CN113330117A

CN113330117A - Nucleic acid, composition containing nucleic acid, conjugate, preparation method and application

Info

Publication number: CN113330117A
Application number: CN202080009787.1A
Authority: CN
Inventors: 张鸿雁; 高山; 康代武
Original assignee: Suzhou Ruibo Biotechnology Co Ltd
Current assignee: Suzhou Ruibo Biotechnology Co Ltd
Priority date: 2019-01-18
Filing date: 2020-01-17
Publication date: 2021-08-31
Also published as: WO2020147847A1

Abstract

The present disclosure provides a siRNA inhibiting Factor XII (Factor XII, FXII) gene expression, pharmaceutical compositions and conjugates containing the siRNA. Each nucleotide in the siRNA is a modified or unmodified nucleotide independently, the siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence I, the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 1 in length and has NO more than 3 nucleotide differences, and the antisense strand comprises a nucleotide sequence II, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 2 in length and has NO more than 3 nucleotide differences. The siRNA and the pharmaceutical composition and conjugate thereof provided by the disclosure can effectively treat and/or prevent Hereditary Angioedema (HAE) and/or thrombosis.

Description

Nucleic acid, composition containing nucleic acid, conjugate, preparation method and application

Technical Field

The present disclosure relates to nucleic acids capable of inhibiting the expression of factor XII gene and compositions and conjugates containing the same. The disclosure also relates to methods of making and uses of these nucleic acids, compositions and conjugates.

Background

Hereditary Angioedema (HAE) is a rare disease characterized by recurrent episodes of severe swelling. The most common areas of swelling of the body are the extremities, the face, the intestines and the airways. The onset may be spontaneous or may be due to physical trauma or stress. Laryngeal (airway) edema can be life threatening as it can lead to asphyxia death.

Factor XII is a serine protease expressed primarily in the liver and found in the blood, having dual functions in the endogenous coagulation pathway and the kinin-kallikrein (kinin-kallikrein) system. The kinin-kallikrein system plays a role in inflammation, blood pressure control, coagulation and pain. The active form of factor XII (also known as FXII, F12 or Hageman factor) binds to and cleaves prekallikrein in the factor XI and kallikrein systems in the coagulation cascade, producing the active forms FXI and kallikrein, respectively.

Factor XII is one of the key targets for the treatment of HAE. By inhibiting the expression of factor XII, the occurrence of HAE can be effectively inhibited. Therefore, silencing gene expression from the gene level, blocking factor XII production, would certainly be the most desirable therapeutic approach. Small interfering RNAs (sirnas) can inhibit or block the expression of any target gene of interest in a sequence-specific manner based on the mechanism of RNA interference (RNAi), thereby achieving the purpose of treating diseases.

Suitable siRNA sequences and modifications and their delivery systems are two key technologies in small RNA drug development.

Disclosure of Invention

In some embodiments, the present disclosure provides an siRNA conjugate having the structure represented by formula (308):

wherein the content of the first and second substances,

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

each m1, m2 and m3 is independently an integer selected from 2 to 10;

each R₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄And R₁₅Each independently is H, or is selected from the group consisting of: c₁-C ₁₀Alkyl radical, C₁-C ₁₀Haloalkyl and C₁-C ₁₀An alkoxy group;

R ₃a group of the structure shown in formula a 59:

wherein E is₁Is OH, SH or BH₂；

Nu is siRNA having a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, the sense strand comprising a nucleotide sequence I, and the antisense strand comprising a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially complementary in reverse to form a double-stranded region, wherein the nucleotide sequence I and the nucleotide sequence II are selected from the group consisting of I) to v) as follows:

i) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 1 in length and has NO more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 2 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence I comprises a position corresponding to Z ₁Nucleotide Z of₃The nucleotide sequence II comprises a position corresponding to Z₂Nucleotide Z of₄Z is the same as₄Is the first nucleotide at the 5' end of the antisense strand;

II) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 61 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 62 in length and has NO more than 3 nucleotide differences, wherein the nucleotide sequence I comprises a position corresponding to Z₅Nucleotide Z of₇The nucleotide sequence II comprises a position corresponding to Z₆Nucleotide Z of₈Z is the same as₈Is the first nucleotide at the 5' end of the antisense strand;

iii) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 121 in length and has NO more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 122 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence I comprises a position corresponding to Z₁₃Nucleotide Z of₁₅The nucleotide sequence II comprises a position corresponding to Z₁₄Nucleotide Z of₁₆Z is the same as₁₆Is the first nucleoside at the 5' end of the antisense strandAn acid;

iv) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 181 and has NO more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 182 and has NO more than 3 nucleotide differences, and the position of the nucleotide sequence I is corresponding to Z ₁₇Nucleotide Z of₁₉The nucleotide sequence II comprises a position corresponding to Z₁₈Nucleotide Z of₂₀Z is the same as₂₀Is the first nucleotide at the 5' end of the antisense strand;

v) the nucleotide sequence I is equal to the nucleotide sequence shown by SEQ ID NO. 241 in length and has NO more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown by SEQ ID NO. 242 in length and has NO more than 3 nucleotide differences, and the position of the nucleotide sequence I is corresponding to Z₂₁Nucleotide Z of₂₃The nucleotide sequence II comprises a position corresponding to Z₂₂Nucleotide Z of₂₄Z is the same as₂₄Is the first nucleotide at the 5' end of the antisense strand;

R ₂is a straight chain 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、C ₂-C ₁₀Alkenylene radical, C₂-C ₁₀Alkynylene, C₆-C ₁₀Arylene radical, C₃-C ₁₈Heterocyclylene and C₅-C ₁₀A heteroarylene group; and wherein R₂May optionally have a substituent of any one or more of the group consisting of: c₁-C ₁₀Alkyl radical, C₆-C ₁₀Aryl radical, C₅-C ₁₀Heteroaryl group, C₁-C ₁₀Haloalkyl, -OC₁-C ₁₀Alkyl, -OC₁-C ₁₀Alkylphenyl, -C₁-C ₁₀alkyl-OH, -OC ₁-C ₁₀Haloalkyl, -SC₁-C ₁₀Alkyl, -SC₁-C ₁₀Alkylphenyl, -C₁-C ₁₀alkyl-SH, -SC₁-C ₁₀Haloalkyl, halogen substituents, -OH, -SH, -NH₂、-C ₁-C ₁₀alkyl-NH₂、-N(C ₁-C ₁₀Alkyl) (C₁-C ₁₀Alkyl), -NH (C)₁-C ₁₀Alkyl), -N (C)₁-C ₁₀Alkyl) (C₁-C ₁₀Alkylphenyl), -NH (C)₁-C ₁₀Alkylphenyl), cyano, nitro, -CO₂H、-C(O)O(C ₁-C ₁₀Alkyl), -CON (C)₁-C ₁₀Alkyl) (C₁-C ₁₀Alkyl), -CONH (C)₁-C ₁₀Alkyl), -CONH₂、-NHC(O)(C ₁-C ₁₀Alkyl), -NHC (O) (phenyl), -N (C)₁-C ₁₀Alkyl radical C (O) (C)₁-C ₁₀Alkyl), -N (C)₁-C ₁₀Alkyl group C (O) (phenyl), -C (O) C₁-C ₁₀Alkyl, -C (O) C₁-C ₁₀Alkylphenyl, -C (O) C₁-C ₁₀Haloalkyl, -OC (O) C₁-C ₁₀Alkyl, -SO₂(C ₁-C ₁₀Alkyl), -SO₂(phenyl), -SO₂(C ₁-C ₁₀Haloalkyl), -SO₂NH ₂、-SO ₂NH(C ₁-C ₁₀Alkyl), -SO₂NH (phenyl), -NHSO₂(C ₁-C ₁₀Alkyl), -NHSO₂(phenyl) and-NHSO₂(C ₁-C ₁₀Haloalkyl);

each L₁Independently a linear alkylene group of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH ═ N, S (O)₂、C ₂-C ₁₀Alkenylene radical, C₂-C ₁₀Alkynylene, C₆-C ₁₀Arylene radical, C₃-C ₁₈Heterocyclylene and C₅-C ₁₀A heteroarylene group; and wherein L₁May optionally have a substituent of any one or more of the group consisting of: c₁-C ₁₀Alkyl radical, C₆-C ₁₀Aryl radical, C₅-C ₁₀Heteroaryl group, C ₁-C ₁₀Haloalkyl, -OC₁-C ₁₀Alkyl, -OC₁-C ₁₀Alkylphenyl, -C₁-C ₁₀alkyl-OH, -OC₁-C ₁₀Haloalkyl, -SC₁-C ₁₀Alkyl, -SC₁-C ₁₀Alkylphenyl, -C₁-C ₁₀alkyl-SH, -SC₁-C ₁₀Haloalkyl, halogen substituents, -OH, -SH, -NH₂、-C ₁-C ₁₀alkyl-NH₂、-N(C ₁-C ₁₀Alkyl) (C₁-C ₁₀Alkyl), -NH (C)₁-C ₁₀Alkyl), -N (C)₁-C ₁₀Alkyl) (C₁-C ₁₀Alkylphenyl), -NH (C)₁-C ₁₀Alkylphenyl), cyano, nitro, -CO₂H、-C(O)O(C ₁-C ₁₀Alkyl), -CON (C)₁-C ₁₀Alkyl) (C₁-C ₁₀Alkyl), -CONH (C)₁-C ₁₀Alkyl), -CONH₂，-NHC(O)(C ₁-C ₁₀Alkyl), -NHC (O) (phenyl), -N (C)₁-C ₁₀Alkyl radical C (O) (C)₁-C ₁₀Alkyl), -N (C)₁-C ₁₀Alkyl group C (O) (phenyl), -C (O) C₁-C ₁₀Alkyl, -C (O) C₁-C ₁₀Alkylphenyl, -C (O) C₁-C ₁₀Haloalkyl, -OC (O) C₁-C ₁₀Alkyl, -SO₂(C ₁-C ₁₀Alkyl), -SO₂(phenyl), -SO₂(C ₁-C ₁₀Haloalkyl), -SO₂NH ₂、-SO ₂NH(C ₁-C ₁₀Alkyl), -SO₂NH (phenyl), -NHSO₂(C ₁-C ₁₀Alkyl), -NHSO₂(phenyl) and-NHSO₂(C ₁-C ₁₀Haloalkyl);

represents the site of covalent attachment of a group;

M ₁represents a targeting group.

In some embodiments, the present disclosure provides an siRNA comprising a sense strand and an antisense strand, each nucleotide in the sense strand and the antisense strand being independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide; the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, the fluorinated modified nucleotide is positioned in the nucleotide sequence I and the nucleotide sequence II, and in the direction from 5 'end to 3' end, in the sense strand, the 7 th, 8 th and 9 th nucleotides of the nucleotide sequence I are fluorinated modified nucleotides, and the rest nucleotides in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5 'end to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14, 16 of the nucleotide sequence II are fluorine-modified nucleotides, the nucleotides at the remaining positions in the antisense strand are non-fluorine-modified nucleotides, and,

i) The nucleotide sequence I has the same length with the nucleotide sequence shown in SEQ ID NO. 1 and has NO more than 3 nucleotide differences, the nucleotide sequence II has the same length with the nucleotide sequence shown in SEQ ID NO. 2 and has NO more than 3 nucleotide differences, and the nucleotide sequence I comprises a position corresponding to Z₁Nucleotide Z of₃The nucleotide sequence II comprises a position corresponding to Z₂Nucleotide Z of₄Z is the same as₄Is the first nucleotide at the 5' end of the antisense strand; alternatively, the first and second electrodes may be,

II) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 61 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 62 in length and has NO more than 3 nucleotide differences, wherein the nucleotide sequence I comprises a position corresponding to Z₅Nucleotide Z of₇The nucleotide sequence II comprises a position corresponding to Z₆Nucleotide Z of₈Z is the same as₈Is the first nucleotide at the 5' end of the antisense strand; alternatively, the first and second electrodes may be,

iii) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 121 in length and has NO more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 122 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence I comprises a position corresponding to Z ₁₃Nucleotide Z of₁₅The nucleotide sequence II comprises a position corresponding to Z₁₄Nucleotide Z of₁₆Z is the same as₁₆Is the first nucleotide at the 5' end of the antisense strand; alternatively, the first and second electrodes may be,

iv) the nucleotide sequence I and SEQ ID NO 181 is equal in length and not more than 3 nucleotides different, and the nucleotide sequence II is equal in length and not more than 3 nucleotides different from the nucleotide sequence shown in SEQ ID NO. 182, and the nucleotide sequence I comprises a nucleotide sequence with a position corresponding to Z₁₇Nucleotide Z of₁₉The nucleotide sequence II comprises a position corresponding to Z₁₈Nucleotide Z of₂₀Z is the same as₂₀Is the first nucleotide at the 5' end of the antisense strand; alternatively, the first and second electrodes may be,

v) the nucleotide sequence I is equal to the nucleotide sequence shown by SEQ ID NO. 241 in length and has NO more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown by SEQ ID NO. 242 in length and has NO more than 3 nucleotide differences, and the position of the nucleotide sequence I is corresponding to Z₂₁Nucleotide Z of₂₃The nucleotide sequence II comprises a position corresponding to Z₂₂Nucleotide Z of₂₄Z is the same as₂₄Is the first nucleotide at the 5' end of the antisense strand.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising an siRNA of the present disclosure and a pharmaceutically acceptable carrier.

In some embodiments, the present disclosure provides an siRNA conjugate comprising an siRNA provided by the present disclosure and a conjugate group conjugated to the siRNA.

In some embodiments, the present disclosure provides the use of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the manufacture of a medicament for the treatment and/or prevention of hereditary angioedema HAE and/or thrombosis.

In some embodiments, the present disclosure provides a method of treating and/or preventing HAE and/or thrombosis, the method comprising administering to a subject having HAE an effective amount of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.

In some embodiments, the present disclosure provides a method of inhibiting FXII gene expression in a hepatocyte, the method comprising contacting the hepatocyte with an effective amount of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.

In some embodiments, the present disclosure provides a kit comprising an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.

Is 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.

Advantageous effects

The siRNA, the composition containing the siRNA and the siRNA conjugate provided by the disclosure have good stability and higher gene inhibition activity, and/or can remarkably treat or relieve HAE symptoms.

In some embodiments, the siRNA, the composition comprising the siRNA, or the siRNA conjugate provided by the present disclosure exhibits excellent target gene inhibitory activity in an in vitro cell assay. In some embodiments, at a dose of 50nM, the sirnas provided by the present disclosure exhibit a rate of inhibition of FXII mRNA expression in human liver primary cells of up to 78.70%. In some embodiments, at a dose of 50nM, the sirnas provided by the present disclosure exhibit up to 70.09% inhibition of FXII mRNA expression in C57 mouse liver primary cells.

In some embodiments, the siRNA conjugates of the present disclosure exhibit up to 98.7% inhibition of FXII mRNA expression in C57 mice at a 5mg/kg dose.

In some embodiments, the siRNA, composition comprising the siRNA, or siRNA conjugate provided by the present disclosure does not exhibit significant off-target effects. The off-target effect can be, for example, inhibition of normal expression of a gene other than the target gene. It is believed that off-target effects are not significant if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20% or 10% compared to the effect on the target gene.

Therefore, the siRNA, the pharmaceutical composition and the siRNA conjugate provided by the disclosure can inhibit the expression of FXII gene, effectively treat and/or prevent HAE symptoms, and have good application prospects.

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

Drawings

FIGS. 1 and 2 are scattergrams of FXII mRNA expression (relative to GAPDH) in liver tissue of C57 mice after administration of PBS to C57 mice and different doses of each conjugate, respectively.

Detailed Description

The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

In the present disclosure, FXII mRNA refers to a sequence as shown in Genbank accession No. NM _ 000505.3. Further, unless otherwise specified, the term "target gene" used in the present disclosure refers to a gene expressing the above FXII mRNA, and the term "target mRNA" refers to the above FXII mRNA.

Definition of

In the above and below, capital C, G, U, A represents the base composition of nucleotides, unless otherwise specified; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates a phosphorothioate-based linkage between two nucleotides adjacent to the left and right of the letter s; p1 indicates that the adjacent nucleotide to the right of P1 is a 5 '-phosphate nucleotide or a 5' -phosphate analogue modified nucleotide. In some embodiments, P1 is a VP, Ps, or P that represents a specific modification, wherein the letter combination VP represents that one nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate modified nucleotide, the letter combination Ps represents that one nucleotide adjacent to the right of the letter combination Ps is a phosphorothioate modified nucleotide, and the capital letter P represents that one nucleotide adjacent to the right of the letter P is a 5' -phosphate nucleotide.

In the above and below, the "fluorine-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-fluorine-modified nucleotide" refers to a nucleotide or a nucleotide analog 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 can replace 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 a heteronucleotide, a bridged nucleotide (BNA for short) or an acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group.

In the present context, the expressions "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to the person 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) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair comprises a purine and a pyrimidine. Two strands are considered to be complementary to each other when adenine on one strand always pairs with thymine (or uracil) on the other strand and guanine always pairs with cytosine, and the sequence of that strand can be deduced from the sequence of its complementary strand. Accordingly, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at the corresponding positions are not paired in a complementary fashion.

In the above and below, 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 no more than 1 base mismatch exists between two nucleotide sequences; "completely reverse complementary" means that there is no base mismatch between two nucleotide sequences.

In the above and below, the "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the former has a change in the base type of the nucleotide at the same position as compared with the latter, for example, in the latter, when one nucleotide base is A, in the case where the corresponding nucleotide base at the same position of the former is U, C, G or T, it is considered that there is a nucleotide difference between the two nucleotide sequences at that position. In some embodiments, when a nucleotide in situ is replaced with a nucleotide without a base or its equivalent, it is also believed that a nucleotide difference is created at that position.

In the above and the following, particularly in describing the preparation method of the siRNA, the siRNA-containing composition or the siRNA conjugate of the present disclosure, unless otherwise specified, the Nucleoside monomer (Nucleoside monomer) means modified or unmodified Nucleoside phosphoramidite monomers (sometimes referred to as Nucleoside phosphoramidites) used in solid phase synthesis of phosphoramidites, depending on the kind and order of nucleotides in the siRNA or siRNA conjugate to be prepared. Solid phase phosphoramidite synthesis is a method used in RNA synthesis well known to those skilled in the art. Nucleoside monomers for use in the present disclosure are all commercially available.

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

As herein describedAs used, a dash ("-") that is not between two letters or two symbols is used to indicate a point of attachment for a substituent. For example: structural formula' -C₁-C ₁₀alkyl-NH₂"the leftmost bar means 66 passes through C₁-C ₁₀Alkyl groups are attached.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted" alkyl "includes" alkyl "and" substituted alkyl "as defined below. It will be understood by those skilled in the art that, for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically non-feasible, and/or inherently unstable.

As used herein, "alkyl" refers to straight and branched chains having the specified number of carbon atoms, typically from 1 to 20 carbon atoms, for example from 1 to 10 carbon atoms, such as from 1 to 8 or from 1 to 6 carbon atoms. E.g. C₁-C ₆Alkyl groups include straight and branched chain alkyl groups of 1 to 6 carbon atoms. When referring to an alkyl residue having a particular 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 and refers to the same residue as alkyl but with 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 the adjacent carbon atom 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: a vinyl group; 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, e.g., but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-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 and refers to the same residue as alkenyl, but with 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: an ethynyl group; propynyl groups, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl groups 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, and in other embodiments 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to the same residue as alkynyl, but with two points of attachment.

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

As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring, typically having from 3 to 7 ring carbon atoms. The rings may be saturated or have one or more carbon-carbon double bonds. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl, as well as bridged and caged ring groups, such as norbornane (norbonane).

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

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

"heterocyclyl" refers to a stable 3-to 18-membered non-aromatic ring radical 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, and may include fused or bridged ring systems. The heteroatoms in the heterocyclic group may be optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. Heterocyclyl groups are partially or fully saturated. The heterocyclyl group may be attached to the remainder of the molecule through any ring atom. Examples of such heterocyclic groups include, but are not limited to: dioxanyl, thienyl [1,3] dithioyl (thienyl [1,3] dithianyl), decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithioyl (trithiofuranyl), tetrahydropyranyl, thiomorpholinyl (thiomorpholinyl), 1-oxothiomorpholinyl (1-oxo-thiomorpholinyl), and 1, 1-dioxothiomorpholinyl (1, 1-dioxothiomorpholinyl).

"heteroaryl" refers to a group derived from a 3-to 18-membered aromatic ring radical containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, a heteroaryl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., comprises a cyclic delocalized (4n +2) pi-electron system according to huckel theory. Heteroaryl includes fused or bridged ring systems. The heteroatoms in the heteroaryl group are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heteroaryl group is attached to the remainder of the molecule through any ring atom. Examples of heteroaryl groups include, but are not limited to: azacyclotrienoyl, acridinyl, benzimidazolyl, benzindolyl, 1, 3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo [ d ] thiazolyl, benzothiadiazolyl, benzo [ b ] [1,4] dioxepinyl (benzo [ b ] [1,4] dioxepinyl), benzo [ b ] [1,4] oxazinyl (benzo [ b ] [1,4] oxazinyl), 1,4-benzodioxanyl (1,4-benzodioxanyl), benzonaphthofuranyl, benzoxazolyl, benzodioxolyl (benzodioxolyl), benzodioxinyl (benzodioxanyl), benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothiophenyl, benzothieno [3,2-d ] pyrimidinyl, benzotriazolyl, benzo [4,6] imidazo [1,2-a ] pyridinyl, Carbazolyl, 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 (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-hexahydrocycloocta [ d ] pyrimidinyl, 5,6,7,8,9, 10-hexahydrocycloocta [ d ] pyridazinyl, 5,6,7,8,9, 10-hexahydrocycloocta [ d ] pyridazinyl, 7,8,9, 10-hexahydrocycloocta [ d ] pyridyl, isothiazolyl, imidazolyl, indazolyl (indazolyl), indolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5, 8-methanol-5, 6,7,8-tetrahydroquinazolinyl (5,8-methano-5,6,7,8-tetrahydroquinazolinyl), naphthyridinyl (naphthyridinyl), 1,6-naphthyridinonyl (1,6-naphthyridinonyl), oxadiazolyl, 2-oxazepinyl (2-oxoazepinyl), oxazolyl, oxacyclopropane (oxacinnanyl), 5,6,6a,7,8,9,10,10 a-octahydrobenzo [ H ] quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, and oxazolyl, Phthalazinyl (phthalazinyl), pteridinyl (pteridinyl), purinyl, pyrrolyl, pyrazolyl, pyrazolo [3,4-d ] pyrimidinyl, pyridyl, pyrido [3,2-d ] pyrimidinyl, pyrido [3,4-d ] pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, 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-cyclohepta [4,5] thieno [2,3-d ] pyrimidinyl, 5,6,7, 8-tetrahydropyrido [4,5-c ] pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridinyl, Triazinyl, thieno [2,3-d ] pyrimidinyl, thieno [3,2-d ] pyrimidinyl, thieno [2,3-c ] pyridinyl (thieno [2,3-c ] pridinyl) and thienyl (thiophenyl/thiophenyl).

Various hydroxyl protecting groups may be used in the present disclosure. In general, protecting groups render a chemical functional group insensitive to particular reaction conditions, and can be added to and removed from that functional group in a molecule without substantially damaging the remainder of the molecule. Representative hydroxyl protecting Groups are disclosed in Beaucage et al, Tetrahedron 1992,48,2223-2311, and Greenea and Wuts, Protective Groups in Organic Synthesis, Chapter 2,2d ed, John Wiley & Sons, New York, 1991, which are incorporated herein by reference in their entirety. In some embodiments, the protecting group is stable under basic conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups that may be used herein include Dimethoxytrityl (DMT), 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, e.g., a mammal or a marsupial. 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, cows, sheep, rats, and any species of poultry.

As used herein, "treat," "alleviate," or "improve" may be used interchangeably herein. These terms refer to methods of achieving beneficial or desired results, including but not limited to therapeutic benefits. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is achieved by eradicating or ameliorating one or more 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, "prevent" and "prevention" are used interchangeably. These terms refer to methods of achieving beneficial or desired results, including but not limited to prophylactic benefits. To obtain a "prophylactic benefit," the conjugate or composition can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.

The sirnas of the present disclosure contain a nucleotide group as a basic structural unit, which is well known to those skilled in the art, and the nucleotide group contains a phosphate group, a ribose group and a base, which are not described in detail herein.

First siRNA

According to the present disclosure, the siRNA may be a first siRNA.

The first siRNA comprises a sense strand and an antisense strand, each nucleotide in the first siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 1 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 2 in length and has NO more than 3 nucleotide differences:

5'-GGAACUCAAUAAAGUGCUZ ₁-3'(SEQ ID NO:1)；

5'-Z ₂AGCACUUUAUUGAGUUCC-3'(SEQ ID NO:2)，

wherein Z is₁Is U, Z₂The content of the compound is A,

and, the position contained in the nucleotide sequence I corresponds to Z₁Nucleotide Z of₃The nucleotide sequence II comprises a position corresponding to Z₂Nucleotide Z of₄Z is the same as₄Is the first nucleotide at the 5' end of the antisense strand.

In the above and below, "positional correspondence" means that they are at the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the 1 st nucleotide from the 3 'end of the nucleotide sequence I is the nucleotide whose position corresponds to the 1 st nucleotide from the 3' end of SEQ ID NO. 1.

In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.

In some embodiments, the nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 1, and/or the nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 2.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO. 2 comprises Z₄A difference at position, and Z₄Selected from U, C or G. In some embodiments, the nucleotide difference is Z₄Difference in position, Z₄Selected from U, C or G. In some embodiments, Z₃Is a reaction of with Z₄A complementary nucleotide. These nucleotide differences did not significantly reduce the target base of the siRNA conjugatessiRNA conjugates comprising these nucleotide differences due to inhibitory ability are also within the scope of the present disclosure.

In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary. The term "substantially reverse complementary" in the context of the present disclosure means that there are no more than 3 base mismatches between two nucleotide sequences; "substantially reverse complementary" means that no more than 1 base mismatch occurs between two nucleotide sequences; "completely reverse complementary" means that there is no base mismatch between two nucleotide sequences.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO. 3, nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 4:

5'-GGAACUCAAUAAAGUGCUZ ₃-3'(SEQ ID NO:3)；

5'-Z ₄AGCACUUUAUUGAGUUCC-3'(SEQ ID NO:4)，

wherein, Z is₄Is the first nucleotide at the 5' end of the antisense strand, Z₃Selected from A, U, G or C, and Z₄Is a reaction of with Z₃A complementary nucleotide; in some embodiments, Z₃Is U, Z₄Is A;

and the lengths of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides.

In some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected at the 5 'end of the nucleotide sequence I, and the nucleotide sequence IV is connected at the 3' end of the nucleotide sequence II. In some embodiments, the nucleotide sequence IV is substantially reverse complementary or fully reverse complementary to a second nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 1 in the target mRNA and that is the same length as the nucleotide sequence IV.

In some embodiments, the length of each of the nucleotide sequence III and the nucleotide sequence IV is 1 nucleotide in the 5'-3' direction, the base of the nucleotide sequence III is a, and the base of the nucleotide sequence IV is U; in this case, the length ratio of the sense strand to the antisense strand was 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and according to the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is CA, and the base composition of the nucleotide sequence IV is UG; in this case, the length ratio of the sense strand to the antisense strand was 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and according to the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is GCA, and the base composition of the nucleotide sequence IV is UGC; in this case, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is CGCA according to the direction from the 5 'end to the 3' end, and the base composition of the nucleotide sequence IV is UGCG; in this case, the length ratio of the sense strand to the antisense strand was 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and in the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is CA, and the base composition of the nucleotide sequence IV is UG; in this case, the length ratio of the sense strand to the antisense strand was 21/21.

In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are fully complementary in reverse orientation, such that, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are defined.

Second siRNA

According to the present disclosure, the siRNA may be a second siRNA.

The second siRNA comprises a sense strand and an antisense strand, each nucleotide in the second siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 61 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 62 in length and has NO more than 3 nucleotide differences:

5'-CUCAAUAAAGUGCUUUGAZ ₅-3'(SEQ ID NO:61)；

5'-Z ₆UCAAAGCACUUUAUUGAG-3'(SEQ ID NO:62)，

wherein Z is₅Is A, Z₆Is U;

and, the position contained in the nucleotide sequence I corresponds to Z₅Nucleotide Z of₇The nucleotide sequence II comprises a position corresponding to Z₆Nucleotide Z of₈Z is the same as₈Is the first nucleotide at the 5' end of the antisense strand.

In some embodiments, the nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 61, and/or the nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 62.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:62 comprises Z₈A difference at position, and Z₈Selected from A, C or G. In some embodiments, the nucleotide difference is Z₈Difference in position, Z₈Selected from A, C or G. In some embodiments, Z₇Is a reaction of with Z₈A complementary nucleotide. These nucleotide differences do not significantly reduce the target gene inhibition ability of the siRNA conjugates, and the siRNA conjugates comprising the nucleotide differences are also within the scope of the present disclosureWithin.

In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO. 63, nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 64:

5'-CUCAAUAAAGUGCUUUGAZ ₇-3'(SEQ ID NO:63)；

5'-Z ₈UCAAAGCACUUUAUUGAG-3'(SEQ ID NO:64)，

Wherein, Z is₈Is the first nucleotide at the 5' end of the antisense strand, Z₇Selected from A, U, G or C, and Z₈Is a reaction of with Z₇A complementary nucleotide; in some embodiments, Z₇Is A, Z₈Is U;

In some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary with a second nucleotide sequence, and the second nucleotide sequence refers to a nucleotide sequence which is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 61 in the target mRNA and has the same length as the nucleotide sequence IV.

In some embodiments, the length of each of the nucleotide sequence III and the nucleotide sequence IV is 1 nucleotide in the 5'-3' direction, the base of the nucleotide sequence III is a, and the base of the nucleotide sequence IV is U; in this case, the length ratio of the sense strand to the antisense strand was 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, the base composition of the nucleotide sequence III is AA, and the base composition of the nucleotide sequence IV is UU according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is GAA and the base composition of the nucleotide sequence IV is UUC according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is GGAA, and the base composition of the nucleotide sequence IV is UUCC according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and the base composition of the nucleotide sequence III is AA and the base composition of the nucleotide sequence IV is UU in the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21.

Third type of siRNA

According to the present disclosure, the siRNA may be a third siRNA.

The third siRNA comprises a sense strand and an antisense strand, each nucleotide in the third siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 121 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 122 in length and has NO more than 3 nucleotide differences:

5'-GGAGCCCAAGAAAGUGAAZ ₁₃-3'(SEQ ID NO:121)；

5'-Z ₁₄UUCACUUUCUUGGGCUCC-3'(SEQ ID NO:122)，

wherein Z is₁₃Is A, Z₁₄Is U;

and, the position contained in the nucleotide sequence I corresponds to Z₁₃Nucleotide Z of₁₅The nucleotide sequence II comprises a position corresponding to Z₁₄Nucleotide Z of₁₆Z is the same as₁₆Is the first nucleotide at the 5' end of the antisense strand.

In some embodiments, the nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 121, and/or the nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 122.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:122 comprises Z₁₆A difference at position, and Z₁₆Selected from A, C or G. In some embodiments, the nucleotide difference is Z₁₆Difference in position, Z₁₆Selected from A, C or G. In some embodiments, Z₁₅Is a reaction of with Z₁₆A complementary nucleotide. These nucleotide differences do not significantly reduce the target gene inhibition ability of the siRNA conjugates, and siRNA conjugates comprising the nucleotide differences are also within the scope of the present disclosure.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO. 123, nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 124:

5'-GGAGCCCAAGAAAGUGAAZ ₁₅-3'(SEQ ID NO:123)；

5'-Z ₁₆UUCACUUUCUUGGGCUCC-3'(SEQ ID NO:124)，

Wherein, Z is₁₆Is the first nucleotide at the 5' end of the antisense strand, Z₁₅Selected from A, U, G or C, and Z₁₆Is a reaction of with Z₁₅A complementary nucleotide; in some embodiments, Z₁₅Is A, Z₁₆Is U;

In some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary with a second nucleotide sequence which is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 121 in the target mRNA and has the same length as the nucleotide sequence IV.

In some embodiments, the length of each of the nucleotide sequence III and the nucleotide sequence IV is 1 nucleotide, the base of the nucleotide sequence III is U, the base of the nucleotide sequence IV is a; in this case, the length ratio of the sense strand to the antisense strand was 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, the base composition of the nucleotide sequence III is UU and the base composition of the nucleotide sequence IV is AA according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is UUUU and the base composition of the nucleotide sequence IV is AAA according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is GUUU according to the direction from the 5 'end to the 3' end, and the base composition of the nucleotide sequence IV is AAAC; in this case, the length ratio of the sense strand to the antisense strand was 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and the base composition of the nucleotide sequence III is AA and the base composition of the nucleotide sequence IV is UU in the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21.

Fourth siRNA

In accordance with the present disclosure, the siRNA can be a fourth siRNA.

The fifth siRNA comprises a sense strand and an antisense strand, each nucleotide in the fifth siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 181 and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 182 and has NO more than 3 nucleotide differences:

5'-AGCCCAAGAAAGUGAAAGZ ₁₇-3'(SEQ ID NO:181)；

5'-Z ₁₈CUUUCACUUUCUUGGGCU-3'(SEQ ID NO:182)，

wherein Z is₁₇Is A, Z₁₈Is a group of U, and the number of U,

and, the position contained in the nucleotide sequence I corresponds to Z₁₇Nucleotide Z of₁₉The nucleotide sequence II comprises a position corresponding to Z₁₈Nucleotide Z of₂₀Z is the same as₂₀Is the first nucleotide at the 5' end of the antisense strand.

In some embodiments, the nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 181 and/or the nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 182.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:182 comprises Z₂₀A difference at position, and Z₂₀Selected from A, C or G. In some embodiments, the nucleotide difference is Z₂₀Difference in position, Z₂₀Selected from A, C or G. In some embodiments, Z₁₉Is a reaction of with Z₂₀A complementary nucleotide. These nucleotide differences do not significantly reduce the target gene inhibition ability of the siRNA conjugates, and siRNA conjugates comprising the nucleotide differences are also within the scope of the present disclosure.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:183, nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 184:

5'-AGCCCAAGAAAGUGAAAGZ ₁₉-3'(SEQ ID NO:183)；

5'-Z ₂₀CUUUCACUUUCUUGGGCU-3'(SEQ ID NO:184)，

Wherein, Z is₂₀Is the first nucleotide at the 5' end of the antisense strand, Z₁₉Selected from A, U, G or C, and Z₂₀Is a reaction of with Z₁₉A complementary nucleotide; in some embodiments, Z₁₉Is A, Z₂₀Is U;

In some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary with a second nucleotide sequence, and the second nucleotide sequence refers to a nucleotide sequence which is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 181 in the target mRNA and has the same length as the nucleotide sequence IV.

In some embodiments, the length of each of the nucleotide sequence III and the nucleotide sequence IV is 1 nucleotide, the base of the nucleotide sequence III is G, the base of the nucleotide sequence IV is C; in this case, the length ratio of the sense strand to the antisense strand was 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, the base composition of the nucleotide sequence III is GG and the base composition of the nucleotide sequence IV is CC according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is UGG, and the base composition of the nucleotide sequence IV is CCA according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is UUGG according to the direction from the 5 'end to the 3' end, and the base composition of the nucleotide sequence IV is CCAA; in this case, the length ratio of the sense strand to the antisense strand was 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and in the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is GG, and the base composition of the nucleotide sequence IV is CC; in this case, the length ratio of the sense strand to the antisense strand was 21/21.

Fifth siRNA

According to the present disclosure, the siRNA may be a fifth siRNA.

The fifth siRNA comprises a sense strand and an antisense strand, each nucleotide in the fifth siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO:241 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO:242 in length and has NO more than 3 nucleotide differences:

5'-CCAAGAAAGUGAAAGACCZ ₂₁-3'(SEQ ID NO:241)；

5'-Z ₂₂GGUCUUUCACUUUCUUGG-3'(SEQ ID NO:242)，

wherein Z is₂₁Is A, Z₂₂Is U;

the nucleotide sequence I comprises a position corresponding to Z₂₁Nucleotide Z of₂₃The nucleotide sequence II comprises a position corresponding to Z₂₂Nucleotide Z of₂₄Z is the same as₂₄Is the first nucleotide at the 5' end of the antisense strand.

In some embodiments, the nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 241 and/or the nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 242.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:242 comprises Z₂₄A difference at position, and Z₂₄Selected from A, C or G. In some embodiments, the nucleotide difference is Z₂₄Difference in position, Z₂₄Selected from A, C or G. In some embodiments, Z₂₃Is a reaction of with Z₂₄A complementary nucleotide. These nucleotide differences do not significantly reduce the target gene inhibition ability of the siRNA conjugates, and siRNA conjugates comprising the nucleotide differences are also within the scope of the present disclosure.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO. 303, nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 304:

5'-CCAAGAAAGUGAAAGACCZ ₂₃-3'(SEQ ID NO:243)；

5'-Z ₂₄GGUCUUUCACUUUCUUGG-3'(SEQ ID NO:244)，

Wherein, Z is₁₆Is the first nucleotide at the 5' end of the antisense strand, Z₂₃Selected from A, U, G or C, and Z₂₄Is a reaction of with Z₂₃A complementary nucleotide; in some embodiments, Z₂₃Is A, Z₂₄Is U;

In some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary with a second nucleotide sequence, and the second nucleotide sequence refers to a nucleotide sequence which is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 241 and has the same length with the nucleotide sequence IV in the target mRNA.

In some embodiments, the length of each of the nucleotide sequence III and the nucleotide sequence IV is 1 nucleotide, the base of the nucleotide sequence III is C, the base of the nucleotide sequence IV is G; in this case, the length ratio of the sense strand to the antisense strand was 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GC and the base composition of the nucleotide sequence IV is GC according to the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, according to the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is AGC, and the base composition of the nucleotide sequence IV is GCU; in this case, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, according to the direction from 5 'end to 3' end, the base composition of the nucleotide sequence III is GAGC, and the base composition of the nucleotide sequence IV is GCUC; in this case, the length ratio of the sense strand to the antisense strand was 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and the base composition of the nucleotide sequence III is GC and the base composition of the nucleotide sequence IV is GC in the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21.

Pendant end and modification of siRNA

Hereinafter, the description of the nucleotide sequence V, the nucleic acid sequence, the nucleotide modification in the siRNA, and the modified sequence is applicable to any one of the first to fifth sirnas described above. That is, if not specified, the following description of siRNA shall be regarded as that the first siRNA, the second siRNA, the third siRNA, the fourth siRNA and the fifth siRNA are described one by one. For example, the phrase "the siRNA further contains a nucleotide sequence V" means that "the first siRNA, the second siRNA, the third siRNA, the fourth siRNA or the fifth siRNA further contains a nucleotide sequence V" unless a specific siRNA is specifically indicated.

In some embodiments, the sense strand and the antisense strand are different in length, and the antisense strand further comprises a nucleotide sequence V, 1 to 3 nucleotides in length, attached at the 3 'end of the antisense strand to form a 3' overhang of the antisense strand. Thus, the present disclosure provides siRNA sense and antisense strands that can have a length ratio of 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23, 21/24, 22/23, 22/24, 22/25, 23/24, 23/25, or 23/26. In some embodiments, the nucleotide sequence V is 2 nucleotides in length, and thus, the ratio of the lengths of the sense and antisense strands of the sirnas provided by the present disclosure may be 19/21, 21/23, or 23/25.

Each nucleotide in the nucleotide sequence V can be any nucleotide, and for the convenience of synthesis and the saving of synthesis cost, the nucleotide sequence V is continuous 2 thymidylate ribonucleotides (dTdT) or continuous 2 uracil ribonucleotides (UU); alternatively, to increase the affinity of the siRNA antisense strand to the target mRNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. Thus, in some embodiments, the siRNA of the present disclosure has a ratio of the length of the sense strand to the length of the antisense strand of 19/21 or 21/23, where the siRNA of the present disclosure has better mRNA silencing activity.

The nucleotide at the corresponding position of the target mRNA refers to a nucleotide or a nucleotide sequence adjacent to the 5' -end of a nucleotide sequence of the target mRNA, which is substantially reverse-complementary or completely reverse-complementary to the nucleotide sequence II, or a nucleotide sequence consisting of the nucleotide sequence II and the nucleotide sequence IV.

In some embodiments, for the first siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 5 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 6:

5'-GGAACUCAAUAAAGUGCUZ ₃-3'(SEQ ID NO:5)；

5'-Z ₄AGCACUUUAUUGAGUUCCUG-3'(SEQ ID NO:6)；

Or the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 7, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 8:

5'-CAGGAACUCAAUAAAGUGCUZ ₃-3'(SEQ ID NO:7)；

5'-Z ₄AGCACUUUAUUGAGUUCCUGCG-3'(SEQ ID NO:8)；

wherein, Z is₄Is the first nucleotide at the 5' end of the antisense strand, Z₃Selected from A, U, G or C, and Z₄Is a reaction of with Z₃A complementary nucleotide.

In some embodiments, for the second siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 65 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 66:

5'-CUCAAUAAAGUGCUUUGAZ ₇-3'(SEQ ID NO:65)；

5'-Z ₈UCAAAGCACUUUAUUGAGUU-3'(SEQ ID NO:66)，

or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 67, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 68:

5'-AACUCAAUAAAGUGCUUUGAZ ₇-3'(SEQ ID NO:67)；

5'-Z ₈UCAAAGCACUUUAUUGAGUUCC-3'(SEQ ID NO:68)，

wherein, Z is₈Is the first nucleotide at the 5' end of the antisense strand, Z₇Selected from A, U, G or C, and Z₈Is a reaction of with Z₇A complementary nucleotide.

In some embodiments, for the third siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO 125 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO 126:

5'-GGAGCCCAAGAAAGUGAAZ ₁₅-3'(SEQ ID NO:125)；

5'-Z ₁₆UUCACUUUCUUGGGCUCCAA-3'(SEQ ID NO:126)，

or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 127, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 128:

5'-UUGGAGCCCAAGAAAGUGAAZ ₁₅-3'(SEQ ID NO:127)；

5'-Z ₁₆UUCACUUUCUUGGGCUCCAAAC-3'(SEQ ID NO:128)，

Wherein, Z is₁₆Is the first nucleotide at the 5' end of the antisense strand, Z₁₅Selected from A, U, G or C, and Z₁₆Is a reaction of with Z₁₅A complementary nucleotide.

In some embodiments, for the fourth siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 185 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 186:

5'-AGCCCAAGAAAGUGAAAGZ ₁₉-3'(SEQ ID NO:185)；

5'-Z ₂₀CUUUCACUUUCUUGGGCUCC-3'(SEQ ID NO:186)；

or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 187, and the antisense strand contains the nucleotide sequence shown as SEQ ID NO. 188:

5'-GGAGCCCAAGAAAGUGAAAGZ ₁₉-3'(SEQ ID NO:187)；

5'-Z ₂₀CUUUCACUUUCUUGGGCUCCAA-3'(SEQ ID NO:188)；

wherein, Z is₂₀Is the first nucleotide at the 5' end of the antisense strand, Z₁₉Selected from A, U, G or C, and Z₂₀Is a reaction of with Z₁₉A complementary nucleotide.

In some embodiments, for the fifth siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:245 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 246:

5'-CCAAGAAAGUGAAAGACCZ ₂₃-3'(SEQ ID NO:245)；

5'-Z ₂₄GGUCUUUCACUUUCUUGGGC-3'(SEQ ID NO:246)；

or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 247, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 248:

5'-GCCCAAGAAAGUGAAAGACCZ ₂₃-3'(SEQ ID NO:247)；

5'-Z ₂₄GGUCUUUCACUUUCUUGGGCUC-3'(SEQ ID NO:248)；

wherein, Z is₂₄Is the first nucleotide at the 5' end of the antisense strand, Z₂₃Selected from A, U, G or C, and Z ₂₄Is a reaction of with Z₂₃A complementary nucleotide.

In some embodiments, the siRNA of the present disclosure is siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1, or siFXIIf 2:

siFXIIa1

sense strand: 5'-GGAACUCAAUAAAGUGCUU-3' (SEQ ID NO:9)

Antisense strand: 5'-AAGCACUUUAUUGAGUUCCUG-3' (SEQ ID NO:10)

siFXIIa2

Sense strand: 5'-CAGGAACUCAAUAAAGUGCUU-3' (SEQ ID NO:11)

Antisense strand: 5'-AAGCACUUUAUUGAGUUCCUGCG-3' (SEQ ID NO:12)

siFXIIb1

Sense strand: 5'-CUCAAUAAAGUGCUUUGAA-3' (SEQ ID NO:69)

Antisense strand: 5'-UUCAAAGCACUUUAUUGAGUU-3' (SEQ ID NO:70)

siFXIIb2

Sense strand: 5'-AACUCAAUAAAGUGCUUUGAA-3' (SEQ ID NO:71)

Antisense strand: 5'-UUCAAAGCACUUUAUUGAGUUCC-3' (SEQ ID NO: 72).

siFXIId1

Sense strand: 5'-GGAGCCCAAGAAAGUGAAA-3' (SEQ ID NO:129)

Antisense strand: 5'-UUUCACUUUCUUGGGCUCCAA-3' (SEQ ID NO:130)

siFXIId2

Sense strand: 5'-UUGGAGCCCAAGAAAGUGAAA-3' (SEQ ID NO:131)

Antisense strand: 5'-UUUCACUUUCUUGGGCUCCAAAC-3' (SEQ ID NO:132)

siFXIIe1

Sense strand: 5'-AGCCCAAGAAAGUGAAAGA-3' (SEQ ID NO:189)

Antisense strand: 5'-UCUUUCACUUUCUUGGGCUCC-3' (SEQ ID NO:190)

siFXIIe2

Sense strand: 5'-GGAGCCCAAGAAAGUGAAAGA-3' (SEQ ID NO:191)

Antisense strand: 5'-UCUUUCACUUUCUUGGGCUCCAA-3' (SEQ ID NO:192)

siFXIIf1

Sense strand: 5'-CCAAGAAAGUGAAAGACCA-3' (SEQ ID NO:249)

Antisense strand: 5'-UGGUCUUUCACUUUCUUGGGC-3' (SEQ ID NO:250)

siFXIIf2

Sense strand: 5'-GCCCAAGAAAGUGAAAGACCA-3' (SEQ ID NO:251)

Antisense strand: 5'-UGGUCUUUCACUUUCUUGGGCUC-3' (SEQ ID NO:252)

In some embodiments, the siRNA has a nucleotide sequence (i.e., a sequence of nucleobases) set forth in siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1, or siFXIIf 2.

As previously described, the nucleotides in the sirnas of the present disclosure are each independently modified or unmodified nucleotides. In some embodiments, the nucleotides in the sirnas of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the sirnas of the present disclosure are modified nucleotides, and such modifications on the nucleotide groups do not result in a significant impairment or loss of function of the siRNA conjugates of the present disclosure to inhibit FXII gene expression.

In some embodiments, the sirnas of the present disclosure contain at least 1 modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" is used to refer to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is replaced with another group, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotides do not result in significant impairment or loss of the function of the siRNA to inhibit gene expression. For example, one can select the modified nucleotides disclosed in J.K.Watts, G.F.Delevay, and M.J.Damha, chemical modified siRNA: tools and applications.drug discovery Today,2008,13(19-20): 842-55.

In some embodiments, at least one nucleotide in the sense strand or the antisense strand of an siRNA provided by the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate group having a modifying group; in other words, at least a portion of the phosphate groups and/or ribosyl 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 and/or ribosyl groups having a modifying group.

In some embodiments, all of the nucleotides in the sense strand and/or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the sirnas provided by the present disclosure is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

The inventors of the present disclosure surprisingly found that the sirnas described in the present disclosure achieved a high balance of stability in plasma and gene silencing efficiency in animal experiments.

In some embodiments, the fluoro-modified nucleotide is located in nucleotide sequence I and nucleotide sequence II, and the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluoro-modified nucleotides in the direction from the 5 'end to the 3' end; the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II are fluorinated and modified nucleotides according to the direction from the 5 'end to the 3' end.

In some embodiments, the fluoro-modified nucleotide is located in nucleotide sequence I and nucleotide sequence II, the fluoro-modified nucleotide is no more than 5 in the nucleotide sequence I, and the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluoro-modified nucleotides in the direction from the 5 'end to the 3' end; the number of the fluorinated modified nucleotides in the nucleotide sequence II is not more than 7, and the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II are fluorinated modified nucleotides.

In some embodiments, in the direction from the 5 'end to the 3' end, in the sense strand, the 7 th, 8 th, 9 th or 5 th, 7 th, 8 th, 9 th nucleotide of the nucleotide sequence I is a fluorinated modified nucleotide, and the remaining nucleotides in the sense strand are non-fluorinated modified nucleotides; according to the direction from the 5 'end to the 3' end, in the antisense strand, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions or the nucleotides at the 2 nd, 6 th, 8 th, 9 th, 14 th and 16 th positions of the nucleotide sequence II are fluorine-modified nucleotides, and the nucleotides at the rest positions in the antisense strand are non-fluorine-modified nucleotides.

In the context of the present disclosure, "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, which has a structure represented by the following formula (7). "non-fluorinated 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 a non-fluorinated group, or a nucleotide analog. In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group.

The nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group is substituted with a non-fluorine group is known to those skilled in the art, and the nucleotide may be one selected from the group consisting 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.

In some embodiments, the 2 '-alkoxy modified nucleotide is a methoxy modified nucleotide (2' -OMe), as shown in formula (8). In some embodiments, the 2' -substituted alkoxy modified nucleotide, for example, can be a 2' -O-methoxyethyl modified nucleotide (2' -MOE), as shown in formula (9). In some embodiments, 2 '-amino modified nucleotides (2' -NH)₂) As shown in equation (10). In some embodiments, the 2' -Deoxynucleotide (DNA) is according to formula (11):

a nucleotide analog refers to a group that can replace a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, the nucleotide analog can be a heteronucleotide, a bridged nucleotide (BNA for short), or an acyclic nucleotide.

BNA refers to a constrained or inaccessible nucleotide. BNAs may contain five-membered, six-membered, or seven-membered ring bridged structures with "fixed" C3' -endo-sugar pull-down. The bridge is typically incorporated at the 2'-, 4' -position of the ribose to provide a 2',4' -BNA nucleotide. In some embodiments, BNA may be LNA, ENA, cET BNA, etc., where LNA is as shown in equation (12), ENA is as shown in equation (13), and cET BNA is as shown in equation (14):

acyclic nucleotides are a class of nucleotides in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide can be an Unlocked Nucleic Acid (UNA) or a Glycerol Nucleic Acid (GNA), wherein UNA is represented by formula (15) and GNA is represented by formula (16):

in the above formulae (15) and (16), R is selected from H, OH or an alkoxy group (O-alkyl group).

An isonucleotide is a compound formed by changing the position of a base in a nucleotide on a ribose ring. In some embodiments, the isonucleotides can be compounds in which the base moves from the 1' -position to the 2' -position or the 3' -position of the ribose ring, as shown in formula (17) or (18):

in the above-mentioned compounds of formula (7) to formula (18), Base represents a nucleic acid 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 a heteronucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each of the non-fluorinated modified nucleotides is a methoxy modified nucleotide, which refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group, both supra and infra.

In the above and hereinafter, "fluoro-modified nucleotide", "2 '-fluoro-modified nucleotide", "nucleotide in which 2' -hydroxyl group of ribose group is substituted with fluorine" and "nucleotide having 2 '-fluoro-ribosyl group" have the same meaning, and all refer to a compound having a structure represented by formula (7) in which 2' -hydroxyl group of nucleotide is substituted with fluorine; the terms "methoxy-modified nucleotide", "2 '-methoxy-modified nucleotide", "nucleotide in which 2' -hydroxyl group of ribose group is substituted with methoxy group" and "nucleotide having 2 '-methoxy ribosyl group" are the same, and refer to a compound having a structure represented by formula (8) in which 2' -hydroxyl group of ribose group of nucleotide is substituted with methoxy group.

In some embodiments, the siRNA of the present disclosure is an siRNA with the following modifications: in the direction from the 5 'end to the 3' end, in the sense strand, the nucleotides at the 7 th, 8 th and 9 th positions or the 5 th, 7 th, 8 th and 9 th positions of the nucleotide sequence I are fluorine-modified nucleotides, and the nucleotides at the rest positions in the sense strand are methoxy-modified nucleotides; in the antisense strand, the 2 nd, 6 th, 14 th, 16 th or 2 nd, 6 th, 8 th, 9 th, 14 th, 16 th nucleotide of the nucleotide sequence II is a fluoro-modified nucleotide, and the rest nucleotides in the antisense strand are methoxy-modified nucleotides.

In some embodiments, the siRNA of the present disclosure is an siRNA with the following modifications: the nucleotides at the 5 th, 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy-modified nucleotides, and the nucleotides at the 2 nd, 6 th, 8 th, 9 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy-modified nucleotides, in the direction from the 5 'end to the 3' end;

or, according to the direction from 5 'end to 3' end, the nucleotides at the 5 th, 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are fluorine modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy modified nucleotides, and, according to the direction from 5 'end to 3' end, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluorine modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy modified nucleotides;

or, according to the direction from 5 'end to 3' end, the nucleotides at the 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are-fluoro modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy modified nucleotides, and, according to the direction from 5 'end to 3' end, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluoro modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy modified nucleotides.

In some embodiments, the siRNA provided by the present disclosure is any one of siFXIIa-M, siFXIIb-M, siFXIId-M, siFXIIe-M, siFXIIf-M, and siFXIIf-M:

siFXIIa1-M1

sense strand:

5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:13)

antisense strand:

5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO:14)

siFXIIa1-M2

sense strand:

5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:15)

antisense strand:

5'-AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO:16)

siFXIIa1-M3

sense strand:

5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:17)

antisense strand:

5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO:18)

siFXIIa2-M1

sense strand:

5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:19)

antisense strand:

5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO:20)

siFXIIa2-M2

sense strand:

5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:21)

antisense strand:

5'-AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO:22)

siFXIIa2-M3

sense strand:

5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:23)

antisense strand:

5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO:24)

siFXIIb1-M1

sense strand:

5'-CmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:73)

antisense strand:

5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO:74)

siFXIIb1-M2

sense strand:

5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:75)

antisense strand:

5'-UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO:76)

siFXIIb1-M3

sense strand:

5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:77)

antisense strand:

5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO:78)

siFXIIb2-M1

sense strand:

5'-AmAmCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:79)

antisense strand:

5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'

(SEQ ID NO:80)

siFXIIb2-M2

sense strand:

5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:81)

antisense strand:

5'-UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'

(SEQ ID NO:82)

siFXIIb2-M3

sense strand:

5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:83)

antisense strand:

5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'

(SEQ ID NO:84)

siFXIId1-M1

sense strand:

5'-GmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:133)

antisense strand:

5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO:134)

siFXIId1-M2

sense strand:

5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:135)

antisense strand:

5'-UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO:136)

siFXIId1-M3

sense strand:

5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:137)

antisense strand:

5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO:138)

siFXIId2-M1

sense strand:

5'-UmUmGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:139)

antisense strand:

5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'

(SEQ ID NO:140)

siFXIId2-M2

sense strand:

5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:141)

antisense strand:

5'-UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'

(SEQ ID NO:142)

siFXIId2-M3

sense strand:

5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:143)

antisense strand:

5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'

(SEQ ID NO:144)

siFXIIe1-M1

sense strand:

5'-AmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:193)

antisense strand:

5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO:194)

siFXIIe1-M2

sense strand:

5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:195)

antisense strand:

5'-UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO:196)

siFXIIe1-M3

sense strand:

5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:197)

Antisense strand:

5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO:198)

siFXIIe2-M1

sense strand:

5'-GmGmAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:199)

antisense strand:

5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'

(SEQ ID NO:200)

siFXIIe2-M2

sense strand:

5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:201)

antisense strand:

5'-UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'

(SEQ ID NO:202)

siFXIIe2-M3

sense strand:

5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:203)

antisense strand:

5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'

(SEQ ID NO:204)

siFXIIf1-M1

sense strand:

5'-CmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:253)

antisense strand:

5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO:254)

siFXIIf1-M2

sense strand:

5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:255)

antisense strand:

5'-UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO:256)

siFXIIf1-M3

sense strand:

5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:257)

antisense strand:

5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO:258)

siFXIIf2-M1

sense strand:

5'-GmCmCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:259)

antisense strand:

5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'

(SEQ ID NO:260)

siFXIIf2-M2

sense strand:

5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:261)

antisense strand:

5'-UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'

(SEQ ID NO:262)

siFXIIf2-M3

sense strand:

5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:263)

antisense strand:

5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'

(SEQ ID NO:264)

wherein, the capital letters C, G, U, A represent the base composition of nucleotides; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that the nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide.

The modified siRNA is low in cost, and can ensure that ribonuclease in blood does not easily cut nucleic acid, so that the stability of the nucleic acid is improved, and the nucleic acid has stronger resistance to nuclease hydrolysis. Meanwhile, the modification does not significantly reduce the inhibition performance of the siRNA.

In some embodiments, the present disclosure provides sirnas wherein at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands are phosphate groups having a modifying group. In some embodiments, the phosphate group having a modifying group is a phosphorothioate group formed by substituting at least one oxygen atom in a phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, the phosphate group having a modifying group is a phosphorothioate group having a structure as shown in formula (1):

The modification can stabilize the double-stranded structure of siRNA and maintain the high specificity and high affinity of base pairing.

In some embodiments, the present disclosure provides sirnas wherein the phosphorothioate-based linkage is present at least one of the group consisting of: between the first and second nucleotides at either end of the sense or antisense strand; between the second and third nucleotides at either end of the sense or antisense strand; or any combination of the above. In some embodiments, phosphorothioate-based linkages are present at all of the above positions except at the 5' end of the sense strand. In some embodiments, phosphorothioate-based linkages are present at all of the above positions except at the 3' end of the sense strand. In some embodiments, the phosphorothioate-based linkage is present in at least one of the following positions:

between the 1 st and 2 nd nucleotides of the 5' terminus of the sense strand;

between the 2 nd and 3 rd nucleotides at the 5' end of the sense strand;

between the 1 st and 2 nd nucleotides of the 3' terminus of the sense strand;

between the 2 nd and 3 rd nucleotides at the 3' terminus of the sense strand;

between the 1 st and 2 nd nucleotides of the 5' terminus of the antisense strand;

Between the 2 nd and 3 rd nucleotides of the 5' terminus of the antisense strand;

between the 1 st and 2 nd nucleotides of the 3' terminus of the antisense strand; and

between the 2 nd and 3 rd nucleotides of the 3' terminus of the antisense strand.

In some embodiments, the siRNA provided by the present disclosure is any one of siFXIIa-M1, siFXIIa-M2, siFXIIa-M3, siFXIIb-M1, siFXIIb-M2, siFXIIb-M3, siFXIId-M1, siFXIId-M2, siFXIId-M3, siFXIIe-M1, siFXIIe-M2, siFXe-M3, siFXIIf-M1, siFXIIf-IIf-M2, siFXIIf-M3, siFXIIf-M1, siFXIIf-M2, siFXIIf-M3, siFXIIf-M1, siFXIIb-M2, siFXIIb-M3, siFXIIb-M1, siFXIIb-M2, siFXIIb-3, siFXIIb-M2, siFXIIb-M1, siFXIIb-M2, siFXIIb-M2, siFXIIb-3, siFXIIb-M1, siFXIIb-3, siFXIIb-M1, siFXIId-3, siFXIId-M1, siFXIId-M2, siFXIId-M1, siFXIId-M2, siFXIId, siFXIIb-M, siFXIId, siFXIIb, siFXIId, siFXIIb-M2, siFXIIb-M2, siFXIIb-M1, siFXIIb, siFXIId, siFXIIb, siFXIId, si:

siFXIIa1-M1S

sense strand:

5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:25)

antisense strand:

5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO:26)

siFXIIa1-M2S

sense strand:

5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:27)

antisense strand:

5'-AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO:28)

siFXIIa1-M3S

sense strand:

5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:29)

antisense strand:

5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO:30)

siFXIIa2-M1S

sense strand:

5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:31)

antisense strand:

5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO:32)

siFXIIa2-M2S

sense strand:

5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:33)

antisense strand:

5'-AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO:34)

siFXIIa2-M3S

sense strand:

5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:35)

antisense strand:

5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO:36)

siFXIIb1-M1S

sense strand:

5'-CmsUmsCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:85)

antisense strand:

5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO:86)

siFXIIb1-M2S

sense strand:

5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:87)

antisense strand:

5'-UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO:88)

siFXIIb1-M3S

sense strand:

5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:89)

antisense strand:

5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO:90)

siFXIIb2-M1S

sense strand:

5'-AmsAmsCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:91)

antisense strand:

5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'

(SEQ ID NO:92)

siFXIIb2-M2S

sense strand:

5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:93)

antisense strand:

5'-UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'

(SEQ ID NO:94)

siFXIIb2-M3S

Sense strand:

5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:95)

antisense strand:

5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'

(SEQ ID NO:96)

siFXIId1-M1S

sense strand:

5'-GmsGmsAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:145)

antisense strand:

5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'

(SEQ ID NO:146)

siFXIId1-M2S

sense strand:

5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:147)

antisense strand:

5'-UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmsAmsAm-3'

(SEQ ID NO:148)

siFXIId1-M3S

sense strand:

5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:149)

antisense strand:

5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'

(SEQ ID NO:150)

siFXIId2-M1S

sense strand:

5'-UmsUmsGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:151)

antisense strand:

5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'

(SEQ ID NO:152)

siFXIId2-M2S

sense strand:

5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:153)

antisense strand:

5'-UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'

(SEQ ID NO:154)

siFXIId2-M3S

sense strand:

5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:155)

antisense strand:

5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'

(SEQ ID NO:156)

siFXIIe1-M1S

sense strand:

5'-AmsGmsCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:205)

antisense strand:

5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO:206)

siFXIIe1-M2S

sense strand:

5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:207)

antisense strand:

5'-UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO:208)

siFXIIe1-M3S

sense strand:

5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:209)

antisense strand:

5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO:210)

siFXIIe2-M1S

sense strand:

5'-GmsGmsAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:211)

antisense strand:

5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'

(SEQ ID NO:212)

siFXIIe2-M2S

sense strand:

5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:213)

antisense strand:

5'-UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'

(SEQ ID NO:214)

siFXIIe2-M3S

sense strand:

5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:215)

antisense strand:

5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'

(SEQ ID NO:216)

siFXIIf1-M1S

sense strand:

5'-CmsCmsAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:265)

antisense strand:

5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO:266)

siFXIIf1-M2S

sense strand:

5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:267)

antisense strand:

5'-UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO:268)

siFXIIf1-M3S

sense strand:

5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:269)

antisense strand:

5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO:270)

siFXIIf2-M1S

sense strand:

5'-GmsCmsCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:271)

antisense strand:

5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'

(SEQ ID NO:272)

siFXIIf2-M2S

sense strand:

5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:273)

antisense strand:

5'-UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'

(SEQ ID NO:274)

siFXIIf2-M3S

sense strand:

5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:275)

antisense strand:

5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'

(SEQ ID NO:276)

wherein, the capital letters C, G, U, A represent the base composition of nucleotides; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates a phosphorothioate-based linkage between the two nucleotides to the left and right of the letter.

In some embodiments, the 5' terminal nucleotide of the siRNA antisense strand is a 5' -phosphate nucleotide or a 5' -phosphate analog modified nucleotide.

The commonly used 5' -phosphate nucleotide or 5' -phosphate analogue modified nucleotide is well known to those skilled in the art, and for example, the 5' -phosphate nucleotide may have a structure represented by the following formula (2):

For another example, The following 4 5' -phosphate analogue modified nucleotides are disclosed in Anastasia Khvorova and Jonathan K.Watts, The chemical evolution of oligonucleotide therapeutics of clinical utility, Nature Biotechnology,2017,35(3): 238-48:

wherein R is selected from H, OH, methoxy and fluorine; base represents a nucleobase selected from A, U, C, G or T.

In some embodiments, the nucleotide 5 '-phosphate is a nucleotide containing a 5' -phosphate modification represented by formula (2), and the nucleotide 5 '-phosphate analog modification is a nucleotide containing a vinyl phosphate (5' - (E) -vinylphosphonate, E-VP) modification, represented by formula (3), or a phosphorothioate modification, represented by formula (5).

In some embodiments, the siRNA provided by the present disclosure is siFXIIa-M1P, siFXIIa-M2P, siFXIIa-M3P, siFXIIa-M1 SP, siFXIIa-M2 SP, siFXIIa-M3 SP, siFXIIa1-M1P, siIIa 1-M2P, siFXIIa1-M3P, siFXIIa2-M1P, siFXIIa2-M2P, siFXIIa2-M3P, siFXIIa1-M1SP, siFXIIa1-M2SP, siFXIIa2-M1SP, siFXIIa 2-IIa 2-M2SP, siFXIIa 2-IIa 3-M3P, siFXIIa1-M3SP, siFXIIa2-M1SP, siFXIIa2-M3SP, siFXIIa 3-Si 3-IIa, siFXIIa 3-M3-Si 3-IIB, siFXd, siFXIIa 3-Si-IIa, siFXIIB, siFXIIa 3-Si-M3-Si-IIa, siFXIIa, siFXIIB IIB, siFXIIB IIB, siFXIIB IIB, siFXIIB 3-M3, siFXIIB, si, Any one of siFXIId-M2P, siFXIId-M3P, siFXIId-M1 SP, siFXIId-M2 SP, siFXIId-M3 SP, siFXIIe-M1P, siFXIIe-M2P, siFXIIe-M3P, siFXIIe-M1 SP, siFXIIe-M2 SP, siFXe-M3 SP, siFXIIe-M1 SP, siFXIIe-M2 SP, siFXIIe-M3 SP, siFXIIf-M1 SP, siFXIIf-M2P, siFXIIf-M3P, siFXIIf-M1 SP, siFXIIf-M2 SP, siFXIIf-M3 SP, siFXIIf-Si-M1 SP, siFXIIf-M3 SP, siFXIIf-Si-M2 SP, siFXIIf-M3 SP, siFXIIf-Si-M1 SP, siFXIIf-M2 SP, siFXIIf-M3 SP, siFXIIf-Si-M2 SP, siFXIIf-Si-M3 SP, siFXIIf-Si-M2 SP, siFXIIf-M3 SP, siFXIIf-Si-3 SP, Si-M2 SP, Si-3 SP, Si-3 SP, Si-M2 SP, Si-M2 SP:

siFXIIa1-M1P1

Sense strand:

5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:37)

antisense strand:

5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO:38)

siFXIIa1-M2P1

sense strand:

5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:39)

antisense strand:

5'-P1AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO:40)

siFXIIa1-M3P1

sense strand:

5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:41)

antisense strand:

5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO:42)

siFXIIa2-M1P1

sense strand:

5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:43)

antisense strand:

5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO:44)

siFXIIa2-M2P1

sense strand:

5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:45)

antisense strand:

5'-P1AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO:46)

siFXIIa2-M3P1

sense strand:

5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:47)

antisense strand:

5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO:48)

siFXIIa1-M1SP1

sense strand:

5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:49)

antisense strand:

5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO:50)

siFXIIa1-M2SP1

sense strand:

5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:51)

antisense strand:

5'-P1AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO:52)

siFXIIa1-M3SP1

sense strand:

5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:53)

antisense strand:

5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO:54)

siFXIIa2-M1SP1

sense strand:

5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:55)

antisense strand:

5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO:56)

siFXIIa2-M2SP1

sense strand:

5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:57)

antisense strand:

5'-P1AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO:58)

siFXIIa2-M3SP1

sense strand:

5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:59)

antisense strand:

5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO:60)

siFXIIa1U-M1P1

sense strand:

5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:335)

antisense strand:

5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO:336)

siFXIIa1U-M2P1

sense strand:

5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:337)

antisense strand:

5'-P1UmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO:338)

siFXIIa1U-M3P1

sense strand:

5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:339)

antisense strand:

5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO:340)

siFXIIa2U-M1P1

sense strand:

5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:341)

antisense strand:

5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO:342)

siFXIIa2U-M2P1

sense strand:

5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:343)

antisense strand:

5'-P1UmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO:344)

siFXIIa2U-M3P1

sense strand:

5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:345)

antisense strand:

5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO:346)

siFXIIa1U-M1SP1

sense strand:

5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:347)

antisense strand:

5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO:348)

siFXIIa1U-M2SP1

sense strand:

5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:349)

antisense strand:

5'-P1UmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO:350)

siFXIIa1U-M3SP1

sense strand:

5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:351)

antisense strand:

5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO:352)

siFXIIa2U-M1SP1

sense strand:

5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:353)

antisense strand:

5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO:354)

siFXIIa2U-M2SP1

sense strand:

5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:355)

antisense strand:

5'-P1UmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO:356)

siFXIIa2U-M3SP1

sense strand:

5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:357)

antisense strand:

5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO:358)

siFXIIb1-M1P1

sense strand:

5'-CmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:97)

antisense strand:

5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO:98)

siFXIIb1-M2P1

sense strand:

5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:99)

antisense strand:

5'-P1UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO:100)

siFXIIb1-M3P1

sense strand:

5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:101)

antisense strand:

5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO:102)

siFXIIb2-M1P1

sense strand:

5'-AmAmCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:103)

antisense strand:

5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'

(SEQ ID NO:104)

siFXIIb2-M2P1

sense strand:

5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:105)

antisense strand:

5'-P1UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'

(SEQ ID NO:106)

siFXIIb2-M3P1

sense strand:

5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:107)

antisense strand:

5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'

(SEQ ID NO:108)

siFXIIb1-M1SP1

sense strand:

5'-CmsUmsCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:109)

antisense strand:

5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO:110)

siFXIIb1-M2SP1

sense strand:

5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:111)

antisense strand:

5'-P1UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO:112)

siFXIIb1-M3SP1

sense strand:

5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:113)

antisense strand:

5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO:114)

siFXIIb2-M1SP1

sense strand:

5'-AmsAmsCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:115)

antisense strand:

5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'

(SEQ ID NO:116)

SiFXIIb2-M2SP1

sense strand:

5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:117)

antisense strand:

5'-P1UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'

(SEQ ID NO:118)

SiFXIIb2-M3SP1

sense strand:

5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:119)

antisense strand:

5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'

(SEQ ID NO:120)

siFXIId1-M1P1

sense strand:

5'-GmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:157)

antisense strand:

5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO:158)

siFXIId1-M2P1

sense strand:

5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:159)

antisense strand:

5'-P1UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO:160)

siFXIId1-M3P1

sense strand:

5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:161)

antisense strand:

5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO:162)

siFXIId2-M1P1

sense strand:

5'-UmUmGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:163)

Antisense strand:

5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'

siFXIId2-M2P1

sense strand:

5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:165)

antisense strand:

5'-P1UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'

(SEQ ID NO:166)

siFXIId2-M3P1

sense strand:

5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:167)

antisense strand:

5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'

(SEQ ID NO:168)

siFXIId1-M1SP1

sense strand:

5'-GmsGmsAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:169)

antisense strand:

5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'

(SEQ ID NO:170)

siFXIId1-M2SP1

sense strand:

5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:171)

antisense strand:

5'-P1UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmsAmsAm-3'

(SEQ ID NO:172)

siFXIId1-M3SP1

sense strand:

5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:173)

antisense strand:

5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'

(SEQ ID NO:174)

siFXIId2-M1SP1

sense strand:

5'-UmsUmsGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:175)

antisense strand:

5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'

(SEQ ID NO:176)

siFXIId2-M2SP1

sense strand:

5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:177)

antisense strand:

5'-P1UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'

(SEQ ID NO:178)

siFXIId2-M3SP1

sense strand:

5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:179)

antisense strand:

5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'

(SEQ ID NO:180)

siFXIIe1-M1P1

sense strand:

5'-AmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:217)

antisense strand:

5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO:218)

siFXIIe1-M2P1

sense strand:

5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:219)

antisense strand:

5'-P1UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO:220)

siFXIIe1-M3P1

sense strand:

5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:221)

antisense strand:

5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO:222)

siFXIIe2-M1P1

sense strand:

5'-GmGmAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:223)

antisense strand:

5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'

(SEQ ID NO:224)

siFXIIe2-M2P1

sense strand:

5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:225)

antisense strand:

5'-P1UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'

(SEQ ID NO:226)

siFXIIe2-M3P1

sense strand:

5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:227)

antisense strand:

5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'

(SEQ ID NO:228)

siFXIIe1-M1SP1

sense strand:

5'-AmsGmsCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:229)

antisense strand:

5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO:230)

siFXIIe1-M2SP1

sense strand:

5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:231)

antisense strand:

5'-P1UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO:232)

siFXIIe1-M3SP1

sense strand:

5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:233)

antisense strand:

5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO:234)

siFXIIe2-M1SP1

sense strand:

5'-GmsGmsAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:235)

antisense strand:

5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'

(SEQ ID NO:236)

siFXIIe2-M2SP1

sense strand:

5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:237)

antisense strand:

5'-P1UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'

(SEQ ID NO:238)

siFXIIe2-M3SP1

sense strand:

5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:239)

antisense strand:

5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'

(SEQ ID NO:240)

siFXIIf1-M1P1

sense strand:

5'-CmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:277)

antisense strand:

5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO:278)

siFXIIf1-M2P1

sense strand:

5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:279)

antisense strand:

5'-P1UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO:280)

siFXIIf1-M3P1

sense strand:

5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:281)

antisense strand:

5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO:282)

siFXIIf2-M1P1

sense strand:

5'-GmCmCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:283)

antisense strand:

5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'

(SEQ ID NO:284)

siFXIIf2-M2P1

sense strand:

5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:285)

antisense strand:

5'-P1UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'

(SEQ ID NO:286)

siFXIIf2-M3P1

sense strand:

5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:287)

antisense strand:

5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'

(SEQ ID NO:288)

siFXIIf1-M1SP1

sense strand:

5'-CmsCmsAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:289)

antisense strand:

5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO:290)

siFXIIf1-M2SP1

sense strand:

5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:291)

antisense strand:

5'-P1UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO:292)

siFXIIf1-M3SP1

sense strand:

5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:293)

antisense strand:

5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO:294)

siFXIIf2-M1SP1

sense strand:

5'-GmsCmsCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:295)

antisense strand:

5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'

(SEQ ID NO:296)

siFXIIf2-M2SP1

sense strand:

5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:297)

antisense strand:

5'-P1UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'

(SEQ ID NO:298)

siFXIIf2-M3SP1

sense strand:

5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:299)

antisense strand:

5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'

(SEQ ID NO:300)

wherein, the capital letters C, G, U, A represent the base composition of nucleotides; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates a phosphorothioate-based linkage between the two nucleotides to the left and right of the letter; p1 indicates that the adjacent nucleotide to the right of P1 is a 5 '-phosphate nucleotide or a 5' -phosphate analogue modified nucleotide. In some embodiments, P1 is a VP, Ps, or P that represents a particular modification, wherein a letter combination VP represents that the adjacent nucleotide to the right of the letter combination VP is a vinyl phosphate (5'- (E) -vinylphosphonate, E-VP) modified nucleotide, a letter combination Ps represents that the adjacent nucleotide to the right of the letter combination Ps is a phosphorothioate modified nucleotide, and an uppercase letter P represents that the adjacent nucleotide to the right of the letter P is a 5' -phosphate nucleotide.

The inventors of the present disclosure have surprisingly found that the sirnas provided by the present disclosure not only have significantly enhanced plasma and lysosomal stability, but also retain very high gene suppression activity.

The siRNA provided by the present disclosure can be obtained by methods conventional in the art for siRNA preparation, such as methods of solid phase synthesis and solution phase synthesis. Among them, solid phase synthesis has been commercially available as a custom service. Modified nucleotide groups can be introduced into the sirnas described in the present disclosure by using nucleotide monomers with corresponding modifications, and methods of preparing nucleotide monomers with corresponding modifications and methods of introducing modified nucleotide groups into sirnas are also well known to those skilled in the art.

Pharmaceutical composition

The present disclosure provides a pharmaceutical composition comprising the siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier can be a carrier conventionally used in the art of siRNA administration, such as, but not limited to, magnetic nanoparticles (e.g., Fe-based)₃O ₄Or Fe₂O ₃Nanoparticles of (a), carbon nanotubes (carbon nanotubes), mesoporous silicon (mesopore silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), Polyethyleneimine (PEI), polyamidoamine (pamam) dendrimer), polylysine (L-lysine), PLL), chitosan (chitosan), 1, 2-dioleoyl-3-trimethyolpropane (1, 2-dioleoyl-3-trimethyoronium-propane, DOTAP), poly-D or L-type lactic acid/glycolic acid copolymer (D) glycolic acid copolymer (PEI) &L-lactic/glycolic acid) copolymer, PLGA), poly (aminoethyl ethylene phosphate) (poly (2-aminoethylene phosphate),PPEEA) and poly (N, N-dimethylaminoethyl methacrylate) (poly (2-dimethylamino methyl methacrylate), PDMAEMA) and one or more of their derivatives.

In some embodiments, the content of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly required, and in some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier may be 1 (1-500), and in some embodiments, the above 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 protective agent, and an osmotic pressure regulator.

The pH buffer may be a tris hydrochloride buffer at a pH of 7.5 to 8.5 and/or a phosphate buffer at a pH of 5.5 to 8.5, for example a phosphate buffer at a pH of 5.5 to 8.5.

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

The osmotic pressure regulator may be sodium chloride and/or potassium chloride. The content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmol/kilogram (mOsm/kg). The content of the osmolality adjusting agent can be easily determined by the skilled person, depending on the desired osmolality.

In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection solution; or can be lyophilized powder for injection, and can be mixed with liquid adjuvant to make into liquid preparation. The liquid preparation can be used for subcutaneous, intramuscular or intravenous injection, and can also be used for spraying administration to the lung or spraying administration to other organ tissues (such as 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 liposome 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, helper lipid, and pegylated lipid may be selected from one or more of the amine-containing transfection compounds described in CN103380113A (herein incorporated by reference in its entirety), or a pharmaceutically acceptable salt or derivative thereof, helper lipid, and pegylated lipid, respectively.

In some embodiments, the organic amine may be a compound described in CN103380113A as shown in formula (201) or a pharmaceutically acceptable salt thereof:

wherein:

each X₁₀₁And X₁₀₂Each independently O, S, N-A or C-A, wherein A is hydrogen or a C1-C20 hydrocarbon chain;

each Y₁₀₁And Z₁₀₁Each independently is C O, C S, S O, CH OH or SO₂；

Each R₁₀₁、R ₁₀₂、R ₁₀₃、R ₁₀₄、R ₁₀₅、R ₁₀₆And R₁₀₇Each independently is hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or linear aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic group, a substituted or unsubstituted, branched or linear acyl group, a substituted or unsubstituted, branched or linear aryl group, a substituted or unsubstituted, branched or linear heteroaryl group;

x is an integer from 1 to 10;

n is an integer of 1 to 3, m is an integer of 0 to 20, p is 0 or 1; wherein if m ═ p ═ 0, then R₁₀₂Is hydrogen;

and, if at least one of n or m is 2, then R₁₀₃And the nitrogen in formula (201) forms a structure as shown in formula (202) or formula (203):

wherein g, e and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each xn represents a nitrogen atom in formula (201).

In some embodiments, R₁₀₃Is a polyamine. In other embodiments, R₁₀₃Is a ketal. In some embodiments, R in formula (201)₁₀₁And R₁₀₂Each of which 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, then R₁₀₃May be any of the following formulae (204) to (213):

wherein, in formula (204) -formula (213), g, e and f are each independently an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, and each indicates R₁₀₃A possible point of attachment to the nitrogen atom in formula (201), wherein each H at any x position may be replaced to achieve attachment to the nitrogen atom in formula (201).

Among them, the compound represented by formula (201) can be prepared according to the description in CN 103380113A.

In some embodiments, the organic amine is an organic amine according to formula (214) and/or an organic amine according to formula (215):

the helper lipid is cholesterol, cholesterol analogue and/or cholesterol derivative;

The pegylated lipid is 1, 2-dipalmitoamide-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, (50-70): (20-40): (3-20).

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

In some embodiments, the weight ratio (weight/weight ratio) of siRNA to total lipid (e.g., organic amine, helper lipid, and/or pegylated lipid) in the pharmaceutical composition formed from siRNA of the present disclosure and the above-described amine-containing transfection reagent is in a range 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, for example, the weight ratio of siRNA 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 1: 18.

In some embodiments, the pharmaceutical compositions may be sold with the components present separately and may be in the form of a liquid formulation for use. In some embodiments, the pharmaceutical composition of the siRNA provided by the present disclosure and the above pharmaceutically acceptable carrier can be prepared according to various known methods, except that the siRNA provided by the present disclosure is used to replace the existing siRNA; in some embodiments, the following methods may be used:

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

The siRNA provided by the present disclosure is dissolved in a buffered salt solution to obtain an siRNA aqueous solution. The concentration of the buffered salt solution is 0.05-0.5M, such as 0.1-0.2M, the pH of the buffered salt solution is adjusted to 4.0-5.5, such as 5.0-5.2, and the amount of buffered salt solution is such that the concentration of siRNA does not exceed 0.6mg/mL, such as 0.2-0.4 mg/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.

The lipid solution and the aqueous siRNA solution are mixed, and the resulting mixture is incubated at 40-60 ℃ for at least 2 minutes, which may be, for example, 5-30 minutes, to obtain a post-incubation liposome preparation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1: (2-5).

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

Wherein the concentration or dilution may be performed before, after or simultaneously with the removal of the impurities. The impurities can be removed by various methods, such as ultrafiltration using a cut-phase flow system and a hollow fiber column under 100K Da conditions, and the ultrafiltration exchange solution is Phosphate Buffered Saline (PBS) with pH 7.4. The sterilization can be carried out by various methods, for example, by filtration sterilization on a 0.22 μm filter.

siRNA conjugates

The present disclosure provides an siRNA conjugate comprising the above siRNA and a conjugate group conjugated to the siRNA.

Generally, the conjugate group comprises at least one targeting group that is pharmaceutically acceptable and optionally a linker (linker), and the siRNA, the linker and the targeting group are linked in sequence. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The siRNA 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 siRNA to the conjugate group may be at the 3' end or 5' end of the sense strand of the siRNA, or at the 5' end of the antisense strand, or within the internal sequence of the siRNA. In some embodiments, the site of conjugation of the siRNA to the conjugate group is at the 3' end of the sense strand of the siRNA.

In some embodiments, the conjugate group may be attached to the phosphate group, the hydroxyl group at the 2' -position, or the base of the nucleotide. In some embodiments, the conjugate group may also be attached to the hydroxyl group at the 3' -position, in which case 2' -5' phosphodiester linkages are used between nucleotides. When a conjugate group is attached to the end of the siRNA 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 the siRNA, the conjugate group is typically attached to a ribose sugar ring or base. Various ways of attachment can be found in the literature: siRNA conjugates and subsequent assembled tertiary N-acetyl amino acids in vivo in contexts ACS Chemical biology 2015,10(5):1181-7.

In some embodiments, the siRNA may be attached to the conjugate group via acid labile, or reducible, chemical bonds that may degrade under the acidic environment of the cellular endosome, thereby leaving the siRNA in a free state. For non-degradable conjugation, a conjugation group can be attached to the sense strand of the siRNA, thereby minimizing the effect of conjugation on siRNA activity.

In some embodiments, the pharmaceutically acceptable 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, the pharmaceutically acceptable targeting group may be selected from one or more of the following ligands formed by targeting molecules or derivatives thereof: lipophilic molecules such as cholesterol, bile acids, vitamins (e.g. vitamin E), lipid molecules of varying chain length; polymers, such as polyethylene glycol; polypeptides, such as membrane-penetrating peptides; an aptamer; an antibody; quantum dots; sugars such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); ligands for receptors expressed by parenchymal hepatocytes, such as asialoglycoprotein, asialoglycoresidues, lipoproteins (e.g., high density lipoproteins, low density lipoproteins, etc.), glucagon, neurotransmitters (e.g., epinephrine), growth factors, transferrin, and the like.

In some embodiments, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to the liver surface asialoglycoprotein receptor (ASGPR). These ligand classes are known to those skilled in the art and generally function to bind to specific receptors on the surface of target cells and mediate the delivery of siRNA linked to the ligand to the target cell.

In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to asialoglycoprotein receptor (ASGPR) on the surface of a mammalian liver cell. In some embodiments, each ligand is independently an asialoglycoprotein, such as asialo-orosomucoid (ASOR) or asialo-fetuin (ASF). In some embodiments, the ligand is a sugar or a derivative of a sugar.

In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, disaccharide or trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. In some embodiments, each ligand is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or a monosaccharide derivative. In some embodiments, each or at least one ligand is selected from the group consisting of: glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives and sialic acid.

In some embodiments, each of the ligands can be independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannofuranose, beta-D-mannofuranose, alpha-D-mannopyranose, beta-D-mannopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-fructosyl, beta-fructooligosaccharides, and beta-fructooligosaccharides, and beta-fructooligosaccharides, beta-fructooligosaccharides, beta-fructooligosaccharides, and beta-fructooligosaccharides, beta-fructooligosaccharides, and beta-fructooligosaccharides, beta-fructooligosaccharides, beta-fructooligosaccharides, and beta-fructooligosaccharides, beta-fructooligosaccharides, beta-fructooligosaccharides, beta-fructooligosaccharides, and, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [ (R) -1-carboxyethyl ] -2-deoxy-beta-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, beta-galactofuranose, glucosamine, N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [ (R) -1-carboxyethyl ] -2-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4-dideoxy-4-carboxamido-2, 3-O-methyl-D-mannopyranose, D-glucopyranose, beta-galactopyranose, and, N-glycolyl-alpha-neuraminic acid, 5-thio-beta-D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranoside methyl ester, 4-thio-beta-D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio-alpha-D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allositrile, ribose, D-4-thioribose, L-ribose or L-4-thioribose. Other options for such ligands can be found, for example, in the disclosure of CN105378082A, the entire disclosure of which is incorporated herein by reference.

In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent values as described herein refer to the molar ratio of the siRNA group to the galactose or N-acetylgalactosamine group in the siRNA conjugate after the siRNA molecule and the conjugate molecule containing the galactose or N-acetylgalactosamine group as the targeting group form the siRNA conjugate, respectively, of 1:1, 1:2, 1:3, or 1: 4. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described in the present disclosure is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA of the present disclosure is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

The targeting group can be attached to the siRNA molecule via a suitable linker, which one skilled in the art can select depending on the particular type of targeting group. The identity of these linkers, targeting groups, and the manner of attachment to the siRNA can be found in the disclosure of WO2015006740a2, which is incorporated by reference herein in its entirety.

In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be of the structure shown in formula (301):

wherein the content of the first and second substances,

k is an integer of 1 to 3;

L ^Ais a chain part containing amido bond with the structure as shown in formula (302), and each L^AWith one of said targeting groups and said L at each end thereof^CThe moieties are linked by an ether linkage:

L ^Bis a chain part containing N-acyl pyrrolidine with a structure shown as a formula (303), wherein the chain part has carbonyl at one end and is connected with the L^CPart is connected through amido bond, the other end has oxygen group and is connected with the siRNA through phosphate bond:

L ^Cis a 2-4 valent linking group based on hydroxymethylaminomethane, dimethylolaminomethane or trimethylolpropane, said L^CVia an oxygen atom with each of said L^AThe moieties being linked by an ether bond and being linked to the L via a nitrogen atom ^BThe moieties are linked by amide bonds.

In some embodiments, when n is 3, L^CIn the case of a 4-valent linking group based on tris (hydroxymethyl) aminomethane, the linker is composed of^A) ₃Tris-hydroxymethyl aminomethane-L^B-linking the N-acetylgalactosamine molecule and the siRNA molecule to form an siRNA conjugate, which has the following structure (304):

In the formula, the double helix structure represents siRNA.

Similarly, the conjugation site of the siRNA to the conjugate group can be at the 3' end or 5' end of the sense strand of the siRNA, also at the 5' end of the antisense strand, and also in the internal sequence of the siRNA.

In some embodiments, the 3' end of the sense strand of the sirnas of the present disclosure is linked to the sense strand of the siRNA through a linker- (L)^A) ₃Tris-hydroxymethyl aminomethane-L^B-covalent conjugation with three molecules of N-acetylgalactosamine (GalNAc) to obtain a siRNA conjugate with a molar ratio of siRNA molecule to GalNAc molecule of 1:3, hereinafter also referred to as (GalNAc)₃-siRNA, having the structure represented by the following formula (305):

wherein the double helix structure represents the siRNA and the linker is attached to the 3' end of the sense strand of the siRNA.

In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be of the structure shown in formula (306):

wherein the content of the first and second substances,

l is an integer of 0 to 3;

^*represents a site on the linker attached to the targeting group by an ether linkage;

^#indicates the site on the linker to which the siRNA is attached via a phosphoester bond.

In some embodiments, when l ═ 2, the siRNA conjugate has the structure shown in formula (307):

The above conjugates can be synthesized by methods that have been described in detail in the prior art. For example, methods for the preparation of various conjugates are 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. As a method for preparing the structure of formula (305) is described in WO2014025805A1, Rajeev et al in ChemBiochem 2015,16,903-908 describe the structure of formula (307).

In some embodiments, the siRNA conjugate has a structure as shown in formula (308):

wherein:

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

each m1, m2 and m3 is independently an integer selected from 2 to 10;

each R₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄And R ₁₅Each independently is H, or is selected from the group consisting of: c₁-C ₁₀Alkyl radical, C₁-C ₁₀Haloalkyl and C₁-C ₁₀An alkoxy group;

R ₃a group of the structure shown in formula a 59:

wherein E is₁Is OH, SH or BH₂Nu is the siRNA of the present disclosure described previously;

R ₂is a straight chain alkylene group of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH ═ N, S (O) ₂、C ₂-C ₁₀Alkenylene radical, C₂-C ₁₀Alkynylene, C₆-C ₁₀Arylene radical, C₃-C ₁₈Heterocyclylene and C₅-C ₁₀A heteroarylene group; and wherein R₂May optionally have a substituent of any one or more of the group consisting of: c₁-C ₁₀Alkyl radical, C₆-C ₁₀Aryl radical, C₅-C ₁₀Heteroaryl group, C₁-C ₁₀Haloalkyl, -OC₁-C ₁₀Alkyl, -OC₁-C ₁₀Alkylphenyl, -C₁-C ₁₀alkyl-OH, -OC₁-C ₁₀Haloalkyl, -SC₁-C ₁₀Alkyl, -SC₁-C ₁₀Alkylphenyl, -C₁-C ₁₀alkyl-SH, -SC₁-C ₁₀Haloalkyl, halogen substituents, -OH, -SH, -NH₂、-C ₁-C ₁₀alkyl-NH₂、-N(C ₁-C ₁₀Alkyl) (C₁-C ₁₀Alkyl), -NH (C)₁-C ₁₀Alkyl), -N (C)₁-C ₁₀Alkyl) (C₁-C ₁₀Alkylphenyl), -NH (C)₁-C ₁₀Alkylphenyl), cyano, nitro, -CO₂H、-C(O)O(C ₁-C ₁₀Alkyl), -CON (C)₁-C ₁₀Alkyl) (C₁-C ₁₀Alkyl), -CONH (C)₁-C ₁₀Alkyl), -CONH₂，-NHC(O)(C ₁-C ₁₀Alkyl), -NHC (O) (phenyl), -N (C)₁-C ₁₀Alkyl radical C (O) (C)₁-C ₁₀Alkyl), -N (C)₁-C ₁₀Alkyl group C (O) (phenyl), -C (O) C₁-C ₁₀Alkyl, -C (O) C₁-C ₁₀Alkylphenyl, -C (O) C₁-C ₁₀Haloalkyl, -OC (O) C₁-C ₁₀Alkyl, -SO₂(C ₁-C ₁₀Alkyl), -SO₂(phenyl), -SO₂(C ₁-C ₁₀Haloalkyl), -SO₂NH ₂、-SO ₂NH(C ₁-C ₁₀Alkyl), -SO₂NH (phenyl), -NHSO₂(C ₁-C ₁₀Alkyl), -NHSO₂(phenyl) and-NHSO₂(C ₁-C ₁₀Haloalkyl);

each L₁Is a straight chain alkylene group of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH ═ N, S (O) ₂、C ₂-C ₁₀Alkenylene radical, C₂-C ₁₀Alkynylene, C₆-C ₁₀Arylene radical, C₃-C ₁₈Heterocyclylene and C₅-C ₁₀A heteroarylene group; and wherein L₁May optionally have a substituent of any one or more of the group consisting of: c₁-C ₁₀Alkyl radical, C₆-C ₁₀Aryl radical, C₅-C ₁₀Heteroaryl group, C₁-C ₁₀Haloalkyl, -OC₁-C ₁₀Alkyl, -OC₁-C ₁₀Alkylphenyl, -C₁-C ₁₀alkyl-OH, -OC₁-C ₁₀Haloalkyl, -SC₁-C ₁₀Alkyl, -SC₁-C ₁₀Alkylphenyl, -C₁-C ₁₀alkyl-SH, -SC₁-C ₁₀Haloalkyl, halogen substituents, -OH, -SH, -NH₂、-C ₁-C ₁₀alkyl-NH₂、-N(C ₁-C ₁₀Alkyl) (C₁-C ₁₀Alkyl), -NH (C)₁-C ₁₀Alkyl), -N (C)₁-C ₁₀Alkyl) (C₁-C ₁₀Alkylphenyl), -NH (C)₁-C ₁₀Alkylphenyl), cyano, nitro, -CO₂H、-C(O)O(C ₁-C ₁₀Alkyl), -CON (C)₁-C ₁₀Alkyl) (C₁-C ₁₀Alkyl), -CONH (C)₁-C ₁₀Alkyl), -CONH₂，-NHC(O)(C ₁-C ₁₀Alkyl), -NHC (O) (phenyl), -N (C)₁-C ₁₀Alkyl radical C (O) (C)₁-C ₁₀Alkyl), -N (C)₁-C ₁₀Alkyl group C (O) (phenyl), -C (O) C₁-C ₁₀Alkyl, -C (O) C₁-C ₁₀Alkylphenyl, -C(O)C ₁-C ₁₀Haloalkyl, -OC (O) C₁-C ₁₀Alkyl, -SO₂(C ₁-C ₁₀Alkyl), -SO₂(phenyl), -SO₂(C ₁-C ₁₀Haloalkyl), -SO₂NH ₂、-SO ₂NH(C ₁-C ₁₀Alkyl), -SO₂NH (phenyl), -NHSO₂(C ₁-C ₁₀Alkyl), -NHSO₂(phenyl) and-NHSO₂(C ₁-C ₁₀Haloalkyl).

In some embodiments, L₁Can be selected from the group consisting of A1-A26 groups or any combination thereof, wherein the structures and definitions of A1-A26 are shown below:

wherein j1 is an integer from 1 to 20; j2 is an integer from 1 to 20;

R' is C₁-C ₁₀An alkyl group;

ra is selected from the group consisting of groups of formula A27-A45:

rb is C₁-C ₁₀An alkyl group;

indicates the site at which the group is covalently attached.

The skilled person will understand that although for convenience L is used₁Is defined as a linear alkylene group, but it may not be a linear group or differ in name, for example, by an amine or an alkenyl group resulting from the above substitution and/or displacement. For purposes of this disclosure, L₁Is the number of atoms in the chain connecting the two points of attachment. For this purpose, a ring (e.g., a heterocyclylene or heteroarylene) obtained by substituting a carbon atom of the linear alkylene group is counted as one atom.

M ₁Refers to targeting groups, which are defined and alternative to the same scope as the targeting groups described above. In some embodiments, each M is₁Independently selected from one of the ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells.

When M is₁In the case of ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells, n1 can be an integer from 1 to 3 and n3 can be an integer from 0 to 4 in some embodiments, providing that M is an integer from 0 to 4 in the conjugate₁The number of targeting groups is at least 2; in some embodiments, n1+ n3 ≧ 2, which can result in M ₁The number of targeting groups is at least 3, such that M₁The targeting group binds more readily to the hepatic surface asialoglycoprotein receptor, thereby facilitating entry of the conjugate into cells by endocytosis. Experiments show that when M is used₁When the number of targeting groups is more than 3, M₁The increased ease of binding of the targeting group 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 is 2 to 3, all taken together from the aspects of ease of synthesis, structure/process cost, and delivery efficiency.

In some embodiments, when each of M1, M2, and M3 is independently selected from an integer of 2 to 10, a plurality of M may be used₁Spatial position between targeting groups is adapted to M₁In order to make the conjugates provided by the present disclosure simpler, easier to synthesize, and/or less costly, the binding of the targeting group to the liver surface asialoglycoprotein receptor, in some embodiments each of m1, m2, and m3 is independently an integer from 2 to 5, in some embodiments, m1 ═ m2 ═ m 3.

As will be understood by those skilled in the art, when each R is₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄And R₁₅Each independently selected from H, C₁-C ₁₀Alkyl radical, C₁-C ₁₀Haloalkyl, and C ₁-C ₁₀One of the alkoxy groups, without altering the properties of the conjugates of the present disclosure, can achieve the objectives of the present disclosure. In some embodiments, each R is₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄And R₁₅Each independently selected from H, methyl and ethyl. In some embodiments, each R is₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄And R₁₅Are all H.

R ₃A group of the structure shown as formula A59, wherein E₁Is OH, SH or BH₂In some embodiments, E is based on considerations of ready availability of starting materials for preparation₁Is OH or SH.

R ₂Is selected to effect attachment to the N atom of the nitrogen-containing backbone to a 59. In the context of the present disclosure, "nitrogen-containing backbone" means a linkage with R₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄And R₁₅A chain structure in which carbon atoms and N atoms are linked to each other. Thus, R₂Can be any ofA linking group linking the a59 group to an N atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, where the siRNA conjugate represented by formula (308) is prepared by a process of solid phase synthesis, R is₂The group desirably contains both a linking site to the N atom of the nitrogen-containing skeleton and a linking site to R₃The P atom in (a) to which the linking site is attached. In some embodiments, R₂Wherein the site attached to the N atom of the nitrogen-containing backbone forms an amide bond with N, said amide bond with R ₃The site to which the P atom is attached forms a phosphoester bond with the P atom; in some embodiments, R₂May be B5, B6, B5 'or B6':

wherein the content of the first and second substances,

indicating the site of covalent attachment of the group.

q ₂Can be an integer from 1 to 10, and in some embodiments, q is₂Is an integer of 1 to 5.

L ₁Has the effect of mixing M₁The targeting group is linked to the N atom on the nitrogen-containing backbone to provide a liver targeting function for the siRNA conjugate shown in formula (308). In some embodiments, L₁One or more connecting combinations selected from the group of the formulas A1-A26. In some embodiments, L₁A combination of one or more linkages selected from a1, a4, a5, a6, A8, a10, a11, and a 13. In some embodiments, L₁A linked combination of at least 2 selected from a1, a4, A8, a10, and a 11. In some embodiments, L₁At least 2 connecting combinations selected from A1, A8 and A10.

In some embodiments, L₁May be 3-25 atoms in length3-20 atoms, 4-15 atoms or 5-12 atoms. In some embodiments, L₁The length of (a) 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 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 C₁-C ₄Alkyl, and in some embodiments, R' is one of methyl, ethyl, and isopropyl. Ra is one of a27, a28, a29, a30, and a31, and in some embodiments, Ra is a27 or a 28. Rb is C₁-C ₅In some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', Ra, Rb are each selected in formulas A1-A26 to achieve M₁The targeting group is linked to N on the nitrogen-containing backbone and M is attached₁The spatial position between the targeting groups is more suitable for M₁The targeting group binds to the hepatic surface asialoglycoprotein receptor.

In some embodiments, the conjugate has 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, for example, the P atom in formula a59 can be attached to any one nucleotide of the sense or 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 or 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 of the sense strand or the antisense strand from one end thereof. In some embodiments, the P atom in formula a59 is attached to the end of the sense or 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 linking to the above position of the sense strand of siRNA, after the siRNA conjugate shown in formula (308) enters the cell, upon unwinding, the siRNA antisense strand alone can be released to block the process of translation of protein by FXII mRNA, inhibiting Factor XII (FXII) gene expression.

In some embodiments, the P atom in formula a59 can be attached to any possible position on a nucleotide in the siRNA, e.g., 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 attached to the nucleotide in the siRNA at the 2' position, 3' position, or 5' position by forming a phosphodiester bond. In some embodiments, the P atom in formula a59 is attached to an oxygen atom formed after dehydrogenation of the 3' hydroxyl group of the 3' terminal nucleotide of the siRNA sense strand (in which case the P atom in a59 can also be considered to be the P atom in the phosphate group contained in the siRNA), or the P atom in formula a59 is attached to the nucleotide by substitution of a hydrogen in the 2' -hydroxyl group of one nucleotide in the siRNA sense strand, or the P atom in formula a59 is attached to the nucleotide by substitution of a hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide in the siRNA sense strand.

The inventors of the present disclosure surprisingly found that the siRNA conjugates of the present disclosure, while having significantly improved stability in plasma, low off-target effect, also show no significantly reduced FXII mRNA silencing activity. Thus, in some embodiments, the siRNA in the siRNA conjugates of the present disclosure may be any one of the sirnas shown in tables 1a, 1b, 1d, 1e, and 1 f.

TABLE 1A first siRNA sequence in conjugates of the disclosure

TABLE 1b second siRNA sequence in conjugates of the present disclosure

TABLE 1d third siRNA sequences in conjugates of the disclosure

TABLE 1e fourth siRNA sequence in conjugates of the present disclosure

TABLE 1f fifth siRNA sequence in conjugates of the disclosure

In the siRNA or siRNA conjugate, each adjacent nucleotide is connected by phosphodiester bond or phosphorothioate diester bond, non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond has negative charge, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond can exist in the form of hydroxyl or sulfhydryl, and hydrogen ions in the hydroxyl or sulfhydryl can be partially or completely replaced by cations. The cation may be any cation, such as a metal cation, ammonium NH₄ ⁺One of organic ammonium cations. For the purpose of enhancing solubility, in one embodiment, the cation is selected from one or more of alkali metal ions, tertiary amine forming ammonium cations, and quaternary ammonium cations. The alkali metal ion may be K⁺And/or Na⁺The cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N, N-diisopropylethylamine. Thus, the siRNA or siRNA conjugate of the present disclosure may be at least partially present in the form of a salt. 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 sirnas or siRNA conjugates of the present disclosure are present as sodium salts or partial sodium salts.

It is clear to the skilled person that modified nucleotide groups can be introduced into the siRNA by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or can be prepared by known methods.

Preparation of siRNA conjugate represented by formula (308)

Any reasonable synthetic route can be used to prepare the siRNA conjugates represented by formula (308).

In some embodiments, the siRNA conjugate represented by formula (308) can be prepared by a method comprising sequentially linking nucleoside monomers in a 3 'to 5' direction according to the nucleotide types and the order of the sense strand and the antisense strand of the siRNA, respectively, under the conditions of phosphoramidite solid phase synthesis, the linking of each nucleoside monomer comprising four steps of deprotection, coupling, capping, oxidation, or sulfurization; separating a sense strand and an antisense strand of the siRNA, and annealing, wherein the siRNA is the siRNA of the present disclosure;

and, the method further comprises contacting the compound represented by formula (321) with a nucleoside monomer or a nucleotide sequence attached to a solid support in the presence of a coupling reagent under coupling reaction conditions to allow the compound represented by formula (321) to be attached to the nucleotide sequence via a coupling reaction. Hereinafter, the compound represented by formula (321) is also referred to as a conjugate molecule.

Wherein:

R ₄is a group capable of binding to the siRNA represented by Nu in the compound represented by the formula (308). In some embodiments, R₄Is a group capable of binding to the siRNA represented by Nu through a covalent bond. In some embodiments, R₄A group which is capable of being conjugated to any functional group of the siRNA represented by Nu through a phosphodiester bond by a reaction;

each S₁Independently is M₁Wherein each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and alkylphenyl; in some embodiments, Y is methyl.

n1、n3、m1、m2、m3、R ₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄、R ₁₅、L ₁、M ₁The respective definitions and alternative ranges are as described above.

R ₄Is selected to achieve attachment to the N atom of the nitrogen-containing backbone and to provide a suitable reaction site for the synthesis of the siRNA conjugate shown in formula (308). In some embodiments, R₄Including R₂Linking groups or protected R₂A linking group, and a functional group that can react with the siRNA to form the structure shown as A59.

In some embodiments, R₄Comprises a 1 st functional group which can form a phosphite ester with a group on the siRNA or nucleoside monomer represented by Nu and a 2 nd functional group which can react with a hydroxyl group or an amino group to form a covalent bond or a solid phase carrier connected by the covalent bond. In some embodiments, the 1 st functional group is a phosphoramidite, a hydroxyl, or a protected hydroxyl. In some embodiments, the 2 nd functional group is a phosphoramidite, a carboxyl, or a carboxylate. In some embodiments, the 2 nd functional group is a solid support attached to the rest of the molecule via a covalent bond formed from a hydroxyl or amino group. In some embodiments, the solid support is linked via a phosphate ester linkage, a carboxylate ester linkage, or an amide linkage. In some embodiments, the solid support is a resin.

In some embodiments, the 1 st functional group contains a hydroxyl group, -OR_kOr a group of formula (C3); the 2 nd functional group has a structure represented by formula (C1), (C2), (C3), (C1') or (C3'):

in the formula, q₁Is an integer of 1 to 4, X is O or NH, M⁺Is a cation, R_kIs a hydroxyl protecting group, SPS represents a solid phase carrier,

indicates the site at which the group is covalently attached.

In some embodiments, the 1 st functional group contains a phosphoramidite group, as shown in formula (C3), which can be coupled to a hydroxyl group at any position on a nucleotide, such as a hydroxyl group at the 2 'position or a hydroxyl group at the 3' position, to form a phosphite, and oxidized or sulfurized to form a phosphodiester or phosphorothioate linkage as shown in formula a59, to conjugate the conjugation molecule to the siRNA. At this time, even if the 2 nd functional group is not present, the compound of formula (321) can be conjugated to a nucleotide without affecting the obtainment of the siRNA conjugate represented by formula (308). In this case, after obtaining the sense strand or the antisense strand of the siRNA via a phosphoramidite solid phase synthesis or the like, the compound of formula (321) is reacted with a hydroxyl group on the terminal nucleotide in the nucleotide sequence and forms a phosphodiester linkage or a phosphorothioate linkage during a subsequent oxidation or sulfurization process, and the compound of formula (321) is conjugated to the siRNA.

In some embodiments, the 1 st functional group contains a protected hydroxyl group. In some embodiments, the 2 nd functional group comprises a group that can react with a solid support, the reaction providing a conjugate molecule comprising a solid support. In some embodiments, the 2 nd functional group contains a carboxyl, carboxylate, or phosphoramidite, as shown in formula (C1), (C2), or (C3), when the 2 nd functional group contains a carboxyl or carboxylate, the compound of formula (321) undergoes an esterification or amidation reaction with a hydroxyl or amino group on a solid support, e.g., a resin, to form a carboxylate-linked conjugate molecule comprising the solid support. When the 2 nd functional group comprises a phosphoramidite functional group, the compound of formula (321) undergoes a coupling reaction with a hydroxyl group on a common solid support, e.g., a resin, and is oxidized to form a phosphodiester linked conjugate molecule comprising a solid support. Subsequently, the nucleoside monomers are sequentially linked according to a phosphoramidite solid phase synthesis method by using the product after the solid phase carrier is linked as the starting material to obtain the sense strand or the antisense strand of the siRNA with the conjugated group. During solid phase phosphoramidite synthesis, deprotection of the 1 st functional group occurs, followed by coupling with a phosphoramidite group on a nucleoside monomer under coupling reaction conditions.

In some embodiments, the 1 st functional group contains a hydroxyl group or a protected hydroxyl group; the 2 nd functional group contains a solid phase carrier connected by a carboxylate bond or an amide bond or a solid phase carrier connected by a phosphate bond, and is shown as a formula (C1') or (C3'). At this time, the nucleoside monomers are sequentially linked according to a phosphoramidite solid phase synthesis method starting from the compound of formula (321) instead of the solid phase carrier to obtain the sense strand or the antisense strand of the siRNA to which the conjugate group is linked.

In some embodiments, the carboxylate may be represented by-COO^-M ⁺Wherein M is⁺Is a cation, e.g. selected from the group consisting of metal cations, ammonium cations NH₄ ⁺One of organic ammonium cations. In one embodiment, the metal ion is selected from one of the alkali metal ions, such as K⁺Or Na⁺. In view of the solubility enhancement and the ease of reaction, in some embodiments, the organic ammonium ion is an ammonium cation formed from a tertiary amine or a quaternary ammonium cation, such as an ammonium ion formed from triethylamine or an ammonium ion formed from N, N-diisopropylethylamine. In some embodiments, the carboxylate is triethylamine carboxylate or N, N-diisopropylethylamine carboxylate.

In some embodiments, R₄Contains a structure represented by formula (B9), (B10), (B9'), (B10'), (B11), (B12), (B11') or (B12'):

wherein q is₁Is an integer of 1 to 4, q₂Is an integer of 1 to 10, X is O or NH, M⁺Is a cation, R_kIs a hydroxyl protecting group, SPS represents a solid phase carrier,

indicates the site at which the group is covalently attached. In some embodiments, q is₁Is 1 or 2. In some embodiments, q is₂Is an integer of 1 to 5. In some embodiments, R₄Contains a structure represented by the formula (B9) or (B10). In some embodiments, R₄Contains a structure represented by the formula (B11) or (B12).

In some embodiments, R_kIs one or more of Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4' -bismethoxytrityl) and TMTr (4,4',4' -trimethoxytrityl). In some embodiments, R_kMay be DMTr, i.e. 4,4'-dimethoxytrityl (4,4' -dimethoxytrityl).

L ₁As defined above.

In some embodiments, L₁Is used for M₁The targeting group is attached to the N atom on the nitrogen-containing backbone, thereby providing a liver targeting function to the siRNA conjugate shown in formula (308). In some embodiments, L₁Comprises any one or the combination of A1-A26.

From the above description, it is easily understood by those skilled in the art that the siRNA conjugate represented by formula (308) that links a conjugate molecule to any possible position of a nucleotide sequence, for example, the conjugate molecule is linked to the end of the nucleotide sequence and the conjugate molecule is linked to the end of the nucleotide sequence, can be obtained through the above-described 1 st functional group and optionally the 2 nd functional group, compared to the solid phase synthesis method of phosphoramidite known in the art. Accordingly, unless otherwise indicated, in the following description relating to the preparation of conjugates and/or conjugate molecules, when reference is made to "deprotection", "coupling", "capping", "oxidation", "sulfurization", etc. reactions, it is to be understood that reaction conditions and reagents involved in solid phase synthesis methods of phosphoramidite nucleic acids known in the art are equally applicable to these reactions. Exemplary reaction conditions and reagents will be described in detail hereinafter.

In some embodiments, each S is₁Independently is M₁. In some embodiments, each S is₁Independently is M₁Wherein at least one active hydroxyl group is protected by a hydroxyl protecting group. In some embodiments, each S is₁Independently is M₁Any active hydroxyl groups present in (a) are all protected by a hydroxyl protecting group. In some embodiments, any hydroxy protecting group known to those skilled in the art may be used to protect M ₁Active hydroxyl group in (1). In some embodiments, the protected hydroxy group may be represented by the formula YCOO-, wherein each Y is independently selected from the group consisting of C₁-C ₁₀Alkyl and C₆-C ₁₀Aryl group, said C₁-C ₁₀Alkyl and C₆-C ₁₀Aryl is optionally substituted with one or more substituents selected from the group consisting of halogen and C₁-C6 alkyl. In some embodiments, each Y is independently selected from the group consisting of: methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and C₁-C ₆An alkyl phenyl group.

In some embodiments, each S is₁Each independently selected from the group consisting of formula A46-A54:

in some embodiments, S₁Is of formula A49 or A50.

In some embodiments, each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; in some embodiments, Y is methyl.

As described above, the preparation method of the siRNA conjugate represented by formula (308) further comprises the steps of: synthesizing the other strand of the siRNA (for example, when the sense strand of the siRNA to which the conjugate molecule is linked is synthesized in the above-mentioned step, synthesizing the antisense strand of the siRNA according to a solid phase synthesis method and vice versa is also included), separating the sense strand and the antisense strand, and annealing. Specifically, in the separation step, the solid support attached to the nucleotide sequence and/or conjugate molecule is cleaved off, while the necessary protecting groups are removed (at this point, each S in the compound of formula (321) ₁Conversion of the group to the corresponding M₁Targeting group) to obtain a sense strand (or antisense strand) and a corresponding antisense strand (or sense strand) of the siRNA linked with the conjugate molecule, the sense strand and the antisense strand annealing to form a double-stranded RNA structure, obtaining the siRNA conjugate shown in formula (308).

In some embodiments, the method of preparing the siRNA conjugate represented by formula (308) comprises the steps of: contacting a compound shown in a formula (321) with a first nucleoside monomer at the 3' end of a sense strand or an antisense strand under a coupling reaction condition and in the presence of a coupling reagent, connecting the first nucleotide in a connecting sequence to the compound shown in the formula (321), and sequentially connecting the nucleoside monomers in the 3' to 5' direction according to the type and the sequence of the nucleotide of the desired sense strand or antisense strand under the condition of phosphoramidite solid phase synthesis to synthesize the sense strand or antisense strand of the siRNA; wherein the compound represented by the formula (321) is R₄The compound contains a 1 st functional group and a 2 nd functional group, wherein the 1 st functional group contains protected hydroxyl, the 2 nd functional group has a structure shown as a formula (C1') or (C3'), and the compound shown as the formula (321) is subjected to deprotection before being connected with a first nucleoside monomer; the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfuration; obtaining a sense or antisense strand of the nucleic acid to which the conjugate group is attached; under the condition of solid phase synthesis of phosphoramidite, nucleoside monomers are connected in sequence according to the nucleotide types and the sequence of an antisense strand or a sense strand and in the 3 'to 5' direction to synthesize the antisense strand or the sense strand of nucleic acid; each nucleoside monomer The connection of (2) comprises four steps of deprotection, coupling, capping, oxidation or sulfuration; removing protecting group, cutting with solid phase carrier, separating and purifying to obtain sense strand and antisense strand, and annealing.

In some embodiments, the method of preparing the siRNA conjugate represented by formula (308) comprises the steps of: according to the nucleotide types and the sequence of a sense strand or an antisense strand in the double-stranded siRNA, nucleoside monomers are sequentially connected in a 3 'to 5' direction to synthesize the sense strand and the antisense strand, wherein the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfuration, and the sense strand connected to a solid phase carrier and the antisense strand connected to the solid phase carrier are obtained; contacting the compound shown in the formula (321) with a sense strand connected to a solid phase carrier or an antisense strand connected to the solid phase carrier in the presence of a coupling reaction condition and a coupling reagent, and connecting the compound shown in the formula (321) to the sense strand or the antisense strand, wherein the compound shown in the formula (321) is R₄A compound of formula (321) having a 1 st functional group, wherein the 1 st functional group is a phosphoramidite group; removing protecting groups, cutting with a solid phase carrier, respectively separating and purifying to obtain a sense strand or an antisense strand of the siRNA, and annealing, wherein the sense strand or the antisense strand of the siRNA is connected with a conjugate group.

In some embodiments, the P atom in formula a59 is attached to the 3' end of the sense strand in the siRNA, and the method of preparing the siRNA conjugate represented by formula (308) comprises:

(1) removing the compound of formula (321) (wherein the compound of formula (321) is R₄Contains a 1 st functional group and a 2 nd functional group, the 1 st functional group contains a protected hydroxyl group OR_kThe 2 nd functional group is a compound having a structure represented by the formula (C1') or (C3')_k(ii) a Under the coupling reaction condition and the existence of a coupling reagent, contacting a product obtained by deprotection with a nucleoside monomer to obtain the nucleoside monomer connected to a solid phase carrier through a conjugation molecule;

(2) synthesizing a sense strand of the siRNA by a phosphoramidite solid phase synthesis method in a 3'-5' direction starting with the nucleoside monomer linked to the solid phase support by the conjugate molecule;

(3) synthesizing an antisense strand of the siRNA by a phosphoramidite solid phase synthesis method;

(4) the sense strand and the antisense strand of the siRNA are isolated and annealed to obtain an siRNA conjugate represented by formula (308).

Wherein, in the step (1), the protecting group R in the compound of the formula (321) is removed_kThe method of (2) comprises contacting a compound of formula (321) with a deprotection reagent under deprotection conditions. Deprotection conditions include temperatures of 0 to 50 deg.C, in some embodiments 15 to 35 deg.C, reaction times of 30 to 300 seconds, in some embodiments 50 to 150 seconds, and the deprotection reagent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments dichloroacetic acid. The molar ratio of deprotecting reagent to compound of formula (321) is from 10:1 to 1000:1, and in some embodiments from 50:1 to 500: 1.

The coupling reaction conditions and coupling reagents may use any conditions and reagents suitable for the above-described coupling reaction. In some embodiments, the same conditions and reagents can be used as for the coupling reaction in the solid phase synthesis method employed.

In some embodiments, the conditions of the coupling reaction include a reaction temperature of from 0 to 50 ℃, in some embodiments from 15 to 35 ℃. The molar ratio of the compound of formula (321) to nucleoside monomer is 1:1 to 1:50, in some embodiments 1:2 to 1: 5; the molar ratio of the compound of formula (321) to the coupling reagent may be in the range of from 1:1 to 1:50, and in some embodiments from 1:3 to 1:10, with a reaction time of from 200 to 3000 seconds, and in some embodiments, from 500 to 1500 seconds. The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, and 5-benzylthio 1H-tetrazole, and in some embodiments is 5-ethylthio 1H-tetrazole. The coupling reaction may be carried out in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, anhydrous dichloromethane, and in some embodiments, anhydrous acetonitrile. The organic solvent is used in an amount of 3 to 50L/mol, and in some embodiments 5 to 20L/mol, relative to the compound of formula (321).

In step (2), the sense strand S of the second siRNA conjugate is synthesized in the 3'-5' direction by a method of solid phase synthesis of phosphoramidite nucleic acid, starting with the nucleoside monomer attached to the solid support by the conjugate molecule prepared in the above step. At this point, the conjugate group is attached to the 3' end of the resulting sense strand.

Other conditions of the solid phase synthesis in the steps (2) and (3) include deprotection conditions of nucleoside monomers, types and amounts of deprotection reagents, coupling reaction conditions, types and amounts of coupling reagents, capping reaction conditions, types and amounts of capping reagents, oxidation reaction conditions, types and amounts of oxidation reagents, vulcanization reaction conditions, and types and amounts of vulcanization reagents, which are various reagents, amounts and conditions conventionally used in the art.

For example, in some embodiments, the solid phase synthesis in steps (2) and (3) may use the following conditions:

the nucleoside monomer deprotection conditions include a temperature of 0 to 50 deg.C, in some embodiments 15 to 35 deg.C, a reaction time of 30 to 300 seconds, in some embodiments 50 to 150 seconds, and the deprotection reagent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments dichloroacetic acid. The molar ratio of deprotecting reagent to 4,4' -dimethoxytrityl protecting group on the solid support can be from 2:1 to 100:1, and in some embodiments from 3:1 to 50: 1.

The coupling reaction conditions include a temperature of 0-50 deg.C, in some embodiments 15-35 deg.C, and a molar ratio of nucleic acid sequence attached to the solid support to nucleoside monomer can be 1:1 to 1:50, in some embodiments 1:5 to 1: 15; the molar ratio of nucleic acid sequence attached to the solid support to coupling reagent is from 1:1 to 1:100, and in some embodiments from 1:50 to 1:80, and the reaction time and choice of coupling reagent are the same as described above.

Capping reaction conditions include a temperature of 0-50 deg.C, in some embodiments 15-35 deg.C, and a reaction time of 5-500 seconds, in some embodiments 10-100 seconds, with the same selection of capping reagents as previously described. The molar ratio of the total amount of capping reagent to the nucleic acid sequence attached to the solid support is 1:100-100:1, and in some embodiments 1:10-10: 1. Where equimolar amounts of acetic anhydride and N-methylimidazole are used as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole and nucleic acid sequence attached to the solid support may be 1:1:10 to 10:10:1, and in some embodiments 1:1:2 to 2:2: 1.

The oxidation reaction conditions include a temperature of from 0 to 50 deg.C, in some embodiments from 15 to 35 deg.C, a reaction time of from 1 to 100 seconds, in some embodiments from 5 to 50 seconds, and the oxidizing agent, in some embodiments, iodine (in some embodiments, provided in the form of iodine water). The molar ratio of oxidizing reagent to nucleic acid sequence attached to the solid support in the coupling step can be from 1:1 to 100:1, and in some embodiments from 5:1 to 50: 1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran, water, pyridine ═ 3:1:1-1:1: 3. The sulfurization reaction conditions include a temperature of from 0 to 50 deg.C, in some embodiments from 15 to 35 deg.C, a reaction time of from 50 to 2000 seconds, in some embodiments 100 and 1000 seconds, and the sulfurizing agent, in some embodiments hydrogenated flavonones. The molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling step is from 10:1 to 1000:1, and in some embodiments from 10:1 to 500: 1. In some embodiments, the sulfurization reaction is carried out in a mixed solvent of acetonitrile and pyridine 1:3-3: 1.

After ligating all nucleoside monomers, the method further comprises isolating the sense and antisense strands of the siRNA prior to annealing. Isolation procedures are well known to those skilled in the art and generally involve cleaving the synthesized nucleotide sequence from the solid support, removing protecting groups on the base, phosphate and ligand, purification and desalting.

The nucleotide sequence obtained by synthesis is cut from the solid phase carrier, and the removal of the protecting groups on the base, the phosphate group and the ligand can be carried out according to the conventional cutting and deprotection method in the siRNA synthesis. For example, the obtained nucleotide sequence with the solid support attached thereto is contacted with concentrated ammonia water; during deprotection, the protecting group YCOO-of the A46-A54 group is converted into a hydroxyl group, S₁Conversion of the group to the corresponding M₁And (c) a group to produce a conjugate shown as formula (308). Wherein, theThe concentrated ammonia water can be 25-30 wt% ammonia water, and the dosage of the concentrated ammonia water can be 0.2 ml/mu mol-0.8 ml/mu mol compared with the target siRNA sequence.

When there is at least one 2'-TBDMS protection on the synthesized nucleotide sequence, the method further comprises contacting the nucleotide sequence with the solid support removed with triethylamine trihydrofluoride to remove the 2' -TBDMS protection. At this time, the corresponding nucleotide in the obtained target siRNA sequence has a free 2' -hydroxyl group. The amount of the triethylamine trihydrofluoride pure product can be 0.4 ml/mu mol-1.0 ml/mu mol compared with the target siRNA sequence. This gave an siRNA conjugate represented by the formula (308).

Methods of purification and desalination are well known to those skilled in the art. For example, purification of nucleic acids can be accomplished by gradient elution with NaBr or NaCl using a preparative ion chromatography purification column; the products can be desalted by adopting a reverse phase chromatographic purification column after being collected and combined.

In the siRNA conjugate represented by the formula (308) thus obtained, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond between nucleotides is substantially bound to sodium ions, and the siRNA conjugate represented by the formula (308) exists substantially in the form of a sodium salt. Other forms of siRNA conjugates represented by formula (308) can be obtained by replacing the sodium ions with hydrogen ions and/or other cations using well known ion exchange methods. The cations are as described above.

The purity and molecular weight of the nucleic acid sequence can be readily determined during synthesis to better control the quality of the synthesis, and such methods are well known to those skilled in the art. For example, nucleic acid purity can be detected by ion exchange chromatography and molecular weight determined by liquid chromatography-mass spectrometry (LC-MS).

Methods of annealing are also well known to those skilled in the art. For example, the synthesized sense strand (S strand) and antisense strand (AS strand) can be simply mixed in equimolar ratio in water for injection and heated to 70-95 ℃ followed by cooling at room temperature to allow formation of a double-stranded structure by hydrogen bonding. This gave an siRNA conjugate represented by the formula (308).

After obtaining the conjugate, in some embodiments, the synthesized siRNA conjugate of formula (308) can also be characterized by molecular weight detection, etc. using methods such as mass spectrometry, etc., to determine that the synthesized siRNA conjugate is the siRNA conjugate of formula (308) designed for the target, and that the sequence of the synthesized siRNA is the sequence of the desired siRNA, e.g., one of the sequences listed in tables 1a, 1b, 1d, 1e, and 1 f.

The compound represented by the formula (321) can be obtained by the following production method: the method comprises the following steps of contacting a compound shown as a formula (313) with a cyclic acid anhydride in an organic solvent under esterification reaction conditions in the presence of a base and an esterification catalyst, carrying out ion exchange, and separating to obtain a compound shown as a formula (321):

wherein, n1, n3, m1, m2, m3 and R₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄、R ₁₅、L _1、S ₁The respective definitions and alternative ranges are as described above;

R ₆to provide R in formula (321)₄A group of (a); in some embodiments, R₆Has a structure represented by formula (A61):

wherein R is_iTo enable connection to N atoms of nitrogen-containing skeleton, to R_kO is linked to and is linked to an optional radical of a free hydroxyl group, R_kIs a hydroxyl protecting group. In this case, R is obtained ₄The compound contains a 1 st functional group and a 2 nd functional group which are used as hydroxyl protecting groups, and the 2 nd functional group contains a compound shown as a formula (321) shown as a formula (C1) or (C2).

The esterification reaction conditions include a reaction temperature of 0-100 ℃ and a reaction time of 8-48 hours, and in some embodiments, the esterification reaction conditions are a reaction temperature of 10-40 ℃ and a reaction time of 20-30 hours.

In some embodiments, the organic solvent comprises one or more of an epoxy-based solvent, an ether-based solvent, a haloalkane-based solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In some embodiments, the epoxy-based solvent is dioxane and/or tetrahydrofuran, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether, and the haloalkane-based 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 3 to 50L/mol, and in some embodiments, 5 to 20L/mol, relative to the compound represented by formula (313).

In some embodiments, the cyclic anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, or pimelic anhydride, and in some embodiments succinic anhydride. The molar ratio of the cyclic anhydride to the compound of formula (313) is from 1:1 to 10:1, and in some embodiments from 2:1 to 5: 1.

The esterification catalyst may be any catalyst that catalyzes the esterification reaction, for example, the catalyst may be 4-dimethylaminopyridine. The molar ratio of the catalyst to the compound of formula (313) is from 1:1 to 10:1, and in some embodiments from 2:1 to 5: 1.

In some embodiments, the base can be any inorganic base, organic base, or combination thereof. The base may be, for example, a tertiary amine in view of solubility and product stability. In some embodiments, the tertiary amine is triethylamine or N, N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound of formula (313) is from 1:1 to 20:1, and in some embodiments from 3:1 to 10: 1.

Said ion exchange being the conversion of the compound of formula (321) into the desired carboxylic acid or carboxylate salt form, the methods of ion exchange being well known to those skilled in the art,suitable ion exchange solutions and exchange conditions can be used to obtain compounds having M⁺The cationic conjugate molecule will not be described in detail. In some embodiments, the ion exchange reaction is carried out using a triethylamine phosphate solution, which has a concentration of 0.2 to 0.8M, in some embodiments 0.4 to 0.6M, in an amount of 3 to 6L/mol, and in further embodiments 4 to 5L/mol, relative to the compound of formula (313).

The compound of formula (321) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, the compound of formula (321) may be isolated by evaporation of the solvent followed by chromatographic methods, e.g., using two chromatographic conditions: (1) normal phase purification of silica gel: 200-mesh 300-mesh silica gel filler, and performing gradient elution by using dichloromethane containing 1 wt% of triethylamine and methanol at a ratio of 100:18-100: 20; or (2) reversed-phase purification: c18, C8 reversed phase packing, eluting with a gradient of methanol to acetonitrile 0.1:1 to 1: 0.1. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (321) which may be used directly in a subsequent reaction.

In some embodiments, the method of preparing the compound of formula (321) further comprises contacting the product of the ion exchange reaction with a solid support comprising an amino group or a hydroxyl group in an organic solvent in the presence of a condensing agent, a condensation catalyst, and a tertiary amine under condensation reaction conditions. In this case, R is obtained₄The compound contains a 1 st functional group and a 2 nd functional group, wherein the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group contains a compound of a formula (321) with a structure shown as a formula (C1').

The solid phase carrier is one of carriers used in solid phase synthesis of siRNA, some of which are well known to those skilled in the art. For example, the solid support may be selected from solid supports containing reactive hydroxyl or amino functional groups, and in some embodiments, the solid support is an amino resin or a hydroxyl resin. In some embodiments, the amino or hydroxyl resin has the following parameters: the particle size is 100-400 meshes (mesh), and the surface amino or hydroxyl loading is 0.2-0.5 mmol/g. The dosage ratio of the compound shown in the formula (321) to the solid phase carrier is 10-400 mu mol of the compound per gram of the solid phase carrier (mu mol/g). In some embodiments, the compound of formula (321) is present in an amount of 50 to 200. mu. mol/g relative to the solid support.

The organic solvent may be any suitable solvent or mixture of solvents known to those skilled in the art. In some embodiments, the organic solvent is one or more of acetonitrile, an epoxy-based solvent, an ether-based solvent, a haloalkane-based solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In some embodiments, the epoxy-based solvent is dioxane and/or tetrahydrofuran, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether, and the haloalkane-based solvent is one or more of dichloromethane, chloroform, and 1, 2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. The organic solvent is used in an amount of 20 to 200L/mol, and in some embodiments 50 to 100L/mol, relative to the compound of formula (321).

In some embodiments, the condensing agent may be benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 3-diethoxyphosphoryl-1, 2, 3-benzole 4(3H) -one, and/or O-benzotriazol-tetramethyluronium hexafluorophosphate, which in some embodiments is O-benzotriazol-tetramethyluronium hexafluorophosphate. The molar ratio of the condensing agent to the compound of formula (321) is 1:1 to 20:1, and in some embodiments 1:1 to 5: 1.

In some embodiments, the tertiary amine is triethylamine and/or N, N-diisopropylethylamine, in some embodiments N, N-diisopropylethylamine; the molar ratio of the tertiary amine to the compound of formula (321) is from 1:1 to 20:1, and in some embodiments from 1:1 to 5: 1.

In some embodiments, the method for preparing the compound of formula (321) may further comprise contacting the resulting condensation product with a capping reagent and an acylation catalyst in an organic solvent under capping reaction conditions to isolate the compound of formula (321). The capping reaction serves to remove any reactive functional groups that have not reacted to completion to avoid the production of unwanted by-products in subsequent reactions. The capping reaction conditions include a reaction temperature of 0 to 50 deg.C, in some embodiments 15 to 35 deg.C, and a reaction time of 1 to 10 hours, in some embodiments 3 to 6 hours. The capping reagent may be one used in solid phase synthesis of siRNA, and the capping reagent used in solid phase synthesis of siRNA is well known to those skilled in the art.

In some embodiments, the capping reagent consists of capping reagent 1(cap1) and capping reagent 2(cap2), wherein capping reagent 1 is N-methyl imidazole, in some embodiments provided as a pyridine/acetonitrile mixed solution of N-methyl imidazole, wherein the volume ratio of pyridine to acetonitrile is 1:10 to 1:1, in some embodiments 1:3 to 1:1, and the volume ratio of the total volume of pyridine to acetonitrile to N-methyl imidazole is 1:1 to 10:1, in some embodiments 3:1 to 7: 1. The capping reagent 2 is acetic anhydride. In some embodiments, the capping reagent 2 is provided as an acetonitrile solution of acetic anhydride, wherein the volume of acetic anhydride and acetonitrile is from 1:1 to 1:10, and in further embodiments from 1:2 to 1: 6.

In some embodiments, the ratio of the volume of the pyridine/acetonitrile mixed solution of N-methylimidazole to the mass of the compound of formula (321) is 5ml/g to 50ml/g, in some embodiments 15ml/g to 30 ml/g. The ratio of the volume of the acetonitrile solution of acetic anhydride to the mass of the compound of formula (321) is from 0.5ml/g to 10ml/g, in some embodiments from 1ml/g to 5 ml/g.

In some embodiments, the capping reagent uses equimolar amounts of acetic anhydride and N-methylimidazole. In some embodiments, the organic solvent is one or more of acetonitrile, an epoxy-based solvent, an ether-based solvent, a haloalkane-based solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile. The organic solvent is used in an amount of 10 to 50L/mol, and in some embodiments, 5 to 30L/mol, relative to the compound of formula (321).

In some embodiments, the acylation catalyst may be selected from any catalyst useful for esterification condensation or amidation condensation, such as a basic heterocyclic compound. In some embodiments, the acylation catalyst is 4-dimethylaminopyridine. The mass ratio of the catalyst to the compound of formula (321) is 0.001:1 to 1:1, and in some embodiments 0.01:1 to 0.1: 1.

In some embodiments, the compound of formula (321) may be isolated from the reaction mixture using any suitable separation method. In some embodiments, the compound of formula (321) may be obtained by washing well with an organic solvent selected from acetonitrile, dichloromethane, methanol, in some embodiments acetonitrile, and filtering to remove unreacted reactants, excess capping reagent, and other impurities.

In some embodiments, a method of preparing a conjugate molecule of formula (321) comprises contacting a compound of formula (313) with a phosphoramidite in an organic solvent under coupling reaction conditions and in the presence of a coupling reagent, and isolating the compound of formula (321). In this case, R is obtained₄The compound contains a 1 st functional group and a 2 nd functional group, wherein the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group contains a compound of a formula (321) with a structure shown as a formula (C3).

In some embodiments, the coupling reaction conditions include a temperature that may range from 0 to 50 ℃, e.g., from 15 to 35 ℃, and a molar ratio of the compound of formula (313) to the phosphoramidite may range from 1:1 to 1:50, e.g., from 1:5 to 1: 15; the molar ratio of the compound of formula (313) to the coupling reagent may be from 1:1 to 1:100, for example from 1:50 to 1: 80; the reaction time may be 200-3000 seconds, for example 500-1500 seconds. The phosphorodiamidite may be, for example, bis (diisopropylamino) (2-cyanoethoxy) phosphine, which is commercially available or synthesized according to a method well known in the art. The coupling reagent is one or more selected from 1H-tetrazole, 5-ethylthio 1H-tetrazole, and 5-benzylthio 1H-tetrazole, such as 5-ethylthio 1H-tetrazole. The coupling reaction can be carried out in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, for example, anhydrous acetonitrile. In some embodiments, the organic solvent is used in an amount of 3 to 50L/mol, for example, 5 to 20L/mol, relative to the compound of formula (313). By carrying out this coupling reaction, the hydroxyl group in the compound of formula (313) reacts with the phosphoramidite to form a phosphoramidite group. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (321) which may be used directly in a subsequent reaction.

In some embodiments, the process for preparing a compound of formula (321) further comprises the steps of: the isolated product is further contacted with a solid support comprising hydroxyl groups under coupling reaction conditions in an organic solvent and in the presence of a coupling reagent. Subsequently, the compound of formula (321) is isolated by capping reaction, oxidation reaction. In this case, R is obtained₄The compound contains a 1 st functional group and a 2 nd functional group, wherein the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group has a structure shown as a formula (C3').

In some embodiments, the solid phase support is a solid phase support known in the art and useful for solid phase synthesis of nucleic acids, e.g., a commercially available general-purpose solid phase support after deprotection reaction (c)

HL UnyLinker ^TM300oligonucleotid Synthesis Support, Kinovate Life Sciences, having the structure shown in formula B80):

deprotection reactions are well known to those skilled in the art. In some embodiments, the deprotection conditions include a temperature of 0 to 50 ℃, e.g., 15 to 35 ℃; the reaction time is from 30 to 300 seconds, for example from 50 to 150 seconds. The deprotection agent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments, the deprotection agent is dichloroacetic acid. The molar ratio of deprotecting reagent to-DMTr (4,4' -dimethoxytrityl) protecting group on the stationary phase is 2:1 to 100:1, for example 3:1 to 50: 1. By carrying out the deprotection, free hydroxyl groups with reactivity are obtained on the surface of the solid phase carrier, so that subsequent coupling reaction is facilitated.

The coupling reaction conditions and the choice of coupling reagents may be as described above. By carrying out this coupling reaction, the free hydroxyl group formed in the deprotection reaction reacts with the phosphoramidite group to form a phosphite linkage.

In some embodiments, capping reaction conditions include a temperature of 0 to 50 ℃, e.g., 15 to 35 ℃, and a reaction time of 5 to 500 seconds, e.g., 10 to 100 seconds, the capping reaction being carried out in the presence of a capping reagent. The selection and amount of capping reagent may be as described above.

The oxidation reaction conditions include a temperature of from 0 to 50 deg.C, for example, from 15 to 35 deg.C, a reaction time of from 1 to 100 seconds, for example, from 5 to 50 seconds, and an oxidizing agent, for example, iodine (in some embodiments, provided in the form of iodine water). In some embodiments, the molar ratio of oxidizing reagent to nucleic acid sequence attached to the solid support is from 1:1 to 100:1, and can be, for example, from 5:1 to 50: 1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran, water, pyridine ═ 3:1:1-1:1: 3.

In some embodiments, R₆Is one of the groups of formula B7 or B8,

wherein q is₂The definition of (a) is as described above,

in this case, the compound represented by formula (313) can be obtained by the following production method: contacting a compound represented by the formula (314) with a compound represented by the formula (A-1) or a compound represented by the formula (A-2) in an organic solvent under amidation reaction conditions in the presence of an amidation reaction condensing agent and a tertiary amine, followed by separation:

Wherein, n1, n3, m1, m2, m3 and R₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄、R ₁₅、L ₁、S ₁、q ₂And R_kThe respective definitions and alternative ranges are as described above.

The amidation reaction conditions may include a reaction temperature of 0 to 100 ℃ and a reaction time of 1 to 48 hours, and in some embodiments, the amidation reaction conditions are a reaction temperature of 10 to 40 ℃ and a reaction time of 2 to 16 hours.

In some embodiments, the organic solvent is one or more of an alcohol solvent, an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. The alcoholic solvent is in some embodiments one or more of methanol, ethanol, propanol, and in some embodiments ethanol. The epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments. The ethereal solvent is, in some embodiments, diethyl ether and/or methyl tert-butyl ether. The haloalkane-based solvent is, in some embodiments, one or more of dichloromethane, trichloromethane and 1, 2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The amount of organic solvent used is in the range of 3 to 50L/mol, and in further embodiments 3 to 20L/mol, relative to the compound of formula (314).

In some embodiments, the amidation reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1, 2, 3-benzazole-4 (3H) -one, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (EEDQ), or O-benzotriazol-tetramethylurea hexafluorophosphate, in further embodiments 3-diethoxyphosphoryl-1, 2, 3-benzazole-4 (3H) -one. The molar ratio of the amidation reaction condensing agent to the compound of formula (314) may be 1:1 to 10:1, and in some embodiments, 2.5:1 to 5: 1.

In some embodiments, the tertiary amine is triethylamine or N, N-diisopropylethylamine, and in further embodiments is N, N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound of formula (314) is from 3:1 to 20:1, and in some embodiments from 5:1 to 10: 1.

In some embodiments, the compounds of formula (A-1) and formula (A-2) may be prepared by any suitable means. For example, when R is_kIn the case of DMTr group, the compound of formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl; similarly, the compound of formula (A-2) may be prepared by first contacting 3-amino-1, 2-propanediol with a cyclic anhydride, which may be a cyclic anhydride having from 4 to 13 carbon atoms, and in some embodiments, from 4 to 8 carbon atoms, and then reacting with DMTrCl. It will be readily understood by those skilled in the art that the selection of the cyclic anhydride corresponds to q in the compound (A-2) ₂Different values of (A), e.g. when the cyclic anhydride is succinic anhydride, q₂When the cyclic anhydride is glutaric anhydride, q is 1₂And so on for 2.

In some variations, the compound of formula (313) may also be prepared by reacting a compound of formula (314) with the cyclic anhydride, 3-amino-1, 2-propanediol, and DMTrCl, in that order. It will be readily understood by those skilled in the art that these modifications do not affect the structure and function of the compound of formula (313), and that these modifications are readily achievable by those skilled in the art based on the above-described methods.

Similarly to the above, any suitable separation method may be used to separate the compound of formula (313) from the reaction mixture. In some embodiments, the compound of formula (313) may be isolated by removal of the solvent by evaporation followed by chromatographic methods, e.g., using two chromatographic conditions: (1) normal phase purification of silica gel: 200-mesh 300-mesh silica gel filler is subjected to gradient elution by using petroleum ether, ethyl acetate, dichloromethane, N-dimethylformamide as the raw materials, wherein the ratio of petroleum ether to ethyl acetate to dichloromethane is 1:1:1:0.5-1:1:1: 0.6; and (2) reversed-phase purification: c18, C8 reversed phase packing, eluting with a gradient of methanol to acetonitrile 0.1:1 to 1: 0.1. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (313), which may be used directly in a subsequent reaction.

In some embodiments, the compound of formula (314) may be prepared by the following method: the method comprises the steps of contacting a compound shown as a formula (320) with a compound shown as a formula (316) in an organic solvent in the presence of an amidation reaction condensing agent and tertiary amine under a condensation reaction condition, and then separating:

wherein, n1, n3, m1, m2, m3 and R₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄、R ₁₅The respective definitions and alternative ranges are as described above.

Compounds of formula (316) may be prepared using, for example, compounds disclosed in j.am. chem.soc.2014,136,169581-16961, or compounds of formula (316) may be prepared by various methods by those skilled in the art, for example, certain compounds of formula (316) may be prepared by methods disclosed in example 1 of U.S. patent US8,106,022B2, the entire contents of which are incorporated herein by reference in their entirety.

In some embodiments, the condensation reaction conditions include a reaction temperature of 0 to 100 ℃ and a reaction time of 0.1 to 24 hours, in some embodiments a reaction temperature of 10 to 40 ℃ and a reaction time of 0.5 to 16 hours.

In view of the structure of the desired product compound of formula (314), the molar ratio of the compound of formula (316) to the compound of formula (320) should be determined based on the sum of n1 and n3 in formula (320). In some embodiments, for example, when n1+ n3 is 3, the molar ratio of the compound of formula (316) to the compound of formula (320) may be 3:1 to 3.5:1, and in some embodiments 3.01:1 to 3.15:1, in order to ensure that the reaction is complete and not excessive.

In some embodiments, the organic solvent is one or more of acetonitrile, an epoxy-based solvent, in some embodiments dioxane and/or tetrahydrofuran, an ether-based solvent, in some embodiments diethyl ether and/or methyl tert-butyl ether, an ether-based solvent, in some embodiments one or more of dichloromethane, chloroform and 1, 2-dichloroethane, an alkyl halide-based solvent, in some embodiments dichloromethane, an ethyl halide-based solvent, in some embodiments dioxane, and N, N-diisopropylethylamine. The organic solvent is used in an amount of 3 to 50L/mol, and in some embodiments, 5 to 20L/mol, relative to the compound of formula (320).

In some embodiments, the amidation reaction condensing agent is one or more of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (DEPBT), O-benzotriazol-tetramethyluronium hexafluorophosphate, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, or 1-hydroxybenzotriazole, in a further embodiment a mixture of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate and 1-hydroxybenzotriazole, wherein benzotriazole-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate and 1-hydroxybenzotriazole are used in equimolar amounts. The molar ratio of the total amidation reaction condensing agent to the compound of formula (316) may be 1:1 to 3:1, and in some embodiments 1.05:1 to 1.5: 1.

The tertiary amine may be N-methylmorpholine, triethylamine or N, N-diisopropylethylamine, in some embodiments N-methylmorpholine; the molar ratio of the tertiary amine to the compound of formula (316) may be 2:1 to 10:1, and in some embodiments 2:1 to 5: 1.

Similarly to the above, the compound of formula (314) may be isolated from the reaction mixture using any suitable separation method. In some embodiments, the compound of formula (314) may be isolated by removal of the solvent by evaporation followed by chromatographic methods, e.g., the isolation may be performed using two chromatographic conditions: (1) normal phase purification of silica gel: 200-300 mesh silica gel filler, and gradient elution is carried out by using dichloromethane and methanol as 100:5-100: 7; and (2) reversed-phase purification: c18, C8 reversed phase packing, eluting with a gradient of methanol to acetonitrile 0.1:1 to 1: 0.1. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (314) which may be used directly in a subsequent reaction.

The compounds of formula (320) are commercially available or obtained by one skilled in the art using known methods. For example, when m1 ═ m2 ═ m3 ═ 3, n1 ═ 1, n3 ═ 2, and each R is₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄、R ₁₅In the case of both H, the compound of formula (320) is commercially available from the company Afahesar.

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, for details see the description above for the pharmaceutical compositions of the present disclosure.

siRNA, pharmaceutical composition containing siRNA and application of conjugate

In some embodiments, the present disclosure provides the use of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the manufacture of a medicament for the treatment and/or prevention of HAE and/or thrombosis.

In some embodiments, the present disclosure provides a method of preventing and/or treating HAE and/or thrombosis comprising administering to a subject in need thereof an effective amount of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.

By administering the siRNA active ingredients of the present disclosure to a subject in need thereof, the prevention and/or treatment of HAE and/or thrombosis can be achieved through the mechanism of RNA interference. Accordingly, the siRNA and/or the pharmaceutical composition and/or the siRNA conjugate of the present disclosure may be used for preventing and/or treating HAE and/or thrombosis, or for preparing a medicament for preventing and/or treating HAE and/or thrombosis.

The term "administering" as used herein refers to placing an siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure into a subject by a method or route that results in at least partially positioning the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure at a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. In general, topical administration results in delivery of more siRNA conjugate to a particular site as compared to the systemic circulation of the subject; whereas systemic administration results in delivery of the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure to the subject's basal systemic circulation. In view of the present disclosure directed to providing a means of preventing and/or treating HAE, in some embodiments a mode of administration capable of delivering the drug to the liver is employed.

Administration to a subject can 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) administration. The frequency of administration may be 1 or more times per day, week, month, quarter, year, or year.

The dosage of the siRNA, pharmaceutical composition or siRNA conjugate described in the present disclosure may be a dosage that is conventional in the art, and the dosage may be determined according to various parameters, particularly age, weight and sex of the subject. Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD₅₀(lethal dose to death of 50% of the population) and ED₅₀(in the quantitative response, the dose which causes 50% of the maximal response intensity, and in the qualitative response, the dose which causes 50% of the subjects to develop positive response). The range of human doses can be derived based on data obtained from cell culture analysis and animal studies.

In administering the siRNA, the pharmaceutical composition, and/or the siRNA conjugate of the present disclosure, for example, for male or female, 6 to 12 weeks old, 18 to 25g weight of C57BL/6J or 30 to 45g ob/ob mice, the ratio, in terms of amount of siRNA: (i) for siRNA conjugates, the amount of siRNA may range from 0.001 to 100mg/kg body weight, in some embodiments from 0.01 to 50mg/kg body weight, in some embodiments from 0.05 to 20mg/kg body weight, in some embodiments from 0.1 to 15mg/kg body weight, and in other embodiments from 0.1 to 10mg/kg body weight; (ii) for pharmaceutical compositions of siRNA and a pharmaceutically acceptable carrier, the amount of siRNA may be from 0.001 to 50mg/kg body weight, in some embodiments from 0.01 to 10mg/kg body weight, in some embodiments from 0.05 to 5mg/kg body weight, and in some embodiments, from 0.1 to 3mg/kg body weight.

In some embodiments, the present disclosure provides a method of inhibiting FXII gene expression in a hepatocyte, comprising contacting the hepatocyte with an effective amount of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure, introducing the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure into the hepatocyte, for the purpose of inhibiting FXII gene expression in the hepatocyte by a mechanism of RNA interference. The liver cell can be selected from liver cancer cell lines such as SMMC-7721, HepG2, Huh7 and the like or isolated liver primary cells. In some embodiments, the cell is a human liver primary cell.

The amount of siRNA used in the provided modified siRNA, pharmaceutical composition and/or siRNA conjugate is typically such that the FXII gene is inhibited in a cell using the methods provided by the present disclosure: it is sufficient to reduce the expression of the target gene and result in an extracellular concentration at the surface of the target cell of 1pM to 1 μ M, or 0.01nM to 100nM, or 0.05nM to 50nM or 0.05nM to about 5 nM. 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, the route of delivery (local versus systemic), and the like. The concentration at the delivery site may be significantly higher than the concentration at the surface of the target cell or tissue.

Reagent kit

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

In some embodiments, the kits described herein can provide modified siRNA in one container. In some embodiments, a kit described herein may comprise one container providing a pharmaceutically acceptable excipient. In some embodiments, the kit may further comprise other ingredients, such as stabilizers or preservatives and the like. In some embodiments, the kits described herein can comprise at least one additional therapeutic agent in a container other than the container providing the modified siRNA described herein. In some embodiments, the kit may comprise instructions for mixing the modified siRNA with a pharmaceutically acceptable carrier and/or adjuvant or other ingredients (if any).

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

The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.

Examples

Unless otherwise specified, reagents and media used in the following examples are commercially available, and the procedures for nucleic acid electrophoresis, real-time PCR and the like used therein are performed by the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

Human liver primary cells were prepared by the institute of molecular medicine, university of Beijing, institute of molecular medicine, nucleic acid technology laboratory, thawed in a 37 ℃ water bath prior to use, and then seeded in collagen type I coated glass or plastic coverslips or tissue culture dishes at appropriate density, cultured in RPMI 1460 medium containing 1 Xdouble antibody and 10% FBS at 37 ℃ and cultured in RPMI 1460 medium containing 1 Xdouble antibody5％CO ₂Cells were cultured in an incubator with 95% air.

The C57 mouse liver primary cells were obtained from C57 mice (purchased from showa derivitis) provided by the institute of molecular medicine, university of beijing, nucleic acid technology laboratories. Inoculating appropriate density of cells in type I collagen-coated glass or plastic cover glass or tissue culture dish, culturing the cells in RPMI 1460 medium containing 1 Xdouble antibody and 10% FBS at 37 deg.C in 5% CO₂Culturing the cells in an incubator with 95% air for 15-30 min.

The C57 mice used were 6-8 week old mice purchased from Experimental animals technology, Inc. of Wei Tong Hua, Beijing.

Lipofectamine is used when the siRNA, siRNA conjugate or siRNA, siRNA conjugate as negative control synthesized by the present disclosure aiming at FXII gene transfects cells^TM2000(Invitrogen) as transfection reagent, reference was made to the instructions provided by the manufacturer for the specific procedures.

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

The experimental data are as follows

Data analysis was performed using Graphpad prism statistical analysis software.

Preparation example 1 preparation of conjugate 1

In this preparation example, conjugate 1 was synthesized. The conjugate is formed by conjugating L-9 conjugation molecule with SiFXIIa1M1SPsiRNA listed in Table 3.

(1-1) Synthesis of L-10 Compound

The L-10 compound was synthesized according to the following method:

(1-1-1) Synthesis of conjugated end segment GAL-5

Synthesis of (1-1-1a) GAL-2

100.0g GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS number: 1772-03-8, available from Ningbo Honghong Biochemical company, 463.8mmol) was dissolved in 1000ml of anhydrous pyridine, 540ml of acetic anhydride (available from Enox company, 5565.6mmol) was added under ice-water bath, and the reaction was stirred at room temperature for 1.5 hours. Pouring the reaction solution into 10L of ice water, carrying out suction filtration under reduced pressure, washing a filter cake with 2L of ice water, adding an acetonitrile/toluene mixed solvent (volume ratio of acetonitrile to toluene is 1:1) until the acetonitrile/toluene mixed solvent is completely dissolved, and evaporating the solvent to dryness to obtain a white solid product GAL-2130.0 g.

Synthesis of (1-1-1b) GAL-3

GAL-2(35.1g, 90.0mmol) obtained in step (1-1-1a) was dissolved in 213ml of anhydrous 1, 2-dichloroethane, and 24.0g of TMSOTf (CAS No.: 27607-77-8, available from Michael corporation, 108.0mmol) was added under ice water bath and nitrogen protection, and reacted at room temperature overnight.

The reaction solution was diluted with 400ml of dichloromethane, filtered through celite, and then 1L of saturated aqueous sodium bicarbonate was added, stirred well, the organic phase was separated, the aqueous phase was extracted twice with 300ml of dichloroethane, the organic phases were combined, washed with 300ml of saturated aqueous sodium bicarbonate and 300ml of saturated brine, respectively, the organic phase was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to obtain light yellow viscous syrup product GAL-326.9 g.

(1-1-1c) Synthesis of GAL-4

GAL-3(26.9g, 81.7mmol) obtained in step (1-1-1b) was dissolved in 136ml of anhydrous 1, 2-dichloroethane, and dried

30g of molecular sieve powder, 9.0g of 5-hexen-1-ol (CAS number: 821-41-0, from Adamas-beta, 89.9mmol) were added, the mixture was stirred at room temperature for 30 minutes, and 9.08g of TM was added under protection of nitrogen in an ice bathSOTf (40.9mmol), the reaction was stirred at room temperature overnight. Filtering to remove

Molecular sieve powder, adding 300ml dichloromethane into filtrate for dilution, filtering with diatomite, adding 500ml saturated sodium bicarbonate aqueous solution, stirring for 10 minutes for washing, separating an organic phase, extracting an aqueous phase once with 300ml dichloroethane, combining the organic phases, respectively washing with 300ml saturated sodium bicarbonate aqueous solution and 300ml saturated saline solution, separating the organic phase, drying with anhydrous sodium sulfate, and evaporating the solvent under reduced pressure to obtain a yellow syrup-like product GAL-441.3 g, wherein the next oxidation reaction is directly carried out without purification.

Synthesis of (1-1-1d) GAL-5

GAL-4(14.9g, 34.7 mmol) obtained by the method described in step (1-1-1c) was dissolved in a mixed solvent of 77ml of methylene chloride and 77ml of acetonitrile, 103ml of deionized water and 29.7g of sodium periodate (CAS No.: 7790-28-5, available from Alantin, 138.8mmol) were added, respectively, stirred for 10 minutes in an ice water bath, ruthenium trichloride (CAS No.: 14898-67-0, available from Annona, 238mg, 1.145mmol) was added, and the reaction was allowed to proceed overnight at room temperature. The reaction mixture was diluted with 300ml of water and stirred, saturated sodium bicarbonate was added to adjust the pH to about 7.5, the organic phase was separated and discarded, and the aqueous phase was extracted three times with 200ml portions of dichloromethane and the organic phase was discarded. The aqueous phase was adjusted to pH about 3 with citric acid solid, extracted three times with 200ml each time with dichloromethane, the organic phases combined, dried over anhydrous sodium sulfate and the solvent evaporated under reduced pressure to dryness to give GAL-56.85 g as a white foamy solid product.¹H NMR(400MHz,DMSO)δ12.01(br,1H),7.83(d,J＝9.2Hz,1H),5.21(d,J＝3.2Hz,1H),4.96(dd,J＝11.2,3.2Hz,1H),4.49(d,J＝8.4Hz,1H),4.07–3.95(m,3H),3.92–3.85(m,1H),3.74–3.67(m,1H),3.48– 3.39(m,1H),2.20(t,J＝6.8Hz,2H),2.11(s,3H),2.00(s,3H),1.90(s,3H),1.77(s,3H),1.55–1.45(m,4H).

(1-1-2) Synthesis of L-8:

j-0(9.886g, 52.5mmol, commercially available from Afahesa) and GAL-5(72.819g, 162.75mmol, obtained from combining the various batches) obtained in step (1-1-1) were dissolved in 525ml of dichloromethane, diisopropylethylamine (DIEA, 44.782g, 346.50mmol), benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP, 90.158g, 173.25mmol) and hydroxybenzotriazole (HOBt, 23.410g, 173.25mmol) were added, reacted at room temperature for 4h, 20ml of saturated sodium bicarbonate and 200ml of saturated brine were added and the aqueous phase was extracted 2 times with dichloromethane, 100ml each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and the solvent was evaporated to dryness under reduced pressure to give the crude product. Purifying by using 200-mesh 300-mesh normal phase silica gel, neutralizing the acidity of the silica gel with 10 wt% of triethylamine, balancing a column with 1 wt% of triethylamine, performing gradient elution with dichloromethane and methanol at a ratio of 100:25-100:40, collecting product eluent, and evaporating the solvent under reduced pressure to obtain a pure product L-838.8 g. ¹H NMR(400MHz,DMSO)δ7.84(d,J＝9.0Hz,3H),7.27–7.23(m,1H),7.13–7.18(m,1H),5.22(d,J＝3.1Hz,3H),4.97(dd,J＝11.3,3.1Hz,3H),4.48(d,J＝8.4Hz,3H),4.09–3.98(m,9H),3.88(dd,J＝19.3,9.3Hz,3H),3.75–3.66(m,3H),3.44–3.38(m,3H),3.17–3.30(m,4H),3.10–2.97(m,4H),2.35–2.20(m,6H),2.15–2.08(m,9H),2.07–1.98(m,13H),1.94–1.87(m,9H),1.81–1.74(m,9H),1.65–1.42(m,18H).MS m/z：C ₈₅H ₁₁₉N ₇O ₃₀，[M+H] ⁺Theory: 1477.59, actually measuring: 1477.23.

(1-1-3a) Synthesis of A-1

Dissolving DMTrCl (4,4' -bis (methoxy) trityl chloride, 101.65g, 300mmol) in 1000ml of anhydrous pyridine, adding DL-calcium glycerate hydrate (28.63g, 100mmol), reacting at 45 ℃ for 20h, filtering the reaction solution, rinsing the filter cake with 200ml of DCM, concentrating the filtrate under reduced pressure to dryness, and using 500ml of dichloro chloride as the residueRedissolving methane, washing with 0.5M triethylamine phosphate (pH 7-8) for 2 times, 200ml each time, extracting the aqueous phase with dichloromethane for 2 times, 200ml each time, combining organic phases, drying with anhydrous sodium sulfate, filtering, evaporating the solvent under reduced pressure, purifying with a 200-mesh 300-mesh normal phase silica gel column, gradient-eluting with petroleum ether, ethyl acetate, dichloromethane, methanol, 1:1:0.35-1:1:1:0.55, collecting the product eluate, evaporating the solvent under reduced pressure, redissolving 600ml dichloromethane, washing with 200ml 0.5M triethylamine phosphate for 1 time, extracting the aqueous phase with 200ml dichloromethane for 1 time, combining organic phases, drying with anhydrous sodium sulfate, filtering, evaporating the solvent under reduced pressure, and reducing the pressure of an oil pump overnight to obtain a white solid product A-150.7 g.¹H NMR(400MHz,DMSO-d6)δ7.46(ddd,J＝6.5,2.3,1.1Hz,1H),7.40–7.28(m,7H),6.89–6.81(m,4H),4.84(d,J＝5.0Hz,1H),4.36–4.24(m,1H),4.29(s,6H),3.92(dd,J＝12.4,7.0Hz,1H),3.67(dd,J＝12.3,7.0Hz,1H),2.52(q,J＝6.3Hz,6H),1.03(t,J＝6.3Hz,9H).MS m/z：C ₂₄H ₂₃O ₆，[M-H] ^-Theory: 407.15, actually measuring: 406.92.

(1-1-3b) Synthesis of L-7:

mixing L-8(40g, 27.09mmol, obtained by combining several batches of product) obtained in step (1-1-2) and A-1(41.418g, 81.27mmol) obtained in step (1-1-3a), dissolving in 271ml of dichloromethane, adding 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (DEPBT) (24.318g, 81.37mmol), adding diisopropylethylamine (21.007g, 162.54mmol), stirring at 25 ℃ for 1.5H, washing the organic phase with 800ml of saturated sodium bicarbonate, extracting the aqueous phase 3 times with dichloromethane, 50ml each time, the organic phase was washed with 150ml of saturated brine, the aqueous phase was extracted 1 time with 50ml of dichloromethane, the organic phases were combined and dried over anhydrous sodium sulfate, filtered and the solvent was evaporated under reduced pressure, foamed and dried overnight with a vacuum oil pump to give the crude product. Column purification 2kg of 200-mesh 300 mesh normal phase silica gel was used, and the silica gel acidity was neutralized with 200ml of triethylamine to contain 1 wt% And (3) carrying out petroleum ether equilibrium column chromatography on triethylamine, carrying out gradient elution on the petroleum ether, ethyl acetate, dichloromethane and N, N-dimethylformamide by a ratio of 1:1:1:0.5-1:1:1:0.6, collecting product eluent, and evaporating the solvent under reduced pressure to obtain a pure product L-740.4 g.¹H NMR(400MHz,DMSO)δ7.90–7.78(m,4H),7.75–7.64(m,1H),7.38–7.18(m,9H),6.91–6.83(m,4H),5.25–5.10(m,4H),4.97(dd,J＝11.2,3.2Hz,3H),4.48–4.30(m,4H),4.02(s,9H),3.93–3.84(m,3H),3.76–3.66(m,9H),3.45–3.35(m,3H),3.24–2.98(m,10H),2.30–2.20(m,2H),2.11–1.88(m,31H),1.80–1.40(m,28H).MS m/z：C ₉₀H ₁₂₈N ₇O ₃₅，[M-DMTr] ⁺Theory: 1564.65, actually measuring: 1564.88.

(1-1-4) Synthesis of L-9:

mixing L-7(40g, 21.4247mmol) obtained in step (1-1-3b), succinic anhydride (4.288g, 42.8494mmol) and 4-dimethylaminopyridine (DMAP, 5.235g, 42.8494mmol) and dissolving in 215ml of dichloromethane, adding diisopropylethylamine (DIEA, 13.845g, 107.1235mmol), stirring at 25 ℃ for 24h, washing the reaction solution with 800ml of 0.5M triethylamine phosphate, extracting the aqueous phase with dichloromethane 3 times, 5ml each time, combining the organic phases and evaporating to dryness under reduced pressure to obtain a crude product. The column purification uses 1kg of 200-300 mesh normal phase silica gel, 1 wt% of triethylamine is used to neutralize the acidity of the silica gel, the column is equilibrated with dichloromethane, the product eluent is collected by gradient elution with 1 wt% of triethylamine in dichloromethane and methanol being 100:18-100:20, the solvent is evaporated under reduced pressure to obtain 31.0g of pure L-9 conjugate molecule.¹H NMR(400MHz,DMSO)δ8.58(d,J＝4.2Hz,1H),7.94–7.82(m,3H),7.41–7.29(m,5H),7.22(d,J＝8.1Hz,5H),6.89(d,J＝8.3Hz,4H),5.49–5.37(m,1H),5.21(d,J＝3.0Hz,3H),4.97(d,J＝11.1Hz,3H),4.49(d,J＝8.2Hz,3H),4.02(s,9H),3.88(dd,J＝19.4,9.4Hz,3H),3.77–3.65(m,9H),3.50–3.39(m,6H),3.11–2.90(m,5H),2.61–2.54(m,4H),2.47–2.41(m,2H),2.26–2.17(m,2H),2.15–1.95(m,22H),1.92–1.84(m,9H),1.80–1.70(m,10H),1.65–1.35(m,17H),1.31–1.19(m,4H),0.96(t,J＝7.1Hz,9H).MS m/z：C ₉₄H ₁₃₂N ₇O ₃₈，[M-DMTr] ⁺Theory: 1664.72, actually measuring: 1665.03.

(1-1-5) Synthesis of L-10 Compound:

in this step, the L-10 compound is prepared by attaching the L-9 conjugate molecule to a solid support.

Mixing the L-9 conjugate molecule (22.751g, 11mmol) obtained in step (1-1-4), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 6.257g, 16.5mmol) and diisopropylethylamine (DIEA, 2.843g, 22mmol), dissolving in 900ml acetonitrile, stirring for 5 minutes at room temperature, adding aminomethyl resin (88g, 100-mesh 200-mesh, with an amino load of 400 mu mol/g, purchased from Nankai Okazai Kagaku Co., Ltd.) into the reaction solution, carrying out shaking table reaction at 25 ℃ at a rotation speed of 150 rpm, reacting for 18 hours, filtering, leaching the filter cake with DCM for 2 times (300 ml each time), leaching acetonitrile for 3 times (300 ml each time), drying for 18 hours by a vacuum oil pump, and then adding raw materials (CapA, CapB, 4-Dimethylaminopyridine (DMAP) and acetonitrile) according to the feeding ratio shown in Table 2 to carry out capping reaction. Placing the mixture on a shaking bed at 25 ℃, rotating at 150 revolutions per minute, reacting for 5 hours, filtering reaction liquid, leaching a filter cake for 3 times by using acetonitrile, wherein each time is 300ml, evaporating the solvent to dryness under reduced pressure, and drying overnight under reduced pressure by using a vacuum oil pump to obtain 102g of an L-10 compound (namely L-9 conjugated molecule connected with a solid phase carrier) with the loading capacity of 90.8 mu mol/g.

TABLE 2 charging ratio for cap reaction

Raw materials	Dosage of	Specification of	Batch number	Manufacturer of the product
CapA	1980ml	——	——	——
CapB	220ml	——	——	——
DMAP	1.100g	Analytical purity	I1422139	Aladdin
Acetonitrile	220ml	Pure spectrum	O15161001	Shanghai xing can

Wherein, the CapA and the CapB are capping reagent solutions, the CapA is a pyridine/acetonitrile mixed solution of 20 volume percent of N-methylimidazole, and the volume ratio of the pyridine to the acetonitrile is 3: 5; CapB is 20% acetic anhydride in acetonitrile.

(1-2) Synthesis of sense Strand of conjugate 1

The nucleoside monomers are connected one by one from the 3'-5' direction according to the arrangement sequence of sense strand nucleotides by a solid phase phosphoramidite method and by utilizing the L-10 compound prepared by the steps to start circulation. Each attachment of a nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, oxidation or sulfurization. When two nucleotides are connected by adopting phosphate ester, and the next nucleoside monomer is connected, four-step reactions including deprotection, coupling, capping and oxidation are carried out. When two nucleotides are connected by phosphorothioate, and the latter nucleoside monomer is connected, the four-step reaction of protection, coupling, capping and sulfuration is included. The synthesis conditions are given as follows:

the nucleoside monomer was supplied as a 0.1M acetonitrile solution, the deprotection conditions were the same for each step, i.e., temperature was 25 deg.C, reaction time was 70 seconds, the deprotection reagent was dichloroacetic acid in dichloromethane (3% v/v), and the molar ratio of dichloroacetic acid to 4,4' -dimethoxytrityl protecting group on the solid support was 5: 1.

The coupling reaction conditions in each step are the same, and the coupling reaction conditions comprise that the temperature is 25 ℃, the molar ratio of the nucleic acid sequence connected on the solid phase carrier to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence connected on the solid phase carrier to the coupling reagent is 1:65, the reaction time is 600 seconds, and the coupling reagent is a 0.5M acetonitrile solution of 5-Ethylthio-1H-tetrazole (5- (ethyhio) -1H-tetrazole, ETT).

The capping conditions were the same for each step, including a temperature of 25 ℃ and a reaction time of 15 seconds. The capping reagent solution is a mixed solution of CapA and CapB with a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence connected to the solid phase carrier is acetic anhydride, N-methylimidazole and the nucleic acid sequence connected to the solid phase carrier is 1:1: 1.

The oxidation reaction conditions in each step are the same, including the temperature of 25 ℃, the reaction time of 15 seconds, and the oxidizing agent of 0.05M iodine water. The molar ratio of iodine to nucleic acid sequence attached to the solid support in the coupling step is 30: 1. The reaction was carried out in a mixed solvent of tetrahydrofuran, water and pyridine in a ratio of 3:1: 1.

The conditions of each step of sulfuration reaction are the same, including the temperature of 25 ℃, the reaction time of 300 seconds, and the sulfuration reagent of hydrogenization xanthogen. The molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling step is 120: 1. The reaction was carried out in a mixed solvent of acetonitrile and pyridine in a ratio of 1: 1.

Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence with the attached vector was added to 25 wt% ammonia water in an amount of 0.5 ml/. mu.mol, reacted at 55 ℃ for 16 hours, the liquid was removed, and the residue was concentrated to dryness in vacuo.

Purification and desalting: purification of nucleic acids was achieved by gradient elution with NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the method comprises the following steps: eluent A: 20mM sodium phosphate (pH 8.1) in water/acetonitrile 9:1 (volume ratio); eluent B: 1.5M sodium chloride, 20mM sodium phosphate (pH 8.1) and solvent water/acetonitrile 9:1 (volume ratio); elution gradient: eluting with eluent A and eluent B in gradient of 100:0-50: 50. Collecting product eluates, mixing, desalting with reverse phase chromatography purification column, specifically desalting with Sephadex column, and eluting with deionized water, wherein the filler is Sephadex G25(Sephadex G25).

And (3) detection: purity was checked using ion exchange chromatography (IEX-HPLC) and molecular weight was analyzed using liquid chromatography-mass spectrometry (LC-MS). Observed values are consistent with theoretical values, indicating that the sense strand S, 3' end conjugated to the L-9 conjugate molecule, was synthesized.

(1-3) Synthesis of antisense strand of conjugate 1

By the solid phase phosphoramidite method, using a universal solid phase carrier (UnyLinker)^TM loaded

HL Solid Supports, Kinovate Life Sciences) initiated the cycle, synthesizing the antisense strand AS of conjugate 1. The conditions of deprotection, coupling, capping, oxidation or sulfuration reaction, cutting, deprotection, purification and desalination in the solid phase synthesis method are the same as those of the synthesis of a sense chain.

When the target sequence has a 5-P modification at the first nucleotide of the 5' -terminal end of the antisense strand, in the preparation of the antisense strand according to the solid phase phosphoramidite method, after the last nucleoside monomer of the antisense strand is connected, CPR-I monomer (Cat #13-2601-XX, Suzhou Jima) is connected to the 5' -terminal end of the antisense strand through four steps of deprotection, coupling, capping and oxidation to form a 5' -phosphate modification.

In the connection, the universal solid phase carrier is used, and the conditions of deprotection, coupling, capping, oxidation or sulfuration reaction, cutting, deprotection, purification and desalination are the same as those of the synthesis of a sense chain.

And (3) detection: detecting purity by ion exchange chromatography (IEX-HPLC); the molecular weight of the resulting product was analyzed by liquid chromatography-mass spectrometry (LC-MS). As a result, the observed value was matched with the theoretical value, indicating that the antisense strand AS having the target sequence was synthesized.

(1-4) Synthesis of conjugate 1

For conjugate 1, S chain and AS chain are respectively dissolved in water for injection to obtain 40mg/mL solution, the solution is mixed according to an equal molar ratio, heated at 50 ℃ for 15min, cooled at room temperature to obtain an annealed product, and freeze-dried powder is obtained. After the conjugate was diluted to a concentration of 0.2mg/mL using ultrapure water (Milli-Q ultrapure water meter, resistivity 18.2 M.OMEGA.. multidot.cm (25 ℃)), molecular weight measurement was performed using a Liquid chromatograph-Mass Spectrometry (LC-MS, available from Waters, Inc., model: LCT Premier). Observed values are consistent with theoretical values, indicating that the synthesized conjugate 1 is a target designed double-stranded nucleic acid sequence with an L-9 conjugate molecule. The structure is shown as formula (403).

Preparation example 2 preparation of conjugates 2 to 6

Conjugate 2, conjugate 3, conjugate 4, conjugate 5 and conjugate 6 were synthesized, respectively, in the same manner as in preparation example 1, except that the sirnas in the conjugates had sense and antisense strands corresponding to conjugate 2, conjugate 3, conjugate 4, conjugate 5 and conjugate 6, respectively, shown in table 3, and thus in order to synthesize these conjugates, the corresponding sense and antisense strands were synthesized in accordance with the sense and antisense strand sequences of the sirnas shown in table 3, respectively, when preparing the sense and antisense strands. The molecular weights of the obtained conjugates 2-6 were respectively detected by LC-MS, and the measured values are consistent with the theoretical values, which indicates that the synthesized conjugates are the target designed double-stranded nucleic acid sequences with L-9 conjugate molecules. The structure of conjugate 2-6 is shown in formula (403).

TABLE 3 siRNA conjugates

Wherein, the capital letters C, G, U, A represent the base composition of nucleotides; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that the phosphorothioate linkage is between the two nucleotides to the left and right of the letter s; the capital letter P indicates that the nucleotide adjacent to the right side of the letter P is a nucleotide modified with a 5' -phosphate nucleotide.

In the above conjugate, the sense strand and the antisense strand comprise a nucleotide base difference (5'-3' direction, G-a difference at 2 nd nucleotide of the sense strand, and C-U difference at 18 th nucleotide of the antisense strand) between the siRNA in the conjugate 3 and the siRNA in the conjugate 1, the siRNA antisense strand of the conjugate 1 is completely reverse-complementary to human FXII mRNA, and the siRNA antisense strand of the conjugate 3 is completely reverse-complementary to mouse FXII mRNA. The siRNA antisense strands of the remaining conjugates 2, 4, 5 and 6 were fully reverse complementary to both human FXII mRNA and mouse FXII mRNA.

Preparation example 3 Synthesis of siRNA sequences

The siRNA listed in table 4 was obtained as sense and antisense strands by a solid phase synthesis method, and the siRNA listed in table 4 was obtained as lyophilized powder by dissolving equimolar mixtures of sense and antisense strands in water using DEPC, followed by annealing to form siRNA duplexes, and lyophilizing.

TABLE 4 siRNA sequences

Wherein, the capital letters C, G, U, A represent the base composition of nucleotides; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that the phosphorothioate linkage is between the two nucleotides to the left and right of the letter s; dT represents thymidine.

In the preparation of the above sequence, when an unmodified nucleotide is contained in the target sequence, the product is dissolved with 0.4 ml/. mu.mol of N-methylpyrrolidone, and then 0.3 ml/. mu.mol of triethylamine and 0.6 ml/. mu.mol of triethylamine trihydrofluoride are added to remove the 2' -TBDMS protection on ribose after ammonia treatment in the cleavage and deprotection conditions, relative to the amount of single-stranded nucleic acid.

Experimental example 4 inhibition efficiency of FXII mRNA expression amount by siRNA in human liver primary cells was examined.

Lipofectamine was used according to the instructions provided by the supplier^TM2000 siRNA to be tested (solutions of siRNA7, 8, 10, 11 and 12 and a reference sequence NC, wherein NC is a negative control sequence having no significant sequence correlation with FXII mRNA, and the solution is an aqueous solution prepared by re-dissolving siRNA at a desired concentration in DEPC water before an experiment) were transfected into human liver primary cells respectively, each siRNA having a final concentration of 50nM and 2 replicate wells per concentration.

The expression level of FXII mRNA in the human liver primary cells transfected with each siRNA was detected by Real-Time fluorescent Quantitative PCR (Quantitative Real-Time PCR). The method comprises the following specific steps: after culturing the transfected cells for 24 hours, total RNA was extracted from the cells using Trizol (Thermo Fisher Co.) according to standard procedures for total RNA extraction; mu.g of each total RNA was collected and subjected to reverse transcription using a reverse transcription kit (Promega corporation, cat. No. A3500) in accordance with the protocol described in the specification to obtain cDNA. The FXII mRNA expression level was detected using 2X Ultra SYBR Mixed (with ROX) (Beijing Kang is a century Biotechnology Co., Ltd., product No. CW0956) kit by the procedure of the instruction manual using cDNA as a template. Among them, PCR primers for amplifying FXII and GAPDH as reference genes are shown in Table 5.

TABLE 5 primer information

The FXII mRNA expression level was calculated as follows: FXII mRNA expression level ═ 100% (expression level of FXII mRNA in test group/expression level of GAPDH mRNA in test group)/(expression level of FXII mRNA in control group/expression level of GAPDH mRNA in control group).

The mRNA inhibition rate was (1-FXII mRNA expression amount) × 100%. Wherein, each test group is human liver primary cells treated by siRNA with each concentration, and the control group is cells treated by comparison sequence NC. The results are shown in Table 6.

TABLE 6 inhibition of FXII mRNA in human liver primary cells

siRNA	Numbering	mRNA inhibition%
siRNA 7	siFXIIa1M1S	38.01
siRNA 8	siFXIIb1M1S	45.81
siRNA 10	siFXIId1M1S	73.83
siRNA 11	siFXIIe1M1S	77.61
siRNA 12	siFXIIf1M1S	78.70
NC	-	-0.08

As can be seen from the results in Table 6, the modified siRNA provided by the present disclosure shows higher FXII mRNA inhibitory activity in human liver primary cells, wherein the inhibition rate of 50nM siRNA12 on the expression level of FXII mRNA can reach 78.70%.

Experimental example 5siRNA was examined for the inhibitory efficiency of FXII mRNA expression level in primary liver cells of C57 mice.

The assay was performed in the same manner as in experimental example 4, except that the mRNA inhibition rates of siRNA7, siRNA8, siRNA10, siRNA11 and siRNA12 were measured in C57 mouse liver primary cells, while PCR primers for amplifying FXII and GAPDH as an internal reference gene shown in table 7 were used in place of the primers shown in table 5.

TABLE 7 primer information

The inhibitory activity of each siRNA in mouse liver primary cells is shown in table 8.

TABLE 8 inhibition of FXII mRNA in liver primary cells of C57 mice

siRNA	Numbering	mRNA inhibition%
siRNA 7	siFXIIa1M1S	65.99
siRNA 8	siFXIIb1M1S	69.27
siRNA 10	siFXIId1M1S	54.84
siRNA 11	siFXIIe1M1S	69.05
siRNA 12	siFXIIf1M1S	70.09
NC	-	-0.24

As can be seen from the results in Table 8, the siRNA provided by the present disclosure shows higher FXII mRNA inhibitory activity in primary liver cells of C57 mice, wherein the inhibition rate of 50nM siRNA12 on FXII mRNA expression can reach 70.09%.

Experimental example 6 inhibition efficiency of siRNA conjugate against FXII mRNA expression level in C57 mouse

In this experimental example, the inhibition rate of the conjugates 1 to 6 on FXII mRNA in liver tissue in C57 mice was examined by two experiments.

(6-A) inhibition Rate of FXII mRNA in liver tissue by conjugates 1, 2 and 3 in C57 mice

6-8 week-old C57 mice were randomly divided into 7 groups of 5 mice each, and each group was administered with a solution of conjugate 1, 2 or 3 (each conjugate corresponds to 2 groups of different doses) or control sequence NC (1 group), respectively (the solution was formed by re-dissolving siRNA conjugates at the desired concentrations in 1 XPBS (pH7.4) buffer before the experiment), and a PBS blank group (15 mice, administered with 1 XPBS). All animals calculate the dosage according to the body weight, and are singly administrated in a subcutaneous injection mode, the administration dosage (based on the amount of siRNA) of the siRNA conjugate is respectively two dosage groups of 5mg/kg and 1mg/kg, the administration volume is 10mL/kg, and the concentration of the drug which is required to be prepared by each siRNA conjugate is calculated according to the administration dosage and the administration volume.

Mice were sacrificed 7 days after administration, livers were collected and stored with RNA laters (Sigma Aldrich company); the liver tissue was then homogenized with a tissue homogenizer and total RNA was extracted using Trizol (Thermo Fisher Co.) according to standard procedures for total RNA extraction.

Detecting the expression quantity of FXII mRNA in the liver tissue by adopting real-time fluorescent quantitative PCR (polymerase chain reaction), specifically: the cDNA was obtained by reverse transcription using a reverse transcription kit (Promega corporation, cat. No. A3500) according to the protocol described in the specification. The detection of the expression amount of FXII mRNA was performed using SYBR Select Master Mix (Applied biosystem, cat # 4472897) kit with cDNA as a template according to the procedure of the specification, and the inhibition rate of the siRNA conjugate on the expression amount of FXII mRNA was calculated. Among them, PCR primers for amplifying FXII and GAPDH as reference genes are shown in Table 7.

The FXII mRNA expression level, i.e., the residual expression level, was calculated according to the following equation: FXII mRNA expression level ═ 100% (expression level of FXII mRNA in test group/expression level of GAPDH mRNA in test group)/(expression level of FXII mRNA in control group/expression level of GAPDH mRNA in control group).

The inhibition rate of the siRNA conjugate on the FXII mRNA expression level was (1-FXII mRNA expression level). times.100%. The control group was mice administered with PBS in this experiment, and each test group was mice administered with different siRNA conjugates.

FIG. 1 is a scattergram of FXII mRNA expression (relative to GAPDH as an internal reference) in liver tissue of C57 mice after administration of PBS (C57 mice) or different doses of conjugates 1, 2 or 3. As can be seen from fig. 1, at day 7 post-administration, the control sequence NC did not show any inhibition; meanwhile, the inhibition rate of siRNA conjugate 2 at 5mg/kg dose on FXII mRNA expression level was as high as 94.9%, and the inhibition rate of siRNA conjugate 2 at 1mg/kg dose on FXII mRNA expression level was also high at 74.8%; as for siRNA conjugate 3, a high inhibition rate of 96.7% was shown at a dose of 1mg/kg, and the inhibition rate of FXII mRNA expression at a dose of 5mg/kg was even as high as 98.7%; in addition, although siRNA conjugate 1 did not show a high inhibition rate of FXII mRNA expression in mice, it can be expected that it is highly likely to show a high inhibition rate of FXII mRNA expression in humans as well, considering that siRNA contained in the conjugate is siRNA corresponding to human FXII mRNA, and that siRNA conjugate 3 having the same target mRNA site as it shows unexpectedly high inhibitory activity.

(6-B) inhibition Rate of FXII mRNA expression amount in liver tissue by conjugates 1, 3, 4, 5 and 6 in C57 mice

In this experiment, the inhibition rate of the FXII mRNA expression level in liver tissues was examined for conjugates 1, 3, 4, 5 and 6 in C57 mice using the same experimental conditions and procedures as (6-a), except that the siRNA conjugates used were conjugates 1, 3, 4, 5 and 6, and 10 mice were used for the PBS group.

FIG. 2 is a scatter plot of FXII mRNA expression (relative to GAPDH as an internal reference) in liver tissue of C57 mice after administration of PBS or different doses of conjugates 1, 3, 4, 5 and 6 to C57 mice. As can be seen from the results of fig. 2, conjugate 1 and conjugate 3 showed similar inhibitory effects to (6-a), particularly conjugate 3 showed high inhibition rates of FXII mRNA expression of 95.4% and 97.4% at 1mg/kg and 5mg/kg doses, respectively; conjugates 4, 5 and 6 showed inhibition rates of FXII mRNA expression of 54.9%, 61.8% and 41.2% at the dose of 1mg/kg, respectively, and high inhibition rates of FXII mRNA expression of 83.9%, 87.0% and 83.8% at the dose of 3mg/kg, respectively.

From the results of the above experimental examples 4, 5, (6-A) and (6-B), it is clear that the siRNA and siRNA conjugate of the present disclosure showed excellent FXII mRNA inhibitory activity in both the in vitro human/mouse liver primary cell experiment and the mouse in vivo experiment.

Some embodiments of the present disclosure are described in detail above, however, the present disclosure is not limited to the specific details in the above embodiments, and many simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in some embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not further described.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Claims

An siRNA conjugate, said conjugate having a structure represented by formula (308):

wherein the content of the first and second substances,

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

each m1, m2 and m3 is independently an integer selected from 2 to 10;

each R₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄And R₁₅Each independently is H, or is selected from the group consisting of: c₁-C ₁₀Alkyl radical, C₁-C ₁₀Haloalkyl and C₁-C ₁₀An alkoxy group;

R ₃A group of the structure shown in formula a 59:

wherein E is₁Is OH, SH or BH₂；

Nu is siRNA, the siRNA has a sense strand and an antisense strand, each nucleotide in the siRNA is a modified or unmodified nucleotide independently, the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-strand region, wherein the nucleotide sequence I and the nucleotide sequence II are selected from the group consisting of I) to v),

i) the nucleotide sequence I has the same length with the nucleotide sequence shown in SEQ ID NO. 1 and has NO more than 3 nucleotide differences, the nucleotide sequence II has the same length with the nucleotide sequence shown in SEQ ID NO. 2 and has NO more than 3 nucleotide differences, and the nucleotide sequence I comprises a position corresponding to Z₁Nucleotide Z of₃The nucleotide sequence II comprises a position corresponding to Z₂Nucleotide Z of₄Z is the same as₄Is the first nucleotide at the 5' end of the antisense strand;

II) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 61 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 62 in length and has NO more than 3 nucleotide differences, wherein the nucleotide sequence I comprises a position corresponding to Z ₅Nucleotide Z of₇The nucleotide sequence II comprises a position corresponding to Z₆Nucleotide Z of₈Z is the same as₈Is the first nucleotide at the 5' end of the antisense strand;

iii) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 121 in length and has NO more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 122 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence I comprises a position corresponding to Z₁₃Nucleotide Z of₁₅The nucleotide sequence II comprises a position corresponding to Z₁₄Nucleotide Z of₁₆Z is the same as₁₆Is the first nucleotide at the 5' end of the antisense strand;

iv) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 181 and has NO more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 182 and has NO more than 3 nucleotide differences, and the position of the nucleotide sequence I is corresponding to Z₁₇Nucleotide Z of₁₉The nucleotide sequence II comprises a position corresponding to Z₁₈Nucleotide Z of₂₀Z is the same as₂₀Is the first nucleotide at the 5' end of the antisense strand;

v) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 241 in length and has NO more than 3 nucleotide differences, and the nucleus The nucleotide sequence II has the same length with the nucleotide sequence shown in SEQ ID NO. 242 and has NO more than 3 nucleotide differences, and the nucleotide sequence I comprises a position corresponding to Z₂₁Nucleotide Z of₂₃The nucleotide sequence II comprises a position corresponding to Z₂₂Nucleotide Z of₂₄Z is the same as₂₄Is the first nucleotide at the 5' end of the antisense strand;

R ₂is a straight chain 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、C ₂-C ₁₀Alkenylene radical, C₂-C ₁₀Alkynylene, C₆-C ₁₀Arylene radical, C₃-C ₁₈Heterocyclylene and C₅-C ₁₀A heteroarylene group; and wherein R₂May optionally have a substituent of any one or more of the group consisting of: c₁-C ₁₀Alkyl radical, C₆-C ₁₀Aryl radical, C₅-C ₁₀Heteroaryl group, C₁-C ₁₀Haloalkyl, -OC₁-C ₁₀Alkyl, -OC₁-C ₁₀Alkylphenyl, -C₁-C ₁₀alkyl-OH, -OC₁-C ₁₀Haloalkyl, -SC₁-C ₁₀Alkyl, -SC₁-C ₁₀Alkylphenyl, -C₁-C ₁₀alkyl-SH, -SC₁-C ₁₀Haloalkyl, halogen substituents, -OH, -SH, -NH₂、-C ₁-C ₁₀alkyl-NH₂、-N(C ₁-C ₁₀Alkyl) (C₁-C ₁₀Alkyl), -NH (C)₁-C ₁₀Alkyl), -N (C)₁-C ₁₀Alkyl) (C₁-C ₁₀Alkylphenyl), -NH (C)₁-C ₁₀Alkylphenyl), cyano, nitro, -CO₂H、-C(O)O(C ₁-C ₁₀Alkyl), -CON (C)₁-C ₁₀Alkyl) (C ₁-C ₁₀Alkyl), -CONH (C)₁-C ₁₀Alkyl), -CONH₂、-NHC(O)(C ₁-C ₁₀Alkyl), -NHC (O) (phenyl), -N (C)₁-C ₁₀Alkyl radical C (O) (C)₁-C ₁₀Alkyl), -N (C)₁-C ₁₀Alkyl group C (O) (phenyl), -C (O) C₁-C ₁₀Alkyl, -C (O) C₁-C ₁₀Alkylphenyl, -C (O) C₁-C ₁₀Haloalkyl, -OC (O) C₁-C ₁₀Alkyl, -SO₂(C ₁-C ₁₀Alkyl), -SO₂(phenyl), -SO₂(C ₁-C ₁₀Haloalkyl), -SO₂NH ₂、-SO ₂NH(C ₁-C ₁₀Alkyl), -SO₂NH (phenyl), -NHSO₂(C ₁-C ₁₀Alkyl), -NHSO₂(phenyl) and-NHSO₂(C ₁-C ₁₀Haloalkyl);

each L₁Independently a linear alkylene group of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH ═ N, S (O)₂、C ₂-C ₁₀Alkenylene radical, C₂-C ₁₀Alkynylene, C₆-C ₁₀Arylene radical, C₃-C ₁₈Heterocyclylene and C₅-C ₁₀A heteroarylene group; and wherein L₁May optionally have a substituent of any one or more of the group consisting of: c₁-C ₁₀Alkyl radical, C₆-C ₁₀Aryl radical, C₅-C ₁₀Heteroaryl group, C₁-C ₁₀Haloalkyl, -OC₁-C ₁₀Alkyl, -OC₁-C ₁₀Alkylphenyl, -C₁-C ₁₀alkyl-OH, -OC₁-C ₁₀Haloalkyl, -SC₁-C ₁₀Alkyl, -SC₁-C ₁₀Alkylphenyl, -C₁-C ₁₀alkyl-SH, -SC₁-C ₁₀Haloalkyl, halogen substituents, -OH, -SH, -NH₂、-C ₁-C ₁₀alkyl-NH₂、-N(C ₁-C ₁₀Alkyl) (C₁-C ₁₀Alkyl), -NH (C)₁-C ₁₀Alkyl), -N (C)₁-C ₁₀Alkyl) (C₁-C ₁₀Alkylphenyl), -NH (C) ₁-C ₁₀Alkylphenyl), cyano, nitro, -CO₂H、-C(O)O(C ₁-C ₁₀Alkyl), -CON (C)₁-C ₁₀Alkyl) (C₁-C ₁₀Alkyl), -CONH (C)₁-C ₁₀Alkyl), -CONH₂，-NHC(O)(C ₁-C ₁₀Alkyl), -NHC (O) (phenyl), -N (C)₁-C ₁₀Alkyl radical C (O) (C)₁-C ₁₀Alkyl), -N (C)₁-C ₁₀Alkyl group C (O) (phenyl), -C (O) C₁-C ₁₀Alkyl, -C (O) C₁-C ₁₀Alkylphenyl, -C (O) C₁-C ₁₀Haloalkyl, -OC (O) C₁-C ₁₀Alkyl, -SO₂(C ₁-C ₁₀Alkyl), -SO₂(phenyl), -SO₂(C ₁-C ₁₀Haloalkyl), -SO₂NH ₂、-SO ₂NH(C ₁-C ₁₀Alkyl), -SO₂NH (phenyl), -NHSO₂(C ₁-C ₁₀Alkyl), -NHSO₂(phenyl) and-NHSO₂(C ₁-C ₁₀Haloalkyl);

represents the site of covalent attachment of a group;

M ₁represents a targeting group.
The siRNA conjugate of claim 1, wherein each L is₁Independently selected from the group consisting of groups represented by formulas (A1) - (A26) and any combination thereof:

wherein each j1 is independently an integer from 1-20; each j2 is independently an integer from 1-20;

each R' is independently C1-C10 alkyl;

each Ra is selected from the group consisting of groups represented by formulas (a27) - (a45) and any combination thereof:

each Rb is independently C₁-C ₁₀An alkyl group;

or, L₁Is a combination of one or more of groups A1, A4, A5, A6, A8, A10, A11 and A13;

or, L₁Is a linked combination of at least 2 of the groups A1, A4, A8, A10 and A11;

or, L ₁Is a linked combination of at least 2 of the groups A1, A8, A10;

or, L₁Is 3-25 atoms in length;

or, L₁Is 4-15 atoms in length.
The siRNA conjugate of claim 2, wherein j1 is an integer from 2 to 10, j2 is an integer from 2 to 10, and R' is C₁-C ₄Alkyl, Ra is one of A27, A28, A29, A30 and A31, and Rb is C₁-C ₅An alkyl group;

or j1 is an integer of 3-5, j2 is an integer of 3-5, R' is one of methyl, ethyl and isopropyl, Ra is a group represented by formula (A27) or a group represented by formula (A28), and Rb is one of methyl, ethyl, isopropyl and butyl.
The siRNA conjugate according to any one of claims 1 to 3, wherein n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1+ n3 is 2 to 3.
The siRNA conjugate of any one of claims 1 to 4, wherein each of m1, m2 and m3 is independently an integer from 2 to 5, and/or m1 ═ m2 ═ m 3.
The siRNA conjugate of any of claims 1-5, wherein each of said targeting groups is independently a ligand that has affinity for asialoglycoprotein receptors on the surface of mammalian hepatocytes;

or each of said targeting groups is independently an asialoglycoprotein or a saccharide;

Or each of said targeting groups is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannofuranose, beta-D-mannofuranose, alpha-D-mannopyranose, beta-D-mannopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructopyranose, beta-D-galactopyranose, beta-D-galactopyranose, beta-fructooligosaccharides, and combinations thereof, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [ (R) -1-carboxyethyl ] -2-deoxy-beta-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, beta-galactofuranose, glucosamine, N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [ (R) -1-carboxyethyl ] -2-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4-dideoxy-4-carboxamido-2, 3-O-methyl-D-mannopyranose, D-glucopyranose, beta-galactopyranose, and, N-glycolyl-alpha-neuraminic acid, 5-thio-beta-D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranoside methyl ester, 4-thio-beta-D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio-alpha-D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allositrile, ribose, D-4-thioribose, L-ribose, L-4-thioribose;

Alternatively, at least one or each of the targeting groups is galactose or N-acetylgalactosamine.
The siRNA conjugate of any of claims 1-6, wherein each R₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄And R₁₅Independently H, methyl or ethyl;

or, each R₁₀、R ₁₁、R ₁₂、R ₁₃、R ₁₄And R₁₅Are all H.
The siRNA conjugate of any of claims 1-7, wherein R₂Containing both a linking site to the N atom of the nitrogen-containing skeleton and a linking site to R₃The attachment site to which the P atom in (a) is attached;

or, R₂The site to which the N atom of the nitrogen-containing skeleton is bonded forms an amide bond with the N atom, and the site forms an amide bond with R₃The site to which the P atom is attached forms a phosphoester bond with the P atom;

or, R₂Selected from the group consisting of those represented by the formula (B5), (B6), (B5') or (B6').
The siRNA conjugate according to any one of claims 1 to 8, wherein the conjugate has 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).
The siRNA conjugate of any of claims 1 to 9, wherein the P atom in formula a59 is attached to the end of the sense or antisense strand of the siRNA, said end referring to the first 4 nucleotides of said sense or antisense strand, taken from one end thereof;

Alternatively, the P atom in formula a59 is attached to the end of the sense or antisense strand of the siRNA; or the P atom in formula a59 is attached to the 3' end of the siRNA sense strand;

alternatively, the P atom in formula a59 is attached to the nucleotide in the siRNA at the 2' position, 3' position, or 5' position by forming a phosphodiester bond.
The siRNA conjugate of claim 1, wherein I) said nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 1 and/or said nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 2; or II) NO more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO. 61 and/or NO more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 62; or, iii) NO more than 1 nucleotide difference between said nucleotide sequence I and the nucleotide sequence depicted in SEQ ID NO. 121, and/or NO more than 1 nucleotide difference between said nucleotide sequence II and the nucleotide sequence depicted in SEQ ID NO. 122; or, iv) NO more than 1 nucleotide difference between said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO. 181, and/or NO more than 1 nucleotide difference between said nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 182; or v) NO more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO. 241 and/or NO more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 242.
The siRNA conjugate of claim 1, wherein i) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO. 2 comprises Z₄A difference at position, and Z₄Selected from A, C or G; or II) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:62 comprises Z₈A difference at position, and Z₈Selected from A, C or G; or iii) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 122 comprises Z₁₆A difference at position, and Z₁₆Selected from A, C or G; orIv) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 182 comprises Z₂₀A difference at position, and Z₂₀Selected from A, C or G; or v) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:242 comprises Z₂₄A difference at position, and Z₂₄Selected from A, C or G.
The siRNA conjugate of claim 12, wherein Z₃Is a reaction of with Z₄A complementary nucleotide; or Z₇Is a reaction of with Z₈A complementary nucleotide; or Z₁₅Is a reaction of with Z₁₆A complementary nucleotide; or Z₁₉Is a reaction of with Z₂₀A complementary nucleotide; or Z₂₃Is a reaction of with Z₂₄A complementary nucleotide.
The siRNA conjugate of any of claims 11 to 13, wherein said nucleotide sequence I and said nucleotide sequence II are substantially reverse complementary, or fully reverse complementary; by substantially reverse complementary is meant that no more than 3 base mismatches occur between two nucleotide sequences; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; the complete reverse complementarity means that there is no mismatch between the two nucleotide sequences.
The siRNA conjugate of any of claims 11 to 14, wherein said sense strand further comprises a nucleotide sequence III, said antisense strand further comprises a nucleotide sequence IV, said nucleotide sequence III and said nucleotide sequence IV being each independently 1 to 4 nucleotides in length, said nucleotide sequence III being linked at the 5 'end of nucleotide sequence I, said nucleotide sequence IV being linked at the 3' end of nucleotide sequence II, said nucleotide sequence III and said nucleotide sequence IV being of equal length and being substantially reverse complementary or fully reverse complementary; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; the complete reverse complementarity means that there is no mismatch between the two nucleotide sequences.
The siRNA conjugate of claim 15, wherein said nucleotide sequence I is equal to and NO more than 3 nucleotides different from the nucleotide sequence shown in SEQ ID NO. 1, and wherein said nucleotide sequences III and IV are each 1 nucleotide in length, and wherein the base of said nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is CA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is GCA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is CGCA according to the direction from the 5 'end to the 3' end;

Or the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 61 in length and has NO more than 3 nucleotide differences, the nucleotide sequences III and IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is AA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is GAA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is GGAA according to the direction from the 5 'end to the 3' end;

or the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 121 in length and has NO more than 3 nucleotide differences, the nucleotide sequences III and IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is U; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is UU according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is UUUU according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is GUUU according to the direction from the 5 'end to the 3' end;

Or the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 181 and has NO more than 3 nucleotide differences, the nucleotide sequences III and IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is G; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GG according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is UGG according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is UUGG according to the direction from the 5 'end to the 3' end;

or the nucleotide sequence I is equal to the nucleotide sequence shown by SEQ ID NO. 241 in length and has NO more than 3 nucleotide differences, the nucleotide sequences III and IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is C; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is AGC according to the direction from the 5 'end to the 3' end; or, the nucleotide sequences III and IV are both 4 nucleotides in length, and the base composition of the nucleotide sequence III is GAGC from the 5 'end to the 3' end.
The siRNA conjugate of claim 16, wherein said nucleotide sequences III and IV are fully reverse complementary.
The siRNA conjugate of any of claims 11 to 17, wherein said antisense strand further comprises a nucleotide sequence V, having a length of 1 to 3 nucleotides, attached at the 3 'end of said antisense strand to form the 3' overhang of the antisense strand; or the nucleotide sequence V is 2 nucleotides in length; or the nucleotide sequence V is two continuous thymine deoxyribonucleotides or two continuous uracil ribonucleotides; or the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA.
The siRNA conjugate according to any one of claims 11 to 18, wherein the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID No. 5 and the antisense strand comprises a nucleotide sequence shown as SEQ ID No. 6; or the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 7, and the antisense strand contains the nucleotide sequence shown as SEQ ID NO. 8; or the sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 65, and the antisense strand contains a nucleotide sequence shown as SEQ ID NO. 66; or the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 67, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 68; or the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 125, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 126; or the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 127, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 128; or the sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 185, and the antisense strand contains a nucleotide sequence shown as SEQ ID NO. 186; or the sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 187, and the antisense strand contains a nucleotide sequence shown as SEQ ID NO. 188; or the sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 245, and the antisense strand contains a nucleotide sequence shown as SEQ ID NO. 246; or the sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 247, and the antisense strand contains a nucleotide sequence shown as SEQ ID NO. 248.
The siRNA conjugate of any of claims 11 to 19, wherein said siRNA is siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1, or siFXIIf 2.
The siRNA conjugate of any of claims 11 to 20, wherein at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide and/or at least one phosphate group is a phosphate group having a modifying group.
The siRNA conjugate of claim 21, wherein each nucleotide in the sense strand and the antisense strand is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide;

or the fluorinated modified nucleotide is positioned in the nucleotide sequence I and the nucleotide sequence II, and the nucleotides at the 7 th, the 8 th and the 9 th positions of the nucleotide sequence I are fluorinated modified nucleotides according to the direction from the 5 'end to the 3' end; the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II are fluorine-modified nucleotides according to the direction from the 5 'end to the 3' end;

or according to the direction from 5 'end to 3' end, in the sense strand, the nucleotides at positions 7, 8 and 9 or 5, 7, 8 and 9 of the nucleotide sequence I are fluorine-modified nucleotides, and the nucleotides at the rest positions in the sense strand are non-fluorine-modified nucleotides; according to the direction from the 5 'end to the 3' end, in the antisense strand, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions or the nucleotides at the 2 nd, 6 th, 8 th, 9 th, 14 th and 16 th positions of the nucleotide sequence II are fluorine-modified nucleotides, and the nucleotides at the rest positions in the antisense strand are non-fluorine-modified nucleotides.
The siRNA conjugate according to claim 21 or 22, wherein each of the non-fluorinated modified nucleotides is independently selected from one of a nucleotide or a nucleotide analogue in which a hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorine group;

or the nucleotide formed by substituting the hydroxyl at the 2 '-position of the ribosyl of the nucleotide by a non-fluorine group is selected from one of 2' -alkoxy modified nucleotide, 2 '-substituted alkoxy modified nucleotide, 2' -alkyl modified nucleotide, 2 '-substituted alkyl modified nucleotide, 2' -amino modified nucleotide, 2 '-substituted amino modified nucleotide, 2' -deoxynucleotide; the nucleotide analogue is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA;

or each of the non-fluorinated modified nucleotides is a methoxy-modified nucleotide, which means a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group.
The siRNA conjugate according to any one of claims 21 to 23, wherein nucleotides at positions 5, 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides, in the direction from 5 'end to 3' end, and nucleotides at positions 2, 6, 8, 9, 14 and 16 of the nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides, and nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides, in the direction from 5 'end to 3' end;

Or, according to the direction from 5 'end to 3' end, the nucleotides at the 5 th, 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are fluorine modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy modified nucleotides, and, according to the direction from 5 'end to 3' end, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluorine modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy modified nucleotides;

or, according to the direction from 5 'end to 3' end, the nucleotides at the 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are-fluoro modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy modified nucleotides, and, according to the direction from 5 'end to 3' end, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluoro modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy modified nucleotides.
The siRNA conjugate of any one of claims 11-24, wherein said siRNA is any one of siFXIIa-M, siFXIIb-M, siFXIId-M, siFXIIe-M, siFXIIf-M, siFXf-M, siFXiif-M, siFXIIf-M.
The siRNA conjugate according to any one of claims 11 to 25, wherein the phosphate group having the modifying group is a phosphorothioate group in which at least one oxygen atom in a phosphodiester bond in the phosphate group is substituted with a sulfur atom; or the phosphate group with the modification group is a thiophosphate group with the structure shown in the formula (1):
the siRNA conjugate of claim 26, wherein in said siRNA, a phosphorothioate-based linkage is present at least one of the group consisting of:

between the 1 st and 2 nd nucleotides of the 5' terminus of the sense strand;

between the 2 nd and 3 rd nucleotides at the 5' end of the sense strand;

between the 1 st and 2 nd nucleotides of the 3' terminus of the sense strand;

between the 2 nd and 3 rd nucleotides at the 3' terminus of the sense strand;

between the 1 st and 2 nd nucleotides of the 5' terminus of the antisense strand;

between the 2 nd and 3 rd nucleotides of the 5' terminus of the antisense strand;

between the 1 st and 2 nd nucleotides of the 3' terminus of the antisense strand; and

between the 2 nd and 3 rd nucleotides of the 3' terminus of the antisense strand.
The siRNA conjugate according to any one of claims 11 to 27, wherein the siRNA is any one of siFXIIa-M1, siFXIIa-M2, siFXIIa-M3, siFXIIb-M1, siFXIIb-M2, siFXIIb-M3, siFXIId-M1, siIId-M2, siFXIId-M3, siFXIId-M1, siFXIId-M2, siFXIId-M3, siFXIIe-M1, siFXe-M2, siFXIIe-M3, siFXIIe-M1, siFXe-M2, siFXe-M3, siFXIIf-IIf-M1, siFXIIf-M2, siFXIIf-M3, siFXIIf-M1, siFXIIf-M2, siFXIIf-M3, siFXIIb-M1, siFXIIb-M2, siFXIIb-3, siFXIIf-3, siFXIIb-3, or siFXIIb-M1.
The siRNA conjugate of any of claims 11 to 28, wherein the 5' terminal nucleotide of the siRNA antisense strand is a 5' -phosphate nucleotide or a 5' -phosphate analogue modified nucleotide;

or the 5 '-phosphate nucleotide is a nucleotide with a structure shown in the formula (2), the 5' -phosphate analogue modified nucleotide is selected from nucleotides with a structure shown in any one of the formulas (3) to (6),

wherein R is selected from H, OH, methoxy or fluorine; base represents a nucleobase selected from A, U, C, G or T.
The siRNA conjugate according to any one of claims 11 to 29, wherein the siRNA is siFXIIa-M1P, siFXIIa-M2P, siFXIIa-M3P, siFXIIa-M1 SP, siFXIIa-M2 SP, siFXIIa-M3 SP, siFXIIa1-M1P, siFXIIa1-M2P, siFXIIa1-M3P, siFXIIa2-M1P, siFXIIa2-M2P, siFX2-M3P, siFXIIa1-M1SP, siFXIIa1-M3SP, siFXIIa2-M1SP, siFXIIa 2-IIB 2-IIM 2SP, siFXIIa 3-M3 SP, siFXIIa 2-M2-IIB, siFXIIB 3-Si-M1-M2 SP, siFXIIa, siFXIIB 3-Si-M3 SP, siFXIIa, siFXIIB-M2-M3 SP, siFXIIB, siFXIIa-M3 SP, siFXIIB, siFXIIa-M2-M3 SP, siFXIIB, siFXIIa, siFXIIB 3-M2-M3 SP, siFXIIB, siFXIIa, siFXIIB, siFXIIa-M3-M2-M3, siFXIIB, siFXIIa-M2-M3, siFXIIB, siFXIIa, siFXIIB IIB, siFXIIB IIB, siFXIIB IIB, siFXIIB, siFXII, One of siFXIId-M3P, siFXIId-M1P, siFXIId-M2P, siFXIId-M3P, siFXIId-M1 SP, siFXIId-M2 SP, siFXIId-M3 SP, siFXIIe-M1P, siFXIIe-M2P, siFXIIe-M3P, siFXIIe-M1 SP, siFXIIe-M2 SP, siFXIIe-M3 SP, siFXIIf-M1P, siFXIIf-M2 SP, siFXIIf-M3 SP, siFXIIf-Si-M3 SP, siFXIIf-Si-M3 SP, siFXIIf, siFXIIe-M3 SP, siFXIIe-M2 SP, siFXIIe-M3 SP, siFXIIf, siFXIIe-M3 SP, siFXIIf, siFXIIe-M3 SP, siFXIIf, siFXIIe-M3 SP, siFXIIe-M3 SP, siFXIIf, siFXIIe-M3 SP, siFXIIe, siFXIIf, siFXIIe-M3 SP, siFXIIe-M2 SP, siFXIIe-M3 SP, siFXIIe-M3 SP, siFXIIe-M3 SP, siFXIIe, si.
An siRNA comprising a sense strand and an antisense strand, each nucleotide in said sense strand and said antisense strand being independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide; the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, the fluorinated modified nucleotide is positioned in the nucleotide sequence I and the nucleotide sequence II, and according to the direction from 5 'end to 3' end, in the sense strand, the nucleotides at the 7 th, 8 th and 9 th positions of the nucleotide sequence I are fluorinated modified nucleotides, and the nucleotides at the rest positions in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5 'end to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14, 16 of the nucleotide sequence II are fluorine-modified nucleotides, the nucleotides at the remaining positions in the antisense strand are non-fluorine-modified nucleotides, and,

i) the nucleotide sequence I has the same length with the nucleotide sequence shown in SEQ ID NO. 1 and has NO more than 3 nucleotide differences, the nucleotide sequence II has the same length with the nucleotide sequence shown in SEQ ID NO. 2 and has NO more than 3 nucleotide differences, and the nucleotide sequence I comprises a position corresponding to Z ₁Nucleotide Z of₃The nucleotide sequence II comprises a position corresponding to Z₂Nucleotide Z of₄Z is the same as₄Is the first nucleotide at the 5' end of the antisense strand; alternatively, the first and second electrodes may be,

II) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 61 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 62 in length and has NO more than 3 nucleotide differences, wherein the nucleotide sequence I comprises a position corresponding to Z₅Nucleotide Z of₇The nucleotide sequence II comprises a position corresponding to Z₆Nucleotide Z of₈Z is the same as₈Is the first nucleotide at the 5' end of the antisense strand; alternatively, the first and second electrodes may be,

iii) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 121 in length and has NO more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 122 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence I comprises a position corresponding to Z₁₃Nucleotide Z of₁₅The nucleotide sequence II comprises a position corresponding to Z₁₄Nucleotide Z of₁₆Z is the same as₁₆Is the first nucleotide at the 5' end of the antisense strand; alternatively, the first and second electrodes may be,

iv) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO:181 in length and has NO more than 3 nucleotide differences In addition, the nucleotide sequence II has the same length with the nucleotide sequence shown in SEQ ID NO. 182 and has NO more than 3 nucleotide differences, and the nucleotide sequence I comprises a position corresponding to Z₁₇Nucleotide Z of₁₉The nucleotide sequence II comprises a position corresponding to Z₁₈Nucleotide Z of₂₀Z is the same as₂₀Is the first nucleotide at the 5' end of the antisense strand; alternatively, the first and second electrodes may be,

v) the nucleotide sequence I is equal to the nucleotide sequence shown by SEQ ID NO. 241 in length and has NO more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown by SEQ ID NO. 242 in length and has NO more than 3 nucleotide differences, and the position included in the nucleotide sequence I corresponds to Z₂₁Nucleotide Z of₂₃The nucleotide sequence II comprises a position corresponding to Z₂₂Nucleotide Z of₂₄Z is the same as₂₄Is the first nucleotide at the 5' end of the antisense strand.
The siRNA of claim 31, wherein each non-fluorinated modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group.
The siRNA of claim 32, wherein the nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide is substituted with a non-fluorine group is selected from one of 2' -alkoxy-modified nucleotide, 2 '-substituted alkoxy-modified nucleotide, 2' -alkyl-modified nucleotide, 2 '-substituted alkyl-modified nucleotide, 2' -amino-modified nucleotide, 2 '-substituted amino-modified nucleotide, 2' -deoxynucleotide; the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA.
The siRNA of any one of claims 31 to 33, wherein each of the non-fluorinated modified nucleotides is a methoxy modified nucleotide, which is a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group.
The siRNA of any one of claims 31-34, wherein I) said nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 1 and/or said nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 2; or II) NO more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO. 61 and/or NO more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 62; or, iii) NO more than 1 nucleotide difference between said nucleotide sequence I and the nucleotide sequence depicted in SEQ ID NO. 121, and/or NO more than 1 nucleotide difference between said nucleotide sequence II and the nucleotide sequence depicted in SEQ ID NO. 122; or, iv) NO more than 1 nucleotide difference between said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO. 181, and/or NO more than 1 nucleotide difference between said nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 182; or v) NO more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO. 241 and/or NO more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 242.
The siRNA of any of claims 31-35, wherein i) the nucleotide difference between said nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO. 2 comprises Z₄A difference at position, and Z₄Selected from A, C or G; or II) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:62 comprises Z₈A difference at position, and Z₈Selected from A, C or G; or iii) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 122 comprises Z₁₆A difference at position, and Z₁₆Selected from A, C or G; or iv) the nucleotide sequence II is identical to the nucleotide sequence of SEQ ID NO:182 comprises Z₂₀A difference at position, and Z₂₀Selected from A, C or G; or v) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:242 comprises Z₂₄A difference at position, and Z₂₄Selected from A, C or G.
The siRNA of claim 36, wherein Z is₃Is a reaction of with Z₄A complementary nucleotide; or Z₇Is a reaction of with Z₈A complementary nucleotide; or Z₁₅Is a reaction of with Z₁₆A complementary nucleotide; or Z₁₉Is a reaction of with Z₂₀A complementary nucleotide; or Z₂₃Is a reaction of with Z₂₄A complementary nucleotide.
The siRNA of any of claims 31 to 37, wherein said nucleotide sequence I and said nucleotide sequence II are substantially reverse complementary, or fully reverse complementary; by substantially reverse complementary is meant that no more than 3 base mismatches occur between two nucleotide sequences; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; the complete reverse complementarity means that there is no mismatch between the two nucleotide sequences.
The siRNA of any of claims 31 to 38, wherein said sense strand further comprises a nucleotide sequence III and said antisense strand further comprises a nucleotide sequence IV, said nucleotide sequence III and said nucleotide sequence IV being each independently 1 to 4 nucleotides in length, said nucleotide sequence III being linked at the 5 'end of nucleotide sequence I and said nucleotide sequence IV being linked at the 3' end of nucleotide sequence II, said nucleotide sequence III and said nucleotide sequence IV being of equal length and being substantially reverse complementary or fully reverse complementary; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; the complete reverse complementarity means that there is no mismatch between the two nucleotide sequences.
The siRNA of any one of claims 31-39, wherein said nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO. 1 and differs by NO more than 3 nucleotides, and wherein said nucleotide sequences III and IV are each 1 nucleotide in length and the base of said nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is CA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is GCA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is CGCA according to the direction from the 5 'end to the 3' end;

Or the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 61 in length and has NO more than 3 nucleotide differences, the nucleotide sequences III and IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is AA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is GAA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is GGAA according to the direction from the 5 'end to the 3' end;

or the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 121 in length and has NO more than 3 nucleotide differences, the nucleotide sequences III and IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is U; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is UU according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is UUUU according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is GUUU according to the direction from the 5 'end to the 3' end;

Or the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 181 and has NO more than 3 nucleotide differences, the nucleotide sequences III and IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is G; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GG according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is UGG according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is UUGG according to the direction from the 5 'end to the 3' end;

or the nucleotide sequence I is equal to the nucleotide sequence shown by SEQ ID NO. 241 in length and has NO more than 3 nucleotide differences, the nucleotide sequences III and IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is C; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is AGC according to the direction from the 5 'end to the 3' end; or, the nucleotide sequences III and IV are both 4 nucleotides in length, and the base composition of the nucleotide sequence III is GAGC from the 5 'end to the 3' end.
The siRNA of claim 40, wherein said nucleotide sequences III and IV are fully reverse complementary.
The siRNA of any of claims 31-40, wherein said antisense strand further comprises a nucleotide sequence V, having a length of 1 to 3 nucleotides, attached to the 3 'terminus of said antisense strand to form the 3' overhang of the antisense strand; or the nucleotide sequence V is 2 nucleotides in length; or the nucleotide sequence V is two continuous thymine deoxyribonucleotides or two continuous uracil ribonucleotides; or the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA.
The siRNA of any of claims 31-42, wherein the sense strand of said siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 5 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO. 6; or the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 7, and the antisense strand contains the nucleotide sequence shown as SEQ ID NO. 8; or the sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 65, and the antisense strand contains a nucleotide sequence shown as SEQ ID NO. 66; or the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 67, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 68; or the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 125, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 126; or the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 127, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 128; or the sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 185, and the antisense strand contains a nucleotide sequence shown as SEQ ID NO. 186; or the sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 187, and the antisense strand contains a nucleotide sequence shown as SEQ ID NO. 188; or the sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 245, and the antisense strand contains a nucleotide sequence shown as SEQ ID NO. 246; or the sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 247, and the antisense strand contains a nucleotide sequence shown as SEQ ID NO. 248.
The siRNA of any of claims 31-43, wherein said siRNA has the nucleotide sequence of siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1, or siFXIIf 2.
The siRNA of any one of claims 31-44, wherein said siRNA is any one of siFXIIa1-M1, siFXIIa2-M1, siFXIIb1-M1, siFXIIb2-M1, siFXIId1-M1, siFXIId2-M1, siFXIIe1-M1, siFXIIe2-M1, siFXIIf1-M1, or siFXIIf 2-M1.
The siRNA of any of claims 31-45, wherein at least one phosphate group in said sense strand or said antisense strand is a phosphate group having a modifying group.
The siRNA of claim 46, wherein said phosphate group having a modifying group is a phosphorothioate group in which at least one oxygen atom in a phosphodiester bond in the phosphate group is replaced by a sulfur atom; or the phosphate group with the modification group is a thiophosphate group with the structure shown in the formula (1):
the siRNA of claim 47, wherein in said siRNA, a phosphorothioate-based linkage is present at least one position in the group consisting of:

Between the 1 st and 2 nd nucleotides of the 5' terminus of the sense strand;

between the 2 nd and 3 rd nucleotides at the 5' end of the sense strand;

between the 1 st and 2 nd nucleotides of the 3' terminus of the sense strand;

between the 2 nd and 3 rd nucleotides at the 3' terminus of the sense strand;

between the 1 st and 2 nd nucleotides of the 5' terminus of the antisense strand;

between the 2 nd and 3 rd nucleotides of the 5' terminus of the antisense strand;

between the 1 st and 2 nd nucleotides of the 3' terminus of the antisense strand; and

between the 2 nd and 3 rd nucleotides of the 3' terminus of the antisense strand.
The siRNA of any of claims 31-48, wherein said siRNA is any of siFXIIa1-M1S, siFXIIa2-M1S, siFXIIb1-M1S, siFXIIb2-M1S, siFXIId1-M1S, siFXIId2-M1S, siFXIIe1-M1S, siFXIIe2-M1S, siFXIIf1-M1S, siFXIIf 2-M1S.
The siRNA of any of claims 31-49, wherein the 5' terminal nucleotide of the antisense strand of said siRNA is a nucleotide that is 5' -phosphate or a nucleotide modified by a 5' -phosphate analog;

or the 5 '-phosphate nucleotide is a nucleotide with a structure shown in the formula (2), the 5' -phosphate analogue modified nucleotide is selected from nucleotides with a structure shown in any one of the formulas (3) to (6),

Wherein R is selected from H, OH, methoxy or fluorine; base represents a nucleobase selected from A, U, C, G or T.
The siRNA of any one of claims 31-50, wherein said siRNA is selected from siFXIIa1-M1P1, siFXIIa2-M1P1, siFXIIa1 1-M1P1, siFXIIa2 1-M1P1, siFXIIa1 1-M1SP1, siFXIIa2 1-M1SP1, siFXIIb1-M1P1, siFXIId1-M1, siFXIIe1-M1P1, siFXIIf1-M1, siFXIIa 1-SP 1P1, siFXIIb 36IIP 1, siFXIIB 36IIP 1, siFXIIB 36IIB 1, siFXIIB1-M1, siFXIIB 1-1, siFXIIB 36IIB 1, siFXIIB 363636IIB 1, siFXIIB 36IIB 1, siFXIIB 36IIB 1, siFXIIB 36IIB 1, siFXIIB 36IIB 1, siFXIIB 36IIB 1, siFXIIB1-M1, siFXIIB1, or siFXIIB 1-1.
A pharmaceutical composition comprising the siRNA of any one of claims 31-51 and a pharmaceutically acceptable carrier.
An siRNA conjugate comprising an siRNA of any one of claims 31 to 51 and a conjugate group conjugated to the siRNA.
Use of an siRNA conjugate according to any of claims 1 to 30 and 53, an siRNA according to any of claims 31 to 51 and/or a pharmaceutical composition according to claim 52 for the manufacture of a medicament for the treatment and/or prevention of hereditary angioedema HAE and/or thrombosis.
A method of treating and/or preventing hereditary angioedema HAE and/or thrombosis, wherein the method comprises administering to a subject having hereditary angioedema HAE an effective amount of the siRNA conjugate of any one of claims 1 to 30 and 53, the siRNA of any one of claims 31 to 51 and/or the pharmaceutical composition of claim 52.
A method of inhibiting FXII gene expression in a hepatocyte, the method comprising contacting the hepatocyte with an effective amount of an siRNA conjugate of any one of claims 1-30 and 53, an siRNA of any one of claims 31-51 and/or a pharmaceutical composition of claim 52.
A kit comprising an siRNA conjugate according to any one of claims 1 to 30 and 53, an siRNA according to any one of claims 31 to 51 and/or a pharmaceutical composition according to claim 52.