CN115137743A

CN115137743A - RNA plasmid delivery system for treating pulmonary fibrosis

Info

Publication number: CN115137743A
Application number: CN202210325040.8A
Authority: CN
Inventors: 张辰宇; 陈熹; 付正; 李菁; 张翔; 周心妍; 张丽; 余梦超; 郭宏源
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2021-03-30
Filing date: 2022-03-29
Publication date: 2022-10-04
Also published as: WO2022206778A1

Abstract

The present application provides an RNA plasmid delivery system for the treatment of pulmonary fibrosis. The system comprises a plasmid carrying RNA fragments capable of being used for treating pulmonary fibrosis, wherein the plasmid can be enriched in organ tissues of a host and endogenously and spontaneously form a composite structure containing the RNA fragments in the organ tissues of the host, and the composite structure can deliver RNA capable of inhibiting pulmonary fibrosis into the lung so as to realize treatment of pulmonary fibrosis. The safety and reliability of the RNA delivery system for treating pulmonary fibrosis are fully verified, the medicine property is very good, the universality is strong, and the economic benefit and the application prospect are excellent.

Description

RNA plasmid delivery system for treating pulmonary fibrosis

Technical Field

The application relates to the technical field of biomedicine, in particular to an RNA plasmid delivery system for treating pulmonary fibrosis.

Background

Pulmonary fibrosis is the terminal change of a large group of lung diseases characterized by fibroblast proliferation and massive extracellular matrix aggregation with inflammatory injury and tissue structure destruction, namely structural abnormality (scar formation) caused by abnormal repair after normal alveolar tissues are damaged. Pulmonary fibrosis seriously affects the respiratory function of the human body, manifested as dry cough and progressive dyspnea (insufficient conscious qi), and the respiratory function of the patient is continuously worsened with the aggravation of the disease condition and the lung injury. The incidence and mortality of idiopathic pulmonary fibrosis increases year by year, with a mean survival after diagnosis of only 2.8 years, with a mortality rate higher than that of most tumors, known as a "neoplastic-like disease".

RNA interference (RNAi) therapy has been considered a promising strategy for the treatment of human diseases since its invention, but in clinical practice a number of problems have been encountered, the progress of which has fallen far behind expectations.

It is generally considered that RNA cannot exist stably for a long period outside cells because RNA is degraded into fragments by RNase which is abundant outside cells, and therefore, a method for stably existing RNA outside cells and allowing targeted entry into a specific tissue must be found to highlight the effect of RNAi therapy.

Many patents related to siRNA are focused on the following aspects: 1. siRNA with medical effect is designed. 2. The siRNA is chemically modified, so that the stability of the siRNA in an organism is improved, and the yield is improved. 3. Various artificial vectors (such as lipid nanoparticles, cationic polymers, and viruses) are increasingly designed to increase the efficiency of siRNA delivery in vivo. Many of these patents in aspect 3 are based on the fact that researchers have recognized the lack of suitable siRNA delivery systems to deliver siRNA to target tissues safely, accurately and efficiently, which has become a central problem in RNAi therapy.

Chinese patent publication No. CN108624590A discloses an siRNA capable of inhibiting DDR2 gene expression; chinese patent with publication number CN108624591A discloses a siRNA capable of silencing ARPC4 gene, and the siRNA is modified by alpha-phosphorus-selenium; chinese patent with publication number CN108546702A discloses siRNA of targeting long-chain non-coding RNA DDX11-AS 1. Chinese patent publication No. CN106177990A discloses a siRNA precursor that can be used for various tumor treatments. These patents have designed specific sirnas and are directed to certain diseases caused by genetic changes.

Chinese patent publication No. CN108250267A discloses a polypeptide, polypeptide-siRNA induced co-assembly, using polypeptide as a carrier of si RNA. Chinese patent publication No. CN108117585A discloses a polypeptide for promoting breast cancer cell apoptosis by targeted introduction of siRNA, and the polypeptide is also used as a carrier of siRNA. Chinese patent publication No. CN108096583A discloses a nanoparticle carrier, which contains chemotherapeutic drugs and also can be loaded with siRNA with breast cancer therapeutic effect. These patents are all the inventions on siRNA vectors, but the technical scheme has a common feature that the vector and siRNA are pre-assembled in vitro and then introduced into a host. In fact, most of the current designs for delivery technology do so. However, such delivery systems have a common problem in that these artificially synthesized exogenous delivery systems are easily cleared by the host's circulatory system, may elicit an immunogenic response, and may even be toxic to specific cell types and tissues.

The research team of the present invention finds that endogenous cells can selectively encapsulate miRNAs into exosomes (exosomes) which can deliver miRNAs into recipient cells, and the secreted miRNAs can powerfully block the expression of target genes at relatively low concentrations. Exosomes are biocompatible with the host immune system and have the innate ability to protect and transport miRNA across biological barriers in vivo, thus becoming a potential solution to overcome problems associated with siRNA delivery. For example, chinese patent publication No. CN110699382A discloses a method for preparing exosomes for delivering siRNA, and discloses a technique for isolating exosomes from plasma and encapsulating siRNA into exosomes by electroporation.

However, such technologies for in vitro separation or preparation of exosomes usually require a large amount of exosomes obtained by cell culture, and a step of sirna encapsulation, which causes the clinical cost of large-scale application of the product to be very high and cannot be borne by general patients; more importantly, the complex production/purification process of exosomes makes it almost impossible to comply with GMP standards.

The drug taking exosome as an active ingredient has not been approved by CFDA so far, and the core problem is that the consistency of exosome products cannot be ensured, and the problem directly results in that the products cannot obtain the drug production license. If the problem can be solved, the method has no significance on promoting RNAi therapy of pulmonary fibrosis.

Therefore, the development of a safe, accurate and efficient siRNA delivery system is a crucial link for improving the RNAi therapeutic effect of pulmonary fibrosis and promoting RN Ai therapy.

Disclosure of Invention

In view of the above, the embodiments of the present application provide an RNA plasmid delivery system for treating pulmonary fibrosis, so as to solve the technical defects existing in the prior art.

The application provides an RNA plasmid delivery system for treating pulmonary fibrosis, which comprises a plasmid, wherein the plasmid carries RNA fragments capable of being used for treating pulmonary fibrosis, the plasmid can be enriched in organ tissues of a host, and endogenously and spontaneously forms a composite structure containing the RNA fragments in the organ tissues of the host, and the composite structure can send RNA capable of inhibiting pulmonary fibrosis into the lung so as to realize the treatment of pulmonary fibrosis. After the RNA segment is sent into the lung, the expression of the gene matched with the RNA segment can be inhibited, so that the development of pulmonary fibrosis is inhibited, and the treatment of pulmonary fibrosis is realized.

Optionally, the RNA segment comprises 1, two or more specific RNA sequences of medical interest, said RNA sequences being siRNA, shRNA or miRNA sequences of medical interest capable of inhibiting or hindering the development of pulmonary fibrosis.

FIG. 16 shows that the plasmid delivery system alone carries 6 RNA sequences, carries RNA fragments with any 2 compositions, and carries RNA fragments with any 3 compositions, and has the effects of in vivo enrichment, self-assembly and treatment on pulmonary fibrosis.

Optionally, the plasmid further comprises a promoter and a targeting tag, wherein the targeting tag is capable of forming a targeting structure of the composite structure in the organ tissue of the host, the targeting structure is located on the surface of the composite structure, and the composite structure is capable of finding and binding to the target tissue through the targeting structure to deliver the RNA fragment into the target tissue.

Optionally, the plasmid comprises any one or a combination of several of the following lines: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; each plasmid comprises at least one RNA segment and one targeting label, and the RNA segment and the targeting label are positioned in the same line or different lines.

It is shown by FIGS. 17-19 that plasmid delivery systems constructed with 1-2 RNA fragments and 1-2 targeting tags all have in vivo enrichment, self-assembly and therapeutic effects on pulmonary fibrosis.

Optionally, the plasmid further comprises flanking sequences, compensating sequences and loop sequences capable of folding the lines into the correct structure and expressing, the flanking sequences comprising a 5 'flanking sequence and a 3' flanking sequence;

the plasmid comprises any one line or combination of lines as follows: 5' -promoter-5 ' flanking sequence-RNA fragment-loop sequence-compensating sequence-3 ' flanking sequence, 5' -promoter-targeting tag or 5' -promoter-targeting tag-5 ' flanking sequence-RNA fragment-loop sequence-compensating sequence-3 ' flanking sequence.

Optionally, the 5' flanking sequence is ggatcctggaggcttgctgagaggctgtatgctgaattc or a sequence having more than 80% homology thereto;

the loop sequence is gtttggccactgactgac or a sequence with homology more than 80 percent;

<xnotran> 3' accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag 80% ; </xnotran>

The compensation sequence is a reverse complementary sequence of the RNA segment, and any 1-5 bases in the RNA segment are deleted. The base at position 1 to 5 of the reverse complement sequence of RNA is deleted in order to prevent the sequence from being expressed.

FIG. 20 shows that plasmids containing multiple 5 'flanking sequences, loop sequences, 3' flanking sequences and homologous sequences have the effects of in vivo enrichment, self-assembly and treatment of pulmonary fibrosis. Preferably, the complementing sequence is the reverse complement of the RNA fragment, and any 1-3 bases in the RNA fragment are deleted.

More preferably, the complementary sequence is the reverse complement of the RNA fragment, and any 1-3 consecutive bases in the complementary sequence are deleted.

Most preferably, the complementing sequence is the reverse complement of the RNA fragment, and the 9 th and/or 10 th base is deleted.

Alternatively, in the case where at least two lines are present in the plasmid, adjacent lines are connected by a sequence consisting of sequences 1 to 3 (sequence 1-sequence 2-sequence 3);

wherein, the sequence 1 is CAGATC, the sequence 2 is a sequence consisting of 5-80 bases, and the sequence 3 is TGGATC.

FIG. 21 shows that when the RNA plasmid delivery system carries multiple strands, the junction sequence between adjacent strands, i.e., sequence 2, can be composed of multiple bases, and the plasmid injection also has an enrichment effect.

Alternatively, where at least two lines are present in the plasmid, adjacent lines are connected by sequence 4 or a sequence having greater than 80% homology to sequence 4;

wherein the sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACGACCAGTGGAC.

As shown in FIG. 22, when the connecting sequence is SEQ ID No. 4 or at least 3 sequences with homology of more than 80% to SEQ ID No. 4, the constructed plasmid delivery system also has the effects of in vivo enrichment, self-assembly and treatment of pulmonary fibrosis.

Optionally, the organ tissue is liver and the composite structure is exosome.

Optionally, the targeting tag is selected from a targeting peptide or a targeting protein having a targeting function.

Optionally, the targeting peptide comprises an RVG targeting peptide, a GE11 targeting peptide, a PTP targeting peptide, a TCP-1 targeting peptide, an MSP targeting peptide;

the target protein comprises RVG-LAMP2B fusion protein, GE11-LAMP2B fusion protein, PTP-LAMP2B fusion protein, TCP-1-LAMP2B fusion protein and MSP-LAMP2B fusion protein.

Optionally, the RNA sequence is 15-25 nucleotides in length. For example, the RNA sequence can be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides in length. Preferably, the RNA sequence is 18-22 nucleotides in length.

It is shown in FIG. 23 that the RNA sequences in the plasmid delivery system are 18, 19, and 21 in length, respectively, and have the effects of in vivo enrichment, self-assembly, and treatment of pulmonary fibrosis.

Optionally, the RNA sequence is selected from any one or several of: an antisense strand of miRNA-21, an siRNA of TGF-beta 1 gene, or an RNA sequence having a homology of greater than 80% with the above sequence, or a nucleic acid molecule encoding the above RNA. It should be noted that the RNA sequence in the "nucleic acid molecule encoding the RNA sequence" also includes RNA sequences having a homology of more than 80% for each RNA.

Optionally, the siRNA of TGF- β 1 gene comprises acggaauaccuagaugggc, ugacuugucauaguucgu, UUGAAGAACAUAUAUAUGCUG, ucauaacaguaaguguuccc, ucucaggucucuggccucag, other sequences that inhibit TGF- β 1 gene expression, and sequences that have more than 80% homology with the above sequences.

FIG. 24 shows that the gene circuit has the in vivo enrichment, self-assembly and therapeutic effect on pulmonary fibrosis under the condition that the gene circuit comprises the antisense strand of miRNA-21 and the 5 TGF-beta 1 gene siRNAs (siRNA-1, siRNA-2, siRNA-3, siRNA-4 and siRNA-5).

Optionally, the RNA fragment comprises an RNA sequence body and a modified RNA sequence obtained by ribose modification of the RNA sequence body. That is, the RNA fragment may consist of only at least one RNA sequence entity, may consist of only at least one modified RNA sequence, or may consist of both the RNA sequence entity and the modified RNA sequence.

In the present invention, the isolated nucleic acids also include variants and derivatives thereof. The nucleic acids can be modified by one of ordinary skill in the art using conventional methods. Modifications include (but are not limited to): methylation modification, alkyl modification, glycosylation modification (such as 2-methoxy-glycosyl modification, alkyl-glycosyl modification, sugar ring modification and the like), nucleic acid modification, peptide segment modification, lipid modification, halogen modification, nucleic acid modification (such as 'TT' modification) and the like. In one embodiment of the invention, the modification is an internucleotide linkage, for example selected from: thiophosphate, 2'-O Methoxyethyl (MOE), 2' -fluoro, alkyl phosphonate, dithiophosphate, alkyl thiophosphonate, phosphoramidate, carbamate, carbonate, phosphotriester, acetamide ester, carboxymethyl ester, and combinations thereof. In one embodiment of the invention, the modification is a modification to a nucleotide, for example selected from: peptide Nucleic Acids (PNA), locked Nucleic Acids (LNA), arabinose-nucleic acids (FANA), analogs, derivatives and combinations thereof. Preferably, the modification is a 2' fluoropyrimidine modification. The 2 '-fluoropyrimidine modification is to replace 2' -OH of pyrimidine nucleotide on RNA with 2'-F, and the 2' -F can make RNA not be easily recognized by RNase in vivo, thereby increasing the stability of RNA fragment in vivo delivery.

Optionally, the delivery system is a delivery system for use in a mammal, including a human.

The present application also provides the use of an RNA plasmid delivery system as described above for the treatment of pulmonary fibrosis in medicine.

Optionally, the drug is a drug for treating pulmonary fibrosis and related diseases thereof, where the related diseases refer to related diseases or complications, sequelae, etc. occurring during the formation or development of the above-mentioned pulmonary fibrosis, or other diseases having a certain correlation with pulmonary fibrosis.

Optionally, the drug includes the above plasmid, specifically, the plasmid herein refers to a plasmid carrying an RNA fragment, or a plasmid carrying an RNA fragment and a targeting tag, and can enter a host body, and can be enriched at a liver site, self-assemble to form a complex structure exosome, and the complex structure can deliver the RNA fragment to a lung, so that the RNA fragment is expressed in the lung, and further, the expression of a gene matched with the RNA fragment is inhibited, thereby achieving the purpose of treating pulmonary fibrosis.

The dosage form of the medicine can be tablets, capsules, powder, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes and the like.

Optionally, the administration mode of the drug comprises oral administration, inhalation, subcutaneous injection, intramuscular injection and intravenous injection. Intravenous injection is preferred.

The technical effects of this application do:

the RNA delivery system for treating pulmonary fibrosis provided by the application takes the plasmid as a carrier and takes the plasmid as a mature injectant, and has safety and reliabilityThe property is fully verified, and the drug property is very good. The RNA sequence which finally exerts the effect is encapsulated and conveyed by the endogenous exosome, no immune reaction exists, and the safety of the exosome does not need to be verified. The delivery system can deliver various small-molecule RNAs and has strong universality. Moreover, the preparation of the plasmid is much cheaper than that of exosome or substances such as protein, polypeptide and the like, and the economy is good. The RNA delivery systems provided herein are capable of self-assembly with an AGO in vivo ₂ Tightly combined and enriched into a composite structure (exosome), not only can prevent the exosome from being degraded prematurely and maintain the stability of the exosome in circulation, but also is beneficial to the absorption of receptor cells, the intracytoplasmic release and the escape of lysosomes, and the required dosage is low.

The RNA delivery system for treating pulmonary fibrosis is applied to medicines, namely, a medicine delivery platform is provided, the treatment effect of pulmonary fibrosis can be greatly improved, the research and development basis of more RNA medicines can be formed through the platform, and the RNA medicine research and development and use are greatly promoted.

Drawings

FIG. 1 is a graph comparing plasmid distribution and metabolism in mice provided by an embodiment of the present application;

FIG. 2 is a graph comparing the expression levels of proteins in mice provided by an example of the present application;

FIG. 3 is a graph comparing relevant siRNA levels in mice provided by an example of the present application;

FIG. 4 is a graph comparing absolute siRNA levels in various tissues of a mouse as provided in an example of the present application;

FIG. 5 is a graph comparing the effect of plasmid dose on mouse siRNA levels as provided in one embodiment of the present application;

FIG. 6 is a graph comparing the metabolism of precursors and matures in the liver of mice injected with plasmids, provided by an embodiment of the present application;

FIG. 7 is a graph comparing the kinetics and distribution of siRNA in different tissues of a mouse, as provided by an example of the present application;

FIG. 8 is a graph comparing the effect of different promoters on siRNA provided in one embodiment of the present application;

FIG. 9 is a graph comparing the eGFP fluorescence intensity in different tissues of a mouse, as provided in an example of the present application;

FIG. 10 is a graph comparing the levels of glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, total bilirubin, blood urea nitrogen, serum alkaline phosphatase, creatinine content, and thymus weight, spleen weight, and percentage of peripheral blood cells in mice provided by an example of the present application;

FIG. 11 is a graph comparing the hydroxyproline content in mice provided in an example of the present application;

FIG. 12 is a graph of fluorescence staining of mouse lungs provided by an embodiment of the present application;

FIG. 13 is a Masson trichrome staining of mouse lungs as provided in an example of the present application;

FIG. 14 is a graph of HE staining of mouse lungs provided by an embodiment of the present application;

FIG. 15 is a graph comparing the levels of partial mouse protein and mRNA provided in an example of the present application.

Fig. 16 is a data diagram illustrating the therapeutic effect on pulmonary fibrosis of a plasmid delivery system containing RNA fragments according to an embodiment of the present application, in which a is the result of measuring the relative amount of mRNA of PTP1B after injection of the plasmid delivery system containing 6 RNA sequences, RNA fragments consisting of any 2 of the 6 RNA sequences, and RNA fragments consisting of any 3 of the 6 RNA sequences, and B is the result of measuring the relative amount of protein of PTP1B after injection of the plasmid delivery system containing 6 RNA sequences, RNA fragments consisting of any 2 of the 6 RNA sequences, and RNA fragments consisting of any 3 of the 6 RNA sequences.

FIG. 17 is the result of the metabolic distribution of CMV-siRNA-1+2 after intravenous injection provided in an example of the present application, where A is the enrichment effect in lung and B is the enrichment effect in blood.

FIG. 18 shows the results of the metabolic distribution of CMV-GE11-siRNA-1+2 and (CMV-GE 11-siRNA-1+ CMV-GE 11-siRNA-2) after intravenous injection with targeting label GE11 provided in an embodiment of the present application, wherein A and C are the enrichment effects of CMV-GE11-siRNA-1+2 in lung and plasma, and B and D are the enrichment effects of CMV-GE11-siRNA-1+ CMV-GE11-siRNA-2 in lung and plasma, respectively.

FIG. 19 is a data graph of therapeutic effect on pulmonary fibrosis after intravenous injection of CMV-GE11-siRNA-1, CMV-GE11-siRNA-1+2 and CMV-GE11-siRNA-1+ CMV-GE11-siRNA-2 with targeting label GE11 provided in an example of the present application, in which A and C are results of protein content and mRNA content of TGFb1 of CMV-GE11-siRNA-1 and CMV-GE11-siRNA-1+2, respectively, and B and D are results of protein content and mRNA content of TGFb1 of CMV-GE11-siRNA-1 and CMV-GE11-siRNA-1+ CMV-GE11-siRNA-2, respectively.

FIG. 20 is a graph of the enrichment effect (expressed as siRNA content) in blood following injection of a plasmid containing 3 different sequence segments of the 5 'flanking sequence, loop sequence and 3' flanking sequence provided in an example of the present application.

FIG. 21 is a graph showing the effect of enrichment in blood (in terms of siRNA content) after injection of a plasmid delivery system comprising a plurality of linker sequences (SEQ ID NO: 2) of different numbers of bases as provided in an example of the present application.

FIG. 22 is a graph showing the effect of enrichment in blood (expressed as siRNA content) after injection of a plasmid delivery system comprising a plurality of linker sequences having a homology of greater than 80% (SEQ ID NO: 4) according to an embodiment of the present application, in which the abscissa sequence 4-1 is the base sequence 4 and the sequences 4-2/4-3/4-4 are homologous sequences having a homology of greater than 80% to the sequence 4-1 (SEQ ID NO: 4), respectively.

Fig. 23 shows the therapeutic effect of pulmonary fibrosis with RNA sequence lengths of 18, 19 and 21 in the plasmid delivery system provided in an embodiment of the present application, where a is the mRNA content result of TGFb1 and B is the protein content result of TGFb 1.

FIG. 24 shows the result of hydroxyproline content detected by the gene circuit provided in one embodiment of the present application under the condition of including the antisense strand of miRNA-21 and 5 siRNA of TGF-beta 1 gene.

Detailed Description

The following description of specific embodiments of the present application refers to the accompanying drawings.

First, terms, test methods, and the like according to the present invention will be explained.

Hematoxylin-eosin staining (HE staining) is short for hematoxylin-eosin staining. HE staining is one of the most basic and widely used technical methods in histology, pathology teaching and scientific research.

The hematoxylin staining solution is alkaline, and can stain basophilic structures (such as ribosome, nucleus, ribonucleic acid in cytoplasm and the like) of tissues into bluish purple; eosin is an acid dye that stains the tissue's eosinophilic structures (e.g., intracellular and intercellular proteins, including lewy bodies, alcohol bodies, and most of the cytoplasm) pink, leaving the entire tissue morphology clearly visible.

The HE staining method comprises the following specific steps: fixing and slicing sample tissues; deparaffinizing the tissue sample; hydrating the tissue sample; staining tissue sections with hematoxylin, differentiating and turning blue; eosin staining and dehydrating the tissue section; air-drying the tissue sample slices and sealing the slices; finally, the film was observed under a microscope and photographed.

Masson staining gives collagen fibers either a blue (stained with aniline blue) or green (stained with brilliant green) color and muscle fibers a red (stained with acid fuchsin and ponceau red) color, depending on the size of the anionic dye molecules and the permeability of the tissue. Fixed tissue is stained with a series of anionic water-soluble dyes, either sequentially or in combination, and it is found that red blood cells are stained with the smallest anionic dye, muscle fibers and cytoplasm are stained with the medium-sized anionic dye, and collagen fibers are stained with the larger anionic dye. This demonstrates that erythrocytes have minimal permeability to anionic dyes, muscle fibers and cytoplasm, while collagen fibers have maximal permeability. Type I and type III collagens are green (GBM, TBM, mesangial matrix and renal interstitium are green), and the eosinophilic proteins, tubule cytoplasm, and erythrocytes are red.

The Masson staining method specifically comprises the following steps:

fixing the tissue in Bouin's fluid, flushing with running water for one night, and conventionally dehydrating and embedding; slicing and dewaxing to water (dewaxing in xylene for 10min × 3 times, blotting liquid with absorbent paper, 100% ethanol for 5min × 2 times, blotting liquid with absorbent paper, 95% ethanol for 5min × 2 times, blotting liquid with absorbent paper, flowing for 2min, blotting water with absorbent paper); weiger's ferrohematoxylin staining for 5-10min; slightly washing with running water; differentiating with 0.5% hydrochloric acid alcohol for 15s; washing with running water for 3min; dyeing the ponceau acid fuchsin liquid for 8min; slightly washing with distilled water; treating with 1% phosphomolybdic acid water solution for about 5min; without washing, the fabric is directly re-dyed with aniline blue solution or brilliant green solution for 5min; treating with 1% glacial acetic acid for 1min; dehydrating with 95% ethanol for 5min × 2 times, and drying with absorbent paper; 100% ethanol for 5min × 2 times, and drying the liquid with absorbent paper; transparent in xylene for 5min × 2 times, and sucking the liquid with absorbent paper; and (5) sealing the neutral gum.

The Western immunoblotting (Western Blot) is carried out by transferring the protein to a membrane and detecting the protein with an antibody.

Western Blot was performed by polyacrylamide gel electrophoresis, and the test substance was a protein, "probe" was an antibody, "and" secondary antibody for color development "was labeled. Transferring the protein sample separated by PAGE to a solid phase carrier (such as nitrocellulose film), adsorbing the protein by the solid phase carrier in a non-covalent bond form, keeping the type and biological activity of the electrophoretically separated polypeptide unchanged, taking the protein or polypeptide on the solid phase carrier as an antigen, carrying out immunoreaction with a corresponding antibody, then reacting with an enzyme or isotope labeled second antibody, and carrying out substrate chromogenic or autoradiography to detect the protein component expressed by the specific target gene separated by electrophoresis. The method mainly comprises the following steps: protein extraction, protein quantification, glue preparation and electrophoresis, membrane transfer, immune labeling and development.

Immunohistochemistry, which is the principle of antigen-antibody reaction, i.e., the specific binding of antigen and antibody, determines the antigens (polypeptides and proteins) in tissue cells by developing color-developing agents (fluorescein, enzyme, metal ions, isotopes) of labeled antibodies through chemical reaction, and performs localized, qualitative and relatively quantitative studies on the antigens, is called immunohistochemistry (immunohistochemistry) or immunocytochemistry (immunocytochemistry).

The main steps of immunohistochemistry include: soaking the slices, airing overnight, dewaxing xylene, dewaxing gradient alcohol (100%, 95%, 90%, 80%, 75%, 70%, 50%, 3min each time), double-distilling with water, dropping 3% hydrogen peroxide solution to remove catalase, washing with water, repairing antigen, dropping 5% BSA, sealing for 1h, diluting primary antibody, washing with PBS buffer solution, incubating secondary antibody, washing with PBS buffer solution, developing with developing solution, washing with water, dyeing with hematoxylin, dehydrating with gradient ethanol, and sealing with neutral gum.

The detection of siRNA level, protein content and mRNA content in the invention is realized by injecting RNA delivery system into mouse body to establish mouse stem cell in vitro model. mRNA and siRNA expression levels in cells and tissues were examined by qRT-PCR. The absolute quantification of siRNA was determined by plotting a standard curve against the standard. The expression amount of each siRNA or mRNA relative to the internal reference can be expressed by 2- Δ CT, where Δ CT = C sample-C internal reference. The internal reference gene is U6 snRNA (in tissues) or miR-16 (in serum and exosomes) molecules when siRNA is amplified, and the time base for mRNA amplification is GAPDH or 18s RNA. Western blotting experiment is used for detecting the expression level of protein in cells and tissues, and ImageJ software is used for carrying out quantitative analysis on the protein.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the reagents, materials and procedures used herein are those that are widely used in the corresponding fields.

Example 1

The present embodiment provides an RNA plasmid delivery system for the treatment of pulmonary fibrosis, comprising a plasmid carrying RNA fragments that can be used for the treatment of pulmonary fibrosis, said plasmid being capable of enriching in the host organ tissue and spontaneously forming endogenously in said host organ tissue a complex structure containing said RNA fragments, said complex structure being capable of delivering RNA capable of inhibiting pulmonary fibrosis into the lungs to effect the treatment of pulmonary fibrosis.

In this example, the plasmid also includes a promoter and a targeting tag. The plasmid comprises any one line or combination of lines as follows: the plasmid comprises a promoter-RNA sequence, a promoter-targeting label and a promoter-RNA sequence-targeting label, wherein each plasmid at least comprises an RNA segment and a targeting label, and the RNA segment and the targeting label are positioned in the same line or different lines. In other words, the plasmid may include only the promoter-RNA sequence-targeting tag, or may include a combination of the promoter-RNA sequence, the promoter-targeting tag, or a combination of the promoter-targeting tag, the promoter-RNA sequence-targeting tag.

In FIGS. 17-19, the in vivo enrichment, self-assembly and therapeutic effect on pulmonary fibrosis are shown when 1-2 RNA fragments and 1-2 targeting tags are included in the plasmid delivery system, respectively.

Further, the plasmid may also include flanking sequences, including 5 'flanking sequences and 3' flanking sequences, and loop sequences that enable the lines to be folded into the correct structure and expressed; the plasmid comprises any one line or combination of lines as follows: 5 '-promoter-5' flanking sequence-RNA sequence-loop sequence-compensation sequence-3 'flanking sequence, 5' -promoter-targeting label-5 'flanking sequence-RNA sequence-loop sequence-compensation sequence-3' flanking sequence.

As shown in fig. 20, the plasmid containing 3 different fragments of 5 'flanking sequence, loop sequence and 3' flanking sequence also has the effects of in vivo enrichment, self-assembly and treatment of pulmonary fibrosis, and the sequences are:

1. 3 5' flanking sequences with homology of more than 80%;

2. 3 loop sequences with homology more than 80%;

3. 3' flanking sequences with homology greater than 80%. The sequences are specifically shown in Table 2 below.

Wherein, the 5' flanking sequence is preferably ggatcctggaggcttgctgtgagaggctgtatgctgaattgaattc or a sequence with homology of more than 80 percent with the ggatcctggaggcttgctgagagctgctgtatgctgaattc, including sequences with homology of 85 percent, 90 percent, 92 percent, 95 percent, 98 percent, 99 percent and the like.

The loop sequence is preferably gttttgggccactgactgac or a sequence with homology of more than 80 percent, and comprises sequences with homology of 85 percent, 90 percent, 92 percent, 95 percent, 98 percent and 99 percent with gttttgggccactgactgac and the like.

<xnotran> 3' accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag 80% , accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag 85%, 90%, 92%, 95%, 98%, 99% . </xnotran>

The compensation sequence is a reverse complementary sequence of the RNA segment, and any 1-5 bases in the RNA segment are deleted. When the RNA fragment contains only one RNA sequence, the complementary sequence may be a reverse complement of the RNA sequence in which any 1-5 bases are deleted.

Preferably, the complementing sequence is the reverse complement of the RNA fragment, and any 1-3 bases in the RNA fragment are deleted. When the RNA fragment contains only one RNA sequence, the complementary sequence may be a reverse complement of the RNA sequence from which any 1 to 3 bases are deleted.

More preferably, the complementary sequence is the reverse complement of the RNA fragment, and any 1-3 consecutive bases in the complementary sequence are deleted. When the RNA fragment contains only one RNA sequence, the complementary sequence may be a reverse complement of the RNA sequence in which any of the bases arranged in sequence at positions 1 to 3 is deleted.

Most preferably, the complementing sequence is the reverse complement of the RNA fragment, and the 9 th and/or 10 th base is deleted. When the RNA fragment contains only one RNA sequence, the complementary sequence may be a reverse complement of the RNA sequence in which position 9 and/or position 10 is deleted. The deletion of the 9 th and 10 th bases is most effective.

The flanking sequence, the compensating sequence and the loop sequence are not randomly selected, but are determined based on a large amount of theoretical research and experiments, and the expression rate of the RNA fragment can be improved to the maximum extent under the coordination of the specific flanking sequence, the compensating sequence and the loop sequence.

In the case of plasmids carrying two or more strands, adjacent strands may be connected by the sequences 1-2-3; among them, the sequence 1 is preferably CAGATC, the sequence 2 may be a sequence consisting of 5 to 80 bases, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 bases, preferably a sequence consisting of 10 to 50 bases, more preferably a sequence consisting of 20 to 40 bases, and the sequence 3 is preferably TGGATC.

When the RNA plasmid delivery system carries multiple strands, the connecting sequence between adjacent strands, i.e., sequence 2, can be composed of multiple bases, and the plasmid has an enrichment effect after injection, as shown in fig. 21.

The sequence 2 is specifically shown in the following Table 3.

More preferably, in the case of plasmids carrying two or more strands, adjacent strands are connected by sequence 4 or a sequence having a homology of greater than 80% to sequence 4; wherein the sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACGACCAGTGGAC.

When the connecting sequence is sequence 4 or at least 3 sequences with homology of more than 80% with sequence 4, the constructed plasmid delivery system also has the effects of in vivo enrichment, self-assembly and pulmonary fibrosis treatment, as shown in figure 22.

The specific sequence is shown in the following table 4, wherein the sequence 4-1 is the above sequence 4, and the sequences 4-2/4-3/4-4 are homologous sequences with homology of more than 80% of the sequence 4-1.

The RNA fragments described above comprise 1, two or more specific RNA sequences of medical interest, which are capable of being expressed in the target receptor and the complementing sequences are not capable of being expressed in the target receptor. The RNA sequence can be an siRNA sequence, an shRNA sequence or an miRNA sequence, and is preferably an siRNA sequence.

An RNA sequence is 15-25 nucleotides (nt), preferably 18-22nt, such as 18nt, 19nt, 20nt, 21nt, 22 nt. The range of the sequence length is not arbitrarily selected, but determined by trial and error. A large number of experiments prove that under the condition that the length of the RNA sequence is less than 18nt, particularly less than 15nt, the RNA sequence is mostly ineffective and can not play a role, and under the condition that the length of the RNA sequence is more than 22nt, particularly more than 25nt, the cost of a line is greatly improved, the effect is not better than that of the RNA sequence with the length of 18-22nt, and the economic benefit is poor. Therefore, when the length of the RNA sequence is 15 to 25nt, particularly 18 to 22nt, both cost and action can be achieved, and the effect is the best.

The in vivo enrichment, self-assembly and therapeutic effect on pulmonary fibrosis at RNA sequence lengths of 18, 19, 21, respectively, in the plasmid delivery system are shown in figure 23.

The specific sequences are shown in Table 5 below.

21nt sequence	TATCTTTGCTGTCACAAGAGC
		19nt sequence	TAAAGTCAATGTACAGCTG
18nt sequence	TTCATGTCATGGATGGTG

The RNA sequence is selected from any one or more of the following: an antisense strand of miRNA-21, an siRNA of TGF-beta 1 gene, or an RNA sequence having a homology of greater than 80% with the above sequence, or a nucleic acid molecule encoding the above RNA.

The siRNA of the TGF-beta 1 gene comprises ACGGAAAUAACCUAGAGUGGGC, UGAACUUGUCAUAGAUUUCGUCU, UUGAACAUAUAUAUGCUG, UCUAACUACAGUAGUGUGUUCCCC, UCUCUCAGACUCUGGGGCCUCAG, other sequences which can inhibit the expression of the TGF-beta 1 gene and sequences with homology of more than 80 percent with the sequences.

The number of RNA sequences in the RNA fragment is 1, 2 or more. For example, the antisense strand of miRNA-21 alone or siRNA of TGF-. Beta.1 gene may be used on the same plasmid vector, or the antisense strand of miRNA-21 and siRNA of TGF-. Beta.1 gene may be used in combination on the same plasmid vector.

The gene circuit has the in vivo enrichment, self-assembly and pulmonary fibrosis treatment effects under the condition that the gene circuit comprises an miRNA-21 antisense chain and the 5 TGF-beta 1 gene siRNAs (siRNA-1, siRNA-2, siRNA-3, siRNA-4 and siRNA-5), as shown in figure 24.

Taking the combined use of "siRNA1" and "siRNA2" on the same plasmid vector as an example, the functional structural region of the plasmid vector can be represented as: (promoter-siRNA 1) -joining sequence- (promoter-siRNA 2) -joining sequence- (promoter-targeting tag), or (promoter-targeting tag-siRNA 1) -joining sequence- (promoter-targeting tag-siRNA 2), or (promoter-siRNA 1) -joining sequence- (promoter-targeting tag-siRNA 2), and the like.

More specifically, the functional structural region of the plasmid vector can be represented as: (5 ' -promoter-5 ' flanking sequence-siRNA 1-loop sequence-compensating sequence-3 ' flanking sequence) -linking sequence- (5 ' -promoter-5 ' flanking sequence-siRNA 2-loop sequence-compensating sequence-3 ' flanking sequence) -linking sequence- (5 ' -promoter-targeting tag) -5' flanking sequence-siRNA 1-loop sequence-compensating sequence-3 ' flanking sequence) -linking sequence- (5 ' -promoter-targeting tag-5 ' flanking sequence-siRNA 2-loop sequence-compensating sequence-3 ' flanking sequence), or (5 ' -promoter-5 ' flanking sequence-siRNA 1-loop sequence-compensating sequence-3 ' flanking sequence) -linking sequence- (5 ' -promoter-targeting tag-5 ' flanking sequence-siRNA 2-loop sequence-compensating sequence-3 ' flanking sequence), (5 ' -promoter-targeting tag-5 ' flanking sequence-1-siRNA 2-loop sequence-compensating sequence-3 ' flanking sequence), etc. In other cases, the same can be analogized, and the description is omitted here. The above linker sequence may be "sequence 1-sequence 2-sequence 3" or "sequence 4", and a bracket indicates a complete line (circuit).

Preferably, the RNA may also be obtained by modifying the RNA sequence (siRNA, shRNA or miRNA) in RNA, preferably 2' fluoropyrimidine. The 2 '-fluoropyrimidine modification is to replace 2' -OH of pyrimidine nucleotide on siRNA, shRNA or miRNA with 2'-F, wherein the 2' -F can make RNA enzyme in human body not easily recognize the siRNA, shRNA or miRNA, so that the stability of RNA in vivo transmission can be increased.

Specifically, the liver phagocytoses exogenous plasmids, up to 99% of the exogenous plasmids enter the liver, so that when the plasmids are used as a vector, the exogenous plasmids can be enriched in liver tissues without specific design, then the exogenous plasmids are opened to release RNA molecules (siRNA, shRNA or miRNA), the liver tissues spontaneously wrap the RNA molecules into exosomes, and the exosomes become RNA delivery mechanisms.

Preferably, in order to make the RNA delivery mechanism (exosome) have the ability of "precise targeting", we design a targeting tag in the plasmid injected into the body, and the targeting tag will also be assembled into the exosome by the liver tissue, especially when selecting some specific targeting tags, the targeting tag can be inserted into the exosome surface, thereby becoming a targeting structure capable of guiding exosome, which greatly improves the accuracy of the RNA delivery mechanism of the present invention, on one hand, the amount of exogenous plasmid to be introduced can be greatly reduced, and on the other hand, the efficiency of potential drug delivery can be greatly improved.

The targeting label is selected from one of peptides, proteins or antibodies with targeting functions, the selection of the targeting label is a process requiring creative labor, on one hand, an available targeting label needs to be selected according to target tissues, and on the other hand, the targeting label is ensured to be stably present on the surface of exosomes, so that the targeting function is achieved. The currently screened targeting peptides include, but are not limited to, RVG targeting peptide (nucleotide sequence is shown in SEQ ID No: 1), GE11 targeting peptide (nucleotide sequence is shown in SEQ ID No: 2), PTP targeting peptide (nucleotide sequence is shown in SEQ ID No: 3), TCP-1 targeting peptide (nucleotide sequence is shown in SEQ ID No: 4), MSP targeting peptide (nucleotide sequence is shown in SEQ ID No: 5); the target protein includes, but is not limited to, RVG-LAMP2B fusion protein (nucleotide sequence is shown in SEQ ID No: 6), GE11-LAMP2B fusion protein (nucleotide sequence is shown in SEQ ID No: 7), PTP-LAMP2B fusion protein (nucleotide sequence is shown in SEQ ID No: 8), TCP-1-LAMP2B fusion protein (nucleotide sequence is shown in SEQ ID No: 9), MSP-LAMP2B fusion protein (nucleotide sequence is shown in SEQ ID No: 10). The GE11 targeting peptide, GE11-LAMP2B fusion protein, is preferably used.

The RNA described above comprises 1, two or more specific RNA sequences of medical interest, which are capable of being expressed in the target recipient, and the complementing sequences are not capable of being expressed in the target recipient.

In addition, for the purpose of precise delivery, we tested various plasmid vector loading schemes, and developed another optimized scheme: the plasmid vector may also be composed of multiple plasmids with different structures, wherein one plasmid contains a promoter and a targeting tag, and the other plasmid contains a promoter and an RNA fragment. The targeting effect of the two plasmid vectors is not inferior to that generated by loading the same targeting tag and RNA fragment in one plasmid vector.

More preferably, when two different plasmid vectors are injected into a host, the plasmid vector with the RNA sequence can be injected first, and the plasmid vector with the targeting tag can be injected after 1-2 hours, so that a better targeting effect can be achieved.

The delivery systems described above may be used in mammals including humans.

As shown in FIG. 1A, in order to understand the distribution of plasmids in vivo, we performed a plate test on mice, sampled at time points (1 h, 3h, 6h, 9h, 12h, 24h, 72h, 168h, 720 h) after injection of the plasmids, transformed with plasmids extracted with spectinomycin, and observed the number of clones in liver, plasma, lung, brain, kidney, spleen, and as a result, as shown in FIGS. 1B, 1C, and 1D, it can be seen that the plasmids were distributed most in the liver of the mice, and reached a peak around 3h after injection, and were substantially metabolized after 12h after injection.

C57BL/6J mice were injected intravenously with CMV eGFP siR co-expressing eGFP protein and EGFR siRNA ^E The line shows that the eGFP fluorescence in the mouse liver gradually increases with time, reaches a peak value in about 12 hours, and decreases to a background level in 48 hours, and no eGFP signal is observed in other tissues, as shown in fig. 2.

Mice were injected with control plasmid (CMV-scrR), plasmid expressing EGFR siRNA (CMV-siR), respectively ^E ) And establishing a mouse hepatocyte in vitro model, and respectively detecting the injected CMV-scrR and CMV-siR ^E The results are shown in FIG. 3A, and it can be seen that CMV-siR was injected ^E The mouse hepatocyte exosomes of (a) have expression of siRNA.

We generally consider that binding to Ago2 protein is a necessary condition for siRNA function, i.e. siRNA in exosomes can bind to Ago2 protein, so we performed Ago2 immunoprecipitation experiments, and the results are shown in fig. 3B, fig. 3C. Wherein, input represents a sample which directly cracks and detects the exosome without immunoprecipitation, and represents a positive control.

The distribution of mature siRNA in different tissues after intravenous injection of plasmid into mice is shown in FIG. 4. As can be seen from FIG. 4A, EGFR-siRNA levels in plasma, exosomes, plasma without exosomes were time-dependent; as can be seen from FIG. 4B, the accumulation of mouse EGFR-siRNA in liver, lung, pancreas, spleen, kidney is time-dependent.

Injecting into mice respectivelyControl plasmid (CMV-scrR), 0.05mg/kg CMV-siR ^E Plasmid, 0.5mg/kg CMV-siR ^E Plasmid, 5mg/kg CMV-siR ^E Plasmid for detecting mouse liver, spleen, heart, lung, kidney, pancreas, brain, skeletal muscle and CD4 ⁺ Absolute siRNA (EGFR siRNA) levels in cells, the results are shown in FIG. 5A, and it can be seen that no siRNA expression was observed in the tissues of mice injected with control plasmids, and CMV-siR was injected ^E Level of siRNA expression in mouse tissues of plasmid vs CMV-siR ^E Plasmid concentrations were positively correlated. As shown in FIG. 5B, fluorescence In Situ Hybridization (FISH) also confirmed the level of siRNA expression and CMV-siR ^E The plasmid concentration is in positive correlation, namely the tissue distribution of EGFRsiRNA is dose-dependent.

Since the plasmid will express the Precursor (Precurror) after entering into the body and then be processed into the mature body (siRNA), the metabolism of the Precursor (Precurror) and the mature body (siRNA) in the liver after the plasmid is injected into the mouse is detected, and the result is shown in FIG. 6. It can be seen that the expression levels of the Precursor (precorsor) and the mature body (siRNA) in the liver of the mouse reached the peak at the time node of 6 hours after the injection of the plasmid, the metabolism of the mature body (siRNA) in the liver of the mouse was completed 36 hours after the injection of the plasmid, and the metabolism of the Precursor (precorsor) in the liver of the mouse was completed 48 hours after the injection of the plasmid.

The results of detecting the absolute siRNA levels in the plasma (exosome-free), exosome (exosome) and plasma of mice after injecting exogenous siRNA into common bile duct of mice are shown in fig. 7A. After the mice are injected with exogenous siRNA into common bile duct, the spleen, heart, lung, kidney, pancreas, brain, skeletal muscle and CD4 of the mice are respectively detected ⁺ Levels of siRNA in cells, the results are shown in FIG. 7B. These two graphs reflect that the kinetics of siRNA are almost the same in different tissues, and the distribution of siRNA is significantly different in different tissues.

The results of intravenous injection of siRNA using albumin ALB as a promoter, siRNA using CMV as a promoter, and siRNA without any promoter into mice were shown in fig. 8, in which the absolute siRNA levels in the mice were measured at 0h, 3h, 6h, 9h, 12h, 24h, 36h, and 48h after injection. As can be seen, the level of siRNA using CMV as a promoter in mice is the highest, namely the CMV as the promoter has the best effect.

We observed the inhibition of eGFP levels in mice by self-assembled eGFP sirnas by fluorescence assay as follows: eGFP transgenic mice were injected intravenously with PBS or 5mg/kg CMV-siR ^G Or CMV-RVG-siR ^G Plasmid, mice sacrificed 24 hours after treatment and eGFP fluorescence levels detected in frozen sections, fig. 9A shows representative fluorescence microscopy images, where green indicates a positive eGFP signal, blue indicates DAPI stained nuclei, scale bar: 100 μm, as seen by CMV-RVG-siR ^G The plasmid has more obvious inhibition effect on the mouse eGFP; intravenous injection of PBS or CMV-scrR or CMV-siR into eGFP transgenic mice ^E Plasmid, 24 hours after treatment mice were sacrificed and eGFP fluorescence levels were measured in frozen sections, FIG. 9B is PBS, CMV-siR injections ^E 、CMV-RVG-siR ^E The Fluorescence intensity (Fluorescence intensity) column comparison graphs of the heart, the lung, the kidney, the pancreas, the brain and the skeletal muscle of the mouse show that the Fluorescence intensity comparison of the mouse at the liver, the spleen, the lung and the kidney is more obvious.

For injections of PBS, CMV-scrR, CMV-siR, respectively ^E The mice (a) were tested for their glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), total Bilirubin (TBIL), blood Urea Nitrogen (BUN), serum alkaline phosphatase (ALP), creatinine (CREA) content, thymus weight, spleen weight, and peripheral blood cell percentage (percent of peripheral blood cells), and the results are shown in FIG. 10, in which 10A-F are PBS, mouse CMV-scrR, CMV-siR cells, and 10A-F are injected into the mice, respectively ^E The comparison graphs of glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase, total bilirubin, blood urea nitrogen, serum alkaline phosphatase and creatinine content are shown in the figure 10G, the comparison graphs of tissues of liver, lung, spleen and kidney of mice are shown in the figure 10H-I, the comparison graphs of tissues of thymus and spleen of mice are shown in the figure 10J, and the comparison graphs of percentage of peripheral blood cells (peripheral in peripheral blood cells) of mice are shown in the figure 10J.

The results showed that PBS, CMV-scrR, CMV-siR were injected ^E The contents of ALT, AST and the like of mice, the weight of thymus gland, the weight of spleen and the percentage of peripheral blood cells are almost the same, and CMV-siR is injected ^E The mice had no tissue damage to the liver, lung, spleen and kidney as compared with the mice injected with PBS.

The above experiments can show that the safety and reliability of the RNA delivery system provided by the application are fully verified and the druggability is very good by taking the plasmid as a carrier and taking the plasmid as a mature injectant. The RNA sequence which finally exerts the effect is encapsulated and conveyed by the endogenous exosome, no immune reaction exists, and the safety of the exosome does not need to be verified. The delivery system can deliver various small-molecule RNAs and has strong universality. Moreover, the preparation of the plasmid is much cheaper than that of exosome or substances such as protein, polypeptide and the like, and the economy is good. The RNA delivery systems provided herein are capable of self-assembly with an AGO in vivo ₂ Tightly combined and enriched into a composite structure (exosome), not only can prevent the exosome from being degraded prematurely and maintain the stability of the exosome in circulation, but also is beneficial to the absorption of receptor cells, the intracytoplasmic release and the escape of lysosomes, and the required dosage is low.

Example 2

On the basis of example 1, this example provides a drug. The medicament comprises a plasmid carrying an RNA fragment to be delivered, wherein the plasmid can be enriched in organ tissues of a host and can endogenously and spontaneously form a composite structure containing the RNA fragment in the organ tissues of the host, and the composite structure can enter and combine with target tissues so as to deliver the RNA fragment into the target tissues. Wherein, the RNA segment to be delivered is an RNA segment with a therapeutic effect on pulmonary fibrosis, and the target tissue is lung.

Optionally, the RNA fragment comprises 1, two or more specific RNA sequences of medical interest, said RNA sequences being siRNA, shRNA or miRNA of medical interest.

In the plasmid delivery system, in the case of carrying RNA fragments, there are in vivo enrichment, spontaneous formation of complex structure and therapeutic effect on pulmonary fibrosis, as shown in fig. 16, the grouping cases of RNA fragments include but are not limited to:

1) siRNA1 alone, siRNA2 alone, shRNA1 alone, shRNA2 alone, miRNA1 alone and miRNA2 alone;

2) RNA fragments containing any 2 RNA sequences in 1) above;

3) The RNA fragment of 1) above, which comprises any 3 RNA sequences.

The specific sequences are shown in table 1 below.

Optionally, the plasmid further comprises a promoter and a targeting tag, wherein the targeting tag is capable of forming a targeting construct of the composite structure in an organ tissue of the host, the targeting construct is located on the surface of the composite structure, and the composite structure is capable of targeting and binding to a target tissue through the targeting construct to deliver the RNA fragment into the target tissue.

For the explanation of the above plasmids, RNA fragments, targeting tags, etc., reference can be made to example 1, which is not repeated herein.

Further, the RNA capable of treating pulmonary fibrosis is selected from any one or more of the following RNAs: an antisense strand of miRNA-21, an siRNA of TGF-. Beta.1 gene, or a nucleic acid molecule encoding the above RNA.

The drug can be delivered to target tissues by the RNA delivery system described in example 1 after entering a human body by oral administration, inhalation, subcutaneous injection, intramuscular injection or intravenous injection, and then exert a therapeutic effect.

The medicament of this embodiment may further comprise a pharmaceutically acceptable carrier including, but not limited to, diluents, buffers, emulsions, encapsulating agents, excipients, fillers, adhesives, sprays, transdermal absorbents, humectants, disintegrants, absorption enhancers, surfactants, colorants, flavors, adjuvants, desiccants, adsorbent carriers, and the like.

The dosage form of the drug provided in this embodiment may be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes, and the like.

The drug can also be used in combination with other drugs for treating pulmonary fibrosis to improve the treatment effect, such as: glucocorticoids, immunosuppressive agents, anticoagulants, and the like.

The medicine provided by the embodiment takes the plasmid as a carrier and the plasmid as a mature injectant, the safety and reliability of the medicine are fully verified, and the medicine performance is very good. The RNA sequence which finally exerts the effect is encapsulated and conveyed by the endogenous exosome, no immune reaction exists, and the safety of the exosome does not need to be verified. The medicine can deliver various small molecular RNAs and has strong universality. Moreover, the preparation of the plasmid is much cheaper than that of exosome or substances such as protein, polypeptide and the like, and the economy is good. The drugs provided herein are capable of self-assembling with an AGO in vivo ₂ Tightly combined and enriched into a composite structure (exosome), not only can prevent the exosome from being degraded prematurely and maintain the stability of the exosome in circulation, but also is beneficial to the absorption of receptor cells, the intracytoplasmic release and the escape of lysosomes, and the required dosage is low.

Example 3

On the basis of example 1 or 2, the present example provides the use of the RNA plasmid delivery system for the treatment of pulmonary fibrosis in a medicament for the treatment of pulmonary fibrosis. This example specifically illustrates the use of the RNA delivery system in the treatment of pulmonary fibrosis in conjunction with the following assay.

This example sets up 8 test groups and 3 control groups. The test groups are respectively an Anti-miR-21 (1 mg/kg) group, an Anti-miR-21 (5 mg/kg) group, an Anti-miR-21 (10 mg/kg) group, a TGF-beta 1siRNA (1 mg/kg) group, a TGF-beta 1siRNA (5 mg/kg) group, a TGF-beta 1siRNA (10 mg/kg) group, an Anti-miR-21 TGF-beta 1siRNA (10 mg/kg) group and a Pirfenidone (300 mg/kg) group, and the control groups are respectively a Normal group, a PBS group and a scrRNA group.

Wherein, the Anti-miR-21 (1 mg/kg) group, the Anti-miR-21 (5 mg/kg) group and the Anti-miR-21 (10 mg/kg) group are respectively injecting 1mg/kg, 5mg/kg and 10mg/kg of miR-21siRNA plasmids, 1mg/kg, 5mg/kg and 10mg/kg of TGF-beta 1siRNA (1 mg/kg) group, 5mg/kg of TGF-beta 1siRNA (5 mg/kg) group and 10mg/kg of TGF-beta 1siRNA (10 mg/kg) group into the tail vein of mice with pulmonary fibrosis, respectively injecting 10mg/kg of Anti-miR-21 and TGF-beta 1siRNA plasmids, 300mg/kg of Pirfenidone (300 mg/kg) group into the tail vein of mice with pulmonary fibrosis PBS group, injecting 10mg/kg of Pirfenidone-miR-21 and TGF-beta 1siRNA plasmids into the tail vein of mice with pulmonary fibrosis, respectively injecting 300mg/kg of Pirfenidone-PBS group into the tail vein of mice with pulmonary fibrosis, respectively injecting Normal control rRNA group and Normal control group into the mice with pulmonary fibrosis.

The hydroxyproline content of each group of mice was measured, and the results are shown in fig. 11. Hydroxyproline is the main component of collagen, the content of hydroxyproline reflects the degree of pulmonary fibrosis, and as can be seen from fig. 11, the hydroxyproline content of mice in the group of Anti-miR-21 (5 mg/kg), the group of Anti-miR-21 (10 mg/kg), the group of TGF-beta 1siRNA (10 mg/kg) and the group of Anti-miR-21+ TGF-beta 1siRNA (10 mg/kg) is relatively low, and the pulmonary fibrosis is inhibited.

The lung of each group of mice was fluorescence stained, and the results are shown in FIG. 12, in which the green part represents type I Collagen (Collagen I), the red part represents α -SMA, and the blue part represents DAPI. It can be seen that the PBS group and the scrRNA group mice have more collagen I and alpha-SMA contents, while the experimental group mice have relatively less collagen I and alpha-SMA contents, especially the Anti-miR-21 (5 mg/kg) group, the Anti-miR-21 (10 mg/kg) group and the Anti-miR-21+ TGF-beta 1siRNA (10 mg/kg) group have almost no expression of collagen I and alpha-SMA.

The lungs of each group of mice were individually Masson's trichrome stained, and the results are shown in fig. 13. It can be seen that alveolar spaces of PBS group and scrna group mice were severely damaged, resulting in interstitial lung collagen, while the experimental group significantly reduced these phenomena.

The H & E staining of the lungs of each group of mice was performed individually and the results are shown in FIG. 14. It can be seen that the PBS group and the scrRNA group mice have widened alveolar space, inflammatory cells are infiltrated, and alveolar structures are damaged, while the lung tissues of the test group are normal.

The Normal group, PBS group, scrRNA group, TGF-. Beta.1 siRNA (1 mg/kg) group, TGF-. Beta.1 siRNA (5 mg/kg) group, TGF-. Beta.1 siRNA (10 mg/kg) group, pirfenidone (300 mg/kg) group mouse TGF-. Beta.1 protein level, TGF-. Beta.1 mRNA level were detected by western blot, respectively, and the results are shown in FIG. 15A to FIG. 15C, and it can be seen that mouse TGF-. Beta.1 protein level and TGF-. Beta.1 mRNA level were lowest in TGF-. Beta.1 siRNA (10 mg/kg) group. This demonstrates that TGF-. Beta.1 can be successfully delivered to the lung for function after tail vein injection of the corresponding siRNA expression plasmid.

Relative miR-21 levels of mice in a Normal group, a PBS group, a scrRNA group, an Anti-miR-21 (1 mg/kg) group, an Anti-miR-21 (5 mg/kg) group and an Anti-miR-21 (10 mg/kg) group were respectively detected, and as a result, as shown in FIG. 15D, the relative miR-21 level of the mice in the Anti-miR-21 (10 mg/kg) group was the highest. This demonstrates that the antisense strand of miR-21 can be successfully delivered to the lung for function after tail vein injection of the corresponding antisense strand expression plasmid.

The above experiments demonstrate that CMV-siR is encapsulated using liver-compatible plasmids ^miR-21 、CMV-siR ^TGF-β1 、CMV-siR ^miR ^-21+TGF-β1 The circuit can obviously relieve the degree of pulmonary fibrosis, and has great drug potential and clinical research value.

In this document, "upper", "lower", "front", "rear", "left", "right", and the like are used only to indicate relative positional relationships between relevant portions, and do not limit absolute positions of the relevant portions.

In this document, "first", "second", and the like are used only for distinguishing one from another, and do not indicate the degree and order of importance, the premise that each other exists, and the like.

In this context, "equal," "same," and the like are not strictly mathematical and/or geometric limitations, but also encompass errors that may be understood by one skilled in the art and that may be allowed for manufacturing or use, etc.

Unless otherwise indicated, numerical ranges herein include not only the entire range within its two endpoints, but also several sub-ranges subsumed therein.

The preferred embodiments and examples of the present application have been described in detail with reference to the accompanying drawings, but the present application is not limited to the embodiments and examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application.

Claims

1. An RNA plasmid delivery system for the treatment of pulmonary fibrosis, comprising a plasmid carrying RNA fragments that can be used for the treatment of pulmonary fibrosis, said plasmid being capable of enriching in a host's organ tissue and spontaneously forming endogenously in said host's organ tissue a complex structure containing said RNA fragments, said complex structure being capable of delivering into the lungs RNA capable of inhibiting pulmonary fibrosis, so as to effect the treatment of pulmonary fibrosis.

2. The RNA plasmid delivery system for the treatment of pulmonary fibrosis of claim 1, wherein said RNA segment comprises 1, two or more specific RNA sequences of medical significance which are siRNA, shRNA or miRNA sequences of medical significance capable of inhibiting or hindering the development of pulmonary fibrosis.

3. The RNA plasmid delivery system of claim 1, wherein the plasmid further comprises a promoter and a targeting tag, wherein the targeting tag is capable of forming a targeting construct of the complex structure in an organ tissue of the host, wherein the targeting construct is located on the surface of the complex structure, and wherein the complex structure is capable of seeking and binding a target tissue through the targeting construct to deliver the RNA fragment into the target tissue.

4. The RNA plasmid delivery system for the treatment of pulmonary fibrosis of claim 3, wherein said plasmid comprises any one or a combination of the following lines: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; each plasmid at least comprises an RNA segment and a targeting label, and the RNA segment and the targeting label are positioned in the same line or different lines.

5. The RNA plasmid delivery system for the treatment of pulmonary fibrosis of claim 4, wherein the plasmid further comprises flanking sequences, compensating sequences and loop sequences that enable the lines to fold into the correct structure and be expressed, the flanking sequences comprising a 5 'flanking sequence and a 3' flanking sequence;

6. The RNA plasmid delivery system for the treatment of pulmonary fibrosis of claim 5, wherein the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with greater than 80% homology thereto;

The compensation sequence is a reverse complementary sequence of the RNA segment, and any 1-5 bases in the RNA segment are deleted.

7. The RNA plasmid delivery system for the treatment of pulmonary fibrosis of claim 4, wherein, in the presence of at least two lines in the plasmid, adjacent lines are connected by a sequence consisting of sequences 1-3;

8. The RNA plasmid delivery system for the treatment of pulmonary fibrosis of claim 7, wherein, in the presence of at least two lines in the plasmid, adjacent lines are connected by sequence 4 or a sequence having greater than 80% homology to sequence 4;

wherein the sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACGACCAGTGGAC.

9. The RNA plasmid delivery system for the treatment of pulmonary fibrosis of claim 1, wherein said organ tissue is the liver and said complex structure is an exosome.

10. The RNA plasmid delivery system for the treatment of pulmonary fibrosis of claim 3, wherein said targeting tag is selected from a targeting peptide or a targeting protein having a targeting function.

11. The RNA plasmid delivery system for the treatment of pulmonary fibrosis of claim 10, wherein said targeting peptide comprises RVG targeting peptide, GE11 targeting peptide, PTP targeting peptide, TCP-1 targeting peptide, MSP targeting peptide;

12. The RNA plasmid delivery system for the treatment of pulmonary fibrosis of claim 10, wherein the targeting tag is a GE11 targeting peptide or a GE11-LAMP2B fusion protein.

13. The RNA plasmid delivery system for the treatment of pulmonary fibrosis of claim 2, wherein said RNA sequence is 15-25 nucleotides in length.

14. The RNA plasmid delivery system for the treatment of pulmonary fibrosis of claim 13, wherein said RNA sequence is selected from any one or more of: an antisense strand of miRNA-21, an siRNA of TGF-beta 1 gene, or an RNA sequence having a homology of greater than 80% with the above sequence, or a nucleic acid molecule encoding the above RNA.

15. The RNA plasmid delivery system of claim 14, wherein the siRNA of TGF- β 1 gene comprises acggaaauaacccuagugggc, ugacuugucauaguucgu, uugaagaacauauauauaugcug, ucauaacaguaguguuccc, ucucagacuuggggccucag, other sequences with a sequence that inhibits expression of TGF- β 1 gene and sequences with greater than 80% homology to the above sequences.

16. The RNA plasmid delivery system for the treatment of pulmonary fibrosis of claim 1, wherein said delivery system is a delivery system for use in mammals, including humans.

17. Use of an RNA plasmid delivery system according to any of claims 1 to 16 for the treatment of pulmonary fibrosis in medicine.

18. The use of claim 17, wherein the medicament is for the treatment of pulmonary fibrosis and related disorders, and the administration of the medicament comprises oral administration, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection.