mRNA)的过表达也可以旨在在转染或转导的细胞所驻留或使其驻留的生物体的背景下引起免疫应答。实例是抗原呈递细胞(如树突细胞)的遗传修饰，以使其呈递抗原用于疫苗接种目的。其它实例是肿瘤中细胞因子的过表达，以引起肿瘤特异性免疫应答。此外，引入的外源核糖核酸(优选mRNA)的过表达也可以旨在产生用于细胞疗法的体内或离体瞬时遗传修饰的细胞，如修饰的T细胞或前体或者干细胞或其它用于再生医学的细胞。 In general, the present invention can achieve the effect of disease treatment, disease prevention and disease diagnosis through the interaction of ribonucleic acid with cell molecules and organelles. This interaction can activate the innate immune system alone, such as certain CpG oligonucleotides and sequences designed to interact specifically with toll-like receptors and other extracellular or intracellular receptors. In addition, the intake or introduction of ribonucleic acid (preferably mRNA) in the cell can be intended to cause nucleotide sequences, such as the expression of genes included in ribonucleic acid (preferably mRNA); it can be intended to be used for the expression of endogenous genes caused by the presence of introduced exogenous nucleic acid in the cell, silencing or knocking down; or it can be intended for the modification of endogenous nucleic acid sequences, such as the repair, excision, insertion or exchange of the whole stretches of the selected base or endogenous nucleic acid sequence; or it can be intended for the presence and interaction of introduced exogenous ribonucleic acid (preferably mRNA) in the cell and actually interfere with any cellular process. The overexpression of the exogenous ribonucleic acid (preferred mRNA) introduced can be intended to compensate or complement endogenous gene expression, particularly in the case of endogenous gene defect or silence, causing gene expression product to be absent, insufficient or defective or malfunctioning, multiple metabolic and hereditary diseases, such as cystic fibrosis, hemophilia or muscular dystrophy, etc. are often such situations. The overexpression of the exogenous ribonucleic acid (preferred mRNA) introduced can also be intended to make expression product interact with any endogenous cell process or interfere with any endogenous cell process, such as the regulation, signal transduction and other cell processes of gene expression. The overexpression of the exogenous ribonucleic acid (preferred mRNA) introduced can also be intended to cause immune response in the background of the organism in which the cell of transfection or transduction resides or resides. Example is the genetic modification of antigen presenting cells (such as dendritic cells) so that it presents antigens for vaccination purposes. Other examples are the overexpression of cytokines in tumors, so as to cause tumor-specific immune response. Furthermore, overexpression of introduced exogenous ribonucleic acid (preferably mRNA) can also be aimed at generating transiently genetically modified cells in vivo or ex vivo for cell therapy, such as modified T cells or precursors or stem cells or other cells for regenerative medicine.
除此之外，本发明还可以通过RNA干扰(RNAi)、使用核酶、反义寡核苷酸、tRNA、长双链RNA来实现内源基因表达的下调、沉默或敲低以用于治疗目的。内源或预先存在的基因表达的下调、沉默或敲低可以用于治疗获得性、遗传性或自发性疾病，包括病毒感染和癌症。还可以设想，可以将核酸引入细胞中作为预防措施来实践，以预防例如病毒感染或瘤形成。类似地，可以在基因组水平和mRNA水平(包括外显子跳跃)实现基因修复、碱基或序列改变。碱基或序列改变可以例如通过RNA引导的位点特异性DNA切割、通过利用反式剪接、反式剪接核酶、嵌合修复体(chimeraplasts)、剪接体介导的RNA反式剪接的剪切和粘贴机制、或通过利用II类或重新靶向的内含子、或通过利用由病毒介导的插入诱变或利用使用原核、真核或病毒整合酶系统的靶向基因组插入来实现。In addition, the present invention can also realize the downregulation, silencing or knocking down of endogenous gene expression for therapeutic purposes by RNA interference (RNAi), using ribozymes, antisense oligonucleotides, tRNA, long double-stranded RNA. The downregulation, silencing or knocking down of endogenous gene expression of endogenous or pre-existing gene expression can be used to treat acquired, hereditary or spontaneous diseases, including viral infection and cancer. It can also be imagined that nucleic acid can be introduced into cells as a preventive measure to practice, to prevent, for example, viral infection or tumor formation. Similarly, gene repair, base or sequence changes can be realized at the genome level and mRNA level (including exon skipping). Base or sequence changes can be realized, for example, by RNA-guided site-specific DNA cutting, by utilizing trans-splicing, trans-splicing ribozymes, chimeric repair bodies (chimeraplasts), the shear and paste mechanism of RNA trans-splicing mediated by spliceosomes, or by utilizing class II or re-targeted introns, or by utilizing viral-mediated insertion mutagenesis or utilizing the targeted genome insertion using prokaryotic, eukaryotic or viral integrase systems.
此外，由单一基因的突变引起的多种遗传障碍是已知的，并且是RNA，优选mRNA治疗方法的候选者。关于某种性状将在后代中出现的可能性，单一基因突变引起的障碍，如囊性纤维化、血友病和多种其它疾病可以是显性或隐性的。相比之下，多基因障碍由两个或更多个基因引起，并且相应疾病的表现形式常常是变数并且与环境因素相关。多基因障碍的实例是高血压、胆固醇水平升高、癌症、神经退行性疾病、精神疾病等。同样在这些情况下，代表这些基因中的一种或多种的治疗性RNA，优选mRNA可以对那些患者有益。此外，遗传障碍不一定是从亲本基因传递，也可能由新的突变引起。同样在这些情况下，代表正确基因序列的治疗性RNA，优选mRNA可以对患者有益。In addition, the multiple genetic disorders caused by the mutation of a single gene are known and are candidates for RNA, preferably mRNA therapeutic methods. About the possibility that a certain trait will occur in offspring, the disorder caused by a single gene mutation, such as cystic fibrosis, hemophilia and multiple other diseases can be dominant or recessive. In contrast, polygenic disorders are caused by two or more genes, and the manifestation of the corresponding disease is often variable and related to environmental factors. The example of polygenic disorder is hypertension, elevated cholesterol levels, cancer, neurodegenerative diseases, mental illness, etc. In these cases as well, the therapeutic RNA, preferably mRNA, representing one or more of these genes can be beneficial to those patients. In addition, genetic disorders are not necessarily transmitted from parental genes, and may also be caused by new mutations. In these cases as well, the therapeutic RNA, preferably mRNA, representing the correct gene sequence can be beneficial to patients.
可以在ONIM(在线人类孟德尔遗传数据库(Online Mendelian Inheritance in Man))网址(http://onim.org)获得在线目录，其中具有22,993个人类基因和遗传障碍条目以及其相应基因和其表型的描述；每个序列都可以从Uniprot数据库(http://www.uniprot.org)中获得。An online catalogue with 22,993 entries for human genes and genetic disorders with descriptions of the corresponding genes and their phenotypes is available at ONIM (Online Mendelian Inheritance in Man) (http://onim.org); each sequence is available from the Uniprot database (http://www.uniprot.org).
在一些实施方式中，本发明的RNA，优选mRNA的编码序列可以被转录和翻译成部分或全长蛋白，该部分或全长蛋白包含水平等于或大于天然蛋白的水平的细胞活性。In some embodiments, the coding sequence of the RNA, preferably mRNA, of the present invention can be transcribed and translated into a partial or full-length protein comprising a level of cellular activity equal to or greater than that of the native protein.
在一些实施方案中，可涉及遗传性疾病，例如其影响肺部，如SPB(表面活性蛋白B)缺乏、ABCA3缺乏、囊性纤维化(CF)、原发性纤毛运动障碍(Primary cliliary dyskinesia)、哮喘、慢性阻塞性肺病(COPD)和α1-抗胰蛋白酶缺乏，或者其影响血浆蛋白质(例如先天性血色素沉着病(铁调素 (hepcidin)缺乏)、血栓性血小板减少性紫癜(TPP，ADAMTS 13缺乏)并引起凝血缺陷(例如血友病a和b)和补体缺陷(例如C蛋白缺乏)、免疫缺陷诸如例如SCID(引起在不同基因如RAG1、RAG2、JAK3、IL7R、CD45、CD3δ、CD3ε的突变)或缺乏、由于缺乏腺苷脱氨酶例如(ADA-SCID)、败血性肉芽肿病(例如由gp-91-phox基因p47-phox基因、p67-phox基因或p33-phox基因的突变引起)和贮积病如高歇氏病、法布里氏病、克腊比氏病、MPS I、MPS II(亨特综合征)、MPS VI、II型糖原贮积病或muccopolysacchaidoses。In some embodiments, a genetic disease may be involved, for example, one that affects the lungs, such as SPB (surfactant protein B) deficiency, ABCA3 deficiency, cystic fibrosis (CF), primary ciliary dyskinesia, asthma, chronic obstructive pulmonary disease (COPD), and alpha 1-antitrypsin deficiency, or one that affects plasma proteins, such as congenital hemochromatosis (hepcidin Deficiency of the immune system, such as hemophilia a and b, and complement deficiencies (such as protein C deficiency), immunodeficiencies such as SCID (caused by mutations in various genes such as RAG1, RAG2, JAK3, IL7R, CD45, CD3δ, CD3ε) or deficiency, due to a lack of adenosine deaminase, such as (ADA-SCID), septic granulomatosis (caused by mutations in the gp-91-phox gene, p47-phox gene, p67-phox gene or p33-phox gene) and storage diseases such as Gaucher's disease, Fabry's disease, Krabbe's disease, MPS I, MPS II (Hunter's syndrome), MPS VI, glycogen storage disease type II or muccopolysacchaidoses.
本发明的RNA，优选mRNA对其可发挥治疗作用的其它疾病包括如SMN1相关的脊髓性肌萎缩(SMA)；肌萎缩性侧索硬化(ALS)；GALT相关的半乳糖血症；SLC3A1相关的障碍，包括胱氨酸尿症；COL4A5相关的障碍，包括阿尔波特综合征；半乳糖脑苷脂酶缺乏；X连锁脑白质肾上腺萎缩症和肾上腺脊神经病；弗里德赖希氏共济失调；佩-梅二氏病；TSC1和TSC2相关的结节性硬化症；桑菲列普综合征B综合征(MPS IIIB)；CTNS相关的胱氨酸病；FMR1相关的障碍，包括脆性X染色体综合征、脆性X关联性震颤/共济失调综合症和脆性X卵巢早衰综合征；普-韦二氏综合征；遗传性出血性毛细血管扩张症(AT)；C1型尼-皮二氏病；神经元蜡样脂褐质沉积症相关的疾病，包括少年神经元蜡样脂褐质沉积症(JNCL)、少年巴腾病、Santavuori-Haltia病、Jansky-Bielschowsky病、和PTT-1和TPP1缺乏；EIF2B1、EIF2B2、EIF2B3、EIF2B4和EIF2B5相关的童年共济失调伴中枢神经系统髓鞘形成减少(hypomyelination)/消失白质；CACNA1A和CACNB4相关的发作性共济失调2型；MECP2相关的障碍，包括经典雷特综合征、MECP2相关的严重新生儿脑病和PPM-X综合征；CDKL5相关的非典型雷特综合征；肯尼迪氏病(SBMA)；Notch-3相关脑的常染色体显性动脉病伴皮层下梗死和脑白质病(CADASIL)；SCN1A和SCN1B相关的发作障碍；聚合酶G相关的障碍，包括Alpers-Huttenlocher综合征、POLG相关的感觉共济失调神经病、发音困难、和眼肌瘫痪(ophthalmoparesis)、和常染色体显性和隐性渐进性外眼肌麻痹伴线粒体DNA缺失；X连锁肾上腺发育不全；X连锁无丙种球蛋白血症；法布里病；和威尔逊氏病。Other diseases for which the RNA, preferably mRNA, of the present invention may exert a therapeutic effect include, for example, SMN1-related spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-related galactosemia; SLC3A1-related disorders, including cystinuria; COL4A5-related disorders, including Alport syndrome; galactocerebrosidase deficiency; X-linked leukoreflex atrophy and adrenoleukodystrophy; Friedreich's ataxia; Perelman-Merzbacher disease; TSC1 and TSC2-related tuberous sclerosis; Sanfilip B syndrome MPS IIIB; CTNS-related cystinosis; FMR1-related disorders, including fragile X syndrome, fragile X-linked tremor/ataxia syndrome, and fragile X premature ovarian failure syndrome; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia (AT); Nieto-Philosophy disease type C1; neuronal ceroid lipofuscinosis-related disorders, including juvenile neuronal ceroid lipofuscinosis (JNCL), juvenile Batten disease, Santavuori-Haltia disease, Jansky-Bielschowsky disease, and PT T-1 and TPP1 deficiency; childhood ataxia with hypomyelination/vanishing white matter in the central nervous system associated with EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5; episodic ataxia type 2 associated with CACNA1A and CACNB4; MECP2-related disorders, including classic Rett syndrome, MECP2-related severe neonatal encephalopathy, and PPM-X syndrome; CDKL5-related atypical Rett syndrome; Kennedy's disease (SBMA); Notch- 3-related cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); SCN1A and SCN1B-related seizure disorders; polymerase G-related disorders, including Alpers-Huttenlocher syndrome, POLG-related sensory ataxia neuropathy, dysphonia, and ophthalmoparesis, and autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions; X-linked adrenal dysgenesis; X-linked agammaglobulinemia; Fabry disease; and Wilson's disease.
在所有这些疾病中，蛋白质，例如酶，是有缺陷的，其可以通过用本发明的编码任一种上述蛋白质的RNA，优选mRNA的治疗进行治疗，这使得由缺陷的基因或其功能性片段编码的蛋白质可获得。转录物替代疗法/酶替代疗法不影响潜在的遗传缺陷，但是增加患者缺乏的酶的浓度。作为实例，在蓬珀 氏病中，转录物替代疗法/酶替代疗法替代缺乏的溶酶体酶酸性α-葡糖苷酶(GAA)。In all these diseases, a protein, such as an enzyme, is defective, which can be treated by treatment with the RNA, preferably mRNA, of the invention encoding any of the above proteins, which makes available the protein encoded by the defective gene or a functional fragment thereof. Transcript replacement therapy/enzyme replacement therapy does not affect the underlying genetic defect, but increases the concentration of the enzyme that the patient lacks. As an example, in Pompeii In Schizophrenia, transcript replacement therapy/enzyme replacement therapy replaces the deficient lysosomal enzyme acid alpha-glucosidase (GAA).
因此，可由本发明的mRNA编码的蛋白质的非限制性实例是促红细胞生成素(EPO)、生长激素(growth hormone)(生长激素(somatotropin)、hGH)、囊性纤维化跨膜传导调节蛋白(CFTR)、生长因子如GM-SCF、G-CSF、MPS、蛋白C、铁调素、ABCA3和表面活性蛋白B。可以用根据本发明的RNA治疗的疾病的其它实例是血友病A/B、法布里氏病、CGD、ADAMTS13、胡尔勒氏病、X染色体介导的A-γ-球蛋白血症、腺苷脱氨酶相关的免疫缺陷和新生儿中的呼吸窘迫综合征，其与SP-B相关。特别优选地，根据本发明的RNA，优选mRNA含有囊性纤维化跨膜传导调节蛋白(CFTR)、α1-抗胰蛋白酶、动力蛋白轴突中间链1(Dynein axonemal intermediate chain 1,DNAI1)、表面活性蛋白B(SP-B)或促红细胞生成素的编码序列。可以由根据本发明的本发明的RNA，优选mRNA编码的蛋白质的其它实例是生长因子，如人生长激素hGH、BMP-2或血管生成因子。Thus, non-limiting examples of proteins that can be encoded by the mRNA according to the invention are erythropoietin (EPO), growth hormone (somatotropin, hGH), cystic fibrosis transmembrane conductance regulator (CFTR), growth factors such as GM-SCF, G-CSF, MPS, protein C, hepcidin, ABCA3 and surfactant protein B. Other examples of diseases that can be treated with the RNA according to the invention are hemophilia A/B, Fabry disease, CGD, ADAMTS13, Hurler's disease, X-chromosome-mediated A-gammaglobulinemia, adenosine deaminase-related immunodeficiency and respiratory distress syndrome in newborns, which is associated with SP-B. Particularly preferably, the RNA according to the invention, preferably the mRNA, contains a coding sequence for cystic fibrosis transmembrane conductance regulator (CFTR), α1-antitrypsin, Dynein axonemal intermediate chain 1 (DnaI1), surfactant protein B (SP-B) or erythropoietin. Further examples of proteins that can be encoded by the inventive RNA, preferably mRNA, according to the invention are growth factors, such as the human growth hormone hGH, BMP-2 or angiogenic factors.
可选地，RNA优选mRNA可以含有编码全长抗体或纳米抗体(例如，重链和轻链两者)的核糖核苷酸序列，该全长抗体或纳米抗体可以用于治疗环境(therapeutic settings)，例如赋予对象免疫力。相应抗体及其治疗应用(一种或多种)是本领域已知的。Alternatively, the RNA, preferably mRNA, may contain a ribonucleotide sequence encoding a full-length antibody or nanobody (e.g., both heavy and light chains), which may be used in therapeutic settings, such as to confer immunity to a subject. Corresponding antibodies and their therapeutic application(s) are known in the art.
在另一实施方案中，RNA优选mRNA可以编码功能性单克隆或多克隆抗体，功能性单克隆或多克隆抗体可以用于靶向生物学目标(例如，刺激性细胞因子，如肿瘤坏死因子)和/或使其失活。类似地，RNA，优选mRNA序列可以编码例如用于治疗II型膜性增生性肾小球性肾炎或急性溶血性尿毒症综合征的功能性抗肾病因子抗体，或者可选地可以编码用于治疗VEGF介导的疾病(如癌症)的抗血管内皮生长因子(VEGF)抗体。In another embodiment, the RNA, preferably mRNA, can encode a functional monoclonal or polyclonal antibody that can be used to target and/or inactivate a biological target (e.g., a stimulatory cytokine such as tumor necrosis factor). Similarly, the RNA, preferably mRNA sequence can encode a functional anti-nephrotic factor antibody, for example, for the treatment of type II membranoproliferative glomerulonephritis or acute hemolytic uremic syndrome, or alternatively can encode an anti-vascular endothelial growth factor (VEGF) antibody for the treatment of VEGF-mediated diseases such as cancer.
在另一实施方案中，RNA优选mRNA可以含有编码可以用于基因组编辑技术的多肽或蛋白质的核糖核苷酸序列。利用不同多肽或蛋白质的多种基因组编辑系统是本领域已知的，即，例如CRISPR-Cas系统、大范围核酸酶(归巢核酸内切酶，meganucleases)、锌指核酸酶(ZFN)和基于转录激活因子样效应物的核酸酶(TALEN)。Trends in Biotechnology,2013,31(7),397-405中综述了用于基因组工程的方法。 In another embodiment, RNA, preferably mRNA, may contain a ribonucleotide sequence encoding a polypeptide or protein that can be used for genome editing technology. A variety of genome editing systems using different polypeptides or proteins are known in the art, i.e., for example, CRISPR-Cas systems, large-range nucleases (homing nucleases, meganucleases), zinc finger nucleases (ZFNs), and nucleases (TALENs) based on transcription activator-like effectors. Trends in Biotechnology, 2013, 31 (7), 397-405 summarizes methods for genome engineering.
因此，在优选的实施方案中，RNA，优选mRNA可以含有编码Cas(CRISPR相关蛋白)蛋白家族的多肽或蛋白质——优选Cas9(CRISPR相关蛋白9)——的核糖核苷酸序列。可以在基于CRISPR/Cas9的方法和/或CRISPR/Cas9基因组编辑技术中使用Cas蛋白家族的蛋白质，优选Cas9。Nat.Biotechnol.,2014,32(4):347-355中综述了用于基因组编辑、调控和靶向的CRISPR-Cas系统。Therefore, in a preferred embodiment, RNA, preferably mRNA, may contain a ribonucleotide sequence encoding a polypeptide or protein of the Cas (CRISPR-associated protein) protein family, preferably Cas9 (CRISPR-associated protein 9). Proteins of the Cas protein family, preferably Cas9, may be used in CRISPR/Cas9-based methods and/or CRISPR/Cas9 genome editing techniques. Nat. Biotechnol., 2014, 32(4): 347-355 reviews CRISPR-Cas systems for genome editing, regulation and targeting.
在另一优选的实施方案中，RNA，优选mRNA可以含有编码转录激活因子样效应物核酸酶(TALEN)的核糖核苷酸序列。在另一优选的实施方案中，RNA，优选mRNA含有编码锌指核酸酶(ZFN)的核糖核苷酸序列。在另一优选的实施方案中，RNA，优选mRNA可以含有编码大范围核酸酶的核糖核苷酸序列。In another preferred embodiment, the RNA, preferably the mRNA, may contain a ribonucleotide sequence encoding a transcription activator-like effector nuclease (TALEN). In another preferred embodiment, the RNA, preferably the mRNA, may contain a ribonucleotide sequence encoding a zinc finger nuclease (ZFN). In another preferred embodiment, the RNA, preferably the mRNA, may contain a ribonucleotide sequence encoding a meganuclease.
作为上述的可选方式，RNA含有不被表达成蛋白质或多肽的核糖核苷酸序列。因此，术语RNA不应仅仅理解为意指任何这样的多核苷酸分子：其如果被引入细胞中，则可翻译成多肽/蛋白质或其片段。确切地说，还考虑了RNA含有仅被转录成(功能性)RNA的核糖核苷酸序列，其中所述RNA是最终产物(并且因此，不需要被翻译)。在这种背景下，设想RNA含有核糖核苷酸序列，其优选地提供siRNA序列或另一期望的核糖核苷酸序列的遗传信息。As the above-mentioned optional mode, RNA contains the ribonucleotide sequence that is not expressed into protein or polypeptide.Therefore, the term RNA should not be merely interpreted as meaning any such polynucleotide molecule: if it is introduced into a cell, it can be translated into polypeptide/protein or its fragment.To be precise, it is also considered that RNA contains the ribonucleotide sequence that is only transcribed into (functional) RNA, wherein the RNA is the final product (and therefore, does not need to be translated).In this context, it is envisioned that RNA contains the ribonucleotide sequence, which preferably provides the genetic information of the siRNA sequence or another desired ribonucleotide sequence.
在某些实施方案中，本发明提供了一种用于制备包含递送特定抗原和/或编码特定抗原的核酸的本文所述的聚合物-脂质的治疗组合物的方法。其中，所述抗原可以是来自细菌、病毒、真菌或癌细胞的抗原。In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising a polymer-lipid as described herein for delivering a specific antigen and/or a nucleic acid encoding a specific antigen, wherein the antigen may be an antigen from bacteria, virus, fungus or cancer cell.
术语“抗原”表示刺激动物中保护性免疫反应的肽或核苷酸型生物材料(天然的、重组的或合成的)。适合于本发明的抗原可以是氨基酸序列如肽或蛋白质，或核酸序列如基因组DNA、cDNA、mRNA、saRNA、circRNA、tRNA、rRNA、小干扰RNA(iRNA)杂交序列，或改性或未改性的合成的或半合成的寡核昔酸序列。The term "antigen" refers to a peptide or nucleotide-based biological material (natural, recombinant or synthetic) that stimulates a protective immune response in an animal. Antigens suitable for the present invention can be amino acid sequences such as peptides or proteins, or nucleic acid sequences such as genomic DNA, cDNA, mRNA, saRNA, circRNA, tRNA, rRNA, small interfering RNA (iRNA) hybridization sequences, or modified or unmodified synthetic or semisynthetic oligonucleotide sequences.
适合于本发明的抗原可从选自由细菌、病毒、寄生虫、立克次氏体、原生动物和癌细胞组成的组的生物体中获得。Antigens suitable for the present invention may be obtained from an organism selected from the group consisting of bacteria, viruses, parasites, rickettsiae, protozoa and cancer cells.
本发明的聚合物-脂质组合物可用于治疗或预防大量疾病和紊乱，例如：The polymer-lipid compositions of the invention can be used to treat or prevent a number of diseases and disorders, such as:
涉及下述病毒的疾病和紊乱：逆转录病毒科(例如人类免疫缺陷性病毒，包括HIV-1)；黄病毒科(例如登革热病毒、脑炎病毒、黄热病毒)；冠状病毒科(例如冠状病毒)；弹状病毒科(例如水疱性口腔炎病毒、狂犬病病毒)；纤丝病毒科(例如埃博拉病毒)；副粘病毒科(例如副流感病毒、腮腺炎病 毒、麻疹病毒、呼吸道合胞体病毒)；正粘病毒科(例如流感病毒)；呼肠孤病毒科(例如呼肠孤病毒、环状病毒和轮状病毒)；双核糖核酸病毒科；肝脫氧核糖核酸病毒科(乙肝病毒)；细小病毒科(细小病毒)；疱疹病毒科(单纯性疱疹病毒(HSV)1和2、水痘带状疱疹病毒、细胞肥大病毒(CMV)、疱渗病毒；痘病毒科(Poxyiridae)(猴痘病毒、天花病毒、痘苗病毒、痘病毒)；和虹彩病毒科(例如非洲猪瘟病毒)；和未分类的病毒(例如海绵状脑病的病原体，丁型病毒性肝炎的试剂(被认为是乙肝病毒的缺陷卫星)、HCV病毒(引起非甲、非乙型肝炎)；Norwalk及相关病毒和星状病毒)。特别优选HIV、甲肝、乙肝、丙肝、冠状病毒、狂犬病病毒、脊髓灰质炎病毒、流感病毒、脑膜炎病毒、麻疹病毒、腮腺炎病毒、风疹、百日咳、脑炎病毒、乳头状瘤病毒、黄热病毒、呼吸道合胞体病毒、细小病毒、切昆贡亚热病毒、出血热病毒和疱疹病毒(特别是水痘)、细胞肥大病毒和埃-巴二氏病毒。在上述实施方式中，选择用于聚合物-脂质组合物的抗原源自于存在于天然存在的病毒中(或在感染期间表达/诱导)的那些抗原(或参考那些抗原而设计)。Diseases and disorders involving the following viruses: Retroviridae (e.g., human immunodeficiency virus, including HIV-1); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus); virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Reoviridae (e.g., reovirus, orbivirus, and rotavirus); Binaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus); Poxviridae (monkeypox virus, smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the causative agent of spongiform encephalopathy, the agent of hepatitis delta virus (thought to be a defective satellite of hepatitis B virus), HCV virus (causing non-A, non-B hepatitis); Norwalk and related viruses and astroviruses). HIV, hepatitis A, hepatitis B, hepatitis C, coronavirus, rabies virus, polio virus, influenza virus, meningitis virus, measles virus, mumps virus, rubella, pertussis, encephalitis virus, papilloma virus, yellow fever virus, respiratory syncytial virus, parvovirus, Chikungunya virus, hemorrhagic fever virus and herpes virus (especially varicella), cytomegalovirus and Epstein-Barr virus are particularly preferred. In the above embodiments, the antigens selected for the polymer-lipid composition are derived from those antigens present in naturally occurring viruses (or expressed/induced during infection) (or designed with reference to those antigens).
涉及下述革兰氏阴性和革兰氏阳性细菌的疾病和紊乱：幽门螺杆菌(Helicobacter pylori)、嗜肺军团菌(Legionella pneumophilia)、分枝杆菌属(例如肺结核分枝杆菌(M.tuberculosis)、金黄色葡萄球菌(Staphylococcus aureus)、淋病奈瑟氏菌(Neisseria gonorrhoeae)、脑膜炎奈瑟氏球菌(Neisseria meningitidis)、单核细胞增多性李氏菌(Listeria monocytogenes)、化脓性链球菌(Streptococcus pyogenes)(A组链球菌属)、无乳链球菌(Streptococcus agalactiae)(B组链球菌属)、草绿色链球菌(Streptococcus viridans)、肺炎链球菌(Streptococcus pneumoniae)、克雷伯氏杆菌属(Klebsiella spp)(包括克雷伯氏肺炎菌(K.pneumoniae))、立克次氏体属(Rickettsia spp.)和放线菌属(Actinomyces spp.)(包括衣氏放线菌(A.israelii))。在上述实施方式中，选择用于聚合物-脂质组合物的抗原源自于存在于天然存在的细菌中(或在感染期间表达/诱导)的那些抗原(或参考那些抗原而人工设计)。Diseases and disorders involving the following Gram-negative and Gram-positive bacteria: Helicobacter pylori, Legionella pneumophilia, Mycobacterium (e.g., M. tuberculosis, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Streptococcus agalactiae) (Group B Streptococcus), Streptococcus viridans, Streptococcus pneumoniae, Klebsiella spp. (including K. pneumoniae), Rickettsia spp., and Actinomyces spp. (including A. israelii). In the above embodiments, the antigens selected for the polymer-lipid composition are derived from those antigens present in naturally occurring bacteria (or expressed/induced during infection) (or artificially designed with reference to those antigens).
涉及下述真菌的疾病和紊乱：新型隐球菌(Cryptococcus neoformans)、荚膜组织胞浆菌(Histoplasma capsulatum)、粗球抱子菌(Coccidioides immitis)、皮炎芽生菌(Blastomyces dermatitidis)、沙眼衣原体(Chlamydia trachomatis)和白色念珠菌(Candida albicans)，在上述实施方式中，选择用于疫苗的抗原源自于存 在于天然存在的真菌中(或在感染期间表达/诱导)的那些抗原(或参考那些抗原而设计)。Diseases and disorders involving the following fungi: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans, in which the antigen selected for the vaccine is derived from an existing Those antigens that exist in naturally occurring fungi (or are expressed/induced during infection) (or designed with reference to those antigens).
涉及下述原生动物的疾病和紊乱：疟原虫属(Plasmodium spp.)(包括恶性疟原虫(Plasmodium falciparum)、三日疟原虫(Plasmodium malariae)、卵形疟原虫(Plasmodium ovale)和间日疟原虫(Plasmodium vivax))、弓形体属(Toxoplasma spp.)(包括刚地弓形虫(T.gondii)和T.cruzii)和利什曼虫属(Leishmania spp.)。Diseases and disorders involving the following protozoa: Plasmodium spp. (including Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax), Toxoplasma spp. (including T. gondii and T. cruzii), and Leishmania spp.
涉及下述癌细胞的疾病和紊乱：血液和淋巴系统癌症(包括霍奇金病、白血病、淋巴瘤、多发性骨髓瘤和病)、黑素瘤(包括眼睛的黑素瘤)、腺瘤、肉瘤、实体组织的癌、黑素瘤、肺癌、甲状腺癌、唾液腺癌、腿癌、舌癌、唇癌、胆管癌、骨盆癌、纵隔腔癌、尿道癌、卡波西肉瘤(例如与AIDS有关时)；皮肤癌(包括恶性黑素瘤)、消化道癌症(包括头和颈癌、食道癌、胃癌、胰腺癌、肝癌、结肠和直肠癌、肛门癌)、生殖和泌尿系统的癌症(包括肾癌、膀胱癌、睾丸癌、前列腺癌)、女性癌症(包括乳房癌、子宫颈癌、卵巢癌、妇科癌症和绒毛膜癌)以及脑癌、骨类癌瘤、鼻咽癌、腹膜后肿瘤、甲状腺癌、软组织瘤和未知的主要部位癌症。在上述实施方式中，选择用于疫苗的抗原为存在于恶性细胞和/或组织中的同源性新抗原或肿瘤相关的抗原。Diseases and disorders involving cancer cells such as: Cancers of the blood and lymphatic system (including Hodgkin's disease, leukemia, lymphoma, multiple myeloma, and Diseases), melanoma (including melanoma of the eye), adenoma, sarcoma, cancer of solid tissue, melanoma, lung cancer, thyroid cancer, salivary gland cancer, leg cancer, tongue cancer, lip cancer, bile duct cancer, pelvic cancer, mediastinal cancer, urethral cancer, Kaposi's sarcoma (e.g., when associated with AIDS); skin cancer (including malignant melanoma), digestive tract cancer (including head and neck cancer, esophageal cancer, stomach cancer, pancreatic cancer, liver cancer, colon and rectal cancer, anal cancer), reproductive and urinary system cancer (including kidney cancer, bladder cancer, testicular cancer, prostate cancer), female cancer (including breast cancer, cervical cancer, ovarian cancer, gynecological cancer and choriocarcinoma) and brain cancer, bone carcinoid tumor, nasopharyngeal cancer, retroperitoneal tumor, thyroid cancer, soft tissue tumor and unknown primary site cancer. In the above embodiment, the antigen selected for the vaccine is a homologous neoantigen or tumor-associated antigen present in malignant cells and/or tissues.
涉及多细胞寄生虫如蠕虫(例如血吸虫属(Schistosoma spp))的疾病和紊乱。Diseases and disorders involving multicellular parasites such as helminths (e.g. Schistosoma spp.
在一个优选实施方式中，可用于本发明的抗原是mRNA。In a preferred embodiment, the antigen useful in the present invention is mRNA.
抗原以致免疫的有效量自然地与本发明的聚合物-脂质组合物结合。“致免疫的有效量”表示当向个体施用包含本发明的聚合物-脂质组合物和抗原(例如，mRNA)时，抗原包含足以保护动物免于目标疾病的浓度的保护性成分。作为抗原的致免疫的有效量的实例，可提及0.01-100μg的量。The antigen is naturally combined with the polymer-lipid composition of the present invention in an immunogenic effective amount. "Immunogenic effective amount" means that when a polymer-lipid composition of the present invention and an antigen (e.g., mRNA) are administered to an individual, the antigen contains protective components at a concentration sufficient to protect the animal from the target disease. As an example of an immunogenic effective amount of an antigen, an amount of 0.01-100 μg can be mentioned.
在某些实施方案中，本发明提供了一种用于制备包含递送免疫调节剂和/或编码免疫调节剂的mRNA的本文所述的组合物的方法。所述免疫调节剂包括但不限于，例如，白介素2(IL-2)、白介素12(IL-12)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白介素23(IL-23)、C-C结构域趋化因子配体28(CCL28)、白介素36γ(IL-36γ)、干扰素基因(STING)蛋白刺激物的组成型活性变体等。In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an immunomodulator and/or an mRNA encoding an immunomodulator. The immunomodulator includes, but is not limited to, for example, interleukin 2 (IL-2), interleukin 12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 23 (IL-23), C-C domain chemokine ligand 28 (CCL28), interleukin 36γ (IL-36γ), constitutively active variants of interferon gene (STING) protein stimulators, etc.
将理解的是，用于本发明的背景下的颗粒可以包含单一类型的RNA，但是可以可选地包含两种或更多种类型的RNA的组合，例如单一颗粒中包含两 种或更多种类型的RNA的颗粒形式，或者其中所含RNA类型不同的颗粒混合物的形式。It will be appreciated that particles for use in the context of the present invention may comprise a single type of RNA, but may alternatively comprise a combination of two or more types of RNA, e.g. two or more types of RNA in a single particle. The present invention may be in the form of particles containing one or more types of RNA, or in the form of a mixture of particles containing different types of RNA.
蛋白protein
在一些实施方案中，所述聚合物-脂质组合物可以进一步包含一种或多种蛋白。一些蛋白可以包括酶诸如核酸酶。本文所述的组合物可以包含一种或多种CRISPR有关的蛋白(例如CRISPR酶)，包括Cas蛋白。这些酶是已知的；例如，化脓链球菌(S.pyogenes)Cas9蛋白的氨基酸序列可以在SwissProt数据库中在登录号Q99ZW2下找到。In some embodiments, the polymer-lipid composition may further comprise one or more proteins. Some proteins may include enzymes such as nucleases. The compositions described herein may include one or more CRISPR-related proteins (e.g., CRISPR enzymes), including Cas proteins. These enzymes are known; for example, the amino acid sequence of Streptococcus pyogenes (S. pyogenes) Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.
本文所述的组合物中的蛋白可以是Cas9(例如，来自化脓链球菌或肺炎链球菌(S.pneumonia))。CRISPR酶可以指导一条或两条链在靶序列位置处的切割，诸如在靶序列内和/或在靶序列的互补序列内。可以相对于相应的野生型酶使CRISPR酶突变，使得突变的CRISPR酶缺乏切割含有靶序列的靶多核苷酸的一条或两条链的能力。例如，来自化脓链球菌的Cas9的RuvC I催化结构域中的天冬氨酸‐至‐丙氨酸置换(D10A)将Cas9从切割两条链的核酸酶转化成切口酶(切割单链)。在一些实施方案中，Cas9切口酶可以与指导序列(例如两个指导序列)组合使用，所述指导序列分别靶向DNA靶标的有义链和反义链。这种组合允许两条链都被切口并用于诱导NHEJ或HDR。The protein in the compositions described herein can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae (S. pneumonia)). The CRISPR enzyme can direct the cleavage of one or both strands at the location of the target sequence, such as within the target sequence and/or within the complement of the target sequence. The CRISPR enzyme can be mutated relative to the corresponding wild-type enzyme so that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaving a single strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence (e.g., two guide sequences) that target the sense and antisense strands of a DNA target, respectively. This combination allows both strands to be nicked and used to induce NHEJ or HDR.
在一些实施方案中，本公开内容提供了含有一种或多种治疗性蛋白的化合物。可以被包括在组合物中的治疗性蛋白包括宽范围的分子诸如细胞因子、趋化因子、白介素、干扰素、生长因子、凝固因子、抗凝剂、血液因子、骨形态形成蛋白、免疫球蛋白和酶。特定治疗性蛋白的一些非限制性实例包括促红细胞生成素(EPO)、粒细胞集落刺激因子(G‐CSF)、α‐半乳糖苷酶A、α‐L‐艾杜糖醛酸酶、促甲状腺素α、N‐乙酰基半乳糖胺‐4‐硫酸酯酶(rhASB)、阿法链道酶、组织型纤溶酶原激活物(TPA)Activase、葡糖脑苷脂酶、干扰素(IF)β‐1a、干扰素β‐1b、干扰素γ、干扰素α、TNF‐α、IL‐1至IL‐36、人生长激素(rHGH)、人胰岛素(BHI)、人绒毛膜促性腺激素α、达依泊汀α、促卵泡激素(FSH)和因子VIII。In some embodiments, the present disclosure provides compounds containing one or more therapeutic proteins. Therapeutic proteins that can be included in the composition include a wide range of molecules such as cytokines, chemokines, interleukins, interferons, growth factors, coagulation factors, anticoagulants, blood factors, bone morphogenic proteins, immunoglobulins, and enzymes. Some non-limiting examples of specific therapeutic proteins include erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), α-galactosidase A, α-L-iduronidase, thyrotropin α, N-acetylgalactosamine-4-sulfatase (rhASB), dornase alfa, tissue-type plasminogen activator (TPA) Activase, glucocerebrosidase, interferon (IF) β-1a, interferon β-1b, interferon γ, interferon α, TNF-α, IL-1 to IL-36, human growth hormone (rHGH), human insulin (BHI), human chorionic gonadotropin α, darbepoetin α, follicle-stimulating hormone (FSH), and factor VIII.
两亲性嵌段共聚物 Amphiphilic block copolymers
各种两亲性嵌段共聚物可用于实践本发明。在一些实施方案中，两亲性嵌段共聚物也称为“表面活性剂”或“非离子表面活性剂”或“非离子两亲性嵌段共聚物”或“嵌段共聚物”或“两亲性聚合物”或“聚合物”。A variety of amphiphilic block copolymers can be used to practice the present invention. In some embodiments, the amphiphilic block copolymers are also referred to as "surfactants" or "nonionic surfactants" or "nonionic amphiphilic block copolymers" or "block copolymers" or "amphiphilic polymers" or "polymers".
本文中，术语“嵌段共聚物”是指包含至少两个聚合的单体单元的组或嵌段的聚合物。“嵌段”是指通过单体聚合获得的基序，其可在聚合物内重复。嵌段共聚物必须包含至少两个不同种类的聚合单体的嵌段。As used herein, the term "block copolymer" refers to a polymer comprising at least two groups or blocks of polymerized monomer units. "Block" refers to a motif obtained by polymerization of monomers, which may be repeated within the polymer. A block copolymer must contain blocks of at least two different types of polymerized monomers.
本文中，术语“两亲性嵌段共聚物”是指包含至少一个亲水性嵌段和至少一个疏水性嵌段的嵌段共聚物。在一些实施方案中，所述嵌段是非离子的，即不包含形成离子的部分。As used herein, the term "amphiphilic block copolymer" refers to a block copolymer comprising at least one hydrophilic block and at least one hydrophobic block. In some embodiments, the blocks are nonionic, ie, do not comprise ion-forming moieties.
在一些实施方案中，两亲性嵌段共聚物为四官能的两亲性嵌段共聚物，其中所述四官能的两亲性嵌段共聚物包含各自包含至少一个亲水性嵌段和至少一个疏水性嵌段的四个支链的嵌段共聚物，或所述两亲性嵌段共聚物为线性的两亲性嵌段共聚物，其中所述线性的两亲性嵌段共聚物包含至少一个亲水性嵌段和至少一个疏水性嵌段的嵌段共聚物。In some embodiments, the amphiphilic block copolymer is a tetrafunctional amphiphilic block copolymer, wherein the tetrafunctional amphiphilic block copolymer comprises four branched block copolymers each comprising at least one hydrophilic block and at least one hydrophobic block, or the amphiphilic block copolymer is a linear amphiphilic block copolymer, wherein the linear amphiphilic block copolymer comprises a block copolymer of at least one hydrophilic block and at least one hydrophobic block.
在一些实施方案中，所述亲水性嵌段选自聚氧化烯、聚乙烯醇、聚乙烯吡咯烷酮、聚(2-甲基-2-噁唑啉)和糖，和/或所述疏水性嵌段选自聚氧化烯、脂肪链、亚烷基聚酯、具有苄基聚醚端部的聚乙二醇和胆固醇，优选地，所述亲水性嵌段包含聚氧化乙烯单元，且所述疏水性嵌段包含聚氧化丙烯单元。In some embodiments, the hydrophilic block is selected from polyoxyalkylenes, polyvinyl alcohol, polyvinyl pyrrolidone, poly(2-methyl-2-oxazoline) and sugars, and/or the hydrophobic block is selected from polyoxyalkylenes, fatty chains, alkylene polyesters, polyethylene glycol with benzyl polyether ends and cholesterol. Preferably, the hydrophilic block comprises polyethylene oxide units and the hydrophobic block comprises polypropylene oxide units.
在一些优选的实施方案中，所述两亲性嵌段共聚物包含选自以下的至少一种：泊洛沙胺(poloxamine或)、泊洛沙姆(poloxamer或)、聚氧乙二醇脱水醇烷基酯(聚山梨醇酯)、聚乙烯吡咯烷酮(PVP)、聚乙二醇醚(BRIJ)、聚氧乙烯脂肪酸酯、聚氧乙烯脂肪醇醚、脱水山梨糖醇以及它们的衍生物。In some preferred embodiments, the amphiphilic block copolymer comprises at least one selected from the group consisting of poloxamine or ), poloxamer or ), polyoxyethylene glycol dehydrated alcohol alkyl esters (polysorbates), polyvinyl pyrrolidone (PVP), polyethylene glycol ethers (BRIJ), polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, sorbitan and their derivatives.
在一些实施方案中，所述两亲性嵌段共聚物占所述组合物的0.1％-98.0％重量百分比，例如0.5％-95.0％重量百分比、5.0％-85.0％重量百分比、10.0％-80.0％重量百分比、20.0％-75.0％重量百分比、30.0％-70.0％重量百分比或40.0％-60.0％重量百分比。In some embodiments, the amphiphilic block copolymer comprises 0.1%-98.0% by weight of the composition, e.g., 0.5%-95.0% by weight, 5.0%-85.0% by weight, 10.0%-80.0% by weight, 20.0%-75.0% by weight, 30.0%-70.0% by weight, or 40.0%-60.0% by weight.
泊洛沙胺(poloxamine或)Poloxamine (or )
适于本发明的泊洛沙胺，也被称为四官能团的非离子两亲性嵌段共聚物。 The poloxamines suitable for the present invention are also known as tetrafunctional nonionic amphiphilic block copolymers.
本发明的四官能的非离子两亲性嵌段共聚物优选包含亲水性嵌段/疏水性嵌段比例为0.5至1.5、优选0.8至1.3、更优选0.8至1.2的亲水性嵌段和疏水性嵌段。The tetrafunctional nonionic amphiphilic block copolymer of the present invention preferably comprises a hydrophilic block and a hydrophobic block in a hydrophilic block/hydrophobic block ratio of 0.5 to 1.5, preferably 0.8 to 1.3, more preferably 0.8 to 1.2.
可用于本发明的四官能的非离子两亲性四官能的嵌段共聚物可以是(A-B)n-C支链嵌段共聚物，其中A表示亲水性嵌段，B表示疏水性嵌段，C表示连接部分，和n为4和表示连接至C的(A-B)基团的数量。The tetrafunctional nonionic amphiphilic tetrafunctional block copolymer useful in the present invention can be a (A-B)n-C branched block copolymer, wherein A represents a hydrophilic block, B represents a hydrophobic block, C represents a connecting portion, and n is 4 and represents the number of (A-B) groups connected to C.
优选地，所述亲水性嵌段A为聚氧化乙烯嵌段(PEO)，所述疏水性嵌段B为聚氧化丙烯嵌段(PPO)。Preferably, the hydrophilic block A is a polyethylene oxide block (PEO), and the hydrophobic block B is a polypropylene oxide block (PPO).
所述连接部分C可以是亚烷基二胺部分，优选为亚乙基二胺部分。The linking moiety C may be an alkylenediamine moiety, preferably an ethylenediamine moiety.
可用于本发明的四官能的非离子两亲性嵌段共聚物可以是下式的结构：
The tetrafunctional nonionic amphiphilic block copolymer useful in the present invention may be of the following structure:
其中RA、RB、RC、RD彼此独立地表示
Where RA , RB , RC and RD represent independently
其中in
i的值为约2至约225，特别是约10至约100，更特别是约10至约60，和j的值为2至约185，特别是约10至约50，特别是约10至约20，更特别是等于或大于13，i has a value of about 2 to about 225, particularly about 10 to about 100, more particularly about 10 to about 60, and j has a value of 2 to about 185, particularly about 10 to about 50, particularly about 10 to about 20, more particularly equal to or greater than 13,
R*为具有2-6个碳的亚烷基，具有5-8个碳的环亚烷基或亚苯基，并且优选为亚乙基，R* is an alkylene group having 2 to 6 carbons, a cycloalkylene group having 5 to 8 carbons or a phenylene group, and is preferably an ethylene group,
对于R1和R2，或者(a)两个均为氢，或(b)一个为氢，另一个为甲基，For R1 and R2 , either (a) both are hydrogen, or (b) one is hydrogen and the other is methyl,
对于R3和R4，或者(a)两个均为氢，或者(b)一个为氢，另一个为甲基，和 For R3 and R4 , either (a) both are hydrogen, or (b) one is hydrogen and the other is methyl, and
如果R3和R4均为氢，则R5和R6中一个为氢，另一个为甲基，或如果R3和R4之一为甲基，则R5和R6均为氢。If R3 and R4 are both hydrogen, then one of R5 and R6 is hydrogen and the other is methyl, or if one of R3 and R4 is methyl, then R5 and R6 are both hydrogen.
更优选地，可用于本发明的非离子两亲性四官能的嵌段共聚物可以是下式的结构：
More preferably, the nonionic amphiphilic tetrafunctional block copolymer useful in the present invention may be of the following structure:
其中in
i的值为约2至约225，特别是约10至约100，更特别是约10至约60，和The value of i is from about 2 to about 225, particularly from about 10 to about 100, more particularly from about 10 to about 60, and
j的值为约2至约185，特别是约10至约50，特别是约10至约20，更特别是等于或大于13，The value of j is about 2 to about 185, particularly about 10 to about 50, particularly about 10 to about 20, and more particularly equal to or greater than 13,
和其中对于各R1、R2对，一个应为氢，另一个应为甲基。and wherein for each R1 , R2 pair, one shall be hydrogen and the other shall be methyl.
优选地，i可以为约5至约125，特别是约10至约100，更特别是约10至约60，和j可以是约5至约50，特别是约10至约25，特别是约10至约20，更特别是等于或大于13。Preferably, i may be about 5 to about 125, particularly about 10 to about 100, more particularly about 10 to about 60, and j may be about 5 to about 50, particularly about 10 to about 25, particularly about 10 to about 20, more particularly equal to or greater than 13.
在一些实施方案中，本文所述非离子两亲性四官能团的嵌段共聚物具有下式的结构：
In some embodiments, the nonionic amphiphilic tetrafunctional block copolymers described herein have the structure of the following formula:
其中in
i的值为约2至约225，特别是约10至约100，更特别是约10至约60，和The value of i is from about 2 to about 225, particularly from about 10 to about 100, more particularly from about 10 to about 60, and
j的值为约2至约185，特别是约10至约50，特别是约10至约20，更特别 是等于或大于13，The value of j is about 2 to about 185, particularly about 10 to about 50, particularly about 10 to about 20, and more particularly is equal to or greater than 13,
和其中对于各R1、R2对，一个应为氢，另一个应为甲基。and wherein for each R1 , R2 pair, one shall be hydrogen and the other shall be methyl.
优选地，i可以为约5至约125，特别是约10至约100，更特别是约10至约60，和j可以是约5至约50，特别是约10至约25，特别是约10至约20，更特别是等于或大于13。Preferably, i may be about 5 to about 125, particularly about 10 to about 100, more particularly about 10 to about 60, and j may be about 5 to about 50, particularly about 10 to about 25, particularly about 10 to about 20, more particularly equal to or greater than 13.
本发明所述非离子两亲性四官能团的嵌段共聚物的分子量可以是1000至35000，特别是4500至30000，更特别是5000至25000。The molecular weight of the nonionic amphiphilic tetrafunctional block copolymer of the present invention may be 1,000 to 35,000, particularly 4,500 to 30,000, and more particularly 5,000 to 25,000.
作为本发明的优选实施方案的非离子两亲性四官能团的嵌段共聚物，可以更具体地提及分子量为1650g/mol，PEO/PPO比为15:16(例如泊洛沙胺304)；或分子量为5500g/mol，PEO/PPO比为50:56(例如泊洛沙胺As a preferred embodiment of the present invention, the nonionic amphiphilic tetrafunctional block copolymer can be more specifically mentioned as having a molecular weight of 1650 g/mol and a PEO/PPO ratio of 15:16 (e.g., Poloxamine 304); or having a molecular weight of 5500 g/mol and a PEO/PPO ratio of 50:56 (e.g., Poloxamine
704)；或分子量为6700g/mol，PEO/PPO比为61:68(例如泊洛沙胺904))704); or a molecular weight of 6700 g/mol and a PEO/PPO ratio of 61:68 (e.g., poloxamine 904))
本发明的非离子两亲性四官能团的嵌段共聚物可包含，优选由约40％、特别是约45％、特别是约45％至约80％、特别是约45-70％，更特别是约45-60％，更优选约50％的氧化乙烯单元含量组成。The nonionic amphiphilic tetrafunctional block copolymer of the present invention may comprise, preferably consists of, an ethylene oxide unit content of about 40%, particularly about 45%, particularly about 45% to about 80%, particularly about 45-70%, more particularly about 45-60%, more preferably about 50%.
许多本发明的四官能的非离子两亲性嵌段共聚物，特别是非离子两亲性四官能的嵌段共聚物，可以通用商品名″poloxamines″或商购。Many of the tetrafunctional nonionic amphiphilic block copolymers of the present invention, particularly the nonionic amphiphilic tetrafunctional block copolymers, are generally known by the trade names "poloxamines" or Commercially available.
适合用于本发明的泊洛沙胺(poloxamines)的更多细节可见于SurfactantSystems，Eds.Attwood和Florence，Chapman and Hall，London 1983，p 356-361；TheCondensed Encyclopaedia of Surfactants，Ed.Ash和Ash，Edward Arnold，London，1989；Non-ionic Surfactants，pp.300-371，Ed.Nace，Dekker，New York，1996；Santon，Am.Perfumer Cosmet.72(4)：54-58(1958)；(Dekker，N.Y.，1967)；或US6,353,055。Further details of poloxamines suitable for use in the present invention may be found in Surfactant Systems, Eds. Attwood and Florence, Chapman and Hall, London 1983, p 356-361; The Condensed Encyclopaedia of Surfactants, Ed. Ash and Ash, Edward Arnold, London, 1989; Non-ionic Surfactants, pp. 300-371, Ed. Nace, Dekker, New York, 1996; Santon, Am. Perfumer Cosmet. 72(4): 54-58 (1958); (Dekker, N.Y., 1967); or US6,353,055.
在一些实施方案中，合适的泊洛沙胺为泊洛沙胺304。在一些实施方案中，合适的泊洛沙胺为泊洛沙胺701。在一些实施方案中，合适的泊洛沙胺为泊洛沙胺704。在一些实施方案中，合适的泊洛沙胺为泊洛沙胺901。在一些实施方案中，合适的泊洛沙胺为泊洛沙胺904。在一些实施方案中，合适的泊洛沙胺为泊洛沙胺908。在一些实施方案中，合适的泊洛沙胺为泊洛沙胺1107。在一些实施方案中，合适的泊洛沙胺为泊洛沙胺1301。在一些实施方案中，合适的泊洛沙胺为泊洛沙胺1304。在一些实施方案中，合适的泊洛沙胺为泊洛沙胺1307。在一些实施方案中，合适的泊洛沙胺为泊洛沙胺 90R4。在一些实施方案中，合适的泊洛沙胺为泊洛沙胺150R1。在一些实施方案中，合适的泊洛沙胺为它们的组合。In some embodiments, a suitable poloxamine is poloxamine 304. In some embodiments, a suitable poloxamine is poloxamine 701. In some embodiments, a suitable poloxamine is poloxamine 704. In some embodiments, a suitable poloxamine is poloxamine 901. In some embodiments, a suitable poloxamine is poloxamine 904. In some embodiments, a suitable poloxamine is poloxamine 908. In some embodiments, a suitable poloxamine is poloxamine 1107. In some embodiments, a suitable poloxamine is poloxamine 1301. In some embodiments, a suitable poloxamine is poloxamine 1304. In some embodiments, a suitable poloxamine is poloxamine 1307. In some embodiments, a suitable poloxamine is poloxamine 90R4. In some embodiments, the suitable poloxamine is poloxamine 150R1. In some embodiments, the suitable poloxamine is a combination thereof.
在一些实施方案中，本发明的两亲性嵌段共聚物(例如，泊洛沙胺poloxamine)可通过化学结构修饰改善其靶向特征，例如糖基化修饰、蛋白靶向配体修饰、抗体修饰、多肽修饰、叶酸修饰、生长因子修饰、细胞因子修饰、维生素修饰以及整合素修饰等。以糖基化修饰为例，糖基化修饰的两亲性嵌段共聚物包含至少一个与糖基部分共轭的末端嵌段，优选一个末端亲水性嵌段，更优选糖基化修饰的两亲性嵌段共聚物的至少25％、特别是至少50％、特别是至少75％、更特别是至少100％的末端嵌段与糖基部分共轭。糖基部分可通过在所述糖基部分的一个官能团与所述嵌段共聚物的一个官能团之间形成的共价键来共轭至本发明的嵌段共聚物。所述共价键可由本身改性至反应性的两个官能团之间的反应来形成，糖基部分可直接共轭至嵌段共聚物。或者，糖基部分可通过间隔基共轭至嵌段共聚物。In some embodiments, the amphiphilic block copolymers of the present invention (e.g., poloxamine) can improve their targeting characteristics by chemical structure modification, such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification. Taking glycosylation modification as an example, the glycosylated amphiphilic block copolymer comprises at least one terminal block conjugated to a glycosyl moiety, preferably a terminal hydrophilic block, and more preferably at least 25%, especially at least 50%, especially at least 75%, and more especially at least 100% of the terminal blocks of the glycosylated amphiphilic block copolymer are conjugated to the glycosyl moiety. The glycosyl moiety can be conjugated to the block copolymer of the present invention by a covalent bond formed between a functional group of the glycosyl moiety and a functional group of the block copolymer. The covalent bond can be formed by a reaction between two functional groups modified to be reactive, and the glycosyl moiety can be directly conjugated to the block copolymer. Alternatively, the glycosyl moiety can be conjugated to the block copolymer through a spacer.
泊洛沙姆(Poloxamer或)Poloxamer (Poloxamer or )
在一些实施方案中，合适的两亲性聚合物为泊洛沙姆。例如，合适的泊洛沙姆具有以下结构(PEO-PPO-PEO，即聚环氧乙烷-聚环氧丙烷-聚环氧乙烷结构)：
In some embodiments, suitable amphiphilic polymers are poloxamer. For example, suitable poloxamer has the following structure (PEO-PPO-PEO, i.e., polyethylene oxide-polypropylene oxide-polyethylene oxide structure):
或其盐或异构体，其中a为10与150之间的整数，并且b为20与60之间的整数。例如，a为约12并且b为约20，或者a为约80并且b为约27，或者a为约64并且b为约37，或者a为约141并且b为约44，或者a为约101并且b为约56。or a salt or isomer thereof, wherein a is an integer between 10 and 150, and b is an integer between 20 and 60. For example, a is about 12 and b is about 20, or a is about 80 and b is about 27, or a is about 64 and b is about 37, or a is about 141 and b is about 44, or a is about 101 and b is about 56.
在一些实施方案中，适于本发明的泊洛沙姆具有约10至约150个环氧乙烷单元。在一些实施方案中，泊洛沙姆具有约10至约100个环氧乙烷单元。 In some embodiments, poloxamers suitable for the present invention have from about 10 to about 150 ethylene oxide units. In some embodiments, poloxamers have from about 10 to about 100 ethylene oxide units.
在一些实施方案中，与泊洛沙姆具有类似结构的“反相-泊洛沙姆”(PPO-PEO-PPO，即具有聚环氧丙烷-聚环氧乙烷-聚环氧丙烷结构的泊洛沙姆)同样适用于本发明。例如，合适的“反相-泊洛沙姆”具有以下结构(PPO-PEO-PPO)：
In some embodiments, "reverse-poloxamer" (PPO-PEO-PPO, i.e., poloxamer having a polypropylene oxide-polyethylene oxide-polypropylene oxide structure) having a similar structure to poloxamer is also suitable for use in the present invention. For example, a suitable "reverse-poloxamer" has the following structure (PPO-PEO-PPO):
或其盐或异构体，其中a为10与150之间的整数，并且b为20与60之间的整数。or a salt or isomer thereof, wherein a is an integer between 10 and 150, and b is an integer between 20 and 60.
在一些实施方案中，适于本发明的“反相-泊洛沙姆”具有约10至约150个环氧乙烷单元。在一些实施方案中，“反相-泊洛沙姆”具有约10至约100个环氧乙烷单元。In some embodiments, "reverse-poloxamers" suitable for the present invention have from about 10 to about 150 ethylene oxide units. In some embodiments, "reverse-poloxamers" have from about 10 to about 100 ethylene oxide units.
在一些实施方案中，合适的泊洛沙姆为泊洛沙姆84。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆101。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆105。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆108。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆122。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆123。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆124。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆181。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆182。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆183。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆184。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆185。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆188。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆212。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆215。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆217。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆231。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆234。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆235。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆237。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆238。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆282。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆284。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆288。在一 些实施方案中，合适的泊洛沙姆为泊洛沙姆304。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆331。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆333。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆334。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆335。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆338。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆401。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆402。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆403。在一些实施方案中，合适的泊洛沙姆为泊洛沙姆407。在一些实施方案中，合适的泊洛沙姆为它们的组合。In some embodiments, a suitable poloxamer is poloxamer 84. In some embodiments, a suitable poloxamer is poloxamer 101. In some embodiments, a suitable poloxamer is poloxamer 105. In some embodiments, a suitable poloxamer is poloxamer 108. In some embodiments, a suitable poloxamer is poloxamer 122. In some embodiments, a suitable poloxamer is poloxamer 123. In some embodiments, a suitable poloxamer is poloxamer 124. In some embodiments, a suitable poloxamer is poloxamer 181. In some embodiments, a suitable poloxamer is poloxamer 182. In some embodiments, a suitable poloxamer is poloxamer 183. In some embodiments, a suitable poloxamer is poloxamer 184. In some embodiments, a suitable poloxamer is poloxamer 185. In some embodiments, a suitable poloxamer is poloxamer 188. In some embodiments, a suitable poloxamer is poloxamer 212. In some embodiments, a suitable poloxamer is poloxamer 215. In some embodiments, a suitable poloxamer is poloxamer 217. In some embodiments, a suitable poloxamer is poloxamer 231. In some embodiments, a suitable poloxamer is poloxamer 234. In some embodiments, a suitable poloxamer is poloxamer 235. In some embodiments, a suitable poloxamer is poloxamer 237. In some embodiments, a suitable poloxamer is poloxamer 238. In some embodiments, a suitable poloxamer is poloxamer 282. In some embodiments, a suitable poloxamer is poloxamer 284. In some embodiments, a suitable poloxamer is poloxamer 288. In one In some embodiments, a suitable poloxamer is poloxamer 304. In some embodiments, a suitable poloxamer is poloxamer 331. In some embodiments, a suitable poloxamer is poloxamer 333. In some embodiments, a suitable poloxamer is poloxamer 334. In some embodiments, a suitable poloxamer is poloxamer 335. In some embodiments, a suitable poloxamer is poloxamer 338. In some embodiments, a suitable poloxamer is poloxamer 401. In some embodiments, a suitable poloxamer is poloxamer 402. In some embodiments, a suitable poloxamer is poloxamer 403. In some embodiments, a suitable poloxamer is poloxamer 407. In some embodiments, suitable poloxamers are combinations thereof.
在一些实施方案中，合适的泊洛沙姆具有约4,000g/mol至约20,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约1,000g/mol至约50,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约1,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约2,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约3,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约4,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约5,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约6,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约7,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约8,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约9,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约10,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约20,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约30,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约40,000g/mol的平均分子量。在一些实施方案中，合适的泊洛沙姆具有约50,000g/mol的平均分子量。In some embodiments, suitable poloxamers have an average molecular weight of about 4,000 g/mol to about 20,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 1,000 g/mol to about 50,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 1,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 2,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 3,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 4,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 5,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 6,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 7,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 8,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 9,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 10,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 20,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 30,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 40,000 g/mol. In some embodiments, suitable poloxamers have an average molecular weight of about 50,000 g/mol.
在一些实施方案中，本发明的两亲性嵌段共聚物(例如，泊洛沙姆poloxamer)可通过化学结构修饰改善其靶向特征，例如糖基化修饰、蛋白靶向配体修饰、抗体修饰、多肽修饰、叶酸修饰、生长因子修饰、细胞因子修饰、维生素修饰以及整合素修饰等。以糖基化修饰为例，糖基化修饰的两亲性嵌段共聚物包含至少一个与糖基部分共轭的末端嵌段，优选一个末端亲水性嵌段，更优选糖基化修饰的两亲性嵌段共聚物的至少25％、特别是至少50％、 特别是至少75％、更特别是至少100％的末端嵌段与糖基部分共轭。糖基部分可通过在所述糖基部分的一个官能团与所述嵌段共聚物的一个官能团之间形成的共价键来共轭至本发明的嵌段共聚物。所述共价键可由本身改性至反应性的两个官能团之间的反应来形成，糖基部分可直接共轭至嵌段共聚物。或者，糖基部分可通过间隔基共轭至嵌段共聚物。In some embodiments, the amphiphilic block copolymers of the present invention (e.g., poloxamer) can improve their targeting characteristics by chemical structure modification, such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification. Taking glycosylation modification as an example, the glycosylated amphiphilic block copolymer comprises at least one terminal block conjugated to a glycosyl portion, preferably a terminal hydrophilic block, and more preferably at least 25%, especially at least 50%, of the glycosylated amphiphilic block copolymer. In particular, at least 75%, more particularly at least 100% of the terminal blocks are conjugated to the glycosyl moiety. The glycosyl moiety may be conjugated to the block copolymer of the invention via a covalent bond formed between one functional group of the glycosyl moiety and one functional group of the block copolymer. The covalent bond may be formed by a reaction between two functional groups which are themselves modified to be reactive, and the glycosyl moiety may be conjugated directly to the block copolymer. Alternatively, the glycosyl moiety may be conjugated to the block copolymer via a spacer.
其他两亲性嵌段共聚物Other amphiphilic block copolymers
在一些实施方案中，两亲性嵌段共聚物为聚乙烯基吡咯烷酮(PVP)，诸如具有3kDa、10kDa或29kDa分子量的PVP。In some embodiments, the amphiphilic block copolymer is polyvinylpyrrolidone (PVP), such as PVP having a molecular weight of 3 kDa, 10 kDa, or 29 kDa.
在一些实施方案中，两亲性嵌段共聚物是聚乙二醇醚(BRIJ)、聚山梨醇酯、脱水山梨糖醇以及它们的衍生物。在一些实施方案中，两亲性聚合物为聚山梨醇酯，诸如PS 20。In some embodiments, the amphiphilic block copolymer is polyethylene glycol ether (BRIJ), polysorbate, sorbitan and their derivatives. In some embodiments, the amphiphilic polymer is a polysorbate, such as PS 20.
在一些实施方案中，两亲性嵌段共聚物为聚乙二醇醚。在一些实施方案中，合适的聚乙二醇醚为式(S-1)的化合物：
In some embodiments, the amphiphilic block copolymer is a polyethylene glycol ether. In some embodiments, a suitable polyethylene glycol ether is a compound of formula (S-1):
或其盐或异构体，其中t为1与100之间的整数；R1BRIJ独立地为C10-40烷基、C10-40烯基或C10-40炔基；并且任选地，R5PEG的一个或多个亚甲基基团独立地用C3-10亚碳环基、4至10元亚杂环基、C6‐10亚芳基、4至10元亚杂芳基、‐N(RN)‐、‐O‐、‐S‐、‐C(O)‐、‐C(O)N(RN)‐、‐NRNC(O)‐、‐NR C(O)N(R)‐、‐C(O)O‐‐OC(O)‐、‐OC(O)O‐‐OC(O)N(RN)‐、‐NRNC(O)O‐‐C(O)S‐‐SC(O)‐、‐C(＝NRN)‐、‐C(＝NR)N(R)‐、‐NRNC(＝NRN)‐‐NRNC(＝NRN)N(RN)‐、‐C(S)‐、‐C(S)N(RN)‐、‐NRNC(S)‐、‐NRNC(S)N(RN)‐、‐S(O)‐、‐OS(O)‐、‐S(O)O‐‐OS(O)O‐‐OS(O)2‐‐S(O)2O‐‐OS(O)2O‐‐N(RN)S(O)‐、‐S(O)N(RN)‐‐N(RN)S(O)N(RN)‐‐OS(O)N(RN)‐‐N(RN)S(O)O‐‐S(O)2‐‐N(RN)S(O)2‐‐S(O)2N(RN)‐、‐N(RN)S(O)2N(RN)‐‐OS(O)2N(RN)‐或‐N(RN)S(O)2O‐替代；并且RN的每个实例独立地为氢、C1-6烷基或氮保护基团。 or a salt or isomer thereof, wherein t is an integer between 1 and 100; R1BRIJ is independently C10-40 alkyl, C10-40 alkenyl or C10-40 alkynyl; and optionally, one or more methylene groups of R5PEG are independently replaced by C3-10 carbocyclylene, 4- to 10-membered heterocyclylene, C6-10 arylene, 4- to 10-membered heteroarylene, -N( RN )-, -O-, -S-, -C(O)-, -C(O)N( RN )-, -NRNC(O)-, -NRC ( O)N(R)-, -C(O)O--OC(O)-, -OC(O)O--OC(O)N( RN )-, -NRNC ( O)O--C(O)S--SC(O)-, -C(＝ NRN )‐、‐C(＝NR)N(R)‐、‐NR N C(＝NR N )‐‐NR N C(＝NR N )N(R N )‐、‐C(S)‐、‐C(S)N(R N )‐、‐NR N C(S)‐、‐NR N C(S)N(R N )‐、‐S(O)‐、‐OS(O)‐、‐S(O)O‐‐OS(O)O‐‐OS(O) 2 ‐‐S(O) 2 O‐‐OS(O) 2 O‐‐N(R N )S(O)‐、‐S(O)N(R N )‐‐N(R N )S(O)N(R N )‐‐OS(O)N(R N )‐‐N(R N )S(O)O‐‐S(O) 2 ‐‐N(R N )S(O) 2 ‐‐S(O) 2 N(R N )-, -N( RN )S(O) 2N ( RN )-, -OS(O) 2N ( RN )-, or -N( RN )S(O) 2O- ; and each instance of RN is independently hydrogen, C1-6 alkyl, or a nitrogen protecting group.
在一些实施方案中，R1BRIJ为C烷基。例如，聚乙二醇醚为式(S-1a)的化合物：
In some embodiments, R 1BRIJ is C alkyl. For example, the polyethylene glycol ether is a compound of formula (S-1a):
或其盐或异构体，其中S为1与100之间的整数。or a salt or isomer thereof, wherein S is an integer between 1 and 100.
在一些实施方案中，R1BRIJ为C烯基。例如，合适的聚乙二醇醚为式(S-1b)的化合物：
In some embodiments, R 1BRIJ is C alkenyl. For example, a suitable polyethylene glycol ether is a compound of formula (S-1b):
或其盐或异构体，其中S为1与100之间的整数。or a salt or isomer thereof, wherein S is an integer between 1 and 100.
在一些实施方案中，本发明的两亲性嵌段共聚物(例如，PVP、BRIJ、聚山梨醇酯、脱水山梨糖醇等)可通过化学结构修饰改善其靶向递送特征，例如糖基化修饰、蛋白靶向配体修饰、抗体修饰、多肽修饰、叶酸修饰、生长因子修饰、细胞因子修饰、维生素修饰以及整合素修饰等。以糖基化修饰为例，糖基化修饰的两亲性嵌段共聚物包含至少一个与糖基部分共轭的末端嵌段，优选一个末端亲水性嵌段，更优选糖基化修饰的两亲性嵌段共聚物的至少25％、特别是至少50％、特别是至少75％、更特别是至少100％的末端嵌段与糖基部分共轭。糖基部分可通过在所述糖基部分的一个官能团与所述嵌段共聚物的一个官能团之间形成的共价键来共轭至本发明的嵌段共聚物。所述共价键可由本身改性至反应性的两个官能团之间的反应来形成，糖基部分可直接共轭至嵌段共聚物。或者，糖基部分可通过间隔基共轭至嵌段共聚物。In some embodiments, the amphiphilic block copolymers of the present invention (e.g., PVP, BRIJ, polysorbate, sorbitan, etc.) can improve their targeted delivery characteristics by chemical structure modification, such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification. Taking glycosylation modification as an example, the glycosylated amphiphilic block copolymer comprises at least one terminal block conjugated to a glycosyl portion, preferably a terminal hydrophilic block, and more preferably at least 25%, especially at least 50%, especially at least 75%, and more especially at least 100% of the terminal blocks of the glycosylated amphiphilic block copolymer are conjugated to the glycosyl portion. The glycosyl portion can be conjugated to the block copolymer of the present invention by a covalent bond formed between a functional group of the glycosyl portion and a functional group of the block copolymer. The covalent bond can be formed by a reaction between two functional groups that are modified to be reactive, and the glycosyl portion can be directly conjugated to the block copolymer. Alternatively, the glycosyl moiety can be conjugated to the block copolymer via a spacer.
阳离子脂质 Cationic lipids
如本文所用，术语“阳离子脂质”是指在选定pH(诸如生理pH)下具有净正电荷的许多脂质和类脂质物类中的任一者。本发明所述阳离子脂质包含选自以下的至少一种：永久阳离子脂质、可电离的阳离子脂质、胆固醇衍生的阳离子脂质和树枝状聚合物或树枝块(dendron)。这样的脂质包括但不限于，DOTMA、DOSPA、DOTAP、ePC、DODAP、DODMA、DDAB、DSDMA、DODAC、DOAP、DMRIE、DOGS、DMOBA、HGT5000、HGT5001、HGT5002、HGT4001、HGT4002、HGT4003、HGT4005、DLin-MC3-DMA、DLin-KC2-DMA、Acuitas ALC-0315、Acuitas A9、Acuitas Lipid 2,2、Moderna Lipid H(SM-102)、Moderna Lipid 5、A2-Iso5-2DC18、BAME-O16B、9A1P9、C12-200、cKK-E12、OF-Deg-Lin、306Oi10、TT3、FTT5、Lipid319、5A2-SC8、Genevant CL1、DLinDMA、DLenDMA、ClinDMA、CpLinDMA、咪唑胆固醇酯(ICE)、DC‐Choi(N,N‐二甲基‐N‐乙基甲酰胺胆固醇)、1,4-双(3‐N‐油基氨基‐丙基)哌嗪、N4-精氨胆固醇羰酰氨(GL67)、与碱性氨基酸序列偶联的胆固醇衍生物、RE-1、RE-2、RE-3和GL-67、5A2-SC8、Acuitas A9、Arcturus Lipid 2,2(8,8)4C CH3、OF-02、A18-Iso5-2DC18、BAME-O16B、A6、98N12-5、L319、L343、304O13、306O138、306O12B、306-O12B、LP01、G0-C14、7C1、Cephalin、Dlin-EG-DMA、DLinAP、DLin-MPZ、DLin-C-DAP、DLin-2-DMAP、Dlin-S-DMA、DLinDAP、DLin-MA、DLin-DAC、DLin-K-DMA、DLin-K-MPZ、DLin-K-DMA、DLin-K6-C4-DMA、DLin-K-C4-DMA、DLin-K-C3-DMA、CpLinDMA、DOcarbDAP、DLincarbDAP、C12-(2-3-2)、Genevant Lipid CL1、XTC、ALNY-100、NC98-5，及它们的混合物。许多这些脂质和相关类似物已经记述在美国专利公开号20220160633、20220168234、20220062175、20220370624、20210137840、20210378980、20210316008、20210369866、20180153822、20170151333、20100036115、20120202871、20130064894、20130129785、20130150625、20130178541、20130225836、20060083780、和20060240554；美国专利号11045418、10245229、10130649、10821186、9801944、8058069、9364435、9567296、10980895、10233148、10961188、9365610、9404127、10940207、5208036、5264618、5279833、5283185、5753613、7893302、7404969、8283333、8466122、5785992和PCT公开号WO2022032154、WO2022040641、WO2022235935、WO20220378702、WO20220389422、WO2021216577、WO2021226463、WO2021141944、WO2021016430、 WO2021222801、WO2020097520、WO2020051220、WO2019246203、WO2019141814、WO2018183901、WO2017201091、WO2017048789、WO2017201076、WO2017075531、WO2015061467、WO2015199952、WO2013149140、WO2013086373、WO2013086354、WO2013116126、WO20130225836、WO96/10390、WO2012170889、WO2012170930、WO2012040184、WO2012061259、WO2012054365、WO2012044638、WO2011153120、WO2011149733、WO2011090965、WO2011043913、WO2011022460、WO2010080724、WO201021865、WO2008103276、WO2010054401中，通过引用将其公开内容整体分别引入本文中用于全部目的。另外，可获得许多商业阳离子脂质制剂并可以用于本发明。这些包括，例如，(可商购的阳离子脂质体，其包含DOTMA和DOPE,来自GIBCO/BRL,Grand Island,纽约，美国)；(可商购的阳离子脂质体，其包含DOSPA和DOPE，来自GIBCO/BRL)；和(可商购的阳离子脂质体,其包含DOGS,来自Promega Corp.,Madison,威斯康星州，美国)。As used herein, the term "cationic lipid" refers to any of a number of lipid and lipidoid species having a net positive charge at a selected pH, such as physiological pH. The cationic lipid of the present invention comprises at least one selected from the group consisting of permanent cationic lipids, ionizable cationic lipids, cholesterol-derived cationic lipids, and dendritic polymers or dendrons. Such lipids include, but are not limited to, DOTMA, DOSPA, DOTAP, ePC, DODAP, DODMA, DDAB, DSDMA, DODAC, DOAP, DMRIE, DOGS, DMOBA, HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4005, DLin-MC3-DMA, DLin-KC2-DMA, Acuitas ALC-0315, Acuitas A9, Acuitas Lipid 2,2, Moderna Lipid H (SM-102), Moderna Lipid 5, A2-Iso5-2DC18, BAME-016B, 9A1P9, C12-200, cKK-E12, OF-Deg-Lin, 306Oi10, TT3, FTT5, Lipid319, 5A2-SC8, Genevan CL1, DLinDMA, DLenDMA, ClinDMA, CpLinDMA, imidazole cholesterol ester (ICE), DC-Choi (N,N-dimethyl-N-ethylformamide cholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, N4-arginine cholesterol carbonylamide (GL67), cholesterol derivatives coupled to basic amino acid sequences, RE-1, RE-2, RE-3 and GL-67, 5A2-SC8, Acuitas A9, Arcturus Lipid 2,2(8,8)4C CH3, OF-02, A18-Iso5-2DC18, BAME-O16B, A6, 98N12-5, L319, L343, 304O13, 306O138, 306O12B, 306-O12B, LP01, G0-C14, 7C1, Cephalin, Dlin-EG-DMA, DLinAP, DLin-MPZ, DLin-C-DAP, DLin-2-DMAP, Dlin-S-DMA, DLinDAP, DLin-MA, DLin-DAC, DLin-K-DMA, DLin-K-MPZ, DLin-K-DMA, DLin-K6-C4-DMA, DLin-K-C4-DMA, DLin-K-C3-DMA, CpLinDMA, DOcarbDAP, DLincarbDAP, C12-(2-3-2), Genevan Lipid CL1, XTC, ALNY-100, NC98-5, and mixtures thereof. Many of these lipids and related analogs have been described in U.S. Patent Publication Nos. 20220160633, 20220168234, 20220062175, 20220370624, 20210137840, 20210378980, 20210316008, 20210369866, 20180153822, 20170151333, 2 0100036115, 20120202871, 20130064894, 20130129785, 20130150625, 20130178541, 20130225836, 20060083780, and 20060240554; U.S. Patent Nos. 11045418, 10245229, 10130649, 108211 86, 9801944, 8058069, 9364435, 9567296, 10980895, 10233148, 10961188, 9365610, 9404127, 10940207, 5208036, 5264618, 5279833, 5283185, 5753613, 7893302, 7404969, 828 3333, 8466122, 5785992 and PCT Publication Nos. WO2022032154, WO2022040641, WO2022235935, WO20220378702, WO20220389422, WO2021216577, WO2021226463, WO2021141944, WO2021016430, WO2021222801、WO2020097520、WO2020051220、WO2019246203、WO2019141814、WO2018183901、WO2017201091、WO2017048789、WO2017201076、WO2017075531、WO2015061467、WO2015199952、WO2013149140、WO2013086373、WO2013086354、WO2013116126、WO20130225836、WO96/ 10390, WO2012170889, WO2012170930, WO2012040184, WO2012061259, WO2012054365, WO2012044638, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2010080724, WO201021865, WO2008103276, WO2010054401, the disclosures of which are incorporated herein by reference in their entirety for all purposes. In addition, many commercial cationic lipid preparations are available and can be used in the present invention. These include, for example, (commercially available cationic liposomes comprising DOTMA and DOPE from GIBCO/BRL, Grand Island, New York, USA); (commercially available cationic liposomes comprising DOSPA and DOPE from GIBCO/BRL); and (Commercially available cationic liposomes containing DOGS are from Promega Corp., Madison, Wisconsin, USA).
在某些实施方案中，本发明的组合物和方法包括的阳离子脂质可定义为具有下式结构的化合物或其盐：
In certain embodiments, the cationic lipids included in the compositions and methods of the present invention can be defined as compounds having the following structure or salts thereof:
其中，in,
R1和R2独立选择并且是H或C1-C3烷基； R1 and R2 are independently selected and are H or C1 - C3 alkyl;
R3和R4独立选择并且是具有约10-约20个碳原子的烷基，且R3和R4中至少一个包括至少2个不饱和位点。 R3 and R4 are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3 and R4 includes at least 2 sites of unsaturation.
在某些实例中，R3和R4均相同，即，R3和R4均是亚油基(C18)等。在某些其他实例中，R3和R4不同，即，R3是十四碳三烯基(tetradectrienyl)(C14)且R4是亚油基(C18)。在优选的实施方案中，式I的阳离子脂质是对称的，即，R3和R4均相同。在另一个优选实施方案中，R3和R4均包括至少2个不饱和位点。在一些实施方案中，R3和R4独立选自由十二碳二烯基(dodecadienyl)、十四碳二烯基(tetradecadienyl)、十六碳二烯基(hexadecadienyl)、亚油基、和二十碳二烯基(icosadienyl)组成的组。在优选的实施方案中，R3和R4均是亚油 基。在一些实施方案中，R3和R4包括至少3个不饱和位点且独立选自，例如，十二碳三烯基(dodecatrienyl)，十四碳三烯基，十六碳三烯基(hexadecatrienyl)，亚麻基(linolenyl)，和二十碳三烯基(icosatrienyl)。In some instances, R 3 and R 4 are the same, that is, R 3 and R 4 are linoleyl (C 18 ) and the like. In some other instances, R 3 and R 4 are different, that is, R 3 is tetradectrienyl (C 14 ) and R 4 is linoleyl (C 18 ). In a preferred embodiment, the cationic lipid of Formula I is symmetrical, that is, R 3 and R 4 are the same. In another preferred embodiment, R 3 and R 4 both include at least 2 unsaturated sites. In some embodiments, R 3 and R 4 are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In a preferred embodiment, R 3 and R 4 are linoleyl. In some embodiments, R3 and R4 include at least 3 sites of unsaturation and are independently selected from, for example, dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.
在优选的实施方案中，式I的阳离子脂质是1,2-二亚油基氧基-N,N-二甲基氨基丙烷(DLinDMA)或1,2-二亚麻基氧基-N,N-二甲基氨基丙烷(DLenDMA)。In a preferred embodiment, the cationic lipid of Formula I is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or 1,2-dilinolenoyloxy-N,N-dimethylaminopropane (DLenDMA).
此外，具有以下式结构的阳离子脂质(或其盐)可有效用于本发明中。
In addition, a cationic lipid having the following structure (or a salt thereof) can be effectively used in the present invention.
其中,in,
R1和R2相同或不同并独立地是任选取代的C12-C24烷基，任选取代的C12-C24烯基，任选取代的C12-C24炔基,或任选取代的C12-C24酰基； R1 and R2 are the same or different and are independently optionally substituted C12 - C24 alkyl, optionally substituted C12 - C24 alkenyl, optionally substituted C12 - C24 alkynyl, or optionally substituted C12 - C24 acyl;
R3和R4相同或不同并独立地是任选取代的C1-C6烷基，任选取代的C1-C6烯基，或任选取代的C1-C6炔基或R3和R4可以结合形成任选取代的4-6个碳原子和1或2个选自氮和氧的杂原子的杂环； R3 and R4 are the same or different and are independently optionally substituted C1 - C6 alkyl, optionally substituted C1 - C6 alkenyl, or optionally substituted C1 - C6 alkynyl, or R3 and R4 may combine to form an optionally substituted heterocyclic ring of 4-6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen;
R5不存在或是氢或C1-C6烷基以提供季胺；R 5 is absent or is hydrogen or C 1 -C 6 alkyl to provide a quaternary amine;
m,n,和p相同或不同并独立地是0或1，条件是m,n,和p不同时为0；q是0,1,2,3,或4；且Y和Z相同或不同并独立地是0,S,或NH。m, n, and p are the same or different and are independently 0 or 1, provided that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are the same or different and are independently 0, S, or NH.
在一些实施方案中，具有上式结构的阳离子脂质是2,2-二亚油基-4-(2-二甲基氨乙基)-[1,3]-二氧环戊烷(DLin-K-C2-DMA)、2,2-二亚油基-4-(3-二甲基氨基丙基-二氧环戊烷(DLin-K-C3-DMA)、2,2-二亚油基-4-(4-二甲基氨基丁基)-[1,3]-二氧环戊烷(DLin-K-C4-DMA)、2,2-二亚油基-5-二甲基氨基甲基-[1,3]-二噁烷(DLin-K6-DMA)、2,2-二亚油基-4-N-甲基pepiazino-[l,3]-二氧环戊烷(DLin-K-MPZ)、2,2-二亚油基-4-二甲基氨基甲基-[1,3]-二氧环戊烷(DLin-K-DMA)、1,2-二亚油基氨甲酰基氧基-3-二甲基氨基丙烷(DLin-C-DAP)、1,2-二亚油氧基-3-(二甲基氨基)乙酰氧基丙烷(DLin-DAC)、1,2-二亚油氧基-3-吗咻代丙烷(DLin-MA)、1,2-二亚油酰基-3-二甲基氨基丙烷(DLinDAP)、1,2-二亚油基硫代-3-二甲基氨基丙烷(DLin-S-DMA)、1- 亚油酰基-2-亚油基氧基-3-二甲基氨基丙烷(DLin-2-DMAP)、氯化1,2-二亚油基氧基-3-三甲基氨基丙烷盐(DLin-TMA.Cl)、氯化1,2-二亚油酰基-3-三甲基氨基丙烷盐(DLin-TAP.Cl)、1,2-二亚油基氧基-3-(N-甲基哌嗪并)丙烷(DLin-MPZ)、3-(N,N-二亚油基氨基)-1,2-丙二醇(DLinAP)、3-(N,N-二油基氨基)-1,2-丙二醇(DOAP)、1,2-二亚油基氧代-3-(2-N-二甲基氨基)乙氧基丙烷(DLin-EG-DMA)，或其混合物。在优选的实施方案中，具有上式结构的阳离子脂质是DLin-K-C2-DMA。In some embodiments, the cationic lipid having the above structure is 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino- [1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyl-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyl-3-morphocyanopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1- Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazine) propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N-dimethylamino)ethoxypropane (DLin-EG-DMA), or a mixture thereof. In a preferred embodiment, the cationic lipid having the above structure is DLin-K-C2-DMA.
在某些实施方案中，本发明的组合物和方法所述永久阳离子脂质包含季铵离子。在某些实施方案中，这类永久阳离子脂质具有以下通式：
In certain embodiments, the compositions and methods of the present invention include permanent cationic lipids comprising quaternary ammonium ions. In certain embodiments, such permanent cationic lipids have the following general formula:
其中,in,
R1和R2各自独立地是C8‐C24烷基、C8‐C24烯基或任一个基团的被取代形式； R1 and R2 are each independently C8 - C24 alkyl, C8 - C24 alkenyl, or a substituted form of either group;
R3、R3'和R3”各自独立地是烷基(C≤6)或被取代的烷基(C≤6)；R 3 , R 3 ′ and R 3 ″ are each independently alkyl (C≤6) or substituted alkyl (C≤6) ;
X‐是单价阴离子。X ‐ is a monovalent anion.
在某些实施方案中，本发明的组合物和方法所述阳离子脂质进一步定义为具有下式结构的化合物或其盐：
In certain embodiments, the cationic lipids of the compositions and methods of the present invention are further defined as compounds having the following structure or salts thereof:
其中,in,
R1和R2各自独立地是C8‐C24烷基、C8‐C24烯基或任一个基团的被取代形式； R1 and R2 are each independently C8 - C24 alkyl, C8 - C24 alkenyl, or a substituted form of either group;
R3、R3'和R3”各自独立地是烷基(C≤6)或被取代的烷基(C≤6)；R 3 , R 3 ′ and R 3 ″ are each independently alkyl (C≤6) or substituted alkyl (C≤6) ;
R4是烷基(C≤6)或被取代的烷基(C≤6)； R4 is alkyl (C≤6) or substituted alkyl (C≤6) ;
且X‐是单价阴离子。and X ‐ is a monovalent anion.
在某些实施方案中，本发明的组合物和方法所述阳离子脂质进一步定义为具有下式结构的化合物或其盐：
In certain embodiments, the cationic lipids of the compositions and methods of the present invention are further defined as compounds having the following structure or salts thereof:
其中,in,
R1和R2独立选择并且是H或C1-C3烷基； R1 and R2 are independently selected and are H or C1 - C3 alkyl;
R3和R4独立选择并且是具有约10-约20个碳原子的烷基，且R3和R4中至少一个包括至少2个不饱和位点。 R3 and R4 are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3 and R4 includes at least 2 sites of unsaturation.
在某些实例中，R3和R4均相同，即，R3和R4均是亚油基(C18),等。在某些其他实例中，R3和R4不同，即，R3是十四碳三烯基(C14)且R4是亚油基(C18)。在优选的实施方案中，本发明的阳离子脂质是对称的，即，R3和R4均相同。在另一个优选实施方案中，R3和R4均包括至少2个不饱和位点。在一些实施方案中，R3和R4独立选自由十二碳二烯基、十四碳二烯基、十六碳二烯基、亚油基和二十碳二烯基组成的组。在优选的实施方案中，R3和R4均是亚油基。在一些实施方案中，R3和R4包括至少3个不饱和位点且独立选自，例如，十二碳三烯基、十四碳三烯基、十六碳三烯基、亚麻基和二十碳三烯基。In some examples, R 3 and R 4 are all the same, that is, R 3 and R 4 are all linoleyl (C 18 ), etc. In some other examples, R 3 and R 4 are different, that is, R 3 is tetradecatrienyl (C 14 ) and R 4 is linoleyl (C 18 ). In a preferred embodiment, the cationic lipid of the present invention is symmetrical, that is, R 3 and R 4 are all the same. In another preferred embodiment, R 3 and R 4 both include at least 2 unsaturated sites. In some embodiments, R 3 and R 4 are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl and eicosadienyl. In a preferred embodiment, R 3 and R 4 are all linoleyl. In some embodiments, R 3 and R 4 include at least 3 unsaturated sites and are independently selected from, for example, dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linoleyl and eicosadienyl.
在某些实施方案中，本发明的组合物和方法所述阳离子脂质进一步定义为具有下式结构的化合物或其盐：
In certain embodiments, the cationic lipids of the compositions and methods of the present invention are further defined as compounds having the following structure or salts thereof:
其中,in,
R4和R4'各自独立地是C6‐C24烷基、C6‐C24烯基或任一个基团的被取代形式；R 4 and R 4 'are each independently C 6 -C 24 alkyl, C 6 -C 24 alkenyl, or a substituted form of either group;
R4”是烷基(C≤24)、烯基(C≤24)或任一个基团的被取代形式；R 4 ″ is alkyl (C≤24) , alkenyl (C≤24) , or a substituted form of either group;
R4”'是C1‐C8烷基、C2‐C8烯基或任一个基团的被取代形式； R 4 ″′ is C 1 -C 8 alkyl, C 2 -C 8 alkenyl, or a substituted form of either group;
且X2是单价阴离子。and X2 is a monovalent anion.
在某些实施方案中，本发明的组合物和方法所述永久阳离子脂质进一步定义为具有下式结构的化合物或其盐：
In certain embodiments, the permanent cationic lipid of the compositions and methods of the present invention is further defined as a compound having the following structure or a salt thereof:
其中，Y1、Y2或Y3各自独立地是X1C(O)R1或X2N+R3R4R5；只要Y1、Y2和Y3中的至少一个是X2N+R3R4R5；wherein Y 1 , Y 2 or Y 3 are each independently X 1 C(O)R 1 or X 2 N + R 3 R 4 R 5 ; provided that at least one of Y 1 , Y 2 and Y 3 is X 2 N + R 3 R 4 R 5 ;
R1是C1‐C24烷基、C1‐C24取代的烷基、C1‐C24烯基、C1‐C24取代的烯基； R1 is C1 - C24 alkyl, C1 - C24 substituted alkyl, C1 - C24 alkenyl, C1 - C24 substituted alkenyl;
X1是O或NRa，其中Ra是氢、C1‐C4烷基或C1‐C4取代的烷基； X1 is O or NR a , wherein R a is hydrogen, C 1 ‐C 4 alkyl or C 1 ‐C 4 substituted alkyl;
X2是C1‐C6烷二基或C1‐C6取代的烷二基； X2 is C1 - C6 alkanediyl or C1 - C6 substituted alkanediyl;
R3、R4和R5各自独立地是C1‐C24烷基、C1‐C24取代的烷基、C1‐C24烯基、C1‐C24取代的烯基；R 3 , R 4 and R 5 are each independently C 1 -C 24 alkyl, C 1 -C 24 substituted alkyl, C 1 -C 24 alkenyl, C 1 -C 24 substituted alkenyl;
A1是阴离子，其具有的电荷等于化合物中X2N+R3R4R5基团的数目。 A1 is an anion having a charge equal to the number of X2N + R3R4R5 groups in the compound.
用于本发明的组合物和方法的合适的阳离子脂质包括如国际专利公开WO 2010/144740以及美国专利8,058,069中所述的阳离子脂质，该专利公开以引用的方式并入本文。Suitable cationic lipids for use in the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO 2010/144740 and U.S. Patent 8,058,069, which are incorporated herein by reference.
用于本发明的组合物和方法的其他合适的阳离子脂质包括如国际专利公开WO2013/149140中所述的可电离阳离子脂质，该专利公开以引用的方式并入本文。在一些实施方案中，本发明的组合物和方法包括下式之一的阳离子脂质：
Other suitable cationic lipids for the compositions and methods of the present invention include ionizable cationic lipids as described in International Patent Publication WO2013/149140, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid of one of the following formulas:
或其药学上可接受的盐，or a pharmaceutically acceptable salt thereof,
其中R1和R2各自独立地选自由以下项组成的组：氢、任选地取代的不同饱和或不饱和的C1‐C20烷基和任选地取代的不同饱和或不饱和的C6‐C20酰基；其中L1和L2各自独立地选自由以下项组成的组：氢、任选地取代的C1‐C30烷基、任选地取代的不同不饱和的C1‐C30烯基和任选地取代的C1‐C30炔基；其中m和 o各自独立地选自由以下项组成的组：零和任何正整数(例如，其中m为三)；并且其中n为零或任何正整数(例如，其中n为一)。wherein R 1 and R 2 are each independently selected from the group consisting of hydrogen, optionally substituted different saturated or unsaturated C 1 -C 20 alkyl groups, and optionally substituted different saturated or unsaturated C 6 -C 20 acyl groups; wherein L 1 and L 2 are each independently selected from the group consisting of hydrogen, optionally substituted C 1 -C 30 alkyl groups, optionally substituted different unsaturated C 1 -C 30 alkenyl groups, and optionally substituted C 1 -C 30 alkynyl groups; wherein m and o are each independently selected from the group consisting of: zero and any positive integer (eg, where m is three); and where n is zero or any positive integer (eg, where n is one).
在一些实施方案中，本发明提供的聚合物-脂质组合物包含下式的阳离子脂质：
In some embodiments, the polymer-lipid composition provided herein comprises a cationic lipid of the formula:
或其药学上可接受的盐，or a pharmaceutically acceptable salt thereof,
其中，p是1至9之间的整数，包括端值；Wherein, p is an integer between 1 and 9, inclusive;
R2的每个实例独立地为氢或任选取代的C1‐6烷基；Each instance of R 2 is independently hydrogen or optionally substituted C 1-6 alkyl;
R6和R7的每个实例独立地为式(i)、(ii)或(iii)的基团；Each instance of R 6 and R 7 is independently a group of formula (i), (ii) or (iii);
式(i)、(ii)和(iii)为：
Formulas (i), (ii) and (iii) are:
其中，R’的每个实例独立地为氢或任选取代的烷基；wherein each instance of R' is independently hydrogen or optionally substituted alkyl;
X为O、S或NRX，其中RX为氢、任选取代的烷基、任选取代的烯基、任选取代的炔基、任选取代的碳环基、任选取代的杂环基、任选取代的芳基、任选取代的杂芳基、或氮保护基团；X is O, S or NR x , wherein RX is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;
Y为O、S或NRY，其中RY为氢、任选取代的烷基、任选取代的烯基、任选取代的炔基、任选取代的碳环基、任选取代的杂环基、任选取代的芳基、任选取代的杂芳基、或氮保护基团；Y is O, S or NR Y , wherein RY is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group;
RP为氢、任选取代的烷基、任选取代的烯基、任选取代的炔基、任选取代的碳环基、任选取代的杂环基、任选取代的芳基、任选取代的杂芳基、连接至氧原子的氧保护基团、连接至硫原子的硫保护基团、或连接至氮原子的氮 保护基团；以及RL为任选取代的C1‐50烷基、任选取代的C2‐50烯基、任选取代的C2‐50炔基、任选取代的杂C1‐50烷基、任选取代的杂C2‐50烯基、任选取代的杂C2‐50炔基、或聚合物。R is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group attached to an oxygen atom, a sulfur protecting group attached to a sulfur atom, or a nitrogen protecting group attached to a nitrogen atom. protecting group; and RL is optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted heteroC1-50 alkyl, optionally substituted heteroC2-50 alkenyl, optionally substituted heteroC2-50 alkynyl, or a polymer.
在一些实施方案中，本发明的组合物和方法包括具有下式的阳离子脂质：
In some embodiments, the compositions and methods of the invention include a cationic lipid having the formula:
或其药学上可接受的盐，其中每个X独立地是O或S；每个Y独立地是O或S；每个m独立地为0至20；每个n独立为1至6；每个RA独立地是氢、任选地取代的C1‐50烷基、任选地取代的C2‐50烯基、任选地取代的C2‐50炔基、任选地取代的C3‐10碳环基、任选地取代的3‐14元杂环基、任选地取代的C6‐14芳基、任选地取代的5‐14元杂芳基或卤素；并且每个RB独立地是氢、任选地取代的C1‐50烷基、任选地取代的C2‐50烯基、任选地取代的C2‐50炔基、任选地取代的C3‐10碳环基、任选地取代的3‐14元杂环基、任选地取代的C6‐14芳基、任选地取代的5‐14元杂芳基或卤素。or a pharmaceutically acceptable salt thereof, wherein each X is independently O or S; each Y is independently O or S; each m is independently 0 to 20; each n is independently 1 to 6; each RA is independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl, or halogen; and each RB is independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl, or halogen.
用于本发明的组合物和方法的其他合适的阳离子脂质包括如PCT申请公开号WO2020097384A1中所述的阳离子脂质，该PCT申请的公开内容以引用的方式并入本文。在一些实施方案中，本发明的组合物和方法包括具有下式的阳离子脂质：
Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in PCT Application Publication No. WO2020097384A1, the disclosure of which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the formula:
或其药学上可接受的盐，其中每个R1和R2独立地是H或C1‐C6脂族；每个m独立地为具有1至4的值的整数；每个A独立地是共价键或亚芳基；每个L1独立地是酯、硫酯、二硫键或酸酐基团；每个L2独立地是C2‐C10脂族；每个X1独立地是H或OH；以及每个R3独立地是C6‐C20脂族。or a pharmaceutically acceptable salt thereof, wherein each R 1 and R 2 are independently H or C 1 -C 6 aliphatic; each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or an arylene group; each L 1 is independently an ester, a thioester, a disulfide bond or an anhydride group; each L 2 is independently C 2 -C 10 aliphatic; each X 1 is independently H or OH; and each R 3 is independently C 6 -C 20 aliphatic.
用于本发明的组合物和方法的其他合适的阳离子脂质包括如国际专利公开WO2017/075531中所述的阳离子脂质，该专利公开以引用的方式并入本文。在一些实施方案中，本发明的组合物和方法包括具有下式的阳离子脂质：
Other suitable cationic lipids for the compositions and methods of the present invention include cationic lipids as described in International Patent Publication WO2017/075531, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include cationic lipids having the formula:
或其药学上可接受的盐，其中L1或L2中的一者是‐O(C＝O)‐、‐(C＝O)O‐、‐C(＝O)‐、‐O‐、‐S(O)x、‐S‐S‐、‐C(＝O)S‐、‐SC(＝O)‐、‐NRaC(＝O)‐、‐C(＝O)NRa‐、NRaC(＝O)NRa‐、‐OC(＝O)NRa‐或‐NRaC(＝O)O‐；并且L1或L2中的另一者是‐O(C＝O)‐、‐(C＝O)O‐、‐C(＝O)‐、‐O‐、‐S(O)x、‐S‐S‐、‐C(＝O)S‐、SC(＝O)‐、‐NRaC(＝O)‐、‐C(＝O)NRa‐、NRaC(＝O)NRa‐、‐OC(＝O)NRa‐或‐NRaC(＝O)O‐或直接键；G1和G2各自独立地是未取代的C1‐C12亚烷基或C1‐C12亚烯基；G3是C1‐C24亚烷基、C1‐C24亚烯基、C3‐C8亚环烷基、C3‐C8亚环烯基；Ra是H或C1‐C12烷基；R1和R2各自独立地是C6‐C24烷基或C6‐C24烯基；R3是H、OR5、CN、‐C(＝O)OR4、‐OC(＝O)R4或‐NR5C(＝O)R4；R4是C1‐C12烷基；R5是H或C1‐C6烷基；并且x为0、1或2。or a pharmaceutically acceptable salt thereof, wherein one of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -S-S-, -C(=O)S- , -SC(=O)-, -NRaC (=O)-, -C(=O) NRa- , NRaC (=O)NRa-, -OC(=O) NRa- , or -NRaC (=O)O-; and the other of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -S-S-, -C(=O)S-, SC(=O)-, -NRaC (=O)-, -C(=O)NRa- , NRaC(=O) NRa- , -OC(=O) NRa -or -NR a C(＝O)O- or a direct bond; G 1 and G 2 are each independently unsubstituted C 1 －C 12 alkylene or C 1 －C 12 alkenylene; G 3 is C 1 －C 24 alkylene, C 1 －C 24 alkenylene, C 3 －C 8 cycloalkylene, C 3 －C 8 cycloalkenylene; Ra is H or C 1 －C 12 alkyl; R 1 and R 2 are each independently C 6 －C 24 alkyl or C 6 －C 24 alkenyl; R 3 is H, OR 5 , CN, -C(＝O)OR 4 , -OC(＝O)R 4 or -NR 5 C(＝O)R 4 ; R 4 is C 1 －C 12 alkyl; R 5 is H or C 1 －C 6 alkyl; and x is 0, 1 or 2.
用于本发明的组合物和方法的其他合适的阳离子脂质包括如国际专利公开WO2018/089790和WO2022/032154中所述的基于胆固醇的阳离子脂质，该专利公开以引用的方式并入本文。Other suitable cationic lipids for use in the compositions and methods of the invention include cholesterol-based cationic lipids as described in International Patent Publications WO2018/089790 and WO2022/032154, which are incorporated herein by reference.
在某些实施方案中，本发明的组合物和方法包括具有下式结构的化合物：
B-L1-S，In certain embodiments, the compositions and methods of the invention include a compound having the following structure:
BL 1 -S,
或其药学上可接受的盐，or a pharmaceutically acceptable salt thereof,
其中，in,
B是碱性官能团，其中所述质子化形式具有不超过约8.0的pKa； B is a basic functional group, wherein the protonated form has a pKa of no more than about 8.0;
L1是任选经取代的连接基团，其为C1-C20亚烷基或2至20元亚杂烷基；L 1 is an optionally substituted linking group, which is a C 1 -C 20 alkylene group or a 2 to 20-membered heteroalkylene group;
且S是固醇。And S is a sterol.
优选地，B是任选经取代的5元或6元含氮杂芳基。B是选自吡咯基、咪唑基、吡唑基、三唑基、四唑基、吡啶基、嘧啶基、吡嗪基和哒嗪基的基团，其中的每一个任选经取代。Preferably, B is an optionally substituted 5-membered or 6-membered nitrogen-containing heteroaryl. B is a group selected from pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl, each of which is optionally substituted.
优选地，L1为-X1-C(X3)-X2、-(C1-C19亚烷基)-X1-C(X3)-X2、-X1-C(X3)-X2(C1-C19亚烷基)-、-(C1-C19亚烷基)-X1-、-X1-(C1-C19亚烷基)-，其中每个X1和X2独立地为共价键、-O-、-S-或-NH-；X3独立地为＝O、＝S或＝NH；且其中所述C1-C19亚烷基任选经取代。Preferably, L 1 is -X 1 -C(X 3 )-X 2 , -(C 1 -C 19 alkylene)-X 1 -C(X 3 )-X 2 , -X 1 -C(X 3 )-X 2 (C 1 -C 19 alkylene)-, -(C 1 -C 19 alkylene)-X1-, -X1-(C1-C19 alkylene)-, wherein each X 1 and X 2 is independently a covalent bond, -O-, -S- or -NH-; X 3 is independently =O, =S or =NH; and wherein the C 1 -C 19 alkylene is optionally substituted.
更优选地，L1包含为酯基、酰胺基、氨基甲酸酯基、碳酸酯基或脲基的部分。且L1不包含具有结构-N(R')2的取代基，或其带正电形式，其中每个R'独立地为氢或任选经取代的C1-C20烷基。More preferably, L1 comprises a moiety that is an ester, amide, carbamate, carbonate or urea group, and L1 does not comprise a substituent having the structure -N(R') 2 , or a positively charged form thereof, wherein each R' is independently hydrogen or an optionally substituted C1 - C20 alkyl group.
优选地，其中S是动物固醇，或其氧化或还原形式；S是植物固醇，或其氧化或还原形式。S是合成固醇，或其氧化或还原形式。Preferably, S is a zoosterol, or an oxidized or reduced form thereof; S is a phytosterol, or an oxidized or reduced form thereof; S is a synthetic sterol, or an oxidized or reduced form thereof.
更优选地，S是选自胆固醇、胆固醇的氧化形式、胆固醇的还原形式、烷基石胆酸盐、豆固醇、豆固烷醇、菜油固醇、麦角固醇和谷固醇的固醇。More preferably, S is a sterol selected from the group consisting of cholesterol, an oxidized form of cholesterol, a reduced form of cholesterol, an alkyl lithochlate, stigmasterol, stigmasterol, campesterol, ergosterol and sitosterol.
在某些实施方案中，本发明的组合物和方法包括具有下式结构的化合物，
In certain embodiments, the compositions and methods of the invention include a compound having the structure,
或其药学上可接受的盐，or a pharmaceutically acceptable salt thereof,
其中，n是0或1，优选地是0，wherein n is 0 or 1, preferably 0,
R1是基团–(CH2)q-NH2或基团–(CH2)r-NH-(CH2)s-NH2，R 1 is a group –(CH 2 ) q -NH 2 or a group –(CH 2 ) r -NH-(CH 2 ) s -NH 2 ,
其中q、r和s独立地是2至6的整数，wherein q, r and s are independently integers from 2 to 6,
R2是基团–(CH2)t-NH2或基团–(CH2)u-NH-(CH2)w-NH2，R 2 is a group –(CH 2 ) t -NH 2 or a group –(CH 2 ) u -NH-(CH 2 ) w -NH 2 ,
其中t、u和w独立地是2至6的整数，wherein t, u and w are independently integers from 2 to 6,
R3是具有1至4个碳原子的直链烷二基； R3 is a straight chain alkanediyl group having 1 to 4 carbon atoms;
在一些实施方案中，作为本文所述的阳离子脂质的代替或除本文所述的阳离子脂质之外，可以使用基于胆固醇(甾醇)的阳离子脂质。合适的基于甾醇的阳离子脂质是含二烷基氨基、咪唑、碱性氨基酸序列和胍的基于甾醇的阳离子脂质。例如，某些实施方案涉及包含一种或多种包含咪唑的基于甾醇的阳离子脂质的组合物，例如咪唑胆固醇酯或“ICE”脂质(3S,10R,13R,17R)‐10,13‐二甲基‐17‐((R)‐6‐甲基庚烷‐2‐基)‐2,3,4,7,8,9,10,11,12,13,14,15,16,17‐十四氢‐1H‐环戊二烯并[a]菲‐3‐基3‐(1H‐咪唑‐4‐基)丙酸酯。In some embodiments, as a replacement of cationic lipids as described herein or in addition to cationic lipids as described herein, cationic lipids based on cholesterol (sterol) can be used. Suitable cationic lipids based on sterols are cationic lipids based on sterols containing dialkylamino, imidazoles, basic amino acid sequences and guanidine. For example, some embodiments relate to compositions comprising one or more cationic lipids based on sterols comprising imidazoles, such as imidazole cholesterol ester or "ICE" lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopentadien[a]phenanthrene-3-yl 3-(1H-imidazole-4-yl) propionate.
在本发明的组合物的一些实施方案中，所述胆固醇衍生的阳离子脂质包含选自以下的至少一种：DC‐Choi(N,N‐二甲基‐N‐乙基甲酰胺胆固醇)、1,4-双(3‐N‐油基氨基‐丙基)哌嗪(Gao,等人.Biochem.Biophys.Res.Comm.179,280(1991)；Wolf等人.BioTechniques 23,139(1997)；美国专利号5,744,335)、N4-精氨胆固醇羰酰氨(GL67)、与碱性氨基酸序列偶联的胆固醇衍生物、咪唑胆固醇酯(ICE)以及它们的衍生物。In some embodiments of the composition of the present invention, the cholesterol-derived cationic lipid comprises at least one selected from the following: DC-Choi (N,N-dimethyl-N-ethylformamide cholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Patent No. 5,744,335), N4-arginine cholesterol carbonylamide (GL67), cholesterol derivatives coupled to basic amino acid sequences, imidazole cholesterol ester (ICE) and their derivatives.
用于本发明的组合物和方法的其他合适的阳离子脂质包括如国际专利公开WO2012/170889中所述的可切割阳离子脂质，该专利公开以引用的方式并入本文。在一些实施方案中，本发明的组合物和方法包括具有下式的阳离子脂质：
Other suitable cationic lipids for use in the compositions and methods of the present invention include cleavable cationic lipids as described in International Patent Publication WO2012/170889, which is incorporated herein by reference. In some embodiments, the compositions and methods of the present invention include a cationic lipid having the formula:
其中R1选自由以下项组成的组：咪唑、胍、氨基、亚胺、烯胺、任选地取代的烷基氨基(例如，烷基氨基，诸如二甲基氨基)和吡啶基；其中R2选自由以下两个通式之一组成的组：
wherein R 1 is selected from the group consisting of imidazole, guanidine, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino such as dimethylamino) and pyridyl; wherein R 2 is selected from the group consisting of one of the following two general formulae:
并且其中R3和R4各自独立地选自由以下项组成的组：任选地取代的不同饱和或不饱和的C6‐C20烷基和任选地取代的不同饱和或不饱和的C6‐C20酰基； 并且其中n为零或任何正整数(例如，一、二、三、四、五、六、七、八、九、十、十一、十二、十三、十四、十五、十六、十七、十八、十九、二十或更多)。and wherein R 3 and R 4 are each independently selected from the group consisting of: optionally substituted different saturated or unsaturated C 6 _C 20 alkyl groups and optionally substituted different saturated or unsaturated C 6 _C 20 acyl groups; and wherein n is zero or any positive integer (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more).
用于本发明的组合物和方法的其他合适的阳离子脂质包括可降解的阳离子脂质，如PCT申请公开号WO2019222424A1中所述，该PCT申请以引用的方式并入本文。在某些实施方案中，本发明的组合物和方法包括阳离子脂质，所述阳离子脂质为WO2019222424A1中所述的通式中的任一者或者结构(1a)‐(21a)和(1b)‐(21b)和(22)‐(237)。在某些实施方案中，本发明的组合物和方法包括具有根据下式的结构的阳离子脂质，
Other suitable cationic lipids for the compositions and methods of the present invention include degradable cationic lipids, as described in PCT Application Publication No. WO2019222424A1, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention include cationic lipids, which are any of the general formulas described in WO2019222424A1 or structures (1a)-(21a) and (1b)-(21b) and (22)-(237). In certain embodiments, the compositions and methods of the present invention include cationic lipids having a structure according to the formula,
及其药学上可接受的盐。其中RX独立地为‐H、‐L1‐R1或–L5A‐L5B‐B’；L1、L2和L3各自独立为共价键、‐C(O)‐、‐C(O)O‐、‐C(O)S‐或‐C(O)NRL‐；每个L4A和L5A独立地为‐C(O)‐、‐C(O)O‐或‐C(O)NRL‐；每个L4B和L5B独立地为C1‐C20亚烷基；C2‐C20亚烯基；或C2‐C20亚炔基；每个B和B’是NR4R5或5‐10元含氮杂芳基；每个R1、R2和R3独立地为C6‐C30烷基、C6‐C30链烯基或C6‐C30炔基；每个R4和R5独立地为氢、C1‐C10烷基；C2‐C10链烯基；或C2‐C10炔基；并且每个RL独立地为氢，C1‐C20烷基、C2‐C20链烯基或C2‐C20炔基。and pharmaceutically acceptable salts thereof. wherein RX is independently -H, -L1-R1 or -L5A-L5B-B'; L1 , L2 and L3 are each independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S- or -C(O)NRL-; each L4A and L5A are independently -C(O)-, -C(O)O- or -C(O)NRL-; each L4B and L5B are independently C1 -C20 alkylene; C2 - C20 alkenylene; or C2 -C20 alkynylene; each B and B' is NR4R5 or a 5-10 membered nitrogen-containing heteroaryl group; each R1, R2 and R3 are independently C6 - C30 alkyl, C6-C30 alkenyl or C6- C30 alkynyl; each R4 and R5 are independently C6- C30 alkyl, C6 - C30 alkenyl or C6 -C30 alkynyl; each R4 and R5 are independently C1 -C20 alkyl, C2 -C20 alkenyl or C2 - C20 alkynyl. 5 is independently hydrogen, C 1 -C 10 alkyl; C 2 -C 10 alkenyl; or C 2 -C 10 alkynyl; and each RL is independently hydrogen, C 1 -C 20 alkyl, C 2 -C 20 alkenyl or C 2 -C 20 alkynyl.
在某些实施方案中，本发明的组合物和方法所述阳离子脂质是如国际专利公开WO2020051220A1中所述的树枝状聚合物或树枝块(dendron)，该专利公开以引用的方式并入本文。In certain embodiments, the cationic lipid in the compositions and methods of the present invention is a dendritic polymer or dendron as described in International Patent Publication No. WO2020051220A1, which is incorporated herein by reference.
在某些实施方案中，本发明的组合物和方法所述阳离子脂质本包含一种或多种具有如下通式的脂质修饰精胺衍生物：
In certain embodiments, the cationic lipid compositions and methods of the present invention comprise one or more lipid-modified spermine derivatives having the following general formula:
其中，X1为-(CH2)n-或羰基，其中n为1，2或3；wherein X 1 is -(CH 2 ) n - or a carbonyl group, wherein n is 1, 2 or 3;
X2选自-(CH2)-、酯基、酰胺基、氧或硫；X 2 is selected from -(CH 2 )-, an ester group, an amide group, oxygen or sulfur;
R1和R2独立地选自C6-C18烷基、含有烯键的C6-C18烷基或亲脂性胆固醇类分子； R1 and R2 are independently selected from C6 - C18 alkyl, C6 - C18 alkyl containing olefinic bonds or lipophilic cholesterol molecules;
进一步地，X1和X2均为-(CH2)-，R1和R2独立地选自C10-C18烷基。Further, X1 and X2 are both -( CH2 )-, and R1 and R2 are independently selected from C10 - C18 alkyl groups.
进一步地，X1为-(CH2)2-，X2为氧，R1和R2为相同的C12-C18烷基或含有烯键的C6-C18烷基。Further, X 1 is -(CH 2 ) 2 -, X 2 is oxygen, and R 1 and R 2 are the same C 12 -C 18 alkyl group or a C 6 -C 18 alkyl group containing an olefinic bond.
优选地，X1为羰基，X2为-(CH2)-，R1和R2为相同的C12-C18烷基或含有烯键的C6-C18烷基。Preferably, X 1 is a carbonyl group, X 2 is -(CH 2 )-, and R 1 and R 2 are the same C 12 -C 18 alkyl group or a C 6 -C 18 alkyl group containing an olefinic bond.
优选地，X1为-(CH2)2-，X2为酯基，R1和R2为相同的C12-C18烷基或含有烯键的C6-C18烷基。Preferably, X 1 is -(CH 2 ) 2 -, X 2 is an ester group, and R 1 and R 2 are the same C 12 -C 18 alkyl group or a C 6 -C 18 alkyl group containing an olefinic bond.
优选地，X1为-(CH2)-，X2为酰胺基，R1和R2为相同的C12-C18烷基或含有烯键的C6-C18烷基。Preferably, X 1 is -(CH 2 )-, X 2 is an amide group, and R 1 and R 2 are the same C 12 -C 18 alkyl group or a C 6 -C 18 alkyl group containing an olefinic bond.
或其药学上可接受的盐。or a pharmaceutically acceptable salt thereof.
本发明的脂质修饰精胺衍生物由精胺和油醇通过不同的化学键偶联组成。精胺作为带正电荷的头基，而油醇链接在位于中间的两个三级胺基。The lipid-modified spermine derivative of the invention is composed of spermine and oleyl alcohol coupled by different chemical bonds, wherein spermine is used as a head group with positive charge, and oleyl alcohol is linked to two tertiary amine groups in the middle.
申请公布号为CN104876831的专利教导了可用于本公开内容中的脂质修饰精胺衍生物的一些非限制性例子，所述文献通过引用并入本文。Patent application publication number CN104876831 teaches some non-limiting examples of lipid-modified spermine derivatives that can be used in the present disclosure, and the patent application publication number CN104876831 is incorporated herein by reference.
在一些实施方案中，本发明的组合物和方法包括阳离子脂质N‐[l‐(2,3‐二油烯基氧基)丙基]‐N,N,N‐三甲基氯化铵(“DOTMA”)(Feigner等人，Proc.Natl.Acad.Sci.84,7413(1987)；美国专利号4,897,355，该专利以引用方式并入本文)。适用于本发明的组合物和方法的其他阳离子脂质包括例如5‐羧基精胺基甘氨酸二十八烷基酰胺(“DOGS”)；2,3‐二油烯基氧基‐N‐[2‐(精胺‐羧胺)乙基]‐N,N‐二甲基‐1‐丙铵(“DOSPA”)(Behr等人，Proc.Natl.Acad.Sci.86,6982(1989)，美国专利号5,171,678；美国专利号5,334,761)；1,2‐二油酰基‐3‐二甲基铵‐丙烷(“DODAP”)；1,2‐二油酰基‐3‐三甲基铵‐丙烷(“DOTAP”)。In some embodiments, the compositions and methods of the invention include the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA") (Feigner et al., Proc. Natl. Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, which is incorporated herein by reference). Other cationic lipids suitable for use in the compositions and methods of the present invention include, for example, 5-carboxysperminylglycine dioctadecylamide ("DOGS"); 2,3-dioleyloxy-N-[2-(spermine-carboxyamide)ethyl]-N,N-dimethyl-1-propylammonium ("DOSPA") (Behr et al., Proc. Natl. Acad. Sci. 86, 6982 (1989), U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761); 1,2-dioleoyl-3-dimethylammonium-propane ("DODAP"); 1,2-dioleoyl-3-trimethylammonium-propane ("DOTAP").
适用于本发明的组合物和方法的另外的示例性阳离子脂质还包括：1,2‐二硬脂酰基氧基‐N,N‐二甲基‐3‐氨基丙烷(“DSDMA”)；1,2‐二油烯基氧基‐N,N‐二甲基‐3‐氨基丙烷(“DODMA”)；1,2‐二亚油基氧基‐N,N‐二甲基‐3‐氨基丙烷(“DLinDMA”)；1,2‐二亚油烯基氧基‐N,N‐二甲基‐3‐氨基丙烷(“DLenDMA”)；N‐二油烯基‐N,N‐二甲基氯化铵(“DODAC”)；N,N‐二硬脂酰基‐N,N‐二甲基溴化铵(“DDAB”)；N‐(1,2‐二肉豆蔻基氧基丙‐3‐基)‐N,N‐二甲基‐N‐羟乙基溴化铵(“DMRIE”)；3‐二甲基氨基‐2‐(胆甾‐5‐烯‐3‐β‐氧基丁烷‐4‐氧基)‐1‐(顺式,顺式‐9,12‐十八碳二烯氧基)丙烷(“CLinDMA”)；2‐[5'‐(胆甾‐5‐烯‐3‐β‐氧基)‐3'‐氧杂戊氧基)‐3‐二甲基‐1‐(顺式,顺式‐9',12'‐十八碳二烯氧基)丙烷(“CpLinDMA”)； N,N‐二甲基‐3,4‐二油烯基氧基苄胺(“DMOBA”)；1,2‐N,N'‐二油烯基氨甲酰基‐3‐二甲基氨基丙烷(“DOcarbDAP”)；2,3‐二亚油酰基氧基‐N,N‐二甲基丙基胺(“DLinDAP”)；1,2‐N,N'‐二亚油基氨甲酰基‐3‐二甲基氨基丙烷(“DLincarbDAP”)；1,2‐二亚油酰基氨甲酰基‐3‐二甲基氨基丙烷(“DLinCDAP”)；2,2‐二亚油基‐4‐二甲基氨基甲基‐[1,3]‐二氧戊环(“DLin‐K‐DMA”)；2‐((8‐[(3P)‐胆甾‐5‐烯‐3‐基氧基]辛基)氧基)‐N,N‐二甲基‐3‐[(9Z,12Z)‐十八碳‐9,12‐二烯‐1‐基氧基]丙烷‐1‐胺(“辛基‐CLinDMA”)；(2R)‐2‐((8‐[(3β)‐胆甾‐5‐烯‐3‐基氧基]辛基)氧基)‐N,N‐二甲基‐3‐[(9Z,12Z)‐十八碳‐9,12‐二烯‐1‐基氧基]丙烷‐1‐胺(“辛基‐CLinDMA(2R)”)；(2S)‐2‐((8‐[(3P)‐胆甾‐5‐烯‐3‐基氧基]辛基)氧基)‐N,fsl‐二甲基3‐[(9Z,12Z)‐十八碳‐9,12‐二烯‐1‐基氧基]丙烷‐1‐胺(“辛基‐CLinDMA(2S)”)；2,2‐二亚油基‐4‐二甲基氨基乙基‐[l,3]‐二氧戊环(“DLin‐K‐XTC2‐DMA”)；和2‐(2,2‐二((9Z,12Z)‐十八碳‐9,l2‐二烯‐1‐基)‐l,3‐二氧戊环‐4‐基)‐N,N‐二甲基乙胺(“DLin‐KC2‐DMA”)(参见，WO2010/042877，该专利以引用的方式并入本文；Semple等人.,Nature Biotech.28:172‐176(2010)；Heyes,J.,等人.,J Controlled Release 107:276‐287(2005)；Morrissey,DV.等人.,Nat.Biotechnol.23(8):1003‐1007(2005)；国际专利公开WO2005/121348)。在一些实施方案中，一种或多种阳离子脂质包含咪唑、二烷基氨基或胍鎓部分中的至少一种。Additional exemplary cationic lipids suitable for use in the compositions and methods of the present invention include: 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"); N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N,N-distearoyl- N,N-dimethylammonium bromide ("DDAB"); N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"); 3-dimethylamino-2-(cholest-5-en-3-β-oxybutane-4-oxy)-1-(cis, cis-9,12-octadecadienyloxy)propane ("CLinDMA");2-[5'-(cholest-5-en-3-β-oxy)-3'-oxapentyloxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienyloxy)propane("CpLinDMA"); N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA");1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane("DOcarbDAP"); 2,3-dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP");1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane("DLincarbDAP"); 1,2-dilinoleoylcarbamoyl-3-dimethylaminopropane ("DLinCDAP”); 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (“DLin-K-DMA”); 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (“octyl-CLinDMA”); (2R)-2-((8-[(3β)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (“octyl-CLinDMA”); ((2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,fsl-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-1-amine ("octyl-CLinDMA(2R)"); (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,fsl-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-1-amine ("octyl-CLinDMA(2S)") ; 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane ("DLin-K-XTC2-DMA"); and 2-(2,2-di((9Z,12Z)-octadecane-9,12-dien-1-yl)-l,3-dioxolane-4-yl)-N,N-dimethylethylamine ("DLin-KC2-DMA") (see, WO2010/042877, which is incorporated herein by reference; Semple et al., Nature Biotech. 28: 172-176 (2010); Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, DV. et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); International Patent Publication No. WO2005/121348). In some embodiments, the one or more cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.
在一些实施方案中，适用于本发明的组合物和方法的一种或多种阳离子脂质包括2,2‐二亚油基‐4‐二甲基氨基乙基‐[1,3]‐二氧戊环(“XTC”)；(3aR,5s,6aS)‐N,N‐二甲基‐2,2‐二((9Z,12Z)‐十八碳‐9,12‐二烯基)四氢‐3aH‐环戊二烯并[d][1,3]二氧杂环戊烯‐5‐胺(“ALNY‐100”)和/或4,7,13‐三(3‐氧代‐3‐(十一烷基氨基)丙基)‐N1,N16‐二‐十一烷基‐4,7,10,13‐四氮杂十六烷‐1,16‐二酰胺(“NC98‐5”)。In some embodiments, one or more cationic lipids suitable for use in the compositions and methods of the invention include 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("XTC"); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadecane-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine ("ALNY-100") and/or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-di-undecyl-4,7,10,13-tetraazahexadecane-1,16-diamide ("NC98-5").
在一些实施方案中，本发明的组合物和方法的其他合适的阳离子脂质包括下式所示的化合物及其药学上可接受的盐：
In some embodiments, other suitable cationic lipids for the compositions and methods of the present invention include compounds of the following formula and pharmaceutically acceptable salts thereof:
其中， in,
RCOO选自包含以下的列表：肉豆蔻酰基、α-D-生育酚琥珀酰基、亚油酰基和油酰基；和X选自包含以下的列表：
RCOO is selected from the list comprising: myristoyl, alpha-D-tocopheryl succinyl, linoleoyl, and oleoyl; and X is selected from the list comprising:
用于本发明的组合物和方法的其他合适的阳离子脂质包括在国际专利公开WO2010/053572、WO2013/063468、WO2015/184256、WO2015/199952、WO2015/095340、WO2016/118725、WO2016/205691、WO2016/004202、WO2017/004143、WO2017/117528、WO2017/049245、WO2017/173054中描述的阳离子脂质，该专利公开以引用的方式并入本文。Other suitable cationic lipids for use in the compositions and methods of the invention include those described in International Patent Publications WO2010/053572, WO2013/063468, WO2015/184256, WO2015/199952, WO2015/095340, WO2016/118725, WO2016/205691, WO2016/004202, WO2017/004143, WO2017/117528, WO2017/049245, WO2017/173054, which are incorporated herein by reference.
用于本发明的组合物和方法的其他合适的阳离子脂质包括如J.McClellan,M.C.King,Cell 2010,141,210‐217和Whitehead等人，Nature Communications(2014)5:4277中所述的阳离子脂质，这些文献以引用的方式并入本文。Other suitable cationic lipids for use in the compositions and methods of the present invention include those described in J. McClellan, M.C. King, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference.
在本发明的组合物的一些实施方案中，所述阳离子脂质包含选自以下的至少一种：永久阳离子脂质、可电离的阳离子脂质、胆固醇衍生的阳离子脂质和树枝状聚合物或树枝块(dendron)。在优选的实施方案中，所述阳离子脂质包含可电离的阳离子脂质。In some embodiments of the compositions of the present invention, the cationic lipid comprises at least one selected from the group consisting of permanent cationic lipids, ionizable cationic lipids, cholesterol-derived cationic lipids, and dendrimers or dendrons. In preferred embodiments, the cationic lipid comprises an ionizable cationic lipid.
在本发明的组合物的一些实施方案中，所述阳离子脂质可以含有一个或多个不对称取代的碳或氮原子，并且可以以光学活性的或外消旋的形式分离。因而，除非特别指出具体的立体化学或异构形式，否则意指化学式的所有手性、非对映异构、外消旋形式、差向异构形式和所有几何异构形式。阳离子脂质可以作为外消旋体和外消旋混合物、单一对映异构体、非对映异构体混合物和各非对映异构体存在。在一些实施方案中，获得单一非对映异构体。本发明所述的阳离子脂质的手性中心可以具有S或R构型。此外，预期阳离子脂质中的一种或多种可以作为结构异构体存在。在一些实施方案中，所述化合物具有相同的化学式，但与核心的氮原子的连接性不同。不希望受任何理论约束，据信这样的阳离子脂质存在，因为起始单体首先与伯胺反应，且然后在统计学上与存在的任何仲胺反应。因此，结构异构体可以呈现完全反应的伯胺，且然后呈现反应的仲胺的混合物。 In some embodiments of the composition of the present invention, the cationic lipid may contain one or more asymmetrically substituted carbon or nitrogen atoms, and may be separated in an optically active or racemic form. Thus, unless a specific stereochemistry or isomeric form is specifically indicated, all chirality, diastereoisomerism, racemic form, epimeric form, and all geometric isomeric forms of a chemical formula are meant. Cationic lipids may exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and each diastereomer. In some embodiments, a single diastereomer is obtained. The chiral center of the cationic lipid of the present invention may have an S or R configuration. In addition, it is expected that one or more of the cationic lipids may exist as structural isomers. In some embodiments, the compound has the same chemical formula, but is different from the connectivity of the nitrogen atom of the core. Without wishing to be bound by any theory, it is believed that such a cationic lipid exists because the starting monomer first reacts with a primary amine, and then statistically reacts with any secondary amine present. Therefore, structural isomers may present a fully reacted primary amine, and then present a mixture of reacted secondary amines.
用于表示本发明所述的阳离子脂质的化学式通常将仅显示可能的几种不同互变异构体中的一种。例如，已知许多类型的酮基与相应的烯醇基平衡存在。类似地，许多类型的亚胺基与烯胺基平衡存在。无论对于给定式描绘哪种互变异构体，并且不管哪种互变异构体是最普遍的，意指给定化学式的所有互变异构体。Chemical formula for representing cationic lipid of the present invention will usually only show one of several possible different tautomers.For example, many types of keto groups are known to exist in equilibrium with the corresponding enol groups.Similarly, many types of imino groups exist in equilibrium with the enamine groups.Whether for a given formula, which tautomer is described, and whether which tautomer is the most common, all tautomers of a given chemical formula are meant.
另外，构成本发明所述的阳离子脂质的原子意图包括这样的原子的所有同位素形式。本文中使用的同位素包括具有相同原子序数但是具有不同质量数的那些原子。作为一般示例而非限制性地，氢的同位素包括氚和氘，且碳的同位素包括13C和14C。In addition, the atoms constituting the cationic lipids of the present invention are intended to include all isotopic forms of such atoms. Isotopes used herein include those atoms with the same atomic number but with different mass numbers. As a general example and not limitation, the isotopes of hydrogen include tritium and deuterium, and the isotopes of carbon include 13 C and 14 C.
应当认识到，形成本文所提供的阳离子型可电离脂质的任何盐形式的一部分的特定阴离子或阳离子不是至关重要的，只要该盐整体上是药理学上可接受的即可。药学上可接受的盐的其它例子和它们的制备方法与使用方法呈现于Handbook of Pharmaceutical Salts:Properties,and Use(2002)中，其通过引用并入本文。It should be recognized that the specific anion or cation that forms part of any salt form of the cationic ionizable lipids provided herein is not critical so long as the salt as a whole is pharmacologically acceptable. Other examples of pharmaceutically acceptable salts and methods for their preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.
在一些实施方案中，所述阳离子脂质占所述组合物中存在的总脂质的约23mol％-约83mol％。In some embodiments, the cationic lipid comprises about 23 mol% to about 83 mol% of the total lipids present in the composition.
在一些实施方案中，所述阳离子脂质占所述组合物中存在的总脂质的约25mol％-约80mol％、例如约30mol％-约80mol％、约35mol％-约80mol％、约40mol％-约80mol％、约45mol％-约80mol％、约50mol％-约80mol％、约55mol％-约80mol％、约60mol％-约80mol％、约65mol％-约80mol％、约70mol％-约80mol％或约75mol％-约80mol％。In some embodiments, the cationic lipid accounts for about 25 mol% to about 80 mol%, e.g., about 30 mol% to about 80 mol%, about 35 mol% to about 80 mol%, about 40 mol% to about 80 mol%, about 45 mol% to about 80 mol%, about 50 mol% to about 80 mol%, about 55 mol% to about 80 mol%, about 60 mol% to about 80 mol%, about 65 mol% to about 80 mol%, about 70 mol% to about 80 mol%, or about 75 mol% to about 80 mol% of the total lipids present in the composition.
在一些实施方案中，所述阳离子脂质占所述组合物中存在的总脂质的约25mol％-约70mol％、例如约30mol％-约70mol％、约35mol％-约70mol％、约40mol％-约70mol％、约45mol％-约70mol％、约50mol％-约70mol％、约55mol％-约70mol％、约60mol％-约70mol％或约65mol％-约70mol％。In some embodiments, the cationic lipid accounts for about 25 mol% to about 70 mol%, e.g., about 30 mol% to about 70 mol%, about 35 mol% to about 70 mol%, about 40 mol% to about 70 mol%, about 45 mol% to about 70 mol%, about 50 mol% to about 70 mol%, about 55 mol% to about 70 mol%, about 60 mol% to about 70 mol%, or about 65 mol% to about 70 mol% of the total lipids present in the composition.
在一些实施方案中，所述阳离子脂质占所述组合物中存在的总脂质的约25mol％-约60mol％、例如约30mol％-约60mol％、约35mol％-约60mol％、约40mol％-约60mol％、约45mol％-约60mol％、约50mol％-约60mol％或约55mol％-约60mol％。In some embodiments, the cationic lipid accounts for about 25 mol% to about 60 mol%, such as about 30 mol% to about 60 mol%, about 35 mol% to about 60 mol%, about 40 mol% to about 60 mol%, about 45 mol% to about 60 mol%, about 50 mol% to about 60 mol%, or about 55 mol% to about 60 mol% of the total lipids present in the composition.
在一些实施方案中，所述阳离子脂质占所述组合物中存在的总脂质的约25mol％-约50mol％、例如约30mol％-约50mol％、约35mol％-约50mol％、 约40mol％-约50mol％或约45mol％-约50mol％。In some embodiments, the cationic lipid accounts for about 25 mol% to about 50 mol%, such as about 30 mol% to about 50 mol%, about 35 mol% to about 50 mol%, or about 50 mol% of the total lipids present in the composition. About 40 mol% to about 50 mol% or about 45 mol% to about 50 mol%.
在一些实施方案中，所述阳离子脂质占所述组合物中存在的总脂质的约23mol％、25mol％、约30mol％、约35mol％、约40mol％、约45mol％、约50mol％、约55mol％、约60mol％、约65mol％、约70mol％、约75mol％、约80mol％或约83mol％。In some embodiments, the cationic lipid comprises about 23 mol%, 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, or about 83 mol% of the total lipids present in the composition.
本领域中技术人员应该清楚的是，取决于该颗粒预期的应用，组合物的组分比例可以变化并且具体制剂的递送效率可以利用，例如，内涵体释放参数(ERP)测定或/和体内基因转染效率(如负载核酸的情况下)测定等来测量。It will be clear to those skilled in the art that, depending on the intended application of the particles, the proportions of the components of the composition may vary and the delivery efficiency of a particular formulation may be measured using, for example, endosomal release parameter (ERP) assays and/or in vivo gene transfection efficiency (such as in the case of nucleic acid loading) assays, etc.
非阳离子/辅助脂质Non-cationic/helper lipids
在本发明的包含聚合物-脂质的组合物的一些实施方案中，所述非阳离子脂质包含选自以下的至少一种：阴离子脂质、两性离子脂质和中性脂质，优选地，所述非阳离子脂质包含中性脂质。如本文所用，短语“非阳离子脂质”是指任何中性、两性离子或阴离子脂质。如本文所用，短语“阴离子脂质”是指在选定pH诸如生理pH下携带净负电荷的多种脂质物质中的任何一种。In some embodiments of the compositions comprising polymer-lipids of the present invention, the non-cationic lipid comprises at least one selected from the group consisting of anionic lipids, zwitterionic lipids and neutral lipids, preferably, the non-cationic lipid comprises a neutral lipid. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a variety of lipid species that carry a net negative charge at a selected pH such as a physiological pH.
在一些实施方案中，所述中性脂质占所述组合物中存在的总脂质的19mol％-75mol％，例如25mol％-70mol％、30mol％-70mol％、35mol％-70mol％、40mol％-70mol％、45mol％-70mol％、50mol％-65mol％或55mol％-60mol％。In some embodiments, the neutral lipids account for 19 mol%-75 mol% of the total lipids present in the composition, such as 25 mol%-70 mol%, 30 mol%-70 mol%, 35 mol%-70 mol%, 40 mol%-70 mol%, 45 mol%-70 mol%, 50 mol%-65 mol% or 55 mol%-60 mol%.
在一些实施方案中，所述中性脂质占所述组合物中存在的总脂质的20mol％-65mol％，例如25mol％-65mol％、30mol％-65mol％、35mol％-65mol％、40mol％-65mol％、45mol％-65mol％、50mol％-65mol％或55mol％-65mol％。In some embodiments, the neutral lipids account for 20 mol%-65 mol% of the total lipids present in the composition, e.g., 25 mol%-65 mol%, 30 mol%-65 mol%, 35 mol%-65 mol%, 40 mol%-65 mol%, 45 mol%-65 mol%, 50 mol%-65 mol% or 55 mol%-65 mol%.
在一些实施方案中，所述中性脂质占所述组合物中存在的总脂质的20mol％-60mol％，例如25mol％-60mol％、30mol％-60mol％、35mol％-60mol％、40mol％-60mol％、45mol％-60mol％、50mol％-60mol％或55mol％-60mol％。In some embodiments, the neutral lipids account for 20 mol%-60 mol% of the total lipids present in the composition, e.g., 25 mol%-60 mol%, 30 mol%-60 mol%, 35 mol%-60 mol%, 40 mol%-60 mol%, 45 mol%-60 mol%, 50 mol%-60 mol% or 55 mol%-60 mol%.
在一些实施方案中，所述中性脂质占所述组合物中存在的总脂质的20mol％-55mol％，例如25mol％-55mol％、30mol％-55mol％、35mol％-55mol％、40mol％-55mol％、45mol％-55mol％、50mol％-55mol％，或20mol％-50mol％，例如25mol％-50mol％、30mol％-50mol％、35mol％-50mol％、40mol％-50mol％或45mol％-50mol％。In some embodiments, the neutral lipids account for 20 mol%-55 mol%, e.g., 25 mol%-55 mol%, 30 mol%-55 mol%, 35 mol%-55 mol%, 40 mol%-55 mol%, 45 mol%-55 mol%, 50 mol%-55 mol%, or 20 mol%-50 mol%, e.g., 25 mol%-50 mol%, 30 mol%-50 mol%, 35 mol%-50 mol%, 40 mol%-50 mol% or 45 mol%-50 mol%, of the total lipids present in the composition.
在一些实施方案中，所述中性脂质占所述组合物中存在的总脂质的20 mol％-45mol％，例如25mol％-45mol％、30mol％-45mol％、35mol％-45mol％或40mol％-45mol％，或20mol％-40mol％，例如25mol％-40mol％、30mol％-40mol％或35mol％-40mol％。In some embodiments, the neutral lipids account for 20% of the total lipids present in the composition. mol%-45mol%, for example 25mol%-45mol%, 30mol%-45mol%, 35mol%-45mol% or 40mol%-45mol%, or 20mol%-40mol%, for example 25mol%-40mol%, 30mol%-40mol% or 35mol%-40mol%.
在一些实施方案中，所述中性脂质包含：In some embodiments, the neutral lipid comprises:
胆固醇或胆固醇衍生的中性脂质；Cholesterol or cholesterol-derived neutral lipids;
磷脂；或phospholipids; or
胆固醇或胆固醇衍生的中性脂质和磷脂的混合物。A mixture of cholesterol or cholesterol-derived neutral lipids and phospholipids.
在一些实施方案中，所述胆固醇占所述组合物中的总脂质的14mol％-70mol％，例如20mol％-70mol％、25mol％-70mol％、30mol％-70mol％、35mol％-70mol％、40mol％-70mol％、15mol％-60mol％，20mol％-60mol％、25mol％-60mol％、30mol％-60mol％、35mol％-60mol％、40mol％-60mol％、15mol％-50mol％，20mol％-50mol％、25mol％-50mol％、30mol％-50mol％、35mol％-50mol％、15mol％-40mol％，20mol％-40mol％、25mol％-40mol％、30mol％-40mol％或35mol％-40mol％。In some embodiments, the cholesterol accounts for 14 mol%-70 mol% of the total lipids in the composition, for example, 20 mol%-70 mol%, 25 mol%-70 mol%, 30 mol%-70 mol%, 35 mol%-70 mol%, 40 mol%-70 mol%, 15 mol%-60 mol%, 20 mol%-60 mol%, 25 mol%-60 mol%, 30 mol%-60 mol%, 35 mol%-60 mol%, 40 mol%-60 mol%, 15 mol%-50 mol%, 20 mol%-50 mol%, 25 mol%-50 mol%, 30 mol%-50 mol%, 35 mol%-50 mol%, 15 mol%-40 mol%, 20 mol%-40 mol%, 25 mol%-40 mol%, 30 mol%-40 mol% or 35 mol%-40 mol%.
在一些实施方案中，所述磷脂占所述组合物中的总脂质的约5mol％-约75mol％，例如5mol％-70mol％、8mol％-65mol％、10mol％-60mol％、10mol％-50mol％、10mol％-40mol％、10mol％-30mol％、10mol％-20mol％、15mol％-55mol％、15mol％-50mol％、15mol％-45mol％、15mol％-40mol％、15mol％-35mol％、15mol％-30mol％、15mol％-25mol％、20mol％-45mol％、20mol％-45mol％、20mol％-40mol％、20mol％-35mol％、20mol％-30mol％、20mol％-25mol％、25mol％-40mol％、25mol％-35mol％或25mol％-30mol％、。In some embodiments, the phospholipids account for about 5 mol% to about 75 mol% of the total lipids in the composition, such as 5 mol% to 70 mol%, 8 mol% to 65 mol%, 10 mol% to 60 mol%, 10 mol% to 50 mol%, 10 mol% to 40 mol%, 10 mol% to 30 mol%, 10 mol% to 20 mol%, 15 mol% to 55 mol%, 15 mol% to 50 mol%, 15 mol% to 45 mol%, 10 mol% to 50 mol%, 10 ... %-40mol%, 15mol%-35mol%, 15mol%-30mol%, 15mol%-25mol%, 20mol%-45mol%, 20mol%-45mol%, 20mol%-40mol%, 20mol%-35mol%, 20mol%-30mol%, 20mol%-25mol%, 25mol%-40mol%, 25mol%-35mol% or 25mol%-30mol%.
在本发明的组合物的一些实施方案中，所述磷脂包含选自以下的至少一种：卵磷脂、磷脂酰乙醇胺、溶血卵磷脂、溶血磷脂酰乙醇胺、磷脂酰丝氨酸、二油酰基磷脂酰丝氨酸(DOPS)、磷脂酰肌醇、鞘磷脂、蛋黄鞘磷脂(ESM)、脑磷脂、心磷脂、磷脂酸、脑苷脂、联十六烷基磷酸酯、二硬脂酰磷脂酰胆碱(DSPC)、二油酰磷脂酰乙醇胺(DOPE)、二油酰磷脂酰胆碱(DOPC)、二棕榈酰磷脂酰胆碱(DPPC)、二油酰磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、棕榈酰油酰-磷脂酰胆碱(POPC)、棕榈酰油酰-磷脂酰乙醇胺(POPE)、棕榈酰油酰-磷脂酰甘油(POPG)、二油酰磷脂酰乙醇胺4-(N-马来酰亚胺基甲基)-环己烷-1-甲酸酯(DOPE-mal)、二棕榈酰-磷脂酰乙醇胺(DPPE)、二肉豆蔻酰-磷脂酰乙醇胺(DMPE)、二硬脂酰-磷脂酰 乙醇胺(DSPE)、单甲基-磷脂酰乙醇胺、二甲基-磷脂酰乙醇胺、二反油酰-磷脂酰乙醇胺(DEPE)、硬脂酰油酰-磷脂酰乙醇胺(SOPE)、溶血磷脂酰胆碱、蛋黄磷脂酰胆碱(EPC)和二亚油酰磷脂酰胆碱。1,2-二棕榈酰-sn-甘油-3-O-4'-(N，N，N-三甲基)-高丝氨酸(DGTS)、单半乳糖二酰甘油(MGDG)、二乙酰二酰基甘油(DGDG)、磺胺喹啉二酰基甘油(SQDG)、1-棕榈酰-2-顺式-9,10-亚甲基己基-癸酰基-sn-甘油-3-磷酸胆碱(Cyclo PC)、16‐O‐单甲基PE、16‐O‐二甲基PE、18‐1‐反式PE以及它们的衍生物。In some embodiments of the composition of the present invention, the phospholipid comprises at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, dioleoylphosphatidylserine (DOPS), phosphatidylinositol, sphingomyelin, egg yolk sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylphosphatidylcholine (DPSC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylphosphatidylcholine (DPSC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DPSC), dioleoylphosphatidylcholine (DOPS), dioleoylphosphatidylcholine (DOPS), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DOPS ... Choline (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidyl ethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, di-trans-oleoyl-phosphatidylethanolamine (DEPE), stearoyl-oleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, egg yolk phosphatidylcholine (EPC) and dilinoleoylphosphatidylcholine. 1,2-Dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS), monogalactosyldiacylglycerol (MGDG), diacetyldiacylglycerol (DGDG), sulfaquinolinediacylglycerol (SQDG), 1-palmitoyl-2-cis-9,10-methylenehexyl-decanoyl-sn-glycero-3-phosphocholine (Cyclo PC), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE and their derivatives.
在一些实施方案中，本发明的组合物和方法包括具有下式的非阳离子脂质或其盐：
In some embodiments, the compositions and methods of the invention include a non-cationic lipid having the formula:
其中，R1和R2各自独立地是C8-C24烷基、C8-C24烯基或任一个基团的被取代形式；R3、R3′和R3″各自独立地是烷基(C≤6)或被取代的烷基(C≤6)；且X‐是单价阴离子。wherein R 1 and R 2 are each independently C 8 -C 24 alkyl, C 8 -C 24 alkenyl or a substituted form of either group; R 3 , R 3 ′ and R 3 ″ are each independently alkyl (C≤6) or substituted alkyl (C≤6) ; and X ‐ is a monovalent anion.
在一些实施方案中，R1是C8-C24烯基或被取代的C8-C24烯基。在一些实施方案中，R2是C8-C24烯基或被取代的C8-C24烯基。在其它实施方案中，R1是C8-C24烷基或被取代的C8-C24烷基。在其它实施方案中，R2是C8-C24烷基或被取代的C8-C24烷基。在一些实施方案中，R1和R2两者是相同的。In some embodiments, R 1 is C 8 -C 24 alkenyl or substituted C 8 -C 24 alkenyl. In some embodiments, R 2 is C 8 -C 24 alkenyl or substituted C 8 -C 24 alkenyl. In other embodiments, R 1 is C 8 -C 24 alkyl or substituted C 8 -C 24 alkyl. In other embodiments, R 2 is C 8 -C 24 alkyl or substituted C 8 -C 24 alkyl. In some embodiments, both R 1 and R 2 are the same.
在一些实施方案中，R3、R3′和R3″各自是相同的。在一些实施方案中，R3、R3′和R3″各自是甲基。在一些实施方案中，X‐是卤化物阴离子诸如溴化物或氯化物。In some embodiments, R 3 , R 3 ′, and R 3 ″ are each the same. In some embodiments, R 3 , R 3 ′, and R 3 ″ are each methyl. In some embodiments, X - is a halide anion such as bromide or chloride.
在一些实施方案中，本发明的组合物和方法包括下式的磷酸甘油酯或其盐：
In some embodiments, the compositions and methods of the invention include a phosphoglyceride or a salt thereof of the formula:
其中，R4是具有10至24个碳原子的直链烷基或具有1至3个双键和 10至24个碳原子的直链烯基；wherein R4 is a straight chain alkyl group having 10 to 24 carbon atoms or a straight chain alkyl group having 1 to 3 double bonds and Straight-chain alkenyl groups of 10 to 24 carbon atoms;
R5是具有10至24个碳原子的直链烷基或具有1至3个双键和10至24个碳原子的直链烯基； R5 is a straight chain alkyl group having 10 to 24 carbon atoms or a straight chain alkenyl group having 1 to 3 double bonds and 10 to 24 carbon atoms;
在一些实施方案中，本发明的组合物和方法包括下式的阴离子脂质或其盐：
In some embodiments, the compositions and methods of the invention include an anionic lipid of the formula:
其中，R1和R2各自独立地是烷基(C8‐C24)、烯基(C8‐C24)或任一个基团的被取代形式；R3是氢、烷基(C≤6)或被取代的烷基(C≤6)或‐Y1‐R4，其中：Y1是烷二基(C≤6)或被取代的烷二基(C≤6)；且R4是酰氧基(C≤8‐24)或被取代的酰氧基(C≤8‐24)。Wherein, R1 and R2 are each independently alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of either group; R3 is hydrogen, alkyl (C≤6) or substituted alkyl (C≤6) or -Y1 - R4 , wherein: Y1 is alkanediyl (C≤6) or substituted alkanediyl (C≤6) ; and R4 is acyloxy (C≤8-24) or substituted acyloxy (C≤8-24) .
适合用于本发明的非-阳离子脂质的其他实例包括不含磷的脂质，例如，十八酰胺、十二烷基胺、十六烷基胺、乙酰棕榈酸酯、甘油蓖麻油酸酯、硬脂酸十六烷酯(hexadecyl stereate)、肉豆蔻酸异丙酯、两性丙烯酸聚合物、三乙醇胺-硫酸月桂酯、硫酸烷基-芳基酯聚乙氧化脂肪酸酰胺、双十八基二甲基溴化鞍、神经酰胺、鞘磷脂以及它们的衍生物等。Other examples of non-cationic lipids suitable for use in the present invention include phosphorus-free lipids, such as stearylamide, dodecylamine, hexadecylamine, acetylapalmitate, glyceryl ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic acid polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethoxylated fatty acid amides, distearyl dimethyl bromide, ceramides, sphingomyelin and their derivatives, etc.
在本发明的组合物的一些实施方案中，所述胆固醇衍生的中性脂质包含选自以下的至少一种：胆甾烷醇、胆甾烷酮、胆甾烯酮、粪甾醇、胆固醇基-2'-羟基乙醚、胆固醇基-4'-羟基丁醚、BHEM-胆固醇、β-谷甾醇、20α-羟基胆固醇、多肽/蛋白共价修饰的胆固醇以及它们的衍生物，其中胆固醇基-2'-羟基乙醚的合成记述在在美国专利号8,058,069中，通过引用将其公开内容整体引入本文中用于全部目的。优选地，所述胆固醇衍生脂质包含β-谷甾醇。In some embodiments of the compositions of the present invention, the cholesterol-derived neutral lipid comprises at least one selected from the group consisting of cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, BHEM-cholesterol, β-sitosterol, 20α-hydroxycholesterol, polypeptide/protein covalently modified cholesterol, and derivatives thereof, wherein the synthesis of cholesteryl-2'-hydroxyethyl ether is described in U.S. Pat. No. 8,058,069, the disclosure of which is incorporated herein by reference for all purposes. Preferably, the cholesterol-derived lipid comprises β-sitosterol.
在一些实施方案中，本发明的组合物和方法所述类固醇的环结构包含三个稠合的环己基环和稠合的环戊基环，如下式所示：
In some embodiments, the compositions and methods of the present invention include a steroid having a ring structure comprising three fused cyclohexyl rings and a fused cyclopentyl ring, as shown in the following formula:
在一些实施方案中，类固醇衍生物包含具有一个或多个非烷基取代的上 述环结构。在一些实施方案中，类固醇或类固醇衍生物是甾醇，其中该式进一步定义为：
In some embodiments, the steroid derivative comprises a moiety having one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol, wherein the formula is further defined as:
在一些实施方案中，本发明的组合物和方法所述类固醇或类固醇衍生物是胆甾烷或胆甾烷衍生物。在胆甾烷中，环结构由下式进一步定义：
In some embodiments, the steroid or steroid derivative of the compositions and methods of the present invention is cholestane or a cholestane derivative. In cholestane, the ring structure is further defined by the formula:
如上所述，胆甾烷衍生物包括一个或多个上述环系统的非烷基取代。在一些实施方案中，所述胆甾烷或胆甾烷衍生物是胆甾烯或胆甾烯衍生物或者甾醇或甾醇衍生物。在其它实施方案中，所述胆甾烷或胆甾烷衍生物是胆甾烯(cholestere)和甾醇或其衍生物。As mentioned above, cholestane derivatives include non-alkyl substitutions of one or more of the above ring systems. In some embodiments, the cholestane or cholestane derivative is cholestene or a cholestene derivative or a sterol or a sterol derivative. In other embodiments, the cholestane or cholestane derivative is cholestene and a sterol or a derivative thereof.
在本发明的包含聚合物-脂质的组合物的一些实施方案中，所述组合物还包含脂质缀合物，其中所述脂质缀合物包含选自以下的至少一种：聚(乙二醇)-脂质缀合物(PEG-脂质缀合物或PEG-脂质)、ATTA-脂质缀合物、聚肌氨酸‐脂质缀合物、多肽/蛋白-脂质缀合物以及阳离子-聚合物-脂质缀合物(CPL)，优选地，所述脂质缀合物包含PEG-脂质缀合物。In some embodiments of the polymer-lipid composition of the present invention, the composition further comprises a lipid conjugate, wherein the lipid conjugate comprises at least one selected from the group consisting of a poly(ethylene glycol)-lipid conjugate (PEG-lipid conjugate or PEG-lipid), an ATTA-lipid conjugate, a polysarcosine-lipid conjugate, a polypeptide/protein-lipid conjugate, and a cation-polymer-lipid conjugate (CPL), preferably, the lipid conjugate comprises a PEG-lipid conjugate.
在优选的实施方案中，所述脂质缀合物是PEG-脂质。PEG-脂质的实例包括，但不仅限于，如，例如，PCT公开号WO 05/026372中所述的与二烷氧基丙基偶联的PEG(PEG-DAA)，如记述在，例如，美国专利公开号20030077829和2005008689中的与二酰甘油偶联的PEG(PEG-DAG)，与磷脂诸如磷脂酰乙醇胺偶联的PEG(PEG-PE)，如记述在例如，美国专利号5,885,613中的与神经酰胺偶联的PEG，与胆固醇或其衍生物缀合的PEG，及其混合物。这些专利文件的公开内容通过引用整体结合与此用于全部目的。另外的PEG-脂质包括，但不仅限于，PEG-C-DOMG，DMG-PEG2000((R)-2,3-bis(myristoyloxy)propyl-1-(methoxy poly(ethylene glycol)2000)carbamate, DMG-PEG2K)，ALC-0159(2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide；2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺)，及其混合物。本文所述PEG-脂质可如国际专利PCT/US2016/000129中所述方法合成。在一些实例中，可用于本发明的PEG-脂质可为描述于国际专利WO2012099755中的PEG化脂质，这些专利的公开内容通过引用整体结合与此用于全部目的。In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examples of PEG-lipids include, but are not limited to, PEG coupled to a dialkoxypropyl group (PEG-DAA) as described in, for example, PCT Publication No. WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in, for example, U.S. Patent Publication Nos. 20030077829 and 2005008689, PEG coupled to a phospholipid such as phosphatidylethanolamine (PEG-PE), PEG coupled to ceramide as described in, for example, U.S. Patent No. 5,885,613, PEG conjugated to cholesterol or its derivatives, and mixtures thereof. The disclosures of these patent documents are hereby incorporated by reference in their entirety for all purposes. Additional PEG-lipids include, but are not limited to, PEG-C-DOMG, DMG-PEG2000 ((R)-2,3-bis(myristoyloxy)propyl-1-(methoxy poly(ethylene glycol)2000)carbamate, DMG-PEG2K), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; 2-[(polyethylene glycol)-2000]-N,N-diethylacetamide), and mixtures thereof. The PEG-lipid described herein can be synthesized as described in International Patent PCT/US2016/000129. In some examples, the PEG-lipid that can be used in the present invention can be a PEGylated lipid described in International Patent WO2012099755, the disclosures of which are incorporated herein by reference in their entirety for all purposes.
PEG是具有两个端羟基的乙烯PEG重复单元的线性、水溶性聚合物。PEG由其分子量分类；例如，PEG2000(PEG2K)具有约2,000道尔顿的平均分子量，且PEG5000(PEG5K)具有约5,000道尔顿的平均分子量。PEG可商购自Sigma-Aldrich Chemical Co.(西格玛奥德里奇化学公司)和其他公司并包括，例如，以下：单甲氧基聚乙二醇(MePEG-OH)、单甲氧基聚乙二醇-琥珀酸酯(MePEG-S)、单甲氧基聚乙二醇-琥珀酰亚胺琥珀酸酯(MePEG-S-NHS)、单甲氧基聚乙二醇-胺(MePEG-NH2)，单甲氧基聚乙二醇-三氟乙基磺酸酯(MePEG-TRES)、和单甲氧基聚乙二醇-咪唑基-羧基(MePEG-IM)。其他PEG诸如美国专利号6,774,180和7,053,150中记述的那些(例如，mPEG(20KDa)胺)也有效用于制备本发明的PEG-脂质缀合物。这些专利的公开内容通过引用整体结合与此用于全部目的。另外，单甲氧基聚乙二醇-乙酸(MePEG-CH2COOH)特别有效用于制备PEG-脂质缀合物包括，例如，PEG-DAA缀合物。PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEG is classified by its molecular weight; for example, PEG2000 (PEG2K) has an average molecular weight of about 2,000 Daltons, and PEG5000 (PEG5K) has an average molecular weight of about 5,000 Daltons. PEG is commercially available from Sigma-Aldrich Chemical Co. and other companies and includes, for example, the following: monomethoxy polyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol-succinate (MePEG-S), monomethoxy polyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxy polyethylene glycol-amine (MePEG-NH2), monomethoxy polyethylene glycol-trifluoroethanesulfonate (MePEG-TRES), and monomethoxy polyethylene glycol-imidazolyl-carboxyl (MePEG-IM). Other PEGs such as those described in U.S. Pat. Nos. 6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are also effective for preparing the PEG-lipid conjugates of the present invention. The disclosures of these patents are hereby incorporated by reference in their entirety for all purposes. In addition, monomethoxypolyethylene glycol-acetic acid (MePEG-CH 2 COOH) is particularly effective for preparing PEG-lipid conjugates including, for example, PEG-DAA conjugates.
本文中所述的PEG-脂质缀合物的PEG结构部分可以包括约550道尔顿-约10,000道尔顿范围内的平均分子量。在某些实例中，PEG结构部分具有约750道尔顿-约5,000道尔顿(例如，约1,000道尔顿-约5,000道尔顿、约1,500道尔顿-约3,000道尔顿、约750道尔顿-约3,000道尔顿、约750道尔顿-约2,000道尔顿，等)的平均分子量。在优选的实施方案中，PEG结构部分具有约5,000道尔顿或约2,000道尔顿或约750道尔顿的平均分子量。The PEG moiety of the PEG-lipid conjugate described herein can include an average molecular weight in the range of about 550 Daltons to about 10,000 Daltons. In certain instances, the PEG moiety has an average molecular weight of about 750 Daltons to about 5,000 Daltons (e.g., about 1,000 Daltons to about 5,000 Daltons, about 1,500 Daltons to about 3,000 Daltons, about 750 Daltons to about 3,000 Daltons, about 750 Daltons to about 2,000 Daltons, etc.). In a preferred embodiment, the PEG moiety has an average molecular weight of about 5,000 Daltons or about 2,000 Daltons or about 750 Daltons.
在某些实例中，PEG可以任选地被烷基、烷氧基、酰基或芳基取代。PEG可以与脂质直接缀合或可以与脂质通过连接体结构部分相连接。可以使用任何适合于使PEG与脂质偶联的连接体结构部分包括，例如，不含酯的连接体结构部分和含酯的连接体结构部分。在优选的实施方案中，所述连接体结构部分是不含酯的连接体结构部分。用于本文中时，术语“不含酯的连接体结构部分”指不包含羧酸酯键(-OC(O)-)的连接体结构部分。合适的不含酯的连接体 结构部分包括，但不仅限于，胺基(-C(O)NH-)、氨基(-NR-)、羧基(-C(O)-)、氨基甲酸酯(-NHC(O)O-)、尿素(-NHC(O)NH-)、二硫化物(-S-S-)、醚(-O-)、琥珀酰基(-(O)CCH2CH2C(O)-)、琥珀酰胺基(-NHC(O)CH2CH2C(O)NH-)，及其组合(诸如包含氨基甲酸酯连接体结构部分和胺基连接体结构部分二者的连接体)。在优选的实施方案中，使用氨基甲酸酯连接体来偶联PEG和脂质。In certain instances, PEG can be optionally substituted with alkyl, alkoxy, acyl or aryl groups. PEG can be directly conjugated to the lipid or can be connected to the lipid through a linker moiety. Any linker moiety suitable for coupling PEG to the lipid can be used including, for example, non-ester-containing linker moieties and ester-containing linker moieties. In a preferred embodiment, the linker moiety is a non-ester-containing linker moiety. As used herein, the term "non-ester-containing linker moiety" refers to a linker moiety that does not contain a carboxylate bond (-OC(O)-). Suitable non-ester-containing linkers are preferably non-ester-containing linkers. Moieties include, but are not limited to, amine (-C(O)NH-), amino (-NR-), carboxyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O ) CCH2CH2C ( O)-), succinamido (-NHC(O) CH2CH2C (O)NH-), and combinations thereof (such as linkers comprising both carbamate linker moieties and amine linker moieties). In a preferred embodiment, a carbamate linker is used to couple the PEG and lipid.
在其他实施方案中，使用含酯的连接体结构部分来偶联PEG和脂质。合适的含酯的连接体结构部分包括，例如，碳酸酯(-OC(O)O-)，琥珀酰基、磷酸酯(-O-(O)POH-O-)、磺酸酯，及其组合。In other embodiments, an ester-containing linker moiety is used to couple the PEG and lipid. Suitable ester-containing linker moieties include, for example, carbonate (—OC(O)O—), succinyl, phosphate (—O—(O)POH—O—), sulfonate, and combinations thereof.
具有不同链长度和饱和程度的多种酰基链基团的磷脂酰乙醇胺可以与PEG缀合形成脂质缀合物。这样的磷脂酰乙醇胺可商购，或可以利用本领域中技术人员已知的常规技术分离或合成。优选碳链长度在C10-C20范围内的包含饱和或不饱和脂肪酸的磷脂酰-乙醇胺。也可以使用具有单-或二不饱和脂肪酸以及饱和与不饱和脂肪酸混合物的磷脂酰乙醇胺。合适的磷脂酰乙醇胺包括，但不仅限于，二肉豆蔻酰基-磷脂酰乙醇胺(DMPE)、二棕榈酰基-磷脂酰乙醇胺(DPPE)、二油酰基磷脂酰乙醇胺(DOPE)和二硬脂酰基-磷脂酰乙醇胺(DSPE)。Phosphatidylethanolamines with various acyl chain groups of different chain lengths and degrees of saturation can be conjugated with PEG to form lipid conjugates. Such phosphatidylethanolamines are commercially available, or can be separated or synthesized using conventional techniques known to those skilled in the art. Phosphatidyl-ethanolamines containing saturated or unsaturated fatty acids with a preferred carbon chain length in the range of C10 - C20 . Phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE) and distearoyl-phosphatidylethanolamine (DSPE).
在一些实施方案中，本发明的组合物和方法所述PEG-脂质缀合物包括下式的聚乙二醇化磷酸甘油酯或其盐：
In some embodiments, the PEG-lipid conjugate of the compositions and methods of the present invention comprises a PEGylated phosphoglyceride of the formula:
其中，in,
p是5至200，优选地10至170，并且最优选10至140的整数；p is an integer from 5 to 200, preferably from 10 to 170, and most preferably from 10 to 140;
R6是具有10至20个碳原子的直链烷基或具有1至3个双键和10至20个碳原子的直链烯基； R6 is a straight chain alkyl group having 10 to 20 carbon atoms or a straight chain alkenyl group having 1 to 3 double bonds and 10 to 20 carbon atoms;
R7是具有10至20个碳原子的直链烷基或具有1至3个双键和10至20个碳原子的直链烯基； R7 is a straight chain alkyl group having 10 to 20 carbon atoms or a straight chain alkenyl group having 1 to 3 double bonds and 10 to 20 carbon atoms;
在一些实施方案中，本发明的组合物和方法所述PEG-脂质缀合物具有下式：
In some embodiments, the compositions and methods of the invention, the PEG-lipid conjugate has the formula:
其中，in,
R12和R13各自独立地是烷基(C≤24)、烯基(C≤24)或这些基团中的任一个的取代形式；Re是氢、烷基(C≤8)或被取代的烷基(C≤8)；且x是1‐250。在一些实施方案中，Re是烷基(C≤8)诸如甲基。R12和R13各自独立地是烷基(C≤4‐20)。在一些实施方案中，x是5‐250。在一个实施方案中，x是5‐125或x是100‐250。在一些实施方案中，所述PEG脂质是1,2‐二肉豆蔻酰基‐sn‐甘油、甲氧基聚乙二醇。R 12 and R 13 are each independently alkyl (C≤24) , alkenyl (C≤24) , or a substituted form of any of these groups; Re is hydrogen, alkyl (C≤8) , or substituted alkyl (C≤8) ; and x is 1-250. In some embodiments, Re is alkyl (C≤8) such as methyl. R 12 and R 13 are each independently alkyl (C≤4-20) . In some embodiments, x is 5-250. In one embodiment, x is 5-125 or x is 100-250. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.
在一些实施方案中，本发明的组合物和方法所述脂质缀合物具有下式：
In some embodiments, the lipid conjugate of the compositions and methods of the invention has the formula:
其中，in,
n1是1‐100之间的整数，且n2和n3各自独立地选自1‐29之间的整数。在一些实施方案中，n1是5、10、15、20、25、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、55、60、65、70、75、80、85、90、95或100或其中可导出的任何范围。在一些实施方案中，n1是约30至约50。在一些实施方案中，n2是5‐23。在一些实施方案中，n2是11至约17。在一些实施方案中，n3是5‐23。在一些实施方案中，n3是11至约17。 n1 is an integer between 1-100, and n2 and n3 are each independently selected from an integer between 1-29. In some embodiments, n1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70 , 75, 80, 85, 90, 95 or 100 or any range derivable therein. In some embodiments, n1 is about 30 to about 50. In some embodiments, n2 is 5-23. In some embodiments, n2 is 11 to about 17. In some embodiments, n3 is 5-23. In some embodiments, n3 is 11 to about 17.
术语“ATTA”或“聚酰胺”指，不仅限于，美国专利号6,320,017和6,586,559中记述的化合物，通过引用将其公开内容整体引入本文中用于全部目的。这 些化合物包括具有下式的化合物：
The term "ATTA" or "polyamide" refers to, but is not limited to, the compounds described in U.S. Pat. Nos. 6,320,017 and 6,586,559, the disclosures of which are incorporated herein by reference in their entirety for all purposes. These compounds include compounds having the formula:
其中R是选自由氢、烷基和酰基组成的组中的成员；R1是选自由氢和烷基组成的组中的成员；或任选地，R和R1以及它们连接的氮形成叠氮结构部分；R2是选自由氢、任选取代的烷基、任选取代的芳基和氨基酸侧链组成的组中的成员；R3是选自由氢、卤素、羟基、烷氧基、巯基、肼基、氨基和NR4R5组成的组中的成员，其中R4和R5独立地是氢或烷基；n是4-80；m是2-6；p是1-4；且q是0或1。本领域中技术人员应该清楚的是其他聚酰胺可以用于本发明的化合物中。wherein R is a member selected from the group consisting of hydrogen, alkyl, and acyl; R1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R1 and the nitrogen to which they are attached form an azide moiety; R2 is a member selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, and an amino acid side chain; R3 is a member selected from the group consisting of hydrogen, halogen, hydroxyl, alkoxy, thiol, hydrazine, amino, and NR4R5 , wherein R4 and R5 are independently hydrogen or alkyl; n is 4-80; m is 2-6; p is 1-4; and q is 0 or 1. It will be clear to those skilled in the art that other polyamides can be used in the compounds of the present invention.
术语“二酰甘油”指具有2个脂肪酰基链，R1和R2的化合物，所述R1和R2二者独立地具有2-30个碳，其通过酯键与甘油的1-和2-位结合。酰基可以是饱和的或具有不同的不饱和程度。合适的酰基包括，但不仅限于，月桂基(C12)、肉豆蔻基(C14)、棕榈基(C16)、硬脂基(C18)，和二十烷基(C20)。在优选的实施方案中，R1和R2相同，即，R1和R2均是肉豆蔻基(即，二肉豆蔻基)，R1和R2均是硬脂基(即，二硬脂基)，等。二酰甘油具有以下通式：
The term "diacylglycerol" refers to a compound having two fatty acyl chains, R1 and R2 , each independently having 2-30 carbons, which are bound to the 1- and 2-positions of glycerol via ester bonds. The acyl groups may be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauryl ( C12 ), myristyl ( C14 ), palmityl ( C16 ), stearyl ( C18 ), and eicosyl ( C20 ). In a preferred embodiment, R1 and R2 are the same, i.e., both R1 and R2 are myristyl (i.e., dimyristyl), both R1 and R2 are stearyl (i.e., distearyl), etc. Diacylglycerol has the following general formula:
术语“二烷氧基丙基”指具有2个烷基链，R1和R2的化合物，所述R1和R2二者独立地具有2-30个碳。烷基基团可以是饱和的或具有不同的不饱和 程度。二烷氧基丙基具有以下通式：
The term "dialkoxypropyl" refers to a compound having two alkyl chains, R1 and R2 , each independently having 2-30 carbons. The alkyl group may be saturated or have varying degrees of unsaturation. Dialkoxypropyl has the following general formula:
在优选的实施方案中，PEG-脂质是具有以下通式的PEG-DAA缀合物：
In a preferred embodiment, the PEG-lipid is a PEG-DAA conjugate having the following general formula:
其中R1和R2独立地选择并且是具有约10-约22个碳原子的长链烷基基团；PEG是聚乙二醇；且L是如上所述的不含酯的连接体结构部分或含酯的连接体结构部分。所述长链烷基基团可以是饱和的或不饱和的。合适的烷基基团包括，但不仅限于，月桂基(C12)、肉豆蔻基(C14)、棕榈基(C16)、硬脂基(C18)，和二十烷基(C20)。在优选的实施方案中，R1和R2相同，即，R1和R2均是肉豆蔻基(即，二肉豆蔻基)，R1和R2均是硬脂基(即，二硬脂基)，等。Wherein R 1 and R 2 are independently selected and are long chain alkyl groups having from about 10 to about 22 carbon atoms; PEG is polyethylene glycol; and L is a non-ester-containing linker moiety or an ester-containing linker moiety as described above. The long chain alkyl group may be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, lauryl (C 12 ), myristyl (C 14 ), palmityl (C 16 ), stearyl (C 18 ), and eicosyl (C 20 ). In preferred embodiments, R 1 and R 2 are the same, i.e., both R 1 and R 2 are myristyl (i.e., dimyristyl), both R 1 and R 2 are stearyl (i.e., distearyl), etc.
在上式中，PEG具有约550道尔顿-约10,000道尔顿范围内的平均分子量。在某些实例中，PEG具有约750道尔顿-约5,000道尔顿(例如，约1,000道尔顿-约5,000道尔顿，约1,500道尔顿-约3,000道尔顿，约750道尔顿-约3,000道尔顿，约750道尔顿-约2,000道尔顿，等)的平均分子量。在优选的实施方案中,PEG具有约2,000道尔顿或约750道尔顿的平均分子量。PEG可以任选地用烷基、烷氧基、酰基或芳基取代。在某些实施方案中，末端羟基基团用乙氧基或甲基取代。In the above formula, PEG has an average molecular weight in the range of about 550 daltons to about 10,000 daltons. In some instances, PEG has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons, etc.). In a preferred embodiment, PEG has an average molecular weight of about 2,000 daltons or about 750 daltons. PEG can be optionally substituted with an alkyl, alkoxy, acyl or aryl group. In certain embodiments, the terminal hydroxyl group is substituted with an ethoxy or methyl group.
在优选的实施方案中，“L”是不含酯的连接体结构部分。合适的不含酯的连接体包括，但不仅限于，胺基连接体结构部分、氨基连接体结构部分、羧基连接体结构部分、氨基甲酸酯连接体结构部分、尿素连接体结构部分、醚连接体结构部分、二硫化物连接体结构部分、琥珀酰胺基连接体结构部分，及其组合。在优选的实施方案中，所述不含酯的连接体结构部分是氨基甲酸酯 连接体结构部分(即，PEG-C-DAA缀合物)。在另一个优选实施方案中，所述不含酯的连接体结构部分是胺基连接体结构部分(即，PEG-A-DAA缀合物)。在另一个优选实施方案中，所述不含酯的连接体结构部分是琥珀酰胺基连接体结构部分(即，PEG-S-DAA缀合物)。In a preferred embodiment, "L" is a non-ester-containing linker moiety. Suitable non-ester-containing linkers include, but are not limited to, amine linker moieties, amino linker moieties, carboxyl linker moieties, carbamate linker moieties, urea linker moieties, ether linker moieties, disulfide linker moieties, succinyl linker moieties, and combinations thereof. In a preferred embodiment, the non-ester-containing linker moiety is a carbamate In another preferred embodiment, the non-ester-containing linker moiety is an amine linker moiety (i.e., a PEG-A-DAA conjugate). In another preferred embodiment, the non-ester-containing linker moiety is a succinyl linker moiety (i.e., a PEG-S-DAA conjugate).
在特殊的实施方案中，PEG-脂质缀合物选自：
In particular embodiments, the PEG-lipid conjugate is selected from:
PEG-DAA缀合物利用本领域中技术人员已知的标准技术和试剂来合成。应该认识到PEG-DAA缀合物会包含多种酰胺、胺、醚、硫代、氨基甲酸酯、和尿素键。本领域中技术人员应该认识到用于形成这些键的方法和试剂是公知的且容易获得。参见，例如，March,ADVANCED ORGANIC CHEMISTRY(现代有机化学)(Wiley 1992)；Larock,COMPREHENSIVE ORGANIC TRANSFORMATIONS(综合有机转化)(VCH 1989)；和Furniss,VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY(VOGEL应用有机化学教科书)，第5版(Longman 1989)。还应该理解，存在的任何官能团可能在PEG-DAA缀合物合成中的不同点时需要保护和去保护。本领域中技术人员应该认识到这样的技术是公知的。参见，例如，Green和Wuts,PROTECTIVE GROUPS IN ORGANIC SYNTHESIS(有机合成中的保护基)(Wiley 1991)。The PEG-DAA conjugates are synthesized using standard techniques and reagents known to those skilled in the art. It should be recognized that the PEG-DAA conjugates will contain a variety of amide, amine, ether, thio, carbamate, and urea bonds. Those skilled in the art should recognize that methods and reagents for forming these bonds are well known and readily available. See, for example, March, ADVANCED ORGANIC CHEMISTRY (Wiley 1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); and Furniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th Edition (Longman 1989). It should also be understood that any functional groups present may require protection and deprotection at various points in the synthesis of the PEG-DAA conjugates. Those skilled in the art should recognize that such techniques are well known. See, e.g., Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).
在一些实施方案中，PEG-DAA缀合物是二月桂基氧基丙基(C12)-PEG缀合物，二肉豆蔻基氧基丙基(C14)-PEG缀合物，二棕榈基氧基丙基(C16)-PEG缀合物，或二硬脂基氧基丙基(C18)-PEG缀合物。本领域中技术人员应该容易理解其他二烷氧基丙基可以用于本发明的PEG-DAA缀合物。In some embodiments, the PEG-DAA conjugate is a dilauryloxypropyl (C 12 )-PEG conjugate, a dimyristyloxypropyl (C 14 )-PEG conjugate, a dipalmityloxypropyl (C 16 )-PEG conjugate, or a distearyloxypropyl (C 18 )-PEG conjugate. Those skilled in the art will readily appreciate that other dialkoxypropyl groups can be used in the PEG-DAA conjugates of the present invention.
除前述外，本领域中技术人员应该容易清楚的是，其他亲水聚合物可以 代替PEG使用。可以代替PEG使用的合适聚合物实例包括，但不仅限于，聚乙烯吡咯烷酮、聚甲基噁唑啉、聚乙基噁唑啉、聚羟基丙基甲基丙烯酰胺、聚甲基丙烯酰胺和聚二甲基丙烯酰胺、聚乳酸、聚乙醇酸，泊洛沙姆，泊洛沙胺，衍生的纤维素诸如羟基甲基纤维素或羧乙基纤维素，聚肌氨酸‐脂质缀合物，和聚肌氨酸与类脂质物质的缀合物。其中所述聚肌氨酸‐脂质缀合物或聚肌氨酸与类脂质物质的缀合物可以选自以下的组：聚肌氨酸‐二酰甘油缀合物、聚肌氨酸‐二烷氧基丙基缀合物、聚肌氨酸‐磷脂缀合物、聚肌氨酸‐神经酰胺缀合物，及其混合物。用于本发明的合适聚肌氨酸‐脂质缀合物或聚肌氨酸与类脂质物质的缀合物，和制备和使用聚肌氨酸‐脂质缀合物或聚肌氨酸与类脂质物质的缀合物的方法公开在，例如美国专利号17/281,697和PCT公开号PCT/EP2019/076369中，通过引用将其公开内容整体引入本文中用于全部目的。在一些实施方案中，用于本发明的组合物和方法的合适的聚肌氨酸‐脂质缀合物包括如国际专利公开WO2020070040中所述的聚肌氨酸脂质，该专利公开以引用的方式并入本文。In addition to the foregoing, it should be readily apparent to those skilled in the art that other hydrophilic polymers may be Instead of PEG. Examples of suitable polymers that can be used instead of PEG include, but are not limited to, polyvinyl pyrrolidone, polymethyl oxazoline, polyethyl oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethyl acrylamide, polylactic acid, polyglycolic acid, poloxamer, poloxamine, derivatized cellulose such as hydroxymethyl cellulose or carboxyethyl cellulose, polysarcosine-lipid conjugates, and conjugates of polysarcosine with lipid-like substances. Wherein the polysarcosine-lipid conjugate or the conjugate of polysarcosine with lipid-like substances can be selected from the following group: polysarcosine-diacylglycerol conjugate, polysarcosine-dialkoxypropyl conjugate, polysarcosine-phospholipid conjugate, polysarcosine-ceramide conjugate, and mixtures thereof. Suitable polysarcosine-lipid conjugates or conjugates of polysarcosine with lipid-like substances for use in the present invention, and methods for preparing and using polysarcosine-lipid conjugates or conjugates of polysarcosine with lipid-like substances are disclosed in, for example, U.S. Patent No. 17/281,697 and PCT Publication No. PCT/EP2019/076369, the disclosures of which are incorporated herein by reference in their entirety for all purposes. In some embodiments, suitable polysarcosine-lipid conjugates for use in the compositions and methods of the present invention include polysarcosine lipids as described in International Patent Publication WO2020070040, which is incorporated herein by reference.
除前述组分外，本发明的聚合物-脂质组合物(例如，PoLixNano)还可以包括阳离子-聚合物-脂质缀合物(CPL)(参见，例如，Chen等，Bioconj.Chem.(生物缀合化学)，11:433-437(2000))。用于本发明的合适PoLixNano-CPL,和制备和使用PoLixNano-CPL的方法公开在，例如美国专利号6,852,334和PCT公开号WO 00/62813中，通过引用将其公开内容整体引入本文中用于全部目的。In addition to the aforementioned components, the polymer-lipid composition (e.g., PoLixNano) of the present invention may also include a cation-polymer-lipid conjugate (CPL) (see, e.g., Chen et al., Bioconj. Chem., 11: 433-437 (2000)). Suitable PoLixNano-CPLs for use in the present invention, and methods for preparing and using PoLixNano-CPLs are disclosed in, e.g., U.S. Pat. No. 6,852,334 and PCT Publication No. WO 00/62813, the disclosures of which are incorporated herein by reference in their entirety for all purposes.
在某些实例中，所述聚阳离子结构部分可以具有连接的配体，诸如靶向配体或用于络合钙的螯合结构部分。优选地，当配体连接后，阳离子结构部分保持正电荷。在某些实例中，连接的配体具有正电荷。合适的配体包括，但不仅限于，具有反应性官能团的化合物或装置并且包括脂质、两亲性脂质、载体化合物、生物亲合化合物、生物材料、生物聚合物、生物医学装置、可分析检测的化合物、治疗活性化合物、酶、肽、蛋白、抗体、免疫刺激剂、放射性标记物、荧光团、生物素、药物、半抗原、DNA、RNA、多糖、脂质体、病毒体、微团、免疫球蛋白、官能团、其他靶向结构部分，或毒素。In some instances, the polycationic moiety may have a connected ligand, such as a targeting ligand or a chelating moiety for complexing calcium. Preferably, after the ligand is connected, the cationic moiety maintains a positive charge. In some instances, the connected ligand has a positive charge. Suitable ligands include, but are not limited to, compounds or devices with reactive functional groups and include lipids, amphipathic lipids, carrier compounds, bioaffinity compounds, biomaterials, biopolymers, biomedical devices, compounds that can be analyzed and detected, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immunostimulants, radioactive markers, fluorophores, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, other targeting moieties, or toxins.
在一些实施方案中，本发明还设想了PEG修饰的磷脂和衍生化的脂质(例如衍生化的神经酰胺(PEG‐CER)，包括N‐辛酰基‐鞘氨醇‐l‐[琥珀酰(甲氧基聚乙二醇)‐2000](C8PEG‐2000神经酰胺))的使用。设想的PEG修饰的脂质包括 但不限于长度为最多2kDa、最多3kDa、最多4kDa或最多5kDa的聚乙二醇链，所述聚乙二醇链共价连接至具有C6‐C20长度的烷基链的脂质。在一些实施方案中，PEG修饰的或PEG化的脂质是PEG化的胆固醇。在一些实施方案中，PEG修饰的或PEG化的脂质是1,2-二肉豆蔻酰-sn-甘油基-3-磷酸乙醇胺-PEG(DMPE-PEG)，其中PEG部分含有10至140个重复单元，更优选地是1,2-二肉豆蔻酰-sn-甘油基-3-磷酸乙醇胺-PEG2000(DMPE-PEG2K)。此类组分的添加可防止复杂的聚集，并且还可提供用于增加循环寿命和增加包含聚合物-脂质的组合物向靶组织的递送的方法(Klibanov等人(1990)《FEBS快报(FEBS Letters)》，268(1):235-237)，或其可选择为在体内快速更换制剂(参见美国专利第5,885,613)。特别有用的可交换脂质是具有较短酰基链(例如，C14或C18)的PEG-神经酰胺。用于本发明的组合物和方法的合适的PEG修饰脂质包括如国际专利公开WO 2020061295和美国专利公开US 20220016029中所述的PEG脂质，该专利公开以引用的方式并入本文。In some embodiments, the present invention also contemplates the use of PEG-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-l-[succinyl(methoxypolyethylene glycol)-2000] (C8PEG-2000 ceramide). Contemplated PEG-modified lipids include But not limited to a polyethylene glycol chain of up to 2 kDa, up to 3 kDa, up to 4 kDa, or up to 5 kDa in length, covalently attached to a lipid having an alkyl chain of C 6 -C 20 in length. In some embodiments, the PEG-modified or PEGylated lipid is PEGylated cholesterol. In some embodiments, the PEG-modified or PEGylated lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG (DMPE-PEG), wherein the PEG portion contains 10 to 140 repeating units, more preferably 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG2000 (DMPE-PEG2K). The addition of such components can prevent complex aggregation, and can also provide a method for increasing the circulation life and increasing the delivery of compositions comprising polymer-lipids to target tissues (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or it can be selected as a rapid replacement formulation in vivo (see U.S. Patent No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides with shorter acyl chains (e.g., C 14 or C 18 ). Suitable PEG-modified lipids for the compositions and methods of the present invention include PEG lipids as described in International Patent Publication WO 2020061295 and U.S. Patent Publication US 20220016029, which are incorporated herein by reference.
在一些实施方案中，本发明的脂质缀合物(例如，PEG脂质)可通过化学结构修饰改善其靶向特征，例如糖基化修饰、蛋白靶向配体修饰、抗体修饰、多肽修饰、叶酸修饰、生长因子修饰、细胞因子修饰、维生素修饰以及整合素修饰等。以糖基化修饰为例，糖基化修饰的PEG脂质包含至少一个与糖基部分共轭的末端嵌段，优选一个末端亲水性嵌段，糖基部分可通过在所述糖基部分的一个官能团与所PEG脂质的一个官能团之间形成的共价键来共轭至本发明的PEG脂质。例如商品化的二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(polyethylene glycol2000)-甘露糖(mannose)(DSPE-PEG2K-Mannose)，所述共价键可由本身改性至反应性的两个官能团之间的反应来形成，糖基部分可直接共轭至PEG脂质。或者，糖基部分可通过间隔基共轭至PEG脂质。In some embodiments, the lipid conjugates of the present invention (e.g., PEG lipids) can improve their targeting characteristics by chemical structural modification, such as glycosylation modification, protein targeting ligand modification, antibody modification, polypeptide modification, folic acid modification, growth factor modification, cytokine modification, vitamin modification, and integrin modification. Taking glycosylation modification as an example, the glycosylated PEG lipid comprises at least one terminal block conjugated to a glycosyl moiety, preferably a terminal hydrophilic block, and the glycosyl moiety can be conjugated to the PEG lipid of the present invention by a covalent bond formed between a functional group of the glycosyl moiety and a functional group of the PEG lipid. For example, the commercialized distearoylphosphatidylethanolamine-polyethylene glycol 2000 (polyethylene glycol 2000)-mannose (mannose) (DSPE-PEG2K-Mannose), the covalent bond can be formed by the reaction between two functional groups that are modified to be reactive, and the glycosyl moiety can be directly conjugated to the PEG lipid. Alternatively, the glycosyl moiety can be conjugated to the PEG lipid through a spacer.
在一些实施方案中，本发明的脂质缀合物可以具有连接的配体，合适的配体包括，但不仅限于，具有反应性官能团的化合物或装置并且包括脂质、载体化合物、生物亲合化合物、生物材料、生物聚合物、生物医学装置、可分析检测的化合物、治疗活性化合物、酶、免疫刺激剂、放射性标记物、荧光团、生物素、药物、半抗原、DNA、RNA、多糖、脂质体、病毒体、微团、免疫球蛋白、官能团、毒素或其他靶向结构部分。In some embodiments, the lipid conjugates of the invention may have a ligand attached. Suitable ligands include, but are not limited to, compounds or devices having reactive functional groups and include lipids, carrier compounds, bioaffinity compounds, biomaterials, biopolymers, biomedical devices, analytically detectable compounds, therapeutically active compounds, enzymes, immunostimulants, radioactive labels, fluorophores, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, toxins or other targeting moieties.
在一些实施方案中，所述脂质缀合物占所述组合物中的总脂质的0.1mol％-10.0mol％，例如0.1mol％-10.0mol％、1mol％-10.0mol％、2mol％-10.0 mol％、3mol％-10.0mol％、5mol％-10.0mol％、0.1mol％-5mol％、1mol％-5mol％、2mol％-5mol％或3mol％-5.0mol％。In some embodiments, the lipid conjugate accounts for 0.1 mol%-10.0 mol% of the total lipids in the composition, e.g., 0.1 mol%-10.0 mol%, 1 mol%-10.0 mol%, 2 mol%-10.0 mol%, 3mol%-10.0mol%, 5mol%-10.0mol%, 0.1mol%-5mol%, 1mol%-5mol%, 2mol%-5mol% or 3mol%-5.0mol%.
在一些实施方案中，本文所述的包含聚合物-脂质的组合物含有以摩尔或重量计占总脂质的小于0.5％、小于0.4％、小于0.3％、小于0.2％或小于0.1％的PEG修饰的脂质缀合物或PEG。在一些实施方案中，本文所述的包含聚合物-脂质的组合物含有以摩尔或重量计占总脂质的0.4％或更少的PEG修饰的脂质或PEG、0.3％或更少的PEG修饰的脂质或PEG、0.2％或更少的PEG修饰的脂质或PEG、或0.1％或更少的PEG修饰的脂质或PEG。在一些实施方案中，本文所述的包含聚合物-脂质的组合物含有以摩尔或重量计占总脂质的0.01％或更少的PEG修饰的脂质或PEG。在一些实施方案中，本文所述的聚合物-脂质的组合物不包含脂质缀合物(例如，PEG-脂质)或PEG。In some embodiments, the compositions comprising polymer-lipids described herein contain less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1% of the total lipids by mole or weight. In some embodiments, the compositions comprising polymer-lipids described herein contain 0.4% or less of PEG-modified lipids or PEG, 0.3% or less of PEG-modified lipids or PEG, 0.2% or less of PEG-modified lipids or PEG, or 0.1% or less of PEG-modified lipids or PEG by mole or weight of total lipids. In some embodiments, the compositions comprising polymer-lipids described herein contain 0.01% or less of PEG-modified lipids or PEG by mole or weight of total lipids. In some embodiments, the compositions of polymer-lipids described herein do not include lipid conjugates (e.g., PEG-lipids) or PEG.
本领域中技术人员应该理解脂质缀合物的浓度可以发生变化，取决于所用脂质缀合物和聚合物-脂质的组合物形成融合的速率。It will be appreciated by those skilled in the art that the concentration of the lipid conjugate may vary, depending on the rate at which the lipid conjugate and polymer-lipid composition used form fusions.
通过控制脂质缀合物的组成和浓度，人们可以控制使脂质缀合物从聚合物-脂质组合物中交换出来的速率，和随即使核酸/聚合物-脂质组合物形成融合体的速率。例如，当PEG-磷脂酰乙醇胺缀合物或PEG-神经酰胺缀合物用作脂质缀合物时，使核酸/聚合物-脂质组合物形成融合体的速率可以例如通过改变脂质缀合物浓度，通过改变PEG分子量，或通过改变磷脂酰乙醇胺或神经酰胺上酰基链基团的链长度和饱和程度而变化。另外，其他变量包括，例如，pH、温度、离子强度等可以用于改变和/或控制使核酸/聚合物-脂质组合物形成融合体的速率。本领域中技术人员在阅读本发明公开内容时应该清楚可以用于控制使核酸/聚合物-脂质组合物形成融合体的速率的其他方法。By controlling the composition and concentration of the lipid conjugate, one can control the rate at which the lipid conjugate is exchanged from the polymer-lipid composition, and the rate at which the nucleic acid/polymer-lipid composition forms a fusion. For example, when a PEG-phosphatidylethanolamine conjugate or a PEG-ceramide conjugate is used as a lipid conjugate, the rate at which the nucleic acid/polymer-lipid composition forms a fusion can be varied, for example, by changing the lipid conjugate concentration, by changing the PEG molecular weight, or by changing the chain length and degree of saturation of the acyl chain groups on the phosphatidylethanolamine or ceramide. In addition, other variables include, for example, pH, temperature, ionic strength, etc., which can be used to change and/or control the rate at which the nucleic acid/polymer-lipid composition forms a fusion. Other methods that can be used to control the rate at which the nucleic acid/polymer-lipid composition forms a fusion will be clear to those skilled in the art when reading the disclosure of the present invention.
在本发明的包含聚合物-脂质的组合物的一些实施方案中，所述PEG-脂质缀合物包含选自以下的至少一种：DMG-PEG2K、DMPE-PEG2K、DSPE-PEG2K、DSPE-PEG2K-Mannose、DMG-PEG5K、DMPE-PEG5K、DSPE-PEG5K-Mannose和DSPE-PEG5K。In some embodiments of the polymer-lipid composition of the present invention, the PEG-lipid conjugate comprises at least one selected from the group consisting of DMG-PEG2K, DMPE-PEG2K, DSPE-PEG2K, DSPE-PEG2K-Mannose, DMG-PEG5K, DMPE-PEG5K, DSPE-PEG5K-Mannose and DSPE-PEG5K.
组合物combination
在本发明的组合物的一些实施方案中，所述组合物包含：In some embodiments of the compositions of the present invention, the compositions comprise:
(1)两亲性嵌段共聚物、阳离子脂质、磷脂、胆固醇和脂质缀合物例如PEG-脂质缀合物，其中所述阳离子脂质占所述组合物中存在的总脂质的30.0 mol％-80.0mol％，磷脂占总脂质的5.0mol％-50.0mol％，胆固醇占总脂质的14.0mol％-64.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比；(1) amphiphilic block copolymers, cationic lipids, phospholipids, cholesterol and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipid accounts for 30.0% of the total lipids present in the composition mol%-80.0mol%, phospholipids account for 5.0mol%-50.0mol% of the total lipids, cholesterol accounts for 14.0mol%-64.0mol% of the total lipids, lipid conjugates account for 0.1mol%-8.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% weight percent of the composition;
(2)两亲性嵌段共聚物、阳离子脂质、磷脂、胆固醇和脂质缀合物例如PEG-脂质缀合物，其中所述阳离子脂质占所述组合物中存在的总脂质的23.0mol％-75.0mol％，磷脂占总脂质的10.0mol％-62.0mol％，胆固醇占总脂质的14.0mol％-46.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比；(2) an amphiphilic block copolymer, a cationic lipid, a phospholipid, cholesterol, and a lipid conjugate such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 23.0 mol%-75.0 mol% of the total lipid present in the composition, the phospholipid accounts for 10.0 mol%-62.0 mol% of the total lipid, the cholesterol accounts for 14.0 mol%-46.0 mol% of the total lipid, the lipid conjugate accounts for 0.1 mol%-8.0 mol% of the total lipid, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition;
(3)两亲性嵌段共聚物、胆固醇衍生的阳离子脂质、磷脂和脂质缀合物例如PEG-脂质缀合物，其中所述胆固醇衍生的阳离子脂质占所述组合物中存在的总脂质的29.0mol％-80.0mol％，磷脂占总脂质的19.0mol％-70.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比；(3) amphiphilic block copolymers, cholesterol-derived cationic lipids, phospholipids, and lipid conjugates such as PEG-lipid conjugates, wherein the cholesterol-derived cationic lipids account for 29.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 19.0 mol%-70.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1%-95.0% by weight of the composition;
(4)两亲性嵌段共聚物、阳离子脂质、胆固醇和脂质缀合物例如PEG-脂质缀合物，其中所述阳离子脂质占所述组合物中存在的总脂质的25.0mol％-80.0mol％，胆固醇占总脂质的15.0mol％-50.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比；(4) an amphiphilic block copolymer, a cationic lipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 25.0 mol%-80.0 mol% of the total lipids present in the composition, cholesterol accounts for 15.0 mol%-50.0 mol% of the total lipids, the lipid conjugate accounts for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition;
(5)两亲性嵌段共聚物、阳离子脂质、磷脂和脂质缀合物例如PEG-脂质缀合物，其中所述阳离子脂质占所述组合物中存在的总脂质的30.0mol％-80.0mol％，磷脂占总脂质的10.0mol％-50.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比；(5) amphiphilic block copolymers, cationic lipids, phospholipids and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids account for 30.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 10.0 mol%-50.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1%-95.0% by weight of the composition;
(6)两亲性嵌段共聚物、阳离子脂质、磷脂和胆固醇，其中所述阳离子脂质占所述组合物中存在的总脂质的30.0mol％-80.0mol％，磷脂占总脂质的5.0mol％-50.0mol％，胆固醇占总脂质的15.0mol％-50.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比；或(6) an amphiphilic block copolymer, a cationic lipid, a phospholipid, and cholesterol, wherein the cationic lipid accounts for 30.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 5.0 mol%-50.0 mol% of the total lipids, the cholesterol accounts for 15.0 mol%-50.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition; or
(7)两亲性嵌段共聚物、胆固醇衍生的阳离子脂质和磷脂，其中所述胆固醇衍生的阳离子脂质占所述组合物中存在的总脂质的30.0mol％-70.0mol％,磷脂占总脂质的30.0mol％-70.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比。 (7) an amphiphilic block copolymer, a cholesterol-derived cationic lipid and a phospholipid, wherein the cholesterol-derived cationic lipid accounts for 30.0 mol%-70.0 mol% of the total lipids present in the composition, the phospholipids account for 30.0 mol%-70.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition.
在本发明的组合物的一些实施方案中，所述组合物包含两亲性嵌段共聚物和以下组分：In some embodiments of the composition of the present invention, the composition comprises an amphiphilic block copolymer and the following components:
(1)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的40.0mol％-70.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的8.0mol％-39.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的15.0％-90.0％重量百分比；(1) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 The composition comprises 40.0 mol% to 70.0 mol% of the total lipids, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE comprises 8.0 mol% to 39.0 mol% of the total lipids, cholesterol or β-sitosterol comprises 20.0 mol% to 40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose comprises 0.1 mol% to 5.0 mol% of the total lipids, and the amphiphilic block copolymer comprises 15.0% to 90.0% by weight of the composition;
(2)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的30mol％-60mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的10.0mol％-49.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的20.0％-90.0％重量百分比；(2) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 1 accounts for 30 mol%-60 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 10.0 mol%-49.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 20.0%-90.0% weight percentage of the composition;
(3)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的24.0mol％-40.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的30.0mol％-64.0mol％，胆固醇或β-谷甾醇占总脂质的15.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的20.0％-90.0％重量百分比；(3) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 accounts for 24.0mol%-40.0mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE account for 30.0mol%-64.0mol% of the total lipids, cholesterol or β-sitosterol account for 15.0mol%-40.0mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1mol%-5.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 20.0%-90.0% weight percent of the composition;
(4)DOTAP、DODAP、DOTMA或DOSPA；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG- PEG5K或DSPE-PEG2K-Mannose，其中所述DOTAP、DODAP、DOTMA或DOSPA占所述组合物中存在的总脂质的23.0mol％-60mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的14.0mol％-60.0mol％，胆固醇或β-谷甾醇占总脂质的15.0mol％-50.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的30.0％-90.0％重量百分比；(4) DOTAP, DODAP, DOTMA or DOSPA; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG- PEG5K or DSPE-PEG2K-Mannose, wherein the DOTAP, DODAP, DOTMA or DOSPA accounts for 23.0mol%-60mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 14.0mol%-60.0mol% of the total lipids, cholesterol or β-sitosterol accounts for 15.0mol%-50.0mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1mol%-5.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% weight percent of the composition;
(5)GL67、ICE、或HGT4002；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述GL67、ICE、或HGT4002占所述组合物中存在的总脂质的40.0mol％-80.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的10.0mol％-50.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的30.0％-90.0％重量百分比；(5) GL67, ICE, or HGT4002; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE; and DMG-PEG2K, DMG-PEG5K, or DSPE-PEG2K-Mannose, wherein the GL67, ICE, or HGT4002 accounts for 40.0 mol%-80.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE accounts for 10.0 mol%-50.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K, or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition;
(6)cKK-E12、DLin-MC3-DMA、ALC-0315、SM-102、C12-200、DOTAP、DODAP、DOTMA、DOSPA、HGT5000或HGT5001；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述cKK-E12、DLin-MC3-DMA、ALC-0315、SM-102、C12-200、DOTAP、DODAP、DOTMA、DOSPA、HGT5000或HGT5001占所述组合物中存在的总脂质的45.0mol％-75.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-45.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的30.0％-90.0％重量百分比；(6) cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, C12-200, DOTAP, DODAP, DOTMA, DOSPA, HGT5000 or HGT5001; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, C12-200, DOTAP , DODAP, DOTMA, DOSPA, HGT5000 or HGT5001 account for 45.0mol%-75.0mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 20.0mol%-45.0mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1mol%-5.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% weight percentage of the composition;
(7)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的30.0mol％-65.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的40.0％-90.0％重量百分比；(7) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 account for 30.0 mol%-65.0 mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 20.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% by weight of the composition;
(8)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin- MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的35.0mol％-70.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的10.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的40.0％-90.0％重量百分比；(8) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin- MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 account for 35.0 mol%-70.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE account for 10.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% weight percent of the composition;
(9)GL67、ICE、或HGT4002；和DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE，其中所述GL67、ICE、或HGT4002占所述组合物中存在的总脂质的40.0mol％-80.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的10.0mol％-50.0mol％，且所述两亲性嵌段共聚物占所述组合物的40.0％-90.0％重量百分比；或(9) GL67, ICE, or HGT4002; and DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE, wherein the GL67, ICE, or HGT4002 accounts for 40.0 mol%-80.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM, or DOPE accounts for 10.0 mol%-50.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% weight percent of the composition; or
(10)DLin-MC3-DMA、ALC-0315、SM-102、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；和胆固醇或β-谷甾醇，其中所述DLin-MC3-DMA、ALC-0315、SM-102、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的30.0mol％-60.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的20.0mol％-45.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-45.0mol％，且所述两亲性嵌段共聚物占所述组合物的30.0％-90.0％重量百分比。(10) DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and cholesterol or β-sitosterol, wherein the DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, HGT5000 or HGT5001 accounts for 30.0 mol%-60.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 20.0 mol%-45.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol%-45.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% weight percent of the composition.
在本发明的包含聚合物-脂质的组合物的一些实施方案中，所述组合物包含以下组分：In some embodiments of the polymer-lipid-containing compositions of the invention, the compositions comprise the following components:
(1)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的10.0mol％，胆固醇或β-谷甾醇占总脂质的38.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.5mol％，且所述两亲性嵌段共聚物占所述组合物的89.9％重量百分比；(1) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.0 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 89.9% by weight of the composition;
(2)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的10.6mol％，胆固醇或β-谷甾醇占总脂质的38.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的72.9％重量百分比； (2) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.6 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 72.9% by weight of the composition;
(3)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的12.5mol％，胆固醇或β-谷甾醇占总脂质的36.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.5mol％，且所述两亲性嵌段共聚物占所述组合物的43.0％重量百分比；(3) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 12.5 mol% of the total lipids, cholesterol or β-sitosterol account for 36.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.0% by weight of the composition;
(4)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.5mol％，胆固醇或β-谷甾醇占总脂质的34.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.5mol％，且所述两亲性嵌段共聚物占所述组合物的81.8％重量百分比；(4) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or β-sitosterol account for 34.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 81.8% by weight of the composition;
(5)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.0mol％，胆固醇或β-谷甾醇占总脂质的35.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的69.2％重量百分比；(5) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.0 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 69.2% by weight of the composition;
(6)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.1mol％，胆固醇或β-谷甾醇占总脂质的35.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的43.3％重量百分比；(6) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.3% by weight of the composition;
(7)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.3mol％，胆固醇或β-谷甾醇占总脂质的35.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.7mol％，且所述两亲性嵌段共聚物占所述组合物的48.4％重量百分比；(7) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.3 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.4% by weight of the composition;
(8)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、 DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.5mol％，胆固醇或β-谷甾醇占总脂质的35.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.5mol％，且所述两亲性嵌段共聚物占所述组合物的43.6％重量百分比；(8) Amphiphilic block copolymers; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.6% by weight of the composition;
(9)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的48.5mol％，DSPC、DPPC或DOPE占总脂质的17.0mol％，胆固醇或β-谷甾醇占总脂质的32.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的42.3％重量百分比；(9) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 48.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.3% by weight of the composition;
(10)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.5mol％，DSPC、DPPC或DOPE占总脂质的20.0mol％，胆固醇或β-谷甾醇占总脂质的32.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的52.9％重量百分比；(10) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 52.9% by weight of the composition;
(11)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的49.0mol％，DSPC、DPPC或DOPE占总脂质的20.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的41.9％重量百分比；(11) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition;
(12)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的49.0mol％，DSPC、DPPC或DOPE占总脂质的20.1mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的40.3％重量百分比；(12) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.1 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.3% by weight of the composition;
(13)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所 述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的51.6mol％，DSPC、DPPC或DOPE占总脂质的20.0mol％，胆固醇或β-谷甾醇占总脂质的27.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的42.9％重量百分比；(13) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein The DLin-MC3-DMA, ALC-0315 or SM-102 account for 51.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 27.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.9% by weight of the composition;
(14)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的50.2％重量百分比；(14) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.2% by weight of the composition;
(15)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的66.1％重量百分比；(15) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 66.1% by weight of the composition;
(16)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的74.5％重量百分比；(16) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 74.5% by weight of the composition;
(17)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的79.6％重量百分比；(17) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 79.6% by weight of the composition;
(18)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0 mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的83.0％重量百分比；(18) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 accounts for 46.0% of the total lipids present in the composition. mol%, DSPC, DPPC or DOPE account for 23.0mol% of the total lipids, cholesterol or β-sitosterol account for 30.0mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 83.0% by weight of the composition;
(19)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的88.2％重量百分比；(19) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 88.2% by weight of the composition;
(20)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的43.0mol％，DSPC、DPPC或DOPE占总脂质的23.3mol％，胆固醇或β-谷甾醇占总脂质的33.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.7mol％，且所述两亲性嵌段共聚物占所述组合物的39.1％重量百分比；(20) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 43.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.3 mol% of the total lipids, cholesterol or β-sitosterol account for 33.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 39.1% by weight of the composition;
(21)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的44.0mol％，DSPC、DPPC或DOPE占总脂质的25.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的63.6％重量百分比；(21) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 44.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 63.6% by weight of the composition;
(22)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的39.5mol％，DSPC、DPPC或DOPE占总脂质的25.3mol％，胆固醇或β-谷甾醇占总脂质的34.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.7mol％，且所述两亲性嵌段共聚物占所述组合物的37.2％重量百分比；(22) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.3 mol% of the total lipids, cholesterol or β-sitosterol account for 34.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 37.2% by weight of the composition;
(23)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的41.0mol％，DSPC、DPPC或DOPE占总脂质的28.0mol％，胆固醇或β-谷甾醇占总 脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的66.0％重量百分比；(23) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 41.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, and cholesterol or β-sitosterol account for 28.0 mol% of the total lipids. 30.0 mol% of lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.0 mol% of total lipids, and the amphiphilic block copolymer accounts for 66.0% by weight of the composition;
(24)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的39.0mol％，DSPC、DPPC或DOPE占总脂质的28.0mol％，胆固醇或β-谷甾醇占总脂质的32.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的36.3％重量百分比；(24) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.3% by weight of the composition;
(25)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的39.0mol％，DSPC、DPPC或DOPE占总脂质的30.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的62.9％重量百分比；(25) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 62.9% by weight of the composition;
(26)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的40.6mol％，DSPC、DPPC或DOPE占总脂质的30.1mol％，胆固醇或β-谷甾醇占总脂质的28.4mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的36.9％重量百分比；(26) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.1 mol% of the total lipids, cholesterol or β-sitosterol account for 28.4 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.9% by weight of the composition;
(27)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的34.7mol％，DSPC、DPPC或DOPE占总脂质的40.1mol％，胆固醇或β-谷甾醇占总脂质的24.3mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的32.5％重量百分比；(27) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 34.7 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 40.1 mol% of the total lipids, cholesterol or β-sitosterol account for 24.3 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 32.5% by weight of the composition;
(28)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的29.0mol％，DSPC、DPPC或DOPE占总脂质的50.1mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述 两亲性嵌段共聚物占所述组合物的28.0％重量百分比；(28) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 29.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 50.1 mol% of the total lipids, cholesterol or β-sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the The amphiphilic block copolymer accounts for 28.0% by weight of the composition;
(29)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的35.0mol％，DSPC、DPPC或DOPE占总脂质的17.6mol％，胆固醇或β-谷甾醇占总脂质的46.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的35.9％重量百分比；(29) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 35.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.6 mol% of the total lipids, cholesterol or β-sitosterol account for 46.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 35.9% by weight of the composition;
(30)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的55.0mol％，DSPC、DPPC或DOPE占总脂质的16.9mol％，胆固醇或β-谷甾醇占总脂质的27.2mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的44.6％重量百分比；(30) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 55.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 16.9 mol% of the total lipids, cholesterol or β-sitosterol account for 27.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 44.6% by weight of the composition;
(31)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的60.0mol％，DSPC、DPPC或DOPE占总脂质的14.9mol％，胆固醇或β-谷甾醇占总脂质的24.2mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的46.5％重量百分比；(31) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 60.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.9 mol% of the total lipids, cholesterol or β-sitosterol account for 24.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 46.5% by weight of the composition;
(32)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的65.0mol％，DSPC、DPPC或DOPE占总脂质的14.1mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的21.0％重量百分比；(32) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 65.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or β-sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 21.0% by weight of the composition;
(33)两亲性嵌段共聚物；GL67、ICE或HGT4002；DSPC、DPPC或DOPE；和DMG-PEG2K或DMG-PEG5K，其中所述GL67、ICE或HGT4002占所述组合物中存在的总脂质的70.0mol％，DSPC、DPPC或DOPE占总脂质的28.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.5mol％，且所述两亲性嵌段共聚物占所述组合物的48.1％重量百分比；(33) an amphiphilic block copolymer; GL67, ICE or HGT4002; DSPC, DPPC or DOPE; and DMG-PEG2K or DMG-PEG5K, wherein the GL67, ICE or HGT4002 accounts for 70.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE accounts for 28.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.1% by weight of the composition;
(34)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；胆固醇 或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的61.3mol％，胆固醇或β-谷甾醇占总脂质的37.6mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.1mol％，且所述两亲性嵌段共聚物占所述组合物的41.9％重量百分比；(34) Amphiphilic block copolymers; DLin-MC3-DMA, ALC-0315 or SM-102; cholesterol Or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 61.3 mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 37.6 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.1 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition;
(35)两亲性嵌段共聚物；cKK-E12、DLin-MC3-DMA、ALC-0315、SM-102或C12-200；DOTAP、DODAP、DOTMA或DOSPA；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述cKK-E12、DLin-MC3-DMA、ALC-0315、SM-102或C12-200占所述组合物中存在的总脂质的30.0mol％，DOTAP、DODAP、DOTMA或DOSPA占总脂质的39.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的57.7％重量百分比；或(35) an amphiphilic block copolymer; cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102 or C12-200; DOTAP, DODAP, DOTMA or DOSPA; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102 or C12-200 accounts for 30.0 mol% of the total lipids present in the composition, DOTAP, DODAP, DOTMA or DOSPA accounts for 39.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 57.7% by weight of the composition; or
(36)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；和胆固醇或β-谷甾醇，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的40.0mol％，DSPC、DPPC或DOPE占总脂质的32.0mol％，胆固醇或β-谷甾醇占总脂质的28.0mol％，且所述两亲性嵌段共聚物占所述组合物的50.0％重量百分比。(36) An amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; and cholesterol or β-sitosterol, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 32.0 mol% of the total lipids, cholesterol or β-sitosterol account for 28.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.0% weight percent of the composition.
在本发明的组合物的一些实施方案中，所述组合物中的阳离子脂质中的氮(胺)基团与核酸的磷酸酯基团的摩尔比(N/P比)为为约0.5-约40，约1-约30，约2-约25，约3-约20，约4-约15，约5-约10，约6-约8，约2-约12，约4-约8，约5-约8，约6-约7.8，约6.7至约7.6或约6.8-约7.5。。In some embodiments of the composition of the invention, the molar ratio of nitrogen (amine) groups in the cationic lipid to phosphate groups of the nucleic acid (N/P ratio) in the composition is about 0.5 to about 40, about 1 to about 30, about 2 to about 25, about 3 to about 20, about 4 to about 15, about 5 to about 10, about 6 to about 8, about 2 to about 12, about 4 to about 8, about 5 to about 8, about 6 to about 7.8, about 6.7 to about 7.6 or about 6.8 to about 7.5.
在本发明的组合物的一些实施方案中，所述组合物中的脂质与核酸的比(质量/质量比)为约1(1:1)-约100(100:1)，约5(5:1)-约90(90:1)，约1(1:1)-约50(50:1)，约5(5:1)-约45(45:1)，约10(10:1)-约40(40:1)，约15(15:1)-约35(35:1)，约20(20:1)-约30(30:1)，约1(1:1)-约25(25:1)，约5(5:1)-约30(30:1)，约5(5:1)-约20(20:1)，约5(5:1)-约15(15:1)或约5(5:1)-约10(10:1)。In some embodiments of the compositions of the invention, the ratio of lipid to nucleic acid in the composition (mass/mass ratio) is about 1 (1:1) to about 100 (100:1), about 5 (5:1) to about 90 (90:1), about 1 (1:1) to about 50 (50:1), about 5 (5:1) to about 45 (45:1), about 10 (10:1) to about 40 (40:1), about 15 (15:1) to about 35 (35:1), about 20 (20:1) to about 30 (30:1), about 1 (1:1) to about 25 (25:1), about 5 (5:1) to about 30 (30:1), about 5 (5:1) to about 20 (20:1), about 5 (5:1) to about 15 (15:1) or about 5 (5:1) to about 10 (10:1).
在本发明包含的一些实施方案中，包括由至少第一和第二单独形成的非相同的聚合物-脂质组合物的混合物。[0013] In some embodiments encompassed by the present invention, a mixture of non-identical polymer-lipid compositions formed from at least a first and a second separate entity is included.
每个聚合物-脂质组合物包含mRNA和一种或多种阳离子脂质，其中所述第一聚合物-脂质组合物包括第一阳离子脂质，所述第二聚合物-脂质组合物包含第二阳离子脂质；其中所述第一阳离子脂质和所述第二阳离子脂质不相同；和其中在向受试者施用药物组合物后由mRNA编码的蛋白质或肽的表达 超过了用第一脂质纳米粒子但不使用第二脂质纳米粒子施用的其他相同量的mRNA编码蛋白质或肽表达至少约两倍。Each polymer-lipid composition comprises an mRNA and one or more cationic lipids, wherein the first polymer-lipid composition comprises a first cationic lipid and the second polymer-lipid composition comprises a second cationic lipid; wherein the first cationic lipid and the second cationic lipid are not the same; and wherein expression of a protein or peptide encoded by the mRNA after administration of the pharmaceutical composition to a subject The expression of the mRNA encoding protein or peptide is at least about two-fold greater than that of an otherwise identical amount administered with the first lipid nanoparticle but without the second lipid nanoparticle.
在本发明包含的一些实施方案中，其中所述第一聚合物-脂质组合物和所述第二聚合物-脂质组合物包含一种或多种非阳离子脂质。其中所述第一非阳离子脂质和所述第二非阳离子脂质不相同；和其中在向受试者施用药物组合物后由mRNA编码的蛋白质或肽的表达超过了用第一聚合物-脂质组合物但不使用第二聚合物-脂质组合物施用的其他相同量的mRNA编码蛋白质或肽表达至少约两倍。In some embodiments included in the present invention, wherein the first polymer-lipid composition and the second polymer-lipid composition comprise one or more non-cationic lipids. Wherein the first non-cationic lipid and the second non-cationic lipid are not the same; and wherein the expression of a protein or peptide encoded by an mRNA after administering the pharmaceutical composition to a subject exceeds the expression of an otherwise identical amount of mRNA-encoded protein or peptide administered with the first polymer-lipid composition but not with the second polymer-lipid composition by at least about two times.
在本发明包含的一些实施方案中，其中所述第一聚合物-脂质组合物和所述第二聚合物-脂质组合物包含一种或多种磷脂。其中所述第一磷脂和所述第二磷脂不相同；和其中在向受试者施用药物组合物后由mRNA编码的蛋白质或肽的表达超过了用第一聚合物-脂质组合物但不使用第二聚合物-脂质组合物施用的其他相同量的mRNA编码蛋白质或肽表达至少约两倍。In some embodiments encompassed by the present invention, wherein the first polymer-lipid composition and the second polymer-lipid composition comprise one or more phospholipids. wherein the first phospholipid and the second phospholipid are not identical; and wherein the expression of a protein or peptide encoded by the mRNA after administration of the pharmaceutical composition to a subject exceeds the expression of an otherwise identical amount of mRNA-encoded protein or peptide administered with the first polymer-lipid composition but without the second polymer-lipid composition by at least about two times.
在本发明包含的一些实施方案中，其中所述第一聚合物-脂质组合物和所述第二聚合物-脂质组合物包含一种或多种脂质缀合物。其中所述第一脂质缀合物和所述第二脂质缀合物不相同；和其中在向受试者施用药物组合物后由mRNA编码的蛋白质或肽的表达超过了用第一聚合物-脂质组合物但不使用第二聚合物-脂质组合物施用的其他相同量的mRNA编码蛋白质或肽表达至少约两倍。In some embodiments encompassed by the present invention, wherein the first polymer-lipid composition and the second polymer-lipid composition comprise one or more lipid conjugates. wherein the first lipid conjugate and the second lipid conjugate are not the same; and wherein the expression of a protein or peptide encoded by the mRNA after administration of the pharmaceutical composition to a subject exceeds the expression of an otherwise identical amount of mRNA-encoded protein or peptide administered with the first polymer-lipid composition but without the second polymer-lipid composition by at least about two times.
在本发明包含的一些实施方案中，其中所述第一聚合物-脂质组合物和所述第二聚合物-脂质组合物包含一种或多种两亲性嵌段共聚物。其中所述第一两亲性嵌段共聚物和所述第二两亲性嵌段共聚物不相同；和其中在向受试者施用药物组合物后由mRNA编码的蛋白质或肽的表达超过了用第一聚合物-脂质组合物但不使用第二聚合物-脂质组合物施用的其他相同量的mRNA编码蛋白质或肽表达至少约两倍。In some embodiments included in the present invention, wherein the first polymer-lipid composition and the second polymer-lipid composition comprise one or more amphiphilic block copolymers. Wherein the first amphiphilic block copolymer and the second amphiphilic block copolymer are not the same; and wherein the expression of a protein or peptide encoded by mRNA after administering the pharmaceutical composition to a subject exceeds the expression of an otherwise identical amount of mRNA encoded protein or peptide administered with the first polymer-lipid composition but not with the second polymer-lipid composition by at least about two times.
在本发明的包含的一些实施方案中，其中所述药物组合物中所述第一聚合物-脂质组合物与所述第二聚合物-脂质组合物的比例为约1:1或约2:1或约3:1或约4:1。In some embodiments of the present invention, the ratio of the first polymer-lipid composition to the second polymer-lipid composition in the pharmaceutical composition is about 1:1 or about 2:1 or about 3:1 or about 4:1.
在本发明包含的一些实施方案中，可在聚合物-脂质组合物中额外添加能够分布在聚合物-脂质组合物纳米颗粒外表面的阳离子脂质(或非脂质阳离子剂)。在一些实施方案中，额外添加的阳离子脂质可为固醇胺，例如GL67, ICE等。在一些实施方案中，额外添加的非脂质阳离子剂可为氨丁三醇、氯化苄烷铵、经修饰精氨酸、氯化鲸蜡基吡啶鎓、L-赖氨酸单水合物等。在另一些实施方案中，聚合物-脂质组合物与阳离子脂质(或非脂质阳离子剂)的接触包含将阳离子脂质(或非脂质阳离子剂)溶解于非离子赋形剂中。在一些实施方案中，非离子赋形剂选自聚乙二醇(15)-羟基硬脂酸酯(HS 15)，1,2-二肉豆蔻酰基-外消旋-甘油-3-甲氧基聚乙二醇-2000(DMG-PEG2K)，聚氧乙烯脱水山梨糖醇单油酸酯(吐温-80)，单油酸脱水山梨糖醇酯(司盘-85)，聚氧乙烯脂肪酸酯(卖泽，如Myrij 52)，聚氧乙烯脂肪醇醚(苄泽，如Brij 35)以及α-生育酚聚乙二醇琥珀酸酯(TPGS)。在一些实施方案中，聚合物-脂质组合物与阳离子脂质(或非脂质阳离子剂)的接触包含将阳离子脂质(或非脂质阳离子剂)溶解于缓冲溶液中，例如PBS以及Tris缓冲液等。In some embodiments included in the present invention, cationic lipids (or non-lipid cationic agents) that can be distributed on the outer surface of polymer-lipid composition nanoparticles can be additionally added to the polymer-lipid composition. In some embodiments, the additionally added cationic lipid can be a sterolamine, such as GL67, ICE, etc. In some embodiments, the additional non-lipid cationic agent may be tromethamine, benzalkonium chloride, modified arginine, cetylpyridinium chloride, L-lysine monohydrate, etc. In other embodiments, the contact of the polymer-lipid composition with the cationic lipid (or non-lipid cationic agent) comprises dissolving the cationic lipid (or non-lipid cationic agent) in a non-ionic excipient. In some embodiments, the non-ionic excipient is selected from polyethylene glycol (15)-hydroxystearate ( HS 15), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K), polyoxyethylene sorbitan monooleate (Tween-80), sorbitan monooleate (Span-85), polyoxyethylene fatty acid esters (such as Myrij 52), polyoxyethylene fatty alcohol ethers (such as Brij 35) and α-tocopherol polyethylene glycol succinate (TPGS). In some embodiments, the contacting of the polymer-lipid composition with the cationic lipid (or non-lipid cationic agent) comprises dissolving the cationic lipid (or non-lipid cationic agent) in a buffer solution, such as PBS and Tris buffer.
用于本发明的组合物和方法的合适的聚合物-脂质组合物的混合物包括如国际专利公开WO 2014144196、WO 2022032154和美国专利US 10,130,649中所述的协同增强核酸递送混合制剂，该专利公开以引用的方式并入本文。Suitable mixtures of polymer-lipid compositions for use in the compositions and methods of the present invention include synergistically enhanced nucleic acid delivery mixed formulations as described in International Patent Publications WO 2014144196, WO 2022032154 and U.S. Patent US 10,130,649, which are incorporated herein by reference.
在本发明的组合物的一些实施方案中，所述活性剂或治疗剂进一步包含蛋白质或多肽，在一些实施方案中，所述蛋白是与翻译或转录有关的蛋白。在一些实施方案中，所述蛋白与CRISPR过程有关。在一些实施方案中，所述蛋白是CRISPR有关的蛋白。在一些实施方案中，所述蛋白是Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Cas10、Cas12a、Cas13a、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csfl、Csf2、Csf3、Csf4、其同系物或其修饰形式。在一些实施方案中，所述蛋白是Cas9。In some embodiments of the compositions of the present invention, the active agent or therapeutic agent further comprises a protein or polypeptide, in some embodiments, the protein is a protein related to translation or transcription. In some embodiments, the protein is related to the CRISPR process. In some embodiments, the protein is a CRISPR-related protein. In some embodiments, the protein is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a, Cas13a, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, a homologue thereof, or a modified form thereof. In some embodiments, the protein is Cas9.
在一些实施方案中，所述蛋白质或多肽与所述核酸以约1:1至约1:20的摩尔比存在。在一些实施方案中，所述摩尔比是约1:1至约1:10。在一些实施方案中，所述摩尔比是约1:3至约1:8。In some embodiments, the protein or polypeptide is present in a molar ratio of about 1:1 to about 1:20 to the nucleic acid. In some embodiments, the molar ratio is about 1:1 to about 1:10. In some embodiments, the molar ratio is about 1:3 to about 1:8.
在一些实施方案中，所述治疗剂是蛋白或多肽。在一些实施方案中，所述组合物包含蛋白和核酸两者。在一些实施方案中，所述组合物包含Cas9蛋白和单指导核酸。在一些实施方案中，所述组合物包含Cas9蛋白、单指导核酸和供体DNA。In some embodiments, the therapeutic agent is a protein or polypeptide. In some embodiments, the composition comprises both a protein and a nucleic acid. In some embodiments, the composition comprises a Cas9 protein and a single guide nucleic acid. In some embodiments, the composition comprises a Cas9 protein, a single guide nucleic acid, and a donor DNA.
在一些实施方案中，所述治疗剂是小分子诸如选自以下的小分子：抗癌 剂、抗真菌剂、精神病学药剂诸如镇痛药、意识水平‐改变剂诸如麻醉剂或催眠药、非甾体类抗炎药(NSAIDS)、驱蠕虫药、抗痤疮剂、抗心绞痛药、抗心律不齐药、抗哮喘药、抗细菌剂、抗‐良性前列腺肥大药、抗凝血剂、抗抑郁药、抗糖尿病剂、止吐药、抗癫痫药、抗痛风药、抗高血压剂、抗炎剂、抗疟药、抗偏头痛药、抗毒蕈碱剂、抗肿瘤剂、抗肥胖剂、抗骨质疏松剂、抗帕金森综合征药、抗增殖剂、抗原虫剂、抗甲状腺剂、镇咳剂、抗尿失禁剂、抗病毒剂、抗焦虑剂、食欲抑制剂、β‐阻滞剂、心脏正性肌力剂、化疗药物、认知增强剂、避孕剂、皮质类固醇、Cox‐2抑制剂、利尿剂、勃起功能障碍改善剂、祛痰药、胃肠剂、组胺受体拮抗剂、免疫抑制剂、角质软化剂、脂质调节剂、白三烯抑制剂、大环内酯类、肌肉松弛药、神经安定药、营养剂、麻醉性镇痛剂、蛋白酶抑制剂或镇静剂。In some embodiments, the therapeutic agent is a small molecule such as a small molecule selected from the group consisting of: anticancer anti-inflammatory drugs, anti-fungal drugs, psychiatric drugs such as analgesics, consciousness-altering agents such as anesthetics or hypnotics, nonsteroidal anti-inflammatory drugs (NSAIDS), anthelmintics, anti-acne agents, anti-angina agents, anti-arrhythmic agents, anti-asthma drugs, anti-bacterial agents, anti-benign prostatic hypertrophy agents, anticoagulants, antidepressants, anti-diabetic agents, antiemetics, anti-epileptic drugs, anti-gout drugs, anti-hypertensive agents, anti-inflammatory agents, anti-malarials, anti-migraine drugs, anti-muscarinics, anti-neoplastic agents, anti-obesity agents, anti-osteoporosis agents, anti-Parkinson's syndrome agents, anti- proliferative agents, antiprotozoal agents, antithyroid agents, antitussives, antiincontinence agents, antiviral agents, anxiolytics, appetite suppressants, beta-blockers, cardiac inotropes, chemotherapeutic agents, cognitive enhancers, contraceptives, corticosteroids, Cox-2 inhibitors, diuretics, erectile dysfunction improvers, expectorants, gastrointestinal agents, histamine receptor antagonists, immunosuppressants, keratolytics, lipid regulators, leukotriene inhibitors, macrolides, muscle relaxants, neuroleptics, nutritional agents, narcotic analgesics, protease inhibitors or sedatives.
在本发明的组合物的一些实施方案中，所述组合物进一步包含靶向部分以将所述组合物靶向至受试者中的目标器官、组织或细胞，优选所述靶向部分包含选自以下中的至少一种：糖基、脂质、核酸适配体、小分子治疗剂、维生素、多肽和蛋白质例如抗体。优选地，所述靶向部分优选自下组：上皮细胞配体，特别是呼吸道上皮细胞配体、胃肠道上皮细胞配体、生殖道上皮细胞配体或微折叠细胞配体；免疫细胞配体，特别是树突细胞配体、T细胞配体、B细胞配体或巨噬细胞配体；内皮细胞配体，特别是肺内皮细胞配体或肝内皮细胞配体；肿瘤细胞配体，特别是黑色素瘤、肺或肝组织相关肿瘤细胞配体；和/或皮肤细胞配体，特别是真皮成纤维细胞配体或角质形成细胞配体。In some embodiments of the composition of the present invention, the composition further comprises a targeting moiety to target the composition to a target organ, tissue or cell in a subject, preferably the targeting moiety comprises at least one selected from the following: a glycosyl, a lipid, a nucleic acid aptamer, a small molecule therapeutic agent, a vitamin, a polypeptide and a protein such as an antibody. Preferably, the targeting moiety is preferably selected from the following group: an epithelial cell ligand, in particular a respiratory epithelial cell ligand, a gastrointestinal epithelial cell ligand, a reproductive epithelial cell ligand or a microfolding cell ligand; an immune cell ligand, in particular a dendritic cell ligand, a T cell ligand, a B cell ligand or a macrophage ligand; an endothelial cell ligand, in particular a lung endothelial cell ligand or a liver endothelial cell ligand; a tumor cell ligand, in particular a melanoma, a lung or liver tissue-associated tumor cell ligand; and/or a skin cell ligand, in particular a dermal fibroblast ligand or a keratinocyte ligand.
在本发明的组合物的一些实施方案中，所述组合物还包含佐剂，优选所述佐剂包含选自以下的至少一种：CpG寡脱氧核苷酸、聚肌苷：聚胞苷酸、皂角提取物(QS-21提取物)、铝佐剂、角鲨烯、α-生育酚、吐温(Tween)、司盘(Span)、脂多糖LPS、Pam3CSK4三酰脂肽、环腺苷二磷酸(c-di-AMP)、2′3′-环鸟苷一磷酸腺苷一磷酸(cGAMP)、单磷酰-脂质A、MPL脂质、鞭毛蛋白或免疫调节蛋白如IL-2、IL-12、GM-CSF、TSLP和编码这些免疫调节剂蛋白的核酸。In some embodiments of the composition of the present invention, the composition further comprises an adjuvant, preferably the adjuvant comprises at least one selected from the following: CpG oligodeoxynucleotides, polyinosinic: polycytidylic acid, saponin extract (QS-21 extract), aluminum adjuvant, squalene, α-tocopherol, Tween, Span, lipopolysaccharide LPS, Pam 3 CSK 4 triacyl lipopeptide, cyclic adenosine diphosphate (c-di-AMP), 2′3′-cyclic guanosine monophosphate adenosine monophosphate (cGAMP), monophosphoryl-lipid A, MPL lipid, flagellin or immunomodulatory proteins such as IL-2, IL-12, GM-CSF, TSLP and nucleic acids encoding these immunomodulator proteins.
合适的佐剂可以包括但不限于无机盐(例如，AlK(SO4)2、AlNa(SO4)2、AlNH(SO4)2、二氧化硅、铝、氢氧化铝、Ca3(PO4)2、高岭土或碳)、具有或不具有免疫刺激复合物(ISCOM)的多核苷酸(例如，CpG寡核苷酸，诸如Chuang,T.H.等人，(2002)J.Leuk.Biol.71(3):538-44；AhmadNejad,P.等人 (2002)Eur.J.Immunol.32(7):1958-68中描述的那些)；聚IC或聚AU酸，具有或不具有CpG的聚精氨酸(在本领域中也称为IC31；参见Schella ck,C.等人(2003)Proceedings of the 34th Annual Meeting of the German Society of Immunology；Lingnau,K.等人(2002)Vaccine 20(29-30):3498-508)、JuvaVaxTM(美国专利号6,693,086)、某些天然物质(例如，来自结核分枝杆菌的蜡D，短小棒状杆菌(Cornyebacterium parvum)、百日咳杆菌(Bordetella pertussis)中存在的物质，或布鲁氏菌(Brucella)属成员)、鞭毛蛋白(Toll样受体5配体；参见McSorley,S.J.等人(2002)J.Immunol.169(7):3914-9)、皂苷(诸如QS21、QS17和QS7)(美国专利号5,057,540；5,650,398；6,524,584；6,645,495)、单磷酰基脂质A(具体而言，3-脱-O-酰化单磷酰基脂质A(3D-MPL))、咪喹莫特(在本领域中也称为IQM，并且以商购获得；美国专利号4,689,338；5,238,944；Zuber,A.K.等人(2004)22(13-14):1791-8)；CCR5抑制剂CMPD167(参见Veazey,R.S.等人(2003)J.Exp.Med.198:1551-1562)；以及鲨烯乳剂，即MF59。Suitable adjuvants may include, but are not limited to, inorganic salts (e.g., AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH(SO 4 ) 2 , silica, aluminum, aluminum hydroxide, Ca 3 (PO 4 ) 2 , kaolin or carbon), polynucleotides with or without immune stimulating complexes (ISCOMs) (e.g., CpG oligonucleotides, such as Chuang, TH et al., (2002) J. Leuk. Biol. 71(3):538-44; Ahmad Nejad, P. et al., (2003) J. Leuk. Biol. 71(3):538-44). (2002) Eur. J. Immunol. 32(7):1958-68); polyIC or polyAU acids, polyarginine with or without CpG (also referred to in the art as IC31; see Schlack, C. et al. (2003) Proceedings of the 34th Annual Meeting of the German Society of Immunology; Lingnau, K. et al. (2002) Vaccine 20(29-30):3498-508), JuvaVax™ (U.S. Pat. No. 6,693,086), certain natural substances (e.g., wax D from Mycobacterium tuberculosis, Cornyebacterium parvum, Bordetella pertussis, or members of the genus Brucella), flagellin (a ligand for Toll-like receptor 5; see McSorley, SJ et al. (2002) J. Immunol. 169(7):3914-9), saponins (such as QS21, QS17, and QS7) (U.S. Pat. Nos. 5,057,540; 5,650,398; 6,524,584; 6,645,495), monophosphoryl lipid A (specifically, 3 -de-O-acylated monophosphoryl lipid A (3D-MPL)), imiquimod (also known in the art as IQM and commercially available as; U.S. Pat. Nos. 4,689,338; 5,238,944; Zuber, AK et al. (2004) 22(13-14): 1791-8); the CCR5 inhibitor CMPD167 (see Veazey, RS et al. (2003) J. Exp. Med. 198: 1551-1562); and squalene emulsion, i.e. MF59.
在一些实施方案中，佐剂是另一种RNA。In some embodiments, the adjuvant is another RNA.
过去在人中广泛使用的佐剂是铝。在一些实施方案中，合适的佐剂是磷酸铝。在一些实施方案中，合适的佐剂是氢氧化铝。在一些实施方案中，合适的佐剂是磷酸铝和氢氧化铝的组合。皂苷及其纯化组分Quil A、弗氏完全佐剂和其他佐剂通常被用于研究和兽医应用中；然而，新的化学定义的制备物，诸如胞壁酰二肽、单磷酰基脂质A、磷脂缀合物，诸如Goodman-Snitkoff等人.J.Immunol.147:410-415(1991)描述的那些，该文献以引用的方式并入本文。An adjuvant widely used in humans in the past is aluminum. In some embodiments, a suitable adjuvant is aluminum phosphate. In some embodiments, a suitable adjuvant is aluminum hydroxide. In some embodiments, a suitable adjuvant is a combination of aluminum phosphate and aluminum hydroxide. Saponins and their purified components Quil A, Freund's complete adjuvant, and other adjuvants are commonly used in research and veterinary applications; however, new chemically defined preparations, such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates, such as those described by Goodman-Snitkoff et al. J. Immunol. 147: 410-415 (1991), which is incorporated herein by reference.
在本发明的包含聚合物-脂质的组合物的一些实施方案中，所述组合物还包含转染增效剂，优选所述转染增效剂包含选自以下中的至少一种：肺部表面活性蛋白、细胞穿膜肽、两亲性多肽、黏液溶解酶、1,2-丙二醇、纤维素(如羧甲基纤维素或羟丙基纤维素)、透明质酸盐、海藻酸盐、果胶、聚乙二醇、泊洛沙姆、泊洛沙胺、葡萄糖、果糖、蔗糖、海藻糖、葡聚糖、聚乙烯吡咯烷酮、壳聚糖、聚乙烯醇、聚乙酸乙烯酯、凝集素、聚乳酸、聚羟基丁酸、氨丁三醇、氯化苄烷铵、经修饰精氨酸、氯化鲸蜡基吡啶鎓、L-赖氨酸单水合物，以及聚乳酸-羟基乙酸共聚物或盐溶液。In some embodiments of the composition comprising a polymer-lipid of the present invention, the composition further comprises a transfection enhancer, preferably the transfection enhancer comprises at least one selected from the group consisting of a pulmonary surfactant protein, a cell-penetrating peptide, an amphiphilic polypeptide, a mucolytic enzyme, 1,2-propylene glycol, a cellulose (such as carboxymethyl cellulose or hydroxypropyl cellulose), a hyaluronate, alginate, pectin, polyethylene glycol, a poloxamer, a poloxamine, glucose, fructose, sucrose, trehalose, dextran, polyvinyl pyrrolidone, chitosan, polyvinyl alcohol, polyvinyl acetate, agglutinin, polylactic acid, polyhydroxybutyric acid, tromethamine, benzalkonium chloride, modified arginine, cetylpyridinium chloride, L-lysine monohydrate, and a polylactic-co-glycolic acid copolymer or a salt solution.
在本发明的包含聚合物-脂质的组合物的一些实施方案中，所述组合物为纳米颗粒的形式，所述纳米颗粒具有约1000nm或更小的平均尺寸。在一些 实施方案中，所述纳米颗粒具有约500nm或更小、400nm或更小、300nm或更小、200nm或更小、150nm或更小、125nm或更小、100nm或更小、75nm或更小、或约50nm或更小的平均尺寸。In some embodiments of the polymer-lipid compositions of the invention, the compositions are in the form of nanoparticles having an average size of about 1000 nm or less. In embodiments, the nanoparticles have an average size of about 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 75 nm or less, or about 50 nm or less.
在一些实施方案中，约30％-约100％、约70％-约100％、约90％-约100％、约50％-约90％、约70％-约90％或约80％-约90％的所述纳米颗粒具有包封在其中的活性剂或治疗剂。In some embodiments, about 30% to about 100%, about 70% to about 100%, about 90% to about 100%, about 50% to about 90%, about 70% to about 90%, or about 80% to about 90% of the nanoparticles have the active or therapeutic agent encapsulated therein.
在本发明的包含聚合物-脂质的组合物的一些实施方案中，所述组合物配制为溶液、干粉、雾化或喷雾的形式。在一些实施方案中所述组合物配制为通过气溶胶化、干粉、吸入、雾化或滴注进行所述肺部和/或鼻部施用。In some embodiments of the composition comprising polymer-lipid of the present invention, the composition is formulated as a solution, dry powder, atomization or spray. In some embodiments, the composition is formulated to be administered to the lung and/or nose by aerosolization, dry powder, inhalation, atomization or instillation.
制备方法Preparation
本发明尤其提供了使用本文所述的本发明方法制备的mRNA-聚合物-脂质组合物。在一些实施方案中，本文所述的包封mRNA的方法包括在两亲聚合物(例如poloxamine和/或poloxamer)存在下混合脂质溶液与mRNA溶液的步骤，使得形成包封mRNA的聚合物-脂质纳米颗粒。在一些实施方案中，两亲性聚合物在混合之前存在于mRNA溶液中。在一些实施方案中，两亲性聚合物在混合之前存在于脂质溶液中。在一些实施方案中，在mRNA溶液和脂质溶液的混合期间加入两亲聚合物。在一些实施方案中，在mRNA溶液和脂质溶液的混合后加入两亲聚合物。在一些实施方案中，在两亲聚合物存在下混合脂质溶液与mRNA溶液的步骤，使得形成包封mRNA的聚合物-脂质纳米颗粒，纯化透析后，进一步向聚合物-脂质纳米颗粒的溶液中加入两亲聚合物。The present invention particularly provides mRNA-polymer-lipid compositions prepared using the methods of the present invention as described herein. In some embodiments, the method for encapsulating mRNA as described herein includes a step of mixing a lipid solution with an mRNA solution in the presence of an amphiphilic polymer (e.g., poloxamine and/or poloxamer) to form polymer-lipid nanoparticles that encapsulate mRNA. In some embodiments, the amphiphilic polymer is present in the mRNA solution before mixing. In some embodiments, the amphiphilic polymer is present in the lipid solution before mixing. In some embodiments, an amphiphilic polymer is added during the mixing of the mRNA solution and the lipid solution. In some embodiments, an amphiphilic polymer is added after the mixing of the mRNA solution and the lipid solution. In some embodiments, the step of mixing a lipid solution with an mRNA solution in the presence of an amphiphilic polymer to form polymer-lipid nanoparticles that encapsulate mRNA, after purification and dialysis, an amphiphilic polymer is further added to the solution of the polymer-lipid nanoparticles.
在一些实施方案中，合适的聚合物-脂质中的脂质溶液包含阳离子脂质和非阳离子脂质(也称为辅助脂质)。在一些实施方案中，合适的脂质溶液包含阳离子脂质、非阳离子脂质和PEG修饰的脂质或PEG。在一些实施方案中，合适的脂质溶液包含阳离子脂质、非阳离子脂质、基于胆固醇的脂质和PEG修饰的脂质或PEG。可以将各种脂质以所需的各自的量和/或比例溶解在合适的溶剂中，以制备用于本文所述方法的脂质溶液。可以使用各种方法来制备合适的脂质溶液。示例性方法描述于US 2016/0038432、US 2018/0153822和US 2018/0125989中，其以引用的方式并入本文。 In some embodiments, the lipid solution in a suitable polymer-lipid comprises a cationic lipid and a non-cationic lipid (also referred to as a helper lipid). In some embodiments, a suitable lipid solution comprises a cationic lipid, a non-cationic lipid and a PEG-modified lipid or PEG. In some embodiments, a suitable lipid solution comprises a cationic lipid, a non-cationic lipid, a cholesterol-based lipid and a PEG-modified lipid or PEG. Various lipids can be dissolved in a suitable solvent with the required respective amount and/or ratio to prepare a lipid solution for the methods described herein. A variety of methods can be used to prepare a suitable lipid solution. Exemplary methods are described in US 2016/0038432, US 2018/0153822 and US 2018/0125989, which are incorporated herein by reference.
在一些实施方案中，合适的聚合物-脂质中的脂质溶液包含一种或多种阳离子脂质、非阳离子脂质、胆固醇和/或PEG-修饰的脂质。例如，聚合物-脂质可包含以下阳离子脂质的至少一种：DLin-MC3-DMA、ALC-0315、SM-102、cKK-E12、A2-Iso5-2DC18、BAME-O16B、9A1P9、OF-Deg-Lin、306Oi10、TT3、FTT5、C12-200、DLin-KC2-DMA、DODAP、HGT4003、ICE、GL67、HGT5000或HGT5001等。在一些实施方案中，聚合物-脂质可包含以下非阳离子脂质的至少一种：DSPC(1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱)、DPPC(1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱)、DOPE(1,2-二油基-sn-甘油-3-磷酸乙醇胺)、DPPE(1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺)、DMPE(1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺)、DOPG(2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油))、DPPG(二棕榈酰磷脂酰甘油)、DPSP(二棕榈酰神经鞘磷脂)、POPE(1-棕榈酰基-2-油酰基磷脂酰乙醇)、DOPC(1,2‐二油基-sn-甘油-3-磷脂酰胆碱)、DSPG(二硬脂酰磷脂酰甘油)、DSPE(1,2-二硬脂酰基-sn-丙三基-3-磷脂酰乙醇胺)、DSPA(二硬脂酰磷脂酸)、POPC(棕榈酰油酰磷脂酰胆碱)等。在一些实施方案中，聚合物-脂质可包含胆固醇和/或β-谷甾醇等。在一些实施方案中，聚合物-脂质中包含PEG-修饰/改性的脂质，PEG改性脂质可包括(但非排他地)共价连接至具有C6-C20长度的烷基链的脂质、长度高达5kDa的PEG链。在一些实施方案中，聚合物-脂质中包含DMG-PEG2K、DMPE-PEG2K、DSPE-PEG2K、DSPE-PEG2K-Mannose、DMG-PEG5K、DMPE-PEG5K、DSPE-PEG5K、DSPE-PEG5K-Mannose等。在某些实施方案中，聚合物-脂质中包含以下脂质制剂之一：DLin-MC3-DMA、DSPC、胆固醇、DMG-PEG2K；DLin-MC3-DMA、DPPC、胆固醇、DMG-PEG2K；DLin-MC3-DMA、DOPE、胆固醇、DMG-PEG2K；DLin-MC3-DMA、DSPC、胆固醇、DMG-PEG5K；DLin-MC3-DMA、DPPC、胆固醇、DMG-PEG5K；DLin-MC3-DMA、DOPE、胆固醇、DMG-PEG5K；ALC-0315、DSPC、胆固醇、DMG-PEG2K；ALC-0315、DPPC、胆固醇、DMG-PEG2K；ALC-0315、DOPE、胆固醇、DMG-PEG2K；ALC-0315、DSPC、胆固醇、DMG-PEG5K；ALC-0315、DPPC、胆固醇、DMG-PEG5K；ALC-0315、DOPE、胆固醇、DMG-PEG5K；SM-102、DSPC、胆固醇、DMG-PEG2K；SM-102、DPPC、胆固醇、DMG-PEG2K；SM-102、DOPE、胆固醇、DMG-PEG2K；SM-102、DSPC、胆固醇、DMG-PEG5K；SM-102、DPPC、胆固醇、DMG-PEG5K；SM-102、DOPE、胆固醇、DMG-PEG5K；C12-200、DOPE、胆固醇、 DMG-PEG2K；DODAP、DOPE、胆固醇、DMG-PEG2K；HGT5000、DOPE、胆固醇、DMG-PEG2K；HGT5001、DOPE、胆固醇、DMG-PEG2K；GL67、DPPC、DMG-PEG2K；cKK-E12、DOTAP、胆固醇、DMG-PEG2K；SM-102、DSPC、胆固醇；SM-102、DOPS、胆固醇；SM-102、DOPE、胆固醇；SM-102、胆固醇、DMG-PEG2K；SM-102、DSPC、DMG-PEG2K。In some embodiments, the lipid solution in a suitable polymer-lipid comprises one or more cationic lipids, non-cationic lipids, cholesterol and/or PEG-modified lipids. For example, the polymer-lipid can comprise at least one of the following cationic lipids: DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, A2-Iso5-2DC18, BAME-016B, 9A1P9, OF-Deg-Lin, 306Oi10, TT3, FTT5, C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, GL67, HGT5000 or HGT5001, etc. In some embodiments, the polymer-lipid may include at least one of the following non-cationic lipids: DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (2-dioleoyl-sn-glycero-3-phosphoethanolamine). In some embodiments, the polymer-lipid may include cholesterol and/or β-sitosterol, etc. In some embodiments, the polymer-lipid includes PEG-modified/modified lipids, and the PEG-modified lipids may include (but not exclusively) lipids covalently linked to alkyl chains having a length of C 6 -C 20 , and PEG chains up to 5 kDa. In some embodiments, the polymer-lipid comprises DMG-PEG2K, DMPE-PEG2K, DSPE-PEG2K, DSPE-PEG2K-Mannose, DMG-PEG5K, DMPE-PEG5K, DSPE-PEG5K, DSPE-PEG5K-Mannose, etc. In certain embodiments, the polymer-lipid comprises one of the following lipid formulations: DLin-MC3-DMA, DSPC, cholesterol, DMG-PEG2K; DLin-MC3-DMA, DPPC, cholesterol, DMG-PEG2K; DLin-MC3-DMA, DOPE, cholesterol, DMG-PEG2K; DLin-MC3-DMA, DSPC, cholesterol, DMG-PEG5K; DLin-MC3-DMA, DPPC, cholesterol, DMG-PEG5K; DLin-MC3-DMA, DOPE, cholesterol, DMG-PEG5K; ALC-0315, DSPC, cholesterol, DMG-PEG2K; ALC-0315, DPPC, cholesterol, DMG-PEG2K; ALC-0 315, DOPE, cholesterol, DMG-PEG2K; ALC-0315, DSPC, cholesterol, DMG-PEG5K; ALC-0315, DPPC, cholesterol, DMG-PEG5K; ALC-0315, DOPE, cholesterol, DMG-PEG5K; SM-102, DSPC, cholesterol, DMG-PEG2K; SM-102, DPPC, cholesterol, DMG-PEG2K; SM-102, DOPE, cholesterol, DMG-PEG2K; SM-102, DSPC, cholesterol, DMG-PEG5K; SM-102, DPPC, cholesterol, DMG-PEG5K; SM-102, DOPE, cholesterol, DMG-PEG5K; C12-200, DOPE, cholesterol, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, cholesterol, DMG-PEG2K; HGT5001, DOPE, cholesterol, DMG-PEG2K; GL67, DPPC, DMG-PEG2K; cKK-E12, DOTAP, cholesterol, DMG-PEG2K; SM-102, DSPC, cholesterol; SM-102, DOPS, cholesterol; SM-102, DOPE, cholesterol; SM-102, cholesterol, DMG-PEG2K; SM-102, DSPC, DMG-PEG2K.
在一些实施方案中，所述方法包括：In some embodiments, the method comprises:
(A)在两亲性嵌段共聚物的存在下将包含所述活性剂或治疗剂的溶液和包含所述脂质的溶液混合以形成所述组合物；或(A) mixing a solution comprising the active agent or therapeutic agent and a solution comprising the lipid in the presence of an amphiphilic block copolymer to form the composition; or
(B)将包含所述活性剂或治疗剂的溶液和包含所述脂质的溶液混合以形成包封所述活性剂或治疗剂的脂质纳米颗粒，随后将所述两亲性嵌段共聚物与所述脂质纳米颗粒溶液混合以形成所述组合物。(B) mixing a solution containing the active agent or therapeutic agent and a solution containing the lipid to form lipid nanoparticles encapsulating the active agent or therapeutic agent, and then mixing the amphiphilic block copolymer with the lipid nanoparticle solution to form the composition.
在一些实施方案中，所述方法包括：In some embodiments, the method comprises:
1)将所述两亲性嵌段共聚物加入到包含所述活性剂或治疗剂的溶液中和/或包含所述脂质的溶液中，和1) adding the amphiphilic block copolymer to a solution comprising the active agent or therapeutic agent and/or a solution comprising the lipid, and
2)将所述包含活性剂或治疗剂的溶液和所述包含脂质的溶液混合，2) mixing the solution containing the active agent or therapeutic agent and the solution containing the lipid,
从而形成所述组合物。Thereby forming the composition.
在一些实施方案中，所述方法包括：In some embodiments, the method comprises:
1)在包含所述脂质的溶液中，使得所述脂质预形成无活性剂或治疗剂的脂质纳米颗粒；和1) in a solution comprising the lipid, allowing the lipid to pre-form into lipid nanoparticles without an active agent or therapeutic agent; and
2)将包含所述活性剂或治疗剂和两亲性嵌段共聚物的溶液与所述脂质纳米颗粒溶液混合，2) mixing a solution comprising the active agent or therapeutic agent and the amphiphilic block copolymer with the lipid nanoparticle solution,
从而形成所述组合物。Thereby forming the composition.
在另一些实施方案中，所述方法包括：In other embodiments, the method comprises:
1)将所述脂质和两亲性嵌段共聚物预形成无活性剂或治疗剂的聚合物-脂质纳米颗粒；和1) preforming the lipid and the amphiphilic block copolymer into polymer-lipid nanoparticles without active agent or therapeutic agent; and
2)将包含所述活性剂或治疗剂的溶液与所述聚合物-脂质纳米颗粒溶液混合，2) mixing a solution containing the active agent or therapeutic agent with the polymer-lipid nanoparticle solution,
从而形成所述组合物。Thereby forming the composition.
在一些实施方案中，所述方法进一步包括去除游离的脂质组分和/或两亲性嵌段共聚物的步骤，优选通过透析和/或切向流过滤去除所述游离的脂质组分和/或两亲性嵌段共聚物。 In some embodiments, the method further comprises the step of removing free lipid components and/or amphiphilic block copolymers, preferably by dialysis and/or tangential flow filtration.
在一些实施方案中，所述方法进一步包括在去除游离的脂质组分和/或两亲性嵌段共聚物之后，再次加入两亲性嵌段共聚物的步骤。In some embodiments, the method further comprises the step of adding the amphiphilic block copolymer again after removing the free lipid component and/or the amphiphilic block copolymer.
在一些实施方案中，可以在混合之前将mRNA溶液或脂质溶液、或两者加热至高于环境温度的预定温度。在一些实施方案中，在混合之前将mRNA溶液和脂质溶液分别加热至预定温度。在一些实施方案中，将mRNA溶液和脂质溶液在环境温度下混合，而然后在混合之后加热至预定温度。在一些实施方案中，将脂质溶液加热至预定温度，并在环境温度下与mRNA溶液混合。在一些实施方案中，将mRNA溶液加热至预定温度，并在环境温度下与脂质溶液混合。在一些实施方案中，与其它没有加热步骤的相同方法相比，在所述方法过程中(形成之前、过程中或之后)包括加热步骤可提供更高的mRNA的包封率。在一些实施方案中，可以通过将包含mRNA-聚合物-脂质以及未包封在聚合物-脂质形成溶液中的一些游离mRNA的制剂溶液加热至如本文所述的预定温度来进一步增强聚合物-脂质中mRNA的包封。In some embodiments, mRNA solution or lipid solution, or both can be heated to a predetermined temperature higher than ambient temperature before mixing. In some embodiments, mRNA solution and lipid solution are heated to a predetermined temperature respectively before mixing. In some embodiments, mRNA solution and lipid solution are mixed at ambient temperature, and then heated to a predetermined temperature after mixing. In some embodiments, lipid solution is heated to a predetermined temperature and mixed with mRNA solution at ambient temperature. In some embodiments, mRNA solution is heated to a predetermined temperature and mixed with lipid solution at ambient temperature. In some embodiments, compared with other identical methods without heating steps, a heating step is included in the method process (before, during or after formation) to provide a higher encapsulation rate of mRNA. In some embodiments, the encapsulation of mRNA in polymer-lipid can be further enhanced by heating a formulation solution comprising mRNA-polymer-lipid and some free mRNA not encapsulated in polymer-lipid forming solution to a predetermined temperature as described herein.
在一些实施方案中，通过将在环境温度下的mRNA储备溶液添加至加热的缓冲溶液中以达到所需的预定温度来将mRNA溶液加热至预定温度。In some embodiments, the mRNA solution is heated to a predetermined temperature by adding an mRNA stock solution at ambient temperature to a buffer solution that is heated to reach the desired predetermined temperature.
如本文所用，术语“环境温度”是指房间中的温度，或围绕所关注的对象而不加热或冷却的温度。在一些实施方案中，一种或多种溶液所维持的环境温度为或小于约35℃、30℃、25℃、20℃或16℃。在一些实施方案中，一种或多种溶液所述维持的环境温度在约15‐35℃、约15‐30℃、约15‐25℃、约15‐20℃、约20‐35℃、约25‐35℃、约30‐35℃、约20‐30℃、约25‐30℃或约20‐25℃的范围内。在一些实施方案中，一种或多种溶液所维持的环境温度为20‐25℃。As used herein, the term "ambient temperature" refers to the temperature in a room, or the temperature surrounding an object of interest without heating or cooling. In some embodiments, the ambient temperature maintained by one or more solutions is or is less than about 35°C, 30°C, 25°C, 20°C, or 16°C. In some embodiments, the ambient temperature maintained by one or more solutions is in the range of about 15-35°C, about 15-30°C, about 15-25°C, about 15-20°C, about 20-35°C, about 25-35°C, about 30-35°C, about 20-30°C, about 25-30°C, or about 20-25°C. In some embodiments, the ambient temperature maintained by one or more solutions is 20-25°C.
因此，高于环境温度的预定温度通常大于约25℃。在一些实施方案中，适合于本发明的预定温度为或大于约30℃、37℃、40℃、45℃、50℃、55℃、60℃、65℃、或70℃。在一些实施方案中，适合于本发明的预定温度在约25‐70℃、约30‐70℃、约35‐70℃、约40‐70℃、约45‐70℃、约50‐70℃、或约60‐70℃的范围内。在特定实施方案中，适合于本发明的预定温度为约65℃。Thus, the predetermined temperature above ambient temperature is typically greater than about 25°C. In some embodiments, the predetermined temperature suitable for the present invention is or is greater than about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, the predetermined temperature suitable for the present invention is in the range of about 25-70°C, about 30-70°C, about 35-70°C, about 40-70°C, about 45-70°C, about 50-70°C, or about 60-70°C. In a specific embodiment, the predetermined temperature suitable for the present invention is about 65°C.
可用于制备本发明所述的聚合物-脂质组合物的方法记述在，例如，美国专利号8,058,069；5,753,613；5,785,992；5,705,385；5,976,567；5,981,501；6,110,745；和6,320,017；和PCT公开号WO 2022/032154和WO 96/40964中，通过引用将其公开内容整体分别引入本文中用于全部目的。 Methods that can be used to prepare the polymer-lipid compositions described herein are described, for example, in U.S. Pat. Nos. 8,058,069; 5,753,613; 5,785,992; 5,705,385; 5,976,567; 5,981,501; 6,110,745; and 6,320,017; and PCT Publication Nos. WO 2022/032154 and WO 96/40964, the disclosures of which are each incorporated herein by reference in their entirety for all purposes.
在一些实施方案中，使用泵将mRNA溶液和脂质溶液混合。由于具有此类混合的包封程序可以在各种规模上进行，因此可以使用不同类型的泵来适应所需的规模。然而，通常期望使用无脉冲流量泵。如本文所用，无脉冲流量泵是指能够以稳定的流速建立连续流量的任何泵。合适的泵的类型可以包括但不限于齿轮泵和离心泵。In some embodiments, a pump is used to mix the mRNA solution and the lipid solution. Since the encapsulation procedure with such mixing can be carried out on various scales, different types of pumps can be used to adapt to the required scale. However, it is generally desirable to use a pulseless flow pump. As used herein, a pulseless flow pump refers to any pump that can establish a continuous flow rate at a stable flow rate. The type of suitable pump may include, but is not limited to, a gear pump and a centrifugal pump.
可以各种流速混合mRNA溶液和脂质溶液。通常，可以以比脂质溶液的流速更大的流速混合mRNA溶液。例如，可以以比脂质溶液的流速大至少1倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、15倍或20倍的流速混合mRNA储液。The mRNA solution and lipid solution can be mixed at various flow rates. Generally, the mRNA solution can be mixed at a flow rate greater than the flow rate of the lipid solution. For example, the mRNA storage solution can be mixed at a flow rate greater than at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 times greater than the flow rate of the lipid solution.
可以基于规模确定用于混合的合适的流速。在一些实施方案中，以在约1‐4ml/分钟、2‐6ml/分钟、3‐8ml/分钟、5‐10ml/分钟、10‐20ml/分钟、15‐30ml/分钟、25‐75ml/分钟、20‐50ml/分钟、25‐75ml/分钟、30‐90ml/分钟、40‐100ml/分钟、50‐110ml/分钟、75‐200ml/分钟、200‐350ml/分钟、350‐500ml/分钟、500‐650ml/分钟、650‐850ml/分钟、或850‐1000ml/分钟范围内的流速混合脂质溶液。在一些实施方案中，以在约1000‐2000ml/分钟、2000‐3000ml/分钟、3000‐4000ml/分钟、或4000‐5000ml/分钟范围内的流速混合mRNA溶液。The suitable flow rate for mixing can be determined based on the scale. In some embodiments, the lipid solution is mixed at a flow rate within the range of about 1-4 ml/min, 2-6 ml/min, 3-8 ml/min, 5-10 ml/min, 10-20 ml/min, 15-30 ml/min, 25-75 ml/min, 20-50 ml/min, 25-75 ml/min, 30-90 ml/min, 40-100 ml/min, 50-110 ml/min, 75-200 ml/min, 200-350 ml/min, 350-500 ml/min, 500-650 ml/min, 650-850 ml/min, or 850-1000 ml/min. In some embodiments, the mRNA solution is mixed at a flow rate in the range of about 1000-2000 ml/min, 2000-3000 ml/min, 3000-4000 ml/min, or 4000-5000 ml/min.
在一些实施方案中，本明聚合物-脂质组合物可使用纳米沉淀法制备，纳米沉淀法可通过如下单元操作，其中聚合物-脂质组合物通过动力学混合，随后进行成熟及连续稀释来自其个别脂质组分自组装。该单元操作通常包括三个液体流的连续线上组合以及一个线上成熟步骤:将水性缓冲液与脂质储备溶液混合，经由受控滞留时间进行成熟，及稀释纳米颗粒。纳米沉淀本身发生在适合规模的混合器中，该混合器经设计以允许水溶液与溶解在乙醇中的脂质储备溶液的连续、高能量组合。在整个此操作中，水溶液及脂质储备溶液均同时连续流入混合器中。保持脂质溶解之乙醇含量突然减少，且脂质均彼此沉淀。由此纳米颗粒在混合室中自组装。In some embodiments, the present polymer-lipid composition can be prepared using nanoprecipitation, which can be performed by the following unit operation, wherein the polymer-lipid composition is mixed by kinetics, and then matured and serially diluted from its individual lipid component self-assembly. The unit operation generally includes a continuous online combination of three liquid streams and an online maturation step: an aqueous buffer is mixed with a lipid stock solution, matured via a controlled residence time, and the nanoparticles are diluted. The nanoprecipitation itself occurs in a mixer suitable for scale, which is designed to allow the continuous, high-energy combination of an aqueous solution and a lipid stock solution dissolved in ethanol. Throughout this operation, the aqueous solution and the lipid stock solution are all continuously flowed into the mixer at the same time. The ethanol content of the lipid dissolution is kept to decrease suddenly, and the lipids are all precipitated from each other. Thus the nanoparticles are self-assembled in the mixing chamber.
在某些实施方案中，本发明提供通过连续混合法，例如，包括以下步骤的过程产生的聚合物-脂质组合物：在第一储槽中提供包含核酸诸如mRNA的水溶液(可包含两亲性嵌段聚合物)，在第二储槽中提供有机脂质溶液(可包含两亲性嵌段聚合物)，并混合所述水溶液和有机脂质溶液以使得有机脂质溶液与水溶液混合从而基本立即产生包封该核酸(例如，mRNA)的聚合物- 脂质组合物。该过程和用于执行该过程的装置详细记述在美国专利公开号20040142025中，通过引用将其公开内容整体引入本文中用于全部目的。In certain embodiments, the present invention provides a polymer-lipid composition produced by a continuous mixing method, for example, a process comprising the steps of providing an aqueous solution (which may include an amphiphilic block polymer) containing a nucleic acid such as mRNA in a first reservoir, providing an organic lipid solution (which may include an amphiphilic block polymer) in a second reservoir, and mixing the aqueous solution and the organic lipid solution so that the organic lipid solution mixes with the aqueous solution to substantially immediately produce a polymer-lipid composition encapsulating the nucleic acid (e.g., mRNA). Lipid composition. The process and apparatus for performing the process are described in detail in US Patent Publication No. 20040142025, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
在另一个实施方案中，本发明提供通过直接稀释法产生的聚合物-脂质组合物，所述直接稀释法包括形成脂质溶液和立即并直接将脂质溶液引入至容纳受控量的稀释缓冲液的收集容器中。在优选的方面，收集容器包括一种或多种配置为搅拌收集容器内容物以促进稀释的部件。在一方面，收集容器中存在的稀释缓冲液的量基本等于向其中引入的脂质溶液的体积。作为非限制性实例，处于45％乙醇中的脂质体溶液在引入至含有等体积稀释缓冲液的收集容器中时,将有利地产生较小的颗粒。In another embodiment, the present invention provides polymer-lipid compositions produced by direct dilution method, and the direct dilution method comprises forming lipid solution and immediately and directly introducing lipid solution into the collection container containing controlled amount of dilution buffer. In preferred aspects, the collection container comprises one or more components configured to stir the collection container contents to promote dilution. On the one hand, the amount of dilution buffer present in the collection container is substantially equal to the volume of lipid solution introduced therein. As a non-limiting example, the liposome solution in 45% ethanol will advantageously produce smaller particles when introduced into the collection container containing equal volume of dilution buffer.
在另一个实施方案中，本发明提供通过直接稀释法产生的聚合物-脂质组合物，其中容纳稀释缓冲液的第三储槽与第二混合区流体相连。这些方法和执行这些直接稀释方法的装置详细记述在美国专利公开号20070042031中，通过引用将其公开内容整体引入本文中用于全部目的。In another embodiment, the present invention provides a polymer-lipid composition produced by a direct dilution method, wherein a third reservoir containing a dilution buffer is fluidically connected to the second mixing zone. These methods and devices for performing these direct dilution methods are described in detail in U.S. Patent Publication No. 20070042031, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
与其他制备技术(例如薄膜再水合/挤压)形成对比，乙醇滴加沉淀法(ethanol-drop precipitation)已成为用于制备核酸脂质纳米颗粒的工业标准。沉淀反应因其连续性、可放大性及易用性而受到青睐。这类方法通常使用高能混合器(例如T型接头、受限冲击射流、微流控混合器、涡旋混合器)以可控制方式将脂质(在乙醇中)引入合适反相溶剂(亦即，水)中，从而驱动液体过饱和及自发沉淀成脂质粒子。在一些实施方案中，所用涡旋混合器描述于美国专利申请62/799,636及62/886,592中，该美国专利申请以全文引用的方式并入本文中。在一些实施方案中，所用微流控混合器可采用PCT专利WO/2014/172045中所描述的混合器，该专利以全文引用之方式并入本文中。在一些实施方案中，混合步骤采用T形接头、受限冲击射流、微流控混合器或涡旋混合器进行。在一些实施方案中，装载步骤采用T形接头、受限冲击射流、微流控混合器或涡旋混合器进行。In contrast to other preparation techniques (e.g., film rehydration/extrusion), ethanol-drop precipitation has become the industry standard for preparing nucleic acid lipid nanoparticles. Precipitation reactions are favored for their continuity, scalability, and ease of use. Such methods typically use high-energy mixers (e.g., T-junctions, confined impinging jets, microfluidic mixers, vortex mixers) to introduce lipids (in ethanol) into a suitable reverse phase solvent (i.e., water) in a controlled manner, thereby driving liquid supersaturation and spontaneous precipitation into lipid particles. In some embodiments, the vortex mixer used is described in U.S. patent applications 62/799,636 and 62/886,592, which are incorporated herein by reference in their entirety. In some embodiments, the microfluidic mixer used may employ the mixer described in PCT patent WO/2014/172045, which is incorporated herein by reference in its entirety. In some embodiments, the mixing step is performed using a T-junction, a confined impinging jet, a microfluidic mixer, or a vortex mixer. In some embodiments, the loading step is performed using a T-junction, a confined impinging jet, a microfluidic mixer, or a vortex mixer.
在另一个实施方案中，本发明提供通过“标准”技术和“后插入”技术制备的PoLixNano-CPLs(含阳离子-聚合物-脂质缀合物CPL的聚合物-脂质组合物)和/或PoLixNano-固醇胺(例如，含固醇胺GL67的聚合物-脂质组合物)，其中所述“后插入”技术，即将CPL或GL67插入至，例如，预先形成的聚合物-脂质组合物(PoLixNano)中，和“标准”技术，其中CPL或GL67在例如，PoLixNano形成步骤过程中包括在脂质混合物中。制备PoLixNano-CPL的方 法，在例如美国专利号5,705,385；6,586,410；5,981,501；6,534,484；和6,852,334；美国专利公开号20020072121；和PCT公开号WO 00/62813中阐述，通过引用将其公开内容整体引入本文中用于全部目的。制备PoLixNano-GL67的方法，在PCT公开号WO 2022/032154中阐述，通过引用将其公开内容整体引入本文中用于全部目的。In another embodiment, the present invention provides PoLixNano-CPLs (polymer-lipid compositions containing cation-polymer-lipid conjugates CPL) and/or PoLixNano-sterolamines (e.g., polymer-lipid compositions containing sterolamine GL67) prepared by "standard" techniques and "post-insertion" techniques, wherein the "post-insertion" techniques, i.e., CPL or GL67 is inserted into, e.g., a preformed polymer-lipid composition (PoLixNano), and the "standard" techniques, wherein CPL or GL67 is included in the lipid mixture during, e.g., the PoLixNano formation step. Methods for preparing PoLixNano-CPLs Methods for preparing PoLixNano-GL67 are described in, for example, U.S. Patent Nos. 5,705,385; 6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent Publication No. 20020072121; and PCT Publication No. WO 00/62813, the disclosures of which are incorporated herein by reference in their entirety for all purposes. Methods for preparing PoLixNano-GL67 are described in PCT Publication No. WO 2022/032154, the disclosures of which are incorporated herein by reference in their entirety for all purposes.
如果需要，本发明的聚合物-脂质组合物可以通过任何可用于调节脂质体尺寸的方法来调节尺寸。可以进行尺寸调节，以获得所需的尺寸范围和相对窄的粒度分布。If desired, the polymer-lipid compositions of the present invention can be sized by any method useful for adjusting the size of liposomes. Size adjustment can be performed to obtain a desired size range and a relatively narrow particle size distribution.
存在若干用于将颗粒调节至所需尺寸的技术，例如超声浴槽/探头超声波降解法、均质化法、挤出法等。一种尺寸调节方法，其用于脂质体并同样适用于本发明所述的聚合物-脂质组合物，记述在美国专利号4,737,323中，通过引用将其公开内容整体引入本文中用于全部目的。There are several techniques for adjusting particles to a desired size, such as ultrasonic bath/probe sonication, homogenization, extrusion, etc. One size adjustment method, which is used for liposomes and is also applicable to the polymer-lipid compositions described herein, is described in U.S. Pat. No. 4,737,323, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
在一些实施方案中，预先浓缩聚合物-脂质组合物中的核酸，例如，美国专利申请号09/744,103中所述，通过引用将其公开内容整体引入本文中用于全部目的。In some embodiments, nucleic acids in a polymer-lipid composition are pre-concentrated, for example, as described in U.S. Patent Application No. 09/744,103, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
在其他实施方案中，本发明提供的方法还包括加入转染增效剂，用于实现增强的核酸转染效果。合适的转染增效剂的实例包括，肺部表面活性蛋白、细胞穿膜肽、两亲性多肽、黏液溶解酶、1,2-丙二醇、纤维素(如羧甲基纤维素或羟丙基纤维素)、透明质酸盐、海藻酸盐、果胶、聚乙二醇、泊洛沙姆、泊洛沙胺、葡萄糖、果糖、蔗糖、海藻糖、葡聚糖、蔗糖、海藻糖、甘露糖、聚乙烯吡咯烷酮、壳聚糖、聚乙烯醇、聚乙酸乙烯酯、凝集素、聚乳酸、聚羟基丁酸、氨丁三醇、氯化苄烷铵、经修饰精氨酸、氯化鲸蜡基吡啶鎓、L-赖氨酸单水合物，以及聚乳酸-羟基乙酸共聚物或盐溶液。这些转染增效剂的添加优选在颗粒已经形成后。In other embodiments, the method provided by the present invention also includes adding a transfection enhancer to achieve enhanced nucleic acid transfection effect. Examples of suitable transfection enhancers include pulmonary surfactant proteins, cell-penetrating peptides, amphipathic polypeptides, mucolytic enzymes, 1,2-propylene glycol, cellulose (such as carboxymethyl cellulose or hydroxypropyl cellulose), hyaluronate, alginate, pectin, polyethylene glycol, poloxamer, poloxamine, glucose, fructose, sucrose, trehalose, dextran, sucrose, trehalose, mannose, polyvinyl pyrrolidone, chitosan, polyvinyl alcohol, polyvinyl acetate, lectin, polylactic acid, polyhydroxybutyric acid, tromethamine, benzalkonium chloride, modified arginine, cetyl pyridinium chloride, L-lysine monohydrate, and polylactic acid-glycolic acid copolymer or saline solution. The addition of these transfection enhancers is preferably after the particles have been formed.
在其他实施方案中，该方法应该还包括加入非脂质聚阳离子，其利用本发明的组合物用于实现细胞的脂质转染。合适的非脂质聚阳离子的实例包括，海美溴铵(hexadimethrine bromide)(商品名来自Sigma-Aldrich Chemical Co.)或其他己二甲鞍盐。其他合适的聚阳离子包括但不限于，例如，聚-L-鸟氨酸，聚-L-精氨酸，聚-D-精氨酸，聚-L-赖氨酸，聚-D-赖氨酸，聚烯丙胺，壳聚糖，和聚乙烯亚胺及其药学上可接受的盐。这些聚阳离子的添加优选在颗粒已经形成后。 In other embodiments, the method may further include adding a non-lipid polycation, which is used to achieve lipid transfection of cells using the composition of the present invention. Examples of suitable non-lipid polycations include hexadimethrine bromide (trade name from Sigma-Aldrich Chemical Co.) or other hexadimethrine salts. Other suitable polycations include, but are not limited to, for example, poly-L-ornithine, poly-L-arginine, poly-D-arginine, poly-L-lysine, poly-D-lysine, polyallylamine, chitosan, and polyethyleneimine and pharmaceutically acceptable salts thereof. These polycations are preferably added after the particles have been formed.
通常，本文所述的本发明方法包括去除未形成颗粒的脂质和/或两亲聚合物(例如poloxamine和/或poloxamer)的步骤。在一些实施方案中，可通过缓冲液交换技术如透析去除游离脂质和/或两亲性聚合物。在一些实施方案中，将聚合物-脂质形成溶液交换至构成产品制剂溶液的溶液中。例如，可以在一种或多种制剂溶液中透析含有形成的聚合物-脂质纳米粒的混合物，以去除在聚合物-脂质纳米粒形成期间存在的游离脂质和/或两亲聚合物。Typically, the inventive method as herein described comprises the step of removing lipid and/or amphipathic polymer (for example poloxamine and/or poloxamer) that do not form particles. In some embodiments, free lipid and/or amphipathic polymer can be removed by buffer exchange techniques such as dialysis. In some embodiments, polymer-lipid formation solution is exchanged in the solution constituting the product formulation solution. For example, the mixture of the polymer-lipid nanoparticle formed can be dialyzed in one or more formulation solutions to remove free lipid and/or amphipathic polymer present during the formation of the polymer-lipid nanoparticle.
包含聚合物-脂质纳米粒的溶液从聚合物-脂质形成溶液到制剂溶液的更换可通过本领域已知的多种缓冲液更换技术中的任一种来实现。在一些实施方案中，将聚合物-脂质形成溶液更换成制剂溶液的步骤伴随聚合物-脂质的纯化和/或浓缩。各种方法可用于实现溶液的更换以及聚合物-脂质的纯化或溶液中聚合物-脂质的浓缩。例如，在一些实施方案中，这种溶液更换是通过渗滤实现的。在一些实施方案中，更换溶液，并且使用切向流过滤(TFF，也被称为交叉流过滤)纯化聚合物-脂质。在TFF中，非所需渗透物穿过过滤器，而所需渗余物(聚合物-脂质纳米粒和游离mRNA)沿着过滤器传递且在下游收集。各种TFF技术是已知的，并且可以用于实施本发明。示例性TFF纯化方法描述于US2016/0040154和US2015/0376220中，其以引用的方式并入本文。The solution comprising polymer-lipid nanoparticles can be replaced from polymer-lipid forming solution to formulation solution by any of a variety of buffer exchange techniques known in the art. In some embodiments, the step of replacing the polymer-lipid forming solution into formulation solution is accompanied by the purification and/or concentration of polymer-lipid. Various methods can be used to realize the replacement of solution and the purification of polymer-lipid or the concentration of polymer-lipid in solution. For example, in some embodiments, this solution replacement is realized by diafiltration. In some embodiments, the solution is replaced, and tangential flow filtration (TFF, also referred to as cross-flow filtration) is used to purify polymer-lipid. In TFF, non-desired permeate passes through the filter, and the desired retentate (polymer-lipid nanoparticles and free mRNA) is passed along the filter and collected downstream. Various TFF technologies are known and can be used to implement the present invention. Exemplary TFF purification methods are described in US2016/0040154 and US2015/0376220, which are incorporated herein by reference.
在一些实施方案中，溶液中游离存在的两亲性聚合物(例如poloxamine和/或poloxamer)的原始量的小于约0.1％、0.09％、0.08％、0.07％、0.06％、0.05％、0.04％、0.03％、0.02％或0.01％在去除后保留。在一些实施方案中，两亲性聚合物的残余量在去除后保留在制剂中。如本文所用，残余量意指在组合物中基本上所有物质(本文所述的两亲性聚合物诸如poloxamine和/或poloxamer)被去除之后的剩余量。残余量可使用已知技术定性或定量地检测。使用已知技术可能无法检测到残余量。In some embodiments, less than about 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the original amount of the amphiphilic polymer (e.g., poloxamine and/or poloxamer) free in the solution remains after removal. In some embodiments, the residual amount of the amphiphilic polymer remains in the formulation after removal. As used herein, residual amount means the remaining amount after substantially all substances (amphiphilic polymers such as poloxamine and/or poloxamer described herein) are removed in the composition. The residual amount can be detected qualitatively or quantitatively using known techniques. The residual amount may not be detected using known techniques.
在一些实施方案中，过量的mRNA与mRNA-聚合物-脂质形成期间存在的两亲性聚合物(例如poloxamine和/或poloxamer)一起也被去除。In some embodiments, excess mRNA is also removed along with the amphiphilic polymer (eg, poloxamine and/or poloxamer) present during mRNA-polymer-lipid formation.
在一些实施方案中，药物产品制剂溶液中不存在非水性溶剂诸如乙醇和柠檬酸盐中的一者或两者。在一些实施方案中，合适的制剂溶液仅包含残留的柠檬酸盐。在一些实施方案中，合适的制剂溶液仅包含残留的非水性溶剂，诸如乙醇。在一些实施方案中，合适的制剂溶液含有小于约10mM的柠檬酸盐。在一些实施方案中，合适的制剂溶液含有小于约25％的非水性溶剂，诸如乙醇。在一些实施方案中，合适的制剂溶液在冻干之前不需要任何进一步 的下游处理(例如，缓冲液更换和/或进一步的纯化步骤和/或另外的赋形剂)。在一些实施方案中，合适的制剂溶液在施用至小瓶、注射器或其他容器中的无菌填充物之前不需要任何进一步的下游处理。在一些实施方案中，合适的制剂溶液在向受试者施用之前不需要任何进一步的下游处理。In some embodiments, one or both of non-aqueous solvents such as ethanol and citrate are absent from the drug product formulation solution. In some embodiments, a suitable formulation solution comprises only residual citrate. In some embodiments, a suitable formulation solution comprises only residual non-aqueous solvents such as ethanol. In some embodiments, a suitable formulation solution contains less than about 10 mM citrate. In some embodiments, a suitable formulation solution contains less than about 25% non-aqueous solvents such as ethanol. In some embodiments, a suitable formulation solution does not require any further lyophilization prior to lyophilization. In some embodiments, the formulation solution does not require any further downstream processing (e.g., buffer exchange and/or further purification steps and/or additional excipients) prior to administration to a sterile fill in a vial, syringe, or other container. In some embodiments, the formulation solution does not require any further downstream processing prior to administration to a subject.
在一些实施方案中，在冷冻解冻之后，合适的制剂溶液能够改善或增强加热后聚合物-脂质组合物的mRNA包封量。在一些实施方案中，合适的制剂溶液为10％蔗糖并且可以稳定冷冻。In some embodiments, after freeze-thawing, a suitable formulation solution can improve or enhance the mRNA encapsulation capacity of the polymer-lipid composition after heating. In some embodiments, a suitable formulation solution is 10% sucrose and can be freeze-stable.
在一些实施方案中，加热后的合适的制剂溶液中的聚合物-脂质组合物可以在约1％、约3％、约5％、约7％、约9％、约10％、约13％、约15％、约20％、约25％、约30％、约35％、约40％、约45％或约50％的蔗糖溶液中稳定冷冻(例如，保留增强的包封)。在一些实施方案中，合适的制剂溶液可以不需要任何下游纯化或加工并且可以以冷冻形式稳定地储存。In some embodiments, the polymer-lipid composition in the suitable formulation solution after heating can be stably frozen (e.g., retaining enhanced encapsulation) in about 1%, about 3%, about 5%, about 7%, about 9%, about 10%, about 13%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% sucrose solution. In some embodiments, the suitable formulation solution may not require any downstream purification or processing and can be stably stored in frozen form.
制剂preparation
各种制剂可与本发明结合使用。A variety of formulations may be used in conjunction with the present invention.
在一些实施方案中，合适的制剂溶液可以包括缓冲剂或盐。示例性的缓冲剂可以包括4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)、氯化钙、硫酸铵、硫酸镁、碳酸氢钠、柠檬酸钠、乙酸钠、磷酸钾和磷酸钠。示例性的盐可以包括氯化钠、氯化镁、乙酸锂、氯化锂、甲酸锂、硝酸锂、硫酸锂、丙二酸钠、硝酸钠、硫酸钠和氯化钾。在一些实施方案中，合适的制剂溶液可以包括上述缓冲盐所含阳离子以及阴离子的组合。In some embodiments, suitable formulation solutions may include buffers or salts. Exemplary buffers may include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), calcium chloride, ammonium sulfate, magnesium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. Exemplary salts may include sodium chloride, magnesium chloride, lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, sodium malonate, sodium nitrate, sodium sulfate, and potassium chloride. In some embodiments, suitable formulation solutions may include a combination of cations and anions contained in the above-mentioned buffer salts.
在一些实施方案中，合适的制剂溶液包含一种或多种保护剂，且一或多种保护剂中的每一者独立地为多元醇(例如二醇或三醇，诸如丙二醇(亦即，1,2-丙二醇))、1,3-丙二醇、甘油、(+/-)-2-甲基-2,4-戊二醇、1,6-已二醇、1,2-丁二醇、2,3-丁二醇、乙二醇或二乙二醇)、非去污剂磺基甜菜碱(例如NDSB-201(3-(1-吡啶基)-1-丙烷磺酸盐)、渗透剂(例如，L-脯氨酸或三甲基胺N-氧化物二水合物)、聚合物(例如，聚乙二醇200(PEG 200)、PEG 400、PEG 600、PEG 1000、PEG 2000、PEG 3350、PEG 4000、PEG 8000、PEG10000、PEG 20000、聚乙二醇单甲醚550(mPEG 550)、mPEG 600、mPEG2000、mPEG 3350、mPEG 4000、mPEG 5000、聚乙烯吡咯啶酮(例如，聚乙烯吡咯啶酮K15)、新戊四醇丙氧基化物或聚丙二醇P400)、有机溶剂(例如二甲基亚砜 (DMSO)或乙醇)、糖(如海藻糖、蔗糖、甘露糖、乳糖或山梨糖醇)或其任意组合。In some embodiments, a suitable formulation solution comprises one or more protective agents, and each of the one or more protective agents is independently a polyol (e.g., a diol or triol, such as propylene glycol (i.e., 1,2-propylene glycol)), 1,3-propylene glycol, glycerol, (+/-)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a non-detergent sulfobetaine (e.g., NDSB-201 (3-(1-pyridyl)-1-propanesulfonate), an osmotic agent (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 2000, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinyl pyrrolidone (e.g., polyvinyl pyrrolidone K15), pentaerythritol propoxylate or polypropylene glycol P400), organic solvents (e.g., dimethyl sulfoxide (DMSO) or ethanol), sugars (such as trehalose, sucrose, mannose, lactose or sorbitol), or any combination thereof.
在一些实施方案中，合适的制剂溶液是包含药学上可接受的赋形剂(包括但不限于冷冻保护剂)的水溶液。在一些实施方案中，合适的制剂溶液是包含药学上可接受的赋形剂的水溶液，所述赋形剂包括但不限于糖，诸如海藻糖、蔗糖、甘露糖、乳糖和甘露醇中的一种或多种。在一些实施方案中，合适的制剂溶液包含海藻糖。在一些实施方案中，合适的制剂溶液包含蔗糖。在一些实施方案中，合适的制剂溶液包含甘露糖。在一些实施方案中，合适的制剂溶液包含乳糖。在一些实施方案中，合适的制剂溶液包含甘露醇。In some embodiments, suitable formulation solutions are aqueous solutions comprising pharmaceutically acceptable excipients (including but not limited to cryoprotectants). In some embodiments, suitable formulation solutions are aqueous solutions comprising pharmaceutically acceptable excipients, and the excipients include but are not limited to sugars, such as one or more of trehalose, sucrose, mannose, lactose and mannitol. In some embodiments, suitable formulation solutions include trehalose. In some embodiments, suitable formulation solutions include sucrose. In some embodiments, suitable formulation solutions include mannose. In some embodiments, suitable formulation solutions include lactose. In some embodiments, suitable formulation solutions include mannitol.
在一些实施方案中，合适的制剂溶液是包含1％至20％重量/体积的糖，诸如海藻糖、蔗糖、甘露糖、乳糖和甘露醇的水溶液。在一些实施方案中，合适的制剂溶液是包含1％至20％重量/体积的海藻糖的水溶液。在一些实施方案中，合适的制剂溶液是包含1％至20％重量/体积的蔗糖的水溶液。在一些实施方案中，合适的制剂溶液是包含1％至20％重量/体积的甘露糖的水溶液。在一些实施方案中，合适的制剂溶液是包含1％至20％重量/体积的乳糖的水溶液。在一些实施方案中，合适的制剂溶液是包含1％至20％重量/体积的甘露醇的水溶液。在一些实施方案中，合适的制剂溶液是包含约10％重量/体积的糖，诸如海藻糖、蔗糖、甘露糖、乳糖和甘露醇的水溶液。在一些实施方案中，合适的制剂溶液是包含约10％重量/体积的海藻糖的水溶液。在一些实施方案中，合适的制剂溶液是包含约10％重量/体积的蔗糖的水溶液。在一些实施方案中，合适的制剂溶液是包含约10％重量/体积的甘露糖的水溶液。在一些实施方案中，合适的制剂溶液是包含约10％重量/体积的乳糖的水溶液。在一些实施方案中，合适的制剂溶液是包含约10％重量/体积的甘露醇的水溶液。In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume sugars, such as trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume trehalose. In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume sucrose. In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume mannose. In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume lactose. In some embodiments, a suitable formulation solution is an aqueous solution containing 1% to 20% weight/volume mannitol. In some embodiments, a suitable formulation solution is an aqueous solution containing about 10% weight/volume sugars, such as trehalose, sucrose, mannose, lactose, and mannitol. In some embodiments, a suitable formulation solution is an aqueous solution containing about 10% weight/volume trehalose. In some embodiments, a suitable formulation solution is an aqueous solution containing about 10% weight/volume sucrose. In some embodiments, a suitable formulation solution is an aqueous solution comprising about 10% weight/volume mannose. In some embodiments, a suitable formulation solution is an aqueous solution comprising about 10% weight/volume lactose. In some embodiments, a suitable formulation solution is an aqueous solution comprising about 10% weight/volume mannitol.
在一些实施方案中，合适的制剂溶液具有在pH 4.5与pH 7.5之间的pH。在一些实施方案中，合适的制剂溶液具有在pH 5.0与pH 7.0之间的pH。在一些实施方案中，合适的制剂溶液具有在pH 5.5与pH 7.0之间的pH。在一些实施方案中，合适的制剂溶液具有高于pH 4.5的pH。在一些实施方案中，合适的制剂溶液具有高于pH 5.0的pH。在一些实施方案中，合适的制剂溶液具有高于pH 5.5的pH。在一些实施方案中，合适的制剂溶液具有高于pH 6.0的pH。在一些实施方案中，合适的制剂溶液具有高于pH 6.5的pH。 In some embodiments, suitable formulation solutions have a pH between pH 4.5 and pH 7.5. In some embodiments, suitable formulation solutions have a pH between pH 5.0 and pH 7.0. In some embodiments, suitable formulation solutions have a pH between pH 5.5 and pH 7.0. In some embodiments, suitable formulation solutions have a pH higher than pH 4.5. In some embodiments, suitable formulation solutions have a pH higher than pH 5.0. In some embodiments, suitable formulation solutions have a pH higher than pH 5.5. In some embodiments, suitable formulation solutions have a pH higher than pH 6.0. In some embodiments, suitable formulation solutions have a pH higher than pH 6.5.
合适的制剂溶液的细节描述于，诸如PCT专利WO2022/032154以及WO2020/160397中，上述专利的公开内容通过引用整体引入本文中用于全部目的。Details of suitable formulation solutions are described in, for example, PCT Patents WO2022/032154 and WO2020/160397, the disclosures of which are incorporated herein by reference in their entirety for all purposes.
试剂盒Reagent test kit
本发明还提供采用试剂盒形式的所述聚合物-脂质组合物。所述试剂盒可以包括划分为容纳聚合物-脂质组合物的各种成分(例如，活性剂或治疗剂诸如核酸和颗粒的各种脂质、聚合物成分)的容器。在一些实施方案中，所述试剂盒还包括内体膜去稳定剂(例如，钙离子)。所述试剂盒典型地包含优选采用脱水形式的本发明的聚合物-脂质组合物和它们再水合和施用的说明书。The present invention also provides the polymer-lipid composition in the form of a kit. The kit may include containers divided into various components (e.g., various lipids, polymer components of active agents or therapeutic agents such as nucleic acids and particles) for accommodating the polymer-lipid composition. In some embodiments, the kit also includes an endosomal membrane destabilizer (e.g., calcium ions). The kit typically includes the polymer-lipid composition of the present invention preferably in a dehydrated form and instructions for their rehydration and administration.
如本文中所述，本发明的聚合物-脂质组合物可以定制为优先地靶向特殊的目标组织、器官、或肿瘤。在某些实例中，聚合物-脂质组合物的优先靶向可以通过控制颗粒组分本身来进行。例如，如实施例11以及实施例16中所述，已经发现(PoLixNano+T904)物理混合制剂可以用于优先地靶向肝和脾，而聚合物-脂质组合物制剂可以用于优先地靶向肺。As described herein, polymer-lipid compositions of the present invention can be customized to preferentially target special target tissues, organs or tumors. In some instances, the preferential targeting of polymer-lipid compositions can be carried out by controlling the particle components themselves. For example, as described in Example 11 and Example 16, it has been found that (PoLixNano+T904) physical mixed formulations can be used for preferentially targeting liver and spleen, and polymer-lipid composition formulations can be used for preferentially targeting lungs.
在某些其他实例中，可能需要使靶向结构部分与聚合物-脂质组合物表面连接，从而进一步增强颗粒的靶向性。使靶向结构部分(例如，糖基、多肽、抗体、蛋白等)与脂质或两亲性聚合物(诸如本发明颗粒中使用的那些)相连接的方法是本领域中技术人员已知的。In certain other examples, it may be desirable to attach a targeting moiety to the surface of the polymer-lipid composition to further enhance the targeting of the particle. Methods for attaching a targeting moiety (e.g., a glycosyl, polypeptide, antibody, protein, etc.) to a lipid or amphiphilic polymer (such as those used in the particles of the present invention) are known to those skilled in the art.
聚合物-脂质组合物的施用Administration of polymer-lipid compositions
一旦形成，本发明所述的聚合物-脂质组合物有效用于将活性剂或治疗剂(例如，mRNA)引入至细胞。因此，本发明还提供用于将活性剂或治疗剂诸如核酸(例如，mRNA)引入至细胞中的方法。该方法通过以下步骤体外或体内进行：首先形成如上所述的颗粒，然后使该颗粒与细胞相接触以足以发生活性剂或治疗剂引入至细胞的一段时间。Once formed, the polymer-lipid composition of the present invention is effective for introducing an active agent or therapeutic agent (e.g., mRNA) into a cell. Therefore, the present invention also provides a method for introducing an active agent or therapeutic agent such as a nucleic acid (e.g., mRNA) into a cell. The method is performed in vitro or in vivo by the following steps: first forming a particle as described above, then contacting the particle with a cell to be sufficient to cause an active agent or therapeutic agent to be introduced into a cell for a period of time.
本发明所述的聚合物-脂质组合物可以吸附于几乎任何与其相混合或相接触的细胞类型。一旦吸附，该颗粒可以被细胞的一部分内吞，与细胞膜交换脂质，或与细胞融合。颗粒的活性剂或治疗剂(例如，核酸)部分的转移或结合可以通过这些途径的任意一种发生。具体地，当融合发生时，颗粒膜整合到细胞膜中并且颗粒的内容物与胞内流体结合。 Polymer-lipid compositions of the present invention can be adsorbed on almost any cell type mixed or contacted therewith. Once adsorbed, the particle can be endocytosed by a part of the cell, exchange lipids with the cell membrane, or merge with the cell. Transfer or combination of the active agent or therapeutic agent (e.g., nucleic acid) part of the particle can occur by any of these approaches. Specifically, when fusion occurs, the particle membrane is integrated into the cell membrane and the content of the particle is combined with the intracellular fluid.
本发明所述的聚合物-脂质组合物可以单独地或以与药用载体(例如生理盐水saline或磷酸盐缓冲液PBS)的混合物施用，所述药用载体根据给药途径和标准药物实践来选择。通常，标准缓冲盐水(例如，135-150mM NaCl)应该作为药用载体来使用。其他合适的载体包括，例如，水(无核酶水)、缓冲水、0.4％盐水、0.3％甘氨酸等，包括用于增加稳定性的糖蛋白，诸如白蛋白，脂蛋白，球蛋白等。另外的合适载体记述在，例如，REMINGTON'S PHARMACEUTICAL SCIENCES(雷明顿制药科学)，Mack Publishing Company(马克出版公司)，费城，PA，第17版(1985)中。用于本文中时，“载体”包括任何和全部溶剂、分散介质、媒介物、包衣、稀释剂、抗细菌剂和抗真菌剂、等渗和吸收延迟剂、缓冲剂、载体溶液、混悬液、胶体等。短语“药用的”指在施用于人时不产生变态反应或类似不利反应的分子实体和组合物。The polymer-lipid composition of the present invention can be administered alone or in a mixture with a pharmaceutical carrier (e.g., saline or phosphate buffered saline PBS), which is selected according to the route of administration and standard pharmaceutical practice. Typically, standard buffered saline (e.g., 135-150mM NaCl) should be used as a pharmaceutical carrier. Other suitable carriers include, for example, water (nuclease-free water), buffered water, 0.4% saline, 0.3% glycine, etc., including glycoproteins such as albumin, lipoproteins, globulins, etc. for increasing stability. Other suitable carriers are described in, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th edition (1985). When used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
药用载体一般在颗粒形成后加入。因此，在颗粒形成后，可以将颗粒稀释至药用载体诸如标准缓冲盐水中。The pharmaceutically acceptable carrier is generally added after the particles are formed. Thus, after the particles are formed, the particles can be diluted into a pharmaceutically acceptable carrier such as standard buffered saline.
药物制剂中颗粒的浓度可以广泛变化，通常处于或至少约0.1-30重量％，到至多约10-90重量％，并且主要根据所选择的具体给药模式通过流体体积、粘度等来选择。例如，可以增加浓度以降低与治疗相关的流体负荷。这在具有与动脉粥样硬化相关的充血性心力衰竭或严重高血压的患者中可能特别理想。备选地，可以将由刺激性脂质制成的颗粒稀释至低浓度，从而减轻给药部位处的炎症。The concentration of particles in the pharmaceutical preparation can vary widely, usually at or at least about 0.1-30% by weight, to about 10-90% by weight at most, and is selected mainly by fluid volume, viscosity, etc. according to the specific mode of administration selected. For example, the concentration can be increased to reduce the fluid load associated with the treatment. This may be particularly desirable in patients with congestive heart failure or severe hypertension associated with atherosclerosis. Alternatively, the particles made of irritating lipids can be diluted to low concentrations, thereby alleviating inflammation at the administration site.
本发明的药物组合物可以通过常规、公知灭菌技术来灭菌。水溶液可以包装备用或在无菌条件下过滤并冻干，冻干的制剂在施用前与无菌水溶液结合。所述组合物可以根据需要包含药用助剂物质，以接近生理条件，诸如pH调节和缓冲剂，张力调节剂等。另外，颗粒混悬液可以包括脂质-保护剂，其在存储中保护脂质免受自由基和脂质-过氧化破坏。亲脂性自由基猝灭剂，诸如α生育酚和水溶性离子特异性螯合剂，诸如铁草胺(ferrioxamine)是适合的。The pharmaceutical composition of the present invention can be sterilized by conventional, well-known sterilization techniques. The aqueous solution can be packaged for standby use or filtered and lyophilized under aseptic conditions, and the lyophilized preparation is combined with a sterile aqueous solution before administration. The composition can contain pharmaceutical auxiliary substances as needed to approach physiological conditions, such as pH adjustment and buffers, tension regulators, etc. In addition, the particle suspension can include a lipid-protectant that protects lipids from free radicals and lipid-peroxidation damage during storage. Lipophilic free radical quenchers, such as alpha tocopherol and water-soluble ion-specific chelators, such as ferrioxamine are suitable.
体外施药In vitro administration
为了体外应用，治疗剂诸如核酸(例如，mRNA)可以递送至培养基中生长的任何细胞，无论植物或动物来源，脊椎动物或无脊椎动物，和任何组织或类型。在优选的实施方案中，所述细胞是动物细胞，更优选哺乳动物细胞，且最优选人细胞。 For in vitro applications, therapeutic agents such as nucleic acids (e.g., mRNA) can be delivered to any cell grown in culture, regardless of plant or animal origin, vertebrate or invertebrate, and any tissue or type. In a preferred embodiment, the cell is an animal cell, more preferably a mammalian cell, and most preferably a human cell.
细胞和本发明所述的聚合物-脂质组合物之间的接触，在体外进行时，在生物相容培养基中发生。颗粒浓度广泛地变化，取决于具体应用，但是通常在约1μmol-约10mmol。用聚合物-脂质组合物治疗细胞通常以生理学温度(约37℃)进行约1-48小时，优选约2-6小时的时期。The contact between cells and the polymer-lipid composition of the present invention occurs in a biocompatible culture medium when performed in vitro. The particle concentration varies widely, depending on the specific application, but is generally between about 1 μmol and about 10 mmol. Treatment of cells with the polymer-lipid composition is generally performed at physiological temperature (about 37° C.) for about 1-48 hours, preferably for a period of about 2-6 hours.
在一组优选的实施方案中，将聚合物-脂质组合物混悬液加入至细胞密度为约103-约106细胞/ml，更优选约1x105细胞/ml的60-80％融合率的接种细胞。加入至细胞的混悬液的浓度是优选约0.01-0.4μg/ml，更优选约0.2μg/ml。In a preferred embodiment, the polymer-lipid composition suspension is added to seeded cells at a cell density of about 10 3 to about 10 6 cells/ml, more preferably about 1x10 5 cells/ml, at a confluency of 60-80%. The concentration of the suspension added to the cells is preferably about 0.01-0.4 μg/ml, more preferably about 0.2 μg/ml.
利用内体释放参数(ERP)测定，可以优化本发明所述的聚合物-脂质组合物的递送效率。ERP测定记述在美国专利公开号20030077829中，通过引用将其公开内容整体引入本文中用于全部目的。The delivery efficiency of the polymer-lipid compositions of the present invention can be optimized using the endosomal release parameter (ERP) assay. The ERP assay is described in U.S. Patent Publication No. 20030077829, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
体内施用In vivo administration
为了体内施用，给药可以采用本领域中已知的任何方式，例如，通过注射、滴鼻、吸入(例如雾化或干粉制剂，经鼻内或气管内吸入)、口服给药、经皮施用、生殖道给药、或直肠给药。给药可以通过单次或分次剂量来实现。所述药物组合物可以经肠胃外，即关节内、静脉内、腹膜内、皮下、或肌内施用。在一些实施方案中，所述药物组合物通过推注注射静脉内或腹膜内施用(参见，例如，美国专利号5,286,634)。胞内核酸递送也已经在Straubringer等，Methods Enzymol.(酶学方法)，101：512(1983)；Mannino等，Biotechniques(生物技术)，6：682(1988)；Nicolau等，Crit.Rev.Ther.Drug Carrier Syst.(治疗药物载体系统评述)，6：239(1989)；和Behr,Acc.Chem.Res.(化学研究报告)，26:274(1993)中讨论。施用基于脂质的治疗剂的其他方法记述在，例如，美国专利号3,993,754；4,145,410；4,235,871；4,224,179；4,522,803；和4,588,578中。本发明所述的聚合物-脂质组合物可以通过在疾病部位处直接注射或通过在远离疾病部位的部位处注射来施用(参见，例如，Culver,HUMAN GENE THERAPY(人类基因治疗)，MaryAnn Liebert,Inc.,Publishers,纽约.第70-71页(1994))。上述参考文献的公开内容通过引用整体引入本文中用于全部目的。For in vivo administration, administration can be by any means known in the art, for example, by injection, nasal drops, inhalation (e.g., atomization or dry powder formulations, inhaled intranasally or intratracheally), oral administration, transdermal administration, genital tract administration, or rectal administration. Administration can be achieved by single or divided doses. The pharmaceutical composition can be administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the pharmaceutical composition is administered intravenously or intraperitoneally by push injection (see, e.g., U.S. Patent No. 5,286,634). Intracellular nucleic acid delivery has also been discussed in Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino et al., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. Drug Carrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993). Other methods of administering lipid-based therapeutics are described, for example, in U.S. Pat. Nos. 3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578. The polymer-lipid compositions of the present invention can be administered by direct injection at the disease site or by injection at a site remote from the disease site (see, e.g., Culver, HUMAN GENE THERAPY, Mary Ann Liebert, Inc., Publishers, New York. Pages 70-71 (1994)). The disclosures of the above references are incorporated herein by reference in their entirety for all purposes.
本发明的组合物可为液体形式或固体形式。在一些实施方案中，组合物为液体形式，可经雾化途径递送至肺，通常使用各种市售雾化装置。在一些实施方案中，组合物为固体形式，适用于吸入向呼吸道给予组合物。 The compositions of the present invention may be in liquid form or solid form. In some embodiments, the compositions are in liquid form and can be delivered to the lungs via aerosolization, typically using various commercially available aerosol devices. In some embodiments, the compositions are in solid form and are suitable for inhalation to administer the compositions to the respiratory tract.
可采用合适方法诸如鼻内滴注、气管内滴注及气管内注射向呼吸道递送组成物。在一些实施方案中，通过鼻内、支气管内或肺递送组合物或纳米颗粒。举例而言，采用雾化器或吸入器递送组合物或纳米颗粒。The composition can be delivered to the respiratory tract by suitable methods such as intranasal instillation, intratracheal instillation, and intratracheal injection. In some embodiments, the composition or nanoparticles are delivered intranasally, intrabronchially, or pulmonary. For example, a nebulizer or inhaler is used to deliver the composition or nanoparticles.
在一些实施方案中，采用雾化吸入途径将组合物递送至肺中。吸入可经由个体的鼻及/或口进行。给药可通过主动吸入组合物或凭借呼吸器向个体给予组合物来进行。用于将组合物递送至呼吸道部位的示例性装置包括但不限于干粉吸入器(DPI)、加压计量吸入器(pMDI)、雾化器(nebulizer)及电动气雾剂装置。用于将组合物通过上述装置递送至呼吸道的方法已经记述在PCT专利WO2022/032154，上述专利的公开内容通过引用整体引入本文中用于全部目的。In some embodiments, the composition is delivered to the lungs by a nebulized inhalation route. Inhalation can be performed through the nose and/or mouth of an individual. Administration can be performed by actively inhaling the composition or by administering the composition to an individual via a respirator. Exemplary devices for delivering the composition to the respiratory tract include, but are not limited to, dry powder inhalers (DPI), pressurized metered dose inhalers (pMDI), nebulizers, and electric aerosol devices. The method for delivering the composition to the respiratory tract via the above-mentioned device has been described in PCT Patent WO2022/032154, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
本发明的组合物，可以单独地或与其他合适成分组合地制成气雾制剂(即，它们可以被“雾化”)，从而通过吸入(例如，鼻内或气管内)施用(参见，Brigham等，Am.J.Sci.(美国科学杂志)，298：278(1989)；以及Patel等，Advanced Materials(先进材料杂志)，31(8):e1805116(2019))。气雾制剂可以置于容许加压的推进剂，诸如二氯二氟甲烷，丙烷，氮等中，也可以通过雾化装置(nebulizer)生成。The compositions of the present invention can be made into aerosol preparations (i.e., they can be "nebulized") alone or in combination with other suitable ingredients for administration by inhalation (e.g., intranasal or intratracheal) (see, Brigham et al., Am. J. Sci., 298: 278 (1989); and Patel et al., Advanced Materials, 31(8): e1805116 (2019)). Aerosol preparations can be placed in a propellant that allows pressurization, such as dichlorodifluoromethane, propane, nitrogen, etc., or can be generated by a nebulizer.
本发明的组合物，可以单独地或与其他合适成分组合地制成喷雾剂，从而通过鼻腔吸入使用。在某些实施方案中，所述药物组合物可以通过鼻内喷雾、吸入、和/或其他气雾剂递送媒介物来递送。用于将核酸组合物通过鼻腔气雾剂喷雾直接递送至肺的方法已经记述在，例如，美国专利号5,756,353和5,804,212中。同样地，利用鼻内微粒树脂和溶血磷脂酰-甘油化合物(美国专利5,725,871)的药物递送也是制药领域中公知的。类似地，采用聚四氟乙烯载体基质的形式透黏膜药物递送记述在美国专利号5,780,045中。上述专利的公开内容通过引用整体引入本文中用于全部目的。The composition of the present invention can be made into sprays individually or in combination with other suitable ingredients, so as to be used by nasal inhalation. In certain embodiments, the pharmaceutical composition can be delivered by intranasal spray, inhalation, and/or other aerosol delivery vehicles. The method for directly delivering nucleic acid compositions to the lungs by nasal aerosol spray has been described in, for example, U.S. Patent Nos. 5,756,353 and 5,804,212. Similarly, drug delivery using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (U.S. Patent No. 5,725,871) is also well known in the pharmaceutical field. Similarly, transmucosal drug delivery using a polytetrafluoroethylene carrier matrix is described in U.S. Patent No. 5,780,045. The disclosure of the above patent is incorporated herein by reference as a whole for all purposes.
适合于肠胃外给药，诸如，例如，通过关节内(关节中)、静脉内、肌内、皮内、腹膜内、和皮下途径的制剂包括水性和非水性、等渗无菌注射溶液，其可以包含抗氧化剂、缓冲剂、抑菌剂、和使该制剂与目的接受者的血液等渗的溶质，和可以包括悬浮剂、增溶剂、增稠剂、稳定剂、和防腐剂的水性和非水性无菌混悬液。在本发明的实践中，组合物优选，例如通过静脉内输注、口服、局部、腹膜内、膀胱内、或鞘内施用。Formulations suitable for parenteral administration, such as, for example, by intraarticular (in a joint), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes include aqueous and nonaqueous, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In the practice of the present invention, the composition is preferably administered, for example, by intravenous infusion, oral, topical, intraperitoneal, intravesical, or intrathecal administration.
用于体内治疗的全身递送，例如，通过机体循环系统将治疗性核酸递送 至远隔靶细胞，诸如PCT公开号WO 05/007196，WO 05/121348,WO 05/120152,和WO 04/002453中记述利用核酸-脂质颗粒已经实现了这类目的，通过引用将其公开内容整体引入本文中用于全部目的。本发明还提供完全包封的聚合物-脂质组合物，其保护核酸免于在血清中被核酸酶降解，是非免疫原性的，尺寸很小，并且适合于反复给药。Systemic delivery for in vivo therapy, e.g., delivery of therapeutic nucleic acids through the body's circulatory system To distant target cells, such as PCT Publication Nos. WO 05/007196, WO 05/121348, WO 05/120152, and WO 04/002453 describe the use of nucleic acid-lipid particles to achieve such purposes, and the disclosure thereof is incorporated herein by reference for all purposes. The present invention also provides a fully encapsulated polymer-lipid composition that protects nucleic acids from being degraded by nucleases in serum, is non-immunogenic, is very small in size, and is suitable for repeated administration.
一般地，当静脉内施用时，聚合物-脂质组合物使用合适的药物载体配制。如前所述，许多药用载体可以用于本发明的组合物和方法中，可以使用多种水性载体，例如，水、缓冲水、生理盐水、0.3％甘氨酸等，还可使用用于增强稳定性的糖蛋白，诸如白蛋白，脂蛋白，球蛋白等。这些组合物可以通过常规脂质体灭菌技术诸如过滤来灭菌。所述组合物可以如所需地包含药用助剂物质，以接近生理条件，诸如pH调节和缓冲剂，张力调节剂，润湿剂等，例如乙酸钠、乳酸钠、氯化钠、氯化钾、氯化钙、脱水山梨醇单月桂酸酯、三乙醇胺油酸酯等。这些组合物可以利用上面提到的技术灭菌或，备选地，它们可以在无菌条件下产生。Generally, when intravenously administered, polymer-lipid compositions are formulated using suitable pharmaceutical carriers. As previously mentioned, many pharmaceutical carriers can be used in the compositions and methods of the present invention, and a variety of aqueous carriers can be used, for example, water, buffered water, physiological saline, 0.3% glycine, etc., and glycoproteins for enhancing stability, such as albumin, lipoprotein, globulin, etc., can also be used. These compositions can be sterilized by conventional liposome sterilization techniques such as filtration. The compositions can contain pharmaceutical auxiliary substances as required to approach physiological conditions, such as pH adjustment and buffers, tension regulators, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. These compositions can be sterilized using the above-mentioned techniques or, alternatively, they can be produced under aseptic conditions.
在某些应用中，本文中公开的聚合物-脂质组合物可以经由口服给药递送至个体。颗粒可以与赋形剂结合并以可摄取的片剂、口含片剂、含片(troche)、胶囊剂、丸剂、锭剂(lozenge)、酏剂、漱口药、混悬液、口腔喷雾剂、糖浆剂、糯米纸囊剂等(参见，例如，美国专利号5,641,515，5,580,579，和5,792,451，通过引用将其公开内容整体引入本文中用于全部目的)。这些口服剂型还可以包含以下：粘合剂，明胶；赋形剂，润滑剂，和/或调味剂。当单位剂型是胶囊剂时，其除上述物质外，可以含有脂质载体。多种其他物质可以作为包衣存在或另外对剂量单位的外形进行改性。当然，制备任何单位剂量中使用的任何物质应该是药物纯的且在使用量中是基本无毒的。In some applications, the polymer-lipid composition disclosed herein can be delivered to an individual via oral administration. The particles can be combined with excipients and in the form of ingestible tablets, buccal tablets, troche, capsules, pills, lozenges, elixirs, mouthwashes, suspensions, oral sprays, syrups, wafers, etc. (see, e.g., U.S. Patent Nos. 5,641,515, 5,580,579, and 5,792,451, the disclosure of which is incorporated herein by reference for all purposes). These oral dosage forms may also include the following: adhesives, gelatin; excipients, lubricants, and/or flavorings. When the unit dosage form is a capsule, it may contain a lipid carrier in addition to the above-mentioned substances. A variety of other substances may exist as a coating or additionally modify the appearance of the dosage unit. Of course, any substance used in the preparation of any unit dose should be pharmaceutically pure and substantially nontoxic in the amount used.
典型地，这些口服制剂可以含有至少约0.1％聚合物-脂质组合物或更多，尽管聚合物-脂质组合物的百分比当然可以不同，并可以方便地为总制剂重量或体积约1％或2％-约60％或70％或更多之间。自然地，在可以制备的每种治疗有效的组合物中颗粒的量是这样的方式，即应该在任意给定单位剂量的化合物中获得合适剂量。制备所述药物制剂的领域中的技术人员应该考虑一些因素诸如溶解性、生物利用度、生物半衰期、给药途径、产品保存期、以及其他药理学考虑因素，并且同样地，许多剂量和治疗方案可以是合乎需要的。Typically, these oral formulations can contain at least about 0.1% polymer-lipid composition or more, although the percentage of polymer-lipid composition can certainly be different, and can be conveniently between about 1% or 2% to about 60% or 70% or more of the total formulation weight or volume. Naturally, the amount of particles in each therapeutically effective composition that can be prepared is such that a suitable dose should be obtained in any given unit dose of the compound. The technician in the field of preparing the pharmaceutical preparation should consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and as such, many dosages and treatment regimens may be desirable.
适合于口服给药的制剂可以由以下组成：(a)脂质溶液，诸如悬浮在稀 释剂诸如水、生理盐水、或PEG400中的有效量的包装的治疗剂诸如核酸(例如，mRNA)；(b)胶囊剂，囊剂，或片剂，各自包含预定量的治疗剂诸如核酸(例如，mRNA)，如液体、固体、颗粒、或明胶；(c)处于适当液体中的混悬液；和(d)合适的乳剂。片剂形式可以包括乳糖、蔗糖、甘露糖、山梨糖醇、磷酸钙、玉米淀粉、马铃薯淀粉、微晶纤维素、明胶、胶体二氧化硅、滑石、硬脂酸镁、硬脂酸、和其他赋形剂、着色剂、填充剂、粘合剂、稀释剂、缓冲剂、润湿剂、防腐剂、调味剂、染料、崩解剂、和药物相容载体中的一种或多种。锭剂形式可以包括在调味剂，例如蔗糖中的治疗剂诸如核酸(例如，mRNA)，以及包含处于惰性基质中的治疗剂的软锭剂(pastille)，所述惰性基质诸如明胶和甘油或蔗糖和阿拉伯树胶乳剂，凝胶，以及除了治疗剂以外含有本领域中已知的载体的类似锭剂。Formulations suitable for oral administration may consist of: (a) a lipid solution, such as a suspension in dilute (a) a therapeutic agent such as a nucleic acid (e.g., mRNA) in an effective amount of a packaged therapeutic agent such as a nucleic acid (e.g., mRNA) in a dispensing agent such as water, saline, or PEG400; (b) a capsule, sachet, or tablet, each containing a predetermined amount of a therapeutic agent such as a nucleic acid (e.g., mRNA), such as a liquid, solid, granular, or gelatin; (c) a suspension in an appropriate liquid; and (d) a suitable emulsion. Tablet forms may include lactose, sucrose, mannose, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and one or more of pharmaceutically compatible carriers. Pastille forms can include a therapeutic agent such as a nucleic acid (e.g., mRNA) in a flavoring agent, such as sucrose, as well as pastilles containing the therapeutic agent in an inert matrix such as gelatin and glycerin or sucrose and acacia emulsions, gelatin, and similar pastilles containing in addition to the therapeutic agent carriers known in the art.
在其应用的另一个实例中，聚合物-脂质组合物可以结合到广泛范围的局部剂型中。例如，可以配制含有核酸的聚合物-脂质组合物的混悬液并作为凝胶、油、乳剂、局部霜剂、栓剂、糊剂、膏剂、洗剂、泡沫剂、摩丝剂等施用。In another example of its use, the polymer-lipid composition can be incorporated into a wide range of topical dosage forms. For example, a suspension of the polymer-lipid composition containing nucleic acids can be formulated and applied as a gel, oil, emulsion, topical cream, suppository, paste, ointment, lotion, foam, mousse, etc.
当制备本发明所述聚合物-脂质组合物的药物制剂时，优选使用大量已经纯化以减少或消除空颗粒或具有与外表面缔合的治疗剂诸如核酸的颗粒。When preparing pharmaceutical formulations of the polymer-lipid compositions of the invention, it is preferred to use a plurality of particles that have been purified to reduce or eliminate empty particles or particles having therapeutic agents, such as nucleic acids, associated with the outer surface.
本发明的方法可以在多种宿主中实施。优选的宿主包括哺乳动物物种，诸如灵长类动物(例如，人和黑猩猩以及其他非人灵长类动物)、犬科动物、猫科动物、马科动物、牛科动物、绵羊、山羊、啮齿动物(例如，大鼠和小鼠)、兔形目动物、和猪。The method of the present invention can be implemented in a variety of hosts. Preferred hosts include mammalian species, such as primates (e.g., humans and chimpanzees and other non-human primates), canines, felines, equines, bovines, sheep, goats, rodents (e.g., rats and mice), lagomorphs, and pigs.
施用的制剂量应该取决于治疗剂(例如，核酸)与聚合物-脂质组分的比，所用的具体治疗剂(例如，核酸)，待治疗的疾病或功能障碍，患者的年龄、体重和状况，和临床医生的判断，但是通常应该在约0.01-约50mg/千克体重，优选约0.1-约5mg/千克体重，或约108-1010个颗粒/施用(例如，雾化吸入)。The amount of formulation administered will depend on the ratio of therapeutic agent (e.g., nucleic acid) to polymer-lipid component, the specific therapeutic agent (e.g., nucleic acid) used, the disease or functional disorder to be treated, the age, weight and condition of the patient, and the judgment of the clinician, but will generally be about 0.01 to about 50 mg/kg body weight, preferably about 0.1 to about 5 mg/kg body weight, or about 10 8 -10 10 particles/administration (e.g., nebulized inhalation).
在一些实施方案中，本发明所述药物组合物配制成用于如下施用：吸入的、口服地、脂肪内地、动脉内地、关节内地、颅内地、真皮内地、病灶内地、肌肉内地、鼻内地、眼内地、心包内地、腹膜内地、胸膜内地、前列腺内地、直肠内地、鞘内地、气管内地、肿瘤内地、脐带内地、阴道内地、静脉内地、膀胱内地、玻璃体内地、局部地、黏膜地、胃肠外地、直肠地、结膜下的、皮下地、舌下地、表面局部地(topically)、经颊地、透皮地、阴道地、以乳膏(crèmes)形式、经由导管、经由灌洗、经由连续输注、经由输注、经由吸 入、经由注射、经由局部递送、或经由局部灌注。在一些实施方案中，所述药物组合物配制成用于静脉内或动脉内注射。In some embodiments, the pharmaceutical compositions described herein are formulated for administration by inhalation, orally, intraadipose, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesically, intravitreally, topically, mucosally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, topically, buccally, transdermally, vaginally, in the form of creams, via catheter, via lavage, via continuous infusion, via infusion, via inhalation In some embodiments, the pharmaceutical composition is formulated for intravenous or intra-arterial injection.
用于递送聚合物-脂质组合物的细胞Cells for delivery of polymer-lipid compositions
本发明的组合物和方法用于体内和体外处理广泛多样的细胞类型。合适的细胞包括，例如，肺泡细胞(包括I型肺泡上皮细胞和II型肺泡上皮细胞)、支气管上皮细胞、杯状细胞、纤毛细胞、棒状细胞(club细胞)、未分化的基底细胞、肺部离子细胞(ionocyte)、微褶皱细胞(M细胞)、树突状细胞(DC细胞)、巨噬细胞、T细胞、B细胞、NK细胞、中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、单核细胞、造血前体(干)细胞、成纤维细胞、角化细胞、肝细胞、内皮细胞、骨骼肌和平滑肌细胞、造骨细胞、神经元、静止淋巴细胞、终末分化细胞、缓慢或非循环的原发细胞、实质细胞、淋巴样细胞、上皮细胞、骨细胞等。在优选的实施方案中，活性剂或治疗剂诸如RNA(例如，mRNA)递送至支气管上皮细胞、纤毛细胞、肺部离子细胞(ionocyte)、微褶皱细胞(M细胞)、树突状细胞(DC细胞)、T细胞。在其他优选的实施方案中，活性剂或治疗剂诸如RNA(例如，mRNA)递送至癌症细胞诸如，例如肺癌细胞、结肠癌细胞、直肠癌细胞、肛门癌细胞、胆管癌细胞、小肠癌细胞、胃癌细胞、食道癌细胞、胆囊癌细胞、肝癌细胞、胰腺癌细胞、阑尾癌细胞、乳腺癌细胞、卵巢癌细胞、宫颈癌细胞、前列腺癌细胞、肾癌细胞、中枢神经系统癌细胞、胶质母细胞瘤肿瘤细胞、皮肤癌细胞、淋巴瘤细胞、绒毛膜癌肿瘤细胞、头颈癌细胞、骨源性肉瘤肿瘤细胞、和血癌细胞。The compositions and methods of the present invention are used to treat a wide variety of cell types in vivo and in vitro. Suitable cells include, for example, alveolar cells (including type I alveolar epithelial cells and type II alveolar epithelial cells), bronchial epithelial cells, goblet cells, ciliated cells, club cells, undifferentiated basal cells, pulmonary ionocytes, microfold cells (M cells), dendritic cells (DC cells), macrophages, T cells, B cells, NK cells, neutrophils, eosinophils, basophils, monocytes, hematopoietic precursor (stem) cells, fibroblasts, keratinocytes, hepatocytes, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, resting lymphocytes, terminally differentiated cells, slow or non-circulating primary cells, parenchymal cells, lymphoid cells, epithelial cells, osteocytes, etc. In preferred embodiments, active agents or therapeutic agents such as RNA (e.g., mRNA) are delivered to bronchial epithelial cells, ciliated cells, pulmonary ionocytes, microfold cells (M cells), dendritic cells (DC cells), T cells. In other preferred embodiments, active agents or therapeutic agents such as RNA (e.g., mRNA) are delivered to cancer cells such as, for example, lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, bile duct cancer cells, small intestine cancer cells, gastric cancer cells, esophageal cancer cells, gallbladder cancer cells, liver cancer cells, pancreatic cancer cells, appendix cancer cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, renal cancer cells, central nervous system cancer cells, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteogenic sarcoma tumor cells, and blood cancer cells.
体内递送聚合物-脂质组合物诸如包封RNA(例如，mRNA)的PoLixNano纳米颗粒适合于靶向任何细胞类型的细胞。所述方法和组合物可以与广泛多样的脊椎动物，包括哺乳动物，诸如例如，犬科动物、猫科动物、马科动物、牛科动物、绵羊、山羊、啮齿动物(诸如小鼠、大鼠和豚鼠)、兔形目动物、猪、和灵长类动物(例如，猴、黑猩猩和人)的细胞一起使用。In vivo delivery of polymer-lipid compositions such as PoLixNano nanoparticles encapsulating RNA (e.g., mRNA) is suitable for targeting cells of any cell type. The methods and compositions can be used with a wide variety of vertebrates, including mammals, such as, for example, canines, felines, equines, bovines, sheep, goats, rodents (such as mice, rats and guinea pigs), lagomorphs, pigs, and primates (e.g., monkeys, chimpanzees and humans) cells.
就可能需要细胞的组织培养而言，是本领域中公知的。例如，Freshney,Culture of Animal Cells,a Manual of Basic Technique(动物细胞培养，基础技术手册)，第3版，Wiley-Liss,纽约(1994)，Kuchler等，Biochemical Methods in Cell Culture and Virology(细胞培养和病毒学中的生物化学方法)，Dowden,Hutchinson and Ross,Inc.(1977)，和其中引用的参考文献对细胞培养提供一 般性指导。培养的细胞系统经常采用细胞单层形式，尽管也使用细胞混悬液。To the extent that tissue culture of cells may be required, this is well known in the art. For example, Freshney, Culture of Animal Cells, a Manual of Basic Technique, 3rd ed., Wiley-Liss, New York (1994), Kuchler et al., Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977), and references cited therein provide a general description of cell culture. General Guidance. Cultured cell systems are most often in the form of cell monolayers, although cell suspensions are also used.
检测聚合物-脂质组合物Detection of polymer-lipid compositions
在一些实施方案中，本发明所述的聚合物-脂质组合物可以在约1、2、3、4、5、6、7、8或更多小时在受试者中检测到。在其他实施方案中，本发明所述的聚合物-脂质组合物可以在施用颗粒后约8、12、24、48、60、72或96小时，或约6、8、10、12、14、16、18、19、22、24、25或28天时在受试者中检测到。聚合物-脂质组合物颗粒的存在可以在来自受试者的细胞、组织或其他生物样品中检测到。聚合物-脂质组合物颗粒可以，例如，通过直接检测颗粒、检测治疗性核酸诸如RNA(例如，mRNA)序列，检测其编码的目的蛋白或多肽序列，或其组合来检测。In some embodiments, polymer-lipid composition of the present invention can be detected in a subject at about 1, 2, 3, 4, 5, 6, 7, 8 or more hours. In other embodiments, polymer-lipid composition of the present invention can be detected in a subject at about 8, 12, 24, 48, 60, 72 or 96 hours after applying particles, or about 6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 25 or 28 days. The presence of polymer-lipid composition particles can be detected in cells, tissues or other biological samples from a subject. Polymer-lipid composition particles can, for example, by directly detecting particles, detecting therapeutic nucleic acids such as RNA (e.g., mRNA) sequences, detecting the target protein or polypeptide sequence encoded therein, or a combination thereof.
(1)聚合物-脂质组合物颗粒的检测(1) Detection of polymer-lipid composite particles
本发明的聚合物-脂质组合物颗粒可以利用本领域中任何已知方法来检测。例如，标记物可以利用本领域中公知的方法与聚合物-脂质组合物颗粒的组分直接或间接偶联。可以使用广泛多样的标记物，标记物的选择取决于所需的灵敏度、与聚合物-脂质组合物颗粒组分缀合的容易度、稳定性要求、和可用的仪器和处理规定。合适的标记物包括，但不仅限于，光谱标记物诸如荧光染料(例如，荧光素及衍生物，诸如异硫氧酸荧光素(FITC)和Oregon GreenTM；罗丹明及衍生物，如德克萨斯红，异硫氧酸四甲基罗丹明(TRITC),等，地高辛配基，生物素，藻红蛋白，AMCA，CyDyesTM,等；放射性标记物诸如3H,125I,35S,14C,32P,33P,等；酶诸如辣根过氧化物酶，碱性磷酸酶，等；光谱比色标记物诸如胶体金或有色玻璃或塑料珠诸如聚苯乙烯，聚丙烯，胶乳，等。所述标记物可以利用本领域中任何已知方式来检测。The polymer-lipid composition particles of the present invention can be detected using any known method in the art. For example, a marker can be coupled directly or indirectly to a component of the polymer-lipid composition particle using methods known in the art. A wide variety of markers can be used, and the selection of the marker depends on the required sensitivity, the ease of conjugation with the polymer-lipid composition particle component, stability requirements, and available instruments and processing regulations. Suitable labels include, but are not limited to, spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives, such as fluorescein isothiocyanate (FITC) and Oregon Green ™ ; rhodamine and derivatives, such as Texas Red, tetramethylrhodamine isothiocyanate (TRITC), etc., digoxigenin, biotin, phycoerythrin, AMCA, CyDyes ™ , etc.; radioactive labels such as 3 H, 125 I, 35 S, 14 C, 32 P, 33 P, etc.; enzymes such as horseradish peroxidase, alkaline phosphatase, etc.; spectral colorimetric labels such as colloidal gold or colored glass or plastic beads such as polystyrene, polypropylene, latex, etc. The labels can be detected by any means known in the art.
(2)核酸的检测(2) Detection of nucleic acid
本文中通过许多本领域中技术人员公知的方式的任一种来检测并量化核酸(例如，mRNA)。核酸的检测可以通过公知的方法诸如Southern分析，Northern分析，凝胶电泳，PCR，放射性标记，闪烁计数，和亲合层析来进行。也可以使用另外的分析性生物化学方法诸如分光光度法、X射线照相法、电泳、毛细管电泳、高效液相色谱法(HPLC)、薄层色谱法(TLC)、和高扩散色谱法。 Nucleic acids (e.g., mRNA) are detected and quantified herein by any of a number of means known to those skilled in the art. Detection of nucleic acids can be performed by known methods such as Southern analysis, Northern analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Other analytical biochemical methods such as spectrophotometry, X-ray photography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and high diffusion chromatography can also be used.
治疗用途Therapeutic Uses
在一方面，本公开提供了一种用于预防和/或治疗受试者中的疾病或病症的方法，所述方法包括给有此需要的受试者于体内施用药学有效量的根据本发明的组合物或药物组合物的步骤，其中所述组合物或药物组合物包含所述针对疾病或病症的活性剂或治疗剂。在一些实施方案中，所述疾病或病症选自免疫系统疾病、代谢性疾病、遗传性疾病、癌症、血液疾病、细菌感染或病毒感染。In one aspect, the present disclosure provides a method for preventing and/or treating a disease or condition in a subject, the method comprising administering a pharmaceutically effective amount of a composition or pharmaceutical composition according to the present invention to a subject in need thereof in vivo, wherein the composition or pharmaceutical composition comprises an activating agent or therapeutic agent for the disease or condition. In some embodiments, the disease or condition is selected from an immune system disease, a metabolic disease, a genetic disease, a cancer, a blood disease, a bacterial infection, or a viral infection.
在另一方面，本发明提供了根据本发明的组合物或药物组合物在制备用于预防和/或治疗受试者中的疾病的药物中的用途，其中所述组合物或药物组合物包含所述针对疾病或病症的活性剂或治疗剂。在一些实施方案中，所述疾病或病症选自免疫系统疾病、代谢性疾病、遗传性疾病、癌症、血液疾病、细菌感染或病毒感染。On the other hand, the present invention provides the use of a composition or pharmaceutical composition according to the present invention in the preparation of a medicament for preventing and/or treating a disease in a subject, wherein the composition or pharmaceutical composition comprises the activating agent or therapeutic agent for the disease or condition. In some embodiments, the disease or condition is selected from immune system diseases, metabolic diseases, hereditary diseases, cancer, blood diseases, bacterial infections or viral infections.
在某些实施方案中，所述组合物用于递送至目标器官和/或靶组织和/或靶细胞治疗人受试者。在一些实施方案中，包含本文所述组合物用于递送至受试者的肺或肺细胞中。在一些实施方案中，本文所述组合物用于递送至受试者的肝或肝细胞中。在一些实施方案中，本文所述组合物用于递送至受试者的脾或脾细胞中。在一些实施方案中，包含本文所述组合物用于递送至受试者的肌肉或肌肉细胞中。In certain embodiments, the compositions are used for delivery to target organs and/or target tissues and/or target cells for treating human subjects. In some embodiments, the compositions described herein are used for delivery to the lungs or lung cells of a subject. In some embodiments, the compositions described herein are used for delivery to the liver or liver cells of a subject. In some embodiments, the compositions described herein are used for delivery to the spleen or spleen cells of a subject. In some embodiments, the compositions described herein are used for delivery to the muscles or muscle cells of a subject.
在某些实施方案中，本发明提供了一种用于递送包含编码蛋白质或多肽的mRNA的本文所述组合物的方法，所述治疗组合物用于治疗肺病。在某些实施方案中，本发明可用于制造编码囊性纤维化跨膜传导调整因子CFTR的mRNA(CFTR mRNA)的方法。CFTR mRNA被递送至需要用于治疗囊性纤维化的治疗组合物的受试者的肺部。在某些实施方案中，本发明可用于制造编码α1-胰蛋白酶的mRNA(A1AT mRNA)的方法。A1AT mRNA被递送至需要用于α1-胰蛋白酶缺乏症的治疗组合物的受试者的肺部和或肝脏。在某些实施方案中，本发明可用于制造编码轴索动力蛋白中链和重链的mRNA(DNAI1mRNA以及DNAH5mRNA)的方法。DNAI1mRNA或DNAH5mRNA被递送至需要用于治疗原发性纤毛运动障碍的治疗组合物的受试者的肺部。In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding a protein or polypeptide, the therapeutic composition being used to treat a lung disease. In certain embodiments, the present invention can be used in a method for producing an mRNA encoding the cystic fibrosis transmembrane conductance regulator CFTR (CFTR mRNA). The CFTR mRNA is delivered to the lungs of a subject in need of a therapeutic composition for treating cystic fibrosis. In certain embodiments, the present invention can be used in a method for producing an mRNA encoding α1-trypsin (A1AT mRNA). The A1AT mRNA is delivered to the lungs and or liver of a subject in need of a therapeutic composition for α1-trypsin deficiency. In certain embodiments, the present invention can be used in a method for producing mRNA encoding the medium and heavy chains of axonal dynein (DNAI1 mRNA and DNAH5 mRNA). DNAI1 mRNA or DNAH5 mRNA is delivered to the lungs of a subject in need of a therapeutic composition for treating primary ciliary dyskinesia.
在某些实施方案中，本发明提供了一种用于递送包含编码蛋白质或多肽的mRNA的本文所述的组合物的方法，所述治疗组合物用于治疗肝病或代谢 疾病。此类蛋白和多肽可包括与尿素循环紊乱相关的、与溶酶体贮积紊乱相关的、与糖原贮积紊乱相关的、与氨基酸代谢紊乱相关的、与脂质代谢或纤维化紊乱相关的、与甲基丙二酸血症相关或与任何其他代谢紊乱相关的那些，对于所述代谢紊乱，以富集的全长mRNA递送至或治疗肝脏或肝细胞提供治疗有益效果。In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising mRNA encoding a protein or polypeptide, wherein the therapeutic composition is used to treat liver disease or metabolic disease. Diseases. Such proteins and polypeptides may include those associated with urea cycle disorders, lysosomal storage disorders, glycogen storage disorders, amino acid metabolism disorders, lipid metabolism or fibrosis disorders, methylmalonic acidemia, or any other metabolic disorder for which delivery or treatment of the liver or hepatocytes with enriched full-length mRNA provides a therapeutic benefit.
在某些实施方案中，本发明提供了一种用于递送包含编码与尿素循环障碍相关的蛋白质的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码鸟氨酸转氨甲酰酶(OTC)蛋白质的mRNA的本文所述的组合物的方法。In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a protein associated with a urea cycle disorder. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding an ornithine transcarbamylase (OTC) protein.
在某些实施方案中，本发明提供了一种用于递送包含编码与溶酶体贮积紊乱相关的蛋白质的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码α半乳糖苷酶蛋白质的mRNA的本文所述的组合物的方法。In certain embodiments, the invention provides a method for delivering a composition described herein comprising mRNA encoding a protein associated with a lysosomal storage disorder. In certain embodiments, the invention provides a method for delivering a composition described herein comprising mRNA encoding an alpha galactosidase protein.
在某些实施方案中，本发明提供了一种用于递送包含编码与糖原贮积症相关的蛋白质的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码酸性α-葡糖苷酶蛋白质的mRNA的本文所述的组合物的方法。In certain embodiments, the invention provides a method for delivering a composition as described herein comprising mRNA encoding a protein associated with glycogen storage disease. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising mRNA encoding an acid alpha-glucosidase protein.
在某些实施方案中，本发明提供了一种用于递送包含编码与氨基酸代谢相关的蛋白质的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码苯丙氨酸羟化酶的mRNA的本文所述的组合物的方法。In certain embodiments, the invention provides a method for delivering a composition as described herein comprising mRNA encoding a protein related to amino acid metabolism. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising mRNA encoding phenylalanine hydroxylase.
在某些实施方案中，本发明提供了一种用于递送包含编码与脂质代谢或纤维化疾病相关的蛋白质的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码mTOR抑制剂的mRNA的本文所述的组合物的方法。In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising an mRNA encoding a protein associated with lipid metabolism or fibrotic diseases. In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising an mRNA encoding an mTOR inhibitor.
在某些实施方案中，本发明提供了一种用于递送包含编码与甲基丙二酸血症相关的蛋白质的mRNA的本文所述的组合物的方法。例如，在某些实施方案中，本发明提供了一种用于递送包含编码甲基丙二酰辅酶A突变酶蛋白质的mRNA的本文所述的组合物的方法。In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding a protein associated with methylmalonic acidemia. For example, in certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding a methylmalonyl-CoA mutant enzyme protein.
在某些实施方案中，所述治疗组合物用于递送至或治疗受试者的肝脏或肝细胞。在某些实施方案中，本发明提供了一种用于递送包含编码ATP7B蛋 白(也被称为威尔逊(Wilson)病蛋白)的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码胆色素原脱氨酶的mRNA的本文所述的组合物的方法。在某些实施方案中，在某些实施方案中，本发明提供了一种用于递送包含编码人血色素沉着病(HFE)蛋白质的mRNA的本文所述的组合物的方法。In certain embodiments, the therapeutic composition is used to deliver to or treat the liver or hepatocytes of a subject. In certain embodiments, the invention provides a method for delivering a composition comprising a protein encoding ATP7B. In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising mRNA encoding porphobilinogen deaminase. In certain embodiments, in certain embodiments, the present invention provides a method for delivering a composition as described herein comprising mRNA encoding human hemochromatosis (HFE) protein.
在某些实施方案中，所述治疗组合物用于递送至或治疗受试者的心血管系统或心血管细胞。在某些实施方案中，本发明提供了一种用于递送包含编码血管内皮生长因子A蛋白质的mRNA的本文所述的组合物的方法。In certain embodiments, the therapeutic composition is used to deliver to or treat the cardiovascular system or cardiovascular cells of a subject. In certain embodiments, the invention provides a method for delivering a composition described herein comprising mRNA encoding vascular endothelial growth factor A protein.
在某些实施方案中，所述治疗组合物用于递送至或治疗受试者的肌肉或肌肉细胞。在某些实施方案中，本发明提供了一种用于递送包含编码肌营养不良蛋白质的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码蛋白质或多肽的mRNA的本文所述的组合物的方法，所述治疗组合物用于递送至或治疗受试者的心肌或心肌细胞。在某些实施方案中，本发明提供了一种用于递送包含编码调整肌肉组织或肌肉细胞中钾通道和钠通道中的一者或两者的蛋白质的mRNA的本文所述的组合物的方法。In certain embodiments, the therapeutic composition is used to deliver to or treat a subject's muscle or muscle cells. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a dystrophin. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a protein or polypeptide, the therapeutic composition being used to deliver to or treat a subject's myocardium or myocardial cells. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a protein or polypeptide that modulates one or both of a potassium channel and a sodium channel in a muscle tissue or muscle cell.
在某些实施方案中，所述治疗组合物用于递送至或治疗受试者的神经系统或神经系统细胞。例如，在某些实施方案中，本发明提供了一种用于递送包含编码存活运动神经元1蛋白质的mRNA的本文所述的组合物的方法。In certain embodiments, the therapeutic composition is used to deliver to or treat the nervous system or nervous system cells of a subject.For example, in certain embodiments, the invention provides a method for delivering a composition described herein comprising an mRNA encoding a survival motor neuron 1 protein.
在某些实施方案中，所述治疗组合物用于递送至或治疗受试者的血液或骨髓或者血细胞或骨髓细胞。在某些实施方案中，本发明提供了一种用于递送包含编码β球蛋白的mRNA的本文所述的组合物的方法。In certain embodiments, the therapeutic composition is for delivery to or treatment of the blood or bone marrow, or blood cells or bone marrow cells, of a subject.In certain embodiments, the invention provides a method for delivering a composition described herein comprising mRNA encoding beta globin.
在某些实施方案中，所述治疗组合物用于递送至或治疗受试者的肾或肾细胞。在某些实施方案中，本发明提供了一种用于递送包含编码IV型胶原α5链(COL4A5)蛋白质的mRNA的本文所述的组合物的方法。In certain embodiments, the therapeutic composition is used to deliver to or treat a kidney or renal cell of a subject.In certain embodiments, the invention provides a method for delivering a composition described herein comprising mRNA encoding type IV collagen alpha 5 chain (COL4A5) protein.
在某些实施方案中，所述治疗组合物用于递送至或治疗受试者的眼或眼细胞。在某些实施方案中，本发明提供了一种用于递送包含编码ATP结合盒亚家族A成员4(ABCA4)蛋白质的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码视网膜劈裂素蛋白的mRNA的本文所述的组合物的方法。 In certain embodiments, the therapeutic composition is used to deliver to or treat an eye or ocular cell of a subject. In certain embodiments, the invention provides a method for delivering a composition described herein comprising an mRNA encoding an ATP-binding cassette subfamily A member 4 (ABCA4) protein. In certain embodiments, the invention provides a method for delivering a composition described herein comprising an mRNA encoding a retinoschizin protein.
在某些实施方案中，所述治疗组合物用于向受试者或受试者的细胞递送疫苗或用疫苗治疗。例如，在某些实施方案中，本发明提供了一种用于递送包含编码来自诸如病毒之类的传染源的抗原的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于制备包含递送编码新型冠状病毒(SARS-CoV-2)抗原的mRNA的本文所述的组合物的方法。In certain embodiments, the therapeutic composition is used to deliver a vaccine to a subject or a subject's cell or to treat with a vaccine. For example, in certain embodiments, the invention provides a method for delivering a composition described herein comprising mRNA encoding an antigen from an infectious source such as a virus. In certain embodiments, the invention provides a method for preparing a composition described herein comprising delivering mRNA encoding a novel coronavirus (SARS-CoV-2) antigen.
在某些实施方案中，本发明提供了用于包含编码来自诸如细菌之类的传染源的抗原的mRNA。例如，在某些实施方案中，本发明提供了一种用于制备包含递送编码肺结核杆菌抗原的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于制备包含递送编码铜绿假单胞菌抗原的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于制备包含递送编码肺炎克雷伯杆菌抗原的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于制备包含递送编码金黄色葡萄球菌抗原的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于制备包含递送编码鲍曼不动杆菌抗原的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于制备包含递送编码嗜肺军团菌抗原的mRNA的本文所述的组合物的方法。In certain embodiments, the present invention provides mRNA for containing an antigen from an infectious source such as bacteria. For example, in certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Mycobacterium tuberculosis antigen. In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Pseudomonas aeruginosa antigen. In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Klebsiella pneumoniae antigen. In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Staphylococcus aureus antigen. In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Baumannii antigen. In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Baumannii antigen. In certain embodiments, the present invention provides a method for preparing a composition as described herein comprising delivering an mRNA encoding a Legionella pneumophila antigen.
在某些实施方案中，本发明提供了用于递送编码受试者的癌症相关抗原或从受试者的癌细胞中鉴定的抗原的mRNA的治疗组合物的方法。在某些实施方案中，本发明提供了一种用于制备包含递送编码从受试者自身的癌细胞确定的抗原的mRNA的本文所述的组合物的方法，即提供个性化的癌症疫苗。在某些实施方案中，本发明提供了一种用于制备包含递送编码从突变KRAS基因表达的抗原的mRNA的本文所述的组合物的方法。In certain embodiments, the present invention provides a method for delivering a therapeutic composition encoding a cancer-associated antigen of a subject or an mRNA of an antigen identified from a subject's cancer cells. In certain embodiments, the present invention provides a method for preparing a composition described herein comprising delivering an mRNA encoding an antigen determined from a subject's own cancer cells, i.e., providing a personalized cancer vaccine. In certain embodiments, the present invention provides a method for preparing a composition described herein comprising delivering an mRNA encoding an antigen expressed from a mutant KRAS gene.
在某些实施方案中，本发明提供了一种用于递送包含编码抗体的mRNA的本文所述的组合物的方法。在某些实施方案中，抗体可以是双特异性抗体。在某些实施方案中，抗体可以是融合蛋白的一部分。在一些实施方案中，所述方法中的两个独立的负载mRNA的聚合物-脂质组合物(mRNA-PoLixNano)包含编码抗体轻链和重链的mRNA。在一些实施方案中，本发明的mRNA-PoLixNano组合物可包括含有不同脂质组成的不相同PoLixNano和包封编码抗体轻链或重链的mRNA的组合。在某些实施方案中，本发明提供了一种用于递送包含编码针对OX40的抗体的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码针对VEGF的抗体的 mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码针对组织坏死因子α的抗体的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码针对CD3的抗体的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码针对CD19的抗体的mRNA的本文所述的组合物的方法。In certain embodiments, the present invention provides a method for delivering a composition described herein comprising mRNA encoding an antibody. In certain embodiments, the antibody may be a bispecific antibody. In certain embodiments, the antibody may be part of a fusion protein. In some embodiments, the two independent mRNA-loaded polymer-lipid compositions (mRNA-PoLixNano) in the method comprise mRNA encoding an antibody light chain and a heavy chain. In some embodiments, the mRNA-PoLixNano composition of the present invention may include a combination of different PoLixNano containing different lipid compositions and mRNA encapsulating an antibody light chain or heavy chain. In certain embodiments, the present invention provides a method for delivering a composition described herein comprising mRNA encoding an antibody against OX40. In certain embodiments, the present invention provides a method for delivering a composition comprising mRNA encoding an antibody against VEGF. In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising mRNA encoding an antibody to tissue necrosis factor alpha. In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising mRNA encoding an antibody to CD3. In certain embodiments, the present invention provides a method for delivering a composition as described herein comprising mRNA encoding an antibody to CD19.
在某些实施方案中，本发明提供了一种用于递送包含编码免疫调节剂的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码白介素2(IL-2)的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码白介素12(IL-12)的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码粒细胞-巨噬细胞集落刺激因子(GM-CSF)的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码白介素23(IL-23)的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码C-C结构域趋化因子配体28(CCL28)的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码白介素36γ(IL-36γ)的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含mRNA的本文所述的组合物的方法，该mRNA编码胸腺基质淋巴细胞生成素或其衍生物。在某些实施方案中，本发明提供了一种用于递送包含mRNA的本文所述的组合物的方法，该mRNA编码一种或多种干扰素基因(STING)蛋白刺激物的组成型活性变体。In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding an immunomodulator. In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding interleukin 2 (IL-2). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding interleukin 12 (IL-12). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding granulocyte-macrophage colony stimulating factor (GM-CSF). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding interleukin 23 (IL-23). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding C-C domain chemokine ligand 28 (CCL28). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding interleukin 36γ (IL-36γ). In certain embodiments, the present invention provides a method for delivering a composition described herein comprising an mRNA encoding thymic stromal lymphopoietin or a derivative thereof. In certain embodiments, the invention provides a method for delivering a composition described herein comprising mRNA encoding one or more constitutively active variants of the stimulator of interferon genes (STING) protein.
在某些实施方案中，本发明提供了一种用于递送包含编码核酸内切酶的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码RNA指导的DNA核酸内切酶蛋白(诸如Cas 9蛋白)的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码大范围核酸酶蛋白质的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码转录激活因子样效应子核酸酶蛋白质的mRNA的本文所述的组合物的方法。在某些实施方案中，本发明提供了一种用于递送包含编码锌指核酸酶蛋白质的mRNA的本文所述的组合物的方法。 In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a nuclease. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a DNA endonuclease protein (such as a Cas 9 protein) guided by RNA. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a meganuclease protein. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a transcription activator-like effector nuclease protein. In certain embodiments, the invention provides a method for delivering a composition as described herein comprising an mRNA encoding a zinc finger nuclease protein.
实施例Example
实施例1.体外转录mRNA的制备Example 1. Preparation of in vitro transcribed mRNA
本文所述的制剂可用于递送任何mRNA，特别地是，编码特定抗原或治疗性蛋白质的体外转录mRNA(IVT-mRNA)。本文所述的制剂也可用于递送任何修饰的或未修饰的mRNA、或具有天然存在的序列或密码子优化的mRNA。作为非限制性实例，本文所述制剂递送的mRNA可(但非排他地)编码萤火虫荧光素酶(Fluc)和新冠病毒SARS-CoV-2刺突蛋白受体结合域RBD抗原。The formulations described herein can be used to deliver any mRNA, in particular, in vitro transcribed mRNA (IVT-mRNA) encoding a specific antigen or therapeutic protein. The formulations described herein can also be used to deliver any modified or unmodified mRNA, or mRNA with a naturally occurring sequence or codon optimization. As a non-limiting example, the mRNA delivered by the formulations described herein can (but not exclusively) encode firefly luciferase (Fluc) and the new coronavirus SARS-CoV-2 spike protein receptor binding domain RBD antigen.
作为非限制性实例，本文所述编码萤火虫荧光素酶的mRNA(Fluc-mRNA)和编码新冠病毒SARS-CoV-2刺突蛋白受体结合域RBD抗原(RBD-mRNA)可通过如下合成：构建包含T7启动子、5’UTR、ORF(Fluc或RBD)、3’UTR和长度约100个核苷酸的3’聚(A)尾部的重组DNA质粒；利用酶切或者PCR扩增制备线性化DNA模板；以线性化DNA模板进行体外转录，然后再添加5’帽子结构(Cap 1)制备Fluc-mRNA和RBD-mRNA。纯化后，通过凝胶电泳确定IVT-mRNA条带大小和纯度。As a non-limiting example, the mRNA encoding firefly luciferase (Fluc-mRNA) and encoding the RBD antigen (RBD-mRNA) of the spike protein of the novel coronavirus SARS-CoV-2 described herein can be synthesized by constructing a recombinant DNA plasmid comprising a T7 promoter, 5'UTR, ORF (Fluc or RBD), 3'UTR, and a 3' poly (A) tail of about 100 nucleotides in length; preparing a linearized DNA template by enzyme digestion or PCR amplification; performing in vitro transcription with the linearized DNA template, and then adding a 5' cap structure (Cap 1) to prepare Fluc-mRNA and RBD-mRNA. After purification, the IVT-mRNA band size and purity are determined by gel electrophoresis.
实施例2.制剂的制备Example 2. Preparation of formulations
制剂实例#1.经典LNP制剂的制备(四组分脂质，阳离子脂质+中性脂质+胆固醇+PEG脂质)：Formulation Example #1. Preparation of classic LNP formulation (four-component lipids, cationic lipids + neutral lipids + cholesterol + PEG lipids):
分别将50mg/mL的Dlin-MC3-DMA的乙醇溶液、50mg/mL的DSPC的乙醇溶液、50mg/mL的胆固醇的乙醇溶液和50mg/mL的DMG-PEG2K的乙醇溶液以50:10:38.5:1.5的摩尔比混合，且用乙醇稀释至总脂质浓度为10.88mg/mL。50 mg/mL of Dlin-MC3-DMA in ethanol, 50 mg/mL of DSPC in ethanol, 50 mg/mL of cholesterol in ethanol, and 50 mg/mL of DMG-PEG2K in ethanol were mixed at a molar ratio of 50:10:38.5:1.5, and diluted with ethanol to a total lipid concentration of 10.88 mg/mL.
Fluc-mRNA(或RBD-mRNA)的水性缓冲液(50mM柠檬酸盐缓冲液，pH 4.0)制备：将12.68mg/mL柠檬酸和8.77mg/mL的柠檬酸钠以1:1的体积比混合，混合液pH约为4.0。将Fluc-mRNA(或RBD-mRNA)用上述缓冲液稀释为0.17mg/mL。Preparation of aqueous buffer (50 mM citrate buffer, pH 4.0) for Fluc-mRNA (or RBD-mRNA): 12.68 mg/mL citric acid and 8.77 mg/mL sodium citrate were mixed in a volume ratio of 1:1, and the pH of the mixture was about 4.0. Fluc-mRNA (or RBD-mRNA) was diluted to 0.17 mg/mL with the above buffer.
完成上述溶液配制后，将溶有脂质的乙醇溶液使用微流控混合装置(NanoFac series,四川铄亘科技有限公司)与含有mRNA的柠檬酸盐缓冲液充分混合(水相：有机相体积比为3:1)，总流速为12mL/min。成功收集混 合溶液后，用1×PBS(pH 7.4)于4℃透析(透析袋截留分子量MWCO:8000-14000)20h，0.22μm滤膜过滤后，于2-8℃下贮存。所制备的LNP制剂内包封的Fluc-mRNA和RBD-mRNA最终浓度约为0.12mg/mL。After the above solutions were prepared, the lipid-dissolved ethanol solution was fully mixed with the citrate buffer containing mRNA using a microfluidic mixing device (NanoFac series, Sichuan Shuogen Technology Co., Ltd.) (the volume ratio of aqueous phase to organic phase was 3:1) at a total flow rate of 12 mL/min. After the solution was mixed, it was dialyzed with 1×PBS (pH 7.4) at 4°C (dialysis bag molecular weight cutoff MWCO: 8000-14000) for 20 hours, filtered with a 0.22 μm filter membrane, and stored at 2-8°C. The final concentration of Fluc-mRNA and RBD-mRNA encapsulated in the prepared LNP preparation was about 0.12 mg/mL.
制剂实例#2.聚合物-脂质的组合物PoLixNano制剂的制备：Formulation Example #2. Preparation of Polymer-Lipid Composition PoLixNano Formulation:
水相的制备：采用“制剂实例#1”中所述方法制备50mM柠檬酸盐缓冲液，用该缓冲液将Fluc-mRNA(或RBD-mRNA)和T904溶液分别制备为0.34mg/mL和8mg/mL的储备液，再将上述Fluc-mRNA(或RBD-mRNA)储备液和T904储备液以1:1的体积比混合均匀后待用。Preparation of aqueous phase: Prepare 50 mM citrate buffer by the method described in "Formulation Example #1", use this buffer to prepare Fluc-mRNA (or RBD-mRNA) and T904 solutions into 0.34 mg/mL and 8 mg/mL stock solutions, respectively, then mix the above Fluc-mRNA (or RBD-mRNA) stock solution and T904 stock solution in a volume ratio of 1:1 and set aside.
有机相的制备：分别将50mg/mL的Dlin-MC3-DMA的乙醇溶液、50mg/mL的DSPC的乙醇溶液、50mg/mL的胆固醇的乙醇溶液和50mg/mL的DMG-PEG2K的乙醇溶液以50:14:35:1的摩尔比混合，且用乙醇稀释至总脂质浓度为15.30mg/mL。Preparation of organic phase: 50 mg/mL of Dlin-MC3-DMA in ethanol, 50 mg/mL of DSPC in ethanol, 50 mg/mL of cholesterol in ethanol and 50 mg/mL of DMG-PEG2K in ethanol were mixed in a molar ratio of 50:14:35:1, and diluted with ethanol to a total lipid concentration of 15.30 mg/mL.
使用微流控混合装置(NanoFac series,四川铄亘科技有限公司)将上述水相溶液与有机相溶液充分混合(水相：有机相体积比为3:1，总流速为12mL/min)。收集混合溶液后，采用1×PBS(pH 7.4)于4℃环境中对收集溶液透析(透析袋MWCO:8000-14000)20h，0.22μm滤膜过滤，置于2-8℃下贮存。所制备的PoLixNano制剂内包封的Fluc-mRNA(或RBD-mRNA)的最终浓度约为0.10mg/mL。The aqueous solution and the organic solution were fully mixed using a microfluidic mixing device (NanoFac series, Sichuan Shuogeng Technology Co., Ltd.) (the volume ratio of aqueous phase to organic phase was 3:1, and the total flow rate was 12 mL/min). After the mixed solution was collected, the collected solution was dialyzed (dialysis bag MWCO: 8000-14000) for 20 h in a 4°C environment using 1×PBS (pH 7.4), filtered with a 0.22 μm filter membrane, and stored at 2-8°C. The final concentration of Fluc-mRNA (or RBD-mRNA) encapsulated in the prepared PoLixNano preparation was approximately 0.10 mg/mL.
制剂实例#3.两步法制备的mRNA-LNP/Poloxamine物理混合物制剂的制备(LNP+T904制剂)：Formulation Example #3. Preparation of mRNA-LNP/Poloxamine physical mixture formulation prepared by two-step method (LNP+T904 formulation):
将T904用1×PBS溶液稀释为75mg/mL，0.22μm滤膜过滤，待用。T904 was diluted to 75 mg/mL with 1× PBS solution, filtered through a 0.22 μm filter membrane, and set aside.
将“制剂实例#1”所述方法制备并纯化后的LNP溶液与上述T904溶液以4:1的体积比混合。室温静置20min后，0.22μm滤膜过滤，置于2-8℃下贮存。所制备的LNP+T904制剂内包封的Fluc-mRNA(或RBD-mRNA)的最终浓度约为0.10mg/mL。The LNP solution prepared and purified by the method described in "Formulation Example #1" was mixed with the above T904 solution at a volume ratio of 4:1. After standing at room temperature for 20 minutes, it was filtered with a 0.22 μm filter membrane and stored at 2-8°C. The final concentration of Fluc-mRNA (or RBD-mRNA) encapsulated in the prepared LNP+T904 formulation was about 0.10 mg/mL.
制剂实例#4.两步法制备的PoLixNano/Poloxamine物理混合物制剂的制备(PoLixNano+T904制剂)：Formulation Example #4. Preparation of PoLixNano/Poloxamine physical mixture formulation prepared by two-step method (PoLixNano+T904 formulation):
将T904用1×PBS稀释为15mg/mL，0.22μm滤膜过滤，待用。 T904 was diluted to 15 mg/mL with 1× PBS, filtered through a 0.22 μm filter membrane, and set aside.
将上述“制剂实例#2”制备并纯化后的PoLixNano溶液和上述T904以4:1的体积比充分混合。室温孵育20min后，0.22μm滤膜过滤，置于2-8℃下贮存。所制备的PoLixNano+T904制剂内包封的Fluc-mRNA(或RBD-mRNA)的最终浓度约为0.08mg/mL。The PoLixNano solution prepared and purified in the above "Formulation Example #2" and the above T904 were mixed thoroughly at a volume ratio of 4:1. After incubation at room temperature for 20 minutes, the mixture was filtered through a 0.22 μm filter membrane and stored at 2-8°C. The final concentration of Fluc-mRNA (or RBD-mRNA) encapsulated in the prepared PoLixNano+T904 formulation was about 0.08 mg/mL.
制剂实例#5.两步法制备的PoLixNano/蔗糖物理混合物制剂的制备(PoLixNano+10％sucrose)：Formulation Example #5. Preparation of PoLixNano/sucrose physical mixture formulation prepared by two-step method (PoLixNano + 10% sucrose):
用1×PBS配制50％的蔗糖溶液，0.22μm滤膜过滤，待用。Prepare 50% sucrose solution with 1×PBS, filter through 0.22 μm filter membrane and set aside.
将上述“制剂实例#2”制备并纯化后的PoLixNano溶液和50％的蔗糖溶液以4:1的体积比充分混合。室温孵育20min后，0.22μm滤膜过滤，置于2-8℃下贮存。所制备的PoLixNano+10％sucrose制剂内包封的Fluc-mRNA的最终浓度约为0.08mg/mL。The PoLixNano solution prepared and purified in the above "Formulation Example #2" and 50% sucrose solution were mixed thoroughly at a volume ratio of 4:1. After incubation at room temperature for 20 minutes, the mixture was filtered through a 0.22 μm filter membrane and stored at 2-8°C. The final concentration of Fluc-mRNA encapsulated in the prepared PoLixNano + 10% sucrose formulation was about 0.08 mg/mL.
制剂实例#6.由可电离/阳离子脂质、中性脂质以及胆固醇组成的三组分脂质制备的PoLixNano制剂(即，不含有PEG-脂质组分的PoLixNano)：Formulation Example #6. PoLixNano formulation prepared from three-component lipids consisting of ionizable/cationic lipids, neutral lipids, and cholesterol (i.e., PoLixNano without a PEG-lipid component):
水相的制备：采用50mM柠檬酸盐缓冲液将Fluc-mRNA(或RBD-mRNA)和Poloxamer 407溶液分别稀释为0.34mg/mL和13.33mg/mL的溶液，再将Fluc-mRNA(或RBD-mRNA)溶液和Poloxamer 407溶液以1:1的体积比混合均匀后待用。Preparation of aqueous phase: Use 50 mM citrate buffer to dilute Fluc-mRNA (or RBD-mRNA) and Poloxamer 407 solutions to 0.34 mg/mL and 13.33 mg/mL solutions, respectively, and then mix the Fluc-mRNA (or RBD-mRNA) solution and Poloxamer 407 solution in a volume ratio of 1:1 for standby use.
有机相的制备：将50mg/mL的Dlin-MC3-DMA的乙醇溶液、50mg/mL的DOPE的乙醇溶液和50mg/mL的胆固醇的乙醇溶液以40:32:28的摩尔比混合且用乙醇稀释至总脂质浓度为18.96mg/mL。Preparation of organic phase: 50 mg/mL of Dlin-MC3-DMA in ethanol, 50 mg/mL of DOPE in ethanol, and 50 mg/mL of cholesterol in ethanol were mixed at a molar ratio of 40:32:28 and diluted with ethanol to a total lipid concentration of 18.96 mg/mL.
使用微流控混合装置(NanoFac series,四川铄亘科技有限公司)将上述水相溶液与有机相溶液充分混合(水相：有机相体积比为3:1，总流速为12mL/min)。收集混合溶液后，采用1×PBS(pH 7.4)于4℃环境中对收集溶液透析(透析袋MWCO:8000-14000)20h，0.22μm滤膜过滤，置于2-8℃下贮存。The aqueous phase solution and the organic phase solution were fully mixed using a microfluidic mixing device (NanoFac series, Sichuan Shuogeng Technology Co., Ltd.) (the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min). After the mixed solution was collected, it was dialyzed (dialysis bag MWCO: 8000-14000) for 20 h in a 4°C environment using 1×PBS (pH 7.4), filtered with a 0.22 μm filter membrane, and stored at 2-8°C.
制剂实例#7.由胆固醇衍生阳离子脂质、中性脂质以及PEG-脂质组成的三组分脂质制备的PoLixNano制剂：Formulation Example #7. PoLixNano formulation prepared from three-component lipids consisting of cholesterol-derived cationic lipids, neutral lipids, and PEG-lipids:
水相的制备：采用50mM柠檬酸盐缓冲液将Fluc-mRNA(或RBD- mRNA)和T904溶液分别稀释为0.34mg/mL和8mg/mL的溶液，再将Fluc-mRNA(或RBD-mRNA)溶液和T904溶液以1:1的体积比混合均匀后待用。Preparation of aqueous phase: Fluc-mRNA (or RBD- The Fluc-mRNA (or RBD-mRNA) and T904 solutions were diluted to 0.34 mg/mL and 8 mg/mL solutions, respectively, and then the Fluc-mRNA (or RBD-mRNA) solution and the T904 solution were mixed evenly at a volume ratio of 1:1 for standby use.
有机相的制备：将10mg/mL的GL67的乙醇溶液、10mg/mL的DOPE的乙醇溶液和10mg/mL的DMG-PEG2K的乙醇溶液以70:28.5:1.5的摩尔比混合且用乙醇稀释至总脂质浓度为3.4mg/mL。Preparation of organic phase: 10 mg/mL GL67 ethanol solution, 10 mg/mL DOPE ethanol solution and 10 mg/mL DMG-PEG2K ethanol solution were mixed at a molar ratio of 70:28.5:1.5 and diluted with ethanol to a total lipid concentration of 3.4 mg/mL.
使用微流控混合装置(NanoFac series,四川铄亘科技有限公司)将上述水相溶液与有机相溶液充分混合(水相：有机相体积比为3:1，总流速为12mL/min)。收集混合溶液后，采用1×PBS(pH 7.4)于4℃环境中对收集溶液透析(透析袋MWCO:8000-14000)20h，0.22μm滤膜过滤，置于2-8℃下贮存。The aqueous phase solution and the organic phase solution were fully mixed using a microfluidic mixing device (NanoFac series, Sichuan Shuogeng Technology Co., Ltd.) (the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min). After the mixed solution was collected, it was dialyzed (dialysis bag MWCO: 8000-14000) for 20 h in a 4°C environment using 1×PBS (pH 7.4), filtered with a 0.22 μm filter membrane, and stored at 2-8°C.
制剂实例#8由可电离/阳离子脂质、胆固醇以及PEG-脂质组成的三组分脂质制备的PoLixNano制剂(即，不含有磷脂组分的PoLixNano)：Formulation Example #8 PoLixNano formulation prepared from three-component lipids consisting of ionizable/cationic lipids, cholesterol, and PEG-lipids (i.e., PoLixNano without a phospholipid component):
水相的制备：采用50mM柠檬酸盐缓冲液将Fluc-mRNA(或RBD-mRNA)和T904溶液分别稀释为0.34mg/mL和8mg/mL的溶液，再将Fluc-mRNA(或RBD-mRNA)溶液和T904溶液以1:1的体积比混合均匀后待用。Preparation of aqueous phase: Use 50 mM citrate buffer to dilute Fluc-mRNA (or RBD-mRNA) and T904 solution to 0.34 mg/mL and 8 mg/mL solution, respectively, and then mix the Fluc-mRNA (or RBD-mRNA) solution and T904 solution in a volume ratio of 1:1 for standby use.
有机相的制备：将50mg/mL的CKK-E12的乙醇溶液、50mg/mL的DOTAP的乙醇溶液、50mg/mL的胆固醇的乙醇溶液和50mg/mL的DMG-PEG2K的乙醇溶液以30:39:30:1的摩尔比混合且用乙醇稀释至总脂质浓度为5.86mg/mL。Preparation of organic phase: 50 mg/mL ethanol solution of CKK-E12, 50 mg/mL ethanol solution of DOTAP, 50 mg/mL ethanol solution of cholesterol and 50 mg/mL ethanol solution of DMG-PEG2K were mixed at a molar ratio of 30:39:30:1 and diluted with ethanol to a total lipid concentration of 5.86 mg/mL.
使用微流控混合装置(NanoFac series,四川铄亘科技有限公司)将上述水相溶液与有机相溶液充分混合(水相：有机相体积比为3:1，总流速为12mL/min)。收集混合溶液后，采用1×PBS(pH 7.4)于4℃环境中对收集溶液透析(透析袋MWCO:8000-14000)20h，0.22μm滤膜过滤，置于2-8℃下贮存。The aqueous phase solution and the organic phase solution were fully mixed using a microfluidic mixing device (NanoFac series, Sichuan Shuogeng Technology Co., Ltd.) (the volume ratio of aqueous phase: organic phase was 3:1, and the total flow rate was 12 mL/min). After the mixed solution was collected, it was dialyzed (dialysis bag MWCO: 8000-14000) for 20 h in a 4°C environment using 1×PBS (pH 7.4), filtered with a 0.22 μm filter membrane, and stored at 2-8°C.
实施例3.LNP和PoLixNano制剂的粒径、多分散系数(PDI)、Zeta电Example 3. Particle size, polydispersity index (PDI), and zeta potential of LNP and PoLixNano formulations 位及包封率Bit and Packing Rate
采用激光粒度分析仪测定上述LNP以及PoLixNano制剂的粒径大小、 PDI、电位信息以及包封率。核酸的包封采用Quant-iT RiboGreen RNA assay测定。所述RiboGreen测定方法基本上如Heyes等于Journal of Controlled Released，2005，107:276-287发表的文章中所述。上述理化性质表征结果以及所述制剂的脂质组分和两亲性嵌段共聚物组成总结在表1中。The particle size of the LNP and PoLixNano preparations was determined by a laser particle size analyzer. PDI, potential information and encapsulation efficiency. Encapsulation of nucleic acids was determined using the Quant-iT RiboGreen RNA assay. The RiboGreen assay was essentially as described in the article published by Heyes et al., Journal of Controlled Released, 2005, 107:276-287. The above physicochemical characterization results and the lipid components and amphiphilic block copolymer compositions of the formulations are summarized in Table 1.
表1.LNP和PoLixNano制剂的理化性质特征

Table 1. Physicochemical properties of LNP and PoLixNano preparations

实施例4.施用负载mRNA的LNP和/或PoLixNano制剂Example 4. Administration of mRNA-loaded LNP and/or PoLixNano formulations
本实施例说明施用负载mRNA的相关制剂的示例性方法和用于在体内靶组织中分析递送的mRNA所表达的蛋白质的方法。This example illustrates exemplary methods of administering relevant formulations loaded with mRNA and methods for analyzing proteins expressed by the delivered mRNA in target tissues in vivo.
如无特殊说明，所有研究在每个实验开始时使用约6-8周龄的雌性Balb/c小鼠进行。所有小鼠均饲养在无特定病原体(SPF)级动物房内，经历12小时/12小时的光/暗循环，可自由获取食物和水。在实验开始前，所有实验动物有至少7天的适应环境时间。Unless otherwise specified, all studies were performed using female Balb/c mice approximately 6-8 weeks old at the start of each experiment. All mice were housed in a specific pathogen-free (SPF) animal room with a 12-hour/12-hour light/dark cycle and free access to food and water. All experimental animals had at least 7 days of acclimatization time before the start of the experiment.
复合物的经气管内喷雾(i.t.)方式给药：Administration of the complex by intratracheal spray (i.t.):
在异氟烷(Isothesia)吸入麻醉期间，使用高压MicroSprayer喷雾针(PennCentury,USA)经单只小鼠气管内喷雾mRNA剂量为2.5μg mRNA/50μL的PoLixNano或LNP制剂。如无特殊说明，所有同类型实验均采用该剂量。During isoflurane inhalation anesthesia, a high-pressure MicroSprayer needle (PennCentury, USA) was used to spray the mRNA dose of 2.5 μg mRNA/50 μL of PoLixNano or LNP formulation into the trachea of a single mouse. Unless otherwise specified, this dose was used in all experiments of the same type.
复合物的经鼻途径(i.n.)施用：Administration of the complex by the nasal route (i.n.):
用mRNA剂量为2.0μg mRNA/40μL的PoLixNano或LNP制剂给药至Balb/c小鼠。在异氟烷(Isothesia)吸入麻醉期间，以一滴的形式施加溶液到单只动物的鼻孔上，自然吸入复合物制剂。如无特殊说明，所有同类型实验均采用该剂量。Balb/c mice were administered PoLixNano or LNP formulations at an mRNA dose of 2.0 μg mRNA/40 μL. The solution was applied as a drop to the nostril of a single animal during isoflurane (Isothesia) inhalation anesthesia, and the complex formulation was naturally inhaled. Unless otherwise specified, all experiments of the same type used this dose.
复合物的经雾化吸入途径(nebulization)给药：Administration of the complex via nebulization:
将给药剂量为25μg mRNA/只小鼠的PoLixNano制剂或LNP制剂置于雾化器系统(Aerogen,Dangan,Ireland)中，雾化速度为30μL/10s，雾化时间为15min。如无特殊说明，所有同类型实验均采用该剂量。The PoLixNano formulation or LNP formulation with a dose of 25 μg mRNA/mouse was placed in a nebulizer system (Aerogen, Dangan, Ireland) with a nebulization rate of 30 μL/10 s and a nebulization time of 15 min. Unless otherwise specified, this dose was used in all experiments of the same type.
复合物的经肌肉注射(i.m.)方式给药：Administration of the complex by intramuscular (i.m.) injection:
Balb/c小鼠大腿外侧肌肉部位用75％酒精棉球消毒皮肤表面，使用注射器快速刺入肌肉，推注mRNA剂量为5μg mRNA/100μL的PoLixNano或LNP制剂。如无特殊说明，所有同类型实验均采用该剂量。The skin surface of the outer thigh muscle of Balb/c mice was disinfected with 75% alcohol cotton balls, and the muscle was quickly pierced with a syringe to inject PoLixNano or LNP preparations with a mRNA dose of 5μg mRNA/100μL. Unless otherwise specified, all experiments of the same type used this dose.
IVIS活体成像检测Fluc-mRNA制剂在小鼠体内的基因转染情况： IVIS in vivo imaging detected the gene transfection of Fluc-mRNA preparation in mice:
为了检测Luciferase报告基因在施用Fluc-mRNA制剂的小鼠体内的表达情况，采用IVIS活体成像装置进行考察。首先使用异氟烷麻醉装置以1-3％(通常以2.5％)进行麻醉。将D-萤光素溶液(3mg D-萤光素溶解于100μL PBS)经腹腔注射途径(i.p.)以150mg/kg的剂量施用到每只动物的体内。使荧光素分布10分钟。将动物置于异氟烷室中直至麻醉。将经麻醉的动物放于IVIS成像室的背靠板(dorsal recumbency)处且头部置于通气管中。使用IVIS活体成像系统(Perkin Elmer,USA)进行生物发光成像。在这些实施例中，仪器设置如下：照相机高度处于D水平，F/Stop为f1，像素组合(binning)为8×8，曝光时间为自动。对活体动物成像后，立即通过颈椎脱位(cervical dislocation)处死动物，快速取出肺、肝或脾等器官，对获得的离体器官再次进行生物发光成像。使用Living Image软件(PerkinElmer,USA)，通过测量设定的感兴趣区域(ROI)中的辐射率来分析图片。数值显示为总辐射率[p/s]。In order to detect the expression of Luciferase reporter gene in mice administered with Fluc-mRNA preparation, IVIS in vivo imaging device was used for investigation. First, anesthesia was performed using an isoflurane anesthesia device at 1-3% (usually 2.5%). D-luciferin solution (3 mg D-luciferin dissolved in 100 μL PBS) was administered to each animal via intraperitoneal injection (i.p.) at a dose of 150 mg/kg. Allow the luciferin to distribute for 10 minutes. The animal was placed in an isoflurane chamber until anesthetized. The anesthetized animal was placed in the dorsal recumbency of the IVIS imaging chamber with the head placed in the ventilator. Bioluminescence imaging was performed using the IVIS in vivo imaging system (Perkin Elmer, USA). In these embodiments, the instrument settings were as follows: the camera height was at D level, F/Stop was f1, the pixel combination (binning) was 8×8, and the exposure time was automatic. Immediately after imaging the live animals, the animals were killed by cervical dislocation, and the lungs, liver, or spleen were quickly removed, and the isolated organs were imaged again for bioluminescence. The images were analyzed by measuring the radiance in the set region of interest (ROI) using Living Image software (PerkinElmer, USA). The values are shown as total radiance [p/s].
实施例5.PoLixNano制剂的理化性质及其在不同细胞中的基因转染情况Example 5. Physicochemical properties of PoLixNano preparations and gene transfection in different cells
LNP制剂的制备：采用上述“制剂实例#1”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为49:20.1:30:0.9，N/P为8。Preparation of LNP preparation: Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 49:20.1:30:0.9, and N/P was 8.
PoLixNano制剂的制备：采用上述“制剂实例#2”中所述方法通过不同脂质比例制备含不同浓度T904和T704的PoLixNano制剂。Preparation of PoLixNano formulations: PoLixNano formulations containing different concentrations of T904 and T704 were prepared using the method described in the above "Formulation Example #2" using different lipid ratios.
用透射电子显微镜(TEM)观察LNP及PoLixNano纳米颗粒的物理形态。结果显示，不同脂质比例制备的LNP和PoLixNano制剂均形成大小均一的纳米颗粒。在脂质组分比例为Dlin-Mc3-DMA:DSPC:Chol:PEG-2000-DMG＝49:20.1:30:0.9时，向上述组分中加入终浓度为3mg/mL的泊洛沙胺904(T904)后，PolixNano纳米粒会形成明显的腔室结构(图1A)。且当两亲性嵌段共聚物的种类和浓度发生变化时，腔室的大小和形态也会随之改变。在脂质组分比例为Dlin-Mc3-DMA:DSPC:Chol:PEG-2000-DMG＝39:40.1:20:0.9时，向上述组分中加入终浓度为12mg/mL泊洛沙胺704(T704)后，PolixNano纳米粒形成了多腔室结构(图1A)。有研究表明，这类纳米粒中的腔室结构可以增加mRNA的转染效率(Cheng MHY,Cullis PR,et al.Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency.Adv Mater.2023,35(31):e2303370)。 The physical morphology of LNP and PoLixNano nanoparticles was observed by transmission electron microscopy (TEM). The results showed that LNP and PoLixNano preparations prepared with different lipid ratios formed nanoparticles of uniform size. When the lipid component ratio was Dlin-Mc3-DMA: DSPC: Chol: PEG-2000-DMG = 49: 20.1: 30: 0.9, after adding poloxamine 904 (T904) with a final concentration of 3 mg/mL to the above components, the PolixNano nanoparticles formed a clear chamber structure (Figure 1A). And when the type and concentration of the amphiphilic block copolymer changed, the size and morphology of the chamber would also change accordingly. When the lipid component ratio was Dlin-Mc3-DMA: DSPC: Chol: PEG-2000-DMG = 39: 40.1: 20: 0.9, after adding poloxamine 704 (T704) with a final concentration of 12 mg/mL to the above components, PolixNano nanoparticles formed a multi-chamber structure (Figure 1A). Studies have shown that the chamber structure in this type of nanoparticle can increase the transfection efficiency of mRNA (Cheng MHY, Cullis PR, et al. Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency. Adv Mater. 2023, 35 (31): e2303370).
相较于对应的对照组(LNP，Lipo2000或者brPEI)，含6mg/mL T704的PoLixNano制剂能够被人支气管上皮细胞16HBE、树突状细胞DC2.4和小鼠骨髓来源树突状细胞(BMDC)细胞高效摄取，有效表达Fluc-mRNA，且对细胞无毒性(图1B和C)。相较于Lipo2000，PoLixNano制剂具有较强的穿透粘液层能力(图1D)。相较于LNP制剂组，PoLixNano制剂能够更为有效地刺激BMDC细胞的分化与成熟，更好地介导后续适应性免疫应答(图1E)。Compared with the corresponding control groups (LNP, Lipo2000 or brPEI), the PoLixNano preparation containing 6 mg/mL T704 can be efficiently taken up by human bronchial epithelial cells 16HBE, dendritic cells DC2.4 and mouse bone marrow-derived dendritic cells (BMDC) cells, effectively express Fluc-mRNA, and is non-toxic to cells (Figure 1B and C). Compared with Lipo2000, the PoLixNano preparation has a stronger ability to penetrate the mucus layer (Figure 1D). Compared with the LNP preparation group, the PoLixNano preparation can more effectively stimulate the differentiation and maturation of BMDC cells and better mediate subsequent adaptive immune responses (Figure 1E).
实施例6.不同制剂经气管内喷雾(i.t.)给药6h后的基因转染效果Example 6. Gene transfection effects of different preparations 6 hours after intratracheal spray (i.t.) administration
方法：采用“制剂实例#1”中所述方法制备LNP，采用“制剂实例#2”中所述方法制备含0.025mg/mL T904、3mg/mL T904、0.025mg/mL T704以及6mg/mL T704的PoLixNano制剂。Methods: LNPs were prepared using the method described in Formulation Example #1, and PoLixNano formulations containing 0.025 mg/mL T904, 3 mg/mL T904, 0.025 mg/mL T704, and 6 mg/mL T704 were prepared using the method described in Formulation Example #2.
T904-mRNA制剂(或T704-mRNA制剂)的制备：将4×Tyrode缓冲液和0.24mg/mL Fluc-mRNA以1:1的体积比混合，然后混合液与6mg/mL T904溶液(或T704溶液)以1:1的体积比混合，室温孵育20min，即获得Fluc-mRNA剂量为3μg/50μL的T904-mRNA制剂(或T704-mRNA制剂)。Preparation of T904-mRNA preparation (or T704-mRNA preparation): Mix 4×Tyrode buffer and 0.24 mg/mL Fluc-mRNA in a volume ratio of 1:1, and then mix the mixture with 6 mg/mL T904 solution (or T704 solution) in a volume ratio of 1:1. Incubate at room temperature for 20 minutes to obtain T904-mRNA preparation (or T704-mRNA preparation) with a Fluc-mRNA dose of 3 μg/50 μL.
Naked-mRNA制剂的制备：将Fluc-mRNA用PBS稀释为3μg/50μL。Preparation of Naked-mRNA preparation: Fluc-mRNA was diluted to 3 μg/50 μL with PBS.
结果如图2A所示，在PBS和naked-mRNA给药组小鼠体内未检测到荧光素酶的表达。T904-mRNA制剂组、T704-mRNA制剂组和LNP制剂组在小鼠体内检测到较低量的荧光素酶信号，LNP制剂组中肺组织的表达高于T904-mRNA制剂组和T704-mRNA制剂组。通过T904或者T704制备的PoLixNano制剂组给药活体小鼠(mice)及其离体肺部(lungs)中均检测到显著优于其他对照组的高水平荧光素酶信号(图2B)。与此同时，含痕迹量T904的PoLixNano制剂或含痕迹量T704的PoLixNano制剂经气管内喷雾(i.t.)给药后，相比于LNP制剂，显著地促进了Fluc-mRNA在小鼠体内(尤其是肺部)的基因转染效率(图2C)。并且含痕迹量T704的PoLixNano制剂在小鼠体内介导Fluc-mRNA产生的荧光素酶表达量与含痕迹量T904的PoLixNano制剂组水平相当(图2C)。As shown in Figure 2A, no luciferase expression was detected in mice in the PBS and naked-mRNA groups. Lower amounts of luciferase signals were detected in mice in the T904-mRNA, T704-mRNA and LNP groups, and the expression of lung tissue in the LNP group was higher than that in the T904-mRNA and T704-mRNA groups. PoLixNano preparations prepared by T904 or T704 were administered to mice in vivo and their lungs in vitro, and a high level of luciferase signals significantly superior to other control groups were detected (Figure 2B). At the same time, after intratracheal spray (i.t.) administration of PoLixNano preparations containing trace amounts of T904 or PoLixNano preparations containing trace amounts of T704, compared with LNP preparations, the gene transfection efficiency of Fluc-mRNA in mice (especially lungs) was significantly promoted (Figure 2C). Furthermore, the luciferase expression level mediated by Fluc-mRNA production in mice by the PoLixNano preparation containing trace amounts of T704 was comparable to that of the PoLixNano preparation group containing trace amounts of T904 ( Figure 2C ).
实施例7.含不同类型两亲性聚合物的PoLixNano制剂经气管内喷雾Example 7. Intratracheal spraying of PoLixNano formulations containing different types of amphiphilic polymers (i.t.)给药6h后的转染效果(i.t.) Transfection effect 6h after administration
LNP制剂的制备：采用上述“制剂实例#1”中所述方法，调整Dlin-MC3- DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为50:14:35:1，N/P为8.2。Preparation of LNP formulation: Using the method described in "Formulation Example #1" above, adjust Dlin-MC3- The molar ratio of DMA, DSPC, cholesterol and DMG-PEG2000 was 50:14:35:1, and the N/P was 8.2.
PoLixNano制剂的制备：采用上述“制剂实例#2”中所述方法制备如图3所示含不同类型两亲性聚合物的PoLixNano制剂，图3中两亲性聚合物的浓度为PoLixNano颗粒内的终浓度。Preparation of PoLixNano formulations: PoLixNano formulations containing different types of amphiphilic polymers as shown in FIG3 were prepared using the method described in the above “Formulation Example #2”. The concentration of the amphiphilic polymer in FIG3 is the final concentration in the PoLixNano particles.
结果如图3所示，相较于LNP制剂组，含不同浓度T904和T704的PoLixNano制剂均能显著增加活体小鼠和肺部中Fluc-mRNA产生的荧光素酶水平，特别是含40mg/mL T904和含6mg/mL T704的PoLixNano制剂显著提高了mRNA在肺部中的转染效率(图3A和B)。将T704或者T904替换为Tween 80、其他的Poloxamers或者Poloxamines制备的PoLixNano制剂气管内喷雾(i.t.)给药仍能在肺部高效转染Fluc-mRNA(图3C和D)。The results are shown in Figure 3. Compared with the LNP preparation group, the PoLixNano preparations containing different concentrations of T904 and T704 can significantly increase the luciferase level produced by Fluc-mRNA in living mice and lungs, especially the PoLixNano preparations containing 40 mg/mL T904 and 6 mg/mL T704 significantly improved the transfection efficiency of mRNA in the lungs (Figure 3A and B). The intratracheal spray (i.t.) administration of PoLixNano preparations prepared by replacing T704 or T904 with Tween 80, other Poloxamers or Poloxamines can still efficiently transfect Fluc-mRNA in the lungs (Figure 3C and D).
实施例8.不同N/P比例和脂质摩尔比例制备的PoLixNano制剂经气管Example 8. PoLixNano preparations prepared with different N/P ratios and lipid molar ratios were administered intratracheally 内喷雾(i.t.)给药6h后的转染效果Transfection effect 6 hours after intravenous (i.t.) administration
LNP制剂的制备：采用上述“制剂实例#1”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为50:14:35:1或50:14.3:35:0.7，N/P为8。Preparation of LNP preparations: Using the method described in the above “Formulation Example #1”, the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1 or 50:14.3:35:0.7, and the N/P was 8.
PoLixNano制剂的制备：采用上述“制剂实例#2”中所述方法，保持各脂质组分摩尔比不变，制备N/P比例分别为2、4、5、6、7、8、9、10、12和15的PoLixNano制剂。采用上述“制剂实例#2”中所述方法，调整N/P为8，制备含不同摩尔比例脂质的PoLixNano制剂，其中DMG-PEG2000摩尔比例在0.1到5之间，DSPC摩尔比例在8到50.1之间，Dlin-MC3-DMA摩尔比例在25到70之间，胆固醇摩尔比例在20.2到46.5之间。Preparation of PoLixNano formulations: Using the method described in the above "Formulation Example #2", keeping the molar ratio of each lipid component unchanged, PoLixNano formulations with N/P ratios of 2, 4, 5, 6, 7, 8, 9, 10, 12 and 15 were prepared. Using the method described in the above "Formulation Example #2", adjusting N/P to 8, PoLixNano formulations containing lipids in different molar ratios were prepared, wherein the molar ratio of DMG-PEG2000 was between 0.1 and 5, the molar ratio of DSPC was between 8 and 50.1, the molar ratio of Dlin-MC3-DMA was between 25 and 70, and the molar ratio of cholesterol was between 20.2 and 46.5.
结果显示，相较于LNP制剂组，含不同N/P比例和脂质摩尔比例的PoLixNano制剂均能显著增加活体小鼠和肺部中Fluc-mRNA产生的荧光素酶水平，其中N/P为8和9的PoLixNano制剂极显著提高了mRNA在肺部中的转染效率(图4A)。不同脂质摩尔比例制备的PoLixNano制剂在活体小鼠和肺部具有不同的基因转染能力且均显著高于LNP对照组，特别是PoLixNano制剂(DMG-PEG2000摩尔比例在0.3到1之间，DSPC摩尔比例在10到30.1之间，Dlin-MC3-DMA摩尔比例在35到65之间，胆固醇摩尔比例在21到46.5之间)均具有高效转染Fluc-mRNA的潜力(图4B和C)。 The results showed that compared with the LNP preparation group, PoLixNano preparations containing different N/P ratios and lipid molar ratios could significantly increase the luciferase level produced by Fluc-mRNA in living mice and lungs, among which PoLixNano preparations with N/P of 8 and 9 significantly improved the transfection efficiency of mRNA in the lungs (Figure 4A). PoLixNano preparations prepared with different lipid molar ratios had different gene transfection abilities in living mice and lungs and were significantly higher than the LNP control group, especially PoLixNano preparations (DMG-PEG2000 molar ratio between 0.3 and 1, DSPC molar ratio between 10 and 30.1, Dlin-MC3-DMA molar ratio between 35 and 65, and cholesterol molar ratio between 21 and 46.5) all had the potential for efficient transfection of Fluc-mRNA (Figures 4B and C).
实施例9.不同类型脂质制备的PoLixNano制剂经气管内喷雾(i.t.)给Example 9. PoLixNano formulations prepared with different types of lipids were administered via intratracheal spray (i.t.) 药6h后的转染效果Transfection effect after 6 hours
LNP制剂的制备：采用上述“制剂实例#1”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为50:14:35:1，N/P为8。Preparation of LNP preparation: Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1, and N/P was 8.
PoLixNano制剂的制备：采用上述“制剂实例#2”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为50:14:35:1，N/P为8，将“Dlin-MC3-DMA”分别换为“ALC-0315”、“SM-102”“HGT5000”或者“C12-200”，“DSPC”分别换成“DPPC”、“DOPE”和“ESM”，“胆固醇”换成“β-谷甾醇或者“胆固醇”与“β-谷甾醇”组分摩尔比例为17.5:17.5，“DMG-PEG2000”换成“DSPE-PEG-Mannose”和“DMG-PEG5000”，制备如图4所示不同类型脂质制备的PoLixNano制剂。Preparation of PoLixNano preparations: Using the method described in the above-mentioned "Preparation Example #2", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1, N/P was 8, "Dlin-MC3-DMA" was replaced with "ALC-0315", "SM-102", "HGT5000" or "C12-200", "DSPC" was replaced with "DPPC", "DOPE" and "ESM", "cholesterol" was replaced with "β-sitosterol or the molar ratio of "cholesterol" to "β-sitosterol" was 17.5:17.5, and "DMG-PEG2000" was replaced with "DSPE-PEG-Mannose" and "DMG-PEG5000" to prepare PoLixNano preparations prepared with different types of lipids as shown in Figure 4.
结果如图5所示，相较于LNP制剂组，不同脂质制备的PoLixNano制剂均能显著增加活体小鼠和肺部中Fluc-mRNA产生的荧光素酶水平。相较于Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000制备的PoLixNano制剂组，通过替换不同结构的脂质制备(除了SM-102)的PoLixNano在肺部中Fluc-mRNA产生的荧光素酶水平相当，通过SM-102制备的PoLixNano显著提升了在肺部中Fluc-mRNA的转染效率，这表明通过替换不同结构的脂质制备的PoLixNano制剂经呼吸道给药具有实现相当水平甚至更高效mRNA转染的潜力。The results are shown in Figure 5. Compared with the LNP preparation group, the PoLixNano preparations prepared with different lipids can significantly increase the luciferase level produced by Fluc-mRNA in living mice and lungs. Compared with the PoLixNano preparation groups prepared with Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000, the PoLixNano prepared by replacing lipids with different structures (except SM-102) has a comparable level of luciferase produced by Fluc-mRNA in the lungs, and the PoLixNano prepared by SM-102 significantly improves the transfection efficiency of Fluc-mRNA in the lungs, which indicates that the PoLixNano preparations prepared by replacing lipids with different structures have the potential to achieve comparable or even more efficient mRNA transfection through respiratory administration.
实施例10.含不同类型Poloxamers、Poloxamines和脂质制备的Example 10. Preparation of Poloxamers, Poloxamines and Lipids PoLixNano制剂经鼻途径(i.n.)给药6h后的转染效果Transfection effect of PoLixNano formulation 6 hours after i.n. administration
LNP制剂的制备：采用上述“制剂实例#1”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为49:20.1:30:0.9，N/P为8。Preparation of LNP preparation: Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 49:20.1:30:0.9, and N/P was 8.
PoLixNano制剂的制备：采用上述“制剂实例#5”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为49:20.1:30:0.9，N/P为8，制备如图6所示含不同类型Poloxamers、Poloxamines和脂质的PoLixNano+10％sucrose制剂，Poloxamers和Poloxamines的浓度为 PoLixNano颗粒内的终浓度。Preparation of PoLixNano formulations: Using the method described in the above "Formulation Example #5", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 49:20.1:30:0.9, and N/P was 8. PoLixNano + 10% sucrose formulations containing different types of poloxamers, poloxamines and lipids were prepared as shown in Figure 6. The concentrations of poloxamers and poloxamines were Final concentration within PoLixNano particles.
结果显示，相较于LNP制剂组，含不同浓度Poloxamers和Poloxamines的PoLixNano制剂能够显著增加肺部中Fluc-mRNA产生的荧光素酶水平，特别是含12mg/mL T704的PoLixNano制剂(图6A)。调整脂质比例(DMG-PEG2000摩尔比例在0.5到4之间，DSPC摩尔比例在5到50之间，Dlin-MC3-DMA摩尔比例在28.9到63.2之间，胆固醇摩尔比例在20到39.1之间)，PoLixNano制剂在肺部中均能高效转染Fluc-mRNA，特别是PoLixNano制剂(DMG-PEG2000摩尔比例在0.5到2之间，DSPC摩尔比例在8到20.1之间，Dlin-MC3-DMA摩尔比例在47.9到56.4之间，胆固醇摩尔比例在30到34.7之间(图6B))。替换Dlin-MC3-DMA、DSPC和DMG-PEG2000的其他制剂组均可在肺部中介导Fluc-mRNA的高效转染，SM-102和ALC-0315的制剂组Fluc-mRNA的表达量显著高于Dlin-MC3-DMA制剂组(图6C)。The results showed that compared with the LNP preparation group, the PoLixNano preparations containing different concentrations of poloxamers and poloxamines could significantly increase the luciferase level produced by Fluc-mRNA in the lungs, especially the PoLixNano preparation containing 12 mg/mL T704 (Figure 6A). By adjusting the lipid ratio (DMG-PEG2000 molar ratio between 0.5 and 4, DSPC molar ratio between 5 and 50, Dlin-MC3-DMA molar ratio between 28.9 and 63.2, and cholesterol molar ratio between 20 and 39.1), the PoLixNano preparations could efficiently transfect Fluc-mRNA in the lungs, especially the PoLixNano preparations (DMG-PEG2000 molar ratio between 0.5 and 2, DSPC molar ratio between 8 and 20.1, Dlin-MC3-DMA molar ratio between 47.9 and 56.4, and cholesterol molar ratio between 30 and 34.7 (Figure 6B)). Other formulation groups replacing Dlin-MC3-DMA, DSPC and DMG-PEG2000 can mediate efficient transfection of Fluc-mRNA in the lungs. The expression levels of Fluc-mRNA in the SM-102 and ALC-0315 formulation groups were significantly higher than that in the Dlin-MC3-DMA formulation group (Figure 6C).
实施例11.物理混合Poloxamers、Poloxamines、多肽和sucrose后获得的Example 11. Physically mixed poloxamers, poloxamines, polypeptides and sucrose to obtain 制剂能够进一步促进肺部Fluc-mRNA的基因转染The preparation can further promote gene transfection of Fluc-mRNA in the lungs
LNP及其物理混合制剂的制备：采用“制剂实例#1”中所述方法制备LNP；采用“制剂实例#3”中所述方法制备图7中相应的LNP物理混合制剂。Preparation of LNP and its physical mixed preparation: LNP was prepared by the method described in "Formulation Example #1"; the corresponding LNP physical mixed preparation in Figure 7 was prepared by the method described in "Formulation Example #3".
PoLixNano及其物理混合制剂的制备：采用上述“制剂实例#4”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为49:20.1:30:0.9，N/P为8，制备PoLixNano；采用上述“制剂实例#4”和“制剂实例#5”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为49:20.1:30:0.9，N/P为8，“制剂实例#4”中“15mg/mL T904”改为“2.5mg/mL KG41(序列：NH2-KETWWETWWTEWWTEWKKKKRRRRRKKKKGACSERSMNFCG-COOH)”，制备如图12中所示相应的PoLixNano物理混合制剂。Preparation of PoLixNano and its physical mixed preparations: Using the method described in the above-mentioned "Preparation Example #4", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 49:20.1:30:0.9, and N/P was 8 to prepare PoLixNano; using the methods described in the above-mentioned "Preparation Example #4" and "Preparation Example #5", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 49:20.1:30:0.9, and N/P was 8, and the "15 mg/mL T904" in "Preparation Example #4" was changed to "2.5 mg/mL KG41 (sequence: NH2-KETWWETWWTEWWTEWKKKKRRRRRKKKKGACSERSMNFCG-COOH)" to prepare the corresponding PoLixNano physical mixed preparation as shown in Figure 12.
结果如图7所示，相较于LNP制剂组，LNP+T904、LNP+KG41和LNP+10％sucrose制剂组经气管内喷雾(i.t.)和鼻途径(i.n.)给药6h后均能够显著促进Fluc-mRNA在活体小鼠、肺部、肝脏和脾部中的转染效率，特别是LNP+10％sucrose制剂组。将含3mg/mL T904的PoLixNano制剂物理混合T904、KG41和sucrose后经气管内喷雾(i.t.)和鼻途径(i.n.)给药6h后在活体小鼠、肺部、肝脏和脾部中均能显著提升Fluc-mRNA产生的荧光素酶 水平。其中最值得注意的是，10％蔗糖溶液能够稳定增强LNP以及PoLixNano制剂在小鼠体内(尤其是肺部)介导IVT-mRNA的转染效率。The results are shown in Figure 7. Compared with the LNP preparation group, the LNP+T904, LNP+KG41 and LNP+10% sucrose preparation groups were able to significantly promote the transfection efficiency of Fluc-mRNA in living mice, lungs, livers and spleens after 6 hours of administration via intratracheal spray (it) and nasal route (in), especially the LNP+10% sucrose preparation group. The PoLixNano preparation containing 3 mg/mL T904 was physically mixed with T904, KG41 and sucrose, and then administered via intratracheal spray (it) and nasal route (in) for 6 hours. It can significantly increase the luciferase produced by Fluc-mRNA in living mice, lungs, livers and spleens. Most notably, 10% sucrose solution can stably enhance the transfection efficiency of LNP and PoLixNano formulations in mice (especially in the lungs) mediated by IVT-mRNA.
实施例12.非常规脂质组分制备的PoLixNano制剂经气管内喷雾(i.t.)Example 12. PoLixNano formulations prepared with unconventional lipid components were sprayed intratracheally (i.t.) 给药6h后的转染效果Transfection effect 6 hours after administration
LNP制剂的制备：采用上述“制剂实例#6”、“制剂实例#7”和“制剂实例#8”中所述方法，分别制备不含Poloxamer和Poloxamine的LNP对照制剂。采用上述“制剂实例#1”中所述方法，调整脂质组分和摩尔比例，N/P为8，制备如图8所示缺DSPC组分的LNP制剂。Preparation of LNP formulations: The LNP control formulations without Poloxamer and Poloxamine were prepared by the methods described in the above "Formulation Example #6", "Formulation Example #7" and "Formulation Example #8", respectively. The LNP formulation lacking the DSPC component was prepared by adjusting the lipid components and molar ratio to 8 N/P using the method described in the above "Formulation Example #1".
PoLixNano制剂的制备：分别采用上述“制剂实例#6”、“制剂实例#7”和“制剂实例#8”中所述方法，调整“制剂实例#2”中所述方法的脂质组分及其摩尔比例(缺DSPC组分组)，N/P为8，制备相应的PoLixNano制剂。Preparation of PoLixNano preparations: The methods described in the above-mentioned "Preparation Example #6", "Preparation Example #7" and "Preparation Example #8" were respectively used to adjust the lipid components and their molar ratios of the method described in "Preparation Example #2" (lacking DSPC component group), with N/P being 8, to prepare the corresponding PoLixNano preparations.
结果显示，相较于LNP对照制剂组，PoLixNano(GL67)和PoLixNano(CKK-E12)制剂经气管内喷雾(i.t.)给药6h后均能够显著增加肺部中Fluc-mRNA产生的荧光素酶水平(图8A和B)。不含DMG-PEG2000或者DSPC的PoLixNano制剂经气管内喷雾(i.t.)给药6h后均有效提升了Fluc-mRNA在肝脏、肺部和脾脏中的转染效率。不含DMG-PEG2000或者DSPC的PoLixNano制剂经鼻途径(i.n.)显著性增加了肺部中Fluc-mRNA产生的荧光素酶(图8C和D)。The results showed that compared with the LNP control group, PoLixNano (GL67) and PoLixNano (CKK-E12) preparations were able to significantly increase the level of luciferase produced by Fluc-mRNA in the lungs after 6 hours of intratracheal spray (i.t.) administration (Figures 8A and B). PoLixNano preparations without DMG-PEG2000 or DSPC effectively increased the transfection efficiency of Fluc-mRNA in the liver, lungs and spleen after 6 hours of intratracheal spray (i.t.) administration. PoLixNano preparations without DMG-PEG2000 or DSPC significantly increased the luciferase produced by Fluc-mRNA in the lungs via the nasal route (i.n.) (Figures 8C and D).
实施例13.雾化前后LNP和PoLixNano制剂的理化性质分析以及含不同Example 13. Analysis of the physicochemical properties of LNP and PoLixNano preparations before and after atomization and the 类型Poloxamers、Poloxamines和脂质的PoLixNano制剂6h后的转染效果Transfection effects of Poloxamers, Poloxamines and lipid-based PoLixNano formulations after 6 hours
LNP制剂的制备：采用上述“制剂实例#1”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为46:23:30:1，N/P为8。Preparation of LNP formulation: Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 46:23:30:1, and N/P was 8.
PoLixNano制剂的制备：采用上述“制剂实例#2”中所述方法，调整Dlin-MC3-DMA(替换为SM-102和ALC-0315)、DSPC(替换为DPPC和DOPE)、胆固醇和DMG-PEG2000的摩尔比例为46:23:30:1，N/P为8，制备图13所示含不同类型Poloxamers或者Poloxamines的PoLixNano制剂，图9B和9D均为10mg/mL 188，图9C中Poloxamers和Poloxamines的浓度为PoLixNano颗粒内的终浓度。 Preparation of PoLixNano preparations: Using the method described in the above-mentioned "Preparation Example #2", the molar ratio of Dlin-MC3-DMA (replaced with SM-102 and ALC-0315), DSPC (replaced with DPPC and DOPE), cholesterol and DMG-PEG2000 was adjusted to 46:23:30:1, and N/P was 8, and PoLixNano preparations containing different types of Poloxamers or Poloxamines as shown in Figure 13 were prepared. Figures 9B and 9D are both 10 mg/mL 188, and the concentrations of Poloxamers and Poloxamines in Figure 9C are the final concentrations in the PoLixNano particles.
使用如图9A所示的雾化装置对PoLixNano制剂或LNP对照制剂进行雾化操作。首先采用透射电子显微镜(TEM)对LNP纳米颗粒以及PoLixNano纳米颗粒雾化前后的形态进行了观察。雾化前的LNP制剂纳米颗粒大小均一，结构完整，而雾化后的LNP制剂纳米颗粒出现聚集，破裂和形变，表现为形态不规则(图9B)。当向上述组分中加入终浓度为10mg/mL的泊洛沙姆188后，所制得的PolixNano制剂颗粒雾化前后粒径大小以及结构形状未发生明显变化(表2及图9B)，说明泊洛沙姆188可以在雾化过程中对PolixNano纳米颗粒发挥保护作用，减轻纳米颗粒受到雾化装置机械作用力的影响，确保其形态结构完整，从而保证PolixNano纳米粒中所负载mRNA的转染效率。The PoLixNano preparation or LNP control preparation was atomized using the atomization device shown in FIG9A. First, the morphology of the LNP nanoparticles and the PoLixNano nanoparticles before and after atomization was observed using a transmission electron microscope (TEM). The LNP preparation nanoparticles before atomization were uniform in size and intact in structure, while the LNP preparation nanoparticles after atomization were aggregated, broken and deformed, showing irregular morphology (FIG9B). When poloxamer 188 was added to the above components at a final concentration of 10 mg/mL, the particle size and structural shape of the prepared PolixNano preparation particles did not change significantly before and after atomization (Table 2 and FIG9B), indicating that poloxamer 188 can protect the PolixNano nanoparticles during the atomization process, reduce the impact of the mechanical force of the atomization device on the nanoparticles, ensure the integrity of their morphological structure, and thus ensure the transfection efficiency of the mRNA loaded in the PolixNano nanoparticles.
表2.PoLixNano制剂雾化前后的理化性质特征
Table 2. Physical and chemical properties of PoLixNano preparations before and after atomization
雾化含不同Poloxamers或者Poloxamines的PoLixNano制剂在肺部转染Fluc-mRNA的能力不一样但均能增加肺部中Fluc-mRNA产生的荧光素酶水平(图9C)。雾化由SM-102和ALC-0315制备的PoLixNano制剂在肺部的转染效率显著优于Dlin-MC3-DMA制备的PoLixNano制剂，DPPC和DOPE制备的PoLixNano制剂具有与DSPC制备的PoLixNano制剂相当水平的转染潜力(图9D)。以上结果表明，使用不同结构的脂质或者不同类型的两亲性聚合物具有增加PoLixNano制剂在肺部的转染潜力。 The ability of aerosolized PoLixNano preparations containing different Poloxamers or Poloxamines to transfect Fluc-mRNA in the lungs is different, but all can increase the level of luciferase produced by Fluc-mRNA in the lungs (Figure 9C). The transfection efficiency of aerosolized PoLixNano preparations prepared by SM-102 and ALC-0315 in the lungs is significantly better than that of PoLixNano preparations prepared by Dlin-MC3-DMA, and PoLixNano preparations prepared by DPPC and DOPE have a comparable level of transfection potential as PoLixNano preparations prepared by DSPC (Figure 9D). The above results show that the use of lipids with different structures or different types of amphiphilic polymers has the potential to increase the transfection potential of PoLixNano preparations in the lungs.
实施例14.含不同脂质比例和剂量的PoLixNano制剂经雾化吸入给药6Example 14. Administration of PoLixNano formulations containing different lipid ratios and doses by aerosol inhalation h后的转染效果Transfection effect after h
LNP制剂的制备：采用上述“制剂实例#1”中所述方法，调整Dlin-MC3-DMA(或SM-102)、DSPC、胆固醇和DMG-PEG2000的摩尔比例为48.5:25:25:1.5，N/P为8。Preparation of LNP preparation: Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA (or SM-102), DSPC, cholesterol and DMG-PEG2000 was adjusted to 48.5:25:25:1.5, and N/P was 8.
PoLixNano制剂的制备：采用上述“制剂实例#2”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例，其中DMG-PEG2000摩尔比例在0.5到3之间，DSPC摩尔比例在14到40之间，Dlin-MC3-DMA摩尔比例在39到63之间，胆固醇摩尔比例在17.5到35之间。Preparation of PoLixNano preparation: Using the method described in the above-mentioned "Preparation Example #2", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted, wherein the molar ratio of DMG-PEG2000 was between 0.5 and 3, the molar ratio of DSPC was between 14 and 40, the molar ratio of Dlin-MC3-DMA was between 39 and 63, and the molar ratio of cholesterol was between 17.5 and 35.
结果显示，相较于LNP制剂组，雾化含不同摩尔比例脂质的PoLixNano制剂均能显著增加肺部中Fluc-mRNA产生的荧光素酶水平(图10A)，特别是SM-102、DSPC、胆固醇和DMG-PEG2000的摩尔比例为48.5:25:25:1.5，含10mg/mL T704的PoLixNano制剂(图10B)。以25μg/只的雾化吸入剂量给药的PoLixNano制剂组在活体小鼠和肺部中均检测到高表达的荧光素酶，50μg/只剂量组Fluc-mRNA产生的荧光素酶水平高于25μg/只剂量组低于150μg/只剂量组，这表明经雾化吸入给药PoLixNano制剂Fluc-mRNA表达量具有剂量依赖性(图10C)。The results showed that compared with the LNP preparation group, the nebulized PoLixNano preparations containing different molar ratios of lipids could significantly increase the level of luciferase produced by Fluc-mRNA in the lungs (Figure 10A), especially the PoLixNano preparation containing 10 mg/mL T704 with a molar ratio of 48.5:25:25:1.5 of SM-102, DSPC, cholesterol and DMG-PEG2000 (Figure 10B). Highly expressed luciferase was detected in both living mice and lungs in the PoLixNano preparation group administered with a nebulized inhalation dose of 25 μg/mouse. The level of luciferase produced by Fluc-mRNA in the 50 μg/mouse group was higher than that in the 25 μg/mouse group and lower than that in the 150 μg/mouse group, indicating that the expression of Fluc-mRNA in the PoLixNano preparation administered by nebulized inhalation was dose-dependent (Figure 10C).
实施例15.PoLixNano制剂经气管内喷雾(i.t.)给药6h后在体内的分Example 15. Analysis of PoLixNano formulation in vivo 6 hours after intratracheal spray (i.t.) administration 布和Fluc-mRNA的表达动力学Expression dynamics of Bu and Fluc-mRNA
PoLixNano制剂的制备：采用上述“制剂实例#2”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为49:20.1:30:0.9，N/P为8，添加DIR到有机相中荧光标记脂质(DIR摩尔数占总脂质摩尔数的2％)。Preparation of PoLixNano preparation: Using the method described in the above "Preparation Example #2", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 49:20.1:30:0.9, N/P was 8, and DIR was added to the organic phase to fluorescently label lipids (the molar number of DIR accounted for 2% of the total molar number of lipids).
结果显示，PoLixNano制剂经气管内喷雾(i.t.)给药6h后，仅在肺部检测到DIR荧光信号和Fluc-mRNA(RT-qPCR法检测)，在脑部、心脏、肝脏、脾脏、肾脏和肠中均未检测到DIR荧光信号和Fluc-mRNA，这表明PoLixNano制剂能特异性靶向到肺部(图11A和B)。相较于肺细胞摄取LNP，支气管上皮细胞(CD326+)、DC细胞(CD11c+)、内皮细胞(CD31+)、I型肺泡细胞(Podoplanin+)和巨噬细胞(F4/80+)可以更有效地摄取PoLixNano(图11C)。 The results showed that 6 hours after the PoLixNano preparation was administered by intratracheal spray (it), DIR fluorescence signals and Fluc-mRNA (RT-qPCR detection) were only detected in the lungs, and no DIR fluorescence signals and Fluc-mRNA were detected in the brain, heart, liver, spleen, kidney and intestine, indicating that the PoLixNano preparation can specifically target the lungs (Figure 11A and B). Compared with the uptake of LNP by lung cells, bronchial epithelial cells (CD326 + ), DC cells (CD11c + ), endothelial cells (CD31 + ), type I alveolar cells (Podoplanin + ) and macrophages (F4/80 + ) can more effectively uptake PoLixNano (Figure 11C).
进一步分析PoLixNano制剂经气管内喷雾(i.t.)给药不同时间Fluc-mRNA和FLuc-circRNA产生的荧光素酶的表达趋势。结果发现，给药6h、12h和24h后在肺部中检测到表达水平相当的荧光素酶，然而给药24h后在肝脏和脾脏中检测到的荧光素酶信号显著低于12h。包封Fluc-circRNA的PoLixNano制剂经气管内喷雾(i.t.)给药，给药6h FLuc-circRNA表达水平最高，随着时间的推移Fluc-circRNA表达水平逐渐下降，给药第九天时仍能检测到微量的荧光素酶信号。The expression trend of luciferase produced by Fluc-mRNA and FLuc-circRNA at different times of administration of PoLixNano preparations by intratracheal spray (i.t.) was further analyzed. The results showed that luciferase with comparable expression levels was detected in the lungs 6h, 12h and 24h after administration, but the luciferase signal detected in the liver and spleen 24h after administration was significantly lower than that at 12h. The PoLixNano preparation encapsulating Fluc-circRNA was administered by intratracheal spray (i.t.). The expression level of FLuc-circRNA was the highest at 6h after administration, and the expression level of Fluc-circRNA gradually decreased over time. A trace amount of luciferase signal could still be detected on the ninth day of administration.
以上结果表明，经气管内喷雾(i.t.)给药，PoLixNano制剂能够介导mRNA和circRNA在小鼠体内高效转染并在一定程度上持续表达。The above results indicate that PoLixNano preparation can mediate efficient transfection of mRNA and circRNA in mice and achieve sustained expression to a certain extent via intratracheal spray (i.t.) administration.
实施例16.PoLixNano制剂经呼吸道途径给药6h后在肺部以及非肺组织Example 16. PoLixNano formulation in lung and non-lung tissues 6 hours after administration via respiratory route 的基因转染Gene transfection
LNP制剂的制备：采用上述“制剂实例#1”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为50:14:35:1(气管内喷雾(i.t.))、49:20.1:30:0.9(鼻途径(i.n.))或者46:23:30:1(雾化吸入)，N/P均为8。Preparation of LNP preparations: Using the method described in the above-mentioned "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1 (intratracheal spray (i.t.)), 49:20.1:30:0.9 (nasal route (i.n.)) or 46:23:30:1 (atomized inhalation), and N/P was 8.
PoLixNano制剂的制备：采用上述“制剂实例#2”和“制剂实例#4”中所述方法，制备LNP制剂相应的含不同Poloxamer、Poloxamine或者Tween 80的PoLixNano制剂。Preparation of PoLixNano preparations: Use the methods described in the above "Preparation Example #2" and "Preparation Example #4" to prepare PoLixNano preparations containing different Poloxamer, Poloxamine or Tween 80 corresponding to the LNP preparations.
结果如图12A所示，经气管内喷雾(i.t.)给药后，LNP制剂组在肺部、肝脏和脾脏均检测到荧光素酶信号；含3mg/mL 188或者3mg/mL T304的PoLixNano制剂，以及先制备含3mg/mL 338的PoLixNano制剂再物理混合1mg/mL 338的PoLixNano制剂组特异性在肺部组织检测到荧光素酶信号。含3mg/mL T904、3mg/mL T704、10mg/mL 407和3mg/mL Tween 80的PoLixNano制剂组经气管内喷雾(i.t.)给药，以及含6mg/mL T704的PoLixNano制剂组经鼻途径(i.n.)给药后在肺部、肝脏和脾脏均检测到高强度的荧光素酶信号(图12C和E)。经雾化吸入给药后，LNP和PoLixNano制剂均介导Fluc-mRNA特异性在肺部的转染且PoLixNano制剂组荧光素酶信号显著高于LNP制剂组(图12E和G)。统计学分析显示，相较于LNP制剂组，PoLixNano制剂组经不同给药方式后能显著提升相应组织荧光素酶的表达水平(图12B、D、F和H)。 As shown in Figure 12A, after intratracheal spray (it) administration, luciferase signals were detected in the lungs, liver and spleen of the LNP preparation group; luciferase signals were specifically detected in the lung tissue of the PoLixNano preparation containing 3mg/mL 188 or 3mg/mL T304, and the PoLixNano preparation containing 3mg/mL 338 was first prepared and then physically mixed with 1mg/mL 338. High-intensity luciferase signals were detected in the lungs, liver and spleen of the PoLixNano preparation group containing 3mg/mL T904, 3mg/mL T704, 10mg/mL 407 and 3mg/mL Tween 80 after intratracheal spray (it) administration, and the PoLixNano preparation group containing 6mg/mL T704 after nasal administration (in) (Figures 12C and E). After administration by nebulized inhalation, both LNP and PoLixNano preparations mediated Fluc-mRNA transfection specifically in the lungs, and the luciferase signal in the PoLixNano preparation group was significantly higher than that in the LNP preparation group (Figure 12E and G). Statistical analysis showed that compared with the LNP preparation group, the PoLixNano preparation group could significantly increase the expression level of luciferase in the corresponding tissues after different administration methods (Figure 12B, D, F and H).
实施例17.PoLixNano制剂经呼吸道途径给药48h后肺组织安全评价Example 17. Safety evaluation of the lung tissue of PoLixNano formulation 48 hours after administration via the respiratory route
LNP制剂的制备：采用上述“制剂实例#1”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为49:20.1:30:0.9，N/P均为8。Preparation of LNP preparation: Using the method described in the above "Preparation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 49:20.1:30:0.9, and N/P was 8.
PoLixNano制剂的制备：采用上述“制剂实例#2”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为49:20.1:30:0.9(气管内喷雾(i.t.)和鼻途径(i.n.))或者46:23:30:1(雾化吸入)，N/P均为8，制备含T704、338或237的PoLixNano制剂。Preparation of PoLixNano preparations: Using the method described in the above-mentioned "Formulation Example #2", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 49:20.1:30:0.9 (intratracheal spray (i.t.) and nasal route (i.n.)) or 46:23:30:1 (nebulized inhalation), and N/P was 8 to prepare PoLixNano preparations containing T704, 338 or 237.
结果显示，PBS组(i.t.)以及含3mg/mL T904(i.t.)、3mg/mL T904(i.n.)和10mg/mL 188(雾化吸入)的PoLixNano制剂组的肺组织结构清晰、无炎性细胞浸润以及肺间质无红细胞渗出，LNP制剂组(i.t.)具有明显的肺损伤和强烈的炎症反应(图13A和B)。已有研究表明，LNP中的可电离脂质/阳离子脂质具有佐剂效应，能够显著刺激机体肺部产生IL-6等促炎因子，进而导致相关器官炎症损伤。因此传统的LNP制剂有过敏反应较为严重的问题，在高剂量给药方案中存在风险。随着237或338的浓度增加，PoLixNano制剂对肺的损伤和引起的炎症反应明显降低(图13B)。上述结果提示：随着PoLixNano制剂中两亲性聚合物组分，有助于屏蔽脂质组分的促炎作用，从而降低肺部的炎症反应及损伤。且随着两亲性聚合物组分的含量增加，降低炎症反应和肺部损伤的效果越明显。The results showed that the lung tissue structure of the PBS group (i.t.) and the PoLixNano preparation group containing 3mg/mL T904 (i.t.), 3mg/mL T904 (i.n.) and 10mg/mL 188 (nebulized inhalation) was clear, there was no inflammatory cell infiltration, and there was no red blood cell exudation in the lung interstitium. The LNP preparation group (i.t.) had obvious lung damage and strong inflammatory response (Figure 13A and B). Studies have shown that the ionizable lipids/cationic lipids in LNP have adjuvant effects, which can significantly stimulate the body's lungs to produce proinflammatory factors such as IL-6, thereby leading to inflammatory damage to related organs. Therefore, traditional LNP preparations have the problem of more serious allergic reactions and are at risk in high-dose dosing regimens. As the concentration of 237 or 338 increases, the damage to the lungs and the inflammatory response caused by the PoLixNano preparation are significantly reduced (Figure 13B). The above results suggest that as the amphiphilic polymer component in the PoLixNano preparation helps to shield the proinflammatory effect of the lipid component, thereby reducing the inflammatory response and damage to the lungs. And as the content of the amphiphilic polymer component increases, the effect of reducing inflammatory response and lung damage becomes more obvious.
此外，含3mg/mL T904(i.t.)的PoLixNano制剂组IL-4、IL-6、IL-17和TNF-α的表达水平与PBS组(i.t.)相当，均显著低于LNP制剂组(i.t.)(图13C)。含3mg/mL T904的PoLixNano制剂组i.t.给药后血清中肝功和肾功指标均正常，在血清中也未检测到T904组分和脂质组分诱导的特异性IgG抗体。In addition, the expression levels of IL-4, IL-6, IL-17 and TNF-α in the PoLixNano preparation group containing 3 mg/mL T904 (i.t.) were comparable to those in the PBS group (i.t.), and were significantly lower than those in the LNP preparation group (i.t.) (Figure 13C). The liver and kidney function indicators in the serum of the PoLixNano preparation group containing 3 mg/mL T904 after i.t. administration were normal, and no specific IgG antibodies induced by the T904 component and lipid component were detected in the serum.
实施例18.PoLixNano制剂经呼吸道途径给药6h后能够促进C57black 6Example 18. PoLixNano formulation can promote C57black 6 after 6 hours of administration via the respiratory route (C57BL/6)小鼠和Sprague-Dawley(SD)大鼠肺部的基因转染Gene transfection in the lungs of C57BL/6 mice and Sprague-Dawley (SD) rats
LNP制剂的制备：采用上述“制剂实例#1”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为49:20.1:30:0.9(气管内喷雾(i.t.)或者鼻途径(i.n.))或者46:23:30:1(雾化吸入)，N/P 均为8。Preparation of LNP formulations: Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 49:20.1:30:0.9 (intratracheal spray (it) or nasal route (in)) or 46:23:30:1 (nebulized inhalation), N/P Both are 8.
PoLixNano制剂的制备：采用上述“制剂实例#2”中所述方法，制备LNP制剂相应的含3mg/mL T904(气管内喷雾(i.t.)或者鼻途径(i.n.))和10mg/mL 188(雾化吸入)的PoLixNano制剂。Preparation of PoLixNano formulations: Using the method described in the above "Formulation Example #2", prepare the corresponding PoLixNano formulations containing 3 mg/mL T904 (intratracheal spray (i.t.) or nasal route (i.n.)) and 10 mg/mL 188 (nebulized inhalation) of the LNP formulation.
结果显示，PoLixNano制剂经气管内喷雾(i.t.)给药C57BL/6小鼠后，在肺部、肝脏和脾脏中均检测高强度的荧光素酶信号。LNP和PoLixNano制剂经鼻途径(i.n.)和雾化吸入给药C57BL/6小鼠后，均只在肺部检测到荧光素酶信号(图14A)。LNP和PoLixNano制剂经气管内喷雾(i.t.)、经鼻途径(i.n.)和雾化吸入给药SD大鼠后均仅在肺部检测到Fluc-mRNA的表达(图14B)。统计学分析表明，在C57BL/6小鼠和SD大鼠中PoLixNano制剂组荧光素酶信号显著高于LNP制剂组。The results showed that after the PoLixNano preparation was administered to C57BL/6 mice by intratracheal spray (i.t.), high-intensity luciferase signals were detected in the lungs, liver, and spleen. After LNP and PoLixNano preparations were administered to C57BL/6 mice by nasal route (i.n.) and nebulized inhalation, luciferase signals were only detected in the lungs (Figure 14A). After LNP and PoLixNano preparations were administered to SD rats by intratracheal spray (i.t.), nasal route (i.n.) and nebulized inhalation, the expression of Fluc-mRNA was only detected in the lungs (Figure 14B). Statistical analysis showed that the luciferase signal in the PoLixNano preparation group was significantly higher than that in the LNP preparation group in C57BL/6 mice and SD rats.
实施例19.PoLixNano制剂包封的RBD-mRNA疫苗经气管内喷雾途径Example 19. PoLixNano formulation encapsulated RBD-mRNA vaccine via intratracheal spray route (i.t.)接种后在小鼠体内诱导产生长期且高效的免疫应答(i.t.) Induces a long-lasting and highly effective immune response in mice after vaccination
采用编码新冠病毒刺突蛋白受体结合域RBD抗原的mRNA(RBD-mRNA)作为疫苗模型，经气管内喷雾途径(i.t.)考察RBD-mRNA/PoLixNano制剂在小鼠体内介导的免疫应答情况。The mRNA encoding the RBD antigen of the SARS-CoV-2 spike protein (RBD-mRNA) was used as a vaccine model, and the immune response mediated by the RBD-mRNA/PoLixNano preparation in mice was investigated via intratracheal spray (i.t.).
LNP制剂的制备：采用上述“制剂实例#1”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为50:14:35:1，N/P为8。制备LNP制剂包封RBD-mRNA的疫苗制剂(以下简称“LNP”)。Preparation of LNP formulation: Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1, and N/P was 8. LNP formulation encapsulating RBD-mRNA vaccine formulation (hereinafter referred to as "LNP") was prepared.
PoLixNano制剂的制备：采用上述“制剂实例#2”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为50:14:35:1，N/P为8，含终浓度为3.0mg/mL的T904制备PoLixNano制剂包封RBD-mRNA的疫苗制剂(以下简称“PoLixNano”)。Preparation of PoLixNano preparation: Using the method described in the above-mentioned "Preparation Example #2", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1, N/P was 8, and the PoLixNano preparation containing T904 at a final concentration of 3.0 mg/mL was prepared to encapsulate RBD-mRNA vaccine preparation (hereinafter referred to as "PoLixNano").
免疫接种剂量为2.5μg RBD-mRNA/只小鼠。以首次接种时间计为“第0天”，并于第21天采用相同接种途径、相同制剂以及相同剂量进行加强免疫。于采集首次免疫28天收集小鼠的血清(Serum)、肺泡灌洗液(BALF)，鼻腔灌洗液(NLF)，肺淋巴细胞(Pulmonary lymphocytes)及脾细胞(Splenocytes)分别并检测机体的体液免疫应答，黏膜免疫应答及适应性免疫应答的情况。The immunization dose was 2.5 μg RBD-mRNA/mouse. The first vaccination time was counted as "day 0", and the same vaccination route, the same preparation and the same dose were used for booster immunization on day 21. Serum, bronchoalveolar lavage fluid (BALF), nasal lavage fluid (NLF), pulmonary lymphocytes and splenocytes were collected from mice 28 days after the first immunization and the humoral immune response, mucosal immune response and adaptive immune response were detected.
采用同等条件下不同接种方式的LNP制剂的小鼠样品作为对照，同时选用同等条件下接种PBS溶液的小鼠样品(以下简称“PBS”组)作为阴性对照。 Mouse samples inoculated with LNP preparations in different ways under the same conditions were used as controls, and mouse samples inoculated with PBS solution under the same conditions (hereinafter referred to as "PBS" group) were used as negative controls.
免疫接种剂量为3μg RBD-mRNA/只小鼠，以首次接种时间计为“第0天”，并于第21天采用相同接种途径、相同制剂以及相同剂量进行加强免疫。首次免疫后14天、28天以及280天分采集小鼠的血清(Serum)，采集28天小鼠的肺泡灌洗液(Bronchoalveolar lavage fluid，BALF)和鼻腔灌洗液(Nasal Lavage Fluid，NLF)分别检测PoLixNano制剂诱导小鼠机体产生体液免疫应答，黏膜免疫应答的能力，同时采集小鼠的纵膈淋巴结细胞(Mediastinal lymphocytes)和脾细胞(Splenocytes)。The immunization dose was 3 μg RBD-mRNA/mouse, and the first vaccination time was counted as "Day 0", and booster immunization was performed on Day 21 using the same vaccination route, the same preparation and the same dose. Serum was collected from mice 14 days, 28 days and 280 days after the first immunization, and bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF) were collected from mice on Day 28 to detect the ability of PoLixNano preparation to induce humoral immune response and mucosal immune response in mice, and mediastinal lymph node cells and splenocytes were collected from mice.
如图15A所示，采用酶联免疫吸附测定法(Enzyme Linked Immunosorbent Assay,ELISA)测定在首次免疫后14天、28天以及280天采集的小鼠血清样品中的RBD抗原的IgG抗体滴度。接种PoLixNano制剂的小鼠血清，在第14天可检测到较高水平IgG抗体滴度，且与肌肉注射接种途径的LNP制剂(LNP-i.m.)诱导产生的IgG抗体滴度无明显差异，但经气管内接种途径的LNP制剂(LNP-i.t.)不能诱导小鼠产生IgG抗体；接种PoLixNano制剂的小鼠血清，在第28天可检测到更高水平IgG抗体滴度，且与肌肉注射接种途径的LNP制剂(LNP-i.m.)诱导产生的IgG抗体滴度无明显差异，但显著高于经气管内喷雾途径接种的LNP制剂(LNP-i.t.)诱导小鼠产生IgG抗体滴度。采用ELISA测定在首次免疫后28天采集的小鼠肺泡灌洗液(Bronchoalveolar lavage fluid，BALF)和鼻腔灌洗液(Nasal Lavage Fluid，NLF)样品中的RBD抗原的IgG抗体滴度，经气管内喷雾途径(i.t.)接种PoLixNano诱导小鼠在BALF和NLF中产生IgG抗体滴度均显著高于LNP-i.t.组。图15B结果显示，采用ELISA测定在首次免疫后28天和280天采集BALF和NLF样品中的RBD抗原的sIgA抗体滴度，在28天以及280天时，经气管内喷雾途径(i.t.)接种PoLixNano诱导小鼠在BALF和NLF中产生sIgA抗体滴度均显著高于LNP-i.t.组。如图15C，将首次免疫后28天采集的血清梯度稀释样本和BALF梯度稀释样本与假病毒混合后加入hACE-293T细胞中感染24h，通过检测相对荧光素酶单位(RLU)来计算假病毒中和滴度。PoLixNano制剂组小鼠血清及BALF中和抗体滴度显著高于采用不同接种方式的LNP制剂组，表明PoLixNano制剂组可以显著提高中和抗体的水平。采用酶联免疫斑点技术(ELISpot)检测免疫接种后28天的小鼠的纵膈淋巴结细胞(Mediastinal lymphocytes)和脾细胞(Splenocytes)IgG及sIgA抗体分泌细胞(ASC)的产生情况，在纵膈淋巴结细胞和脾细胞中PoLixNano诱导产生的IgG抗体分 泌细胞及IgA抗体分泌细胞的数量远超于接种LNP制剂的对照组(图15D)。采用流式细胞术(FCM)检测单次免疫14天后小鼠纵膈淋巴结细胞(Mediastinal lymphocytes)中滤泡辅助性T细胞(Tfh)及生发中心B细胞(GCB)的比率。经气管内喷雾途径(i.t.)接种PoLixNano制剂后，小鼠纵膈淋巴结中的GCB+细胞与Tfh+细胞(图15E)相较于LNP制剂明显提高。As shown in Figure 15A, the IgG antibody titer of RBD antigen in the mouse serum samples collected 14 days, 28 days and 280 days after the first immunization was determined by enzyme linked immunosorbent assay (ELISA). In the serum of mice inoculated with PoLixNano preparation, a higher level of IgG antibody titer can be detected on the 14th day, and there is no significant difference with the IgG antibody titer induced by the LNP preparation (LNP-im) via intramuscular injection, but the LNP preparation (LNP-it) via intratracheal injection cannot induce mice to produce IgG antibodies; in the serum of mice inoculated with PoLixNano preparation, a higher level of IgG antibody titer can be detected on the 28th day, and there is no significant difference with the IgG antibody titer induced by the LNP preparation (LNP-im) via intramuscular injection, but it is significantly higher than the IgG antibody titer induced by the LNP preparation (LNP-it) via intratracheal spray injection. ELISA was used to measure the IgG antibody titer of RBD antigen in the bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF) samples collected from mice 28 days after the first immunization. The IgG antibody titer produced in the BALF and NLF of mice induced by intratracheal spray (it) was significantly higher than that of the LNP-it group. The results of Figure 15B show that the sIgA antibody titer of RBD antigen in the BALF and NLF samples collected 28 days and 280 days after the first immunization was measured by ELISA. At 28 days and 280 days, the sIgA antibody titer produced in the BALF and NLF of mice induced by intratracheal spray (it) PoLixNano was significantly higher than that of the LNP-it group. As shown in Figure 15C, the serum gradient dilution samples and BALF gradient dilution samples collected 28 days after the first immunization were mixed with pseudovirus and added to hACE-293T cells for infection for 24 hours, and the pseudovirus neutralization titer was calculated by detecting the relative luciferase unit (RLU). The neutralizing antibody titers in the serum and BALF of mice in the PoLixNano preparation group were significantly higher than those in the LNP preparation group using different vaccination methods, indicating that the PoLixNano preparation group can significantly improve the level of neutralizing antibodies. The enzyme-linked immunospot technique (ELISpot) was used to detect the production of IgG and sIgA antibody-secreting cells (ASC) in the mediastinal lymph node cells (Mediastinal lymphocytes) and spleen cells (Splenocytes) of mice 28 days after immunization. The IgG antibody produced by PoLixNano in the mediastinal lymph node cells and spleen cells was significantly higher than that in the LNP preparation group using different vaccination methods. The number of secretory cells and IgA antibody secreting cells was much higher than that of the control group inoculated with LNP preparations (Figure 15D). Flow cytometry (FCM) was used to detect the ratio of follicular helper T cells (Tfh) and germinal center B cells (GCB) in the mediastinal lymph node cells of mice 14 days after a single immunization. After inoculation of PoLixNano preparations via intratracheal spray (it), the GCB+ cells and Tfh+ cells in the mediastinal lymph nodes of mice (Figure 15E) were significantly increased compared with LNP preparations.
实施例20.PoLixNano制剂包封的RBD-mRNA疫苗经气管内喷雾途径Example 20. PoLixNano formulation encapsulated RBD-mRNA vaccine via intratracheal spray route (i.t.)接种后在小鼠体内诱导产细胞免疫应答、固有训练免疫应答及长期适应(i.t.) inoculation induces cellular immune response, innate trained immune response and long-term adaptation in mice 性免疫应答Sexual Immune Response
采用编码新冠病毒刺突蛋白受体结合域RBD抗原的mRNA(RBD-mRNA)作为疫苗模型，经气管内喷雾途径(i.t.)考察RBD-mRNA/PoLixNano制剂在小鼠体内介导的免疫应答情况。LNP制剂、PoLixNano制剂制备方法以及免疫接种剂量和方式均与实施例19相同。The mRNA encoding the RBD antigen of the spike protein of the new coronavirus (RBD-mRNA) was used as a vaccine model to investigate the immune response mediated by the RBD-mRNA/PoLixNano preparation in mice via intratracheal spray (i.t.). The preparation methods of the LNP preparation and PoLixNano preparation, as well as the immunization dose and method, were the same as those in Example 19.
于首次免疫后28天、35天以及280天分采集小鼠BALF样品，同时采集小鼠的肺淋巴结细胞(Pulmonary lymphocytes)和脾细胞(Splenocytes)。BALF samples were collected from mice 28 days, 35 days, and 280 days after the first immunization. Pulmonary lymphocytes and spleen cells were also collected from mice.
采用酶联免疫斑点技术(ELISpot)分析首次免疫后28天后的小鼠的脾细胞(Splenocytes)及肺淋巴细胞(Pulmonary lymphocytes)产生细胞因子干扰素-γ(IFN-γ)、白介素-4(IL-4)、白介素-17(IL-17)的能力。经气管内喷雾途径(i.t.)接种PoLixNano制剂后第28天采集小鼠脾细胞和肺淋巴细胞，经RBD重叠肽库(覆盖新冠RBD蛋白的重叠肽库，单条多肽长度为14氨基酸，相邻多肽重叠9个氨基酸)刺激24h，分泌IFN-γ、IL-17和IL-4的淋巴细胞数量显著高于LNP制剂组(图16A)。PoLixNano制剂组诱导小鼠机体产生IFN-γ及IL-17的能力远高于IL-4。上述数据说明，相较于LNP制剂，接种PoLixNano制剂能够诱导小鼠产生更高效的抗原特异性细胞免疫应答(图16A)。采用流式细胞术分析首次免疫后28天后的小鼠的肺淋巴细胞(Pulmonary lymphocytes)中产生细胞因子干扰素-γ(IFN-γ)的能力。如图16B所示，经气管内喷雾途径(i.t.)接种PoLixNano制剂后，于第28天获取小鼠肺淋巴细胞经RBD重叠肽库(覆盖新冠RBD蛋白的重叠肽库，单条多肽长度为14个氨基酸，相邻多肽重叠9个氨基酸)刺激24h后，检测CD4+IFN-γ+细胞及CD8+IFN-γ+细胞含量。与不同接种途径的LNP制剂相比，接种PoLixNano制剂后CD4+IFN-γ+细胞、CD8+IFN-γ+细胞(图16B)明显提升。不同接种方式的LNP制剂组诱导产生CD4+IFN-γ+细胞及CD8+IFN-γ+细胞 与PBS组相似。以上结果表明，相较于LNP制剂，PoLixNano制剂能够显著性提高肺CD4+T细胞及CD8+T细胞IFN-γ分泌水平，PoLixNano制剂能够提高CD4+和CD8+T细胞免疫应答。图16C结果显示，通过流式细胞术分析首次免疫后28天以及280天的小鼠体内的组织驻留记忆T细胞(Trm)亚群，效应记忆T细胞(Tem)亚群、中心记忆T细胞(Tcm)亚群情况，经气管内喷雾途径(i.t.)接种PoLixNano制剂后，通过流式细胞术分析免疫小鼠(于第0天以及第21天接种)体内的Trm、Tem、Tcm亚群情况。结果显示，经气管内喷雾途径(i.t.)接种PoLixNano制剂后，第28天和第280天小鼠肺部CD8+/CD4+Trm细胞、CD8+/CD4+Tem细胞、CD8+/CD4+TCM细胞比例显著高于LNP对照组。以上结果表明，相较于LNP制剂，PoLixNano制剂能够更好促进小鼠肺部组织驻留记忆T细胞的增殖及分化。同时采用流式细胞术检测首次免疫后35天后小鼠肺BALF中肺间质巨噬细胞(IM)及肺泡巨噬细胞(AM)水平。如图16D所示，经气管内喷雾途径(i.t.)接种PoLixNano制剂后，小鼠肺泡灌洗液中的MHCII+IM细胞及MHCII+AM细胞的平均荧光强度(median fluorescence intensity,MFI)均高于同等条件下不同接种方式LNP制剂组。经气管内喷雾途径(i.t.)接种PoLixNano制剂能够诱导小鼠产生更多数量的MHC+IM细胞。以上数据表明，经气管内喷雾途径(i.t.)mRNA/PoLixNano制剂能够诱导小鼠产生较高水平的固有训练免疫应答能。The enzyme-linked immunospot technique (ELISpot) was used to analyze the ability of the splenocytes and pulmonary lymphocytes of mice to produce cytokines interferon-γ (IFN-γ), interleukin-4 (IL-4), and interleukin-17 (IL-17) 28 days after the first immunization. Mouse splenocytes and pulmonary lymphocytes were collected on the 28th day after the PoLixNano preparation was inoculated via intratracheal spray (it), and the number of lymphocytes secreting IFN-γ, IL-17, and IL-4 was significantly higher than that in the LNP preparation group after stimulation for 24 hours by the RBD overlapping peptide library (overlapping peptide library covering the new crown RBD protein, a single polypeptide length of 14 amino acids, and adjacent polypeptides overlapping 9 amino acids) (Figure 16A). The ability of the PoLixNano preparation group to induce the mouse body to produce IFN-γ and IL-17 is much higher than IL-4. The above data show that compared with the LNP preparation, vaccination with the PoLixNano preparation can induce mice to produce more efficient antigen-specific cellular immune responses (Figure 16A). Flow cytometry was used to analyze the ability of pulmonary lymphocytes (Pulmonary lymphocytes) of mice 28 days after the first immunization to produce the cytokine interferon-γ (IFN-γ). As shown in Figure 16B, after the PoLixNano preparation was inoculated via intratracheal spray (it), the mouse lung lymphocytes were obtained on the 28th day and stimulated with the RBD overlapping peptide library (overlapping peptide library covering the new crown RBD protein, a single polypeptide length of 14 amino acids, and adjacent polypeptides overlapping 9 amino acids) for 24 hours, and the content of CD4+IFN-γ+ cells and CD8+IFN-γ+ cells was detected. Compared with LNP preparations with different inoculation routes, CD4+IFN-γ+ cells and CD8+IFN-γ+ cells (Figure 16B) were significantly increased after inoculation with PoLixNano preparations. LNP preparation groups with different inoculation methods induced the production of CD4+IFN-γ+ cells and CD8+IFN-γ+ cells Similar to the PBS group. The above results show that compared with the LNP preparation, the PoLixNano preparation can significantly increase the IFN-γ secretion level of lung CD4+T cells and CD8+T cells, and the PoLixNano preparation can improve the immune response of CD4+ and CD8+T cells. The results of Figure 16C show that the tissue-resident memory T cell (Trm) subpopulation, effector memory T cell (Tem) subpopulation, and central memory T cell (Tcm) subpopulation in mice 28 days and 280 days after the first immunization were analyzed by flow cytometry. After the PoLixNano preparation was inoculated by intratracheal spray (it), the Trm, Tem, and Tcm subpopulations in the immunized mice (inoculated on days 0 and 21) were analyzed by flow cytometry. The results showed that after inoculation of PoLixNano preparations via intratracheal spray (it), the proportions of CD8+/CD4+Trm cells, CD8+/CD4+Tem cells, and CD8+/CD4+TCM cells in the lungs of mice on days 28 and 280 were significantly higher than those in the LNP control group. The above results indicate that PoLixNano preparations can better promote the proliferation and differentiation of resident memory T cells in the lung tissue of mice compared with LNP preparations. At the same time, flow cytometry was used to detect the levels of interstitial macrophages (IM) and alveolar macrophages (AM) in the BALF of the lungs of mice 35 days after the first immunization. As shown in Figure 16D, after inoculation of PoLixNano preparations via intratracheal spray (it), the median fluorescence intensity (MFI) of MHCII+IM cells and MHCII+AM cells in the alveolar lavage fluid of mice was higher than that of LNP preparation groups with different inoculation methods under the same conditions. Inoculation of PoLixNano preparations via intratracheal spray (it) can induce mice to produce more MHC+IM cells. The above data indicate that intratracheal spray (it) mRNA/PoLixNano formulation can induce a higher level of innate trained immune response in mice.
实施例21.PoLixNano制剂包封的RBD-mRNA疫苗经气管内喷雾途径Example 21. RBD-mRNA vaccine encapsulated by PoLixNano formulation via intratracheal spray route (i.t.)接种后攻毒保护效果评价Evaluation of protection effect of challenge after (i.t.) vaccination
采用编码新冠病毒刺突蛋白受体结合域RBD抗原的mRNA(RBD-mRNA)作为疫苗模型，经气管内喷雾途径(i.t.)考察RBD-mRNA/PoLixNano制剂在小鼠体内介导的免疫应答情况。LNP制剂、PoLixNano制剂制备方法以及免疫接种剂量和方式均与实施例19相同。The mRNA encoding the RBD antigen of the spike protein of the new coronavirus (RBD-mRNA) was used as a vaccine model to investigate the immune response mediated by the RBD-mRNA/PoLixNano preparation in mice via intratracheal spray (i.t.). The preparation methods of the LNP preparation and PoLixNano preparation, as well as the immunization dose and method, were the same as those in Example 19.
mRNA PoLixNano疫苗经气管内喷雾途径(i.t.)免疫balb/c小鼠后，可完全保护小鼠免受致死型SARS-CoV-2毒株的攻毒挑战。在第0，21天经经气管内喷雾途径(i.t.)免疫小鼠。初次免疫后28天，通过滴鼻途径对小鼠给予致死剂量的SARS-CoV-2原始株和奥密克戎变异株进行攻毒挑战，并在攻毒后指定时间点收集特定组织以检测病毒载量和肺部病理学。结果表明，mRNA/PoLixNano制剂疫苗表现出显著的保护功效，在预设实验终点时(攻毒后14天)存活率为100％，而新冠病毒原始株对照组的所有小鼠在攻毒后 3-6天内全部死亡(图17A)。如图17所示，且原始株和奥密克戎变异攻毒挑后，第3天时，mRNA/PoLixNano制剂组的肺部并未检测到新冠病毒核衣壳(Nucleocapsid,N)蛋白，而采用呼吸道接种途径的LNP制剂(LNP-i.t.)组的肺部切片中的N蛋白相对较高。综合以上结果，“金标准”LNP制剂难以介导mRNA疫苗在呼吸道黏膜部位产生高效的抗原特异性免疫应答，并且经呼吸道途径接种的mRNA/LNP疫苗在诱导抗原特异性体液免疫应答以及细胞免疫方面的能力极为有限。相比之下，经呼吸道途径给药的PoLixNano制剂能够诱导mRNA疫苗产生强效、均衡且持久的多重抗原特异性免疫应答，包括黏膜免疫应答、体液免疫应答以及细胞免疫应答。经呼吸道途径接种的mRNA/PoLixNano疫苗有望实现对接种者提供强效免疫保护并阻断病原体传播的理想效果。The mRNA PoLixNano vaccine can completely protect balb/c mice from challenge with lethal SARS-CoV-2 strains after immunization with the intratracheal spray route (IT). Mice were immunized by intratracheal spray route (IT) on days 0 and 21. 28 days after the initial immunization, the mice were challenged with a lethal dose of the original strain of SARS-CoV-2 and the Omicron variant strain by nasal drops, and specific tissues were collected at designated time points after the challenge to detect viral load and lung pathology. The results showed that the mRNA/PoLixNano formulation vaccine showed significant protective efficacy, with a survival rate of 100% at the preset experimental endpoint (14 days after the challenge), while all mice in the original strain control group of the new coronavirus died after the challenge. All died within 3-6 days (Figure 17A). As shown in Figure 17, after the original strain and the Omicron variant challenge, on the third day, the new coronavirus nucleocapsid (N) protein was not detected in the lungs of the mRNA/PoLixNano preparation group, while the N protein in the lung sections of the LNP preparation (LNP-it) group using the respiratory tract inoculation route was relatively high. Based on the above results, the "gold standard" LNP preparation is difficult to mediate the mRNA vaccine to produce an efficient antigen-specific immune response in the respiratory mucosa, and the mRNA/LNP vaccine inoculated by the respiratory route is extremely limited in its ability to induce antigen-specific humoral immune responses and cellular immunity. In contrast, the PoLixNano preparation administered by the respiratory route can induce the mRNA vaccine to produce a strong, balanced and lasting multiple antigen-specific immune response, including mucosal immune response, humoral immune response and cellular immune response. The mRNA/PoLixNano vaccine inoculated by the respiratory route is expected to achieve the ideal effect of providing strong immune protection to the vaccine recipients and blocking the spread of pathogens.
实施例22.PoLixNano制剂包封的RBD-mRNA疫苗经鼻途径(i.n.)接种Example 22. RBD-mRNA vaccine encapsulated in PoLixNano formulation was administered via nasal route (i.n.) 后免疫应答评价Post-immune response evaluation
LNP制剂的制备：采用上述“制剂实例#1”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为50:14:35:1，N/P为8。制备LNP制剂包封RBD-mRNA的疫苗制剂。Preparation of LNP formulation: Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1, and N/P was 8. The vaccine formulation of LNP formulation encapsulating RBD-mRNA was prepared.
PoLixNano制剂的制备：采用上述“制剂实例#2”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为50:14:35:1，N/P为8，含终浓度为3.0mg/mL的T904制备PoLixNano制剂包封RBD-mRNA的疫苗制剂。Preparation of PoLixNano preparation: Using the method described in the above "Preparation Example #2", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:14:35:1, N/P was 8, and the PoLixNano preparation containing T904 at a final concentration of 3.0 mg/mL was prepared to encapsulate RBD-mRNA vaccine preparation.
免疫接种剂量为2.0μg Fluc-mRNA/只小鼠在6h，12h，24h，48h以及72h后采用IVIS活体成像技术考察报告基因在小鼠体内的表达情况。在12h和24h时报告基因在小鼠体内表达较高，72h后不在表达(图18A)。The immunization dose was 2.0 μg Fluc-mRNA/mouse, and the expression of the reporter gene in mice was investigated by IVIS live imaging technology at 6h, 12h, 24h, 48h and 72h. The reporter gene was highly expressed in mice at 12h and 24h, but no longer expressed after 72h (Figure 18A).
免疫接种剂量为2.0μg RBD-mRNA/只小鼠，以首次接种时间计为“第0天”，于第21天采用相同接种途径、相同制剂以及相同剂量进行加强免疫。采用ELISA法测定接种PoLixNano制剂的小鼠血清在第14天、21天、28天，35天、42天及49天的RBD抗原特异性IgG，加强免疫后，抗体水平明显提高，经鼻注射接种途径的LNP制剂并未能诱导机体产生IgG(图18B)。28天时，接种PoLixNano制剂的小鼠的BALF、NLF和唾液中产生sIgA抗体水平显著高于LNP组。如图18C所示，进一步采用酶联免疫斑点技术(ELISpot)分析小鼠的肺淋巴血细胞(Splenocytes)产生细胞因子干扰素-γ(IFN-γ)， 白介素-4(IL-4)，白介素-17(IL-17)的能力。经鼻接种途径(i.n.)接种PoLixNano制剂后，第28天获取的小鼠肺淋巴细胞经RBD重叠肽库(覆盖新冠RBD蛋白的重叠肽库，单条多肽长度为14氨基酸，相邻多肽重叠9个氨基酸)刺激后分泌IFN-γ，IL-4,IL-17的能力均远超经鼻途径接种的LNP制剂的对照组。图18D显示，通过流式细胞术分析免疫小鼠(于第0天以及21天接种)小鼠肺组织驻留记忆T细胞(Trm)、效应记忆T细胞(Tem)亚群情况。经鼻接种途径(i.n.)接种PoLixNano制剂后，第28天小鼠肺部CD8+Trm与CD4+Trm细胞的比例远高于经鼻接种途径(i.n.)接种LNP制剂。图18D表明，与LNP-i.n.相比，CD8+Tem与CD4+Tem细胞的比例显著性提高。结果表明，PoLixNano制剂能够诱导小鼠肺组织驻留记忆T细胞及效应记忆T细胞的增值及分化。The immunization dose was 2.0 μg RBD-mRNA/mouse, and the first vaccination time was counted as "Day 0". On the 21st day, booster immunization was performed using the same vaccination route, the same preparation and the same dose. The ELISA method was used to determine the RBD antigen-specific IgG in the serum of mice vaccinated with PoLixNano preparations on days 14, 21, 28, 35, 42 and 49. After booster immunization, the antibody level was significantly increased, and the LNP preparations injected via nasal injection did not induce the body to produce IgG (Figure 18B). At 28 days, the levels of sIgA antibodies produced in the BALF, NLF and saliva of mice vaccinated with PoLixNano preparations were significantly higher than those of the LNP group. As shown in Figure 18C, the enzyme-linked immunospot technique (ELISpot) was further used to analyze the production of cytokine interferon-γ (IFN-γ) by the lung lymphocytes (Splenocytes) of mice. Interleukin-4 (IL-4), interleukin-17 (IL-17) ability. After the PoLixNano preparation was inoculated via the nasal route (in), the mouse lung lymphocytes obtained on the 28th day were stimulated by the RBD overlapping peptide library (covering the overlapping peptide library of the new crown RBD protein, a single polypeptide length of 14 amino acids, and adjacent polypeptides overlapped 9 amino acids) to secrete IFN-γ, IL-4, and IL-17. The ability is far superior to the control group of the LNP preparation inoculated via the nasal route. Figure 18D shows that the resident memory T cells (Trm) and effector memory T cells (Tem) subpopulations of the lung tissue of immunized mice (inoculated on days 0 and 21) were analyzed by flow cytometry. After the PoLixNano preparation was inoculated via the nasal route (in), the ratio of CD8+Trm to CD4+Trm cells in the mouse lungs on the 28th day was much higher than that of the LNP preparation inoculated via the nasal route (in). Figure 18D shows that the ratio of CD8+Tem to CD4+Tem cells was significantly increased compared with LNP-in. The results showed that PoLixNano preparation could induce the proliferation and differentiation of resident memory T cells and effector memory T cells in mouse lung tissue.
实施例23.PoLixNano制剂与LNP经肌注给药途径(i.m.)效果比较Example 23. Comparison of the effects of PoLixNano preparation and LNP via intramuscular injection (i.m.)
LNP制剂的制备：采用上述“制剂实例#1”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为50:10:38.5:1.5，N/P为6；采用上述“制剂实例#6”、“制剂实例#7”和“制剂实例#8”中所述方法，分别制备不含Poloxamer和Poloxamine的LNP对照制剂。采用上述“制剂实例#1”中所述方法，调整脂质组分和摩尔比例，N/P为8，制备如图8所示缺DMG-PEG2000组分的LNP制剂。制备LNP制剂包封mRNA的疫苗制剂。Preparation of LNP preparations: Using the method described in the above "Formulation Example #1", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:10:38.5:1.5, and N/P was 6; using the methods described in the above "Formulation Example #6", "Formulation Example #7" and "Formulation Example #8", LNP control preparations without Poloxamer and Poloxamine were prepared respectively. Using the method described in the above "Formulation Example #1", the lipid components and molar ratios were adjusted, and N/P was 8 to prepare LNP preparations lacking DMG-PEG2000 components as shown in Figure 8. Vaccine preparations encapsulating mRNA in LNP preparations were prepared.
PoLixNano制剂的制备：采用上述“制剂实例#2”中所述方法，调整Dlin-MC3-DMA、DSPC、胆固醇和DMG-PEG2000的摩尔比例为50:10:38.5:1.5，N/P为6，含终浓度为3.0mg/mL的T904制备PoLixNano制剂包封mRNA的疫苗制剂；LNP制剂的制备：采用上述“制剂实例#6”、“制剂实例#7”和“制剂实例#8”中所述方法，分别制备不含Poloxamer和Poloxamine的LNP对照制剂。采用上述“制剂实例#1”中所述方法，调整脂质组分和摩尔比例，N/P为8，制备如图8C所示不含有DMG-PEG2000组分的LNP制剂。Preparation of PoLixNano preparation: Using the method described in the above "Formulation Example #2", the molar ratio of Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 was adjusted to 50:10:38.5:1.5, N/P was 6, and the PoLixNano preparation containing T904 at a final concentration of 3.0 mg/mL was prepared for mRNA vaccine preparation; Preparation of LNP preparation: Using the methods described in the above "Formulation Example #6", "Formulation Example #7" and "Formulation Example #8", LNP control preparations without Poloxamer and Poloxamine were prepared respectively. Using the method described in the above "Formulation Example #1", the lipid components and molar ratio were adjusted, N/P was 8, and the LNP preparation without DMG-PEG2000 component as shown in Figure 8C was prepared.
PoLixNano制剂的制备：分别采用上述“制剂实例#6”、“制剂实例#7”和“制剂实例#8”中所述方法，调整“制剂实例#2”中所述方法的脂质组分及其摩尔比例(不含DSPC组分组)，N/P为8，制备相应的PoLixNano制剂。Preparation of PoLixNano preparations: The methods described in the above-mentioned "Formulation Example #6", "Formulation Example #7" and "Formulation Example #8" were respectively used to adjust the lipid components and their molar ratios (excluding the DSPC component group) of the method described in "Formulation Example #2", and N/P was 8 to prepare the corresponding PoLixNano preparations.
接种剂量为2.5μg Fluc-mRNA/只小鼠，给药后6h采用活体成像技术(IVIS)考察PoLixNano制剂在小鼠体内的转染情况。同等条件下相同接种方式的LNP制剂的小鼠样品作为对照。与LNP相比，经肌注给药途径(i.m.) 给药PoLixNano能够诱导活体小鼠和离体器官产生更高荧光素酶表达(图19A)。当脂质组分中缺少DMG-PEG2000时，与LNP相比，经肌注给药途径(i.m.)给药PoLixNano能够诱导活体小鼠和离体器官产生更高表达的荧光素酶(图19B)。采用ELISA法测定首次免疫后14天，21天，28天，及35天经肌注给药途径(i.m.)给药的PoLixNano制剂的小鼠血清样品中的RBD抗原特异性IgG抗体滴度。与肌肉注射接种途径的LNP制剂(LNP-i.m.)诱导产生的抗原特异性体液免疫应答能力显著提高。如图19D所示，进一步采用ELISpot法分析小鼠的脾细胞(Splenocytes)产生细胞因子、白介素-4(IL-4)和白介素-17(IL-17)的能力。经肌注接种途径(i.m.)给药的PoLixNano制剂，第28天获取小鼠脾细胞经RBD重叠肽库(覆盖新冠RBD蛋白的重叠肽库，单条多肽长度为14氨基酸，相邻多肽重叠9个氨基酸)刺激后分泌IL-4和IL-17的细胞数量显著高于肌注接种的LNP对照制剂组。 The inoculation dose was 2.5 μg Fluc-mRNA/mouse. In vivo imaging technology (IVIS) was used to investigate the transfection of PoLixNano preparations in mice 6 hours after administration. Mouse samples of LNP preparations inoculated in the same way under the same conditions were used as controls. Compared with LNP, the intramuscular administration route (im) Administration of PoLixNano can induce higher luciferase expression in living mice and isolated organs (Figure 19A). When DMG-PEG2000 is missing in the lipid component, compared with LNP, administration of PoLixNano via intramuscular administration (im) can induce higher expression of luciferase in living mice and isolated organs (Figure 19B). The ELISA method was used to determine the RBD antigen-specific IgG antibody titer in the serum samples of mice administered with PoLixNano preparations via intramuscular administration (im) 14 days, 21 days, 28 days, and 35 days after the first immunization. The antigen-specific humoral immune response ability induced by the LNP preparation (LNP-im) via intramuscular injection was significantly improved. As shown in Figure 19D, the ELISpot method was further used to analyze the ability of mouse spleen cells (Splenocytes) to produce cytokines, interleukin-4 (IL-4) and interleukin-17 (IL-17). The PoLixNano preparation was administered by intramuscular injection (im). On the 28th day, the number of cells secreting IL-4 and IL-17 in mouse spleen cells obtained after stimulation with the RBD overlapping peptide library (overlapping peptide library covering the new coronavirus RBD protein, with a single polypeptide length of 14 amino acids and adjacent polypeptides overlapping by 9 amino acids) was significantly higher than that in the LNP control preparation group administered by intramuscular injection.

Claims (45)

  1. 一种包含聚合物-脂质的组合物，所述组合物包含：A polymer-lipid composition comprising:
    (A)活性剂或治疗剂，优选所述活性剂或治疗剂包含核酸；(A) an active agent or therapeutic agent, preferably the active agent or therapeutic agent comprises a nucleic acid;
    (B)两亲性嵌段共聚物；(B) amphiphilic block copolymers;
    (C)阳离子脂质；和(C) a cationic lipid; and
    (D)非阳离子脂质，(D) non-cationic lipids,
    其中所述组合物配制为用于通过生物体黏膜部位(Mucosa)递送，例如呼吸道递送、口腔黏膜递送、胃肠道递送、眼部黏膜递送、耳部黏膜递送、尿道递送、或生殖道递送，优选所述组合物配制为用于通过呼吸道递送。Wherein the composition is formulated for delivery through a mucosal site of an organism, such as the respiratory tract, oral mucosa, gastrointestinal tract, ocular mucosa, ear mucosa, urethra, or reproductive tract, preferably the composition is formulated for delivery through the respiratory tract.
  2. 权利要求1所述的组合物，其中所述核酸包含选自以下的至少一种：信使RNA(mRNA)、自我扩增RNA(saRNA)、环状RNA(circRNA)、小干扰RNA(siRNA)、短发夹RNA(shRNA)和微小RNA(miRNA)、初级‐miRNA、反义寡核苷酸(ASO)、转运RNA(tRNA)、质粒DNA(pDNA)、单链DNA(ssDNA)、双链DNA(dsDNA)、脱氧核酶(DNAzyme)、核酶(RNAzyme)、核酸适配体(aptamer)、成簇的规律间隔的短回文重复序列(CRISPR)相关的核酸、单指导RNA(sgRNA)、CRISPR‐RNA(crRNA)、反式活化crRNA(tracrRNA)、指导RNA、单链RNA(ssRNA)和双链RNA(dsRNA)。The composition of claim 1, wherein the nucleic acid comprises at least one selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) and micro RNA (miRNA), primary-miRNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), plasmid DNA (pDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), deoxyribozyme (DNAzyme), ribozyme (RNAzyme), nucleic acid aptamer (aptamer), clustered regularly interspaced short palindromic repeats (CRISPR)-related nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA, single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA).
  3. 权利要求2所述的组合物，其中所述核酸是治疗性核酸，优选其中所述核酸包含mRNA。The composition of claim 2, wherein the nucleic acid is a therapeutic nucleic acid, preferably wherein the nucleic acid comprises mRNA.
  4. 权利要求1-3中任一项所述的组合物，其中所述两亲性嵌段共聚物占所述组合物的至少0.1％重量百分比，优选0.1％-98.0％重量百分比，例如1％-90.0％重量百分比、40.0％-80.0％重量百分比、50.0％-70.0％重量百分比或50.0％-60.0％重量百分比。The composition of any one of claims 1 to 3, wherein the amphiphilic block copolymer accounts for at least 0.1% by weight of the composition, preferably 0.1% to 98.0% by weight, such as 1% to 90.0% by weight, 40.0% to 80.0% by weight, 50.0% to 70.0% by weight or 50.0% to 60.0% by weight.
  5. 权利要求1-4中任一项所述的组合物，其中所述阳离子脂质包含选自以下的至少一种：永久阳离子脂质、可电离的阳离子脂质、胆固醇衍生的阳离子脂质和树枝状聚合物或树枝块(dendron)，优选地，所述阳离子脂质包含 可电离的阳离子脂质。The composition of any one of claims 1 to 4, wherein the cationic lipid comprises at least one selected from the group consisting of permanent cationic lipids, ionizable cationic lipids, cholesterol-derived cationic lipids and dendritic polymers or dendrons, preferably, the cationic lipid comprises Ionizable cationic lipids.
  6. 权利要求1-5中任一项所述的组合物，所述阳离子脂质占所述组合物中存在的总脂质的23.0mol％-83.0mol％，优选30.0mol％-80.0mol％、30.0mol％-70mol％或40.0mol％-60mol％。The composition of any one of claims 1-5, wherein the cationic lipid accounts for 23.0 mol%-83.0 mol%, preferably 30.0 mol%-80.0 mol%, 30.0 mol%-70 mol% or 40.0 mol%-60 mol% of the total lipids present in the composition.
  7. 权利要求1-6中任一项所述的组合物，其中所述非阳离子脂质包含选自以下的至少一种：阴离子脂质、两性离子脂质和中性脂质，优选地，所述非阳离子脂质包含中性脂质，优选所述中性脂质占所述组合物中存在的总脂质的19.0mol％-75.0mol％。The composition of any one of claims 1 to 6, wherein the non-cationic lipid comprises at least one selected from the group consisting of anionic lipids, zwitterionic lipids and neutral lipids, preferably, the non-cationic lipid comprises a neutral lipid, preferably the neutral lipid accounts for 19.0 mol%-75.0 mol% of the total lipids present in the composition.
  8. 权利要求7所述的组合物，其中所述中性脂质包含：The composition of claim 7, wherein the neutral lipid comprises:
    胆固醇或胆固醇衍生的中性脂质；Cholesterol or cholesterol-derived neutral lipids;
    磷脂；或phospholipids; or
    胆固醇或胆固醇衍生的中性脂质和磷脂的混合物。A mixture of cholesterol or cholesterol-derived neutral lipids and phospholipids.
  9. 根据权利要求8所述的组合物，其中所述胆固醇占所述组合物中的总脂质的14.0mol％-70.0mol％。The composition according to claim 8, wherein the cholesterol accounts for 14.0 mol%-70.0 mol% of the total lipids in the composition.
  10. 根据权利要求8所述的组合物，其中所述磷脂占所述组合物中的总脂质的5.0mol％-75.0mol％。The composition according to claim 8, wherein the phospholipids account for 5.0 mol%-75.0 mol% of the total lipids in the composition.
  11. 权利要求1-10中任一项所述的组合物，其还包含脂质缀合物，其中所述脂质缀合物包含选自以下的至少一种：PEG-脂质缀合物、ATTA-脂质缀合物、聚肌氨酸‐脂质缀合物、多肽/蛋白-脂质缀合物、阳离子-聚合物-脂质缀合物(CPL)以及它们的衍生物，优选地，所述脂质缀合物包含PEG-脂质缀合物，优选所述脂质缀合物占所述组合物中的总脂质的0.1mol％-10.0mol％。The composition of any one of claims 1 to 10, further comprising a lipid conjugate, wherein the lipid conjugate comprises at least one selected from the group consisting of: PEG-lipid conjugates, ATTA-lipid conjugates, polysarcosine-lipid conjugates, polypeptide/protein-lipid conjugates, cation-polymer-lipid conjugates (CPL) and derivatives thereof, preferably, the lipid conjugate comprises a PEG-lipid conjugate, preferably, the lipid conjugate accounts for 0.1 mol%-10.0 mol% of the total lipids in the composition.
  12. 权利要求1-3中任一项所述的组合物，其中所述组合物包含：The composition of any one of claims 1 to 3, wherein the composition comprises:
    (1)两亲性嵌段共聚物、阳离子脂质、磷脂、胆固醇和脂质缀合物例如PEG-脂质缀合物，其中所述阳离子脂质占所述组合物中存在的总脂质的30.0 mol％-80.0mol％，磷脂占总脂质的5.0mol％-50.0mol％，胆固醇占总脂质的14.0mol％-64.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比；(1) amphiphilic block copolymers, cationic lipids, phospholipids, cholesterol and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipid accounts for 30.0% of the total lipids present in the composition mol%-80.0mol%, phospholipids account for 5.0mol%-50.0mol% of the total lipids, cholesterol accounts for 14.0mol%-64.0mol% of the total lipids, lipid conjugates account for 0.1mol%-8.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% weight percent of the composition;
    (2)两亲性嵌段共聚物、阳离子脂质、磷脂、胆固醇和脂质缀合物例如PEG-脂质缀合物，其中所述阳离子脂质占所述组合物中存在的总脂质的23.0mol％-75.0mol％，磷脂占总脂质的10.0mol％-62.0mol％，胆固醇占总脂质的14.0mol％-46.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比；(2) an amphiphilic block copolymer, a cationic lipid, a phospholipid, cholesterol, and a lipid conjugate such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 23.0 mol%-75.0 mol% of the total lipid present in the composition, the phospholipid accounts for 10.0 mol%-62.0 mol% of the total lipid, the cholesterol accounts for 14.0 mol%-46.0 mol% of the total lipid, the lipid conjugate accounts for 0.1 mol%-8.0 mol% of the total lipid, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition;
    (3)两亲性嵌段共聚物、胆固醇衍生的阳离子脂质、磷脂和脂质缀合物例如PEG-脂质缀合物，其中所述胆固醇衍生的阳离子脂质占所述组合物中存在的总脂质的29.0mol％-80.0mol％，磷脂占总脂质的19.0mol％-70.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比；(3) amphiphilic block copolymers, cholesterol-derived cationic lipids, phospholipids, and lipid conjugates such as PEG-lipid conjugates, wherein the cholesterol-derived cationic lipids account for 29.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 19.0 mol%-70.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1%-95.0% by weight of the composition;
    (4)两亲性嵌段共聚物、阳离子脂质、胆固醇和脂质缀合物例如PEG-脂质缀合物，其中所述阳离子脂质占所述组合物中存在的总脂质的25.0mol％-80.0mol％，胆固醇占总脂质的15.0mol％-50.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比；(4) an amphiphilic block copolymer, a cationic lipid, cholesterol, and a lipid conjugate, such as a PEG-lipid conjugate, wherein the cationic lipid accounts for 25.0 mol%-80.0 mol% of the total lipids present in the composition, cholesterol accounts for 15.0 mol%-50.0 mol% of the total lipids, the lipid conjugate accounts for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition;
    (5)两亲性嵌段共聚物、阳离子脂质、磷脂和脂质缀合物例如PEG-脂质缀合物，其中所述阳离子脂质占所述组合物中存在的总脂质的30.0mol％-80.0mol％，磷脂占总脂质的10.0mol％-50.0mol％，脂质缀合物占总脂质的0.1mol％-8.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比；(5) amphiphilic block copolymers, cationic lipids, phospholipids and lipid conjugates such as PEG-lipid conjugates, wherein the cationic lipids account for 30.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 10.0 mol%-50.0 mol% of the total lipids, the lipid conjugates account for 0.1 mol%-8.0 mol% of the total lipids, and the amphiphilic block copolymers account for 0.1%-95.0% by weight of the composition;
    (6)两亲性嵌段共聚物、阳离子脂质、磷脂和胆固醇，其中所述阳离子脂质占所述组合物中存在的总脂质的30.0mol％-80.0mol％，磷脂占总脂质的5.0mol％-50.0mol％，胆固醇占总脂质的15.0mol％-50.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比；或(6) an amphiphilic block copolymer, a cationic lipid, a phospholipid, and cholesterol, wherein the cationic lipid accounts for 30.0 mol%-80.0 mol% of the total lipids present in the composition, the phospholipids account for 5.0 mol%-50.0 mol% of the total lipids, the cholesterol accounts for 15.0 mol%-50.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition; or
    (7)两亲性嵌段共聚物、胆固醇衍生的阳离子脂质和磷脂，其中所述胆固醇衍生的阳离子脂质占所述组合物中存在的总脂质的30.0mol％-70.0mol％,磷脂占总脂质的30.0mol％-70.0mol％，且所述两亲性嵌段共聚物占所述组合物的0.1％-95.0％重量百分比。 (7) an amphiphilic block copolymer, a cholesterol-derived cationic lipid and a phospholipid, wherein the cholesterol-derived cationic lipid accounts for 30.0 mol%-70.0 mol% of the total lipids present in the composition, the phospholipids account for 30.0 mol%-70.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 0.1%-95.0% by weight of the composition.
  13. 权利要求1-12中任一项所述的组合物，其中所述两亲性嵌段共聚物为四官能的两亲性嵌段共聚物，其中所述四官能的两亲性嵌段共聚物包含各自包含至少一个亲水性嵌段和至少一个疏水性嵌段的四个支链的嵌段共聚物，或所述两亲性嵌段共聚物为线性的两亲性嵌段共聚物，其中所述线性的两亲性嵌段共聚物包含至少一个亲水性嵌段和至少一个疏水性嵌段的嵌段共聚物。The composition of any one of claims 1 to 12, wherein the amphiphilic block copolymer is a tetrafunctional amphiphilic block copolymer, wherein the tetrafunctional amphiphilic block copolymer comprises a block copolymer of four branches each comprising at least one hydrophilic block and at least one hydrophobic block, or the amphiphilic block copolymer is a linear amphiphilic block copolymer, wherein the linear amphiphilic block copolymer comprises a block copolymer of at least one hydrophilic block and at least one hydrophobic block.
  14. 权利要求13所述的组合物，其中所述亲水性嵌段选自聚氧化烯、聚乙烯醇、聚乙烯吡咯烷酮、聚(2-甲基-2-噁唑啉)和糖，和/或所述疏水性嵌段选自聚氧化烯、脂肪链、亚烷基聚酯、具有苄基聚醚端部的聚乙二醇和胆固醇，优选地，所述亲水性嵌段包含聚氧化乙烯单元，且所述疏水性嵌段包含聚氧化丙烯单元。The composition of claim 13, wherein the hydrophilic block is selected from polyoxyalkylenes, polyvinyl alcohol, polyvinyl pyrrolidone, poly(2-methyl-2-oxazoline) and sugars, and/or the hydrophobic block is selected from polyoxyalkylenes, fatty chains, alkylene polyesters, polyethylene glycol with benzyl polyether ends and cholesterol, preferably, the hydrophilic block comprises polyethylene oxide units and the hydrophobic block comprises polypropylene oxide units.
  15. 权利要求1-14中任一项所述的组合物，其中所述两亲性嵌段共聚物包含选自以下的至少一种：泊洛沙胺(poloxamine或)、泊洛沙姆(poloxamer或)、聚氧乙二醇脱水醇烷基酯(聚山梨醇酯)、聚乙烯吡咯烷酮(PVP)、聚乙二醇醚(BRIJ)、聚氧乙烯脂肪酸酯、聚氧乙烯脂肪醇醚、脱水山梨糖醇酯以及它们的衍生物。The composition of any one of claims 1 to 14, wherein the amphiphilic block copolymer comprises at least one selected from the group consisting of poloxamine or ), poloxamer or ), polyoxyethylene glycol dehydrated alcohol alkyl esters (polysorbates), polyvinyl pyrrolidone (PVP), polyethylene glycol ethers (BRIJ), polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, dehydrated sorbitan esters and their derivatives.
  16. 权利要求1‐14中任一项所述的组合物，其中所述两亲性嵌段共聚物包含泊洛沙胺(poloxamine或)，例如所述泊洛沙胺选自泊洛沙胺304、泊洛沙胺701、泊洛沙胺704、泊洛沙胺901、泊洛沙胺904、泊洛沙胺908、泊洛沙胺1107、泊洛沙胺1301、泊洛沙胺1304、泊洛沙胺1307、泊洛沙胺90R4、泊洛沙胺150R1或其组合。The composition of any one of claims 1 to 14, wherein the amphiphilic block copolymer comprises poloxamine or ), for example, the poloxamine is selected from poloxamine 304, poloxamine 701, poloxamine 704, poloxamine 901, poloxamine 904, poloxamine 908, poloxamine 1107, poloxamine 1301, poloxamine 1304, poloxamine 1307, poloxamine 90R4, poloxamine 150R1 or a combination thereof.
  17. 权利要求1‐14中任一项所述的组合物，其中所述两亲性嵌段共聚物包含泊洛沙姆(poloxamer或)，例如所述泊洛沙姆选自泊洛沙姆84、泊洛沙姆101、泊洛沙姆105、泊洛沙姆108、泊洛沙姆122、泊洛沙姆123、泊洛沙姆124、泊洛沙姆181、泊洛沙姆182、泊洛沙姆183、泊洛沙姆184、泊洛沙姆185、泊洛沙姆188、泊洛沙姆212、泊洛沙姆215、泊洛沙姆217、泊洛沙姆231、泊洛沙姆234、泊洛沙姆235、泊洛沙姆237、泊洛沙姆238、泊洛沙姆 282、泊洛沙姆284、泊洛沙姆288、泊洛沙姆304、泊洛沙姆331、泊洛沙姆333、泊洛沙姆334、泊洛沙姆335、泊洛沙姆338、泊洛沙姆401、泊洛沙姆402、泊洛沙姆403、泊洛沙姆407或其组合。The composition of any one of claims 1 to 14, wherein the amphiphilic block copolymer comprises poloxamer or ), for example, the poloxamer is selected from poloxamer 84, poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 304, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer 407, or a combination thereof.
  18. 权利要求1-17中任一项所述的组合物，其中所述阳离子脂质包含选自以下的至少一种：DOTMA、DOSPA、DOTAP、ePC、DODAP、DODMA、DDAB、DSDMA、DODAC、DOAP、DMRIE、DOGS、DMOBA、HGT5000、HGT5001、HGT5002、HGT4001、HGT4002、HGT4003、HGT4005、DLin-MC3-DMA、DLin-KC2-DMA、Acuitas ALC-0315、Acuitas A9、Acuitas Lipid 2,2、Moderna Lipid H(SM-102)、Moderna Lipid 5、A2-Iso5-2DC18、BAME-O16B、9A1P9、C12-200、cKK-E12、OF-Deg-Lin、306Oi10、TT3、FTT5、Lipid319、5A2-SC8、Genevant CL1、DLinDMA、DLenDMA、ClinDMA、CpLinDMA、咪唑胆固醇酯(ICE)、RE-1、RE-2、RE-3、GL-67、5A2-SC8、Acuitas A9、Arcturus Lipid 2,2(8,8)4C CH3、OF-02、A18-Iso5-2DC18、BAME-O16B、A6、98N12-5、L319、L343、304O13、306O138、306O12B、306-O12B、LP01、G0-C14、7C1、Cephalin、Dlin-EG-DMA、DLinAP、DLin-MPZ、DLin-C-DAP、DLin-2-DMAP、Dlin-S-DMA、DLinDAP、DLin-MA、DLin-DAC、DLin-K-DMA、DLin-K-MPZ、DLin-K-DMA、DLin-K6-C4-DMA、DLin-K-C4-DMA、DLin-K-C3-DMA、CpLinDMA、DOcarbDAP、DLincarbDAP、C12-(2-3-2)、Genevant Lipid CL1、XTC、ALNY-100、NC98-5以及它们的衍生物。The composition of any one of claims 1-17, wherein the cationic lipid comprises at least one selected from the group consisting of DOTMA, DOSPA, DOTAP, ePC, DODAP, DODMA, DDAB, DSDMA, DODAC, DOAP, DMRIE, DOGS, DMOBA, HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4005, DLin-MC3-DMA, DLin-KC2-DMA, Acuitas ALC-0315, Acu itas A9、Acuitas Lipid 2,2、Moderna Lipid H(SM-102)、Moderna Lipid 5、A2-Iso5-2DC18、BAME-O16B、9A1P9、C12-200、cKK-E12、OF-Deg-Lin、306Oi10、TT3、FTT5、Lipid319、5A2-SC8、Genevant CL1、DLinDMA、DLenDMA、ClinDMA、CpLinDMA、Imidazole cholesteryl ester (ICE), R E-1, RE-2, RE-3, GL-67, 5A2-SC8, Acuitas A9, Arcturus Lipid 2,2(8,8)4C CH3, OF-02, A18-Iso5-2DC18, BAME-O16B, A6, 98N12-5, L319, L343, 304O13, 306O138, 306O12B, 306-O12B, LP01, G0-C14, 7C1, Cephalin, Dlin-EG-DMA, DLinAP, DLin-MPZ, DLin-C-DA P, DLin-2-DMAP, Dlin-S-DMA, DLinDAP, DLin-MA, DLin-DAC, DLin-K-DMA, DLin-K-MPZ, DLin-K-DMA, DLin-K6-C4-DMA, DLin-K-C4-DMA, DLin-K-C3-DMA, CpLinDMA, DOcarbDAP, DLincarbDAP, C12-(2-3-2), Genevan Lipid CL1, XTC, ALNY-100, NC98-5 and their derivatives.
  19. 权利要求5-18中任一项所述的组合物，其中所述胆固醇衍生的阳离子脂质包含选自以下的至少一种：DC‐Choi(N,N‐二甲基‐N‐乙基甲酰胺胆固醇)、1,4-双(3‐N‐油基氨基‐丙基)哌嗪、N4-精氨胆固醇羰酰氨(GL67)、与碱性氨基酸序列偶联的胆固醇衍生物、咪唑胆固醇酯(ICE)以及它们的衍生物。The composition of any one of claims 5-18, wherein the cholesterol-derived cationic lipid comprises at least one selected from the group consisting of DC-Choi (N,N-dimethyl-N-ethylformamide cholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, N4-arginine cholesterol carbonylamide (GL67), cholesterol derivatives coupled to basic amino acid sequences, imidazole cholesterol ester (ICE) and their derivatives.
  20. 权利要求8-19中任一项所述的组合物，其中所述磷脂包含选自以下的至少一种：卵磷脂、磷脂酰乙醇胺、溶血卵磷脂、溶血磷脂酰乙醇胺、磷脂酰丝氨酸、二油酰基磷脂酰丝氨酸(DOPS)、磷脂酰肌醇、鞘磷脂、蛋黄鞘磷脂(ESM)、脑磷脂、心磷脂、磷脂酸、脑苷脂、联十六烷基磷酸酯、二硬脂酰磷脂酰胆碱(DSPC)、二油酰磷脂酰乙醇胺(DOPE)、二油酰磷脂酰 胆碱(DOPC)、二棕榈酰磷脂酰胆碱(DPPC)、二油酰磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、棕榈酰油酰-磷脂酰胆碱(POPC)、棕榈酰油酰-磷脂酰乙醇胺(POPE)、棕榈酰油酰-磷脂酰甘油(POPG)、二油酰磷脂酰乙醇胺4-(N-马来酰亚胺基甲基)-环己烷-1-甲酸酯(DOPE-mal)、二棕榈酰-磷脂酰乙醇胺(DPPE)、二肉豆蔻酰-磷脂酰乙醇胺(DMPE)、二硬脂酰-磷脂酰乙醇胺(DSPE)、单甲基-磷脂酰乙醇胺、二甲基-磷脂酰乙醇胺、二反油酰-磷脂酰乙醇胺(DEPE)、硬脂酰油酰-磷脂酰乙醇胺(SOPE)、溶血磷脂酰胆碱、蛋黄磷脂酰胆碱(EPC)、二亚油酰磷脂酰胆碱、1,2-二棕榈酰-sn-甘油-3-O-4'-(N，N，N-三甲基)-高丝氨酸(DGTS)、单半乳糖二酰甘油(MGDG)、二乙酰二酰基甘油(DGDG)、磺胺喹啉二酰基甘油(SQDG)、1-棕榈酰-2-顺式-9,10-亚甲基己基-癸酰基-sn-甘油-3-磷酸胆碱(Cyclo PC)、16‐O‐单甲基PE、16‐O‐二甲基PE、18‐1‐反式PE以及它们的衍生物。The composition of any one of claims 8 to 19, wherein the phospholipid comprises at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, dioleoylphosphatidylserine (DOPS), phosphatidylinositol, sphingomyelin, egg yolk sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), ... dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DOPS), dioleo Choline (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidyl Ethanolamine, dimethyl-phosphatidylethanolamine, di-trans-oleoyl-phosphatidylethanolamine (DEPE), stearoyl-oleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, egg yolk phosphatidylcholine (EPC), dilinoleoylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS), monogalactosyldiacylglycerol (MGDG), diacetyldiacylglycerol (DGDG), sulfaquinolinediacylglycerol (SQDG), 1-palmitoyl-2-cis-9,10-methylenehexyl-decanoyl-sn-glycero-3-phosphocholine (Cyclo PC), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE and their derivatives.
  21. 权利要求8-20中任一项所述的组合物，其中所述胆固醇衍生的中性脂质包含选自以下的至少一种：胆甾烷醇、胆甾烷酮、胆甾烯酮、粪甾醇、胆固醇基-2'-羟基乙醚、胆固醇基-4’-羟基丁醚、BHEM-胆固醇、β-谷甾醇、20α-羟基胆固醇、与多肽/蛋白共价连接的胆固醇以及它们的衍生物，优选所述胆固醇衍生的中性脂质包含β-谷甾醇。The composition of any one of claims 8-20, wherein the cholesterol-derived neutral lipid comprises at least one selected from the group consisting of cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, BHEM-cholesterol, β-sitosterol, 20α-hydroxycholesterol, cholesterol covalently linked to a polypeptide/protein, and derivatives thereof, preferably the cholesterol-derived neutral lipid comprises β-sitosterol.
  22. 权利要求11-21中任一项所述的组合物，其中所述PEG-脂质缀合物包含选自以下的至少一种：DMG-PEG2K、DMPE-PEG2K、DSPE-PEG2K、DSPE-PEG2K-Mannose、DMG-PEG5K、DMPE-PEG5K、DSPE-PEG5K-Mannose和DSPE-PEG5K。The composition of any one of claims 11-21, wherein the PEG-lipid conjugate comprises at least one selected from the group consisting of DMG-PEG2K, DMPE-PEG2K, DSPE-PEG2K, DSPE-PEG2K-Mannose, DMG-PEG5K, DMPE-PEG5K, DSPE-PEG5K-Mannose and DSPE-PEG5K.
  23. 权利要求1-3中任一项所述的组合物，其中所述组合物包含两亲性嵌段共聚物和以下组分：The composition of any one of claims 1 to 3, wherein the composition comprises an amphiphilic block copolymer and the following components:
    (1)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的40.0mol％-70.0mol％，DSPC、 DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的8.0mol％-39.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的15.0％-90.0％重量百分比；(1) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 account for 40.0 mol% to 70.0 mol% of the total lipids present in the composition, and DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE account for 8.0mol%-39.0mol% of the total lipids, cholesterol or β-sitosterol account for 20.0mol%-40.0mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1mol%-5.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 15.0%-90.0% weight percentage of the composition;
    (2)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的30mol％-60mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的10.0mol％-49.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的20.0％-90.0％重量百分比；(2) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 1 accounts for 30 mol%-60 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 10.0 mol%-49.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 20.0%-90.0% weight percentage of the composition;
    (3)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的24.0mol％-40.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的30.0mol％-64.0mol％，胆固醇或β-谷甾醇占总脂质的15.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的20.0％-90.0％重量百分比；(3) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 accounts for 24.0mol%-40.0mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE account for 30.0mol%-64.0mol% of the total lipids, cholesterol or β-sitosterol account for 15.0mol%-40.0mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1mol%-5.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 20.0%-90.0% weight percent of the composition;
    (4)DOTAP、DODAP、DOTMA或DOSPA；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DOTAP、DODAP、DOTMA或DOSPA占所述组合物中存在的总脂质的23.0mol％-60mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的14.0mol％-60.0mol％，胆固醇或β-谷甾醇占总脂质的15.0mol％-50.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的30.0％-90.0％重量百分比；(4) DOTAP, DODAP, DOTMA or DOSPA; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DOTAP, DODAP, DOTMA or DOSPA accounts for 23.0 mol%-60 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 14.0 mol%-60.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 15.0 mol%-50.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% by weight of the composition;
    (5)GL67、ICE、或HGT4002；DSPC、DPPC、DOPS、SOPE、DOPG、 DSPE、ESM或DOPE；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述GL67、ICE、或HGT4002占所述组合物中存在的总脂质的40.0mol％-80.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的10.0mol％-50.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的30.0％-90.0％重量百分比；(5) GL67, ICE, or HGT4002; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the GL67, ICE, or HGT4002 accounts for 40.0 mol%-80.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 10.0 mol%-50.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% weight percent of the composition;
    (6)cKK-E12、DLin-MC3-DMA、ALC-0315、SM-102、C12-200、DOTAP、DODAP、DOTMA、DOSPA、HGT5000或HGT5001；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述cKK-E12、DLin-MC3-DMA、ALC-0315、SM-102、C12-200、DOTAP、DODAP、DOTMA、DOSPA、HGT5000或HGT5001占所述组合物中存在的总脂质的45.0mol％-75.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-45.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的30.0％-90.0％重量百分比；(6) cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, C12-200, DOTAP, DODAP, DOTMA, DOSPA, HGT5000 or HGT5001; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102, C12-200, DOTAP , DODAP, DOTMA, DOSPA, HGT5000 or HGT5001 account for 45.0mol%-75.0mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 20.0mol%-45.0mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1mol%-5.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% weight percentage of the composition;
    (7)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；胆固醇或β-谷甾醇；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的30.0mol％-65.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的40.0％-90.0％重量百分比；(7) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; cholesterol or β-sitosterol; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001 account for 30.0 mol%-65.0 mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 20.0 mol%-40.0 mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose account for 0.1 mol%-5.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% by weight of the composition;
    (8)DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；和DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315、SM-102、C12-200、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的35.0mol％-70.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的10.0mol％-40.0mol％，DMG-PEG2K、DMG-PEG5K或DSPE-PEG2K-Mannose占总脂质的0.1mol％-5.0mol％，且所述两亲性嵌段共聚物占所述组合物的40.0％-90.0％重量百分比；(8) DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315, SM-102, C12-200, cKK-E12, HGT50 00 or HGT5001 accounts for 35.0mol%-70.0mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 10.0mol%-40.0mol% of the total lipids, DMG-PEG2K, DMG-PEG5K or DSPE-PEG2K-Mannose accounts for 0.1mol%-5.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% weight percentage of the composition;
    (9)GL67、ICE、或HGT4002；和DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE，其中所述GL67、ICE、或HGT4002占所述组合物中存 在的总脂质的40.0mol％-80.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的10.0mol％-50.0mol％，且所述两亲性嵌段共聚物占所述组合物的40.0％-90.0％重量百分比；或(9) GL67, ICE, or HGT4002; and DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE, wherein the GL67, ICE, or HGT4002 accounts for 40.0 mol%-80.0 mol% of the total lipids, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE account for 10.0 mol%-50.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.0%-90.0% weight percent of the composition; or
    (10)DLin-MC3-DMA、ALC-0315、SM-102、cKK-E12、HGT5000或HGT5001；DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE；和胆固醇或β-谷甾醇，其中所述DLin-MC3-DMA、ALC-0315、SM-102、cKK-E12、HGT5000或HGT5001占所述组合物中存在的总脂质的30.0mol％-60.0mol％，DSPC、DPPC、DOPS、SOPE、DOPG、DSPE、ESM或DOPE占总脂质的20.0mol％-45.0mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％-45.0mol％，且所述两亲性嵌段共聚物占所述组合物的30.0％-90.0％重量百分比。(10) DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, HGT5000 or HGT5001; DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE; and cholesterol or β-sitosterol, wherein the DLin-MC3-DMA, ALC-0315, SM-102, cKK-E12, HGT5000 or HGT5001 accounts for 30.0 mol%-60.0 mol% of the total lipids present in the composition, DSPC, DPPC, DOPS, SOPE, DOPG, DSPE, ESM or DOPE accounts for 20.0 mol%-45.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 20.0 mol%-45.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 30.0%-90.0% weight percent of the composition.
  24. 权利要求1-3中任一项所述的组合物，其中所述组合物包含以下组分：The composition of any one of claims 1 to 3, wherein the composition comprises the following components:
    (1)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的10.0mol％，胆固醇或β-谷甾醇占总脂质的38.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.5mol％，且所述两亲性嵌段共聚物占所述组合物的89.9％重量百分比；(1) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.0 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 89.9% by weight of the composition;
    (2)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的10.6mol％，胆固醇或β-谷甾醇占总脂质的38.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的72.9％重量百分比；(2) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 10.6 mol% of the total lipids, cholesterol or β-sitosterol account for 38.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 72.9% by weight of the composition;
    (3)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的12.5mol％，胆固醇或β-谷甾醇占总脂质的36.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.5mol％，且所述两亲性嵌段共聚物占所述组合物的43.0％重量百分比；(3) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 12.5 mol% of the total lipids, cholesterol or β-sitosterol account for 36.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.0% by weight of the composition;
    (4)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、 DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.5mol％，胆固醇或β-谷甾醇占总脂质的34.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.5mol％，且所述两亲性嵌段共聚物占所述组合物的81.8％重量百分比；(4) Amphiphilic block copolymers; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or β-sitosterol account for 34.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 81.8% by weight of the composition;
    (5)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.0mol％，胆固醇或β-谷甾醇占总脂质的35.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的69.2％重量百分比；(5) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.0 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 69.2% by weight of the composition;
    (6)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.1mol％，胆固醇或β-谷甾醇占总脂质的35.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的43.3％重量百分比；(6) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.3% by weight of the composition;
    (7)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.3mol％，胆固醇或β-谷甾醇占总脂质的35.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.7mol％，且所述两亲性嵌段共聚物占所述组合物的48.4％重量百分比；(7) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.3 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.4% by weight of the composition;
    (8)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的50.0mol％，DSPC、DPPC或DOPE占总脂质的14.5mol％，胆固醇或β-谷甾醇占总脂质的35.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.5mol％，且所述两亲性嵌段共聚物占所述组合物的43.6％重量百分比；(8) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 50.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.5 mol% of the total lipids, cholesterol or β-sitosterol account for 35.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 43.6% by weight of the composition;
    (9)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所 述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的48.5mol％，DSPC、DPPC或DOPE占总脂质的17.0mol％，胆固醇或β-谷甾醇占总脂质的32.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的42.3％重量百分比；(9) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein The DLin-MC3-DMA, ALC-0315 or SM-102 account for 48.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.3% by weight of the composition;
    (10)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.5mol％，DSPC、DPPC或DOPE占总脂质的20.0mol％，胆固醇或β-谷甾醇占总脂质的32.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的52.9％重量百分比；(10) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 52.9% by weight of the composition;
    (11)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的49.0mol％，DSPC、DPPC或DOPE占总脂质的20.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的41.9％重量百分比；(11) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition;
    (12)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的49.0mol％，DSPC、DPPC或DOPE占总脂质的20.1mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的40.3％重量百分比；(12) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 49.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.1 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 40.3% by weight of the composition;
    (13)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的51.6mol％，DSPC、DPPC或DOPE占总脂质的20.0mol％，胆固醇或β-谷甾醇占总脂质的27.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的42.9％重量百分比；(13) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 51.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 20.0 mol% of the total lipids, cholesterol or β-sitosterol account for 27.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 42.9% by weight of the composition;
    (14)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0 mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的50.2％重量百分比；(14) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 accounts for 46.0% of the total lipids present in the composition. mol%, DSPC, DPPC or DOPE account for 23.0mol% of the total lipids, cholesterol or β-sitosterol account for 30.0mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.2% by weight of the composition;
    (15)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的66.1％重量百分比；(15) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 66.1% by weight of the composition;
    (16)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的74.5％重量百分比；(16) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 74.5% by weight of the composition;
    (17)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的79.6％重量百分比；(17) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 79.6% by weight of the composition;
    (18)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的83.0％重量百分比；(18) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 83.0% by weight of the composition;
    (19)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DSPE-PEG2K-Mannose，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的46.0mol％，DSPC、DPPC或DOPE占总脂质的23.0mol％，胆固醇或β-谷 甾醇占总脂质的30.0mol％，DMG-PEG2K或DSPE-PEG2K-Mannose占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的88.2％重量百分比；(19) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DSPE-PEG2K-Mannose, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 46.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.0 mol% of the total lipids, and cholesterol or β-sitosterol account for 23.0 mol% of the total lipids. Sterol accounts for 30.0 mol% of the total lipids, DMG-PEG2K or DSPE-PEG2K-Mannose accounts for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 88.2% by weight of the composition;
    (20)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的43.0mol％，DSPC、DPPC或DOPE占总脂质的23.3mol％，胆固醇或β-谷甾醇占总脂质的33.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.7mol％，且所述两亲性嵌段共聚物占所述组合物的39.1％重量百分比；(20) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 43.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 23.3 mol% of the total lipids, cholesterol or β-sitosterol account for 33.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 39.1% by weight of the composition;
    (21)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的44.0mol％，DSPC、DPPC或DOPE占总脂质的25.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的63.6％重量百分比；(21) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 44.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 63.6% by weight of the composition;
    (22)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的39.5mol％，DSPC、DPPC或DOPE占总脂质的25.3mol％，胆固醇或β-谷甾醇占总脂质的34.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.7mol％，且所述两亲性嵌段共聚物占所述组合物的37.2％重量百分比；(22) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.5 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 25.3 mol% of the total lipids, cholesterol or β-sitosterol account for 34.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.7 mol% of the total lipids, and the amphiphilic block copolymer accounts for 37.2% by weight of the composition;
    (23)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的41.0mol％，DSPC、DPPC或DOPE占总脂质的28.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的66.0％重量百分比；(23) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 41.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 66.0% by weight of the composition;
    (24)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的39.0mol％，DSPC、DPPC或DOPE占总脂质的28.0mol％，胆固醇或β-谷甾醇占总脂质的32.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述 两亲性嵌段共聚物占所述组合物的36.3％重量百分比；(24) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 28.0 mol% of the total lipids, cholesterol or β-sitosterol account for 32.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the The amphiphilic block copolymer accounts for 36.3% by weight of the composition;
    (25)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的39.0mol％，DSPC、DPPC或DOPE占总脂质的30.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的62.9％重量百分比；(25) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 39.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.0 mol% of the total lipids, cholesterol or β-sitosterol account for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 62.9% by weight of the composition;
    (26)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的40.6mol％，DSPC、DPPC或DOPE占总脂质的30.1mol％，胆固醇或β-谷甾醇占总脂质的28.4mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的36.9％重量百分比；(26) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.6 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 30.1 mol% of the total lipids, cholesterol or β-sitosterol account for 28.4 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 36.9% by weight of the composition;
    (27)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的34.7mol％，DSPC、DPPC或DOPE占总脂质的40.1mol％，胆固醇或β-谷甾醇占总脂质的24.3mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的32.5％重量百分比；(27) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 34.7 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 40.1 mol% of the total lipids, cholesterol or β-sitosterol account for 24.3 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 32.5% by weight of the composition;
    (28)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的29.0mol％，DSPC、DPPC或DOPE占总脂质的50.1mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的28.0％重量百分比；(28) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 29.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 50.1 mol% of the total lipids, cholesterol or β-sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 28.0% by weight of the composition;
    (29)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的35.0mol％，DSPC、DPPC或DOPE占总脂质的17.6mol％，胆固醇或β-谷甾醇占总脂质的46.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的35.9％重量百分比； (29) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 35.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 17.6 mol% of the total lipids, cholesterol or β-sitosterol account for 46.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 35.9% by weight of the composition;
    (30)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的55.0mol％，DSPC、DPPC或DOPE占总脂质的16.9mol％，胆固醇或β-谷甾醇占总脂质的27.2mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的44.6％重量百分比；(30) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 55.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 16.9 mol% of the total lipids, cholesterol or β-sitosterol account for 27.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 44.6% by weight of the composition;
    (31)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的60.0mol％，DSPC、DPPC或DOPE占总脂质的14.9mol％，胆固醇或β-谷甾醇占总脂质的24.2mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的46.5％重量百分比；(31) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 60.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.9 mol% of the total lipids, cholesterol or β-sitosterol account for 24.2 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 46.5% by weight of the composition;
    (32)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的65.0mol％，DSPC、DPPC或DOPE占总脂质的14.1mol％，胆固醇或β-谷甾醇占总脂质的20.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的0.9mol％，且所述两亲性嵌段共聚物占所述组合物的21.0％重量百分比；(32) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 65.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 14.1 mol% of the total lipids, cholesterol or β-sitosterol account for 20.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 0.9 mol% of the total lipids, and the amphiphilic block copolymer accounts for 21.0% by weight of the composition;
    (33)两亲性嵌段共聚物；GL67、ICE或HGT4002；DSPC、DPPC或DOPE；和DMG-PEG2K或DMG-PEG5K，其中所述GL67、ICE或HGT4002占所述组合物中存在的总脂质的70.0mol％，DSPC、DPPC或DOPE占总脂质的28.5mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.5mol％，且所述两亲性嵌段共聚物占所述组合物的48.1％重量百分比；(33) an amphiphilic block copolymer; GL67, ICE or HGT4002; DSPC, DPPC or DOPE; and DMG-PEG2K or DMG-PEG5K, wherein the GL67, ICE or HGT4002 accounts for 70.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE accounts for 28.5 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.5 mol% of the total lipids, and the amphiphilic block copolymer accounts for 48.1% by weight of the composition;
    (34)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的61.3mol％，胆固醇或β-谷甾醇占总脂质的37.6mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.1mol％，且所述两亲性嵌段共聚物占所述组合物的41.9％重量百分比；(34) an amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 61.3 mol% of the total lipids present in the composition, cholesterol or β-sitosterol account for 37.6 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K account for 1.1 mol% of the total lipids, and the amphiphilic block copolymer accounts for 41.9% by weight of the composition;
    (35)两亲性嵌段共聚物；cKK-E12、DLin-MC3-DMA、ALC-0315、SM-102或C12-200；DOTAP、DODAP、DOTMA或DOSPA；胆固醇或β-谷甾醇；和DMG-PEG2K或DMG-PEG5K，其中所述cKK-E12、DLin-MC3-DMA、ALC- 0315、SM-102或C12-200占所述组合物中存在的总脂质的30.0mol％，DOTAP、DODAP、DOTMA或DOSPA占总脂质的39.0mol％，胆固醇或β-谷甾醇占总脂质的30.0mol％，DMG-PEG2K或DMG-PEG5K占总脂质的1.0mol％，且所述两亲性嵌段共聚物占所述组合物的57.7％重量百分比；或(35) an amphiphilic block copolymer; cKK-E12, DLin-MC3-DMA, ALC-0315, SM-102 or C12-200; DOTAP, DODAP, DOTMA or DOSPA; cholesterol or β-sitosterol; and DMG-PEG2K or DMG-PEG5K, wherein the cKK-E12, DLin-MC3-DMA, ALC- 0315, SM-102 or C12-200 accounts for 30.0 mol% of the total lipids present in the composition, DOTAP, DODAP, DOTMA or DOSPA accounts for 39.0 mol% of the total lipids, cholesterol or β-sitosterol accounts for 30.0 mol% of the total lipids, DMG-PEG2K or DMG-PEG5K accounts for 1.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 57.7% by weight of the composition; or
    (36)两亲性嵌段共聚物；DLin-MC3-DMA、ALC-0315或SM-102；DSPC、DPPC或DOPE；和胆固醇或β-谷甾醇，其中所述DLin-MC3-DMA、ALC-0315或SM-102占所述组合物中存在的总脂质的40.0mol％，DSPC、DPPC或DOPE占总脂质的32.0mol％，胆固醇或β-谷甾醇占总脂质的28.0mol％，且所述两亲性嵌段共聚物占所述组合物的50.0％重量百分比。(36) An amphiphilic block copolymer; DLin-MC3-DMA, ALC-0315 or SM-102; DSPC, DPPC or DOPE; and cholesterol or β-sitosterol, wherein the DLin-MC3-DMA, ALC-0315 or SM-102 account for 40.0 mol% of the total lipids present in the composition, DSPC, DPPC or DOPE account for 32.0 mol% of the total lipids, cholesterol or β-sitosterol account for 28.0 mol% of the total lipids, and the amphiphilic block copolymer accounts for 50.0% weight percent of the composition.
  25. 权利要求1-24中任一项所述的组合物，其中所述组合物中的阳离子脂质中的氮(胺)基团与核酸的磷酸酯基团的摩尔比(N/P比)为约1.0-约30.0，约3.0-约15.0，约4.0-约10.0，约6.0至约8.0。The composition of any one of claims 1-24, wherein the molar ratio of nitrogen (amine) groups in the cationic lipid to phosphate groups of the nucleic acid (N/P ratio) in the composition is about 1.0 to about 30.0, about 3.0 to about 15.0, about 4.0 to about 10.0, about 6.0 to about 8.0.
  26. 权利要求1-25中任一项所述的组合物，其中所述组合物中的脂质与核酸的比(质量/质量比)为约2(2:1)-约100(100:1)，约5(5:1)-约60(60:1)，约15(15:1)-约45(45:1)，或约20(20:1)-约30(30:1)。The composition of any one of claims 1-25, wherein the ratio of lipid to nucleic acid in the composition (mass/mass ratio) is about 2 (2:1) to about 100 (100:1), about 5 (5:1) to about 60 (60:1), about 15 (15:1) to about 45 (45:1), or about 20 (20:1) to about 30 (30:1).
  27. 权利要求1-26中任一项所述的组合物，其中所述活性剂或治疗剂进一步包含蛋白质或多肽，优选所述蛋白质或多肽与所述核酸以约1:1至约1:20的摩尔比存在。The composition of any one of claims 1-26, wherein the active agent or therapeutic agent further comprises a protein or polypeptide, preferably the protein or polypeptide and the nucleic acid are present in a molar ratio of about 1:1 to about 1:20.
  28. 前述权利要求中任一项所述的组合物，其中所述组合物进一步包含靶向部分以将所述组合物靶向至受试者中的目标器官、组织或细胞，优选所述靶向部分包含选自以下中的至少一种：糖基、脂质、核酸适配体、小分子治疗剂、维生素、多肽和蛋白质例如抗体。The composition of any of the preceding claims, wherein the composition further comprises a targeting moiety to target the composition to a target organ, tissue or cell in a subject, preferably the targeting moiety comprises at least one selected from the group consisting of a glycosyl, a lipid, a nucleic acid aptamer, a small molecule therapeutic agent, a vitamin, a polypeptide and a protein such as an antibody.
  29. 前述权利要求中任一项所述的组合物，其中所述组合物还包含佐剂，优选所述佐剂包含选自以下的至少一种：CpG寡脱氧核苷酸、聚肌苷：聚胞苷酸、皂角提取物(QS-21提取物)、铝佐剂、锰佐剂、锌佐剂、角鲨烯、α-生育酚、吐温(Tween)、司盘(Span)、脂多糖LPS、Pam3CSK4三酰脂肽、环 腺苷二磷酸(c-di-AMP)、2′3′-环鸟苷一磷酸腺苷一磷酸(cGAMP)、单磷酰-脂质A、MPL脂质、鞭毛蛋白或免疫调节蛋白如IL-2、IL-12、GM-CSF、TSLP和编码这些免疫调节剂蛋白的核酸。The composition according to any one of the preceding claims, wherein the composition further comprises an adjuvant, preferably the adjuvant comprises at least one selected from the group consisting of: CpG oligodeoxynucleotides, polyinosinic: polycytidylic acid, saponin extract (QS-21 extract), aluminum adjuvant, manganese adjuvant, zinc adjuvant, squalene, α-tocopherol, Tween, Span, lipopolysaccharide LPS, Pam 3 CSK 4 triacyl lipopeptide, cyclohexane Adenosine diphosphate (c-di-AMP), 2′3′-cyclic guanosine monophosphate adenosine monophosphate (cGAMP), monophosphoryl-lipid A, MPL lipid, flagellin or immunomodulatory proteins such as IL-2, IL-12, GM-CSF, TSLP and nucleic acids encoding these immunomodulator proteins.
  30. 前述权利要求中任一项所述的组合物，其中所述组合物还包含转染增效剂，优选所述转染增效剂包含选自以下中的至少一种：肺部表面活性蛋白、细胞穿膜肽、两亲性多肽、黏液溶解酶、1,2-丙二醇、纤维素(如羧甲基纤维素或羟丙基纤维素)、透明质酸盐、海藻酸盐、果胶、聚乙二醇、泊洛沙姆、泊洛沙胺、葡萄糖、果糖、蔗糖、海藻糖、葡聚糖、蔗糖、海藻糖、甘露糖、聚乙烯吡咯烷酮、壳聚糖、聚乙烯醇、聚乙酸乙烯酯、凝集素、聚乳酸、聚羟基丁酸、氨丁三醇、氯化苄烷铵、经修饰精氨酸、氯化鲸蜡基吡啶鎓、L-赖氨酸单水合物，以及聚乳酸-羟基乙酸共聚物或盐溶液。The composition of any of the preceding claims, wherein the composition further comprises a transfection enhancer, preferably the transfection enhancer comprises at least one selected from the group consisting of: pulmonary surfactant protein, cell penetrating peptide, amphiphilic polypeptide, mucolytic enzyme, 1,2-propylene glycol, cellulose (such as carboxymethyl cellulose or hydroxypropyl cellulose), hyaluronate, alginate, pectin, polyethylene glycol, poloxamer, poloxamine, glucose, fructose, sucrose, trehalose, dextran, sucrose, trehalose, mannose, polyvinyl pyrrolidone, chitosan, polyvinyl alcohol, polyvinyl acetate, lectin, polylactic acid, polyhydroxybutyric acid, tromethamine, benzalkonium chloride, modified arginine, cetylpyridinium chloride, L-lysine monohydrate, and polylactic-co-glycolic acid or salt solution.
  31. 前述权利要求中任一项所述的组合物，其中所述组合物为纳米颗粒的形式，所述纳米颗粒具有约1000nm或更小的平均尺寸。The composition of any of the preceding claims, wherein the composition is in the form of nanoparticles having an average size of about 1000 nm or less.
  32. 权利要求31所述的组合物，其中所述纳米颗粒具有约500nm或更小、约400nm或更小、约300nm或更小、约200nm或更小、约150nm或更小、约125nm或更小、约100nm或更小、约75nm或更小、或约50nm或更小的平均尺寸。The composition of claim 31, wherein the nanoparticles have an average size of about 500 nm or less, about 400 nm or less, about 300 nm or less, about 200 nm or less, about 150 nm or less, about 125 nm or less, about 100 nm or less, about 75 nm or less, or about 50 nm or less.
  33. 权利要求31或32的组合物，其中约30％-约100％、约70％-约100％、约90％-约100％、约50％-约90％、约70％-约90％或约80％-约90％的所述纳米颗粒具有包封在其中的活性剂或治疗剂。The composition of claim 31 or 32, wherein about 30% to about 100%, about 70% to about 100%, about 90% to about 100%, about 50% to about 90%, about 70% to about 90%, or about 80% to about 90% of the nanoparticles have an active or therapeutic agent encapsulated therein.
  34. 权利要求1-33中任一项所述的组合物，其中所述组合物配制为溶液、干粉、雾化或喷雾的形式。The composition of any one of claims 1-33, wherein the composition is formulated in the form of a solution, dry powder, atomization or spray.
  35. 权利要求1-34中任一项所述的组合物，所述组合物配制为通过气溶胶化、干粉、吸入、雾化或滴注进行所述呼吸道、肺部、气管、支气管和/或鼻部施用。 The composition of any one of claims 1-34, formulated for administration to the respiratory tract, lungs, trachea, bronchus and/or nose by aerosolization, dry powder, inhalation, nebulization or instillation.
  36. 一种制备权利要求1-35中任一项所述的组合物的方法，所述方法包括：A method for preparing the composition according to any one of claims 1 to 35, the method comprising:
    (A)在两亲性嵌段共聚物的存在下将包含所述活性剂或治疗剂的溶液和包含所述脂质的溶液混合以形成所述组合物；或(A) mixing a solution comprising the active agent or therapeutic agent and a solution comprising the lipid in the presence of an amphiphilic block copolymer to form the composition; or
    (B)将包含所述活性剂或治疗剂的溶液和包含所述脂质的溶液混合以形成包封所述活性剂或治疗剂的脂质纳米颗粒，随后将所述两亲性嵌段共聚物与所述脂质纳米颗粒溶液混合以形成所述组合物。(B) mixing a solution containing the active agent or therapeutic agent and a solution containing the lipid to form lipid nanoparticles encapsulating the active agent or therapeutic agent, and then mixing the amphiphilic block copolymer with the lipid nanoparticle solution to form the composition.
  37. 权利要求36所述的方法，所述方法包括：The method of claim 36, comprising:
    1)将所述两亲性嵌段共聚物加入到包含所述活性剂或治疗剂的溶液中和/或包含所述脂质的溶液中，和1) adding the amphiphilic block copolymer to a solution comprising the active agent or therapeutic agent and/or a solution comprising the lipid, and
    2)将所述包含活性剂或治疗剂的溶液和所述包含脂质的溶液混合，2) mixing the solution containing the active agent or therapeutic agent and the solution containing the lipid,
    从而形成所述组合物。Thereby forming the composition.
  38. 权利要求36所述的方法，所述方法包括：The method of claim 36, comprising:
    1)在包含所述脂质的溶液中，使得所述脂质预形成无活性剂或治疗剂的脂质纳米颗粒；和1) in a solution comprising the lipid, allowing the lipid to pre-form into lipid nanoparticles without an active agent or therapeutic agent; and
    2)将包含所述活性剂或治疗剂和两亲性嵌段共聚物的溶液与所述脂质纳米颗粒溶液混合，2) mixing a solution comprising the active agent or therapeutic agent and the amphiphilic block copolymer with the lipid nanoparticle solution,
    从而形成所述组合物。或Thus forming the composition. Or
    所述方法包括：The method comprises:
    1)将所述脂质和两亲性嵌段共聚物预形成无活性剂或治疗剂的聚合物-脂质纳米颗粒；和1) preforming the lipid and the amphiphilic block copolymer into polymer-lipid nanoparticles without active agent or therapeutic agent; and
    2)将包含所述活性剂或治疗剂的溶液与所述聚合物-脂质纳米颗粒溶液混合，2) mixing a solution containing the active agent or therapeutic agent with the polymer-lipid nanoparticle solution,
    从而形成所述组合物。Thereby forming the composition.
  39. 权利要求36-38中任一项所述的方法，所述方法进一步包括去除游离的脂质组分和/或两亲性嵌段共聚物的步骤，优选通过透析和/或切向流过滤去除所述游离的脂质组分和/或两亲性嵌段共聚物。 The method according to any one of claims 36 to 38, further comprising the step of removing free lipid components and/or amphiphilic block copolymers, preferably removing the free lipid components and/or amphiphilic block copolymers by dialysis and/or tangential flow filtration.
  40. 权利要求39的方法，所述方法进一步包括在去除游离的脂质组分和/或两亲性嵌段共聚物之后，再次加入两亲性嵌段共聚物的步骤。The method of claim 39, further comprising the step of adding the amphiphilic block copolymer again after removing the free lipid components and/or the amphiphilic block copolymer.
  41. 一种药物组合物，其包含：权利要求1‐35中的任一项所述的组合物和药学上可接受的载体和/或赋形剂。A pharmaceutical composition comprising: the composition according to any one of claims 1 to 35 and a pharmaceutically acceptable carrier and/or excipient.
  42. 一种向靶细胞递送活性剂或治疗剂的方法，所述方法包括：在足以造成所述活性剂或治疗剂被摄入所述细胞中的情况下，使所述细胞与根据权利要求1-35中任一项所述的组合物接触，优选所述细胞是哺乳动物细胞。A method for delivering an active agent or therapeutic agent to a target cell, the method comprising: contacting the cell with a composition according to any one of claims 1 to 35 under conditions sufficient to cause the active agent or therapeutic agent to be taken up into the cell, preferably the cell is a mammalian cell.
  43. 一种用于预防和/或治疗哺乳动物的疾病或病症的方法，所述方法包括给有此需要的受试者于体内施用药学有效量的根据权利要求1‐35中任一项所述的组合物或权利要求41所述的药物组合物的步骤，其中所述组合物或药物组合物包含所述针对疾病或病症的活性剂或治疗剂。A method for preventing and/or treating a disease or condition in a mammal, the method comprising the step of administering a pharmaceutically effective amount of a composition according to any one of claims 1 to 35 or a pharmaceutical composition according to claim 41 to a subject in need thereof, wherein the composition or pharmaceutical composition comprises an active agent or therapeutic agent for the disease or condition.
  44. 权利要求1-35中任一项所述的组合物或权利要求41所述的药物组合物在制备用于预防和/或治疗受试者中的疾病的药物中的用途，其中所述组合物或药物组合物包含所述针对疾病或病症的活性剂或治疗剂。Use of the composition of any one of claims 1 to 35 or the pharmaceutical composition of claim 41 in the preparation of a medicament for preventing and/or treating a disease in a subject, wherein the composition or pharmaceutical composition comprises an active agent or therapeutic agent for the disease or condition.
  45. 权利要求43所述的方法或权利要求44所述的用途，其中所述疾病或病症选自免疫系统疾病、代谢性疾病、遗传性疾病、癌症、血液疾病、细菌感染或病毒感染。 The method of claim 43 or the use of claim 44, wherein the disease or condition is selected from an immune system disease, a metabolic disease, a genetic disease, cancer, a blood disease, a bacterial infection or a viral infection.
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