CN114685585B

CN114685585B - Nucleotide sequence, double-stranded oligonucleotide, pharmaceutical composition and conjugate, preparation method and application

Info

Publication number: CN114685585B
Application number: CN202111659092.0A
Authority: CN
Inventors: 梁子才; 杨志伟; 张鸿雁; 曹力强; 李海涛
Original assignee: Suzhou Ruibo Biotechnology Co ltd
Current assignee: Suzhou Ruibo Biotechnology Co ltd
Priority date: 2020-12-31
Filing date: 2021-12-30
Publication date: 2024-06-04
Anticipated expiration: 2041-12-30
Also published as: CN114685585A

Abstract

A nucleotide sequence, each of which is a modified or unmodified nucleotide, wherein the nucleotide sequence comprises a nucleotide sequence I, which is a nucleotide sequence in which at least one of nucleotides 2 to 8 in the 5 '-terminal-3' -terminal direction in the nucleotide sequence a is replaced with an acyclic abasic group, the nucleotide sequence a has 16 to 30 nucleotides, and the nucleotide sequence a is reverse-complementary to at least 14 nucleotides of a stretch of nucleotide sequence in a target mRNA, the acyclic abasic group having a structure as shown in formula (101). The nucleotide sequence, the double-stranded oligonucleotide containing the nucleotide sequence as an antisense strand, and the pharmaceutical composition and the siRNA conjugate containing the double-stranded oligonucleotide can effectively reduce off-target effect and achieve the purpose of reducing toxicity.

Description

Nucleotide sequence, double-stranded oligonucleotide, pharmaceutical composition and conjugate, preparation method and application

Technical Field

The present disclosure relates to a nucleotide sequence, double-stranded oligonucleotides containing the nucleotide sequence, pharmaceutical compositions and conjugates, and methods of making and using the same.

Background

Nucleic acid drugs are increasingly showing excellent research and development and application potential as important novel drugs. Among them, double-stranded oligonucleotides are known as pharmaceutically active ingredients. In recent years, there has been great progress in the field of double-stranded oligonucleotide patent medicine. In the research of double-stranded oligonucleotide patent medicine, off-target effect is one of important effects related to drug toxicity.

However, nucleic acid drugs such as double-stranded oligonucleotides, which exhibit excellent pharmaceutical activity in many preclinical pharmaceutical studies, are difficult to use in practical drug development due to their toxicity resulting from significant off-target effects. In this regard, those skilled in the art are continually striving to develop nucleic acid pharmaceuticals, such as double-stranded oligonucleotides, that are synthesized with good pharmaceutical activity and low off-target effects. Therefore, how to reduce the off-target effect of nucleic acid drugs while maintaining good pharmaceutical activity is an important problem to be solved in the art.

Disclosure of Invention

In order to develop a nucleotide sequence which exhibits a reduced off-target effect while having good pharmaceutical activity, and a double-stranded oligonucleotide comprising the same, the inventors of the present disclosure have unexpectedly found that the use of an acyclic dealkalization group instead of an original nucleotide at a specific position in the nucleotide sequence can effectively reduce the off-target effect of the nucleotide sequence and a nucleic acid drug comprising the nucleotide sequence while maintaining substantially higher pharmaceutical activity. A nucleic acid drug having a good balance of inhibition efficiency and safety of target mRNA is obtained. Thus, the inventors made the following inventions:

In one aspect, the present disclosure provides a nucleotide sequence, each nucleotide in the nucleotide sequence being a modified or unmodified nucleotide, wherein the nucleotide sequence has 16-30 nucleotides; the nucleotide sequence is reversely complementary to a nucleotide sequence in the target mRNA by at least 14 nucleotides; at least one nucleotide between nucleotides 2 to 8 of the nucleotide sequence is replaced with an acyclic dealkalization group according to the direction from the 5 'end to the 3' end.

In another aspect, the present disclosure also provides a double-stranded oligonucleotide comprising an antisense strand formed from the nucleotide sequence of the present disclosure and a sense strand at least partially reverse-complementary thereto.

In yet another aspect, the present disclosure also provides a double-stranded oligonucleotide pharmaceutical composition comprising the present disclosure, the pharmaceutical composition comprising a double-stranded oligonucleotide of the present disclosure and a pharmaceutically acceptable carrier.

In yet another aspect, the present disclosure also provides a conjugate comprising a double-stranded oligonucleotide of the present disclosure, the conjugate comprising a double-stranded oligonucleotide of the present disclosure and a ligand conjugated to the double-stranded oligonucleotide.

In yet another aspect, the present disclosure provides the use of a double-stranded oligonucleotide, pharmaceutical composition or conjugate of the present disclosure in the manufacture of a medicament for the treatment and/or prevention of a pathological condition or disease caused by expression of a specific gene in a hepatocyte.

In yet another aspect, the present disclosure provides a method of treating a pathological condition or disease caused by expression of a specific gene in hepatocytes, the method comprising administering a double-stranded oligonucleotide, pharmaceutical composition or conjugate of the present disclosure to a subject suffering from the disease.

In yet another aspect, the present disclosure provides a method of inhibiting expression of a particular gene in a hepatocyte, the method comprising contacting a double stranded oligonucleotide, pharmaceutical composition or conjugate of the present disclosure with the hepatocyte.

In addition, the present disclosure provides a kit comprising the double-stranded oligonucleotide, pharmaceutical composition or conjugate of the present disclosure.

Incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Drawings

FIG. 1 is a bar graph of the relative expression of APOC3 mRNA in the psi-CHECK system after transfection of conjugates 1-8 or reference conjugate 1, respectively.

FIG. 2 is a bar graph of the relative expression of APOC3 mRNA in the psi-CHECK system after transfection of conjugates 9-11, respectively, or reference conjugates 2-4, respectively.

FIG. 3 is a line graph of serum triglyceride levels in mice over a 36 day period after transfection of conjugates 26-30, or reference conjugate 5, or negative reference conjugate, respectively.

FIG. 4 is a bar graph of the number of genes whose gene expression levels of non-target genes are significantly up-regulated, down-regulated, and unchanged after transfection of conjugate 32, or reference conjugates 15-17, or negative reference conjugates, respectively.

Fig. 5 is a semi-quantitative result of a stability assay of the siRNA conjugates of the present disclosure in lysosomal lysates in vitro.

Advantageous effects

The nucleotide sequences, double-stranded oligonucleotides, pharmaceutical compositions or conjugates of the present disclosure have significantly reduced off-target effects while simultaneously. Also exhibits high target mRNA regulatory activity and good stability, as described below.

First, the nucleotide sequences, double-stranded oligonucleotides, pharmaceutical compositions or conjugates of the present disclosure may have significantly lower off-target effects in vitro or in vivo. For example, in the in vitro psi-CHECK system, the conjugates of the present disclosure exhibit significantly reduced off-target effects over the tested concentration range with no greater than 25% of the inhibitory activity on off-target sequences as compared to siRNA conjugates having the same sequence but containing no acyclic dealkali groups. As another example, the double-stranded oligonucleotides of the present disclosure each have significantly reduced off-target effects as compared to various double-stranded oligonucleotides that do not include different modification schemes of acyclic abasic groups. Also, the siRNA conjugates of the present disclosure with different numbers of acyclic dealkali groups of different steric configurations at different positions all showed significantly reduced off-target effects compared to siRNA conjugates without acyclic dealkali groups. As another example, the conjugates of the present disclosure for modulating HAO1 mRNA inhibitory activity exhibit lower numbers of gene up-or down-regulation than conjugates with other acyclic dealkalization groups, or conjugates without acyclic dealkalization groups, exhibiting significantly lower off-target effects. As another example, the siRNA conjugates of the present disclosure used to modulate ANGPTL3 mRNA do not have so high inhibitory activity on off-target sequences that the value of IC ₅₀ can be calculated, i.e., do not exhibit significant off-target effects

Second, the nucleotide sequences, double-stranded oligonucleotides, pharmaceutical compositions or conjugates of the present disclosure may exhibit higher target mRNA modulating activity in vitro or in vivo, and even higher target mRNA inhibiting activity than siRNA conjugates without an acyclic abasic group. For example, at a concentration of 50nM, the siRNA conjugates of the present disclosure exhibit APOC3mRNA inhibition activity of up to 71.43%, even 77.14%, 9.52% higher than a reference conjugate without an acyclic dealkalization group. For another example, the siRNA conjugates provided by the present disclosure exhibit higher APOC3mRNA inhibition activity in mouse liver primary cells, and at a concentration of 20nM of the siRNA conjugate, the APOC3mRNA inhibition rates all reached 91.5% or more, all higher than the reference conjugate. And, under the condition that the administration dosage is 3mg/kg, the inhibition rate of the siRNA conjugate to the triglyceride is always maintained to be more than 78% and the maximum inhibition rate is up to 84.63% within 36 days after single administration. As another example, the double-stranded oligonucleotides comprising acyclic dealkalization groups of the present disclosure have significantly higher HBV mRNA inhibition activity than double-stranded oligonucleotides comprising other acyclic dealkalization groups. For another example, at an siRNA conjugate concentration of 20nM, the conjugates of the present disclosure all achieved greater than 91.5% inhibition of HAO1 mRNA. At a concentration of 50nM, the conjugates of the present disclosure exhibited an ANGPTL 3mRNA inhibition activity of up to 82.5% greater than the reference conjugate by 28%. And, at an siRNA conjugate concentration of 20nM, the conjugates of the present disclosure all have an inhibition rate on ANGPTL 3mRNA in mice of 92% or more, which is 64.33% and 65.14% higher than the inhibition rate of the reference conjugate.

Third, the nucleotide sequences, double-stranded oligonucleotides, pharmaceutical compositions or conjugates of the present disclosure have good stability, e.g., the conjugates of the present disclosure exhibit very high stability in lysosomal lysates and can be maintained without degradation for long periods of time.

Additional features and advantages of the present disclosure will be set forth in the detailed description which follows.

Detailed Description

The following describes specific embodiments of the present disclosure in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.

In the present disclosure, unless otherwise specified, HBV gene refers to a viral gene of Hepatitis B Virus (HBV), for example, a gene having a sequence as shown in Genbank accession NC_003977.2, HBV mRNA refers to mRNA transcribed from the above HBV gene; APOC3 mRNA refers to mRNA having a sequence as shown in Genbank accession No. NM-000040.3, and APOC3 gene refers to a gene transcribed from the above APOC3 mRNA; ANGPTL3 mRNA refers to an mRNA having a sequence as shown in Genbank accession No. nm_014495.4, the ANGPTL3 gene refers to a gene that transcribes the ANGPTL3 mRNA, HAO1 mRNA refers to an mRNA having a sequence as shown in Genbank accession No. nm_017545.3, and the HAO1 gene refers to a gene that transcribes the HAO1 mRNA.

Definition of the definition

In the above and below, upper case C, G, U, A indicates the base composition of the nucleotide unless otherwise specified; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that phosphorothioate linkages are between two nucleotides adjacent to the letter s; p1 indicates that one nucleotide adjacent to the right of P1 is a 5' -phosphonucleotide or a 5' -phosphoanalog modified nucleotide, the letter composition VP indicates that one nucleotide adjacent to the right of the letter composition VP is a vinyl phosphate modified nucleotide, the letter composition Ps indicates that one nucleotide adjacent to the right of the letter composition Ps is a phosphorothioate modified nucleotide, and the capital letter P indicates that one nucleotide adjacent to the right of the letter P is a 5' -phosphonucleotide. Letter combination (GLY) represents that two nucleotides adjacent to the left and right of (GLY) are linked by an acyclic dealkalization group represented by formula (a 101); (GLY-Ac) represents that two nucleotides adjacent to the left and right of the (GLY-Ac) are linked by an acyclic dealkalization group represented by formula (a 102); (GLY-Ph) two nucleotides adjacent to the (GLY-Ph) side are linked by an acyclic dealkalization group represented by formula (a 103); (GLY-TOS) represents that two nucleotides adjacent to the (GLY-TOS) are linked by an acyclic abasic group represented by formula (a 104); (GLY-iBu) represents that two nucleotides adjacent to the (GLY-iBu) are linked by an acyclic dealkalization group represented by formula (a 105); (GLY-laev) means that two nucleotides adjacent to the left and right of (GLY-laev) are linked by an acyclic dealkalization group represented by formula (a 106); (GLY-Cro) means that two nucleotides adjacent to the (GLY-Cro) are linked by an acyclic dealkalization group represented by the formula (A107).

In the above and below, the "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide is substituted with fluorine, and the "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorine group.

"Nucleotide analog" refers to a group that is capable of replacing a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Such as an iso-nucleotide, a bridged nucleotide (bridged nucleic acid, abbreviated as BNA) or an acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is replaced with a methoxy group.

In the present context, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double stranded nucleic acid molecule, the bases of one strand pair with the bases on the other strand in a complementary manner. In DNA, the purine base adenine (a) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) is always paired with the pyrimidine base cytosine (C). Each base pair includes a purine and a pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary to each other, and the sequence of the strand can be deduced from the sequence of its complementary strand. Accordingly, "mismatch" means in the art that bases at corresponding positions do not exist in complementary pairs in a double-stranded nucleic acid.

In the above and in the following, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there is no more than 1 base mismatch between two nucleotide sequences; "complete reverse complement" means that there is no base mismatch between the two nucleotide sequences.

In the above and below, the "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position is changed as compared with the former, for example, when one nucleotide base is A, when the corresponding nucleotide base at the same position of the former is U, C, G or T, it is determined that there is a nucleotide difference between the two nucleotide sequences at the position. In some embodiments, a nucleotide difference is also considered to occur at an original position when the nucleotide is replaced with an abasic nucleotide or nucleotide analog.

In the foregoing and in the following, and particularly in describing the methods of preparing the double-stranded oligonucleotides, pharmaceutical compositions and/or siRNA conjugates of the present disclosure, the nucleoside monomer (nucleoside monomer) refers to a modified or unmodified nucleoside monomer (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites also referred to as Nucleoside phosphoramidites) used in phosphoramidite solid phase synthesis, depending on the type and order of nucleotides in the double-stranded oligonucleotide, pharmaceutical composition and/or siRNA conjugate to be prepared, unless otherwise specified. Phosphoramidite solid phase synthesis is a method well known to those skilled in the art for use in RNA synthesis. Nucleoside monomers useful in the present disclosure are all commercially available.

In the context of the present disclosure, unless otherwise indicated, "conjugated" means that two or more chemical moieties each having a particular function are linked to each other by covalent linkage; accordingly, "conjugate" refers to a compound formed by covalent linkage between the chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently attaching one or more chemical moieties having specific functions to an siRNA. Hereinafter, the siRNA conjugates of the present disclosure are also sometimes simply referred to as "conjugates". The siRNA conjugate is understood to be a general term of the siRNA conjugate, a general term of the siRNA conjugate shown in formula (305) and formula (307), or an siRNA conjugate shown in formula (305), formula (307), formula (308) according to the context. In the context of the present disclosure, a "conjugate molecule" is understood to be a specific compound that can be conjugated to an siRNA by reaction, ultimately forming the presently disclosed siRNA conjugate.

In the foregoing or hereinafter, a substituted group, such as a substituted alkyl group, a substituted alkoxy group, a substituted amino group, a substituted aliphatic group, a substituted heteroaliphatic group, a substituted acyl group, a substituted aryl group, or a substituted heteroaryl group, unless otherwise indicated, "substituted" group means a group in which a hydrogen atom is replaced with one or more substituents. For example, "substituted alkoxy" refers to a group formed by substitution of one or more hydrogen atoms in an alkoxy group with a substituent. Those skilled in the art will appreciate that various substituents may be included in compounds useful in the application of the present disclosure, as long as the introduction of the substituent does not affect the function of the present disclosure, and can be used in the present disclosure for the purpose of the present disclosure. In some embodiments, the substituents are selected from the group consisting of: c ₁-C₁₀ alkyl, C ₆-C₁₀ aryl, C ₅-C₁₀ heteroaryl, C ₁-C₁₀ haloalkyl, -OC ₁-C₁₀ alkyl, -OC ₁-C₁₀ alkylphenyl, -C ₁-C₁₀ alkyl-OH-OC ₁-C₁₀ haloalkyl, -SC ₁-C₁₀ alkyl, -SC ₁-C₁₀ alkylphenyl, -C ₁-C₁₀ alkyl-SH, -SC ₁-C₁₀ haloalkyl, Halogen substituent, -OH, -SH, -NH ₂、-C₁-C₁₀ alkyl-NH ₂、-N(C₁-C₁₀ alkyl) (C ₁-C₁₀ alkyl), -NH (C ₁-C₁₀ alkyl), -N (C ₁-C₁₀ alkyl) (C ₁-C₁₀ alkylphenyl) -NH (C ₁-C₁₀ alkylphenyl), cyano, nitro, -CO ₂H、-C(O)O(C₁-C₁₀ alkyl), -CON (C ₁-C₁₀ alkyl) (C ₁-C₁₀ alkyl), -CONH (C ₁-C₁₀ alkyl), -CONH ₂,-NHC(O)(C₁-C₁₀ alkyl), -NHC (O) (phenyl), -N (C ₁-C₁₀ alkyl) C (O) (C ₁-C₁₀ alkyl), -N (C ₁-C₁₀ alkyl) C (O) (phenyl), -C (O) C ₁-C₁₀ alkyl, -C (O) C ₁-C₁₀ alkylphenyl, -C (O) C ₁-C₁₀ haloalkyl, -OC (O) C ₁-C₁₀ alkyl, -SO ₂(C₁-C₁₀ alkyl), -SO ₂ (phenyl), -SO ₂(C₁-C₁₀ haloalkyl, -SO ₂NH₂、-SO₂NH(C₁-C₁₀ alkyl), -SO ₂ NH (phenyl), -NHSO ₂(C₁-C₁₀ alkyl), -NHSO ₂ (phenyl) and-NHSO ₂(C₁-C₁₀ haloalkyl). In some embodiments, the substituent is one of C ₁-C₃ alkyl, C ₆-C₈ aryl, -OC ₁-C₃ alkyl, -OC ₁-C₃ alkylphenyl, halogen, -OH, -NH ₂, cyano, or nitro. Those skilled in the art will appreciate that for any group comprising one or more substituents, these groups are not intended to introduce any substitution or pattern of substitution that is sterically impractical, synthetically infeasible, and/or inherently unstable.

As used herein, "alkyl" refers to straight and branched chains having the indicated number of carbon atoms, typically 1 to 20 carbon atoms, for example 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, the C ₁-C₆ alkyl groups contain straight and branched alkyl groups of 1 to 6 carbon atoms. When referring to alkyl residues having a specific number of carbons, it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl groups, referring to residues identical to alkyl groups but having two points of attachment.

As used herein, "alkenyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double bond obtained by removing a molecule of hydrogen from adjacent carbon atoms of the parent alkyl group. The group may be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to: vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylpropan-1-en-1-yl, but-2-en-2-yl, but-1, 3-dien-1-yl, but-1, 3-dien-2-yl, and the like. In certain embodiments, alkenyl groups have 2 to 20 carbon atoms, and in other embodiments 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl groups and refers to residues that are identical to alkenyl groups but have two points of attachment.

As used herein, "alkynyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon triple bond obtained by removing two molecules of hydrogen from adjacent carbon atoms of the parent alkyl group. Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, and the like. In certain embodiments, alkynyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl groups and refers to residues that are identical to alkynyl groups but have two points of attachment.

As used herein, "alkoxy" refers to an alkyl group of the specified number of carbon atoms attached through an oxygen bridge, e.g., methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-methylpentyloxy, and the like. Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by an oxygen bridge.

As used herein, "aryl" refers to a group derived from an aromatic mono-or polycyclic hydrocarbon ring system formed by removal of a hydrogen atom from a ring carbon atom. The aromatic mono-or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, wherein at least one ring of the ring system is fully unsaturated, i.e. it comprises a cyclic, delocalized (4n+2) pi-electron system according to Huckel theory. Aryl groups include, but are not limited to, phenyl, fluorenyl, and naphthyl groups. Arylene is a subset of aryl groups and refers to residues that are identical to aryl groups but have two points of attachment.

As used herein, "halo substituent" or "halo" refers to fluoro, chloro, bromo, or iodo, and the term "halo" includes fluoro, chloro, bromo, or iodo.

As used herein, "haloalkyl" refers to an alkyl group as defined above wherein a specified number of carbon atoms are replaced with one or more up to the maximum allowable number of halogen atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl or pentafluoroethyl.

"Heterocyclyl" means a stable 3-to 18-membered non-aromatic ring group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless otherwise indicated in the specification, heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. The heteroatoms in the heterocyclyl may optionally be oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heterocyclyl groups are partially saturated or fully saturated. The heterocyclyl may be attached to the remainder of the molecule through any ring atom. Examples of such heterocyclyl groups include, but are not limited to: dioxanyl, thienyl [1,3] dithioyl (thienyl [1,3] dithianyl), decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithioyl (trithianyl), tetrahydropyranyl, thiomorpholinyl (thiomorpholinyl), thiomorpholinyl (thiamorpholinyl), 1-oxothiomorpholinyl (1-oxo-thiomorpholinyl) and 1, 1-dioxothiomorpholinyl (1, 1-dioxo-thiomorpholinyl). A heterocyclylene group is a subset of heterocyclyl groups and refers to a residue that is identical to a heterocyclyl group but has two points of attachment.

"Heteroaryl" refers to groups derived from 3-to 18-membered aromatic ring radicals containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, heteroaryl groups may be monocyclic, bicyclic, tricyclic or tetracyclic systems, wherein at least one ring of the ring system is fully unsaturated, i.e. comprises a cyclic delocalized (4n+2) pi-electron system according to huckel theory. Heteroaryl groups include fused or bridged ring systems. The heteroatoms in the heteroaryl group are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. Heteroaryl groups are attached to the remainder of the molecule through any ring atom. Examples of heteroaryl groups include, but are not limited to: azetidinyl, acridinyl, benzimidazolyl, benzindolyl, 1, 3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo [ d ] thiazolyl, benzothiadiazolyl, benzo [ b ] [1,4] dioxaheptyl (benzob ] [1,4] dioxanyl), benzo [ b ] [1,4] oxazinyl (benzob ] [1,4] oxazinyl), 1,4-benzodioxanyl (1, 4-benzodioxanyl), benzonaphtofuranyl, benzoxazolyl, benzodioxolyl (benzodioxolyl), Benzodioxinyl (benzodioxinyl), benzopyranyl, benzopyronyl, benzofuranyl, benzofuranonyl, benzothienyl, benzothieno [3,2-d ] pyrimidinyl, benzotriazolyl, benzo [4,6] imidazo [1,2-a ] pyridinyl, carbazolyl, cinnolinyl (cinnolinyl), cyclopenta [ d ] pyrimidinyl, 6, 7-dihydro-5H-cyclopenta [4,5] thieno [2,3-d ] pyrimidinyl, 5,6-dihydrobenzo [ H ] quinazolinyl (5, 6-dihydrobenzo [ H ] quinazolinyl), 5,6-dihydrobenzo [ H ] cinnolinyl, 6, 7-dihydro-5H-benzo [6,7] cyclohepta [1,2-c ] pyridazinyl, dibenzofuranyl, dibenzothienyl, furanyl, furanonyl, furo [3,2-c ] pyridinyl, 5,6,7,8,9, 10-hexahydrocyclooctano [ d ] pyrimidinyl, 5,6,7,8,9, 10-hexahydrocyclooctano [ d ] pyridazinyl, 5,6,7,8,9, 10-hexahydrocyclooctano [ d ] pyridinyl, isothiazolyl, imidazolyl, Indazolyl (indazolyl), indolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl (indolizinyl), isoxazolyl, 5, 8-methanol-5, 6,7,8-tetrahydroquinazolinyl (5, 8-methano-5,6,7, 8-tetrahydroquinazolinyl), naphthyridinyl (NAPHTHYRIDINYL), 1, 6-naphthyridinyl (1, 6-naphthyridinonyl), oxadiazolyl, 2-oxazepinyl (2-oxoazepinyl), and, Oxazolyl, oxetanyl (oxiranyl), 5, 6a,7,8,9,10 a-octahydrobenzo [ H ] quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl (phthalazinyl), pteridinyl (pteridinyl), purinyl, pyrrolyl, pyrazolyl, pyrazolo [3,4-d ] pyrimidinyl, pyridinyl, pyrido [3,2-d ] pyrimidinyl, pyrido [3,4-d ] pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl (quinoxalinyl), Quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7, 8-tetrahydrobenzo [4,5] thieno [2,3-d ] pyrimidinyl, 6,7,8, 9-tetrahydro-5H-cyclohepto [4,5] thieno [2,3-d ] pyrimidinyl, 5,6,7, 8-tetrahydropyrido [4,5-c ] pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno [2,3-d ] pyrimidinyl, thieno [3,2-d ] pyrimidinyl, thieno [2,3-c ] pyridinyl (thieo [2,3-c ] pridinyl), and thienyl (thiophenyl/thienyl). Heteroarylene is a subset of heteroaryl groups and refers to residues that are identical to heteroaryl groups but have two points of attachment.

Various hydroxyl protecting groups may be used in the present disclosure. In general, protecting groups make chemical functionality insensitive to specific reaction conditions, and can be added and removed from that functionality in the molecule without substantially damaging the rest of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al, tetrahedron 1992,48,2223-2311, and Greeneand Wuts,Protective Groups in Organic Synthesis,Chapter 2,2d ed,John Wiley&Sons,New York,1991, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under alkaline conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Dimethoxytrityl (DMTR), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), and 9- (p-methoxyphenyl) xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4, 4 '-dimethoxytrityl), and TMTr (4, 4',4 "-trimethoxytrityl).

The term "subject" as used herein refers to any animal, such as a mammal or a pouched animal. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, rabbits, and any variety of poultry.

As used herein, "treatment" refers to a method of achieving a beneficial or desired result, including but not limited to therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is obtained by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

As used herein, "prevention" refers to a method of achieving a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," a double-stranded oligonucleotide, pharmaceutical composition, or oligonucleotide conjugate may be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of the disease, even though a diagnosis of the disease may not have been made.

Nucleotide sequence

In one aspect, the present disclosure provides a nucleotide sequence. The nucleotide sequences of the present disclosure contain nucleotide groups as basic structural units, which are well known to those skilled in the art and contain phosphate groups, ribose groups and bases, and are not described in detail herein.

Each nucleotide in the nucleotide sequence of the present disclosure is a modified or unmodified nucleotide, wherein the nucleotide sequence comprises a nucleotide sequence I, which is a nucleotide sequence formed by substitution of at least one nucleotide from the 2 nd to 8 th nucleotides in the 5 'end-3' end direction in the nucleotide sequence a with an acyclic abasic group, the nucleotide sequence a has 16 to 30 nucleotides, and the nucleotide sequence a is reverse complementary to a stretch of nucleotide sequence in the target mRNA by at least 14 nucleotides.

That is, the nucleotide sequence A is a nucleotide having a length of 16-30 nucleotides, and is at least partially reverse-complementary to a stretch of nucleotide sequence in the target mRNA. The nucleotide sequence I is a nucleotide sequence formed by the following modes: at least one of the nucleotides 2 to 8 in the nucleotide sequence A is replaced with an acyclic dealkalization group in the 5 '-3' -direction. In some embodiments, the nucleotide sequence a is more than 85% complementary to a stretch of nucleotides in the target mRNA; in some embodiments, the nucleotide sequence a has no more than 2 base mismatches with a stretch of sequence in the target mRNA.

The acyclic abasic group has a structure as shown in formula (101):

Wherein R ₁、R₂ and R ₃ each independently have a structure represented by formula (201):

r ₄ has a structure as shown in formula (202):

Represents a site of covalent attachment of the groups, each n is independently selected from integers ranging from 0 to 4, each m is independently selected from integers ranging from 1 to 4, E ₁ is selected from OH, SH or BH ₂;

Each R ₁₀₁ is independently selected from the group consisting of H, C ₁-C₅ straight-chain alkyl, C ₁-C₅ alkoxy, C ₁-C₁₀ acyl, C ₁-C₅ alkylsulfonyl, and C ₆-C₁₀ arylsulfonyl;

R ₂₀₁ is selected from OH or NHR ₂₀₂, wherein R ₂₀₂ is selected from the group consisting of H, C ₁-C₅ straight chain alkyl, C ₁-C₁₀ acyl and, C ₁-C₅ alkylsulfonyl and C ₆-C₁₀ arylsulfonyl.

In some embodiments, each n is independently selected from an integer from 0 to 4, and m is independently selected from an integer from 1 to 4. In some embodiments, each n is independently selected from 0 or 1, and m is independently selected from 1 or 2. In some embodiments, n is 0 and m is 1.

In some embodiments, E ₁ is selected from OH, SH, or BH ₂. In some embodiments, E ₁ is selected from OH or SH for easy availability of raw materials.

In some embodiments, each R ₁₀₁ is independently selected from the group consisting of H, C ₁-C₅ straight-chain alkyl, C ₁-C₅ alkoxy, C ₁-C₁₀ acyl, C ₁-C₅ alkylsulfonyl, and C ₆-C₁₀ arylsulfonyl. In some embodiments, each R ₁₀₁ is independently selected from the group consisting of H, methyl, ethyl, and methoxy. In some embodiments, each R ₁₀₁ is independently selected from the group consisting of H, methyl, ethyl, and methoxy.

In some embodiments, R ₂₀₁ is selected from OH or NHR ₂₀₂. In some embodiments, R ₂₀₁ is NHR ₂₀₂ and R ₂₀₂ is selected from the group consisting of H, C ₁-C₅ straight chain alkyl, C ₁-C₁₀ acyl, and C ₁-C₁₀ acyl substituted with sulfonyl. In some embodiments, R ₂₀₂ is selected from the group consisting of H, C ₁-C₅ aliphatic acyl, C ₁-C₅ aromatic acyl, C ₁-C₅ alkylsulfonyl, and C ₆-C₁₀ arylsulfonyl. In some embodiments, R ₂₀₂ is selected from the group consisting of acetyl, isobutyryl, benzoyl, p-toluenesulfonyl, levulinyl, and crotonyl.

In some embodiments, each nucleotide in the nucleotide sequence is independently a modified or unmodified nucleotide. In some embodiments, all nucleotides in the nucleotide sequence are modified nucleotides. In some embodiments, the nucleotides in the nucleotide sequence are all unmodified nucleotides.

In some embodiments, the nucleotide sequence a is between 16-30 nucleotides in length. In some embodiments, the nucleotide sequence A is 19-25, 21-25, 19-21, or 21-23 nucleotides in length. In some embodiments, the nucleotide sequence A is 19-21 nucleotides in length. In some embodiments, the nucleotide sequence a is 19 or 21 nucleotides in length.

In some embodiments, the nucleotide sequence a is reverse complementary to a stretch of nucleotides in the target mRNA by at least 14 nucleotides. In some embodiments, the nucleotide sequence a is reverse complementary to a stretch of nucleotides in the target mRNA by at least 16 nucleotides. In some embodiments, the nucleotide sequence a is reverse complementary to a stretch of nucleotides in the target mRNA by at least 18 nucleotides.

To simplify the nucleotide sequence structure, in some embodiments, nucleotide sequence I is a nucleotide sequence in which at least one of the nucleotides at positions 2,3, 4,5, 6, 7, 8 of nucleotide sequence A in the 5 '-terminal to 3' -terminal direction is replaced with an acyclic abasic group. In some embodiments, nucleotide sequence I is a nucleotide sequence in which at least one of the nucleotides at positions 3, 4,5, 6, 7 in the 5 'to 3' direction in nucleotide sequence a is replaced with an acyclic abasic group. In some embodiments, nucleotide sequence I is a nucleotide sequence in which at least one of the nucleotides 2, 6, 7, 8 of nucleotide sequence a in the 5 'to 3' direction is replaced with an acyclic abasic group. In some embodiments, the nucleotide sequence I is a nucleotide sequence in which one of the nucleotides at positions 4,5, 6 or 7 in the 5 'to 3' direction in the nucleotide sequence a is replaced with an acyclic abasic group. In some embodiments, the nucleotide sequence I is a nucleotide sequence in which one of the nucleotides at positions 6, 7 or 8 of the nucleotide a in the 5 'to 3' direction is replaced with an acyclic abasic group.

In some embodiments, the nucleotide sequence I is a nucleotide sequence in which any 2 nucleotides in the 2-8 positions of the nucleotide sequence a in the 5 'to 3' direction are replaced with acyclic base groups. In some embodiments, the nucleotide sequence I is a nucleotide sequence in which any 2 nucleotides in positions 4,5, 6 or 7 of the nucleotide sequence a in the 5 'to 3' direction are replaced with acyclic abasic groups. In some embodiments, the nucleotide sequence I is a nucleotide sequence in which any 1 nucleotide at position 4,5 or 6 in the 5 'to 3' direction and the seventh nucleotide in the nucleotide sequence a are replaced with acyclic abasic groups.

In some embodiments, each acyclic dealkalization group is independently selected from the group consisting of groups a101-a 107:

wherein the carbon atom marked with "×" indicates that the carbon atom is in the R configuration, S configuration or racemic configuration.

In some embodiments, each nucleotide in the nucleotide sequence a is a modified nucleotide.

In some embodiments, at least two nucleotides in the 2 nd to 16 th positions of the nucleotide sequence a in the 5' end-3 ' end direction are ribosyl 2' positions of the nucleotides having a fluoro-modified nucleotide, and each of the nucleotides at the other positions of the nucleotide sequence a is each independently one of the non-fluoro-modified nucleotides. In some embodiments, at least two of the nucleotides at positions 2, 6, 14, 16 of the nucleotide sequence a in the 5' to 3' direction are ribosyl 2' nucleotides with fluoro modified nucleotides, and each of the nucleotides at the other positions of the nucleotide sequence a is independently one of the non-fluoro modified nucleotides. In some embodiments, at least two nucleotides at positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence a in the 5' to 3' direction are ribosyl 2' nucleotides with fluoro modified nucleotides, and each nucleotide at the other positions of the nucleotide sequence a is independently one of the non-fluoro modified nucleotides.

In the context of the present disclosure and in the context of the present disclosure, the terms "fluoro modified nucleotide", "2 '-fluoro modified nucleotide", "nucleotide with 2' -hydroxy group of ribose group substituted by fluoro" and "2 '-fluoro ribosyl" are the same in meaning, and refer to a compound having a structure as shown in formula (401) formed by substitution of 2' -hydroxy group of nucleotide by fluoro, wherein Base represents a Base selected from C, G, A or U. "methoxy modified nucleotide", "2 '-methoxy modified nucleotide", "nucleotide in which the 2' -hydroxy group of the ribose group is replaced by methoxy" and "2 '-methoxyribosyl" are the same meaning, and each refers to a nucleotide in which the 2' -hydroxy group of the ribose group is replaced by methoxy to form a structure shown in formula (402).

In the context of the present disclosure, "non-fluoro modified nucleotide" refers to a nucleotide analogue or a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is substituted with a non-fluoro group. In some embodiments, each non-fluoro modified nucleotide is independently selected from one of a nucleotide analog or a nucleotide in which the hydroxyl group at the 2' position of the ribosyl of the nucleotide is substituted with a non-fluoro group.

In some embodiments, the 2 '-alkoxy modified nucleotide may be a methoxy modified nucleotide (2' -OMe), as shown in formula (402). In some embodiments, the 2' -substituted alkoxy-modified nucleotide may be a 2' -O-methoxyethyl-modified nucleotide (2 ' -MOE), as shown in formula (403). In some embodiments, the 2 '-amino modified nucleotide (2' -NH ₂) is represented by formula (404). In some embodiments, the 2' -Deoxynucleotide (DNA) is as shown in formula (405).

In some embodiments, the nucleotide formed by substitution of the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide with a non-fluorine group is selected from one of a 2' -alkoxy-modified nucleotide, a 2 '-substituted alkoxy-modified nucleotide, a 2' -alkyl-modified nucleotide, a 2 '-substituted alkyl-modified nucleotide, a 2' -amino-modified nucleotide, a 2 '-substituted amino-modified nucleotide, and a 2' -deoxynucleotide.

Nucleotide analogs refer to groups that can replace nucleotides in a nucleic acid, but differ in structure from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides, above and below the present disclosure. In some embodiments, the nucleotide analog may be an iso-nucleotide, a bridged nucleotide (bridged nucleic acid, abbreviated BNA), or an acyclic nucleotide. BNA refers to a constrained or inaccessible nucleotide. BNA may contain a five-, six-, or seven-membered ring bridging structure with "fixed" C3' -endo-saccharides tucked. The bridge is typically incorporated at the 2'-, 4' -position of the ribose to provide a 2',4' -BNA nucleotide, such as LNA, ENA, cET BNA, etc., where LNA is shown as formula (406), ENA is shown as formula (407), cET BNA is shown as formula (408).

Acyclic nucleotides refer to a class of "open loop" nucleotides formed by the opening of the sugar ring of a nucleotide, such as an Unlocking Nucleic Acid (UNA) or a Glycerolipid Nucleic Acid (GNA), where UNA is represented by formula (409) and GNA is represented by formula (410).

In the above formula (409) and formula (410), R is selected from H, OH or alkoxy (O-alkyl).

An isopucleotide refers to a compound in which the position of a base on the ribose ring is changed in a nucleotide, for example, a compound in which the base is shifted from the 1' -position to the 2' -position or the 3' -position of the ribose ring, as shown in formula (411) or (412).

In the compounds of the above formulae (411) to (412), base represents a Base, for example A, U, G, C or T; r is selected from H, OH, F or a non-fluorine group as described above.

In some embodiments, the nucleotide analog is selected from one of an iso-nucleotide, LNA, ENA, cET BNA, UNA, and GNA.

In some embodiments, the nucleotide sequence consists of the nucleotide sequence I.

In some embodiments, there are a variety of ways in the art that can be used to modify nucleotide sequences, including backbone modifications (e.g., phosphate group modifications) and base modifications, among others, in addition to the ribose group modifications as described above (see, e.g., Watts,J.K.,G.F.Deleavey and M.J.Damha,Chemically modified siRNA:tools and applications.Drug Discov Today,2008.13(19-20)：p.842-55, for incorporated herein by reference in its entirety).

In some embodiments, at least 1 of the phosphate groups in the phosphate-sugar backbone of the nucleotide sequence are phosphate groups having a modifying group. The phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond in the phosphate group with a sulfur atom. In some embodiments, the phosphorothioate group is a phosphorothioate (phosphorthioate) structure as shown in formula (421), replacing the non-bridging oxygen atom in the phosphodiester linkage with one sulfur atom, replacing the phosphodiester linkage with a phosphorothioate linkage, i.e., the linkage between two nucleotides is a phosphorothioate linkage. The modification stabilizes the structure of the nucleotide sequence, maintaining high specificity and high affinity for base pairing.

Double-stranded oligonucleotides, modified or unmodified

In another aspect, the present disclosure also provides a double-stranded oligonucleotide comprising the aforementioned nucleotide sequence capable of modulating gene expression. The double-stranded oligonucleotide contains a sense strand and an antisense strand, the antisense strand comprising a nucleotide sequence as described above, the sense strand being a nucleotide sequence having 16-30 nucleotides, and the sense strand being at least partially reverse-complementary to the antisense strand to form a double-stranded region.

In some embodiments, the antisense strand is substantially reverse complementary or substantially reverse complementary to the sense strand. By substantially reverse complement is meant that there is no more than a 3 base mismatch between the two nucleotide sequences; by substantially reverse complementary is meant that there is no more than 1 base mismatch between the two nucleotide sequences.

In some embodiments, the double-stranded oligonucleotide comprises a nucleotide sequence in which each nucleotide is a modified or unmodified nucleotide.

The double-stranded oligonucleotide also contains a sense strand, wherein the sense strand is a nucleotide sequence with 16-30 nucleotides. In some embodiments, the sense strand is between 16-30 nucleotides in length. In some embodiments, the sense strand is 21-27, 23-27, 21-23, or 23-25 nucleotides in length. In some embodiments, the sense strand is 21-23 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length. The sense strand or the antisense strand may each independently be 19-23 nucleotides in length.

Thus, the ratio of the length of the sense strand and the antisense strand of the double-stranded oligonucleotides provided by the present disclosure may be 19/19、19/20、19/21、19/22、20/20、20/21、20/22、20/23、21/21、21/22、21/23、21/24、22/22、22/23、22/24、22/25、23/23、23/24、23/25 or 23/26. In some embodiments, the double-stranded oligonucleotides provided by the present disclosure have a ratio of the length of the sense strand to the length of the antisense strand of 19/21 or 21/23, at which time the double-stranded oligonucleotides provided by the present disclosure have better silencing activity of the target mRNA.

In some embodiments, at least 17 of the nucleotides at positions 2-19 of the nucleotide sequence a are complementary to the sense strand in a 5 'end to 3' end orientation. In some embodiments, at least 16 nucleotides of nucleotides 2-19 of the nucleotide sequence a are complementary to the sense strand in a 5 'end to 3' end orientation. In some embodiments, at least 14 nucleotides of nucleotides 2-19 of the nucleotide sequence a are complementary to the sense strand in a 5 'end to 3' end orientation.

In some embodiments, each nucleotide in the nucleotide sequence comprised by the double-stranded oligonucleotide is a modified nucleotide. In some embodiments, each nucleotide in the antisense strand comprised by the double-stranded oligonucleotide is a modified nucleotide. In some embodiments, each nucleotide in the sense strand comprised by the double-stranded oligonucleotide is a modified nucleotide.

In some embodiments, the double-stranded oligonucleotides of the present disclosure are double-stranded oligonucleotides having the following modifications: in the sense strand, the nucleotides at positions 7, 8 and 9 or positions 5, 7, 8 and 9 of the nucleotide sequence I are fluoro modified nucleotides, and the nucleotides at the rest positions in the sense strand are methoxy modified nucleotides according to the direction from the 5 'end to the 3' end; in the antisense strand, the nucleotides at the 2, 6, 14 and 16 positions or the 2, 6, 8, 9, 14 and 16 positions of the nucleotide sequence II are fluoro modified nucleotides, and the nucleotides at the rest positions in the antisense strand are methoxy modified nucleotides.

In some embodiments, the double-stranded oligonucleotides of the present disclosure are double-stranded oligonucleotides having the following modifications: according to the direction of the 5' end to the 3' end, the nucleotides at the 5 th, 7 th, 8 th and 9 th positions of the sense strand are ribosyl 2' positions of the nucleotides with fluoro modified nucleotides, and the nucleotides at the rest positions in the sense strand are methoxy modified nucleotides; the nucleotides at positions 2, 6, 8, 9, 14 and 16 of the antisense strand are ribosyl 2' of the nucleotide with fluoro-modified nucleotides, the nucleotides at the remaining positions in the antisense strand are methoxy-modified nucleotides, and the antisense strand comprises the aforementioned nucleotide sequence I, so that 1 or more bases in the antisense strand can be replaced by an acyclic abasic group.

In some embodiments, the double-stranded oligonucleotides of the present disclosure are double-stranded oligonucleotides having the following modifications: according to the direction of the 5' end to the 3' end, the nucleotides at the 7 th, 8 th and 9 th positions of the sense strand are ribosyl 2' positions of the nucleotides with fluoro modified nucleotides, and the nucleotides at the rest positions in the sense strand are methoxy modified nucleotides; the nucleotides at positions 2,6, 14 and 16 of the antisense strand are ribosyl 2' nucleotides with fluoro modifications, the nucleotides at the remaining positions in the antisense strand are methoxy modified nucleotides, and the antisense strand comprises the aforementioned nucleotide sequence I, so that 1 or more bases in the antisense strand can be replaced by an acyclic abasic group.

In some embodiments, the double-stranded oligonucleotides of the present disclosure are double-stranded oligonucleotides having the following modifications: according to the direction of the 5 '-end and the 3' -end, the nucleotides at the 7 th and 8 th positions of the sense strand are ribosyl 2 '-position of the nucleotide and are fluoro-modified nucleotides, the nucleotides at the 5 th and 9 th positions are 2' -deoxynucleotides, and the nucleotides at the rest positions in the sense strand are methoxy-modified nucleotides; the nucleotides at positions 2,6, 14 and 16 of the antisense strand are ribosyl 2' nucleotides with fluoro modifications, the nucleotides at the remaining positions in the antisense strand are methoxy modified nucleotides, and the antisense strand comprises the aforementioned nucleotide sequence I, so that 1 or more bases in the antisense strand can be replaced by an acyclic abasic group.

In some embodiments, the double-stranded oligonucleotides of the present disclosure also contain other modified nucleotide groups that do not result in a significant impairment or loss of the function of the double-stranded oligonucleotides to modulate target gene expression.

Currently, there are a variety of ways available in the art for modifying double-stranded oligonucleotides, including backbone modifications (e.g., phosphate group modifications) and base modifications, etc., in addition to the ribose group modifications mentioned above (see, e.g., Watts,J.K.,G.F.Deleavey and M.J.Damha,Chemically modified siRNA:tools and applications.Drug Discov Today,2008.13(19-20)：p.842-55, for reference herein in its entirety).

In some embodiments, at least 1 of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand are phosphate groups having a modifying group. The phosphate group having a modification group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond in the phosphate group with a sulfur atom, and may be a phosphorothioate (phosphorthioate) structure represented by the formula (421), substitution of one sulfur atom for a non-bridging oxygen atom in the phosphodiester bond, and substitution of a phosphodiester bond with a phosphorothioate diester bond, i.e., a phosphorothioate group linkage is used as a linkage between two nucleotides. The modification stabilizes the structure of double-stranded oligonucleotides, maintaining high specificity and high affinity for base pairing.

In some embodiments, the phosphorothioate linkage is present in the double stranded oligonucleotide at least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above. In some embodiments, phosphorothioate linkages are present at all of the above positions except the 5' end of the sense strand. In some embodiments, thio

Phosphate linkages are present at all of the above positions except the 3' end of the sense strand. In some embodiments, the phosphorothioate linkage is present in at least one of the following positions:

The 5' terminal end of the sense strand is between nucleotide 1 and nucleotide 2;

The 5' terminal end of the sense strand is between nucleotide 2 and nucleotide 3;

the 3' -terminal end of the sense strand is between nucleotide 1 and nucleotide 2;

the 3' -terminal end of the sense strand is between nucleotide 2 and nucleotide 3;

The 5' terminal end of the antisense strand is between nucleotide 1 and nucleotide 2;

the 5' terminal end of the antisense strand is between nucleotide 2 and nucleotide 3;

The 3' -terminal end of the antisense strand is between nucleotide 1 and nucleotide 2; and

The 3' -terminal end of the antisense strand is between nucleotide 2 and nucleotide 3.

In some embodiments, the antisense strand sequence 5' terminal nucleotide of the double stranded oligonucleotide molecule is a 5' -phosphonucleotide or 5' -phosphoanalog modified nucleotide.

In some embodiments, the 5' -phosphate nucleotide has a structure represented by formula (422):

Meanwhile, the kinds of commonly used 5' -phosphate analogue-modified nucleotides are well known to those skilled in the art, for example, the nucleotides represented by the formulae (423) to (426) disclosed in ,Anastasia Khvorova and Jonathan K.Watts,The chemical evolution of oligonucleotide therapies of clinical utility.Nature Biotechnology,2047,35(3):238-48:

wherein R represents a group selected from the group consisting of H, OH, F, and methoxy; base represents a Base selected from A, U, C, G or T.

In some embodiments, the 5' -phosphate analog-modified nucleotide is a vinyl phosphate (E-vinylphosphonate, E-VP) -containing nucleotide of formula (423), or a phosphorothioate-containing nucleotide of formula (425).

The modification schemes disclosed herein may be applicable to a variety of double-stranded oligonucleotides that mediate gene expression. In some embodiments, the double-stranded oligonucleotide is a double-stranded oligonucleotide that inhibits or down-regulates gene expression. In some embodiments, the double stranded oligonucleotide is an siRNA. In some embodiments, the double-stranded oligonucleotide is a double-stranded oligonucleotide that activates or upregulates gene expression. In some embodiments, the double stranded oligonucleotide is a saRNA.

According to some embodiments of the present disclosure, the double stranded oligonucleotide of the present disclosure is an siRNA comprising a sequence as shown in tables 1A-1E:

TABLE 1siRNA sequences

TABLE 1A

TABLE 1B

TABLE 1C

TABLE 1D

TABLE 1E

Wherein S represents a sense strand; AS represents the antisense strand, and capital C, G, U, A represents the base composition of the nucleotide; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a 2' -methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a 2' -fluoro modified nucleotide; the lower case letter s indicates that the linkage between two nucleotides adjacent to the letter s is a phosphorothioate linkage; the lowercase letter d indicates that the adjacent nucleotide to the right of the letter d is a 2' -deoxynucleotide; p1 represents that the one nucleotide adjacent to the right of P1 is a 5' -phosphonucleotide or 5' -phosphoanalog modified nucleotide, in some embodiments a vinyl phosphate modified nucleotide (denoted as VP in the examples below), a 5' -phospho modified nucleotide (denoted as P in the examples below), or a phosphorothioate modified nucleotide (denoted as Ps in the examples below); (GLY) represents that two nucleotides adjacent to the left and right of (GLY) are linked by an acyclic dealkalization group represented by formula (a 101); (GLY-Ac) represents that two nucleotides adjacent to the left and right of the (GLY-Ac) are linked by an acyclic dealkalization group represented by formula (a 102); (GLY-Ph) two nucleotides adjacent to the (GLY-Ph) side are linked by an acyclic dealkalization group represented by formula (a 103); (GLY-TOS) two nucleotides adjacent to the (GLY-TOS) left and right are linked by an acyclic dealkalization group represented by formula (a 104); (GLY-iBu) represents that two nucleotides adjacent to the (GLY-iBu) are linked by an acyclic dealkalization group represented by formula (a 105); (GLY-laev) means that two nucleotides adjacent to the left and right of (GLY-laev) are linked by an acyclic dealkalization group represented by formula (a 106); (GLY-Cro) represents that two nucleotides adjacent to the (GLY-Cro) are linked by an acyclic dealkalide group represented by formula (a 107), and the foregoing acyclic dealkalide groups are each in S configuration; (GLY-S) means that two nucleotides adjacent to each other in the left and right are linked by an acyclic dealkalization group represented by formula (a 101) of S configuration; (GLY-R) means that two nucleotides adjacent to each other in the left and right are linked by an acyclic dealkalization group represented by the formula (A101) in the R configuration.

In some embodiments, the double stranded oligonucleotide is one of siAPO1L、siAPO1、siAPOa1M1SVP2、siAPOb1M1SVP2、siAPOc1M1SVP2、siAPOd1M1SVP2、siAPOe1M1SVP2、siAPOf1M1SVP2、siAPOf1M1SP2、siAPOg1M1SP2、siAPOg1M1SP2-Ac、siAPOg1M1SP2-Ph、siAPOg1M1SP2-TOS、siAPOg1M1SP2-iBu、siAPOg1M1SP2-laev、siAPOg1M1SP2-Cro、siAPOg1M1SVP1R、siAPOg1M1SVP1S、siAPOg1M1SVP2R、siAPOg1M1SVP2S、siAPOg1M1SVP3R、siAPOg1M1SVP3S、siAPOg1M1SVP4R、siAPOg1M1SVP4S、siAPOg1Ph2、siAPOg1Ph3、siAPOg1Ph4、siAPOg1Ph5、siAPOg1Ph6、siAPOg1Ph7、siAPOg1Ph8、siAPOg1Ph4ph7、siAPOg1Ph5ph7、siAPOg1M16ph7、siAPOg1M34ph7、siAPOg1Lph7、siAPOg1Nph7、siHBa1M1SP2、siHBa1M1SVP2、siHBb1M1SVP2、siHBa1M1SVP1-Ac、siHBa1M1SVP2-Ac、siHBa2M1SVP3-Ac、siC5a1M1S2、siC5b1M1S2、siHAOa2M1S2、siHAOa1M1S2、siANGa1M1S2-Ac、siANGa1M1S2-iBu、siANGa1Ph2、siANGa1Ph3、siANGa1Ph4、siANGa1Ph5、siANGa1Ph6、siANGa1Ph7、siANGa1Ph8、siANGa1Ph4ph7、siANGa1Ph5ph7、siANGa1M16ph7、siANGa1M34ph7、siANGa1Lph7 or siANGa Nph 7.

The inventors of the present disclosure have unexpectedly found that these double-stranded oligonucleotides provided by the present disclosure have high stability in blood, high stability in lysosomes, while reducing off-target effects. Meanwhile, the target gene expression regulation activity is not significantly reduced, and an excellent in vivo inhibition effect is shown.

The double-stranded oligonucleotides provided by the present disclosure may be obtained by methods of double-stranded oligonucleotide preparation conventional in the art (e.g., methods of solid phase synthesis and liquid phase synthesis). Among them, solid-phase synthesis already has commercial subscription services. Methods of preparing nucleoside monomers having corresponding modifications and methods of introducing modified nucleotide groups into double-stranded oligonucleotides are also well known to those of skill in the art by using nucleoside monomers having corresponding modifications to introduce modified nucleotide groups into double-stranded oligonucleotides described in the present disclosure.

The nucleotide sequence comprising an acyclic abasic group described in the present disclosure can be prepared according to methods conventional in the art for oligonucleotide preparation, except that the above preparation is performed with an acyclic abasic monomer compound having a structure as shown in formula (110) instead of a nucleoside monomer at the substituted position:

the compounds of formula (110) are commercially available or are obtained by a person skilled in the art using known methods. In some embodiments, the acyclic abasic monomeric compound of formula (110) can be obtained by the following preparation method:

The method comprises the steps of contacting a compound shown in a formula (111) with phosphoramidite shown in a formula (131) in an organic solvent under the condition of condensation reaction and in the presence of tertiary amine organic base and pyridine compound, and separating to obtain the compound shown in the formula (110):

Wherein each of R ₁、R₂、R₃、R₄ is defined and optional ranges are as previously described, each B1 is independently C ₁-C₅ alkyl; b ₂ is selected from one of C ₁-C₅ alkyl, ethyl cyano, propyl cyano and Ding Qingji.

The condensation reaction conditions include a reaction temperature of 0 to 150 ℃ and a reaction time of 0.5 to 72 hours, and in one embodiment, the condensation reaction conditions include a reaction temperature of 10 to 70 ℃ and a reaction time of 1 to 10 hours.

In some embodiments, the organic solvent may be selected from one or more of an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran. In some embodiments, the ether solvent is diethyl ether and/or methyl tert-butyl ether and the haloalkane solvent is one or more of dichloromethane, chloroform, and 1, 2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The organic solvent is used in an amount of 1 to 50L/mol, and in some embodiments 3 to 20L/mol, relative to the compound represented by the formula (111).

In some embodiments, the molar ratio of the phosphoramidite to the compound of formula (111) is from 0.5:1 to 10:1.

In some embodiments, the tertiary amine organic base may be N-methylimidazole, N-methylmorpholine, triethylamine or N, N-diisopropylethylamine. The tertiary amine organic base is in some embodiments N-methylimidazole; the molar ratio of the tertiary amine organic base to the compound of formula (111) may be from 0.3:1 to 20:1, in some embodiments from 0.5:1 to 10:1.

In some embodiments, the pyridine compound may be pyridine trifluoroacetate, 2- (trifluoromethyl) nicotinic acid, 2-amino-6- (trifluoromethyl) pyridine. In some embodiments, the pyridine compound is pyridine trifluoroacetate. The molar ratio of pyridine compound to compound of formula (111) may be from 0.3:1 to 20:1, in some embodiments from 0.5:1 to 10:1.

The compound of formula (110) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, the compound of formula (313) may be removed by evaporation followed by separation by chromatographic methods, e.g., separation may be performed using two sets of chromatographic conditions: (1) normal phase purification: 200-300 mesh silica gel packing, using petroleum ether to ethyl acetate=10:1-1:10 gradient elution, or using dichloromethane to ethyl acetate=10:1-1:10 gradient elution; and (2) reverse phase purification: c ₁₈、C₈ reversed phase packing eluting with methanol: acetonitrile=0.1:1 to 1:0.1 gradient. In some embodiments, the compound of formula (110) may be obtained after evaporation of the solvent and suction filtration under reduced pressure, which may be used directly in the subsequent reaction.

The compounds of formula (111) are commercially available or are obtained by a person skilled in the art using known methods. In some embodiments, the compound of formula (111) may be obtained by the following preparation method: the method comprises the steps of contacting a compound shown in a formula (112) with a halogenated compound in an organic solvent under substitution reaction conditions and in the presence of tertiary amine organic base, and separating to obtain a compound shown in a formula (111):

wherein R ₁、R₂、R₃、R₄ is as defined above and the optional ranges are as defined above.

In some embodiments, the substitution reaction conditions may include a reaction temperature of 0 to 100 ℃ and a reaction time of 1 to 72 hours; in some embodiments the reaction temperature is 10 to 40 ℃ and the reaction time is 5 to 30 hours.

In some embodiments, the organic solvent may be selected from one or more of pyridine, epoxy solvents, ether solvents, haloalkane solvents, dimethyl sulfoxide, N-dimethylformamide, heterocyclic compounds, and N, N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran. In some embodiments, the ether solvent is diethyl ether and/or methyl tert-butyl ether. In some embodiments, the haloalkane solvent is one or more of dichloromethane, chloroform, and 1, 2-dichloroethane. In some embodiments, the heterocyclic compound is one or more of pyridine, pyrrole, and pyridine analogs. In some embodiments, the organic solvent is pyridine. The organic solvent is used in an amount of 0.3 to 50L/mol, and in some embodiments 1 to 20L/mol, relative to the compound represented by the formula (112).

In some embodiments, the halogenated compound is 4,4' -dimethoxytriphenylchloromethane, dithiomethoxycarbonyl, dimethylisopropyl silicon. In some embodiments, the halogenated compound is 4,4' -dimethoxytriphenylchloromethane. The molar ratio of the halogenated compound to the compound shown in the formula (112) is 0.5:1-10:1.

Similarly as described above, the compound of formula (111) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, the compound of formula (111) may be removed by evaporation followed by separation by chromatographic methods, e.g., separation may be performed using two sets of chromatographic conditions: (1) normal phase purification: 200-300 mesh silica gel packing, eluting with methanol: dichloromethane=0.01:1-0.5:1 gradient; or using ethyl acetate: petroleum ether = 0.1:1-1:1 gradient elution; and (2) reverse phase purification: c ₁₈ and C ₈ reversed phase packing eluted with a methanol:acetonitrile=0.1:1-1:0.1 gradient. In some embodiments, the compound of formula (111) may be obtained after evaporation of the solvent, after suction filtration under reduced pressure, which may be used directly in the subsequent reaction.

In some embodiments, R ₄ is-CH ₂ OH, where the compound of formula (112) is glycerol, which is readily available commercially, and where the compound of formula (110) is obtained having the structure shown in formula (121).

The compounds of formula (112) are commercially available or are obtained by a person skilled in the art using known methods. In some embodiments, R ₄ is-CH ₂NHR₂₀₂ and the compound of formula (112) can be obtained by the following preparation method: the method comprises the steps of contacting a compound shown in a formula (113) with an acid shown by R ₂₀₂ OH or an anhydride shown by R ₂₀₂)₂ O in an organic solvent under substitution reaction conditions, and separating to obtain a compound shown in a formula (112):

Wherein R ₁、R₂、R₃ is as defined above and the optional ranges are as defined above.

In some embodiments, the substitution reaction conditions may include a reaction temperature of 0 to 100 ℃ and a reaction time of 1 to 72 hours; in some embodiments the reaction temperature is 10 to 40 ℃ and the reaction time is 3 to 30 hours.

In some embodiments, the organic solvent may be selected from one or more of an alcohol solvent, an ester solvent, an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N-dimethylformamide, a heterocyclic compound, and N, N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran. In some embodiments, the ether solvent is diethyl ether and/or methyl tert-butyl ether. In some embodiments, the haloalkane solvent is one or more of dichloromethane, chloroform, and 1, 2-dichloroethane. In some embodiments, the alcoholic solvent is one or more of methanol, ethanol, propanol, butanol. In some embodiments, the ester solvent is one or more of ethyl acetate, methyl acetate, propyl acetate, butyl acetate. The organic solvent is used in an amount of 0.3 to 50L/mol, and in some embodiments 1 to 20L/mol, relative to the compound represented by the formula (112).

In some embodiments, the acid represented by R ₂₀₂ OH or the anhydride represented by (R ₂₀₂)₂ O) is selected from one or more of acetic anhydride, benzoic anhydride, p-toluenesulfonic anhydride, isobutyric anhydride, levulinic acid, crotonic acid, the molar ratio of the acid represented by R ₂₀₂ OH or the anhydride represented by R ₂₀₂)₂ O to the compound represented by formula (112) is from 0.5:1 to 10:1.

Similar to the above, the compound of formula (112) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, the solvent may be removed by evaporation followed by chromatographic separation of the compound of formula (112), e.g., separation may be performed using two sets of chromatographic conditions: (1) normal phase purification: 200-300 mesh silica gel packing, eluting with methanol: dichloromethane = 0.01:1-1:1 gradient; or gradient elution with ethyl acetate: petroleum ether = 0.1:1-10:1; and (2) reverse phase purification: c ₁₈ and C ₈ reversed phase packing eluted with a methanol:acetonitrile=0.1:1-1:0.1 gradient. In some embodiments, the solvent may be removed by evaporation, followed by suction filtration under reduced pressure to provide a compound of formula (112) product which may be used directly in a subsequent reaction.

The compounds of formula (113) are commercially available or are obtained by a person skilled in the art using known methods. For example, when R ₁、R₂、R₃ is H, the acyclic abasic monomer compound represented by formula (113) is readily commercially available 3-amino-1, 2-propanediol. At this time, the compound of formula (110) obtained has one of the structures shown in formulas (122) to (127).

In some embodiments, the compound of formula (110) prepared by the above method has a structure as shown in one of the following formulas (121) -formula (127):

Pharmaceutical composition

In another aspect, the present disclosure provides a pharmaceutical composition comprising a double-stranded oligonucleotide as described above as an active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be a carrier conventionally used in the field of double-stranded oligonucleotide administration, such as, but not limited to, magnetic nanoparticles (magnetic nanoparticles, such as Fe ₃O₄ or Fe ₂O₃ -based nanoparticles), carbon nanotubes (carbon nanotubes), mesoporous silicon (mesoporous silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), polyethylenimine (PEI), polyamide dendrimers (polyamidoamine (PAMAM) dendrimers), polylysine (L-lysine), PLL), chitosan (chitosan), 1,2-dioleoyl-3-trimethylammonium propane (1, 2-dioleoyl-3-trimethylammonium-propane, DOTAP), poly D-or L-lactic/glycolic acid copolymers (PLGA), poly (aminoethylethylethylene phosphate) (2-ami ETHYL ETHYLENE phospho), PPEEA) and poly (methacrylic acid-N, dimethylaminoethyl methacrylate) or derivatives thereof.

The content of the double-stranded oligonucleotide and the pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly limited, and may be the conventional content of each component. In some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier can be 1 (1-500), and in some embodiments, the weight ratio is 1 (1-50).

In some embodiments, the pharmaceutical composition may further comprise other pharmaceutically acceptable excipients, which may be one or more of various formulations or compounds conventionally employed in the art. For example, the pharmaceutically acceptable additional excipients may include at least one of a pH buffer, a protectant, and an osmolality adjusting agent.

The pH buffer solution can be a tris hydrochloride buffer solution with the pH value of 7.5-8.5 and/or a phosphate buffer solution with the pH value of 5.5-8.5, for example, the pH value of 5.5-8.5.

The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The protective agent may be present in an amount of 0.01 to 30% by weight, based on the total weight of the pharmaceutical composition.

The osmolality adjusting agent may be sodium chloride and/or potassium chloride. The osmolality adjusting agent is present in an amount such that the osmolality of the pharmaceutical composition is 200-700 milliosmoles per kilogram (mOsm/kg). The amount of osmolality adjusting agent can be readily determined by one skilled in the art based on the desired osmolality.

In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection; or freeze-dried powder injection, and is mixed with liquid adjuvant to make into liquid preparation. The liquid formulation may be administered, but is not limited to, for subcutaneous, intramuscular or intravenous injection, and may be administered, but is not limited to, by spraying to the lungs, or by spraying through the lungs to other visceral tissues such as the liver. In some embodiments, the pharmaceutical composition is for intravenous administration.

In some embodiments, the pharmaceutical composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal formulation comprises an amine-containing transfection compound (which may also be referred to hereinafter as an organic amine), a helper lipid, and/or a pegylated lipid. Wherein the organic amine, the helper lipid and the pegylated lipid may be selected from one or more of the amine-containing transfection compounds described in CN103380113a (which is incorporated herein by reference in its entirety) or pharmaceutically acceptable salts or derivatives thereof, the helper lipid and the pegylated lipid, respectively.

In some embodiments, the organic amine may be a compound described in CN103380113a as represented by formula (501) or a pharmaceutically acceptable salt thereof:

Wherein:

Each _X101 and X ₁₀₂ is independently O, S, N-se:Sub>A or C-se:Sub>A, wherein se:Sub>A is hydrogen or se:Sub>A C ₁-C₂₀ hydrocarbon chain;

Each Y and Z is independently c= O, C = S, S = O, CH-OH or SO ₂;

Each R ₃₀₁、R₃₀₂、R₃₀₃、R₃₀₄、R₃₀₅、R₃₀₆ or R ₃₀₇ is independently hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic group, a substituted or unsubstituted, branched or straight chain acyl group, a substituted or unsubstituted, branched or straight chain aryl group, a substituted or unsubstituted, branched or straight chain heteroaryl group;

x is an integer from 1 to 10;

n ₁₀₁ is an integer from 1 to 3, m ₁₀₁ is an integer from 0 to 20, and p is 0 or 1; and wherein, when m ₁₀₁ and p are both 0, R ₃₀₂ is hydrogen;

and, if at least one of n ₁₀₁ or m ₁₀₁ is 2, R ₃₀₃ and nitrogen in formula (501) form a structure as shown in formula (502) or formula (503):

wherein g, e and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each of N represents a nitrogen atom shown in formula (501).

In some embodiments, R ₃₀₃ is a polyamine. In other embodiments, R ₃₀₃ is a ketal. In some embodiments, each of R ₃₀₁ and R ₃₀₂ in formula (501) is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl group having from 3 to about 20 carbon atoms, such as from 8 to about 18 carbon atoms, and from 0 to 4 double bonds, such as from 0 to 2 double bonds.

In some embodiments, if each of n and m independently has a value of 1 or 3, R ₃₀₃ can be any of the following formulas (504) - (513):

Wherein in formulae (504) - (513), g, e and f are each independently integers from 1 to 6, each "HCC" represents a hydrocarbon chain, and each shows a possible point of attachment of R ₃₀₃ to the nitrogen atom in formula (501), wherein each H at any of the positions may be replaced to effect attachment to the nitrogen atom in formula (501).

Wherein the compound of formula (501) may be prepared as described in CN1033113 a.

In some specific embodiments, the organic amine is an organic amine as shown in formula (514) and/or an organic amine as shown in formula (515):

the auxiliary lipid is cholesterol, cholesterol analogues and/or cholesterol derivatives;

The polyethylene glycol lipid is 1, 2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-N- [ methoxy (polyethylene glycol) ] -2000.

In some embodiments, the molar ratio between the organic amine, the helper lipid, and the pegylated lipid in the pharmaceutical composition is (19.7-80): (19.7-80): (0.3-50), for example, may be (50-70): (20-40): (3-20).

In some embodiments, the particles of the pharmaceutical composition formed from the double stranded oligonucleotides of the present disclosure and the amine-containing transfection reagent described above have an average diameter of about 30nm to about 200nm, typically about 40nm to about 135nm, more typically the average diameter of the liposome particles is about 50nm to about 120nm, about 50nm to about 100nm, about 60nm to about 90nm, or about 70nm to about 90nm, e.g., the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160nm.

In some embodiments, the weight ratio (weight/weight ratio) of double-stranded oligonucleotide to total lipid (e.g., organic amine, helper lipid, and/or pegylated lipid) in a pharmaceutical composition formed from a double-stranded oligonucleotide of the present disclosure and an amine-containing transfection reagent as described above is in the range of from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, e.g., the weight ratio of double-stranded oligonucleotide of the present disclosure to total lipid is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 18:18.

In some embodiments, the components of the pharmaceutical composition may be present independently at the point of sale and may be present in liquid formulations at the point of use. In some embodiments, the pharmaceutical compositions of the double-stranded oligonucleotides provided by the present disclosure and the pharmaceutically acceptable carriers described above can be prepared according to various known methods, except that the double-stranded oligonucleotides provided by the present disclosure are substituted for existing double-stranded oligonucleotides; in some embodiments, it may be prepared as follows:

Suspending organic amine, auxiliary lipid and polyethylene glycol lipid in alcohol according to the molar ratio, and uniformly mixing to obtain lipid solution; the amount of alcohol is such that the total mass concentration of the resulting lipid solution is 2-25mg/mL, for example, 8-18mg/mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, e.g., one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, e.g., may be ethanol.

The double-stranded oligonucleotide provided by the present disclosure is dissolved in a buffer salt solution to obtain a double-stranded oligonucleotide aqueous solution. The concentration of the buffer salt solution is 0.05-0.5M, for example, may be 0.1-0.2M, the pH of the buffer salt solution is adjusted to 4.0-5.5, for example, may be 5.0-5.2, and the buffer salt solution is used in such an amount that the concentration of the double-stranded oligonucleotide does not exceed 0.6mg/mL, for example, may be 0.2-0.4mg/mL. The buffer salt is selected from one or more of soluble acetate and soluble citrate, and can be sodium acetate and/or potassium acetate.

Mixing the lipid solution with the double-stranded oligonucleotide aqueous solution, and incubating the mixed product at 40-60 ℃ for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome preparation. The volume ratio of the lipid solution to the double-stranded oligonucleotide aqueous solution is 1 (2-5), and may be 1:4, for example.

Concentrating or diluting the incubated liposome preparation, removing impurities, and sterilizing to obtain the pharmaceutical composition provided by the disclosure, wherein the physical and chemical parameters are that the pH value is 6.5-8, the encapsulation efficiency is not lower than 80%, the particle size is 40-200nm, the polydispersity index is not higher than 0.30, and the osmotic pressure is 250-400mOsm/kg; for example, the physical and chemical parameters can be pH 7.2-7.6, encapsulation efficiency not lower than 90%, particle size 60-100nm, polydispersity index not higher than 0.20, and osmotic pressure 300-400mOsm/kg.

Wherein concentration or dilution may be performed before, after, or simultaneously with removal of impurities. As a method for removing impurities, various methods are available, for example, a tangential flow system, a hollow fiber column, ultrafiltration at 100K Da, and Phosphate Buffer (PBS) of pH7.4 as an ultrafiltration exchange solution can be used. As a method of sterilization, various methods are available, and for example, filtration sterilization on a 0.22 μm filter can be used.

SiRNA conjugates

In another aspect, the present disclosure provides an siRNA conjugate comprising the double-stranded oligonucleotide described above and a conjugate group attached to the double-stranded oligonucleotide.

In the context of the present disclosure, unless otherwise indicated, "conjugated" means that two or more chemical moieties each having a particular function are linked to each other by covalent linkage; accordingly, "conjugate" refers to a compound formed by covalent linkage between the chemical moieties. Further, "siRNA conjugate" means a compound formed by covalent attachment of one or more chemical moieties having specific functions to a double-stranded oligonucleotide. Hereinafter, the siRNA conjugates of the present disclosure are also sometimes simply referred to as "conjugates". More specifically, in the context of the present disclosure, a "conjugate molecule" is understood to be a specific compound that can be conjugated to a double stranded oligonucleotide by reaction, ultimately forming an siRNA conjugate of the present disclosure. The type and manner of attachment of the ligand is well known to those skilled in the art and generally functions to bind to a specific receptor on the surface of a target cell, mediating delivery of the double-stranded oligonucleotide attached to the ligand to the target cell.

Generally, the conjugate group comprises at least one pharmaceutically acceptable targeting group, or further comprises a linker (linker), and the double-stranded oligonucleotide, the linker and the targeting group are sequentially linked. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The double-stranded oligonucleotide molecule may be non-covalently or covalently conjugated to the conjugate group, e.g., may be covalently conjugated to the conjugate group. The conjugation site of the double-stranded oligonucleotide to the conjugation group may be at the 3' end or the 5' end of the sense strand of the double-stranded oligonucleotide, or at the 5' end of the antisense strand, or in the internal sequence of the double-stranded oligonucleotide. In some specific embodiments, the conjugation site of the double-stranded oligonucleotide to the conjugation group is at the 3' end of the sense strand of the double-stranded oligonucleotide.

In some embodiments, the conjugate group may be attached to the phosphate group, the 2' -hydroxyl group, or the base of the nucleotide. In some embodiments, the conjugate group may be attached to the 3' -hydroxyl group, in which case the nucleotides are linked using a 2' -5' phosphodiester linkage. When a conjugate group is attached to the end of a double-stranded oligonucleotide strand, the conjugate group is typically attached to the phosphate group of the nucleotide; when a conjugate group is attached to the internal sequence of a double-stranded oligonucleotide, the conjugate group is typically attached to a ribose sugar ring or base. Various connection modes can be referred to ：Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.ACS Chemical biology,2015,10(5):1181-7.

The targeting group may be attached to the double stranded oligonucleotide molecule via a suitable linker, which may be selected by one skilled in the art depending on the particular type of targeting group. The types of these linkers, targeting groups and the manner of attachment to the double stranded oligonucleotides can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference. In some embodiments, the double-stranded oligonucleotide and the conjugate group may be linked by acid labile, or reducible, chemical bonds that degrade in the acidic environment of the cellular endosome, thereby rendering the double-stranded oligonucleotide free. For non-degradable conjugation means, the conjugation group may be attached to the sense strand of the double-stranded oligonucleotide, thereby minimizing the effect of conjugation on the activity of the double-stranded oligonucleotide.

In some embodiments, the targeting group can be a ligand conventionally used in the art of siRNA administration. In some embodiments, the targeting group may be selected from one or more of the following ligands formed by the targeting molecule or derivative thereof; such as lipophilic molecules, e.g. cholesterol, bile acids, vitamins (e.g. vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; sugars, such as lactose, mannose, galactose, N-acetylgalactosamine (GalNAc); receptor ligands expressed by hepatic parenchymal cells, such as, for example, asialoglycoprotein, asialoglycoresidue, lipoproteins (e.g., high density lipoproteins, low density lipoproteins, etc.), glucagon, neurotransmitters (e.g., epinephrine), growth factors, transferrin, etc. aptamers; an antibody; a quantum dot; polypeptides, such as permeabilizing peptides, or small molecule ligands.

In some embodiments, the targeting group can be a ligand conventionally used in the art of siRNA administration, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.

In some embodiments, at least one or each of the targeting groups is selected from a ligand capable of binding to mammalian liver parenchymal cell surface receptor (ASGPR). In some embodiments, each of the targeting groups is independently a ligand that is affinity to an asialoglycoprotein receptor on the surface of mammalian hepatocytes. In some embodiments, each of the targeting groups is independently an asialoglycoprotein or a sugar. In some embodiments, each of the targeting groups is independently an asialoglycoprotein, such as an asialoglycoprotein of the serotypes (asialoorosomucoid, ASOR) or an asialoglycoprotein of the onset (asialofetuin, ASF). In some embodiments of the present invention, in some embodiments, each of the targeting groups is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylose furanose, L-xylose furanose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannopyranose, beta-D-mannopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructofuranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-isobutyrylgalactosamine, 2-amino-3-O) -1- [ (R ] -2-ethyl ] -2-carboxymethyl ] -2-D-galactopyranose, 4-dimethyl-6-D-amino-6-D-deoxyglucopyranose, 4-dimethyl-4-D-galactopyranose, 2-deoxy-2-sulphonamino-D-glucopyranose, N-glycolyl- α -neuraminic acid, 5-thio- β -D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl- α -D-glucopyranoside methyl ester, 4-thio- β -D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio- α -D-glucoheptopyranoside ethyl ester, 2, 5-anhydro-D-psicosonitrile, ribose, D-4-thioribose, L-ribose, L-4-thioribose. In some embodiments, at least one or each of the targeting groups is galactose or N-acetylgalactosamine.

In some embodiments, the linker in the siRNA conjugates of the present disclosure has a structure as shown in formula (301):

Wherein k is an integer of 1 to 3;

L ^A has a structure containing an amide bond as shown in formula (302), L ^B has a structure containing N-acyl pyrrolidine as shown in formula (303), contains a carbonyl group and an oxygen atom, and L ^C is a linking group based on hydroxymethyl aminomethane, dihydroxymethyl aminomethane or trimethylol aminomethane;

Wherein n ₃₀₂、q₃₀₂ and p ₃₀₂ are each independently integers from 2 to 6, alternatively n ₃₀₂、q₃₀₂ and p ₃₀₂ are each independently 2 or 3; n ₃₀₃ is an integer from 4 to 16, alternatively n ₃₀₃ is an integer from 8 to 12, Indicating the site of covalent attachment of the group.

In the linker, each L ^A is connected with one targeting group through an ether bond and is connected with the L ^C part through an oxygen atom of a hydroxyl group in the L ^C part through an ether bond; l ^B is linked by an amide bond formed by the carbonyl group in formula (303) and the nitrogen atom of the amino group in the L ^C moiety, and is linked by an oxygen atom in formula (303) and the siRNA by an oxygen atom forming a phosphate bond or a phosphorothioate bond.

In some embodiments, the siRNA conjugates provided by the present disclosure have a structure as shown in formula (305):

Where Nu represents the siRNA provided by the present disclosure.

In some embodiments, the linker in the siRNA conjugates of the present disclosure has a structure represented by formula (306):

wherein n ₃₀₆ is an integer from 0 to 3, each p ₃₀₆ is independently an integer from 1 to 6, Represents the site of covalent attachment of the group; the linking group forms an ether bond with the targeting group through an oxygen atom as indicated; the connecting group is formed by connecting at least one of oxygen atoms marked with the number # and the siRNA to form a phosphate bond or a phosphorothioate bond, and the rest oxygen atoms marked with the number # are connected with hydrogen atoms to form hydroxyl groups or are connected with C ₁-C₃ alkyl to form C ₁-C₃ alkoxy groups;

In some embodiments, the siRNA conjugates of the present disclosure have a structure as shown in formula (307):

Where Nu represents the siRNA provided by the present disclosure.

In some embodiments, the conjugate has a structure represented by formula (308):

(308) a step of,

Wherein n1 is an integer selected from 1 to 3, and n3 is an integer selected from 0 to 4;

m1, m2 and m3 are independently integers selected from 2 to 10;

R ₁₀、R₁₁、R₁₂、R₁₃、R₁₄ and R ₁₅ are each independently H, or selected from the group consisting of: c ₁-C₁₀ alkyl, C ₁-C₁₀ haloalkyl, and C ₁-C₁₀ alkoxy;

r ₃ is a group of the structure shown in formula A59:

Wherein E ₁ is OH, SH or BH ₂, and Nu is a double-stranded oligonucleotide;

R ₂ is a linear alkylene group of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by one or more selected from the group consisting of: c (O), NH, O, S, CH = N, S (O) ₂、C₂-C₁₀ alkenylene, C ₂ -C10 alkynylene, C ₆-C₁₀ arylene, C ₃-C₁₈ heterocyclylene, and C ₅-C₁₀ heteroarylene, and wherein R ₂ may optionally have substituents of any one or more of the group consisting of: c ₁-C₁₀ alkyl group, C ₆-C₁₀ aryl, C ₅-C₁₀ heteroaryl, C ₁-C₁₀ haloalkyl, -OC ₁-C₁₀ alkyl, -OC ₁-C₁₀ alkylphenyl, -C ₁-C₁₀ alkyl-OH, -OC ₁-C₁₀ haloalkyl, -SC ₁-C₁₀ alkyl, -SC ₁-C₁₀ alkylphenyl, -C ₁-C₁₀ alkyl-SH, -SC ₁-C₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂、-C₁-C₁₀ alkyl-NH ₂、-N(C₁-C₁₀ alkyl) (C ₁-C₁₀ alkyl), -NH (C ₁-C₁₀ alkyl), cyano, nitro, -CO ₂H、-C(O)O(C₁-C₁₀ alkyl), -CON (C ₁-C₁₀ alkyl) (C ₁-C₁₀ alkyl), -CONH (C ₁-C₁₀ alkyl), -CONH ₂,-NHC(O)(C₁-C₁₀ alkyl), -NHC (O) (phenyl), -N (C ₁-C₁₀ alkyl) C (O) (C ₁-C₁₀ alkyl), -N (C ₁-C₁₀ alkyl) C (O) (phenyl), -C (O) C ₁-C₁₀ alkyl, -C (O) C ₁-C₁₀ alkylphenyl, -C (O) C1-C ₁₀ haloalkyl, -OC (O) C ₁-C₁₀ alkyl, -SO ₂(C₁-C₁₀ alkyl), -SO ₂ (phenyl), -SO ₂(C₁-C₁₀ haloalkyl), -SO ₂NH₂、-SO₂NH(C₁-C₁₀ alkyl), -SO ₂ NH (phenyl), -NHSO ₂(C₁-C₁₀ alkyl), -NHSO ₂ (phenyl) and-NHSO ₂(C₁-C₁₀ haloalkyl);

Each L ₁ is a linear alkylene group of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by one or more selected from the group consisting of: c (O), NH, O, S, CH = N, S (O) ₂、C₂-C₁₀ alkenylene, C ₂-C₁₀ alkynylene, C ₆-C₁₀ arylene, C ₃-C₁₈ heterocyclylene, and C ₅-C₁₀ heteroarylene, and wherein L ₁ may optionally have substituents of any one or more of the group consisting of: c ₁-C₁₀ alkyl group, C ₆-C₁₀ aryl, C ₅-C₁₀ heteroaryl, C ₁-C₁₀ haloalkyl, -OC ₁-C₁₀ alkyl, -OC ₁-C₁₀ alkylphenyl, -C ₁-C₁₀ alkyl-OH, -OC ₁-C₁₀ haloalkyl, -SC ₁-C₁₀ alkyl, -SC ₁-C₁₀ alkylphenyl, -C ₁-C₁₀ alkyl-SH, -SC ₁-C₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂、-C₁-C₁₀ alkyl-NH ₂、-N(C₁-C₁₀ alkyl) (C ₁-C₁₀ alkyl), -NH (C ₁-C₁₀ alkyl), cyano, nitro, -CO ₂H、-C(O)O(C₁-C₁₀ alkyl), -CON (C ₁-C₁₀ alkyl) (C ₁-C₁₀ alkyl), -CONH (C ₁-C₁₀ alkyl), -CONH ₂,-NHC(O)(C₁-C₁₀ alkyl), -NHC (O) (phenyl), -N (C ₁-C₁₀ alkyl) C (O) (C ₁-C₁₀ alkyl), -N (C ₁-C₁₀ alkyl) C (O) (phenyl), -C (O) C ₁-C₁₀ alkyl, -C (O) C ₁-C₁₀ alkylphenyl, -C (O) C1-C ₁₀ haloalkyl, -OC (O) C ₁-C₁₀ alkyl, -SO ₂(C₁-C₁₀ alkyl), -SO ₂ (phenyl), -SO ₂(C₁-C₁₀ haloalkyl), -SO ₂NH₂、-SO₂NH(C₁-C₁₀ alkyl), -SO ₂ NH (phenyl), -NHSO ₂(C₁-C₁₀ alkyl), -NHSO ₂ (phenyl) and-NHSO ₂(C₁-C₁₀ haloalkyl. M1 represents a targeting group.

Represents the site of covalent attachment of the group;

M ₁ represents a targeting group, the definition and optional scope of which are the same as described above. In some embodiments, each M ₁ is independently selected from one of the ligands having an affinity for an asialoglycoprotein receptor on the surface of a mammalian liver cell.

The skilled artisan will appreciate that although L ₁ is defined as a linear alkylene group for convenience, it may not be a linear group or be named differently, such as an amine or alkenyl group resulting from the substitutions and/or substitutions described above. For the purposes of this disclosure, the length of L ₁ is the number of atoms in the chain connecting the two attachment points. For this purpose, the ring (e.g., heterocyclylene or heteroarylene) resulting from substitution of the carbon atom of the linear alkylene group is counted as one atom.

When M ₁ is a ligand having an affinity for an asialoglycoprotein receptor on the surface of a mammalian liver cell, in some embodiments n1 may be an integer from 1 to 3, n3 may be an integer from 0 to 4, ensuring that the number of M ₁ ligands in the conjugate is at least 2; in some embodiments, n1+n3.gtoreq.2, such that the number of M ₁ ligands is at least 3, allows for easier binding of the M ₁ ligand to the hepatic surface asialoglycoprotein receptor, thereby facilitating entry of the conjugate into cells by endocytosis. Experiments have shown that when the number of M ₁ ligands is greater than 3, the increase in ease of binding of the M ₁ ligand to the hepatic surface asialoglycoprotein receptor is not significant, and thus, in some embodiments, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1+n3=2 to 3, from a comprehensive view of ease of synthesis, cost of structure/process, and delivery efficiency.

In some embodiments, where M1, M2, and M3 are independently selected from integers from 2-10, the spatial position between the plurality of M ₁ ligands may be tailored for binding of the M ₁ ligand to the hepatic surface asialoglycoprotein receptor, in order to make the conjugates provided by the present disclosure simpler, easier to synthesize, and/or lower cost, in some embodiments, each of M1, M2, and M3 is independently an integer from 2-5, in some embodiments m1=m2=m3.

Those skilled in the art will appreciate that when R ₁₀、R₁₁、R₁₂、R₁₃、R₁₄ and R ₁₅ are each independently selected from one of H, C ₁-C₁₀ alkyl, C ₁-C₁₀ haloalkyl, and C ₁-C₁₀ alkoxy, the objectives of the present disclosure can be achieved without altering the properties of the conjugates disclosed herein. In some embodiments, R ₁₀、R₁₁、R₁₂、R₁₃、R₁₄ and R ₁₅ are each independently selected from H, methyl, and ethyl. In some embodiments, R ₁₀、R₁₁、R₁₂、R₁₃、R₁₄ and R ₁₅ are both H.

According to the siRNA conjugates provided herein, R ₃ is a group of the structure shown in formula a59, wherein E ₁ is OH, SH, or BH ₂, in some embodiments E ₁ is OH or SH, based on ease of availability of the preparation starting materials.

In some embodiments, R ₂ is selected to achieve a linkage to the N atom on the nitrogen-containing backbone to a 59. In the context of the present disclosure, a "nitrogen-containing backbone" refers to a chain structure in which the carbon atoms to which R ₁₀、R₁₁、R₁₂、R₁₃、R₁₄ and R ₁₅ are attached are interconnected with an N atom. Thus, R ₂ can be any linking group capable of linking the a59 group to the N atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, where the siRNA conjugates of the present disclosure are prepared by a process of solid phase synthesis, it is desirable to have both a linking site in the R ₂ group that is linked to the N atom on the nitrogen-containing backbone and a linking site that is linked to the P atom in R ₃. In some embodiments, the site in R ₂ attached to the N atom on the nitrogen-containing backbone forms an amide bond with the N atom and the site attached to the P atom on R ₃ forms a phosphate bond with the P atom. In some embodiments, R ₂ is B5, B6, B5', or B6':

Wherein, Indicating the site of covalent attachment of the group.

Q ₂ may be an integer in the range of 1 to 10, and in some embodiments q ₂ is an integer in the range of 1 to 5.

The role of L ₁ is to link the M ₁ ligand to N on a nitrogen-containing backbone, providing a targeting function for the siRNA conjugates of the present disclosure. In some embodiments, L ₁ is selected from a linked combination of one or more of the groups of formulas A1-A26. In some embodiments, L ₁ is selected from the group consisting of A1, A4, A5, A6, A8, a10, a11, and a 13; in some embodiments, L ₁ is selected from the group consisting of a linked combination of at least 2 of A1, A4, A8, a10, and a 11; in some embodiments, L ₁ is selected from the group consisting of a linked combination of at least 2 of A1, A8, a 10.

In some embodiments, L ₁ may be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments, L ₁ is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atoms in length.

In some embodiments, j1 is an integer from 2 to 10, and in some embodiments, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3 to 5. R 'is C ₁-C₄ alkyl, in some embodiments, R' is one of methyl, ethyl, and isopropyl. R _a is one of a27, a28, a29, a30, and a31, in some embodiments R _a is a27 or a28.Rb is a C ₁-C₅ alkyl group, in some embodiments, R _b is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', R _a、R_b are each selected in formulas A1-a26 to achieve N-linking of the M ₁ ligand to the nitrogen-containing backbone and to make the spatial position between the M ₁ ligand more suitable for binding of the M ₁ ligand to the hepatic surface asialoglycoprotein receptor.

In some embodiments, the siRNA conjugates of the present disclosure have a structure represented by formula (403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421) or (422):

In some embodiments, the P atom in formula a59 can be attached to any possible position in the siRNA sequence, e.g., the P atom in formula a59 can be attached to any one of the nucleotides of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula a59 is attached to any one nucleotide of the sense strand of the siRNA. In some embodiments, the P atom in formula a59 is attached to the end of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula a59 is attached to the end of the sense strand of the siRNA. The end refers to the first 4 nucleotides from one end of the sense strand or the antisense strand. In some embodiments, the P atom in formula a59 is attached to the end of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula a59 is attached to the 3' end of the sense strand of the siRNA. In the case of the above-described position of the sense strand linked to the siRNA, the conjugates provided by the present disclosure, upon entry into a cell, upon unwinding, can release the separate siRNA antisense strand to inhibit target gene expression by RNAi machinery.

The P atom in formula A59 can be attached to any possible position on the nucleotide in the siRNA, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide, or the base of the nucleotide. In some embodiments, the P atom in formula a59 can be linked to the 2', 3', or 5' position of a nucleotide in the siRNA by formation of a phosphodiester linkage. In some embodiments, the P atom in formula a59 is attached to an oxygen atom formed upon dehydrogenation of the 3' hydroxyl group of the 3' terminal nucleotide of the siRNA sense strand, or the P atom in formula a59 is attached to a nucleotide by substitution of hydrogen in the 2' -hydroxyl group of one nucleotide in the siRNA sense strand, or the P atom in formula a59 is attached to a nucleotide by substitution of hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide of the siRNA sense strand.

In the siRNA or siRNA conjugates described in the present disclosure, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate bond, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the phosphorothioate bond carries a negative charge, and the siRNA or siRNA conjugate can exist in a form of hydroxyl group or sulfhydryl group, and hydrogen ions in the hydroxyl group or sulfhydryl group can also be partially or completely replaced by cations. The cation may be any cation, such as one of a metal cation, ammonium ion NH4 ⁺, and an organic ammonium cation. In some embodiments, the cation is selected from one or more of an alkali metal ion, a tertiary amine-forming ammonium cation, and a quaternary ammonium cation for improved solubility. The alkali metal ions may be K ⁺ and/or Na ⁺, and the tertiary amine-forming cations may be triethylamine-forming ammonium ions and/or N, N-diisopropylethylamine-forming ammonium ions. Thus, the siRNA or siRNA conjugates of the present disclosure may exist at least partially in salt form. In one mode, the non-bridging oxygen or sulfur atoms in the phosphodiester or phosphorothioate linkages are at least partially bound to sodium ions, and the siRNA or siRNA conjugates of the present disclosure are in the form of sodium salts or partial sodium salts.

Preparation of siRNA conjugates of the disclosure

The above siRNA conjugates can be synthesized by methods already described in detail in the prior art. For example, the preparation of various siRNA conjugates is described in detail in WO2015006740A 2. The siRNA conjugates of the present disclosure are obtained by means well known to those skilled in the art. A method for preparing the structure represented by formula (305) is described in WO2014025805A1, and Rajeev et al in ChemBioChem 2015,16,903-908 describes a method for preparing the structure represented by formula (307). Chinese patent application CN110959011a also discloses in detail the method of preparing siRNA conjugates represented by formula (308). The contents of the above documents are incorporated by reference in their entirety.

The siRNA conjugates of the present disclosure may also be combined with other pharmaceutically acceptable excipients, which may be one or more of a variety of formulations or compounds conventionally employed in the art, see the description of the pharmaceutical compositions of the present disclosure above for details.

Nucleotide sequences, double-stranded oligonucleotides, pharmaceutical compositions, and uses of siRNA conjugates of the present disclosure

In some embodiments, the present disclosure provides the use of a nucleotide sequence, a double-stranded oligonucleotide, a pharmaceutical composition, and/or an siRNA conjugate provided by the present disclosure in the manufacture of a medicament for treating and/or preventing a pathological condition or disease caused by expression of a particular gene in a cell.

The modified double-stranded oligonucleotides, pharmaceutical compositions and oligonucleotide conjugates provided herein can be used to modulate abnormal expression of various genes, treating various pathological conditions or diseases caused by abnormal expression of genes. These genes may be various endogenous genes in the human or animal body, or may be pathogen genes that are propagated in the human or animal body. Double-stranded oligonucleotides having specific nucleotide sequences and the modification schemes can be designed and prepared based on mRNA expressed by the target gene. In some embodiments, the mRNA expressed by the target gene is selected from one ：ACE2、ANGPTL3、ApoA、ApoB、ApoC、AR、ASK1、C5、Col1A1、CTGF、Ebola、FOXO1、FTO、FVII、FXI、FXII、GCGR、HBV、HCV、HSD、p53、PCSK9、PNP、PLG、PKK、KNG、SARS-CoV-2、SCD1、SCNN1A、SOD1、STAT3、TIMP-1、TMPRSS6、XO、HAO1 isogene among the mrnas transcribed from the following genes. In some embodiments, the specific gene is selected from the group consisting of HBV gene, ANGPTL3, APOC3 gene, or mRNA expressed by recombinant human hydroxy acid oxidase 1 gene. Accordingly, the disease or condition is selected from diseases or conditions resulting from abnormal gene expression. The disease is selected from chronic liver disease, hepatitis, liver fibrosis disease, liver hyperplasia disease and dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

In some embodiments, the present disclosure provides a method of treating a pathological condition or disease caused by abnormal expression of a particular gene, the method comprising administering to a subject in need thereof an effective amount of a double-stranded oligonucleotide, pharmaceutical composition and/or siRNA conjugate provided by the present disclosure. In some embodiments, the specific gene is selected from one ：ACE2、ANGPTL3、ApoA、ApoB、ApoC、AR、ASK1、C5、Col1A1、CTGF、Ebola、FOXO1、FTO、FVII、FXI、FXII、GCGR、HBV、HCV、HSD、p53、PCSK9、PNP、PLG、PKK、KNG、SARS-CoV-2、SCD1、SCNN1A、SOD1、STAT3、TIMP-1、TMPRSS6、XO、HAO1. of the following genes, in some embodiments, the specific gene is selected from the mRNA expressed by HBV gene, ANGPTL3, APOC3 gene, or recombinant human hydroxy acid oxidase 1 gene. Accordingly, the disease or condition is selected from diseases or conditions resulting from abnormal gene expression. The disease is selected from chronic liver disease, hepatitis, liver fibrosis disease, liver hyperplasia disease and dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis. In some embodiments, the conjugates provided by the present disclosure may also be used to treat other liver diseases, including diseases characterized by unwanted cell proliferation, blood diseases, metabolic diseases, and diseases characterized by inflammation. The proliferative disease of the liver may be a benign or malignant disease, such as cancer, hepatocellular carcinoma (HCC), liver metastasis or hepatoblastoma. The hematological or inflammatory disease of the liver may be a disease involving coagulation factors, complement mediated inflammation or fibrosis. Metabolic diseases of the liver include dyslipidemia and irregularities in glucose regulation. In one embodiment, the disease is treated by administering one or more double stranded oligonucleotides having a high homology to the gene sequences involved in the disease.

In some embodiments, the present disclosure provides a method of inhibiting expression of a particular gene in a cell, the method comprising contacting an effective amount of a double stranded oligonucleotide, pharmaceutical composition, and/or siRNA conjugate provided by the present disclosure with the cell.

By administering the double stranded oligonucleotides, pharmaceutical compositions and/or siRNA conjugates of the present disclosure to a subject in need thereof, the prevention and/or treatment of a pathological condition or disease caused by the expression of a specific gene in a cell can be achieved by a mechanism that regulates gene expression. Thus, the double-stranded oligonucleotides, pharmaceutical compositions and/or siRNA conjugates of the present disclosure may be used for preventing and/or treating the pathological condition or disease, or for the preparation of a medicament for preventing and/or treating the pathological condition or disease described herein.

The term "administration" as used herein refers to placement of a double-stranded oligonucleotide, pharmaceutical composition, and/or siRNA conjugate into a subject by a method or route that results in at least partially positioning the double-stranded oligonucleotide, pharmaceutical composition, and/or siRNA conjugate at a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include topical and systemic administration. In general, topical administration results in more double-stranded oligonucleotides, pharmaceutical compositions, and/or siRNA conjugates being delivered to a particular site than the entire body of the subject; whereas systemic administration results in delivery of the double stranded oligonucleotide, pharmaceutical composition and/or siRNA conjugate to substantially the entire body of the subject. It is contemplated that the present disclosure aims to provide means for preventing and/or treating pathological conditions or diseases caused by the expression of specific genes in hepatocytes, in some embodiments, modes of administration that are capable of delivering drugs to the liver.

The administration to the subject may be by any suitable route known in the art, including but not limited to: oral or parenteral routes such as intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal and topical (including buccal and sublingual). The frequency of administration may be 1 or more times daily, weekly, biweekly, tricyclically, monthly, 2 months, quarterly, semi-annually, or annually.

Dosages of the double stranded oligonucleotides, pharmaceutical compositions and/or siRNA conjugates described in the present disclosure may be dosages conventional in the art, which may be determined according to various parameters, particularly the age, weight and sex of the subject. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the lethal dose to death of 50% of the population) and the ED50 (the dose that causes 50% of the maximal response intensity in the dose response, and the dose that causes 50% of the subjects to develop a positive response in the mass response). The range of doses for human use can be derived based on data obtained from cell culture assays and animal studies.

Upon administration of the double stranded oligonucleotides, pharmaceutical compositions and/or siRNA conjugates of the disclosure, e.g., for male or female, 6-12 weeks old, C57BL/6J or C3H/HeNCrlVr mice weighing 18-25g, based on the amount of double stranded oligonucleotides in the double stranded oligonucleotides, pharmaceutical compositions and/or siRNA conjugates: for siRNA conjugates of the double stranded oligonucleotide and a pharmaceutically acceptable conjugate molecule, the amount of double stranded oligonucleotide may be in the range of 0.001 to 100mg/kg body weight, in some embodiments 0.01 to 50mg/kg body weight, in further embodiments 0.05 to 20mg/kg body weight, in still further embodiments 0.1 to 15mg/kg body weight, and in yet further embodiments 0.1 to 10mg/kg body weight. Such amounts may be preferred when administering the double stranded oligonucleotides, pharmaceutical compositions and/or siRNA conjugates described in the present disclosure.

In addition, by introducing the double stranded oligonucleotide, pharmaceutical composition and/or siRNA conjugate of the present disclosure into a cell, the objective of inhibiting expression of the specific gene in a hepatocyte can also be achieved by RNAi machinery. In some embodiments, the liver cells are hepatitis cells, in some embodiments HEK293A cells or hepg2.2.15 cells. In some embodiments, the liver cells may be selected from liver cancer cell lines such as Hep3B, hepG, huh7, etc., or isolated primary liver cells, in some embodiments Huh7 liver cancer cells.

The amount of double stranded oligonucleotide provided, pharmaceutical compositions and/or siRNA conjugates to be used to inhibit expression of a particular gene in hepatocytes using the methods provided by the present disclosure is readily determinable by one of skill in the art as desired to obtain the effect. For example, in some embodiments, the double stranded oligonucleotide, pharmaceutical composition, and/or siRNA conjugate is an siRNA conjugate, the amount of siRNA in the provided siRNA conjugate is such that: it is sufficient to reduce expression of the target gene and results in an extracellular concentration of 1pM to 1. Mu.M, or 0.01nM to 100nM, or 0.05nM to 50nM, or to about 5nM at the surface of the target cell. The amount required to achieve this local concentration will vary depending on a variety of factors including the method of delivery, the site of delivery, the number of cell layers between the site of delivery and the target cell or tissue, whether the delivery is local or systemic, etc. The concentration at the delivery site may be significantly higher than the concentration at the surface of the target cell or tissue.

Kit for detecting a substance in a sample

The present disclosure provides a kit comprising an effective amount of at least one of the siRNA, pharmaceutical composition and siRNA conjugate of the present disclosure.

In some embodiments, the kits described herein can provide siRNA, pharmaceutical compositions, and/or siRNA conjugates in one container. In some embodiments, the kits described herein can comprise a container that provides a pharmaceutically acceptable excipient. In some embodiments, other ingredients, such as stabilizers or preservatives, and the like, may also be included in the kit. In some embodiments, the kits described herein can comprise at least one additional therapeutic agent in a container other than the container in which the siRNA described herein is provided. In some embodiments, the kit may comprise instructions for mixing the siRNA with a pharmaceutically acceptable carrier and/or adjuvant or other ingredients, if any.

In the kits of the present disclosure, the siRNA and pharmaceutically acceptable carrier and/or adjuvant and the siRNA, pharmaceutical composition and/or siRNA conjugate, and/or pharmaceutically acceptable adjuvant may be provided in any form, such as a liquid form, a dry form or a lyophilized form. In some embodiments, the siRNA and pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or siRNA conjugate and optionally pharmaceutically acceptable adjuvant are substantially pure and/or sterile. In some embodiments, sterile water may be provided in a kit of the present disclosure.

The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereby.

Examples

Unless otherwise specified, reagents and media used in the examples below are commercially available, and the procedures for nucleic acid electrophoresis, real-time PCR, and the like used are carried out by the method described in Molecular Cloning (Cold Spring Harbor LBboratory Press (1989)).

HEK293A cells were supplied from the nucleic acid technology laboratory of the university of Beijing institute of molecular medicine, and cultured in DMEM complete medium (Hyclone) containing 20% fetal bovine serum (FBS, hyclone) and 0.2% by volume of double antibody to Penicillin (Penicillin-Streptomycin, gibco, invitrogen) and incubated in an incubator containing 5% CO2/95% air at 37 ℃.

Unless otherwise indicated, when cells were transfected with the various siRNAs or siRNA conjugates synthesized below, lipofectamine 2000 (Invitrogen) was used as the transfection reagent, with reference to the manufacturer's instructions for specific procedures.

Unless otherwise indicated, the reagent ratios provided below are all calculated as volume ratios (v/v).

The animal model used is as follows:

c57BL/6N mice: 6-8 weeks old, purchased from beijing verdelihua laboratory animal technologies limited, hereinafter abbreviated as C57 mice;

SD rats: is provided by Beijing Vietnam Lihua laboratory animal technology Co., ltd;

HBV transgenic mice C57BL/6-HBV: strain name: B6-Tg HBV/Vst (1.28copy,genotype A), available from Beijing Vietnam Biotechnology Co. Mice with COI >10 ⁴, also referred to below simply as 1.28copy mice, were selected prior to the experiment;

HBV transgenic mice C57BL/6J-Tg (Alb 1 HBV) 44Bri/J: purchased from the department of laboratory animal science, university of Beijing, medical department;

HBV transgenic mice: designated M-TgHBV, purchased from Shanghai public health center animal sector, transgenic mice were prepared as described in Ren J. Et al, J.medical virology.2006, 78:551-560;

AAV-HBV transgenic mice: AAV-HBV models were prepared according to literature methods (Dong Xiaoyan et al, chin J Biotech2010, may 25;26 (5): 679-686), rAAV8-1.3HBV, form D (ayw), purchased from Beijing acanthopanax and molecular medicine research, inc., 1X 10 ¹² viral genome (v.g.), per mL, lot number 2016123011. The experiments were preceded by dilution to 5X 10 ¹¹ v.g./mL with sterile PBS. Each mouse was injected with 200 μl, i.e. 1×10 ¹¹ v.g. per mouse. On day 28 post virus injection, all mice were bled through the orbit (about 100 μl) for collection of serum for detection of HBsAg and HBV DNA;

Low concentration AAV-HBV transgenic mice: the same modeling procedure as described above was used, except that the virus was diluted to 1×10 ¹¹ v.g./mL with sterile PBS prior to the experiment, and 100 μl of virus was injected per mouse, i.e. 1×10 ¹⁰ v.g. per mouse;

BALB/c mice: 6-8 weeks old, purchased from Beijing Vietnam laboratory animal technologies Co., ltd;

ob/ob mice: 6-8 weeks old, purchased from Kwansi laboratory animal Co., ltd;

human APOC3 transgenic mice: b6; CBA-Tg (APOC 3) 3707Bres/J, available from Jackson laboratories, USA;

ˉ

Unless otherwise indicated, the following in vivo/in vitro effect experimental data are all expressed in X+ -SEM, and data analysis uses GRAPHPAD PRISM 6.0.0 statistical analysis software.

Preparation examples 1-48 Synthesis of siRNA conjugates provided by the present disclosure

Conjugates 1 to 48 in the following table 2 were prepared according to the preparation method described in preparation example 1 of CN110959011a, except that the sense strand and the antisense strand of the siRNA contained in the siRNA conjugate are shown in table 2, respectively. The sense strand and the antisense strand of the siRNA were synthesized according to the nucleic acid sequences of the siRNA numbered conjugate 1-conjugate 48 in table 2 below, respectively. The siRNA conjugate was diluted to a concentration of 0.2mg/mL (based on siRNA) using ultra pure water (Milli-Q ultra pure water meter, resistivity 18.2MΩ cm (25 ℃)), and then subjected to molecular weight measurement using a liquid chromatography-Mass spectrometer (LC-MS, liquid Chromatography-Mass SP1ectrometry, available from Waters, model number LCT PREMIER). The actual values were consistent with the theoretical values, indicating that the synthesized conjugates 1-48 were double stranded nucleic acid sequences of the target design. Conjugates 1-48 have a structure represented by formula (403), and the conjugates 1-48 comprise siRNAs having the siRNA sequences corresponding to conjugates 1-48 in Table 2.

TABLE 2siRNA sequences in siRNA conjugates

TABLE 3 reference siRNA sequences in siRNA conjugates

Wherein, the capital letter C, G, U, A represents the base composition of the nucleotide; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a 2' -methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a 2' -fluoro modified nucleotide; the lower case letter s indicates that the linkage between two nucleotides adjacent to the letter s is a phosphorothioate linkage; the lowercase letter d indicates that the adjacent nucleotide to the right of the letter d is a 2' -deoxynucleotide; the letter combination VP represents the nucleotide modified by vinyl phosphate of one nucleotide adjacent to the right side of the VP; capital letter P indicates that the adjacent nucleotide to the right of P is a 5' -phosphate modified nucleotide; (GLY) represents that two nucleotides adjacent to the left and right of (GLY) are linked by an acyclic dealkalization group represented by formula (a 101); (GLY-Ac) represents that two nucleotides adjacent to the left and right of the (GLY-Ac) are linked by an acyclic dealkalization group represented by formula (a 102); (GLY-Ph) two nucleotides adjacent to the (GLY-Ph) side are linked by an acyclic dealkalization group represented by formula (a 103); (GLY-iBu) represents that two nucleotides adjacent to the (GLY-iBu) are linked by an acyclic dealkalization group represented by formula (a 105); (GLY-Cro) represents that two nucleotides adjacent to the (GLY-Cro) are linked by an acyclic dealkalide group represented by formula (a 107), and the foregoing acyclic dealkalide groups are each in S configuration; (GLY-S) means that two nucleotides adjacent to the (GLY-S) side are linked by an acyclic dealkalization group represented by formula (101) of S configuration; (GLY-R) means that two nucleotides adjacent to the left and right of the (GLY-R) are linked by an acyclic abasic group represented by the formula (101) of R configuration (GNA) means that a ribose in a nucleotide adjacent to the left of the GNA is replaced by a GNA to form a monomer; (UNA) means a monomer containing a substitution of ribose in a nucleotide adjacent to the left side of UNA by UNA.

Comparative preparation examples 1-23 Synthesis of reference siRNA conjugates

Reference siRNA conjugates numbered negative reference conjugate and reference conjugates 1 to 23 in table 3 above were prepared according to the preparation method described in CN110959011a preparation example 1, except that the sense strand and the antisense strand of the siRNA contained in the reference siRNA conjugate are as shown in table 3, and the siRNA sequence in the reference siRNA conjugate NC is a negative control sequence having no significant correlation with known genes; the siRNA sequences contained in reference conjugates 1-23 were sequences that possessed the same base ordering as the siRNA sequences in conjugates 1-47 described previously, but did not contain acyclic dealkalization groups. The sense and antisense strands of the siRNAs were synthesized according to the siRNA nucleic acid sequences numbered negative reference conjugates and reference conjugates 1-23, respectively, in Table 3. The siRNA conjugate was diluted to a concentration of 0.2mg/mL (based on siRNA) using ultra pure water (Milli-Q ultra pure water meter, resistivity 18.2MΩ cm (25 ℃)), and then subjected to molecular weight measurement using a liquid chromatography-Mass spectrometer (LC-MS, liquid Chromatography-Mass SP1ectrometry, available from Waters, model number LCT PREMIER). The measured values were consistent with the theoretical values, indicating that the negative reference conjugates and reference conjugates 1-23 synthesized were double stranded nucleic acid sequences of the target design. The conjugate siRNA conjugates have a structure shown in formula (403), and the sirnas contained in each reference siRNA conjugate have siRNA sequences corresponding to negative reference conjugates or reference conjugates 1 to 23 in table 3, respectively.

Experimental example 1

The conjugates of the present disclosure have inhibitory activity against off-target sequences in an in vitro psi-CHECK system

In this experimental example, the inhibitory activity of conjugate 1, conjugate 2, conjugate 3, conjugate 4, conjugate 5, conjugate 6, conjugate 7 and conjugate 8 on the target sequence in the in vitro psi-CHECK system was examined using the in vitro psi-CHECK system.

According to the method described in Kumico Ui-Tei et.al.,Functional dissection of siRNA sequence by systematic DNA substitution:modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.Nucleic Acids Research,2008.36(7),2136-2151, a detection plasmid is constructed, the detection plasmid and the conjugate to be detected are co-transfected into HEK293A cells, and the target sequence inhibition activity of siRNA is reflected through the expression level of the dual luciferase reporter gene. The method comprises the following specific steps:

[1] Construction of the detection plasmid

The psiCHECK ^TM-2(Promega^TM) plasmid is adopted to construct a detection plasmid, the plasmid contains a target sequence 1, the target sequence 1 contains a sequence which is complementary with an siRNA antisense strand part in the conjugate to be detected, and the sequence is repeated 5 times in the target sequence 1, so that the inhibition effect of the conjugate to be detected on the target sequence 1 can reflect the degree of off-target effect. That is, the higher the inhibitory effect, the more likely off-target the conjugate to be detected will occur. The target sequence 1 and its complement were cloned into the Xho I/Not I site of the psiCHECK ^TM -2 plasmid.

For the siRNA conjugates to be tested, target sequence 1 is as follows:

5'-CTCGAGAAACCGCCCTAGGGACAAGAATTGGAAACCGCCC TAGGGACAAGAATTGGAAACCGCCCTAGGGACAAGAATTGGAAAC CGCCCTAGGGACAAGAATTGGAAACCGCCCTAGGGACAAGAA-3'(SEQ ID NO:131)

[2] transfection

HEK293A cells (available from Nanjac hundred Biotechnology Co., ltd.) were cultured in an incubator containing 5% CO ₂/95% air at 37℃in H-DMEM complete medium (HyClone Co.) supplemented with 10% fetal bovine serum (FBS, RMBIO Co.) and 0.2% by volume of green streptomycin double antibody (Penicillin-Streptomycin, hyClone Co.).

HEK293A cells were seeded at 8X 10 ³ cells/well in 96-well plates, and after 16 hours when the cell growth density reached 70%, the culture wells were completely drained and 80. Mu.L opti-MEM medium (GIBCO Co.) was added to each well for further 1.5 hours.

Diluting the above detection plasmid into 20 μm stock solution with PBS; each siRNA conjugate to be tested was formulated with PBS as a working solution of 11 different concentrations of siRNA conjugates, 4. Mu.M, 1. Mu.M, 0.25. Mu.M, 0.0625. Mu.M, 0.015625. Mu.M, 0.003906. Mu.M, 0.0009765. Mu.M, 0.0002441. Mu.M, 0.00006104. Mu.M, 0.00001526. Mu.M, and 0.000003815. Mu.M, respectively, based on the amount of siRNA in the siRNA conjugate. The siRNA conjugates used were conjugate 1, conjugate 2, conjugate 3, conjugate 4, conjugate 5, conjugate 6, conjugate 7 or conjugate 8, respectively, prepared as described above.

For each siRNA conjugate, 1A1-1A11 solutions were prepared, and each 1A1-1A11 solution contained 1. Mu.L of the above 11 concentrations of siRNA working solution, 0.05. Mu.L of the detection plasmid working solution (containing 10ng of the detection plasmid) and 8.95. Mu.L of Opti-MEM medium, respectively, in that order.

A1B solution was prepared containing 0.2. Mu. L LipofectamineTM 2000/serving of 1B solution 2000 and 9.8. Mu.L of Opti-MEM medium.

1C solutions were prepared, each 1C solution containing 0.05. Mu.L of the working solution for the detection plasmid (containing 10ng of the detection plasmid) and 9.95. Mu.L of Opti-MEM medium.

One part of the 1B solution was mixed with one part of the 1A1-1A11 solution of each siRNA conjugate obtained, and incubated at room temperature for 20min, respectively, to obtain the transfection complexes 1X1-1X11 of each siRNA conjugate.

One part of the 1B solution was mixed with one part of the 1C solution and incubated at room temperature for 20min, respectively, to obtain a blank transfection complex 1X12.

In culture wells, each siRNA conjugate of the transfection complexes 1X1-1X11, evenly mixing, adding the amount of 20 u L/hole, each siRNA conjugate of about 40nM、10nM、2.5nM、0.625nM、0.15625nM、0.03906nM、0.009765nM、0.002441nM、0.0006103nM、0.0001526nM、0.00003815nM( based on siRNA amount in siRNA conjugates) of the final concentration of transfection complexes, each siRNA conjugates of the transfection complexes 1X1-1X11 all transfected 3 culture wells, get containing siRNA conjugates of the cotransfection mixture, record as test group.

In the other 3 culture wells, blank transfection complexes 1X12 were added in an amount of 20. Mu.L/well, respectively, to give a transfection mixture without siRNA conjugate, which was designated as a blank control.

After 4 hours of transfection of the cotransfection mixture containing the siRNA conjugate and the transfection mixture without the siRNA conjugate in the culture wells, respectively, 100. Mu.L of H-DMEM complete medium containing 20% FBS was added to each well. The 96-well plate was placed in a CO ₂ incubator for further incubation for 24 hours.

[3] Detection of

The culture medium in the culture wells was aspirated, and 150. Mu.L of Dual-port was added to each wellThe Luciferase reagent and H-DMEM mixed solution (volume ratio is 1:1), fully and uniformly mixing, incubating for 10min at room temperature, transferring 120 mu L of mixed solution to a 96-well ELISA plate, and reading the chemiluminescence values (Fir) of Firefly in each culture well on the 96-well ELISA plate by using a SYNERGY II multifunctional ELISA (BioTek Co); then 60 mu L of Dual-/>, was added to each well of the 96-well ELISA plateStop&After the reagents are fully and uniformly mixed and incubated for 10min at room temperature, the chemiluminescent value (Ren) of Renilla in each culture well on the 96-well ELISA plate is read by using an ELISA reader according to the arrangement mode of reading Fir.

Calculating the luminous Ratio of each hole on the 96-hole ELISA plate=ren/Fir, wherein the luminous Ratio (test) or Ratio (contrast) of each test group or contrast group is the average value of the Ratio of three culture holes; and normalizing the luminous Ratio of each test group by taking the luminous Ratio of the control group as a reference to obtain a Ratio R of Ratio (test)/Ratio (control), thereby representing the relative expression level, namely the residual activity, of the Renilla reporter gene. Inhibition rate of siRNA to target sequence= (1-R) ×100%.

Based on the relative residual activity of Renilla in HEK293A cells transfected with different concentrations of siRNA to be tested, a log (inhibitor) vs. response-Variable slope (four parameters) dose-response curve was fitted using the nonlinear regression analysis function of Graphpad 5.0 software.

Calculating the IC ₂₅ value of the target sequence of the siRNA to be detected according to the function corresponding to the fitted dose-effect curve, wherein the function is as follows,

Wherein:

y is the ratio R, the relative residual activity of Renilla,

X is the logarithmic value of the transfected siRNA concentration,

Bot is the Y value at the bottom of the steady state period,

Top is the Y value at the Top of the steady state period,

X 'is the corresponding X value when Y is halfway between the bottom and top, and HillSlope is the slope of the curve at X'.

From this dose-response curve and the corresponding function, the corresponding X ₂₅ value was determined when y=75% (i.e. 75% residual activity, 25% inhibition), and the calculated IC ₂₅ value=10X ₂₅ (nM) for each siRNA, correspondingly, the larger IC ₂₅, the lower the probability of off-target of the conjugate to be tested. The results showed that the dose-effect curves for conjugate 1, conjugate 2, conjugate 3, conjugate 4, conjugate 5, conjugate 6, conjugate 7, and conjugate 8 showed that conjugates 1-8 did not have more than 25% inhibitory activity on target sequence 1 over the entire concentration range tested, i.e., none of the conjugates of the present disclosure comprising acyclic dealkalization groups at various positions in the sequence exhibited significant off-target effects.

Comparative experiment example 1

Inhibition Activity of siRNA conjugates on off-target sequences in vitro psi-CHECK System

The off-target sequence inhibition activity of reference conjugate 1 was tested in an in vitro psi-CHECK system following the procedure of experimental example 1, with a measured IC ₂₅ value of 1.145nM.

Reference conjugate 1 is a conjugate that has the same siRNA sequence as conjugates 1-8, but does not contain an acyclic abasic group. From the results of experimental example 1 and comparative experimental example 1, it is known that the reference conjugate 1 shows a certain off-target condition for the off-target sequence; while none of the conjugates of the present disclosure containing acyclic dealkali groups at different positions in the siRNA antisense strand showed any off-target effect. It is illustrated that the double-stranded oligonucleotides of the present disclosure all have significantly reduced off-target effects by providing an acyclic abasic group in the antisense strand.

Experimental example 2

The siRNA conjugates of the present disclosure have inhibitory activity on APOC3 mRNA in Huh7 cells in vitro.

[1] Cell culture

Huh7 cells (available from Nanjing Corp. Bai Biotechnology Co.) were cultured in DMEM complete medium (Hyclone) containing 10% fetal bovine serum (FBS, hyclone) and incubated at 37℃in an incubator containing 5% CO ₂/95% air.

[2] Transfection

Huh7 cells were seeded at 1.5X10 ⁵ cells/well in 12-well plates, and after 16 hours when the cell growth density reached 40%, the whole culture medium in the culture well was drained, and 1mL opti-MEM medium (GIBCO Co.) was added to each well for further culture for 1.5 hours.

Each siRNA conjugate to be tested was formulated separately with PBS as an siRNA conjugate working solution at a concentration of 20 μm (based on the amount of siRNA in the siRNA conjugate). The siRNA conjugates used were conjugate 1, conjugate 2, conjugate 3, conjugate 4, conjugate 5, conjugate 6, conjugate 7 or conjugate 8, respectively, prepared as described above.

For each siRNA conjugate, 2A solutions were prepared, and each 2A solution contained 3. Mu.L of the siRNA working solution and 97. Mu.L of Opti-MEM medium, respectively, in sequence.

2B solutions were prepared, each 2B solution containing 2. Mu.L Lipofectamine ^TM, 2000 and 98. Mu.L Opti-MEM medium.

A2C solution was prepared, each 2C solution containing 100. Mu.L of Opti-MEM medium.

One part of the 2B solution was mixed with one part of the obtained 2A solution of each siRNA conjugate, and incubated at room temperature for 20min, respectively, to obtain a transfection complex 2X1-2X8 of each siRNA conjugate.

One part of the 2B solution was mixed with one part of the 2C solution and incubated at room temperature for 20min, respectively, to obtain a blank transfection complex 2X9.

In the culture wells, each siRNA conjugate of the transfection complexes 2X1-2X8, evenly mixing, adding 200 u L/hole, each siRNA conjugate of the final concentration of about 50nM (based on siRNA in siRNA conjugates) transfection complexes, each siRNA conjugate of the transfection complexes 2X1-2X8 were transfected into 2 culture wells, get siRNA conjugates of the cotransfection mixture, record as test group.

In another 2 culture wells, transfection complexes 2X9 were added at 200. Mu.L/well, respectively, to give a transfection mixture without siRNA conjugates, which was designated as a blank.

After 4 hours of transfection of the cotransfection mixture containing the siRNA conjugate and the cotransfection mixture without the siRNA conjugate in the culture wells, respectively, 1mL of H-DMEM complete medium containing 20% FBS was added to each well. The 12-well plate was placed in a CO ₂ incubator for further incubation for 24 hours.

[3] Detection of

Subsequently, total RNA in each well cell was extracted according to the method described in the specification using RNAVzol (available from Wiggares Biotechnology (Beijing) Co., ltd., cat. No. N002).

For each well of cells, 1. Mu.g of total RNA was taken, and the total RNA of each well of cells was reverse transcribed using a reverse transcription kit Goldenstar ^TM RT6 CDNA SYNTHESIS KIT (available from Beijing Optimago Biotechnology Co., ltd., cat. No. TSK 301M) in which Goldenstar ^TM Oligo(dT)₁₇ was selected as a primer, and 20. Mu.l of a reverse transcription reaction system was prepared according to the procedure of the reverse transcription operation in the kit specification. The conditions for reverse transcription are: for each reverse transcription reaction system, the reverse transcription reaction system is placed at 50 ℃ for incubation for 50min, then at 85 ℃ for incubation for 5min, finally at 4 ℃ for incubation for 30s, and 80 μl of DEPC water is added into the reverse transcription reaction system after the reaction is finished, so as to obtain a solution containing cDNA.

For each reverse transcription reaction system, 5. Mu.l of the cDNA-containing solution was used as a template, respectivelySYBR qPCR SuperMix Plus kit (available from offshore protein technologies Co., ltd., cat. No. E096-01B) 20. Mu.l of a qPCR reaction system was prepared, wherein the PCR primer sequences for amplifying the target gene APOC3 and the reference gene GAPDH were as shown in Table 9, and the final concentration of each primer was 0.25. Mu.M. And (3) placing each qPCR reaction system on a ABI StepOnePlus Real-Time PCR instrument, amplifying by using a three-step method, wherein the amplification procedure is that the denaturation is carried out at 95 ℃ for 10min, then the denaturation is carried out at 95 ℃ for 30s, the annealing is carried out at 60 ℃ for 30s, and the extension is carried out at 72 ℃ for 30s, and the denaturation, annealing and extension processes are repeated for 40 times, so that a product W containing the amplified target gene APOC3 and the internal reference gene GAPDH is obtained. And then, sequentially incubating the product W at 95 ℃ for 15s, incubating the product W at 60 ℃ for 1min and incubating the product W at 95 ℃ for 15s, and respectively collecting dissolution curves of the target gene APOC3 and the reference gene GAPDH in the product W by a real-time fluorescence quantitative PCR instrument to obtain Ct values of the target gene APOC3 and the reference gene GAPDH.

TABLE 4 primer information

The relative quantitative calculation of the target gene APOC3 in each test group is carried out by adopting a comparative Ct (delta Ct) method, and the calculation method is as follows:

delta Ct (test group) =ct (test group target gene) -Ct (test group reference gene)

Delta Ct (control) =ct (control target gene) -Ct (control reference gene)

ΔΔct (test group) =Δct (test group) - Δct (control group average)

ΔΔct (control) =Δct (control) - Δct (control average)

Wherein, Δct (control group average) is the arithmetic average of Δct (control group) of each of the three culture wells of the control group. Thus, each culture well of the test and control groups corresponds to one ΔΔct value.

Taking the control group as a reference, normalizing the expression level of the APOC3 mRNA in the test group, defining the expression level of the APOC3 mRNA in the blank control group as 100%,

Test group APOC3 mRNA relative expression level = 2 ^{-ΔΔCt( Test set )} x 100%

Test group APOC3 mRNA inhibition = (1-test group APOC3 mRNA relative expression level) ×100%

FIG. 1 is a bar graph of the relative expression levels of APOC3mRNA in Huh7 cells after transfection of conjugates 1-8 of the present disclosure, respectively.

Comparative experiment example 2

The inhibition activity of the reference conjugate 1 was tested in accordance with the method of experimental example 2, and the results are shown in fig. 1.

The results of fig. 1 show that at a concentration of 50nM, the siRNA conjugates of the present disclosure all exhibit APOC3 mRNA inhibition activity of no less than 50%. In general, the siRNA conjugates of the present disclosure have comparable APOC3 mRNA inhibition activity to the reference conjugate. Of these, reference conjugate 1 exhibited 67.62% inhibitory activity. Conjugate 1, conjugate 4, conjugate 5, conjugate 7, and conjugate 8 exhibited 55.24%, 59.05%, 53.33%, 60%, and 59.05% APOC3 mRNA inhibition activity, respectively, with a slight decrease in activity compared to reference conjugate 1. Conjugate 6 exhibited 65.71% inhibitory activity, i.e., comparable to that of reference conjugate 1. Whereas conjugate 2 and conjugate 3 showed 71.43% and 77.14% inhibitory activity, respectively, i.e., higher inhibitory activity than the reference conjugate 1, especially the highest inhibitory activity of conjugate 3.

From the results of experimental examples 1-2 and comparative experimental examples 1-2, it can be seen that when the antisense strand of the siRNA contains different numbers of acyclic dealkalization groups at different positions, not only the off-target effect is significantly reduced, but also a high inhibition activity is maintained, even a higher target mRNA inhibition activity is exhibited than that of the siRNA conjugate without the acyclic dealkalization groups.

Experimental example 3

The siRNA conjugates of the present disclosure have inhibitory activity against off-target sequences in an in vitro psi-CHECK system

The prepared conjugates 6, 9 and 10 were tested for their inhibitory activity in an in vitro psi-CHECK system, respectively, according to the method of experimental example 1, except that the conjugates 6, 9 and 10 were used instead of the siRNA conjugates tested. Wherein the siRNAs in conjugate 6, conjugate 9 and conjugate 10 are siRNAs having the same base sequence but different nucleic acid modification schemes. The results are shown in Table 5. The results show that the inhibitory activity of conjugate 6 and conjugate 9 on target sequence 1 is above 25% over the whole range of concentrations tested, i.e. conjugate 6 and conjugate 9 do not show a significant off-target effect. The IC ₂₅ value for conjugate 10 was 4.409nM.

Comparative experiment example 3

The prepared reference conjugates 1,2 and 3 were tested for their inhibitory activity in an in vitro psi-CHECK system, respectively, according to the method of experimental example 3. The results are shown in Table 5. Wherein the siRNAs in reference conjugate 1 and reference conjugate 2 are siRNAs having the same modified base sequence as the siRNAs in conjugate 6 and reference conjugate 9, respectively, but which do not contain an acyclic abasic group; the base in the siRNAs in reference conjugate 3 and in conjugate 10 are both unmodified bases and have the same base sequence, but the siRNAs in reference conjugate 3 do not contain siRNAs with acyclic abasic groups.

TABLE 5 IC of conjugates ₂₅

Conjugate numbering	IC₂₅(nM)	Conjugate numbering	IC₂₅(nM)
				Conjugate 6	Without any means for	Reference conjugate 1	1.145
Conjugate 9	Without any means for	Reference conjugate 2	5.235
				Conjugate 10	4.409	Reference conjugate 3	0.269

As can be seen from the results of table 5, for comparison between conjugates having the same base arrangement of the double-stranded oligonucleotides and the same modification method for the rest, both reference conjugates 1 and 2, which did not contain an acyclic dealkalization group, showed a significant off-target effect, whereas neither conjugate 6 nor conjugate 9 of the present disclosure had off-target at all concentrations tested; similarly, conjugate 10 of the present disclosure showed significantly lower off-target effect than reference conjugate 3, which did not contain an acyclic dealkalization group. It can be seen that by providing acyclic abasic groups for double-stranded oligonucleotides with different modification schemes, the resulting double-stranded oligonucleotides of the present disclosure all have significantly reduced off-target effects.

Experimental example 4

The prepared conjugates 6 and 11 were tested for activity on off-target sequences in an in vitro psi-CHECK system, respectively, following the procedure of experimental example 1. The results showed that neither conjugate 6 nor conjugate 11 showed so high inhibitory activity against target sequence 1 that the IC ₂₅ values could be calculated, i.e., neither conjugate 6 nor conjugate 11 exhibited a significant off-target effect.

Comparative experiment example 4

The activity of reference conjugate 1 and reference conjugate 4 on off-target sequences in an in vitro psi-CHECK system was tested, respectively, following the procedure of experimental example 4. The results showed that reference conjugate 1 and reference conjugate 4 exhibited significant off-target effects with IC25 values of 1.145nM and 2.878nM, respectively.

Wherein the siRNAs in conjugate 6 and conjugate 11 are siRNAs targeting the same stretch of target mRNA, but having sense chain lengths of 19 and 21 nucleotides, respectively, and antisense chain lengths of 21 and 23 nucleotides, respectively. The siRNA in reference conjugate 1 and reference conjugate 4 are siRNA having the same base sequence as conjugate 6 and conjugate 11, respectively. As can be seen from the results of experimental example 5 and comparative experimental example 5, the conjugates of the present disclosure containing acyclic dealkalization groups with different sequence lengths each exhibited significantly reduced off-target effects than the reference conjugates without acyclic dealkalization groups.

Experimental example 5

The prepared conjugates 9, 10 and 11 were tested for their inhibitory activity on APOC3 mRNA in Huh7 cells in vitro, respectively, according to the method of experimental example 2. The results are shown in FIG. 2.

Comparative experiment example 5

The prepared reference conjugates 2, 3 and 4 were tested for their inhibitory activity on APOC3 mRNA in Huh7 cells in vitro, respectively, according to the method of experimental example 2. The results are shown in FIG. 2.

The results of fig. 2 show that at 50nM concentrations, the inhibition rates of APOC3 mRNA by conjugate 9, conjugate 10, and conjugate 11 of the present disclosure are as high as 65.72%, 67.14%, and 73.33%. Exhibit higher inhibitory activity than the reference conjugate 2, the reference conjugate 3 and the reference conjugate 4. From the results of experimental examples 3-5 and comparative experimental examples 3-5, it can be seen that the siRNA conjugates of the present disclosure containing an acyclic dealkalization group not only show significantly lower off-target effects than conjugates without an acyclic dealkalization group, but further show higher inhibitory activity relative to the reference conjugates.

Experimental example 6

The prepared conjugates 12 to 19 were tested for their inhibitory activity against off-target sequences in an in vitro psi-CHECK system according to the method of experimental example 1, except that target sequence 2 was substituted for target sequence 1, and that target sequence 2 contained a sequence complementary to the antisense strand portion of the siRNA in the conjugate to be tested, so that the inhibitory effect of the conjugate to be tested on target sequence 2 could reflect the extent of off-target effect. That is, the higher the inhibitory effect, the more likely off-target the conjugate to be detected will occur.

Target sequence 2:

5'-AAACCGCCCTAGGGACAAGAA-3'(SEQ ID NO:136)

in the detection step, from the dose-response curve and the corresponding function, the corresponding X ₅₀ value when y=50% is determined, and the IC ₅₀ value=10x ₅₀ (nM) of each siRNA is calculated, accordingly, the larger the IC ₅₀, the lower the probability of off-target of the conjugate to be detected.

The experimental results show that none of the conjugates 12-19 of the present disclosure have so high inhibitory activity against target sequence 2 that the value of IC ₅₀ can be calculated, i.e., none exhibit a significant off-target effect.

Wherein the conjugates 12 to 19 contain the same base sequence. The difference is that the siRNA in conjugate 12, conjugate 14, conjugate 16, conjugate 18 is an siRNA containing an acyclic dealkalization group of R configuration at the 6, 7, 8 or 6 and 7 positions of the nucleotide in the 5 'end to 3' end direction. The siRNA in conjugate 13, conjugate 15, conjugate 17, and conjugate 19 are siRNA containing an acyclic abasic group of S-configuration at the corresponding positions described above.

Comparative experiment example 6

The prepared reference conjugate 12 was tested for inhibition of off-target sequences in an in vitro psi-CHECK system, following the procedure of experimental example 6. The results showed that the IC ₅₀ of reference conjugate 12 was 0.29nM. Reference conjugate 12 is a conjugate having the same base sequence as conjugates 12-19, but does not contain an acyclic abasic group.

From the results of experimental example 6 and comparative experimental example 6, it can be seen that the conjugates of the present disclosure, having different numbers of acyclic dealkalization groups of different steric configurations at different positions, each exhibit significantly reduced off-target effects compared to conjugates without acyclic dealkalization groups. By providing acyclic abasic groups of different configurations, it is further demonstrated that the various double-stranded oligonucleotides and oligonucleotide conjugates of the present disclosure each exhibit a similar reduced off-target effect.

Experimental example 7

The conjugates 12-19 of the present disclosure were tested for their inhibitory activity on target sequences in vitro in Huh7 cells following the procedure of experimental example 6. The only difference is that target sequence 3 is used instead of target sequence 2:

Target sequence 3:5'-CCCAAUAAAGCUGGACAAGAA-3' (SEQ ID NO: 137);

The target sequence 1 is homologous to a portion of the target mRNA and is the complete complement of the antisense strand in the detected siRNA conjugate, so that the inhibitory effect of each siRNA conjugate on target sequence 3 can reflect the inhibitory capacity of the detected siRNA conjugate to the APOC3 mRNA expressed by the target gene.

The IC ₅₀ values for conjugates 12-19 are shown in Table 6.

Comparative experiment example 7

The reference conjugates 12-19 were tested for their inhibitory activity on target sequences in an in vitro psi-CHECK system following the procedure of experimental example 7, and the results are shown in table 6.

TABLE 6 IC of siRNA conjugates ₅₀

Preparation example number	IC₅₀(nM)
		Conjugate 12	0.015nM
Conjugate 13	0.013nM
		Conjugate 14	0.0078nM
Conjugate 15	0.0044nM
		Conjugate 16	0.021nM
Conjugate 17	0.024nM
		Conjugate 18	0.023nM
Conjugate 19	0.022nM
		Reference conjugate 12	0.011

As can be seen from the results of Table 6 above, conjugates 12-19 all exhibited higher APOC3 mRNA inhibition activities with IC ₅₀ between 0.0044-0.024 nM.

None of the siRNA conjugates containing an acyclic dealkali group of the present disclosure showed a significant decrease in activity compared to the siRNA conjugates containing no acyclic dealkali group. Of these, conjugate 12 and conjugate 13 exhibited APOC3 mRNA inhibition activity substantially comparable to that of reference conjugate 12. Further, conjugates 14 and 15 showed even higher inhibitory activity of APOC3 mRNA than the reference conjugate 12. The results of examples 6-7 and comparative examples 6-7 demonstrate that the double-stranded oligonucleotide conjugates of the present disclosure comprising acyclic dealkalized groups can substantially maintain the inhibitory activity on the target mRNA while having significantly reduced off-target effects, even exhibiting further enhanced inhibitory activity on the target mRNA.

Experimental example 8

Determination of conjugates the conjugates of the present disclosure were assayed for inhibitory activity in an in vitro psi-CHECK system against a target sequence only with respect to conjugates 20, 21, 22, 23, 24 and 25, the target sequences used were target sequences 4, 5, 6, 7, 8 and 9, respectively:

Target sequence 4:

5'-CCCTGAAAGACTACTGGAGCA-3'(SEQ ID NO:138)；

Target sequence 5:

5'-GCTTAAAAGGGACAGTATTCT-3'(SEQ ID NO:139)；

Target sequence 6:

5'-GGACAGTATTCTCAGTGCTCT-3'(SEQ ID NO:140)；

Target sequence 7:

5'-AGTATTCTCAGTGCTCTCCTA -3'(SEQ ID NO:141)；

Target sequence 8:

5'-ACAGTATTCTCAGTGCTCTCC-3'(SEQ ID NO:142)

target sequence 9:

5'-AGGGACAGTATTCTCAGTGCT-3'(SEQ ID NO:143)；

The target sequences 4-9 are homologous to a portion of the target mRNA and are fully complementary to the sequence of the antisense strand in the detected siRNA conjugates, respectively, so that the inhibitory effect of each siRNA conjugate on the corresponding target sequence is responsive to the inhibitory ability of the detected siRNA conjugate on mRNA expressed by the target gene.

The experimental results are shown in table 7.

Comparative experiment example 8

The inhibitory activity of reference conjugates 6-11 on the target sequence in an in vitro psi-CHECK system was determined following the procedure of experimental example 9. For reference conjugates 6, 7, 8, 9, 10 and 11, the target sequences used are target sequences 3, 4, 5, 6, 7 and 8, respectively. The experimental results are shown in table 7.

TABLE 7 siRNA IC of conjugates ₅₀

Conjugate numbering	IC₅₀(nM)	Conjugate numbering	IC₅₀(nM)
				Conjugate 20	0.29	Reference conjugate 6	0.27
Conjugate 21	0.032	Reference conjugate 7	0.032
				Conjugate 22	0.29	Reference conjugate 8	0.12
Conjugate 23	0.019	Reference conjugate 9	0.032
				Conjugate 24	0.017	Reference conjugate 10	0.029
Conjugate 25	0.038	Reference conjugate 11	0.089

As can be seen from the results of table 7, the conjugates of the present disclosure containing an acyclic dealkalization group showed no significant decrease in inhibitory activity compared to the conjugates having the same sequence but differing only in the absence of the acyclic dealkalization group. Further, wherein conjugate 23, conjugate 24 and conjugate 25 also exhibited higher inhibitory activity than the reference conjugate. It is demonstrated that the double-stranded oligonucleotides comprising acyclic abasic groups of the present disclosure have significantly reduced off-target effects while also substantially maintaining the inhibitory activity on the target mRNA even further enhancing the inhibitory activity on the target mRNA for different base sequences.

Experimental example 9

Inhibition ratio assay of APOC 3mRNA by the siRNA conjugates of the present disclosure in mouse liver primary cells.

This experiment examined the determination of the inhibition of APOC3 mRNA in mouse liver primary cells by the prepared conjugates 21, 23 and 25. The specific steps are as follows.

Mouse liver primary cells were obtained from fresh liver tissue of APOC3 transgenic mice, inoculated in type I collagen-coated tissue culture dishes, and cultured in RPMI 1460 medium containing 1 x diabodies and 10% fbs at 37 ℃ in an incubator containing 5% co ₂/95% air for 30min.

The culture medium was discarded, and the mouse liver primary cell density was adjusted to 2X 10 ⁵ cells/mL with opti-MEM to obtain a mouse liver primary cell suspension. The resulting suspensions of the primary cells of mouse liver were then added separately to different culture wells of a 6-well plate, and the primary cells of mouse liver were inoculated into the culture wells. The volume of the added mouse liver primary cell suspension was 2 mL/well, and the number of mouse liver primary cells was 4X 10 ⁵ cells/well.

Each of the following siRNA conjugates, conjugates 21, 23 and 25, were formulated with PBS as a 20 μm siRNA conjugate working solution, respectively.

5. Mu.L of each siRNA conjugate was added to each well and mixed well to give a test set with a final concentration of siRNA conjugate of about 50nM in each well.

In the other 3 culture wells, 5. Mu.L of PBS solution without siRNA conjugate was added, respectively, and the mixture was recorded as a blank control group.

After 4H transfection of the transfection mixture containing siRNA conjugates and the transfection mixture without siRNA conjugates in the culture wells, 1ml of H-DMEM complete medium containing 20% FBS was added to each well. The 6-well plate was placed in a CO ₂ incubator at 37℃for further incubation for 24h.

For each reverse transcription reaction system, 5. Mu.l of the cDNA-containing solution was used as a template, respectivelySYBR qPCR SuperMix Plus kit (available from offshore protein technologies Co., ltd., cat. No. E096-01B) 20. Mu.l of a qPCR reaction system was prepared, wherein the PCR primer sequences for amplifying the target gene APOC3 and the reference gene GAPDH were as shown in Table 8, and the final concentration of each primer was 0.25. Mu.M. And (3) placing each qPCR reaction system on a ABI StepOnePlus Real-Time PCR instrument, amplifying by using a three-step method, wherein the amplification procedure is that the denaturation is carried out at 95 ℃ for 10min, then the denaturation is carried out at 95 ℃ for 30s, the annealing is carried out at 60 ℃ for 30s, and the extension is carried out at 72 ℃ for 30s, and the denaturation, annealing and extension processes are repeated for 40 times, so that a product W containing the amplified target gene APOC3 and the internal reference gene GAPDH is obtained. And then, sequentially incubating the product W at 95 ℃ for 15s, incubating the product W at 60 ℃ for 1min and incubating the product W at 95 ℃ for 15s, and respectively collecting dissolution curves of the target gene APOC3 and the reference gene GAPDH in the product W by a real-time fluorescence quantitative PCR instrument to obtain Ct values of the target gene APOC3 and the reference gene GAPDH.

TABLE 8 primer information

Delta Ct (control) =ct (control target gene) -Ct (control reference gene)

ΔΔct (test group) =Δct (test group) - Δct (control group average)

ΔΔct (control) =Δct (control) - Δct (control average)

The expression level of APOC3 mRNA in the test group is normalized by taking the control group as a reference, and the expression level of APOC3 mRNA in the blank control group is defined as 100%.

Test group APOC3 mRNA inhibition = (1-test group APOC3 mRNA relative expression level) ×100%. The inhibition of APOC3 mRNA by each siRNA conjugate is summarized in table 9.

Comparative experiment example 9

The inhibition ratios of APOC3 mRNA by the prepared reference conjugates 7, 9 and 11 in the primary cells of mouse liver were examined according to the method of experimental example 9, except that the reference conjugates 7, 9 and 11 were used to prepare conjugate working solutions instead of the siRNA conjugates used, respectively, for the test.

The inhibition of APOC3 mRNA by each siRNA conjugate is summarized in table 9. For the same test group siRNA conjugates, APOC3 mRNA inhibition rate was the arithmetic mean of the test group APOC3 mRNA inhibition rates measured for three culture wells.

TABLE 9 inhibition of APOC3 mRNA in mouse liver primary cells

Conjugate numbering	APOC3 mRNA inhibition Rate%
		Conjugate 21	92.08
Conjugate 23	91.54
		Conjugate 25	92.03
Reference conjugate 7	88.77
		Reference conjugate 9	84.34
Reference conjugate 11	89.77

As can be seen from the results in table 9 and fig. 2, the siRNA conjugates provided by the present disclosure show higher APOC3 mRNA inhibition activity in the primary cells of mouse liver, and at 50nM of the siRNA conjugate concentration, the inhibition rates of conjugate 21, conjugate 23 and conjugate 25 on APOC3 mRNA all reached 91.5% or more, which is higher than that of the reference conjugate on APOC3 mRNA. Further, it is demonstrated that the siRNA conjugates provided by the present disclosure can effectively inhibit expression of APOC3 mRNA, thus showing excellent application prospects for treating APOC3 target-related diseases, particularly diseases caused by corresponding dyslipidemia.

Experimental example 10

Effect of siRNA conjugates on blood lipid in CBA-Tg (APOC 3) 3707Bres/J mice this experiment examined the effect of the conjugates 26-30 of the present disclosure on blood lipid levels in mice, as follows:

6-8 week old CBA-Tg (APOC 3) 3707Bres/J mice were randomly grouped, 6 mice per group, and siRNA conjugates (preparations 27-32) and comparative preparation 1 were administered to each group of mice, respectively, as controls. All animals were dosed on a weight basis in a single dose by subcutaneous injection, with an siRNA conjugate dose (based on the amount of siRNA) of 3mg/kg and a dosing volume of 5mL/kg. Each siRNA conjugate was supplied as a physiological saline solution, and the drug concentration to be formulated for the siRNA conjugate was converted according to the dose and the administration volume. Serum blood lipid levels were measured at various time points by taking blood from the orbital venous plexus of mice before dosing (noted as day 0) and at days 8, 15, 22, 29, 36 post-dosing.

The orbital vein is sampled, about 0.1mL of the orbital vein is taken each time, serum is obtained by centrifugation, and the serum is obtained after centrifugation. 20ul of serum was diluted 5-fold with PBS/0.9% physiological saline, and serum samples were sent to Beijing dean center for serum Triglyceride (TG) index.

Normalized blood lipid level= (post-dose test group blood lipid level/pre-dose test group blood lipid level) ×100%.

Inhibition of blood lipid level = (1-post-dose test group blood lipid content/pre-dose test group blood lipid content) ×100%. The results are shown in FIG. 3.

Comparative experiment example 10

The effect of the reference conjugate 5 and the negative reference conjugate on blood lipid levels in mice was examined in the same manner as in experimental example 10, respectively, except that the test was performed using the reference conjugate 5 or the negative reference conjugate instead of each siRNA conjugate, respectively. The results are shown in FIG. 3.

As can be seen from fig. 3, at the condition that the dose is 3mg/kg, at different time points after administration, the inhibition rate of the siRNA conjugate 26-30 to triglyceride is always maintained to be 78% or more for a period of 36 days after a single administration, compared with the negative reference conjugate, for serum triglyceride level, and is similar to that of the reference conjugate 5 to serum triglyceride; the maximum inhibition occurred at day 7 post-dose, with conjugate 26 having an inhibition of up to 84.63% of the triglyceride.

Thus, it is demonstrated that the siRNA conjugates of the present disclosure, which contain different acyclic dealkalization groups, are each capable of significantly reducing serum triglyceride levels and exhibit substantially the same serum triglyceride level-reducing effect as compared to conjugates that do not contain acyclic dealkalization groups.

Experimental example 11

The inhibitory effect of the prepared conjugate 31 on HBV mRNA in an in vitro psi-CHECK system was examined in the same manner as in experimental example 8, except that the test was performed using the conjugate 31 instead of the siRNA conjugate used in experimental example 8.

HEK293A cells used in this experimental example were purchased from Nanjing Corp. Bai biotechnology Co., ltd, and cultured in DMEM complete medium (Hyclone) containing 10% fetal bovine serum (FBS, hyclone Co.) and 0.2% by volume of green streptomycin double antibody (Penicillin-Streptomycin, gibco, invitrogen Co.) at 37℃in an incubator containing 5% CO ₂/95% air.

The difference is that the target sequence used is target sequence 10:

Target sequence 10:5'-GACCTTGAGGCATACTTCAAA-3' (SEQ ID NO: 148)

The target sequence 10 is homologous to a portion of HBV mRNA and is fully complementary to the antisense strand sequence in the siRNA detected, so that the inhibitory effect of the conjugate 31 on the target sequence 10 can be reflected in the inhibitory capacity of the conjugate on HBV mRNA.

The results of the final siRNA conjugates at 10nM, 3.33nM, 1.11nM, 0.370nM, 0.122nM, 0.0407nM, 0.0136nM, 0.0045nM, 0.00150nM (based on siRNA) are summarized in Table 10.

Comparative experiment example 11

The inhibitory effect of reference conjugate 14 on HBV mRNA in an in vitro psi-CHECK system was examined in the same manner as in Experimental example 11. The results are summarized in table 10.

TABLE 10 siRNA IC of conjugates ₅₀

Wherein conjugate 31 is a conjugate of the present disclosure containing an acyclic dealkalization group, and reference conjugate 14 is a conjugate having the same nucleotide sequence as conjugate 31, but containing other acyclic dealkalization groups.

From the results of table 10, conjugate 31 showed significantly higher activity compared to reference conjugate 14, demonstrating that the double stranded oligonucleotides of the present disclosure comprising acyclic dealkalized groups have significantly higher inhibitory activity than other acyclic dealkalized groups. Therefore, the composition has excellent application prospect for treating HBV related diseases, especially hepatitis B.

Experimental example 12

The inhibition ratio of the prepared conjugate 32 and conjugate 33 to HAO1 mRNA in rat liver primary cells was measured in the same manner as in experimental example 9. The only difference is that the primary hepatocytes used in the assay were taken from SD rats; the PCR primer sequences for amplifying the target gene HAO1 and the reference gene beta-actin are shown in Table 11.

TABLE 11 primer information

The relative quantitative calculation of the target gene HAO1 in each test group is carried out by adopting a comparative Ct (delta Ct) method, and the calculation method is as follows:

Delta Ct (control) =ct (control target gene) -Ct (control reference gene)

ΔΔct (test group) =Δct (test group) - Δct (control group average)

ΔΔct (control) =Δct (control) - Δct (control average)

Taking the control group as a reference, normalizing the expression level of the HAO1 mRNA of the test group, defining the expression level of the HAO1 mRNA of the blank control group as 100%,

Relative expression level of HAO1 mRNA in test group = 2 ^{-ΔΔCt( Test set )} x 100%

Test group HAO1 mRNA inhibition = (1-test group HAO1 mRNA relative expression level) ×100%.

The results are summarized in table 12.

Comparative experiment example 12

The inhibition ratio of the reference conjugate 15 and the negative reference conjugate to the HAO1 mRNA in the rat liver primary cells was measured in the same manner as in experimental example 13. The results are summarized in table 12. For the same test group siRNA conjugates, HAO1 mRNA inhibition rate was the arithmetic mean of the test group HAO1 mRNA inhibition rates determined for three culture wells.

TABLE 12 inhibition of HAO1 mRNA in rat liver primary cells

Conjugate numbering	HAO1 mRNA inhibition%
		Conjugate 32	92.77
Conjugate 33	91.66
		Reference conjugate 15	91.75
Negative reference conjugate	23.28

As can be seen from the results of table 12, conjugates 32 and 33 of the present disclosure showed higher HAO1 mRNA inhibition activity in rat liver primary cells, with both conjugates 32 and 33 reaching greater than 91.5% HAO1 mRNA inhibition at siRNA conjugate concentration of 20 nM. Exhibit a considerably higher inhibition rate than the reference conjugate 15. Therefore, the conjugate provided by the disclosure can effectively inhibit the expression of HAO1 mRNA, so that the conjugate has excellent application prospect in treating HAO1 target related diseases.

Experimental example 13

First, the inhibition rate of the conjugate 32 against HAO1 mRNA in rat liver primary cells was measured in the same manner as in Experimental example 12, except that the concentration of the conjugate 32 was 10nM (based on siRNA), and RNAseq differential gene statistics were performed as follows:

performing RNAseq sequencing analysis, performing statistical analysis on HAO1 mRNA expression data, screening genes with significant differences in expression levels of samples in different states, and performing experimental steps as follows:

[1] Normalization of raw readcount (normalization), mainly correction of sequencing depth

[2] Calculation of hypothesis testing probability by using statistical model

[3] Performing multiple hypothesis test correction to obtain FDR value

And screening the threshold value standard by taking (I log2FC I is more than or equal to 0Q value is less than or equal to 0.05) as a screening standard of the differential gene.

The measured inhibition is summarized in table 13 and the results of RNAseq differential gene statistics are summarized in fig. 4.

Comparative experiment example 13

The inhibition rates of HAO1mRNA and RNA seq differential gene statistics of negative reference conjugate, reference conjugate 15, reference conjugate 16 and reference conjugate 17 in rat liver primary cells were determined by the same method as in experimental example 13, respectively. The results are summarized in table 13 and fig. 4, respectively.

TABLE 13 inhibition of HAO1 mRNA in rat liver primary cells

Wherein reference conjugates 16 and 17 are conjugates containing the same nucleotide sequence as conjugate 32, but containing GNA and UNA, respectively. The results in Table 15 show that at a concentration of 10nM, conjugate 32 and reference conjugates 15-17 exhibit substantially comparable inhibitory activity.

FIG. 4 is a bar graph showing the results of RNAseq differential gene analysis of conjugate 32, reference conjugates 15-17 and negative reference conjugates, compared to a blank. As shown in fig. 4, the correlation of the gene of conjugate 32 with HAO1 mRNA is highest, and the number of genes affecting the expression of non-target genes significantly up-or down-regulated is significantly reduced compared to the reference conjugates 15-17, thus it can be seen that conjugates containing the acyclic dealkalization group of the present disclosure exhibit significantly reduced off-target effects while having substantially equivalent inhibitory activity, as compared to conjugates with other acyclic dealkalization groups, or conjugates without acyclic dealkalization groups. This demonstrates that the siRNA conjugates of the present disclosure comprising acyclic dealkalization groups have significantly reduced off-target effects while being able to maintain the inhibitory activity of double-stranded oligonucleotides on HAO1 mRNA.

Experimental example 14

The conjugates 34-42 of the present disclosure were tested for their inhibitory activity against off-target sequences in an in vitro psi-CHECK system in the same manner as in experimental example 1. The difference is that target sequence 11 is used to replace target sequence 1:

target sequence 11:

5'-CTCGAGCTAACCTCTACACAAGAACTATTGGCTAACCTCTACACAAGAACTATTGGCTAACCTCTACACAAGAACTAGCGGCCGC-3'(SEQ ID NO:153)

The target sequence 11 contains a sequence complementary to the siRNA antisense strand part of the conjugate to be detected, and the sequence is repeated 3 times in the target sequence 11, so that the inhibition effect of the conjugate to be detected on the target sequence 11 can reflect the degree of off-target effect. That is, the higher the inhibitory effect, the more likely off-target the conjugate to be detected will occur.

Experimental results show that none of the conjugates 34-42 of the present disclosure have high inhibitory activity against the target sequence 10, i.e., off-target sequence, enough to calculate the IC ₅₀ value. That is, none of the conjugates 34-42 of the present disclosure exhibited a significant off-target effect.

Comparative experiment example 14

The inhibition activity of the reference conjugate 18 on off-target sequences in an in vitro psi-CHECK system was tested in the same manner as in experimental example 14, and an IC ₅₀ value of 1.76nM was measured.

Wherein conjugates 34-42 are conjugates having the same nucleotide base sequence, with different numbers of acyclic dealkalized groups in different positions on the antisense strand in the siRNA. Reference conjugate 18 is a conjugate having the same nucleotide base sequence as conjugates 34-42, but does not contain an acyclic dealkalized group. From the results of experimental example 15 and comparative experimental example 15, the conjugates of the present disclosure containing an acyclic dealkalization group exhibited significantly reduced off-target effect compared to conjugates without an acyclic dealkalization group. Also, conjugates of the present disclosure containing different numbers of acyclic dealkalized groups in different positions all exhibit significantly reduced off-target effects.

Experimental example 15

The inhibitory activity of conjugates 35-42 on ANGPTL3 mRNA in Huh7 cells was measured in the same manner as in experimental example 2, and the results are summarized in table 14.

Comparative experiment example 15

The inhibitory activity of reference conjugate 18 on ANGPTL3 mRNA in Huh7 cells was measured in the same manner as in experimental example 15, and the results are summarized in table 14.

Table 14 siRNA ANGPTL3 mRNA inhibition of conjugates

From the results of table 14, it can be seen that at 50nM concentration, conjugates 35-42 of the present disclosure all exhibited substantially equivalent ANGPTL3 mRNA inhibition compared to reference conjugate 18. Still further, conjugates 36, 37, 38, 39 and 42 exhibited even higher inhibitory activity than the reference conjugate 18, especially conjugate 38, up to 82.5% of the inhibitory activity, 28% higher than the reference conjugate. The results of examples 14-15 and comparative examples 14-15 demonstrate that the double-stranded oligonucleotides of the present disclosure comprising acyclic abasic groups not only have significantly reduced off-target effects, but also have comparable inhibitory activity to the corresponding double-stranded oligonucleotides that do not comprise acyclic abasic groups. Even higher inhibitory activity against ANGPTL3 mRNA was also shown.

Experimental example 16

The inhibitory activity of conjugate 39 and conjugates 43-45 on off-target sequences in an in vitro psi-CHECK system was determined in the same manner as in experimental example 14. The experimental results show that none of the conjugates 43-45 of the present disclosure have so high inhibitory activity against the target sequence 10 that the value of IC ₅₀ can be calculated, i.e., none exhibit a significant off-target effect.

Comparative example 16

The inhibitory activity of reference conjugate 18, reference conjugate 19, reference conjugate 20 and reference conjugate 21 on off-target sequences in an in vitro psi-CHECK system was determined in the same manner as in experimental example 17. The experimental results are summarized in table 15.

TABLE 15 IC of siRNA conjugates ₅₀

Conjugate numbering	IC₅₀
		Reference conjugate 18	1.76
Reference conjugate 19	1.04
		Reference conjugate 20	2.67
Reference conjugate 21	0.32

Wherein the siRNAs in conjugate 39 and conjugates 43-45 are siRNAs having the same base sequence but different base sequence modifications, and these siRNAs contain acyclic dealkalization groups at the same positions in the antisense strand. The siRNAs in reference conjugates 18-21 were siRNAs having the same base modification scheme as the siRNAs in conjugate 39 and conjugates 43-45, respectively, but did not contain an acyclic abasic group. The results of table 15 demonstrate that the siRNA conjugates of the present disclosure comprising an acyclic dealkalization group exhibit significantly reduced off-target effects compared to siRNA conjugates that do not comprise an acyclic dealkalization group. Further, it is demonstrated that by providing acyclic abasic groups, the double stranded oligonucleotides of the present disclosure with different base modification schemes all exhibit significantly reduced off-target effects.

Experimental example 17

The inhibition activity of conjugate 39 and conjugate 46 on off-target sequences in an in vitro psi-CHECK system was determined in the same manner as in experimental example 16, and the experimental results showed that neither the inhibition activity of conjugate 39 nor conjugate 46 on target sequence 10 was high enough to calculate the value of IC ₅₀, i.e., neither showed significant off-target effect.

Comparative experiment example 17

The inhibitory activity of reference conjugate 18 and reference conjugate 22 on off-target sequences in an in vitro psi-CHECK system was determined in the same manner as in experimental example 16, with measured IC ₅₀ values of 1.76nM and 1.347nM, respectively.

Wherein the siRNAs in conjugate 39 and conjugate 46 are siRNAs targeting the same stretch of target mRNA in the ANGPTL3 mRNA, but having sense chain lengths of 19 and 21, respectively, and antisense chain lengths of 21 and 23, respectively. Reference conjugate 18 and reference conjugate 22 are conjugates having the same nucleotide sequence as conjugate 39 and conjugate 46, respectively, but do not contain an acyclic dealkalization group. From the results of experimental example 18 and comparative experimental example 18, it can be seen. Double-stranded oligonucleotides comprising acyclic abasic groups of the present disclosure all exhibit significantly reduced off-target effects for oligonucleotide sequences having different lengths.

Experimental example 18

The inhibitory activity of conjugate 43-46 on ANGTL target mRNA in vitro Huh7 cells was determined in the same manner as in experimental example 15. The experimental results are summarized in table 16.

Comparative experiment example 18

The inhibitory activity of reference conjugates 19-22 on ANGPTL3 target mRNA in vitro Huh7 cells was determined in the same way as in experimental example 18. The experimental results are summarized in table 16.

TABLE 16 ANGPTL3 mRNA inhibition Rate of siRNA conjugates

Conjugate numbering	Inhibition rate%	Conjugate numbering	Inhibition rate%
				Conjugate 43	61.5％	Reference conjugate 19	65.5％
Conjugate 44	79.0％	Reference conjugate 20	72.5％
				Conjugate 45	62.5％	Reference conjugate 21	75.5％
Conjugate 46	66％	Reference conjugate 22	74.5％

From the results of table 16, it can be seen that at a concentration of 50nM, the conjugates of the present disclosure all exhibited an ANGPTL3 mRNA inhibition of no less than 60%. No significant reduction in activity occurred compared to the inhibitory activity of reference conjugates 19-21. In particular conjugate 44, has an inhibition rate as high as 79.0%, and exhibits a higher ANGPTL3 mRNA activity than the reference conjugates 19-21. It can be seen that the siRNA conjugates of the present disclosure comprising acyclic dealkalization groups have significantly reduced off-target effects while also having an inhibition activity on target mRNA that is not reduced, even unexpectedly exhibiting higher target mRNA inhibition activity.

Experimental example 19

The inhibition of ANGPTL3 mRNA by conjugate 47 and conjugate 48 of the present disclosure in mouse liver primary cells was determined following the method of experimental example 9. The only difference was that the conjugate concentration tested was 20nM (based on siRNA).

The only difference is that the primary mouse liver cells were obtained by extraction from fresh liver tissue of normal C57BL/6N mice, cells of appropriate density were inoculated in a type I collagen-coated glass or plastic coverslip or tissue culture dish, cells were cultured in RPMI 1460 medium containing 1 Xdiab and 10% FBS, and cells were cultured at 37℃in an incubator containing 5% CO ₂/95% air for 30min; the PCR primer sequences for amplifying the target gene ANGPTL3 and the internal reference gene GAPDH are shown in Table 17.

TABLE 17 primer information

The relative quantitative calculation of the target gene ANGPTL3 in each test group is carried out by adopting a comparative Ct (delta Ct) method, and the calculation method is as follows:

Delta Ct (control) =ct (control target gene) -Ct (control reference gene)

ΔΔct (test group) =Δct (test group) - Δct (control group average)

ΔΔct (control) =Δct (control) - Δct (control average)

Normalizing the expression level of the ANGPTL3 mRNA of the test group by taking the control group as a reference, defining the expression level of the ANGPTL3 mRNA of the blank control group as 100%,

Test group ANGPTL3 mRNA relative expression level = 2 ^{-ΔΔCt( Test set )} x 100%

Test group ANGPTL3 mRNA inhibition = (1-test group ANGPTL3 mRNA relative expression level) ×100%

The inhibition of ANGPTL3 mRNA by each siRNA conjugate is summarized in table 18. ANGPTL3 mRNA inhibition rate is the arithmetic mean of the assay group ANGPTL3 mRNA inhibition rates determined for three wells for the same assay group siRNA conjugate.

Comparative experiment example 19

The inhibition rate of ANGPTL3 mRNA by reference conjugate 23 and negative reference conjugate in the primary cells of mouse liver was measured in the same manner as in experimental example 19, and the results are shown in table 18.

TABLE 18 inhibition of ANGPTL3 mRNA in mouse liver primary cells

Where conjugates 47 and 48 are the conjugates of the present disclosure containing different acyclic dealkalization groups, while reference conjugate 23 is a conjugate containing other acyclic dealkalization groups.

As can be seen from the results in table 18, the siRNA conjugates provided by the present disclosure showed higher ANGPTL3 mRNA inhibition activity in the primary cells of mouse liver, and at the concentration of the siRNA conjugate of 20nM, the inhibition rate of both conjugate 47 and conjugate 48 on ANGPTL3 mRNA was as high as 92% or more, and the inhibition rate of reference conjugate 23 on ANGPTL3 mRNA was higher by 64.33% and 65.14%. It can be seen that the conjugates of the present disclosure containing acyclic dealkalization groups have significantly higher inhibitory activity against target mRNA than conjugates containing other acyclic dealkalization groups.

Therefore, the siRNA conjugate provided by the disclosure can effectively inhibit the expression of the ANGPTL3mRNA, so that the siRNA conjugate shows excellent application prospect in treating ANGPTL3 target related diseases, particularly diseases caused by corresponding dyslipidemia.

Experimental example 20

Stability of conjugates 12-19 in vitro lysosomal lysates

Test sample preparation by lysosome lysate treatment: conjugates 12-19 were each provided as a 20. Mu.M siRNA concentration in 0.9% sodium chloride aqueous solution, 6. Mu.L each) and mixed with 27.2. Mu.L sodium citrate aqueous solution (pH 5.0), 4.08. Mu.L deionized water, and 2.72. Mu. L Tritosomes (commercially available from Xenotech, cat# R0610LT, lot 1610069). Incubate at 37 ℃. Mu.l of each sample was taken at 0h, 1h, 3h, and 6h, denatured by adding 15. Mu.l of 9M urea, followed by adding 4. Mu.l of 6 Xloading buffer (Soy Corp., cat. 20160830), and immediately freezing to-80℃in a refrigerator. And 0 hours represents the moment when the sample to be tested is immediately taken out after being uniformly mixed with the lysosome lysate.

Reference sample preparation without lysosomal lysate treatment: 1.5. Mu.l of each of the above siRNA conjugates (20. Mu.M) was mixed with 7.5. Mu.L of aqueous sodium citrate (pH 5.0) and 1. Mu.L of deionized water, respectively, and denatured by adding 30. Mu.L of 9M urea solution, followed by adding 8. Mu.L of 6 Xloading buffer, mixing, and immediately freezing to-80℃to terminate the reaction. Each siRNA conjugate reference sample is labeled Con in the electropherogram.

Preparing 16 wt% non-denatured polyacrylamide gel, taking 20 μl of each of the test sample and the reference sample, loading to gel, electrophoresis under 20mA constant current condition for 10min, and continuing electrophoresis under 40mA constant current condition for 30min. After the electrophoresis was completed, the gel was placed on a shaking table and stained with Gelred dye (BioTium, cat. No. 13G 1203) for 10min. The gel was observed and photographed by imaging, and the results are shown in fig. 5.

Comparative experiment example 20

The stability of the reference conjugate 12 in the lysosomal lysate was determined in the same manner as in experimental example 20, and the results are shown in fig. 5.

FIG. 5 shows the results of semi-quantitative detection of the stability of the conjugates 12-19 of the present disclosure tested in vitro lysosomal lysates. The results show that the conjugates of the present disclosure show comparable stability in vitro dissolution solutions compared to reference conjugates that do not contain acyclic dealkalized groups. Can be maintained without degradation for a long time.

The specific embodiments of the present disclosure have been described in detail above, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.

In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.

Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.

Sequence listing

<110> Suzhou Rabo biotechnology Co., ltd

<120> Nucleotide sequence, double-stranded oligonucleotide, pharmaceutical composition and conjugate, preparation method and use

<130> CP1211372/CB

<150> CN202011635152.0

<151> 2020-12-31

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<170> PatentIn version 3.3

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<223> siRNA

<400> 16

uucuugccag cuuuauuggg 20

<210> 17

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 17

uucuugccag cuuuauuggg 20

<210> 18

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 18

uucuugccag cuuuauuggg 20

<210> 19

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 19

uucuugccag cuuuauuggg 20

<210> 20

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 20

uucuugccag cuuuauuggg 20

<210> 21

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 21

uucuugccag cuuuauuggg 20

<210> 22

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 22

uucuuuccag cuuuauuggg 20

<210> 23

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 23

uucuuuccag cuuuauuggg 20

<210> 24

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 24

uucuugccag cuuuauuggg 20

<210> 25

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 25

uucuugccag cuuuauuggg 20

<210> 26

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 26

uucuugucag cuuuauuggg 20

<210> 27

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 27

uucuugucag cuuuauuggg 20

<210> 28

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 28

uucuuccagc uuuauuggg 19

<210> 29

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 29

uucuuccagc uuuauuggg 19

<210> 32

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 32

ucuuguccag cuuuauuggg 20

<210> 33

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 33

uuuuguccag cuuuauuggg 20

<210> 34

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 34

uucuguccag cuuuauuggg 20

<210> 35

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 35

uucuguccag cuuuauuggg 20

<210> 36

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 36

uucuuuccag cuuuauuggg 20

<210> 37

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 37

uucuugccag cuuuauuggg 20

<210> 38

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 38

uucuugucag cuuuauuggg 20

<210> 39

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 39

uucugccagc uuuauuggg 19

<210> 40

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 40

uucugccagc uuuauuggg 19

<210> 41

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 41

caauaaagcu ggacaagaa 19

<210> 42

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 42

caauaaagcu ggacaagaa 19

<210> 43

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 43

uucuugccag cuuuauuggg 20

<210> 44

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 44

cccaauaaag cuggacaaga a 21

<210> 45

<211> 22

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 45

uucuugccag cuuuauuggg uu 22

<210> 46

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 46

caauaaagcu ggacaagaa 19

<210> 47

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 47

uucuugccag cuuuauuggg 20

<210> 48

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 48

ccuugaggca uacuucaaa 19

<210> 49

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 49

uuugaauaug ccucaagguu 20

<210> 50

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 50

uuugaauaug ccucaagguu 20

<210> 51

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 51

ggacaguauu cucagugca 19

<210> 52

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 52

ugcacuagaa uacugucccu 20

<210> 53

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 53

uuugaguaug ccucaagguu 20

<210> 54

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 54

uuugaauaug ccucaagguu 20

<210> 55

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 55

uuugaagaug ccucaagguu 20

<210> 56

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 56

ugacaaaaua acucacuaua a 21

<210> 57

<211> 22

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 57

uuauaggagu uauuuuguca au 22

<210> 58

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 58

acaaaauaac ucacuauaa 19

<210> 59

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 59

uuauaggagu uauuuuguca 20

<210> 60

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 60

gaaugugaaa gucaucgaca a 21

<210> 61

<211> 22

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 61

uugucgugac uuucacauuc ug 22

<210> 62

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 62

augugaaagu caucgacaa 19

<210> 63

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 63

uugucgugac uuucacauuc 20

<210> 64

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 64

ccaagagcac caagaacua 19

<210> 65

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 65

uguucuuggu gcucuuggcu 20

<210> 66

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 66

uauucuuggu gcucuuggcu 20

<210> 67

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 67

uagucuuggu gcucuuggcu 20

<210> 68

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 68

uagucuuggu gcucuuggcu 20

<210> 69

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 69

uaguuuuggu gcucuuggcu 20

<210> 70

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 70

uaguucuggu gcucuuggcu 20

<210> 71

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 71

uaguucuggu gcucuuggcu 20

<210> 72

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 72

uagucuggug cucuuggcu 19

<210> 73

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 73

uagucuggug cucuuggcu 19

<210> 74

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 74

ccaagagcac caagaacua 19

<210> 75

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 75

ccaagagcac caagaacua 19

<210> 76

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 76

uaguucuggu gcucuuggcu 20

<210> 77

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 77

agccaagagc accaagaacu a 21

<210> 78

<211> 22

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 78

uaguucuggu gcucuuggcu uu 22

<210> 79

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 79

ccaagagcac caagaacua 19

<210> 80

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 80

uaguucuggu gcucuuggcu 20

<210> 81

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 81

uaguucuggu gcucuuggcu 20

<210> 82

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 82

uaguucuggu gcucuuggcu 20

<210> 83

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 83

caauaaagcu ggacaagaa 19

<210> 84

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 84

uucuugucca gcuuuauugg g 21

<210> 85

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 85

caauaaagcu ggacaagaa 19

<210> 86

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 86

uucuugucca gcuuuauugg g 21

<210> 87

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 87

caauaaagcu ggacaagaa 19

<210> 88

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 88

uucuugucca gcuuuauugg g 21

<210> 89

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 89

cccaauaaag cuggacaaga a 21

<210> 90

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 90

uucuugucca gcuuuauugg guu 23

<210> 91

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 91

caauaaagcu ggacaagaa 19

<210> 92

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 92

uucuugucca gcuuuauugg g 21

<210> 93

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 93

cugaaagacu acuggagca 19

<210> 94

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 94

ugcuccagua gucuuucagu u 21

<210> 95

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 95

uuaaaaggga caguauuca 19

<210> 96

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 96

ugaauacugu cccuuuuaag c 21

<210> 97

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 97

acaguauucu cagugcuca 19

<210> 98

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 98

ugagcacuga gaauacuguc c 21

<210> 99

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 99

uauucucagu gcucuccua 19

<210> 100

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 100

uaggagagca cugagaauac u 21

<210> 101

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 101

aguauucuca gugcucuca 19

<210> 102

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 102

ugagagcacu gagaauacug u 21

<210> 103

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 103

ggacaguauu cucagugca 19

<210> 104

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 104

ugcacugaga auacuguccc u 21

<210> 105

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 105

caauaaagcu ggacaagaa 19

<210> 106

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 106

uucuugucca gcuuuauugg g 21

<210> 107

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 107

ggacaguauu cucagugca 19

<210> 108

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 108

ugcacugaga auacuguccc u 21

<210> 109

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 109

ccuugaggca uacuucaaa 19

<210> 110

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 110

uuugaaguau gccucaaggu u 21

<210> 111

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 111

augugaaagu caucgacaa 19

<210> 112

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 112

uugucgauga cuuucacauu c 21

<210> 113

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 113

augugaaagu caucgacaa 19

<210> 114

<211> 24

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (9)..(9)

<223> n is a, c, g, or u

<400> 114

uugucgagna ugacuuucac auuc 24

<210> 115

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 115

augugaaagu caucgacaa 19

<210> 116

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 116

uugucgauga cuuucacauu c 21

<210> 117

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 117

ccaagagcac caagaacua 19

<210> 118

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 118

uaguucuugg ugcucuuggc u 21

<210> 119

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 119

ccaagagcac caagaacua 19

<210> 120

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 120

uaguucuugg ugcucuuggc u 21

<210> 121

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 121

ccaagagcac caagaacua 19

<210> 122

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 122

uaguucuugg ugcucuuggc u 21

<210> 123

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 123

ccaagagcac caagaacua 19

<210> 124

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 124

uaguucuugg ugcucuuggc u 21

<210> 125

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 125

agccaagagc accaagaacu a 21

<210> 126

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 126

uaguucuugg ugcucuuggc uuu 23

<210> 127

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 127

ccaagagcac caagaacua 19

<210> 128

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 128

uaguucuugg ugcucuuggc u 21

<210> 129

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 129

uucuccgaac gugucacgu 19

<210> 130

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 130

acgugacacg uucggagaac u 21

<210> 131

<211> 127

<212> DNA

<213> Artificial sequence

<220>

<223> Target sequence

<400> 131

ctcgagaaac cgccctaggg acaagaattg gaaaccgccc tagggacaag aattggaaac 60

cgccctaggg acaagaattg gaaaccgccc tagggacaag aattggaaac cgccctaggg 120

acaagaa 127

<210> 132

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 132

gtgaccgatg gcttcagttc 20

<210> 133

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 133

atggataggc aggtggactt 20

<210> 134

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 134

ggtcggagtc aacggattt 19

<210> 135

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 135

ccagcatcgc cccacttga 19

<210> 136

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Target sequence

<400> 136

aaaccgccct agggacaaga a 21

<210> 137

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Target sequence

<400> 137

cccaauaaag cuggacaaga a 21

<210> 138

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Target sequence

<400> 138

ccctgaaaga ctactggagc a 21

<210> 139

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Target sequence

<400> 139

gcttaaaagg gacagtattc t 21

<210> 140

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Target sequence

<400> 140

ggacagtatt ctcagtgctc t 21

<210> 141

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Target sequence

<400> 141

agtattctca gtgctctcct a 21

<210> 142

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Target sequence

<400> 142

acagtattct cagtgctctc c 21

<210> 143

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Target sequence

<400> 143

agggacagta ttctcagtgc t 21

<210> 144

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 144

gtgaccgatg gcttcagttc 20

<210> 145

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 145

atggataggc aggtggactt 20

<210> 146

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 146

tgcaccacca actgcttag 19

<210> 147

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 147

ggatgcaggg atgatgttc 19

<210> 148

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Target sequence

<400> 148

gaccttgagg catacttcaa a 21

<210> 149

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 149

cctcactgcc cattgttg 18

<210> 150

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 150

gtgcctttcc tgactccc 18

<210> 151

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 151

ccgtgaaaag atgacccaga t 21

<210> 152

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 152

gccaggtcca gacgcagg 18

<210> 153

<211> 85

<212> DNA

<213> Artificial sequence

<220>

<223> Target sequence

<400> 153

ctcgagctaa cctctacaca agaactattg gctaacctct acacaagaac tattggctaa 60

cctctacaca agaactagcg gccgc 85

<210> 154

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 154

gaggagcagc taaccaactt aat 23

<210> 155

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 155

tctgcatgtg ctgttgactt aat 23

<210> 156

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 156

tgcaccacca actgcttag 19

<210> 157

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 157

ggatgcaggg atgatgttc 19

Claims

1. A nucleotide sequence, each nucleotide in the nucleotide sequence being a modified or unmodified nucleotide, wherein the nucleotide sequence comprises a nucleotide sequence I, which is a nucleotide sequence in which one or two nucleotides from the 2 nd to 8 th nucleotides in the 5 'end-3' end direction in the nucleotide sequence a are replaced with an acyclic dealkalized group, the nucleotide sequence a has 19 to 25 nucleotides, and the nucleotide sequence a is reverse-complementary to at least 16 nucleotides of a stretch of nucleotide sequence in a target mRNA, the acyclic dealkalized group having a structure as shown in formula (101):

r ₄ has a structure as shown in formula (202):

Represents the site of covalent attachment of the groups, each n is independently 0, each m is independently selected from 1 or 2, E ₁ is selected from OH, SH or BH ₂;

Each R ₁₀₁ is independently selected from the group consisting of H, methyl, and ethyl;

R ₂₀₁ is selected from OH or NHR ₂₀₂, wherein R ₂₀₂ is selected from the group consisting of C ₁-C₅ aliphatic acyl and C ₇-C₁₀ aromatic acyl.

2. The nucleotide sequence of claim 1, wherein R ₂₀₂ is selected from the group consisting of acetyl, isobutyryl, benzoyl, levulinyl, and crotonyl.

3. The nucleotide sequence according to claim 1, wherein the nucleotide sequence I is a nucleotide sequence in which one or two of the 3 rd to 8 th nucleotides in the 5 '-3' -terminal direction in the nucleotide sequence A are replaced with an acyclic dealkalized group.

4. A nucleotide sequence according to claim 3, wherein the nucleotide sequence I is a nucleotide sequence in which one or two of the 6 th, 7 th and 8 th nucleotides in the 5 'end-3' end direction in the nucleotide sequence a are replaced with an acyclic dealkalized group.

5. The nucleotide sequence according to claim 4, wherein the nucleotide sequence I is a nucleotide sequence in which one of the nucleotides at positions 6, 7 or 8 in the 5 '-3' -terminal direction of the nucleotide A is replaced with an acyclic dealkalized group.

6. A nucleotide sequence according to claim 3, wherein the nucleotide sequence I is a nucleotide sequence in which any 2 nucleotides of nucleotides 4, 5,6 and 7 of the nucleotide sequence a in the 5 'to 3' direction are replaced with an acyclic abasic group.

7. The nucleotide sequence according to claim 6, wherein the nucleotide sequence I is a nucleotide sequence in which any 1 nucleotide at position 4,5 or 6 and the nucleotide at position 7 in the 5 '-3' -terminal direction in the nucleotide sequence A are replaced with acyclic dealkalized groups.

8. The nucleotide sequence according to any one of claims 1 to 7, wherein each acyclic dealkalization group is independently selected from the group consisting of groups a101-a103, a105-a 107:

9. The nucleotide sequence of claim 1, wherein each nucleotide in the nucleotide sequence a is a modified nucleotide.

10. The nucleotide sequence according to claim 9, wherein at least two of the 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 nucleotides in the nucleotide sequence a are fluoro-modified nucleotides at the 2' -position of the ribosyl group of the nucleotide according to the 5' -3' -direction, and wherein the nucleotide sequence I is a nucleotide sequence in which one or two of the 2, 3, 4, 5, 6, 7 or 8 nucleotides in the 5' -3' -direction in the nucleotide sequence a are replaced with an acyclic abasic group.

11. The nucleotide sequence according to claim 10, wherein each non-fluoro modified nucleotide is independently selected from one of a nucleotide analogue or a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluoro group.

12. The nucleotide sequence according to claim 11, wherein the nucleotide formed by substituting the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide with a non-fluorine group is selected from one of a 2' -alkoxy-modified nucleotide, a2 '-substituted alkoxy-modified nucleotide, a 2' -alkyl-modified nucleotide, a2 '-substituted alkyl-modified nucleotide, a 2' -amino-modified nucleotide, a2 '-substituted amino-modified nucleotide, and a 2' -deoxynucleotide; the nucleotide analog is selected from one of an iso nucleotide, LNA, ENA, cET BNA, UNA and GNA.

13. The nucleotide sequence according to claim 1, wherein the nucleotide sequence consists of the nucleotide sequence I.

14. The nucleotide sequence according to claim 13, wherein at least 1 of the phosphate groups in the at least one single-stranded phosphate-sugar backbone in the nucleotide sequence are phosphate groups having a modifying group; the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond in the phosphate group with a sulfur atom.

15. A double-stranded oligonucleotide, wherein the double-stranded oligonucleotide comprises a sense strand and an antisense strand, the antisense strand having the nucleotide sequence of any one of claims 1-14; the sense strand is a nucleotide sequence of 16-30 nucleotides that is at least partially reverse complementary to the antisense strand to form a double-stranded region.

16. The double-stranded oligonucleotide of claim 15, wherein the antisense strand is substantially reverse complementary or substantially reverse complementary to the sense strand.

17. The double-stranded oligonucleotide of claim 16, wherein at least 16 nucleotides of nucleotides 2-19 of the nucleotide sequence a are complementary to the sense strand in a 5 'to 3' end direction.

18. The double stranded oligonucleotide of claim 17, wherein the nucleotide at position 7, 8, or 5, 7, 8, 9, or 9, 10, 11 of the sense strand is a ribosyl 2' nucleotide of the nucleotide having a fluoro modification, each nucleotide at the other positions of the sense strand being independently one of the non-fluoro modified nucleotides.

19. The double-stranded oligonucleotide of claim 18, wherein at least 1 of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand are phosphate groups having a modifying group; the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond in the phosphate group with a sulfur atom.

20. The double stranded oligonucleotide of any one of claims 15-19, wherein the double stranded oligonucleotide is a saRNA or siRNA.

21. The double-stranded oligonucleotide of claim 20, wherein the target mRNA is selected from one of the mrnas expressed by the following genes ：ACE2、ANGPTL3、ApoA、ApoB、ApoC、AR、ASK1、C5、Col1A1、CTGF、Ebola、FOXO1、FTO、FVII、FXI、FXII、GCGR、HBV、HCV、HSD、p53、PCSK9、PNP、PLG、PKK、KNG、SARS-CoV-2、SCD1、SCNN1A、SOD1、STAT3、TIMP-1、TMPRSS6、XO、HAO1.

22. The double-stranded oligonucleotide of claim 21, wherein the target mRNA is selected from one of the mrnas expressed by: HBV, ANGPTL3, APOC, C5 or HAO1.

23. The double-stranded oligonucleotide of claim 21, wherein the double-stranded oligonucleotide is one of siAPO1L、siAPO1、siAPOa1M1SVP2、siAPOb1M1SVP2、siAPOc1M1SVP2、siAPOd1M1SVP2、siAPOe1M1SVP2、siAPOf1M1SVP2、siAPOf1M1SP2、siAPOg1M1SP2、siAPOg1M1SP2-Ac、siAPOg1M1SP2-Ph、siAPOg1M1SP2-TOS、siAPOg1M1SP2-iBu、siAPOg1M1SP2-laev、siAPOg1M1SP2-Cro、siAPOg1M1SVP1R、siAPOg1M1SVP1S、siAPOg1M1SVP2R、siAPOg1M1SVP2S、siAPOg1M1SVP3R、siAPOg1M1SVP3S、siAPOg1M1SVP4R、siAPOg1M1SVP4S、siAPOg1Ph2、siAPOg1Ph3、siAPOg1Ph4、siAPOg1Ph5、siAPOg1Ph6、siAPOg1Ph7、siAPOg1Ph8、siAPOg1Ph4ph7、siAPOg1Ph5ph7、siAPOg1M16ph7、siAPOg1M34ph7、siAPOg1Lph7、siAPOg1Nph7、siHBa1M1SP2、siHBa1M1SVP2、siHBb1M1SVP2、siHBa1M1SVP1-Ac、siHBa1M1SVP2-Ac、siHBa2M1SVP3-Ac、siC5a1M1S2、siC5b1M1S2、siHAOa2M1S2、siHAOa1M1S2、siANGa1M1S2-Ac、siANGa1M1S2-iBu、siANGa1Ph2、siANGa1Ph3、siANGa1Ph4、siANGa1Ph5、siANGa1Ph6、siANGa1Ph7、siANGa1Ph8、siANGa1Ph4ph7、siANGa1Ph5ph7、siANGa1M16ph7、siANGa1M34ph7、siANGa1Lph7 or siANGa1Nph 7.

24. A pharmaceutical composition comprising the nucleotide sequence of any one of claims 1-14 and the double-stranded oligonucleotide of any one of claims 15-23 and a pharmaceutically acceptable carrier.

25. An siRNA conjugate comprising the double stranded oligonucleotide of any one of claims 15-23 and a conjugate group conjugated to the double stranded oligonucleotide.

26. The siRNA conjugate of claim 25, wherein the conjugate group comprises a pharmaceutically acceptable targeting group and a linker, and the double stranded oligonucleotide, the linker and the targeting group are sequentially covalently or non-covalently linked.

27. The siRNA conjugate of claim 25 or 26, wherein each of said targeting groups is independently a ligand that is affinity to an asialoglycoprotein receptor on the surface of mammalian hepatocytes.

28. The siRNA conjugate of claim 27, wherein each targeting group is independently an asialoglycoprotein or a saccharide.

29. The siRNA conjugate of claim 28, wherein at least one or each of said targeting groups is galactose or N-acetylgalactosamine.

30. Use of the nucleotide sequence of any one of claims 1-14, the double stranded oligonucleotide of any one of claims 15-23, the pharmaceutical composition of claim 24 and/or the siRNA conjugate of any one of claims 25-29 in the manufacture of a medicament for the treatment and/or prevention of a disease or symptom caused by abnormal gene expression.

31. A method of inhibiting gene expression in a cell, the method for non-therapeutic purposes, the method comprising contacting an effective amount of the double stranded oligonucleotide of any one of claims 15-23, the pharmaceutical composition of claim 24, and/or the siRNA conjugate of any one of claims 25-29 with the cell.

32. A kit comprising the double stranded oligonucleotide of any one of claims 15-23, the pharmaceutical composition of claim 24, and/or the siRNA conjugate of any one of claims 25-29.