CN113330117B

CN113330117B - Nucleic acid, composition containing nucleic acid, conjugate, preparation method and application

Info

Publication number: CN113330117B
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: 2024-05-28
Anticipated expiration: 2040-01-17
Also published as: CN118562796A; WO2020147847A1; CN113330117A

Abstract

The present disclosure provides an siRNA that inhibits the expression of Factor XII (FXII) gene, pharmaceutical compositions and conjugates containing the siRNA. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, the siRNA comprising a sense strand and an antisense strand, the sense strand comprising nucleotide sequence I equal in length to the nucleotide sequence set forth in SEQ ID No. 1 and not more than 3 nucleotides different, the antisense strand comprising nucleotide sequence II equal in length to the nucleotide sequence set forth in SEQ ID No. 2 and not more than 3 nucleotides different. The siRNA, the pharmaceutical compositions and the conjugates thereof provided by the present disclosure can be effective in treating and/or preventing 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 a nucleic acid capable of inhibiting factor XII gene expression 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, face, intestines and airways. Attacks may be spontaneous or may be caused by physical trauma or stress. Laryngeal (airway) oedema can be life threatening as it can lead to asphyxia death.

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

Factor XII is one of the key targets for the treatment of HAE. By inhibiting factor XII expression, the occurrence of HAE can be effectively inhibited. Thus, blocking factor XII production would certainly be the most desirable therapeutic approach if gene expression could be silenced from the gene level. The small interfering RNA (SMALL INTERFERING RNA, SIRNA) can inhibit or block the expression of any gene of interest in a sequence-specific manner based on the mechanism of RNA interference (RNA INTERFERENCE, RNAI), thereby achieving the goal of treating the disease.

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

Disclosure of Invention

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

Wherein,

N1 is an integer selected from 1-3, n3 is an integer selected from 0-4;

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

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

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

wherein E ₁ is OH, SH or BH ₂;

Nu is an 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 stretch of nucleotide sequence I and the antisense strand comprising a stretch of nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I and the nucleotide sequence II are selected from the group of I) -v):

i) The nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 1 in length and is not more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 2 in length and is not more than 3 nucleotide differences, the nucleotide sequence I comprises a nucleotide Z ₃ with a position corresponding to Z ₁, the nucleotide sequence II comprises a nucleotide Z ₄ with a position corresponding to Z ₂, and the Z ₄ is the first nucleotide at the 5' -end of the antisense strand;

II) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO. 61 and differs by NO more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO. 62 and differs by NO more than 3 nucleotides, the nucleotide sequence I comprises a nucleotide Z ₇ corresponding in position to Z ₅, the nucleotide sequence II comprises a nucleotide Z ₈ corresponding in position to Z ₆, and the Z ₈ is 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 is not more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 122 in length and is not more than 3 nucleotide differences, the nucleotide sequence I comprises a nucleotide Z ₁₅ with a position corresponding to Z ₁₃, the nucleotide sequence II comprises a nucleotide Z ₁₆ with a position corresponding to Z ₁₄, and the Z ₁₆ is the first nucleotide at the 5' -end of the antisense strand;

iv) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:181 and differs by NO more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:182 and differs by NO more than 3 nucleotides, the nucleotide sequence I comprises a nucleotide Z ₁₉ corresponding in position to Z ₁₇, the nucleotide sequence II comprises a nucleotide Z ₂₀ corresponding in position to Z ₁₈, and the Z ₂₀ is the first nucleotide at the 5' -end of the antisense strand;

v) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO. 241 and differs by NO more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO. 242 and differs by NO more than 3 nucleotides, the nucleotide sequence I comprises a nucleotide Z ₂₃ corresponding in position to Z ₂₁, the nucleotide sequence II comprises a nucleotide Z ₂₄ corresponding in position to Z ₂₂, and the Z ₂₄ is the first nucleotide at the 5' -end of the antisense strand;

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

Each L ₁ is independently 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、C₂-C₁₀ alkenylene, C ₂-C₁₀ alkynylene, C ₆-C₁₀ arylene, C ₃-C₁₈ heterocyclylene, and C ₅-C₁₀ heteroarylene; and wherein L ₁ may optionally have substituents of any one or more of the group consisting of: c ₁-C₁₀ alkyl, C ₆-C₁₀ aryl, C ₅-C₁₀ heteroaryl, C ₁-C₁₀ haloalkyl, -OC ₁-C₁₀ alkyl, -OC ₁-C₁₀ alkylphenyl-C ₁-C₁₀ alkyl-OH, -OC ₁-C₁₀ haloalkyl, -SC ₁-C₁₀ alkyl, -SC ₁-C₁₀ alkylphenyl, -C ₁-C₁₀ alkyl-SH, -SC ₁-C₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂、-C₁-C₁₀ alkyl-NH ₂、-N(C₁-C₁₀ alkyl) (C ₁-C₁₀ alkyl), -NH (C ₁-C₁₀ alkyl), -N (C ₁-C₁₀ alkyl) (C ₁-C₁₀ alkylphenyl), -NH (C ₁-C₁₀ alkylphenyl), cyano, nitro, -CO ₂H、-C(O)O(C₁-C₁₀ alkyl), -CON (C ₁-C₁₀ alkyl) (C ₁-C₁₀ alkyl), -CONH (C ₁-C₁₀ alkyl), -CONH ₂,-NHC(O)(C₁-C₁₀ alkyl), -NHC (O) (phenyl), -N (C ₁-C₁₀ alkyl) C (O) (C ₁-C₁₀ alkyl), -N (C ₁-C₁₀ alkyl) C (O) (phenyl), -C (O) C ₁-C₁₀ alkyl, -C (O) C ₁-C₁₀ alkylphenyl, -C (O) C ₁-C₁₀ haloalkyl, -OC (O) C ₁-C₁₀ alkyl, -SO ₂(C₁-C₁₀ alkyl), -SO ₂ (phenyl), -SO ₂(C₁-C₁₀ haloalkyl), -SO ₂NH₂、-SO₂NH(C₁-C₁₀ alkyl), -SO ₂ NH (phenyl), -NHSO ₂(C₁-C₁₀ alkyl), -NHSO ₂ (phenyl) and-NHSO ₂(C₁-C₁₀ haloalkyl);

Represents the site of covalent attachment of the group;

M ₁ represents 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 reversely complementary to form a double-stranded region, the fluoro-modified nucleotide is positioned in the nucleotide sequence I and the nucleotide sequence II, and the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I are fluoro-modified nucleotides in the sense strand according to the direction from the 5 '-end to the 3' -end, and the nucleotides at the rest positions in the sense strand are non-fluoro-modified nucleotides; in the direction from the 5 '-end to the 3' -end, in the antisense strand, the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II are fluoro-modified nucleotides, the nucleotides at the remaining positions in the antisense strand are non-fluoro-modified nucleotides, and

I) The nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 1 in length and is not more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 2 in length and is not more than 3 nucleotide differences, the nucleotide sequence I comprises a nucleotide Z ₃ with a position corresponding to Z ₁, the nucleotide sequence II comprises a nucleotide Z ₄ with a position corresponding to Z ₂, and the Z ₄ is the first nucleotide at the 5' -end of the antisense strand; or alternatively

II) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO. 61 and differs by NO more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO. 62 and differs by NO more than 3 nucleotides, the nucleotide sequence I comprises a nucleotide Z ₇ corresponding in position to Z ₅, the nucleotide sequence II comprises a nucleotide Z ₈ corresponding in position to Z ₆, and the Z ₈ is the first nucleotide at the 5' -end of the antisense strand; or alternatively

Iii) The nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 121 in length and is not more than 3 nucleotide differences, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 122 in length and is not more than 3 nucleotide differences, the nucleotide sequence I comprises a nucleotide Z ₁₅ with a position corresponding to Z ₁₃, the nucleotide sequence II comprises a nucleotide Z ₁₆ with a position corresponding to Z ₁₄, and the Z ₁₆ is the first nucleotide at the 5' -end of the antisense strand; or alternatively

Iv) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:181 and differs by NO more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:182 and differs by NO more than 3 nucleotides, the nucleotide sequence I comprises a nucleotide Z ₁₉ corresponding in position to Z ₁₇, the nucleotide sequence II comprises a nucleotide Z ₂₀ corresponding in position to Z ₁₈, and the Z ₂₀ is the first nucleotide at the 5' -end of the antisense strand; or alternatively

V) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO. 241 and differs by NO more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO. 242 and differs by NO more than 3 nucleotides, the nucleotide sequence I comprises a nucleotide Z ₂₃ corresponding in position to Z ₂₁, the nucleotide sequence II comprises a nucleotide Z ₂₄ corresponding in position to Z ₂₂, and the Z ₂₄ is 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 the siRNA and/or pharmaceutical compositions and/or siRNA conjugates 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 an effective amount of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure to a subject having HAE.

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

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

Incorporated by reference

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

Advantageous effects

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

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

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

In some embodiments, the presently provided siRNA, compositions comprising the siRNA, or siRNA conjugates do not exhibit significant off-target effects. The off-target effect may be, for example, inhibition of normal gene expression of non-target genes. It is believed that the off-target effect is not significant if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20% or 10% compared to the effect at the target gene.

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

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

Drawings

FIGS. 1 and 2 are scatter plots of FXII mRNA expression levels (relative values with GAPDH as an internal reference) of liver tissue of C57 mice after administration of PBS and various doses of each conjugate, respectively.

Detailed Description

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

In the present disclosure, FXII mRNA refers to a sequence as shown in Genbank accession No. nm_ 000505.3. Further, unless otherwise indicated, the term "target gene" as 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 the definition

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

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

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

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

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

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

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

As used herein, a short dash ("-") that is not between two letters or between two symbols is used to indicate a point of attachment for a substituent. For example: the leftmost short traverse in the structural formula "-C ₁-C₁₀ alkyl-NH ₂" means that 66 is linked via a C ₁-C₁₀ alkyl group.

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. Those skilled in the art will appreciate that for any group comprising one or more substituents, these groups are not intended to introduce any substitution or pattern of substitution that is sterically impractical, synthetically infeasible, and/or inherently unstable.

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

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

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

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

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

As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring, typically having 3 to 7 ring carbon atoms. The ring 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 (norbornane).

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 a specified number of carbon atoms are replaced with one or more up to the maximum allowable number of halogen atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl and pentafluoroethyl.

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

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

Various hydroxyl protecting groups may be used in the present disclosure. In general, the protecting group renders the chemical functional group insensitive to specific reaction conditions and can be added and removed from the functional group in the 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 Greeneand 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 alkaline conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Dimethoxytrityl (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, such as a mammal or a pouched animal. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any variety of poultry.

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

As used herein, "prevent" and "prevent" are used interchangeably. These terms refer to methods of achieving a beneficial or desired result, including but not limited to prophylactic benefit. To obtain a "prophylactic benefit," the conjugate or composition may be administered to a subject at risk of suffering from a particular disease, or to a subject reporting one or more physiological symptoms of the disease, even though a diagnosis of the disease may not have been made.

The siRNA of the present disclosure contains 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, and is not described 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 independently a modified or unmodified nucleotide, wherein 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 reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:1 and is not more than 3 nucleotides different, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:2 and is not more than 3 nucleotides different:

5'-GGAACUCAAUAAAGUGCUZ₁-3'(SEQ ID NO:1)；

5'-Z₂AGCACUUUAUUGAGUUCC-3'(SEQ ID NO:2)，

Wherein Z ₁ is U, Z ₂ is A,

And, the nucleotide sequence I comprises a nucleotide Z ₃ with a position corresponding to Z ₁, the nucleotide sequence II comprises a nucleotide Z ₄ with a position corresponding to Z ₂, and the Z ₄ is the first nucleotide at the 5' -end of the antisense strand.

In the above and in the following, "position correspondence" means that the same position in the nucleotide sequence is located from the same end of the nucleotide sequence. For example, nucleotide 1 at the 3 'end of nucleotide sequence I is the nucleotide corresponding in position to nucleotide 1 at 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 from the nucleotide sequence set forth in SEQ ID NO. 1 by NO more than 1 nucleotide, and/or the nucleotide sequence II differs from the nucleotide sequence set forth in SEQ ID NO. 2 by NO more than 1 nucleotide.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO. 2 comprises a difference at position Z ₄, and Z ₄ is selected from U, C or G. In some embodiments, the nucleotide difference is a difference at position Z ₄, Z ₄ is selected from U, C or G. In some embodiments, Z ₃ is a nucleotide complementary to Z ₄. These nucleotide differences do not significantly reduce the target gene inhibition capacity of the siRNA conjugates, and siRNA conjugates comprising these nucleotide differences 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 complement" 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 there is no more than 1 base mismatch between two nucleotide sequences; "complete reverse complement" 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 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 4:

5'-GGAACUCAAUAAAGUGCUZ₃-3'(SEQ ID NO:3)；

5'-Z₄AGCACUUUAUUGAGUUCC-3'(SEQ ID NO:4)，

Wherein, Z ₄ is the first nucleotide at the 5' end of the antisense strand, Z ₃ is selected from A, U, G or C, and Z ₄ is a nucleotide complementary to Z ₃; in some embodiments, Z ₃ is U and Z ₄ is a;

and the length of the sense strand and the length of 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, the antisense strand further comprises nucleotide sequence IV, nucleotide sequence III and nucleotide sequence IV each 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, and the nucleotide sequence IV is connected to 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 and is the same length as the nucleotide sequence IV in the target mRNA.

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

In some embodiments, nucleotide sequence III and nucleotide sequence IV are fully reverse-complementary, thus, the base of nucleotide sequence III is given, and the base of nucleotide sequence IV is also determined.

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 being independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II, which are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:61 and is not more than 3 nucleotides different, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:62 and is not more than 3 nucleotides different:

5'-CUCAAUAAAGUGCUUUGAZ₅-3'(SEQ ID NO:61)；

5'-Z₆UCAAAGCACUUUAUUGAG-3'(SEQ ID NO:62)，

Wherein Z ₅ is A and Z ₆ is U;

And, the nucleotide sequence I comprises a nucleotide Z ₇ with a position corresponding to Z ₅, the nucleotide sequence II comprises a nucleotide Z ₈ with a position corresponding to Z ₆, and the Z ₈ is the first nucleotide at the 5' -end of the antisense strand.

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

In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO. 62 comprises a difference at position Z ₈, and Z ₈ is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at position Z ₈, Z ₈ is selected from A, C or G. In some embodiments, Z ₇ is a nucleotide complementary to Z ₈. These nucleotide differences do not significantly reduce the target gene inhibition capacity of the siRNA conjugates, and these siRNA conjugates comprising the nucleotide differences 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.

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

5'-CUCAAUAAAGUGCUUUGAZ₇-3'(SEQ ID NO:63)；

5'-Z₈UCAAAGCACUUUAUUGAG-3'(SEQ ID NO:64)，

Wherein, Z ₈ is the first nucleotide at the 5' end of the antisense strand, Z ₇ is selected from A, U, G or C, and Z ₈ is a nucleotide complementary to Z ₇; in some embodiments, Z ₇ is a and Z ₈ is U;

In some embodiments, the sense strand further comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, nucleotide sequence III and nucleotide sequence IV each 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 linked at the 5' end of the nucleotide sequence I, the nucleotide sequence IV is linked at the 3' end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse-complementary or completely reverse-complementary to a second nucleotide sequence adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 61 and having the same length as the nucleotide sequence IV in the target mRNA.

In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are each 1 nucleotide in length in the 5'-3' direction, the base of nucleotide sequence III is a, and the base of nucleotide sequence IV is U; at this time, the length ratio of the sense strand to the antisense strand was 20/20; or the length of the nucleotide sequence III and the nucleotide sequence 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; at this time, the length ratio of the sense strand to the antisense strand was 21/21; or the length of the nucleotide sequence III and the nucleotide sequence 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; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or the length of the nucleotide sequence III and the nucleotide sequence 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; at this time, 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, the base composition of the nucleotide sequence III is AA and the base composition of the nucleotide sequence IV is UU in the 5 'end to 3' end direction; at this time, the length ratio of the sense strand to the antisense strand was 21/21.

Third 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 being independently a modified or unmodified nucleotide, wherein the sense strand comprises a stretch of nucleotide sequence I and the antisense strand comprises a stretch of nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence set forth in SEQ ID NO:121 and is NO more than 3 nucleotides different, and the nucleotide sequence II is equal in length to the nucleotide sequence set forth in SEQ ID NO:122 and is NO more than 3 nucleotides different:

5'-GGAGCCCAAGAAAGUGAAZ₁₃-3'(SEQ ID NO:121)；

5'-Z₁₄UUCACUUUCUUGGGCUCC-3'(SEQ ID NO:122)，

Wherein Z ₁₃ is A and Z ₁₄ is U;

And, the nucleotide sequence I comprises a nucleotide Z ₁₅ with a position corresponding to Z ₁₃, the nucleotide sequence II comprises a nucleotide Z ₁₆ with a position corresponding to Z ₁₄, and the Z ₁₆ is the first nucleotide at the 5' -end of the antisense strand.

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

In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO. 122 comprises a difference at position Z ₁₆, and Z ₁₆ is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at position Z ₁₆, Z ₁₆ is selected from A, C or G. In some embodiments, Z ₁₅ is a nucleotide complementary to Z ₁₆. These nucleotide differences do not significantly reduce the target gene inhibition capacity of the siRNA conjugates, and these 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 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 124:

5'-GGAGCCCAAGAAAGUGAAZ₁₅-3'(SEQ ID NO:123)；

5'-Z₁₆UUCACUUUCUUGGGCUCC-3'(SEQ ID NO:124)，

Wherein, Z ₁₆ is the first nucleotide at the 5' end of the antisense strand, Z ₁₅ is selected from A, U, G or C, and Z ₁₆ is a nucleotide complementary to Z ₁₅; in some embodiments, Z ₁₅ is a and Z ₁₆ is U;

In some embodiments, the sense strand further comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, nucleotide sequence III and nucleotide sequence IV each 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 linked at the 5' end of the nucleotide sequence I, the nucleotide sequence IV is linked at the 3' end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse-complementary or completely reverse-complementary to a second nucleotide sequence 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 nucleotide sequence III and the nucleotide sequence IV are each 1 nucleotide in length in the 5'-3' direction, the base of nucleotide sequence III is U and the base of nucleotide sequence IV is a; at this time, the length ratio of the sense strand to the antisense strand was 20/20; or the length of the nucleotide sequence III and the nucleotide sequence 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; at this time, the length ratio of the sense strand to the antisense strand was 21/21; or the length of the nucleotide sequence III and the nucleotide sequence IV is 3 nucleotides, the base composition of the nucleotide sequence III is UU according to the direction from the 5 'end to the 3' end, and the base composition of the nucleotide sequence IV is AAA; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or the length of the nucleotide sequence III and the nucleotide sequence 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; at this time, 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, the base composition of the nucleotide sequence III is AA and the base composition of the nucleotide sequence IV is UU in the 5 'end to 3' end direction; at this time, the length ratio of the sense strand to the antisense strand was 21/21.

Fourth siRNA

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

The fifth siRNA comprises a sense strand and an antisense strand, each nucleotide in the fifth siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II that are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:181 and is not more than 3 nucleotides different, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:182 and is not more than 3 nucleotides different:

5'-AGCCCAAGAAAGUGAAAGZ₁₇ -3'(SEQ ID NO:181)；

5'-Z₁₈CUUUCACUUUCUUGGGCU-3'(SEQ ID NO:182)，

wherein Z ₁₇ is A, Z ₁₈ is U,

And, the nucleotide sequence I comprises a nucleotide Z ₁₉ with a position corresponding to Z ₁₇, the nucleotide sequence II comprises a nucleotide Z ₂₀ with a position corresponding to Z ₁₈, and the Z ₂₀ is the first nucleotide at the 5' -end of the antisense strand.

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

In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO. 182 includes a difference at position Z ₂₀, and Z ₂₀ is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at position Z ₂₀, Z ₂₀ is selected from A, C or G. In some embodiments, Z ₁₉ is a nucleotide complementary to Z ₂₀. These nucleotide differences do not significantly reduce the target gene inhibition capacity of the siRNA conjugates, and these 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 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 184:

5'-AGCCCAAGAAAGUGAAAGZ₁₉ -3'(SEQ ID NO:183)；

5'-Z₂₀CUUUCACUUUCUUGGGCU-3'(SEQ ID NO:184)，

Wherein, Z ₂₀ is the first nucleotide at the 5' end of the antisense strand, Z ₁₉ is selected from A, U, G or C, and Z ₂₀ is a nucleotide complementary to Z ₁₉; in some embodiments, Z ₁₉ is a and Z ₂₀ is U;

In some embodiments, the sense strand further comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, nucleotide sequence III and nucleotide sequence IV each 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 linked at the 5' end of the nucleotide sequence I, the nucleotide sequence IV is linked at the 3' end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse-complementary or completely reverse-complementary to a second nucleotide sequence adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO:181 and having the same length as the nucleotide sequence IV in the target mRNA.

In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are each 1 nucleotide in length in the 5'-3' direction, the base of nucleotide sequence III is G, and the base of nucleotide sequence IV is C; at this time, the length ratio of the sense strand to the antisense strand was 20/20; or the length of the nucleotide sequence III and the nucleotide sequence 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; at this time, the length ratio of the sense strand to the antisense strand was 21/21; or the length of the nucleotide sequence III and the nucleotide sequence 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; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or the length of the nucleotide sequence III and the nucleotide sequence IV is 4 nucleotides, the base composition of the nucleotide sequence III is UUGG, and the base composition of the nucleotide sequence IV is CCAA according to the direction from the 5 'end to the 3' end; at this time, 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, the base composition of the nucleotide sequence III is GG and the base composition of the nucleotide sequence IV is CC in the 5 'end to 3' end direction; at this time, 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 being independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II that are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:241 and is not more than 3 nucleotides different, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:242 and is not more than 3 nucleotides different:

5'-CCAAGAAAGUGAAAGACCZ₂₁ -3'(SEQ ID NO:241)；

5'-Z₂₂GGUCUUUCACUUUCUUGG-3'(SEQ ID NO:242)，

wherein Z ₂₁ is A and Z ₂₂ is U;

The nucleotide sequence I comprises a nucleotide Z ₂₃ which corresponds to the position Z ₂₁, the nucleotide sequence II comprises a nucleotide Z ₂₄ which corresponds to the position Z ₂₂, and the Z ₂₄ is the first nucleotide at the 5' -end of the antisense strand.

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

In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:242 comprises a difference at position Z ₂₄, and Z ₂₄ is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at position Z ₂₄, Z ₂₄ is selected from A, C or G. In some embodiments, Z ₂₃ is a nucleotide complementary to Z ₂₄. These nucleotide differences do not significantly reduce the target gene inhibition capacity of the siRNA conjugates, and these 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 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 304:

5'-CCAAGAAAGUGAAAGACCZ₂₃ -3'(SEQ ID NO:243)；

5'-Z₂₄GGUCUUUCACUUUCUUGG-3'(SEQ ID NO:244)，

Wherein, Z ₁₆ is the first nucleotide at the 5' end of the antisense strand, Z ₂₃ is selected from A, U, G or C, and Z ₂₄ is a nucleotide complementary to Z ₂₃; in some embodiments, Z ₂₃ is a and Z ₂₄ is U;

In some embodiments, the sense strand further comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, nucleotide sequence III and nucleotide sequence IV each 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 linked at the 5' end of the nucleotide sequence I, the nucleotide sequence IV is linked at the 3' end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse-complementary or completely reverse-complementary to a second nucleotide sequence adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO 241 in the target mRNA and has the same length as the nucleotide sequence IV.

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

Overhang ends and modifications 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 above-described first to fifth sirnas. That is, the following description of the sirnas shall be regarded as describing the first, second, third, fourth, and fifth sirnas one by one, if not specified. For example, unless specifically indicated as a specific siRNA, "the siRNA further comprises the nucleotide sequence V" means "the first siRNA, the second siRNA, the third siRNA, the fourth siRNA, or the fifth siRNA further comprises the nucleotide sequence V".

In some embodiments, the sense strand and the antisense strand are different in length, the antisense strand further comprising a nucleotide sequence V of 1 to 3 nucleotides in length attached to the 3 'end of the antisense strand, constituting a 3' overhang of the antisense strand. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure can be 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 length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure may be 19/21, 21/23 or 23/25.

Each of the nucleotides in the nucleotide sequence V may be any nucleotide, and in order to facilitate synthesis and save synthesis costs, the nucleotide sequence V is a continuous 2 thymine deoxyribonucleotides (dTdT) or a continuous 2 uracil ribonucleotides (UU); or to increase the affinity of the antisense strand of the siRNA to the target mRNA, the nucleotide sequence V is complementary to a nucleotide at the corresponding position of the target mRNA. Thus, in some embodiments, the ratio of the length of the sense strand to the antisense strand of the siRNA of the present disclosure is 19/21 or 21/23, at which time 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 stretch of the target mRNA, which stretch of the target mRNA is substantially reverse-complementary or completely reverse-complementary to the nucleotide sequence II or to the 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 a nucleotide sequence as set forth in SEQ ID NO:5 and the antisense strand of the siRNA comprises a nucleotide sequence as set forth in SEQ ID NO: 6:

5'-GGAACUCAAUAAAGUGCUZ₃ -3'(SEQ ID NO:5)；

5'-Z₄AGCACUUUAUUGAGUUCCUG-3'(SEQ ID NO:6)；

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

5'-CAGGAACUCAAUAAAGUGCUZ₃ -3'(SEQ ID NO:7)；

5'-Z₄AGCACUUUAUUGAGUUCCUGCG-3'(SEQ ID NO:8)；

Wherein, Z ₄ is the first nucleotide at the 5' end of the antisense strand, Z ₃ is selected from A, U, G or C, and Z ₄ is a nucleotide complementary to Z ₃.

In some embodiments, for the second siRNA, the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO:65 and the antisense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 66:

5'-CUCAAUAAAGUGCUUUGAZ₇ -3'(SEQ ID NO:65)；

5'-Z₈UCAAAGCACUUUAUUGAGUU-3'(SEQ ID NO:66)，

Or the sense strand of the siRNA comprises a nucleotide sequence as shown in SEQ ID NO. 67, and the antisense strand of the siRNA comprises a nucleotide sequence as shown in SEQ ID NO. 68:

5'-AACUCAAUAAAGUGCUUUGAZ₇ -3'(SEQ ID NO:67)；

5'-Z₈UCAAAGCACUUUAUUGAGUUCC-3'(SEQ ID NO:68)，

Wherein, Z ₈ is the first nucleotide at the 5' end of the antisense strand, Z ₇ is selected from A, U, G or C, and Z ₈ is a nucleotide complementary to Z ₇.

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

5'-GGAGCCCAAGAAAGUGAAZ₁₅-3'(SEQ ID NO:125)；

5'-Z₁₆UUCACUUUCUUGGGCUCCAA-3'(SEQ ID NO:126)，

Or the sense strand of the siRNA comprises a nucleotide sequence as shown in SEQ ID NO:127, and the antisense strand of the siRNA comprises a nucleotide sequence as shown in SEQ ID NO: 128:

5'-UUGGAGCCCAAGAAAGUGAAZ₁₅-3'(SEQ ID NO:127)；

5'-Z₁₆UUCACUUUCUUGGGCUCCAAAC-3'(SEQ ID NO:128)，

Wherein, Z ₁₆ is the first nucleotide at the 5' end of the antisense strand, Z ₁₅ is selected from A, U, G or C, and Z ₁₆ is a nucleotide complementary to Z ₁₅.

In some embodiments, for the fourth siRNA, the sense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO:185 and the antisense strand of the siRNA comprises the nucleotide sequence shown as SEQ ID NO: 186:

5'-AGCCCAAGAAAGUGAAAGZ₁₉ -3'(SEQ ID NO:185)；

5'-Z₂₀CUUUCACUUUCUUGGGCUCC-3'(SEQ ID NO:186)；

Or the sense strand of the siRNA comprises a nucleotide sequence as shown in SEQ ID NO. 187, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO. 188:

5'-GGAGCCCAAGAAAGUGAAAGZ₁₉ -3'(SEQ ID NO:187)；

5'-Z₂₀CUUUCACUUUCUUGGGCUCCAA-3'(SEQ ID NO:188)；

Wherein, Z ₂₀ is the first nucleotide at the 5' end of the antisense strand, Z ₁₉ is selected from A, U, G or C, and Z ₂₀ is a nucleotide complementary to Z ₁₉.

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

5'-CCAAGAAAGUGAAAGACCZ₂₃ -3'(SEQ ID NO:245)；

5'-Z₂₄GGUCUUUCACUUUCUUGGGC-3'(SEQ ID NO:246)；

Or the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO:247, and the antisense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO: 248:

5'-GCCCAAGAAAGUGAAAGACCZ₂₃ -3'(SEQ ID NO:247)；

5'-Z₂₄GGUCUUUCACUUUCUUGGGCUC-3'(SEQ ID NO:248)；

Wherein, Z ₂₄ is the first nucleotide at the 5' end of the antisense strand, Z ₂₃ is selected from A, U, G or C, and Z ₂₄ is a nucleotide complementary to Z ₂₃.

In some embodiments, the siRNA of the present disclosure is siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1, or siFXIIf2:

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 nucleobase sequence) set forth in siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1, or siFXIIf.

As previously described, the nucleotides in the sirnas of the present disclosure are each independently a modified or unmodified nucleotide. In some embodiments, the nucleotides in the siRNA of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNA 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 siRNA of the present disclosure contains 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 analogue 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 nucleotide does not result in a significant impairment or loss of function of the siRNA to inhibit gene expression. For example, the modified nucleotide disclosed in J.K.Watts,G.F.Deleavey,and M.J.Damha,Chemically modified siRNA:tools and applications.Drug Discov Today,2008,13(19-20):842-55 can be selected.

In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate group having a modification group; in other words, at least a portion of the phosphate groups and/or ribose groups in at least one single-stranded phosphate-sugar backbone in the sense strand and the antisense strand are phosphate groups and/or ribose 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 siRNA provided by the present disclosure is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

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

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

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

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

In the context of the present disclosure, a "fluoro-modified nucleotide" refers to a nucleotide formed by substitution of the hydroxyl group at the 2' -position of the ribosyl of the nucleotide with fluorine, which has a structure represented by the following formula (7). "non-fluoro modified nucleotide" refers to a nucleotide, or nucleotide analogue, in which the hydroxyl group at the 2' -position of the ribosyl of the nucleotide is substituted with a non-fluoro group. In some embodiments, each non-fluoro modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by substitution of the hydroxyl group at the 2' position of the ribosyl of the nucleotide with a non-fluoro group.

Nucleotides in which the hydroxyl group at the 2 '-position of the ribosyl group is substituted with a non-fluorine group are well known to those skilled in the art and may be selected from one of 2' -alkoxy-modified nucleotides, 2 '-substituted alkoxy-modified nucleotides, 2' -alkyl-modified nucleotides, 2 '-substituted alkyl-modified nucleotides, 2' -amino-modified nucleotides, 2 '-substituted amino-modified nucleotides, 2' -deoxynucleotides.

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 may be, for example, a 2' -O-methoxyethyl-modified nucleotide (2 ' -MOE), as shown in formula (9). In some embodiments, the 2 '-amino modified nucleotide (2' -NH ₂) is represented by formula (10). In some embodiments, the 2' -Deoxynucleotide (DNA) is represented by formula (11):

nucleotide analogs refer to groups that are capable of replacing nucleotides in a nucleic acid, but that differ in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, the nucleotide analog may be an iso-nucleotide, a bridged nucleotide (bridged nucleic acid, abbreviated BNA), or an acyclic nucleotide.

BNA refers to a constrained or inaccessible nucleotide. BNA may contain a five-, six-, or seven-membered ring bridging structure with "fixed" C3' -endo-saccharides tucked. The bridge is typically incorporated at the 2'-, 4' -position of the ribose to provide a 2',4' -BNA nucleotide. In some embodiments, the BNA may be LNA, ENA, cET BNA, etc., wherein LNA is shown as formula (12), ENA is shown as formula (13), cET BNA is shown as formula (14):

acyclic nucleotides are a class of nucleotides in which the sugar ring of a nucleotide is opened. In some embodiments, the acyclic nucleotide can be an Unlocking 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 formula (15) and formula (16), R is selected from H, OH or alkoxy (O-alkyl).

An isopucleotide refers to a compound in which the position of a base on the ribose ring is changed in a nucleotide. In some embodiments, the isonucleotide may be a compound formed by a base moving 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 compounds of the formulae (7) - (18), base represents a nucleobase, for example A, U, G, C or T; r is selected from H, OH, F or a non-fluorine group as described above.

In some embodiments, the nucleotide analog is selected from one of an iso-nucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluoro modified nucleotide is a methoxy modified nucleotide, which in the foregoing and below refers to a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with a methoxy group.

In the above and in the following, the meaning of "fluoro modified nucleotide", "2 '-fluoro modified nucleotide", "nucleotide with 2' -hydroxyl of ribose group substituted by fluoro" and "nucleotide with 2 '-fluoro ribose group" are the same, and all refer to a compound having a structure as shown in formula (7) formed by substituting 2' -hydroxyl of nucleotide by fluoro; "methoxy-modified nucleotide", "2 '-methoxy-modified nucleotide", "nucleotide in which the 2' -hydroxyl group of the ribose group is replaced by methoxy" and "nucleotide having a2 '-methoxyribosyl" are the same in meaning, and refer to a compound having a structure shown in formula (8) in which the 2' -hydroxyl group of the ribosyl group of the nucleotide is replaced by methoxy.

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

In some embodiments, the siRNA of the present disclosure is an siRNA with modifications of: the nucleotides at positions 5,7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the nucleotides at the rest of the sense strand of the siRNA are methoxy-modified nucleotides, and the 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, the nucleotides at the rest of the antisense strand of the siRNA are methoxy-modified nucleotides in the 5 'to 3' end direction;

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

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

In some embodiments, the present disclosure provides an siRNA of any one of siFXIIa1-M1、siFXIIa1-M2、siFXIIa1-M3、siFXIIa2-M1、siFXIIa2-M2、siFXIIa2-M3、siFXIIb1-M1、siFXIIb1-M2、siFXIIb1-M3、siFXIIb2-M1、siFXIIb2-M2、siFXIIb2-M3、siFXIId1-M1、siFXIId1-M2、siFXIId1-M3、siFXIId2-M1、siFXIId2-M2、siFXIId2-M3、siFXIIe1-M1、siFXIIe1-M2、siFXIIe1-M3、siFXIIe2-M1、siFXIIe2-M2、siFXIIe2-M3、siFXIIf1-M1、siFXIIf1-M2、siFXIIf1-M3、siFXIIf2-M1、siFXIIf2-M2 and siFXIIf 2-M3:

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

The siRNA with the modification has low cost, and can ensure that ribonuclease in blood is not easy to cut nucleic acid, thereby increasing the stability of the nucleic acid and ensuring that the nucleic acid has stronger performance of resisting nuclease hydrolysis. Meanwhile, the above modification does not significantly reduce the inhibition performance of siRNA.

In some embodiments, at least a portion or at least 1 of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA provided by the present disclosure are phosphate groups having a modifying group. In some embodiments, the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of the 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):

This modification stabilizes the double-stranded structure of the siRNA, maintaining high specificity and high affinity for base pairing.

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

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

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

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

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

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

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

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

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

In some embodiments, the present disclosure provides an siRNA of any one of siFXIIa1-M1S、siFXIIa1-M2S、siFXIIa1-M3S、siFXIIa2-M1S、siFXIIa2-M2S、siFXIIa2-M3S、siFXIIb1-M1S、siFXIIb1-M2S、siFXIIb1-M3S、siFXIIb2-M1S、siFXIIb2-M2S、siFXIIb2-M3S、siFXIId1-M1S、siFXIId1-M2S、siFXIId1-M3S、siFXIId2-M1S、siFXIId2-M2S、siFXIId2-M3S、siFXIIe1-M1S、siFXIIe1-M2S、siFXIIe1-M3S、siFXIIe2-M1S、siFXIIe2-M2S、siFXIIe2-M3S、siFXIIf1-M1S、siFXIIf1-M2S、siFXIIf1-M3S、siFXIIf2-M1S、siFXIIf2-M2S and siFXIIf 2-M3S:

Wherein, the capital letter C, G, U, A represents the base composition of the nucleotide; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; lower case letter s indicates that there is a phosphorothioate linkage between the two nucleotides around the letter.

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

Commonly used nucleotides modified by such 5' -phosphonucleotides or 5' -phosphoanalogs are well known to those skilled in the art, e.g., the 5' -phosphonucleotides may have a structure as shown in the following formula (2):

as further disclosed in ,Anastasia Khvorova and Jonathan K.Watts,The chemical evolution of oligonucleotide therapies of clinical utility.Nature Biotechnology,2017,35(3):238-48 are the following 4 5' -phosphate analog modified nucleotides:

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 5 '-phosphate nucleotide is a nucleotide comprising a 5' -phosphate modification shown in formula (2), the 5 '-phosphate analogue modified nucleotide is a nucleotide comprising a vinyl phosphate (5' - (E) -vinylphosphonate, E-VP) modification, as shown in formula (3), or is a phosphorothioate modified nucleotide, as shown in formula (5).

In some embodiments, the present disclosure provides an siRNA of any one of siFXIIa1-M1P1、siFXIIa1-M2P1、siFXIIa1-M3P1、siFXIIa2-M1P1、siFXIIa2-M2P1、siFXIIa2-M3P1、siFXIIa1-M1SP1、siFXIIa1-M2SP1、siFXIIa1-M3SP1、siFXIIa2-M1SP1、siFXIIa2-M2SP1、siFXIIa2-M3SP11、siFXIIa1U-M1P1、siFXIIa1U-M2P1、siFXIIa1U-M3P1、siFXIIa2U-M1P1、siFXIIa2U-M2P11、siFXIIa2U-M3P1、siFXIIa1U-M1SP1、siFXIIa1U-M2SP1、siFXIIa1U-M3SP1、siFXIIa2U-M1SP1、siFXIIa2U-M2SP1、siFXIIa2U-M3SP1、siFXIIb1-M1P1、siFXIIb1-M2P1、siFXIIb1-M3P11、siFXIIb2-M1P1、siFXIIb2-M2P1、siFXIIb2-M3P1、siFXIIb1-M1SP1、siFXIIb1-M2SP1、siFXIIb1-M3SP1、siFXIIb2-M1SP1、siFXIIb2-M2SP1、siFXIIb2-M3SP1、siFXIId1-M1P1、siFXIId1-M2P1、siFXIId1-M3P1、siFXIId2-M1P1、siFXIId2-M2P1、siFXIId2-M3P1、siFXIId1-M1SP1、siFXIId1-M2SP1、siFXIId1-M3SP1、siFXIId2-M1SP1、siFXIId2-M2SP1、siFXIId2-M3SP1、siFXIIe1-M1P1、siFXIIe1-M2P1、siFXIIe1-M3P1、siFXIIe2-M1P1、siFXIIe2-M2P1、siFXIIe2-M3P1、siFXIIe1-M1SP1、siFXIIe1-M2SP1、siFXIIe1-M3SP1、siFXIIe2-M1SP1、siFXIIe2-M2SP1、siFXIIe2-M3SP1、siFXIIf1-M1P1、siFXIIf1-M2P1、siFXIIf1-M3P1、siFXIIf2-M1P1、siFXIIf2-M2P1、siFXIIf2-M3P1、siFXIIf1-M1SP1、siFXIIf1-M2SP1、siFXIIf1-M3SP1、siFXIIf2-M1 SP1、siFXIIf2-M2SP1 and siFXIIf2-M3SP 1:

Wherein, the capital letter C, G, U, A represents the base composition of the nucleotide; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; lowercase letters s represent phosphorothioate linkages between the left and right nucleotides of the letter; p1 represents that one nucleotide adjacent to the right of P1 is a 5 '-phosphonucleotide or a 5' -phosphoanalog modified nucleotide. In some embodiments, P1 is VP, ps, or P representing a particular modification, wherein the letter combination VP represents that one nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate (5 '- (E) -vinylphosphonate, E-VP) 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.

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

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

Pharmaceutical composition

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

The pharmaceutically acceptable carrier may be a carrier conventionally used in the siRNA administration field, for example, but not limited to, magnetic nanoparticles (magnetic nanoparticles such as Fe ₃O₄ or Fe ₂O₃ -based nanoparticles), carbon nanotubes (carbon nanotubes), mesoporous silicon (mesoporous silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), polyethylenimine (PEI), polyamide dendrimers (polyamidoamine (PAMAM) dendrimers), polylysine (L-lysine), PLL), chitosan (chitosan), 1,2-dioleoyl-3-trimethylammonium propane (1, 2-dioleoyl-3-trimethylammonium-propane, DOTAP), poly D-or L-lactic/glycolic acid copolymer (PLGA), poly (aminoethylphosphate) (2-aminoethyl ethylene phosphate), PPEEA) and poly (methylethyl methacrylate) (2-dimethylaminoethyl methacrylate), and one or more of them.

In some embodiments, the amount of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly limited, and in some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier may be 1 (1-500), and in some embodiments, the weight ratio is 1 (1-50).

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

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

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

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

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

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

In some embodiments, the organic amine may be a compound as depicted in formula (201) described in CN103380113a, or a pharmaceutically acceptable salt thereof:

Wherein:

Each X ₁₀₁ and X ₁₀₂ is independently O, S, N-A or C-A, wherein A is hydrogen or se:Sub>A C1-C20 hydrocarbon chain;

Each Y ₁₀₁ and Z ₁₀₁ is independently c= O, C = S, S = O, CH-OH or SO ₂;

Each of R ₁₀₁、R₁₀₂、R₁₀₃、R₁₀₄、R₁₀₅、R₁₀₆ and R ₁₀₇ is independently hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic group, a substituted or unsubstituted, branched or straight chain acyl group, a substituted or unsubstituted, branched or straight chain aryl group, a substituted or unsubstituted, branched or straight chain heteroaryl group;

x is an integer from 1 to 10;

n is an integer from 1 to 3, m is an integer from 0 to 20, and p is 0 or 1; wherein, if m=p=0, then R ₁₀₂ is hydrogen;

and, if at least one of n or m is 2, then R ₁₀₃ and the nitrogen in formula (201) form 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 of N represents a nitrogen atom in formula (201).

In some embodiments, R ₁₀₃ is a polyamine. In other embodiments, R ₁₀₃ is a ketal. In some embodiments, each of R ₁₀₁ and R ₁₀₂ in formula (201) 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 R ₁₀₃ can be any of the following formulas (204) - (213):

Wherein in formulae (204) - (213), g, e and f are each independently integers from 1 to 6, each "HCC" represents a hydrocarbon chain, and each shows a possible point of attachment of R ₁₀₃ to the nitrogen atom in formula (201), wherein each H at any of the positions may be replaced to effect attachment to the nitrogen atom in formula (201).

Wherein the compound of formula (201) may be prepared according to the description in CN103380113 a.

In some embodiments, the organic amine is an organic amine as shown in formula (214) and/or an organic amine as shown in formula (215):

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

The polyethylene glycol lipid is 1, 2-dipalmitoyl amide-sn-glycerin-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 particles of the pharmaceutical composition formed from the siRNA of the present disclosure and the amine-containing transfection reagent described above have an average diameter of about 30nm to about 200nm, typically about 40nm to about 135nm, more typically the average diameter of the liposome particles is about 50nm to about 120nm, about 50nm to about 100nm, about 60nm to about 90nm, or about 70nm to about 90nm, e.g., the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160nm.

In some embodiments, the weight ratio (weight/weight ratio) of siRNA to total lipid (e.g., organic amine, helper lipid, and/or pegylated lipid) in a pharmaceutical composition formed from an siRNA of the present disclosure and an amine-containing transfection reagent as described above is in the range of from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, e.g., the weight ratio of 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 components of the pharmaceutical composition may be present independently at the point of sale and may be present in liquid formulations at the point of use. In some embodiments, the pharmaceutical compositions formed by the sirnas provided by the present disclosure and the pharmaceutically acceptable carriers described above can be prepared according to various known methods, except that the sirnas provided by the present disclosure are used instead of the existing sirnas; in some embodiments, it may be prepared as follows:

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

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

Mixing the lipid solution with siRNA water solution, and incubating the mixed product at 40-60deg.C for at least 2 min, such as 5-30 min, to obtain liposome preparation after incubation. 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 physical and chemical parameters are that the pH value is 6.5-8, the encapsulation efficiency is not lower than 80%, the particle size is 40-200nm, the polydispersity index is not higher than 0.30, and the osmotic pressure is 250-400mOsm/kg; for example, the physical and chemical parameters can be pH 7.2-7.6, encapsulation efficiency not lower than 90%, particle size 60-100nm, polydispersity index not higher than 0.20, and osmotic pressure 300-400mOsm/kg.

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

SiRNA conjugates

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

In general, the conjugate group comprises at least one pharmaceutically acceptable targeting group and optionally a linker (linker), and the siRNA, the linker and the targeting group are sequentially linked. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The 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 conjugation group may be at the 3' end or 5' end of the sense strand of the siRNA, at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugation 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 2' -hydroxyl group, or the base of the nucleotide. In some embodiments, the conjugate group may also be attached to the 3' -hydroxyl group, in which case the nucleotides are linked using a 2' -5' phosphodiester linkage. When a conjugate group is attached to the end of 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 connection modes can be referred to in the literature ：Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.ACS Chemical biology,2015,10(5):1181-7.

In some embodiments, the siRNA and the conjugate group may be linked by acid labile, or reducible, chemical bonds that degrade in the acidic environment of the intracellular inclusion bodies, thereby allowing the siRNA to be in a free state. For non-degradable conjugation, the 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 disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the pharmaceutically acceptable targeting group may be selected from one or more of the following ligands formed by the targeting molecule or derivative thereof: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g. vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as permeabilizing peptides; an aptamer; an antibody; a quantum dot; sugars, such as lactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by hepatic parenchymal cells, such as asialoglycoproteins, asialoglycoresidues, lipoproteins (e.g., high density lipoproteins, low density lipoproteins, etc.), glucagon, neurotransmitters (e.g., epinephrine), growth factors, transferrin, etc.

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 liver surface asialoglycoprotein receptor (ASGPR). The class of these ligands is well known to those skilled in the art and generally functions to bind to specific receptors on the surface of target cells, mediating delivery of siRNA linked to the ligand to the target cells.

In some embodiments, the pharmaceutically acceptable targeting group may be any ligand that binds to an asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently an asialoglycoprotein, such as an asialoglycoprotein serogroup mucin (asialoorosomucoid, ASOR) or an asialogfetuin (asialofetuin, 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, mannans 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 may be independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylose furanose, L-xylose furanose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannopyranose, beta-D-glucopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructofuranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-galactosamine, N-isobutyramide, 2-amino-O-3-carboxyethyl-2-deoxy2-D-deoxygalactopyranose, 2-deoxy2-D-deoxygalactopyranose, 4-D-deoxy2-deoxygalactopyranose 2-deoxy-2-sulphonamino-D-glucopyranose, N-glycolyl- α -neuraminic acid, 5-thio- β -D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl- α -D-glucopyranoside methyl ester, 4-thio- β -D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio- α -D-glucoheptopyranoside ethyl ester, 2, 5-anhydro-D-allose nitrile, ribose, D-4-thioribose, L-ribose or L-4-thioribose. Other choices of the ligand may be found in, for example, 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 may be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule may be monovalent, divalent, trivalent, tetravalent. It should be understood that monovalent, divalent, trivalent, tetravalent, as described herein, refer to the molar ratio of siRNA groups to galactose or N-acetylgalactosamine groups in the siRNA conjugate after the siRNA molecules form an siRNA conjugate with a conjugated molecule containing galactose or N-acetylgalactosamine groups as targeting groups, respectively, being 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 conjugate group comprising N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, the N-acetylgalactosamine molecule is trivalent when the siRNA described in the present disclosure is conjugated to a conjugate group comprising N-acetylgalactosamine.

The targeting group can be attached to the siRNA molecule via a suitable linker, which can be selected by one skilled in the art depending on the particular type of targeting group. The types of these linkers, targeting groups, and attachment to the siRNA can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.

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

Wherein,

K is an integer of 1 to 3;

L ^A is a chain moiety comprising an amide linkage having the structure shown in formula (302), each of said L ^A being linked at both ends thereof to one of said targeting group and said L ^C moiety, respectively, via an ether linkage:

L ^B is a N-acyl pyrrolidine-containing chain moiety having a structure as shown in formula (303) having a carbonyl group at one end thereof and linked to the L ^C moiety through an amide bond and an oxy group at the other end thereof and linked to the siRNA through a phosphate bond:

L ^C is a 2-4 valent linking group based on hydroxymethyl aminomethane, dihydroxymethyl aminomethane or trimethylol aminomethane, said L ^C being linked to each of said L ^A moieties via an oxygen atom by an ether linkage and to said L ^B moiety via a nitrogen atom by an amide linkage.

In some embodiments, when n=3, L ^C is a trimethylol aminomethane-based 4-valent linking group, an siRNA conjugate formed from a- (L ^A)₃ trimethylol aminomethane-L ^B) -linked N-acetylgalactosamine molecule and an siRNA molecule as a linker, having the structure shown in formula (304) below:

in the formula, the double helix structure represents siRNA.

Likewise, the conjugation site of the siRNA to the conjugation group may be at the 3' end or 5' end of the sense strand of the siRNA, at the 5' end of the antisense strand, or in the internal sequence of the siRNA.

In some embodiments, the 3' -terminus of the sense strand of the siRNA of the present disclosure is covalently conjugated to three N-acetylgalactosamine (GalNAc) molecules via a linker- (L ^A)₃ -tris-L ^B), resulting in an siRNA conjugate having a molar ratio of siRNA molecules to GalNAc molecules of 1:3, which may also be referred to hereinafter as (GalNAc) ₃ -siRNA, the structure of which is shown in formula (305) below:

wherein the duplex 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,

L is an integer of 0 to 3;

^* Represents the site on the linker that is linked to the targeting group through an ether linkage;

^# Indicating the site on the linker that is linked to the siRNA via a phosphoester linkage.

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

The conjugates described above can be synthesized by methods already described in detail in the prior art. For example, the preparation of various conjugates is described in detail in WO2015006740 A2. The siRNA conjugates of the present disclosure are obtained by means well known to those skilled in the art. A method for preparing the structure represented by formula (305) is described in WO2014025805A1, and Rajeev et al in ChemBioChem 2015,16,903-908 describes a method for preparing the structure represented by formula (307).

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

Wherein:

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

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

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

Wherein E ₁ is OH, SH or BH ₂, and Nu is siRNA of the present disclosure;

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

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

In some embodiments, L ₁ may be selected from the group consisting of A1-A26 groups, or any combination thereof, wherein the structures and definitions of A1-A26 are as follows:

Wherein j1 is an integer of 1 to 20; j2 is an integer from 1 to 20;

R' is C ₁-C₁₀ alkyl;

Ra is selected from the group consisting of groups of formulae A27-A45 or any combination thereof:

Rb is C ₁-C₁₀ alkyl; indicating the site of covalent attachment of the group.

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

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

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

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

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

R ₃ is a group of the structure shown in formula A59, wherein E ₁ is OH, SH or BH ₂, and in some embodiments E ₁ is OH or SH, based on ease of availability of the starting materials for preparation.

R ₂ is selected to achieve a bond with the N atom on the nitrogen-containing backbone to A59. In the context of the present disclosure, a "nitrogen-containing backbone" refers to a chain structure in which the carbon atoms to which R ₁₀、R₁₁、R₁₂、R₁₃、R₁₄ and R ₁₅ are attached are interconnected with an N atom. Thus, R ₂ can be any linking group capable of linking the a59 group to the N atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, where the siRNA conjugate of formula (308) is prepared by a solid phase synthesis process, it is desirable for the R ₂ group to contain both a linking site to the N atom on the nitrogen-containing backbone and a linking site to the P atom in R ₃. In some embodiments, the site in R ₂ attached to the N atom on the nitrogen-containing backbone forms an amide bond with N and the site attached to the P atom on R ₃ forms a phosphate bond with the P atom; in some embodiments, R ₂ may be B5, B6, B5', or B6':

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

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

L ₁ is used for connecting an M ₁ targeting group with an N atom on a nitrogen-containing framework, and provides liver targeting function for the siRNA conjugate shown in the formula (308). In some embodiments, L ₁ is selected from a linked combination of one or more of the groups of formulas A1-A26. In some embodiments, L ₁ is selected from the group consisting of A1, A4, A5, A6, A8, a10, a11, and a 13. In some embodiments, L ₁ is selected from the group consisting of a linked combination of at least 2 of A1, A4, A8, a10, and a 11. In some embodiments, L ₁ is selected from the group consisting of a linked combination of at least 2 of A1, A8, a 10.

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

In some embodiments, j1 is an integer from 2 to 10, and in some embodiments, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3 to 5. R 'is C ₁-C₄ alkyl, in some embodiments, R' is one of methyl, ethyl, and isopropyl. Ra is one of a27, a28, a29, a30, and a31, and in some embodiments Ra is a27 or a28.Rb is a C ₁-C₅ alkyl group, in 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 N-linking of the M ₁ targeting group to the nitrogen-containing backbone and to make the spatial position between the M ₁ targeting group more suitable for binding of the M ₁ targeting group 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, e.g., the P atom in formula a59 can be attached to any one of the nucleotides of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula a59 is attached to any one nucleotide of the sense strand of the siRNA. In some embodiments, the P atom in formula a59 is attached to the end of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula a59 is attached to the end of the sense strand of the siRNA. The end refers to the first 4 nucleotides from one end of the sense strand or the antisense strand. In some embodiments, the P atom in formula a59 is attached to the end of the sense strand or the antisense strand of the siRNA; in some embodiments, the P atom in formula a59 is attached to the 3' end of the sense strand of the siRNA. In the case of the above-described position of the sense strand attached to the siRNA, the siRNA conjugate represented by formula (308) may release the separate siRNA antisense strand upon unwinding to block the process of translation of FXII mRNA into protein and inhibit Factor XII (FXII) gene expression after entering the cell.

In some embodiments, the P atom in formula a59 can be attached to any possible position on the nucleotide in the siRNA, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide, or the base of the nucleotide. In some embodiments, the P atom in formula a59 can be linked to the 2', 3', or 5' position of a nucleotide in the siRNA by formation of a phosphodiester linkage. In some embodiments, the P atom in formula a59 is attached to an oxygen atom formed after dehydrogenation of the 3' hydroxyl group of the 3' terminal nucleotide of the siRNA sense strand (in this case, the P atom in a59 can also be considered as a P atom in a phosphate group contained in the siRNA), or the P atom in formula a59 is attached to a nucleotide by replacing hydrogen in the 2' -hydroxyl group of one nucleotide in the siRNA sense strand, or the P atom in formula a59 is attached to a nucleotide by replacing hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide of the siRNA sense strand.

The inventors of the present disclosure unexpectedly found that the siRNA conjugates of the present disclosure, while having significantly improved stability in plasma, low off-target effects, also exhibited FXII mRNA silencing activity that was not significantly reduced. 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 sequences in conjugates of the present disclosure

Table 1b second siRNA sequences in conjugates of the disclosure

TABLE 1d third siRNA sequence in the conjugates of the present disclosure

Table 1e fourth siRNA sequences in conjugates of the disclosure

Table 1f fifth siRNA sequences in conjugates of the disclosure

In the siRNA or siRNA conjugates described in the present disclosure, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate bond, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the phosphorothioate bond carries a negative charge, and the siRNA or siRNA conjugate can exist in a form of hydroxyl group or sulfhydryl group, and hydrogen ions in the hydroxyl group or sulfhydryl group can also be partially or completely replaced by cations. The cation may be any cation, such as one of a metal cation, an ammonium ion NH ₄ ⁺, and an organic ammonium cation. In one embodiment, the cation is selected from one or more of an alkali metal ion, a tertiary amine-forming ammonium cation, and a quaternary ammonium cation for improved solubility. The alkali metal ions may be K ⁺ and/or Na ⁺, and the tertiary amine-forming cations may be triethylamine-forming ammonium ions and/or N, N-diisopropylethylamine-forming ammonium ions. Thus, the siRNA or siRNA conjugates of the present disclosure may exist at least partially in salt form. In one mode, the non-bridging oxygen or sulfur atoms in the phosphodiester or phosphorothioate linkages are at least partially bound to sodium ions, and the siRNA or siRNA conjugates of the present disclosure are in the form of sodium salts or partial sodium salts.

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

Preparation of siRNA conjugates represented by formula (308)

The siRNA conjugates of formula (308) may be prepared using any reasonable synthetic route.

In some embodiments, the siRNA conjugates of formula (308) can be prepared by a method comprising sequentially ligating nucleoside monomers in a3 'to 5' direction under conditions of phosphoramidite solid phase synthesis according to the nucleotide species and sequence of the sense strand and the antisense strand, respectively, each nucleoside monomer ligation comprising a deprotection, coupling, capping, oxidation, or sulfidation reaction; isolating the sense strand and the antisense strand of the siRNA, annealing, wherein the siRNA is an siRNA of the disclosure described above;

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, such that the compound represented by formula (321) is 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 ₄ is a group capable of binding to siRNA represented by Nu in the compound represented by formula (308). In some embodiments, R ₄ is a group capable of binding to an siRNA represented by Nu through a covalent bond. In some embodiments, R ₄ is a group capable of reacting to conjugate to any functional group of the siRNA represented by Nu via a phosphodiester bond;

Each S ₁ is independently a group formed by substitution of all active hydroxyl groups in M ₁ with YCOO-groups, wherein each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; in some embodiments, Y is methyl.

n1、n3、m1、m2、m3、R₁₀、R₁₁、R₁₂、R₁₃、R₁₄、R₁₅、L₁、M₁ The respective definitions and optional ranges are as previously described.

R ₄ is selected to achieve attachment to the N atom on the nitrogen-containing backbone and to provide a suitable reaction site for synthesizing the siRNA conjugate of formula (308). In some embodiments, R ₄ includes an R ₂ linker or a protected R ₂ linker, and a functional group that can react with the siRNA to form the structure shown in A59.

In some embodiments, R ₄ comprises the 1 st functional group that can form a phosphite with a group on the siRNA or nucleoside monomer represented by Nu and the 2 nd functional group that can react with a hydroxyl or amino group to form a covalent bond or contains a solid support linked by the covalent bond. In some embodiments, the 1 st functional group is a phosphoramidite, a hydroxyl group, or a protected hydroxyl group. In some embodiments, the 2 nd functional group is a phosphoramidite, a carboxyl group, 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 by a hydroxyl or amino group. In some embodiments, the solid support is linked via a phosphate bond, a carboxylate bond, or an amide bond. In some embodiments, the solid support is a resin.

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

Wherein q ₁ is an integer of 1 to 4, X is O or NH, M ⁺ is a cation, R _k is a hydroxyl protecting group, SPS represents a solid phase carrier, Indicating the site of covalent attachment of the group.

In some embodiments, the 1 st functional group contains a phosphoramidite group, as shown in formula (C3), which can undergo a coupling reaction with 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 ester, and oxidized or sulfided to form a phosphodiester or phosphorothioate linkage shown in formula a59, to conjugate the conjugate 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 obtaining of the siRNA conjugate shown by formula (308). In this case, after obtaining the sense strand or 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 a terminal nucleotide in a nucleotide sequence, and a phosphodiester linkage or phosphorothioate linkage is formed in a subsequent oxidation or vulcanization 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 is reactive with the solid support, the reaction providing a conjugated molecule comprising the solid support. In some embodiments, the 2 nd functional group contains a carboxyl group, carboxylate, or phosphoramidite, as shown in formula (C1), (C2), or (C3), and when the 2 nd functional group contains a carboxyl group or carboxylate, the compound of formula (321) undergoes an esterification reaction or an amidation reaction with a solid support, such as a hydroxyl group or an amino group on a resin, to form a conjugate molecule comprising a solid support linked via a carboxylic acid ester linkage. When the 2 nd functional group comprises a phosphoramidite functional group, the compound of formula (321) is coupled to a common solid support, such as a hydroxyl group on a resin, and oxidized to form a conjugated molecule comprising the solid support linked via a phosphodiester linkage. Subsequently, the above-mentioned product after the solid phase carrier is attached is used as an initial, and nucleoside monomers are sequentially attached according to a phosphoramidite solid phase synthesis method, so as to obtain the sense strand or antisense strand of the siRNA with the attached conjugate group. During the solid phase synthesis of phosphoramidite, the 1 st functional group is deprotected and then coupled to the phosphoramidite group on the 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 linked via a carboxylic ester bond or a solid phase carrier linked via an amide bond, or a solid phase carrier linked via a phosphoric ester bond, as shown in formula (C1 ') or (C3'). At this time, the compound of formula (321) is used as a starting material instead of the solid phase carrier, and nucleoside monomers are sequentially linked according to a phosphoramidite solid phase synthesis method to obtain the sense strand or antisense strand of the siRNA to which the conjugate group is linked.

In some embodiments, the carboxylate may be represented as-COO ^-M⁺, wherein M ⁺ is a cation, for example, one selected from the group consisting of a metal cation, an ammonium cation NH ₄ ⁺, an organic ammonium cation. In one embodiment, the metal ion is selected from one of alkali metal ions, such as K ⁺ or Na ⁺. 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, for reasons of improving solubility and facilitating the reaction. In some embodiments, the carboxylate is triethylamine carboxylate or N, N-diisopropylethylamine carboxylate.

In some embodiments, R ₄ contains a structure represented by formula (B9), (B10), (B9 '), (B10'), (B11), (B12), (B11 ') or (B12'):

Wherein q ₁ is an integer from 1 to 4, q ₂ is an integer from 1 to 10, X is O or NH, M ⁺ is a cation, R _k is a hydroxyl protecting group, SPS represents a solid support, Indicating the site of covalent attachment of the group. In some embodiments, q ₁ is 1 or 2. In some embodiments, q ₂ is an integer from 1 to 5. In some embodiments, R ₄ contains a structure represented by formula (B9) or (B10). In some embodiments, R ₄ contains a structure represented by formula (B11) or (B12).

In some embodiments, R _k is one or more of Tr (trityl), MMTr (4-methoxytrityl), DMTr (4, 4 '-dimethoxytrityl), TMTr (4, 4' -trimethoxytrityl). In some embodiments, R _k can be DMTr, i.e., 4'-dimethoxytrityl (4, 4' -dimethoxytrityl).

L ₁ is as defined above.

In some embodiments, L ₁ is used to attach an M ₁ targeting group to an N atom on a nitrogen-containing backbone, thereby providing liver targeting function to the siRNA conjugate represented by formula (308). In some embodiments, L ₁ comprises any one of A1-A26, or a combination thereof.

From the above description, it will be readily understood by those skilled in the art that the siRNA conjugate represented by formula (308) wherein the conjugate molecule is attached to any possible position of the nucleotide sequence, for example, the conjugate molecule is attached to the end of the nucleotide sequence and the conjugate molecule is attached to the end of the nucleotide sequence, can be obtained by the above-described functional group 1 and optionally functional group 2, as compared to the phosphoramidite solid phase synthesis methods 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," "sulfidation," etc. reactions, it is to be understood that the reaction conditions and reagents involved in solid phase synthesis of phosphoramidite nucleic acids, which are well known in the art, are equally applicable to these reactions. Exemplary reaction conditions and reagents will be described in detail later.

In some embodiments, each S ₁ is independently M ₁. In some embodiments, each S ₁ is independently a group formed in M ₁ in which at least one active hydroxyl group is protected by a hydroxyl protecting group. In some embodiments, each S ₁ is independently a group formed by protecting all of any active hydroxyl groups present in M ₁ with a hydroxyl protecting group. In some embodiments, any hydroxy protecting group known to those skilled in the art may be used to protect the reactive hydroxy group in M ₁. In some embodiments, the protected hydroxyl group may be represented by formula YCOO-wherein each Y is independently selected from the group consisting of C ₁-C₁₀ alkyl and C ₆-C₁₀ aryl, optionally substituted with one or more substituents selected from the group consisting of halogen and C ₁ -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 C ₁-C₆ alkylphenyl.

In some embodiments, each S ₁ is independently selected from the group consisting of formulas a46-a 54:

In some embodiments, S ₁ is formula a49 or a50.

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

As described previously, the preparation method of the siRNA conjugate shown in formula (308) further comprises the steps of: the other strand of the siRNA is synthesized (e.g., when the steps described above synthesize the sense strand of the siRNA to which the conjugate molecule is attached, also include synthesizing the antisense strand of the siRNA according to a solid phase synthesis method, and vice versa), separating the sense strand and the antisense strand, and annealing. Specifically, in the isolation step, the solid support linked to the nucleotide sequence and/or the conjugate molecule is cleaved, while the necessary protecting groups are removed (at this time, each S ₁ group in the compound of formula (321) is converted to a corresponding M ₁ targeting group), resulting in an siRNA sense strand (or antisense strand) and a corresponding antisense strand (or sense strand) linked to the conjugate molecule, which anneals to the antisense strand to form a double-stranded RNA structure, resulting in an siRNA conjugate represented by formula (308).

In some embodiments, the method of preparing the siRNA conjugate of 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 coupling reaction conditions and in the presence of a coupling reagent, connecting the compound shown in the formula (321) with a first nucleotide in the sequence, and sequentially connecting the nucleoside monomers in the 3' to 5' direction under the condition of phosphoramidite solid phase synthesis according to the desired sense strand or antisense strand nucleotide types and sequences to synthesize the sense strand or antisense strand of the siRNA; wherein the compound shown in the formula (321) is a compound which contains a1 st functional group and a 2 nd functional group in R ₄, wherein the 1 st functional group contains a protected hydroxyl group, and the 2 nd functional group has a structure shown as a formula (C1 ') or a formula (C3'), and the compound shown in the formula (321) is deprotected before being connected with a first nucleoside monomer; the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or vulcanization reaction; obtaining a sense strand or an antisense strand of the nucleic acid to which the conjugate group is attached; under the condition of phosphoramidite solid phase synthesis, sequentially connecting nucleoside monomers according to the nucleotide types and sequences of antisense strand or sense strand and the direction from 3 'to 5', and synthesizing the antisense strand or sense strand of the nucleic acid; the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or vulcanization reaction; 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 of formula (308) comprises the steps of: sequentially connecting nucleoside monomers according to the nucleotide types and sequences of a sense strand or an antisense strand in the double-stranded siRNA and the direction from 3 'to 5' 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 reaction to obtain the sense strand connected to a solid carrier and the antisense strand connected to the solid carrier; contacting a compound represented by formula (321) with a sense strand attached to a solid support or an antisense strand attached to a solid support under coupling reaction conditions and in the presence of a coupling reagent, and attaching the compound of formula (321) to the sense strand or the antisense strand, wherein the compound of formula (321) is a compound of formula (321) having a1 st functional group in R ₄ and a1 st functional group being a phosphoramidite group; removing protecting groups, cutting with a solid phase carrier, 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 conjugation 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 of formula (308) comprises:

(1) Removing a compound of formula (321) (wherein the compound of formula (321) is a compound of formula (C1 ') OR (C3 ') in which R ₄ contains a1 st functional group and a2 nd functional group, the 1 st functional group contains a protected hydroxyl group OR _k, and the 2 nd functional group has a hydroxyl protecting group R _k in the formula (C1 '); contacting the deprotected product with a nucleoside monomer under coupling reaction conditions and in the presence of a coupling reagent to obtain a nucleoside monomer attached to a solid support via a conjugate molecule;

(2) Synthesizing the sense strand of the siRNA by a phosphoramidite solid phase synthesis method according to the 3'-5' direction starting from the nucleoside monomer attached to the solid phase carrier through the conjugate molecule;

(3) Synthesizing antisense strand of siRNA through phosphoramidite solid phase synthesis method;

(4) The sense strand and the antisense strand of the siRNA are separated and annealed to obtain the siRNA conjugate shown in formula (308).

Wherein, in step (1), the method for removing the protecting group R _k in the compound of formula (321) above comprises contacting the compound of formula (321) with a deprotection reagent under deprotection conditions. Deprotection conditions include a temperature of 0 to 50 ℃, in some embodiments 15 to 35 ℃, 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, in some embodiments dichloroacetic acid. The molar ratio of deprotecting reagent to compound of formula (321) is from 10:1 to 1000:1, in some embodiments from 50:1 to 500:1.

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

In some embodiments, the conditions of the coupling reaction include a reaction temperature of 0 to 50 ℃, in some embodiments 15 to 35 ℃. The molar ratio of compound of formula (321) to nucleoside monomer is from 1:1 to 1:50, in some embodiments from 1:2 to 1:5; the molar ratio of the compound of formula (321) to the coupling reagent may be from 1:1 to 1:50, in some embodiments from 1:3 to 1:10, and the reaction time from 200 to 3000 seconds, in some embodiments from 500 to 1500 seconds. The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole, and in some embodiments 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, 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 the method of solid phase synthesis of phosphoramidite nucleic acid using the nucleoside monomer attached to the solid support through 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 for the solid phase synthesis described in steps (2) and (3) include deprotection conditions for nucleoside monomers, types and amounts of deprotection reagents, coupling reaction conditions, types and amounts of coupling reagents, conditions for capping reactions, types and amounts of capping reagents, oxidation reaction conditions, types and amounts of oxidizing reagents, sulfidation reaction conditions, types and amounts of sulfidation reagents employing various reagents, amounts and conditions conventionally used in the art.

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

The nucleoside monomer deprotection conditions include a temperature of from 0 to 50 ℃, in some embodiments from 15 to 35 ℃, for a reaction time of from 30 to 300 seconds, in some embodiments from 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 dichloroacetic acid in some embodiments. The molar ratio of deprotection reagent to 4,4' -dimethoxytrityl protecting group on the solid support may be in the range of 2:1 to 100:1, and in some embodiments in the range of 3:1 to 50:1.

Coupling reaction conditions include a temperature of 0 to 50 ℃, in some embodiments 15 to 35 ℃, and the molar ratio of nucleic acid sequence attached to the solid support to nucleoside monomer may be 1:1 to 1:50, in some embodiments 1:5 to 1:15; the molar ratio of nucleic acid sequence to coupling reagent attached to the solid support is 1:1 to 1:100, in some embodiments 1:50 to 1:80, and the reaction time and coupling reagent selection is the same as described above.

The capping reaction conditions include a temperature of 0-50 ℃, in some embodiments 15-35 ℃, a reaction time of 5-500 seconds, in some embodiments 10-100 seconds, and the capping reagent is selected as described above. The molar ratio of the total amount of capping reagent to the nucleic acid sequence attached to the solid support is from 1:100 to 100:1, in some embodiments from 1:10 to 10:1. Where equimolar amounts of acetic anhydride to N-methylimidazole are used for the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole, and nucleic acid sequences attached to the solid support may be 1:1:10 to 10:10:1, in some embodiments 1:1:2 to 2:2:1.

The oxidation reaction conditions include a temperature of 0-50 ℃, in some embodiments 15-35 ℃, a reaction time of 1-100 seconds, in some embodiments 5-50 seconds, and an oxidizing agent, in some embodiments iodine (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 in the coupling step may be in the range of 1:1 to 100:1, in some embodiments in the range of 5:1 to 50:1. In some embodiments, the oxidation reaction is performed in a mixed solvent of tetrahydrofuran: water: pyridine=3:1:1 to 1:1:3. The sulfiding reaction conditions include a temperature of 0-50 ℃, in some embodiments 15-35 ℃, a reaction time of 50-2000 seconds, in some embodiments 100-1000 seconds, and a sulfiding agent, in some embodiments hydrogenation Huang Yuansu. The molar ratio of sulfiding reagent to nucleic acid sequence attached to the solid support during the coupling step is from 10:1 to 1000:1, in some embodiments from 10:1 to 500:1. In some embodiments, the sulfidation reaction is performed in a mixed solvent of acetonitrile: pyridine=1:3-3:1.

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

The nucleotide sequence obtained by synthesis is cut off from the solid phase carrier, and the protecting groups on the base, the phosphate group and the ligand are removed according to the conventional cutting and deprotection method in siRNA synthesis. For example, the obtained nucleotide sequence linked to the solid phase carrier is contacted with concentrated ammonia water; during deprotection, the protecting group YCOO of the A46-A54 group is converted to a hydroxyl group and the S ₁ group is converted to the corresponding M ₁ group, yielding a conjugate of formula (308). Wherein, the ammonia water can be 25-30 wt% ammonia water, and the consumption of the ammonia water can be 0.2 ml/mu mol-0.8 ml/mu mol compared with the target siRNA sequence.

In the presence of at least one 2'-TBDMS protection on the synthesized nucleotide sequence, the method further comprises contacting the solid support-removed nucleotide sequence with triethylamine trihydrofluoride to remove the 2' -TBDMS protection. At this time, the corresponding nucleotide in the resulting target siRNA sequence has a free 2' -hydroxyl group. The amount of pure triethylamine-tricofluoride salt may be 0.4 ml/. Mu.mol to 1.0 ml/. Mu.mol as compared with the target siRNA sequence. Thus, an siRNA conjugate represented by formula (308) was obtained.

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 preparative ion chromatography purification columns; after the product is collected and combined, the desalination can be performed by adopting a reversed phase chromatographic purification column.

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

The purity and molecular weight of the nucleic acid sequence can be detected at any time during the synthesis process, and the quality of the synthesis can be better controlled, and methods for such detection are well known to those skilled in the art. For example, the purity of the nucleic acid can be detected by ion exchange chromatography and the molecular weight can be 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) may simply be mixed in equimolar ratio in water for injection and heated to 70-95℃and then cooled at room temperature to form a double-stranded structure through hydrogen bonding. Thus, an siRNA conjugate represented by formula (308) was obtained.

After obtaining the conjugate, in some embodiments, the synthesized siRNA conjugate of formula (308) may also be characterized by means of molecular weight detection, etc., using methods such as liquid chromatography, etc., to determine that the synthesized siRNA conjugate is the target designed siRNA conjugate of formula (308), and that the sequence of the synthesized siRNA is the sequence of the desired siRNA, such as 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 preparation method: the method comprises the steps of contacting a compound shown in a formula (313) with cyclic anhydride in an organic solvent under esterification reaction conditions and in the presence of alkali and an esterification catalyst, performing ion exchange, and separating to obtain a compound shown in a formula (321):

Wherein ,n1、n3、m1、m2、m3、R₁₀、R₁₁、R₁₂、R₁₃、R₁₄、R₁₅、L_1、S₁ are each defined and selectable

The ranges of (a) are as described above;

R ₆ is a group providing R ₄ in formula (321); in some embodiments, R ₆ has the structure shown in formula (a 61):

Wherein R _i is any group which can realize connection with N atom on a nitrogen-containing framework, is connected with R _k O and is connected with a free hydroxyl, and R _k is a hydroxyl protecting group. At this time, it is obtained that R ₄ contains a1 st functional group and a 2 nd functional group as a hydroxyl protecting group, and the 2 nd functional group contains a compound of formula (321) having a structure as shown in formula (C1) or (C2).

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

In some embodiments, the organic solvent comprises one or more of an epoxy-based solvent, an ether-based solvent, an haloalkane-based solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In some embodiments, the epoxide-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 the formula (313).

In some embodiments, the cyclic anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, or pimelic anhydride, 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, 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, in some embodiments from 2:1 to 5:1.

In some embodiments, the base may 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 tertiary amine to compound of formula (313) is from 1:1 to 20:1, in some embodiments from 3:1 to 10:1.

The ion exchange is to convert the compound of formula (321) to the desired carboxylic acid or carboxylate salt form, and ion exchange methods are well known to those skilled in the art and suitable ion exchange solutions and conditions may be used to provide the conjugated molecule with the M ⁺ cation, not described in detail herein. In some embodiments, the ion exchange reaction is performed using a triethylamine phosphate solution having a concentration of 0.2 to 0.8M, in some embodiments, 0.4 to 0.6M, and in further embodiments, 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 removed by evaporation followed by separation by chromatographic methods, e.g., separation may be performed using two chromatographic conditions: (1) normal phase purification silica gel: 200-300 mesh silica gel packing, using dichloromethane containing 1 wt%o triethylamine, methanol=100:18-100:20 gradient elution; or (2) reverse phase purification: c18, C8 reversed phase packing, eluting with methanol: acetonitrile=0.1:1-1:0.1 gradient. In some embodiments, the solvent may be directly removed to provide a crude compound of formula (321), which may be directly used in subsequent reactions.

In some embodiments, the process for 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 the presence of a condensing agent, a condensation catalyst, and a tertiary amine in an organic solvent under condensation reaction conditions. At this time, obtained is that R ₄ contains a1 st functional group and a2 nd functional group, the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group contains a compound of formula (321) having a structure shown as formula (C1').

The solid support is one of the supports 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 hydroxy resin has the following parameters: particle size of 100-400 mesh, and surface amino or hydroxyl loading of 0.2-0.5mmol/g. The dosage ratio of the compound shown in the formula (321) to the solid carrier is 10-400 mu mol of the compound per gram of the solid carrier (mu mol/g). In some embodiments, the compound of formula (321) is used in an amount of 50 to 200. Mu. Mol/g relative to the solid support.

The organic solvent may be any suitable solvent or mixed solvent 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, an haloalkane-based solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In some embodiments, the epoxide-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-oxy-tripyrrolidinylphosphonium hexafluorophosphate (benzotriazol-yl-oxytripyrrolidino phosphonium hexafluorophosphate, pyBop), 3-diethoxyphosphoryl-1, 2, 3-benzooxazol 4 (3H) -one, and/or O-benzotriazol-tetramethylurea hexafluorophosphate, in some embodiments, the condensing agent is O-benzotriazol-tetramethylurea hexafluorophosphate. The molar ratio of condensing agent to compound of formula (321) is from 1:1 to 20:1, in some embodiments from 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 tertiary amine to compound of formula (321) is from 1:1 to 20:1, in some embodiments from 1:1 to 5:1.

In some embodiments, the method of 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, and isolating the compound of formula (321). The capping reaction serves to remove any reactive functional groups that have not yet reacted to completion, to avoid the production of unwanted byproducts in subsequent reactions. The conditions under which the cap reacts include a reaction temperature of 0-50 ℃, in some embodiments 15-35 ℃, for a period of 1-10 hours, in some embodiments 3-6 hours. Capping reagents used in solid phase synthesis of siRNA can be used and are well known to those skilled in the art.

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

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, and in some embodiments, 15ml/g to 30ml/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 5ml/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, an 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 basic heterocyclic compounds. In some embodiments, the acylation catalyst is 4-dimethylaminopyridine. The mass ratio of the catalyst to the compound of formula (321) is from 0.001:1 to 1:1, in some embodiments from 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 isolation method. In some embodiments, the compound of formula (321) may be obtained by washing thoroughly 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, the preparation method of the conjugate molecule shown in the formula (321) comprises contacting the compound shown in the formula (313) with phosphoramidite under the condition of coupling reaction and in the presence of a coupling reagent, and separating to obtain the compound shown in the formula (321). At this time, obtained is that R ₄ contains a1 st functional group and a2 nd functional group, the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group contains a compound of formula (321) having a structure shown as formula (C3).

In some embodiments, the coupling reaction conditions include a temperature of from 0 to 50 ℃, such as from 15 to 35 ℃, and the molar ratio of the compound of formula (313) to the phosphoramidite may be from 1:1 to 1:50, such as 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 to 3000 seconds, for example 500 to 1500 seconds. The phosphoramidite may be, for example, bis (diisopropylamino) (2-cyanoethoxy) phosphine, which is commercially available or synthetically obtained according to methods well known in the art. The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole and 5-benzylthio 1H-tetrazole, for example, 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, 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 performing 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 directly removed to provide a crude compound of formula (321), which may be directly used in subsequent reactions.

In some embodiments, the method of preparing a compound of formula (321) further comprises the steps of: the isolated product is further contacted with a solid support containing hydroxyl groups under coupling reaction conditions in an organic solvent and in the presence of a coupling reagent. Then, the compound of formula (321) is isolated by capping reaction and oxidation reaction. At this time, obtained is a compound of formula (321) having a structure represented by formula (C3'), wherein R ₄ contains a1 st functional group and a 2 nd functional group, wherein the 1 st functional group contains a hydroxyl protecting group.

In some embodiments, the solid support is a solid support known in the art to be useful in solid phase synthesis of nucleic acids, and may be, for example, a commercially available universal solid support (NittoHL UnyLinker ^TM 300Oligonucleotide Synthesis Support,Kinovate Life Sciences company, structure shown as formula B80):

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

The coupling reaction conditions and the coupling reagents may be selected as described above. By carrying out this coupling reaction, the free hydroxyl groups formed in the deprotection reaction react with phosphoramidite groups to form phosphite linkages.

In some embodiments, the capping reaction conditions include a temperature of 0-50 ℃, e.g., 15-35 ℃, and a reaction time of 5-500 seconds, e.g., 10-100 seconds, with the capping reaction being performed in the presence of a capping reagent. The capping reagent may be selected and used as described above.

The oxidation reaction conditions include a temperature of 0-50 ℃, e.g., 15-35 ℃, a reaction time of 1-100 seconds, e.g., 5-50 seconds, and the oxidizing agent, e.g., iodine (provided in some embodiments 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 1:1 to 100:1, e.g., can be 5:1 to 50:1. In some embodiments, the oxidation reaction is performed in a mixed solvent of tetrahydrofuran: water: pyridine=3:1:1 to 1:1:3.

In some embodiments, R ₆ is one of the groups of formula B7 or B8,

Wherein q ₂ is as defined above,

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

Wherein ,n1、n3、m1、m2、m3、R₁₀、R₁₁、R₁₂、R₁₃、R₁₄、R₁₅、L₁、S₁、q₂ and R _k are each as defined and optional ranges as described above.

The amidation reaction conditions may include a reaction temperature of 0 to 100 ℃ for a reaction time of 1 to 48 hours, and in some embodiments, the amidation reaction conditions are a reaction temperature of 10 to 40 ℃ for 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, an alkyl halide solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. The alcoholic solvent is one or more of methanol, ethanol, propanol in some embodiments, ethanol in some embodiments. The epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments. The ether solvent is diethyl ether and/or methyl tert-butyl ether in some embodiments. The haloalkane-based solvent is in some embodiments one or more of methylene chloride, 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 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-oxy-tripyrrolidinylphosphine hexafluorophosphate, 3-diethoxyphosphoryl-1, 2, 3-benzooxazol 4 (3H) -one, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (EEDQ), or O-benzotriazole-tetramethylurea hexafluorophosphate, in further embodiments 3-diethoxyphosphoryl-1, 2, 3-benzooxazol 4 (3H) -one. The molar ratio of amidation condensing agent to compound of formula (314) may be in the range of 1:1 to 10:1, in some embodiments in the range of 2.5:1 to 5:1.

In some embodiments, the tertiary amine is triethylamine or N, N-diisopropylethylamine, in further embodiments N, N-diisopropylethylamine. The molar ratio of tertiary amine to compound of formula (314) is from 3:1 to 20:1, 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 _k is a DMTr group, the compound of formula (A-1) may be prepared by reacting calcium glycerate with DMTrCl; similarly, 3-amino-1, 2-propanediol can be contacted with a cyclic anhydride, which can be a cyclic anhydride having 4 to 13 carbon atoms, in some embodiments 4 to 8 carbon atoms, followed by reaction with DMTrCl to produce the compound of formula (A-2). It will be readily appreciated by those skilled in the art that the choice of the cyclic anhydride corresponds to different values of q ₂ in the (a-2) compound, for example, q ₂ =1 when the cyclic anhydride is succinic anhydride, q ₂ =2 when the cyclic anhydride is glutaric anhydride, and so on.

In some variations, the compound of formula (313) may also be prepared by reacting the compound of formula (314) with the cyclic anhydride, 3-amino-1, 2-propanediol, and DMTrCl in sequence. It will be readily appreciated 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 as described above, the compound of formula (313) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, the compound of formula (313) may be removed by evaporation followed by separation by chromatographic methods, e.g., separation may be performed using two chromatographic conditions: (1) normal phase purification silica gel: 200-300 mesh silica gel filler, petroleum ether, ethyl acetate, dichloromethane and N, N-dimethylformamide=1:1:0.5-1:1:1:0.6 gradient elution; and (2) reverse phase purification: c18, C8 reversed phase packing, eluting with methanol: acetonitrile=0.1:1-1:0.1 gradient. In some embodiments, the solvent may be directly removed to provide a crude compound of formula (313), which may be directly used in subsequent reactions.

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

Wherein n1, n3, m1, m2, m3, R ₁₀、R₁₁、R₁₂、R₁₃、R₁₄、R₁₅ are each as defined and optionally defined as described above.

The compounds of formula (316) may be prepared using, for example, the compounds disclosed in j.am.chem.soc.2014,136,16958-16961, or the compounds of formula (316) may be prepared by a variety of methods by those skilled in the art, for example, certain compounds of formula (316) may be prepared by reference to the methods disclosed in example 1 of U.S. patent No. 8,106,022B2, the entire contents of which are incorporated herein by reference.

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

Considering 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=3, the molar ratio of the compound of formula (316) to the compound of formula (320) may be 3:1 to 3.5:1, 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, an ether-based solvent, an alkyl halide-based solvent, a dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine, the epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether in some embodiments, the alkyl halide-based solvent is one or more of dichloromethane, chloroform, and 1, 2-dichloroethane in some embodiments, and 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 of formula (320).

In some embodiments, the amidation reaction condensing agent is one or more of benzotriazol-1-yl-oxy-tripyrrolidinylphosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1, 2, 3-benzozol 4 (3H) -one (DEPBT), O-benzotriazole-tetramethylurea hexafluorophosphate, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, or 1-hydroxybenzotriazole, in further embodiments is a mixture of benzotriazol-1-yl-oxy-tripyrrolidinylphosphonium hexafluorophosphate and 1-hydroxybenzotriazole, wherein benzotriazol-1-yl-oxy-tripyrrolidinylphosphonium hexafluorophosphate and 1-hydroxybenzotriazole are in equimolar amounts. The molar ratio of the total amidation condensing agent to the compound of formula (316) may be in the range of 1:1 to 3:1, 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 from 2:1 to 10:1, and in some embodiments, from 2:1 to 5:1.

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

The compounds of formula (320) are commercially available or are obtained by one skilled in the art using known methods. For example, when m1=m2=m3=3, n1=1, n3=2, and each R ₁₀、R₁₁、R₁₂、R₁₃、R₁₄、R₁₅ is H, the compound of formula (320) is commercially available from alfa elsa corporation.

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

SiRNA of the disclosure, pharmaceutical composition containing the same and application of conjugate

In some embodiments, the present disclosure provides the use of the siRNA and/or pharmaceutical compositions and/or siRNA conjugates 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, the method 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 by a mechanism of RNA interference. Thus, the siRNA and/or pharmaceutical compositions and/or siRNA conjugates of the present disclosure are useful for preventing and/or treating HAE and/or thrombosis, or for the manufacture of a medicament for preventing and/or treating HAE and/or thrombosis.

The term "administration" as used herein refers to placement of 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 in part, positioning of the siRNA, pharmaceutical composition and/or siRNA conjugate at a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include topical and systemic administration. In general, local administration results in more siRNA conjugate being delivered to a particular site than the subject's systemic circulation; whereas systemic administration results in delivery of the siRNA, pharmaceutical compositions and/or siRNA conjugates of the present disclosure to the essential systemic circulation of the subject. It is contemplated that the present disclosure is directed to providing means for preventing and/or treating HAE, in some embodiments employing a mode of administration capable of delivering a drug to the liver.

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

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

Upon administration of the sirnas, pharmaceutical compositions, and/or siRNA conjugates described in the present disclosure, for example, for male or female, 6-12 weeks old, C57BL/6J weighing 18-25g or ob/ob mice weighing 30-45g, in terms of siRNA: (i) For siRNA conjugates, the amount of siRNA may be 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 with a pharmaceutically acceptable carrier, the siRNA can be used in an amount of 0.001 to 50mg/kg body weight, in some embodiments 0.01 to 10mg/kg body weight, in some embodiments 0.05 to 5mg/kg body weight, and in some embodiments 0.1 to 3mg/kg body weight.

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 a pharmaceutical composition and/or an siRNA conjugate of the present disclosure, introducing the siRNA and/or the pharmaceutical composition and/or the siRNA conjugate of the present disclosure into the hepatocyte, and inhibiting FXII gene expression in the hepatocyte by a mechanism of RNA interference. The liver cells can be selected from liver cancer cell lines such as SMMC-7721, hepG2, huh7 and the like or isolated primary liver cells. In some embodiments, the cell is a human liver primary cell.

The amount of siRNA in the provided modified siRNA, pharmaceutical compositions and/or siRNA conjugates to inhibit FXII gene expression in a cell using the methods provided by the present disclosure is generally the amount: it is sufficient to reduce expression of the target gene and results in an extracellular concentration of 1pM to 1. Mu.M, or 0.01nM to 100nM, or 0.05nM to 50nM, or 0.05nM to about 5nM at the surface of the target cell. The amount required to achieve this local concentration will vary depending on a variety of factors including the method of delivery, the site of delivery, the number of cell layers between the site of delivery and the target cell or tissue, the route of delivery (local or 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.

Kit for detecting a substance in a sample

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

In some embodiments, the kits described herein can provide modified siRNA in one container. In some embodiments, the kits described herein can comprise a container that provides a pharmaceutically acceptable excipient. In some embodiments, other ingredients, such as stabilizers or preservatives, and the like, may also be included in the kit. In some embodiments, the kits described herein can comprise at least one additional therapeutic agent in a container other than the container in which the modified siRNA described herein is provided. In some embodiments, the kit can 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, as well as 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 liquid form, dry form or 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 a kit of the present disclosure.

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

Examples

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

Human liver primary cells were supplied by the nucleic acid technology laboratory of the university of Beijing institute of molecular medicine, thawed in a 37℃water bath prior to use, followed by seeding of appropriate density of cells in type I collagen-coated glass or plastic coverslips or tissue culture dishes, culturing the cells in RPMI 1460 medium containing 1 Xdiab and 10% FBS, and culturing the cells in an incubator containing 5% CO ₂/95% air at 37 ℃.

The C57 mouse liver primary cells were obtained from C57 mice (purchased from Zhaoyan Co.) as supplied by nucleic acid technology laboratory, university of Beijing molecular medicine research institute. Cells of appropriate density were seeded on type I collagen-coated glass or plastic coverslips or tissue culture dishes, cultured in RPMI 1460 medium containing 1 x diabodies and 10% fbs, and cultured at 37 ℃ in an incubator containing 5% CO ₂/95% air for 15-30min.

The C57 mice used were 6-8 week old mice purchased from Peking Vitre Liwa laboratory animal technologies Co.

When transfected into cells, the siRNA or siRNA conjugate synthesized against FXII gene or as negative control of the present disclosure, lipofectamine ^TM (Invitrogen) was used as a transfection reagent, for specific procedures reference to the instructions provided by the manufacturer.

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

Experimental data are allData analysis is shown using GRAPHPAD PRISM statistical analysis software.

Preparation example 1 preparation of conjugate 1

In this preparation, conjugate 1 was synthesized. The conjugate was formed after conjugation of the L-9 conjugate molecule with the number siFXIIa M1SPsiRNA 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 terminal segment GAL-5

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

100.0G of GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS number 1772-03-8, available from Ningbo paraglider Biochemical Co., 463.8 mmol) was dissolved in 1000ml of anhydrous pyridine, 540ml of acetic anhydride (available from Enox Co., 5565.6 mmol) was added to the solution, and the reaction was stirred at room temperature for 1.5 hours. The reaction solution was poured into 10L of ice water, suction filtration was performed under reduced pressure, after the filter cake was washed with 2L of ice water, acetonitrile/toluene mixed solvent (volume ratio acetonitrile: toluene=1:1) was added until complete dissolution, and the solvent was evaporated to dryness, to obtain a white solid product GAL-2.0 g.

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

GAL-2 (35.1 g,90.0 mmol) obtained in the step (1-1-1 a) was dissolved in 213ml of anhydrous 1, 2-dichloroethane, and 24.0g of TMSOTF (CAS number: 27607-77-8, available from Michael company, 108.0 mmol) was added under an ice-water bath and nitrogen protection, and reacted at room temperature overnight.

400Ml of methylene chloride was added to the reaction solution to dilute it, the mixture was filtered through celite, then 1L of saturated aqueous sodium bicarbonate solution was added thereto and stirred uniformly, the organic phase was separated, the aqueous phase was extracted twice with dichloroethane, 300ml of each time, the organic phases were combined, washed with 300ml of saturated aqueous sodium bicarbonate solution and 300ml of saturated brine, the organic phase was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give GAL-3.9 g as a pale yellow viscous syrup-like product.

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

GAL-3 (26.9 g,81.7 mmol) obtained in step (1-1-1 b) was dissolved in 136ml of anhydrous 1, 2-dichloroethane, and dried was addedMolecular sieve powder 30g, 9.0g of 5-hexen-1-ol (CAS number 821-41-0, available from Adamas-beta, 89.9 mmol) was added, stirred at room temperature for 30 minutes, 9.08g TMSOTF (40.9 mmol) was added under ice-bath and nitrogen protection, and the reaction was stirred at room temperature overnight. Filtration to removeMolecular sieve powder, adding 300ml of dichloromethane into filtrate for dilution, filtering by diatomite, adding 500ml of saturated sodium bicarbonate aqueous solution for stirring and washing for 10 minutes, separating out an organic phase, extracting an aqueous phase once by 300ml of dichloroethane, combining the organic phases and washing by 300ml of saturated sodium bicarbonate aqueous solution and 300ml of saturated saline respectively, separating out the organic phase, drying by anhydrous sodium sulfate, evaporating the solvent under reduced pressure to obtain yellow syrup-shaped product GAL-4.3 g, and directly carrying out the next oxidation reaction without purification.

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

GAL-4 (14.9 g,34.7 mmol) obtained as described in step (1-1-1 c) 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 Aba Ding Gongsi, 138.8 mmol) were added, respectively, and stirred in an ice water bath for 10 minutes, and ruthenium trichloride (CAS No. 14898-67-0, available from Ann Ji Co., 238mg,1.145 mmol) was added, and reacted 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, the aqueous phase was extracted three times with 200ml portions of dichloromethane and the organic phase was discarded. The pH of the aqueous phase was adjusted to about 3 with citric acid solids, extracted three times with 200ml portions of methylene chloride, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give a white foamy solid product GAL-5 6.85g.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＝8.4 Hz,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＝6.8 Hz,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 (9.886 g,52.5mmol, commercially available from Afagaku) and GAL-5 (72.819 g,162.75mmol, obtained from a combination of batches) obtained in step (1-1-1) were dissolved in 525ml of dichloromethane, diisopropylethylamine (DIEA, 44.782g,346.50 mmol), benzotriazol-1-yl-oxy-tripyrrolidinylphosphonium hexafluorophosphate (PyBOP, 90.158g,173.25 mmol) and hydroxybenzotriazole (HOBt, 23.410g,173.25 mmol) were added and the reaction was carried out at room temperature for 4h, washed with 20ml of saturated sodium bicarbonate and 200ml of saturated brine, the aqueous phase was extracted 2 times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulphate, filtered and the solvent was evaporated under reduced pressure to give the crude product. Purifying by using 200-300 mesh normal phase silica gel, neutralizing silica gel acidity by 10wt% of triethylamine, balancing a column by 1wt% of triethylamine, gradient eluting by methylene dichloride:methanol=100:25-100:40, collecting a product eluent, and evaporating the solvent under reduced pressure to obtain a pure product L-8 38.8g.¹H NMR(400 MHz,DMSO)δ7.84(d,J＝9.0 Hz,3H),7.27–7.23(m,1H),7.13–7.18(m,1H),5.22(d,J＝3.1 Hz,3H),4.97(dd,J＝11.3,3.1 Hz,3H),4.48(d,J＝8.4 Hz,3H),4.09–3.98(m,9H),3.88(dd,J＝19.3,9.3 Hz,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：C₈₅H₁₁₉N₇O₃₀,[M+H]⁺, theory: 1477.59, actual measurement: 1477.23.

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

DMTrCl (4, 4' -dimethoxytrityl chloride, 101.65g,300 mmol) was dissolved in 1000ml of anhydrous pyridine, DL-calcium glycerate hydrate (28.63 g,100 mmol) was added, reaction was carried out at 45℃for 20h, the reaction solution was filtered, the filter cake was rinsed with 200ml of DCM, the filtrate was concentrated to dryness under reduced pressure, the residue was redissolved with 500ml of dichloromethane, 0.5M triethylamine phosphate (pH=7-8) was washed 2 times, 200ml each time, the aqueous phase was extracted with dichloromethane 2 times, 200ml each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the solvent was evaporated under reduced pressure, the 200-300 mesh normal phase silica gel column was purified, the product eluent was collected as a gradient of petroleum ether: ethyl acetate: methanol=1:1:0.35-1:1:1:0.55), the solvent was evaporated under reduced pressure, 600ml of dichloromethane was redissolved, the aqueous phase was washed with 200ml of dichloromethane 1 time, the organic phase was combined, anhydrous sodium sulfate, dried, the solvent was evaporated under reduced pressure, and the solvent was distilled under reduced pressure, and the vacuum was A-1 50.7g.¹H NMR(400 MHz,DMSO-d6)δ7.46(ddd,J＝6.5,2.3,1.1 Hz,1H),7.40–7.28(m,7H),6.89–6.81(m,4H),4.84(d,J＝5.0 Hz,1H),4.36–4.24(m,1H),4.29(s,6H),3.92(dd,J＝12.4,7.0 Hz,1H),3.67(dd,J＝12.3,7.0 Hz,1H),2.52(q,J＝6.3 Hz,6H),1.03(t,J＝6.3 Hz,9H).MS m/z：C₂₄H₂₃O₆,[M-H]^-,% by the theory, to obtain a white solid overnight. 407.15, actual measurement: 406.92.

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

L-8 (40 g,27.09mmol, obtained by combining multiple batches of the product) obtained in step (1-1-2) and A-1 (41.418 g,81.27 mmol) obtained in step (1-1-3 a) were mixed, dissolved in 271ml of dichloromethane, 3-diethoxyphosphoryl-1, 2, 3-benzole 4 (3H) -one (DEPBT) (24.318 g,81.37 mmol) was added, diisopropylethylamine (21.007 g,162.54 mmol) was added, the reaction was stirred at 25℃for 1.5H, the organic phase was washed with 800ml of saturated sodium bicarbonate, the aqueous phase was extracted 3 times with dichloromethane, 50ml each time, the organic phase was washed with 150ml of saturated saline, the aqueous phase was extracted 1 time with 50ml of dichloromethane, the organic phase was combined and dried over anhydrous sodium sulfate, the solvent was evaporated after filtration, and foaming and drying was carried out overnight in vacuo to give the crude product. Column purification using 2kg of 200-300 mesh normal phase silica gel, neutralization of silica gel acidity with 200ml of triethylamine, equilibration of the column with petroleum ether containing 1wt% of triethylamine, gradient elution with petroleum ether: ethyl acetate: dichloromethane: N, N-dimethylformamide=1:1:1:0.5-1:1:1:0.6, collection of product eluent, evaporation of solvent under reduced pressure to give pure product L-7 40.4g.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：C₉₀H₁₂₈N₇O₃₅,[M-DMTr]⁺, theory: 1564.65, actual measurement: 1564.88.

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

L-7 (40 g,21.4247 mmol), succinic anhydride (4.284 g,42.8494 mmol) and 4-dimethylaminopyridine (DMAP, 5.235g,42.8494 mmol) obtained in the step (1-1-3 b) were mixed and dissolved in 215ml of dichloromethane, diisopropylethylamine (DIEA, 13.845g,107.1235 mmol) was added thereto, and the reaction solution was washed with 800ml of 0.5M triethylamine phosphate at 25℃with stirring, and the aqueous phase was extracted 3 times with dichloromethane, each time with 5ml of organic phase was combined and evaporated to dryness under reduced pressure to give a crude product. Column purification using 1kg of 200-300 mesh normal phase silica gel, neutralization of silica gel acidity with 1wt% triethylamine, equilibration of column with dichloromethane, gradient elution with 1wt% triethylamine in dichloromethane: methanol=100:18-100:20, collection of product eluent, evaporation of solvent under reduced pressure to give pure product L-9 conjugate molecule 31.0g.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：C₉₄H₁₃₂N₇O₃₈,[M-DMTr]⁺, theory: 1664.72, actual measurement: 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.

L-9 conjugate molecule (22.751 g,11 mmol) obtained in step (1-1-4), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 6.257g,16.5 mmol) and diisopropylethylamine (DIEA, 2.843g,22 mmol) were mixed, dissolved in 900ml of acetonitrile, stirred at room temperature for 5 minutes, aminomethyl resin (88 g,100-200 mesh, amino group loading 400. Mu. Mol/g, available from Nanking and Co.) was added to the reaction solution, shaking reaction was performed at 25℃at 150 rpm, filtration was performed after 18 hours of reaction, the filter cake was rinsed 2 times with DCM, 300ml each time with acetonitrile, 300ml each time with vacuum oil pump dried for 18 hours, and then capping reaction was performed again with the starting materials (CapA, capB, 4-Dimethylaminopyridine (DMAP) and acetonitrile) according to the feed ratios shown in Table 2. Placing the mixture on a shaking table at 25 ℃ at the rotating speed of 150 revolutions per minute, reacting for 5 hours, filtering the reaction liquid, leaching a filter cake with acetonitrile for 3 times, each time 300ml, evaporating the solvent to dryness under reduced pressure, and drying the solvent overnight under reduced pressure of a vacuum oil pump to obtain 102g of an L-10 compound (namely L-9 conjugated molecules connected with a solid phase carrier), wherein the loading capacity is 90.8 mu mol/g.

Table 2 cap reaction batch ratios

Raw materials	Dosage of	Specification of specification	Lot number	Manufacturing factories
					CapA	1980ml	——	——	——
CapB	220ml	——	——	——
					DMAP	1.100g	Analytical grade	I1422139	Aladdin
Acetonitrile	220ml	Spectral purity	O15161001	Starfish-shaped food

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

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

The L-10 compound prepared by the above steps is initially circulated by a solid-phase phosphoramidite method, and nucleoside monomers are sequentially linked from 3'-5' direction according to the nucleotide arrangement sequence of the sense strand. Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. Wherein, when two nucleotides are connected by phosphate, the connection of the latter nucleoside monomer comprises deprotection, coupling, capping and oxidation. When phosphorothioate is adopted to connect two nucleotides, the following nucleoside monomer is connected, and the four steps of protection, coupling, capping and vulcanization are included. The synthesis conditions were given as follows:

The nucleoside monomer was provided as a 0.1M acetonitrile solution, the deprotection conditions were the same for each step, i.e., the temperature was 25 ℃, the reaction time was 70 seconds, the deprotection reagent was a dichloromethane solution of dichloroacetic acid (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, including a temperature of 25 ℃, a molar ratio of the nucleic acid sequence connected to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence connected to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, and a coupling reagent of 5-ethylthio-1H-tetrazole (5- (Ethylthio) -1H-tetrazole, ETT) in 0.5M acetonitrile.

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=1:1:1.

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

The conditions for each step of sulfiding reaction were the same, including a temperature of 25℃and a reaction time of 300 seconds, with the sulfiding reagent being hydrogenated Huang Yuansu. The molar ratio of sulfiding reagent to nucleic acid sequence attached to the solid support in the coupling step was 120:1. The reaction was carried out in a mixed solvent of acetonitrile: pyridine=1:1.

The cleavage and deprotection conditions were as follows: the synthesized carrier-linked nucleotide sequence was added to 25wt% aqueous ammonia at 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.

Purifying 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), solvent water/acetonitrile=9:1 (volume ratio); eluent B:1.5M sodium chloride, 20mM sodium phosphate (pH 8.1), solvent water/acetonitrile=9:1 (volume ratio); elution gradient: eluent a, eluent b=100:0-50:50 gradient elution. Collecting and combining product eluents, desalting by using a reversed phase chromatographic purification column, wherein specific conditions comprise desalting by using a Sephadex column, 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). The actual measurement value is consistent with the theoretical value, which indicates that the sense strand S of the L-9 conjugated molecule is conjugated at the 3' -end.

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

By solid phase phosphoramidite method, a universal solid phase carrier (UnyLinker ^TM loaded NittoHL Solid Supports, kinovate LIFE SCIENCES company) to synthesize the antisense strand AS of conjugate 1. Deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid phase synthesis method are identical to those used for synthesizing the sense strand.

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

In this connection, the general solid support used, deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection, purification and desalting conditions are identical to those used for synthesizing the sense strand.

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 actual measurement value matches the theoretical value, indicating that the antisense strand AS having the target sequence was synthesized.

(1-4) Synthesis of conjugate 1

For the conjugate 1, S chain and AS chain are respectively dissolved in water for injection to obtain 40mg/mL solution, the solution is mixed in an equimolar ratio, the mixture is heated for 15min at 50 ℃, and after cooling at room temperature, annealed products are obtained, and freeze-dried powder is obtained. The conjugate was diluted to a concentration of 0.2mg/mL using ultra pure water (Milli-Q ultra pure water instrument, resistivity 18.2 M.OMEGA..cm (25 ℃ C.), and then subjected to molecular weight measurement using a liquid chromatography-mass spectrometer (LC-MS, liquid Chromatography-Mass Spectrometry, available from Waters, model number LCT PREMIER). The actual measurement value is consistent with the theoretical value, which indicates that the synthesized conjugate 1 is a double-stranded nucleic acid sequence with the L-9 conjugated molecule designed in the target. The structure is shown as a formula (403).

Preparation example 2 preparation of conjugates 2-6

Using the same method as in preparation example 1, conjugate 2, conjugate 3, conjugate 4, conjugate 5 and conjugate 6 were synthesized, respectively, except that the sirnas in the conjugates have sense strand and antisense strand corresponding to those of conjugate 2, conjugate 3, conjugate 4, conjugate 5 and conjugate 6 shown in table 3, respectively, so that in order to synthesize these conjugates, sense strand and antisense strand were synthesized according to the sense strand and antisense strand sequences of the sirnas shown in table 3, respectively. The molecular weights of the obtained conjugates 2 to 6 were detected by LC-MS, respectively, and the measured values were consistent with the theoretical values, indicating that the synthesized conjugates were double-stranded nucleic acid sequences with L-9 conjugated molecules designed as targets. The structure of conjugates 2-6 is shown in formula (403).

TABLE 3 siRNA conjugates

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

Ext> inext> theext> aboveext> conjugatesext>,ext> betweenext> theext> siRNAext> inext> conjugateext> 3ext> andext> conjugateext> 1ext>,ext> theext> senseext> strandext> andext> theext> antisenseext> strandext> eachext> containext> aext> nucleotideext> baseext> differenceext> (ext> 5ext> 'ext> -ext> 3ext>'ext> directionext>,ext> Gext> -ext> Aext> differenceext> atext> nucleotideext> 2ext> ofext> theext> senseext> strandext>,ext> Cext> -ext> Uext> differenceext> atext> nucleotideext> 18ext> ofext> theext> antisenseext> strandext>)ext>,ext> theext> siRNAext> antisenseext> strandext> ofext> conjugateext> 1ext> isext> completelyext> reverseext> -ext> complementaryext> toext> humanext> FXIIext> mRNAext>,ext> andext> theext> siRNAext> antisenseext> strandext> ofext> conjugateext> 3ext> isext> completelyext> reverseext> -ext> complementaryext> toext> mouseext> FXIIext> mRNAext>.ext> 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 sense and antisense strands listed in table 4 were obtained by a solid phase synthesis method, and an equimolar mixture of the sense and antisense strands was dissolved using DEPC water, followed by annealing to form siRNA duplex, freeze-dried, and the siRNA listed in table 4 was obtained as a freeze-dried powder.

TABLE 4siRNA sequences

Wherein, the capital letter C, G, U, A represents the base composition of the nucleotide; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that phosphorothioate linkages are between the two nucleotides around the letter s; dT represents thymidine.

In the preparation of the above sequence, when unmodified nucleotides are contained in the target sequence, after aqueous ammonia treatment, the product is dissolved with 0.4 ml/. Mu.mol N-methylpyrrolidone relative to the amount of single stranded nucleic acid, followed by addition of 0.3 ml/. Mu.mol triethylamine and 0.6 ml/. Mu.mol triethylamine-tricofluoride to remove 2' -TBDMS protection on ribose.

Experimental example 4 detection of inhibition efficiency of siRNA on FXII mRNA expression level in human liver primary cells.

The siRNA to be tested (siRNA 7, 8, 10, 11 and 12 and solutions of the comparison sequences NC, where NC is a negative control sequence with no obvious sequence correlation with FXII mRNA, were each re-solubilized with DEPC-ionized water to form an aqueous solution of the siRNA at the desired concentration, respectively, prior to the experiment) were each transfected into human liver primary cells using Lipofectamine ^TM according to the instructions provided by the supplier, each siRNA at a final concentration of 50nM, each concentration of 2 duplicate wells, respectively.

The amount of FXII mRNA expressed in human primary liver cells transfected with each siRNA was detected by real-Time fluorescent quantitative PCR (Quantitative Real-Time PCR), respectively. The method comprises the following specific steps: after 24 hours of culturing the transfected cells, total RNA was extracted from the cells using Trizol (Thermo Fisher Co.) according to standard procedures for total RNA extraction; mu.g of total RNA was taken and reverse transcribed to obtain cDNA using a reverse transcription kit (Promega Corp., cat. No. A3500) according to the procedure described in the specification. FXII mRNA expression level was measured by the procedure described in the specification using a 2X Ultra SYBR Mixture (with ROX) (Beijing Kao is century Biotechnology Co., ltd., product No. CW 0956) kit and cDNA as a template. Among them, PCR primers for amplifying FXII and GAPDH as an internal reference gene are shown in Table 5.

TABLE 5 primer information

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

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

TABLE 6 inhibition of FXII mRNA in human liver primary cells

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

Experimental example 5 detection of inhibition efficiency of siRNA on FXII mRNA expression level in C57 mouse liver primary cells.

The detection 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 detected in C57 mouse liver primary cells while using PCR primers for amplifying FXII and GAPDH as an internal reference gene shown in table 7 instead 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 primary liver cells of C57 mice

siRNA	Numbering device	MRNA inhibition rate%
			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 inhibition activity in C57 mouse liver primary cells, wherein the inhibition rate of 50nM siRNA12 on FXII mRNA expression amount can reach 70.09%.

Experimental example 6 inhibition efficiency of siRNA conjugate on FXII mRNA expression in C57 mice

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

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

The 6-8 week old C57 mice were randomly divided into 7 groups of 5 mice, each group of mice was given a solution of conjugate 1,2 or 3 (each conjugate corresponds to 2 different doses of group) or control sequence NC (group 1) (the solution refers to a solution formed by re-dissolving siRNA conjugates with 1 XPBS (pH 7.4) buffer at the desired concentration before the experiment), respectively), and PBS blank group (15 mice, given 1 XPBS). All animals calculate the dosage according to the body weight, the dosage is singly dosed by adopting a subcutaneous injection mode, the dosage of the siRNA conjugate (based on the quantity of the siRNA) is respectively 5mg/kg and 1mg/kg, the dosing volume is 10mL/kg, and the concentration of the drug which is required to be prepared by each siRNA conjugate is converted according to the dosing dosage and the dosing volume.

Mice were sacrificed 7 days after dosing, livers were collected and saved with RNA later (SIGMA ALDRICH company); liver tissue was then homogenized using 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 liver tissues by adopting real-time fluorescence quantitative PCR, specifically: cDNA was obtained by reverse transcription using a reverse transcription kit (Promega Corp., cat. No. A3500) according to the procedure described in the specification. Detection of FXII mRNA expression level was performed by using SYBR SELECT MASTER Mix (Applied biosystem, cat. No. 4472897) kit using cDNA as a template according to the procedures of the specification, and the inhibition ratio of siRNA conjugate to FXII mRNA expression level was calculated. Among them, PCR primers for amplifying FXII and GAPDH as an internal reference gene are shown in Table 7.

The FXII mRNA expression level, i.e., the residual expression level, was calculated as follows: FXII mRNA expression level= (expression level of FXII mRNA in test group/expression level of test group GAPDH MRNA)/(expression level of FXII mRNA in control group/expression level of control group GAPDH MRNA) ×100%.

The inhibition ratio of the siRNA conjugate to FXII mRNA expression level was (1-FXII mRNA expression level). Times.100%. Wherein, the control group is a control group mouse to which PBS is applied in the experiment, and each test group is a dosing group mouse to which different siRNA conjugates are respectively applied.

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

(6-B) inhibition of FXII mRNA expression 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 amount of conjugates 1, 3,4, 5 and 6 in C57 mice was examined using the same experimental conditions and procedures as in (6-A), except that 10 mice were used in PBS group as conjugates 1, 3,4, 5 and 6.

FIG. 2 is a scatter plot of FXII mRNA expression levels (relative values with GAPDH as an internal reference) in liver tissue of C57 mice after administration of PBS or various 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), and in particular, conjugate 3 showed high FXII mRNA expression inhibition rates of 95.4% and 97.4% at doses of 1mg/kg and 5mg/kg, respectively; conjugates 4, 5 and 6 showed inhibition of FXII mRNA expression levels of 54.9%, 61.8% and 41.2% at the dose of 1mg/kg, respectively, and higher FXII mRNA expression levels 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 apparent that the siRNA and the siRNA conjugate of the present disclosure show excellent FXII mRNA inhibitory activity both in vitro human/mouse liver primary cell experiments and in vivo experiments in mice.

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

It should be noted that, in the case where the specific features described in the above embodiments are not contradictory, they may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not describe the various possible combinations.

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

Claims

1. 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 substantially reverse complementary or completely reverse complementary to form a double-stranded region, the fluoro-modified nucleotide is positioned in the nucleotide sequence I and the nucleotide sequence II, and the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I are fluoro-modified nucleotides in the sense strand according to the direction from the 5 'end to the 3' end, and the nucleotides at the rest positions in the sense strand are non-fluoro-modified nucleotides; in the direction from the 5 'end to the 3' end, in the antisense strand, the nucleotides at the 2, 6, 14 and 16 positions of the nucleotide sequence II are fluoro modified nucleotides, and the nucleotides at the rest positions in the antisense strand are non-fluoro modified nucleotides; and

I) The nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 1 in length and is not more than 1 nucleotide difference, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 2 in length and is not more than 1 nucleotide difference, the nucleotide sequence I comprises a nucleotide Z ₃ with a position corresponding to Z ₁, the nucleotide sequence II comprises a nucleotide Z ₄ with a position corresponding to Z ₂, and the Z ₄ is the first nucleotide at the 5' -end of the antisense strand; or alternatively

II) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO. 61 and differs by NO more than 1 nucleotide, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO. 62 and differs by NO more than 1 nucleotide, the nucleotide sequence I comprises a nucleotide Z ₇ corresponding in position to Z ₅, the nucleotide sequence II comprises a nucleotide Z ₈ corresponding in position to Z ₆, and the Z ₈ is the first nucleotide at the 5' -end of the antisense strand; or alternatively

Iii) The nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 121 in length and is not more than 1 nucleotide difference, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 122 in length and is not more than 1 nucleotide difference, the nucleotide sequence I comprises a nucleotide Z ₁₅ with a position corresponding to Z ₁₃, the nucleotide sequence II comprises a nucleotide Z ₁₆ with a position corresponding to Z ₁₄, and the Z ₁₆ is the first nucleotide at the 5' -end of the antisense strand; or alternatively

Iv) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:181 and differs by NO more than 1 nucleotide, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:182 and differs by NO more than 1 nucleotide, the nucleotide sequence I comprises a nucleotide Z ₁₉ corresponding in position to Z ₁₇, the nucleotide sequence II comprises a nucleotide Z ₂₀ corresponding in position to Z ₁₈, and the Z ₂₀ is the first nucleotide at the 5' -end of the antisense strand; or alternatively

V) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO. 241 and differs by NO more than 1 nucleotide, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO. 242 and differs by NO more than 1 nucleotide, the nucleotide sequence I comprises a nucleotide Z ₂₃ corresponding in position to Z ₂₁, the nucleotide sequence II comprises a nucleotide Z ₂₄ corresponding in position to Z ₂₂, and the Z ₂₄ is the first nucleotide at the 5' -end of the antisense strand;

the position corresponds to the same position in the nucleotide sequence from the same end of the nucleotide sequence;

by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences;

Wherein i) wherein there is NO nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 2, or the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 2 is a difference at position Z ₄, and Z ₄ is selected from A, C or G; or alternatively

II) wherein there is NO nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 62, or the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 62 is a difference at position Z ₈, and Z ₈ is selected from A, C or G; or alternatively

Iii) Wherein, there is NO nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 122, or the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 122 is a difference at the position Z ₁₆, and Z ₁₆ is selected from A, C or G; or alternatively

Iv) wherein there is NO nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 182, or the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 182 is a difference at position Z ₂₀, and Z ₂₀ is selected from A, C or G; or alternatively

V) wherein there is NO nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 242, or the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 242 is a difference at position Z ₂₄, and Z ₂₄ is selected from A, C or G;

Wherein each of the non-fluoro-modified nucleotides is a methoxy-modified nucleotide in which the 2' -hydroxy group of the ribosyl group is substituted with a methoxy group.

2. The siRNA of claim 1, wherein Z ₃ is a nucleotide complementary to Z ₄; or Z ₇ is a nucleotide complementary to Z ₈; or Z ₁₅ is a nucleotide complementary to Z ₁₆; or Z ₁₉ is a nucleotide complementary to Z ₂₀; or Z ₂₃ is a nucleotide complementary to Z ₂₄.

3. The siRNA of claim 1, wherein said sense strand further comprises a nucleotide sequence III, said antisense strand further comprises a nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being independently 1-4 nucleotides in length, said nucleotide sequence III being linked at the 5 'end of nucleotide sequence I, nucleotide sequence IV being linked at the 3' end of nucleotide sequence II, said nucleotide sequence III and said nucleotide sequence IV being equal in length and substantially reverse complementary or fully reverse complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; by complete reverse complement is meant that there is no mismatch between the two nucleotide sequences.

4. The siRNA according to claim 3, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO. 1 and is not more than 1 nucleotide different, and the nucleotide sequences III and IV are each 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 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 sequence III and the length of the nucleotide sequence IV are 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 is not more than 1 nucleotide difference, the length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is A; or the length of the nucleotide sequence III and the length of the nucleotide sequence IV are 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 sequence III and the length of the nucleotide sequence IV are 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 is not more than 1 nucleotide difference, the length of the nucleotide sequences III and IV is 1 nucleotide, 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 UU according to the direction from the 5 'end to the 3' end; or the length of the nucleotide sequence III and the length of the nucleotide sequence IV are 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 in length and is not more than 1 nucleotide difference, the lengths of the nucleotide sequences III and IV are 1 nucleotide, and the base of the nucleotide sequence III is G; or the length of the nucleotide sequence III and the length of the nucleotide sequence IV are 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 sequence III and the length of the nucleotide sequence IV are 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 in SEQ ID NO. 241 in length and is not more than 1 nucleotide difference, the length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is C; or the length of the nucleotide sequence III and the length of the nucleotide sequence IV are 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 length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is GAGC according to the direction from the 5 'end to the 3' end.

5. The siRNA according to claim 4 wherein said nucleotide sequences III and IV are fully reverse complementary.

6. The siRNA of claim 1 or 3, wherein said antisense strand further comprises a nucleotide sequence V of 1 to 3 nucleotides in length attached to the 3 'end of said antisense strand, constituting the 3' overhanging end of the antisense strand; or the nucleotide sequence V is 2 nucleotides in length; or the nucleotide sequence V is two continuous thymidines or two continuous uracils; or the nucleotide sequence V is complementary to a nucleotide at a corresponding position of the target mRNA.

7. The siRNA according to claim 6, wherein the sense strand of said siRNA comprises a nucleotide sequence as shown in SEQ ID NO. 5 and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO. 6; or the sense strand of the siRNA contains a nucleotide sequence shown as SEQ ID NO. 7, and the antisense strand contains a nucleotide sequence shown as SEQ ID NO. 8; or the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 65, and the antisense strand comprises a nucleotide sequence shown as SEQ ID NO. 66; or the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 67, and the antisense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 68; or the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 125, and the antisense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 126; or the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 127, and the antisense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 128; or the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 185, and the antisense strand comprises a nucleotide sequence shown as SEQ ID NO. 186; or the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 187, and the antisense strand comprises a nucleotide sequence shown as SEQ ID NO. 188; or the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 245, and the antisense strand comprises a nucleotide sequence shown as SEQ ID NO. 246; or the sense strand of the siRNA comprises a nucleotide sequence as shown in SEQ ID NO. 247 and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO. 248.

8. The siRNA of claim 7, wherein the siRNA has any of the following nucleotide sequences 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).

9. The siRNA of claim 8, wherein the siRNA is any one of the following siFXIIa1-M1、siFXIIa2-M1、siFXIIb1-M1、siFXIIb2-M1、siFXIId1-M1、siFXIId2-M1、siFXIIe1-M1、siFXIIe2-M1、siFXIIf1-M1、siFXIIf2-M1:

siFXIIa1-M1

sense strand: 5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 13) antisense strand: 5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3' (SEQ ID NO: 14);

siFXIIa2-M1

Sense strand: 5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 19) antisense strand:

5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO:20)；

siFXIIb1-M1

sense strand: 5'-CmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 73) antisense strand:

5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO:74)；

siFXIIb2-M1

Sense strand:

5'-AmAmCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:79)

siFXIId1-M1

Sense strand: 5'-GmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:133)

Antisense strand:

5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO:134)；

siFXIId2-M1

Sense strand:

5'-UmUmGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:139)

antisense strand:

5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'

(SEQ ID NO:140)；

siFXIIe1-M1

Sense strand: 5'-AmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:193)

Antisense strand:

5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO:194)；

siFXIIe2-M1

Sense strand:

5'-GmGmAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:199)

antisense strand:

5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'

(SEQ ID NO:200)；

siFXIIf1-M1

Sense strand: 5'-CmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:253)

Antisense strand:

5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO:254)；

siFXIIf2-M1

Sense strand:

5'-GmCmCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:259)

antisense strand:

5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'(SEQ ID NO:260)；

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

10. The siRNA of claim 1, wherein at least one phosphate group in the sense strand or the antisense strand is a phosphate group having a modifying group.

11. The siRNA of claim 10, wherein the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond in the phosphate group with a sulfur atom.

12. The siRNA of claim 11, wherein the phosphate group having a modifying group is a phosphorothioate group having a structure as shown in formula (1):

13. the siRNA of claim 12, wherein in the siRNA, phosphorothioate linkage is present at least one of the group consisting of:

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

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

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

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

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

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

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

14. The siRNA of claim 13, wherein the siRNA is any one of the following siFXIIa1-M1S、siFXIIa2-M1S、siFXIIb1-M1S、siFXIIb2-M1S、siFXIId1-M1S、siFXIId2-M1S、siFXIIe1-M1S、siFXIIe2-M1S、siFXIIf1-M1S、siFXIIf2-M1S:

siFXIIa1-M1S

Sense strand: 5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:25)

Antisense strand:

5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO:26)；

siFXIIa2-M1S

Sense strand: 5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3' (SEQ ID NO: 31)

Antisense strand:

5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO:32)；

siFXIIb1-M1S

sense strand: 5'-CmsUmsCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:85)

Antisense strand:

5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO:86)；

siFXIIb2-M1S

Sense strand: 5'-AmsAmsCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3' (SEQ ID NO: 91)

Antisense strand:

5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'

(SEQ ID NO:92)；

siFXIId1-M1S

sense strand: 5'-GmsGmsAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO:145)

Antisense strand:

5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'

(SEQ ID NO:146)；

siFXIId2-M1S

Sense strand: 5'-UmsUmsGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3' (SEQ ID NO: 151)

Antisense strand:

5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'

(SEQ ID NO:152)；

siFXIIe1-M1S

Sense strand: 5'-AmsGmsCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:205)

Antisense strand:

5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO:206)；

siFXIIe2-M1S

sense strand: 5'-GmsGmsAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:211)

Antisense strand:

5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'

(SEQ ID NO:212)；

siFXIIf1-M1S

Sense strand: 5'-CmsCmsAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:265)

Antisense strand:

5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO:266)；

siFXIIf2-M1S

sense strand: 5'-GmsCmsCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3' (SEQ ID NO: 271)

Antisense strand:

5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'

(SEQ ID NO:272)

15. The siRNA of claim 1, wherein the 5' terminal nucleotide of the siRNA antisense strand is a 5' -phosphonucleotide or a 5' -phosphoanalog modified nucleotide.

16. The siRNA according to claim 15, wherein said 5 '-phosphonucleotide is a nucleotide having a structure as shown in formula (2), said 5' -phosphoanalog-modified nucleotide is selected from the group consisting of nucleotides having a structure as shown in any one of formulas (3) - (6),

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

17. The siRNA of claim 16, wherein the siRNA is any one of the following siiFXIIa1-M1P1、siFXIIa2-M1P1、siFXIIa1U-M1P1、siFXIIa2U-M1P1、siFXIIa1U-M1SP1、siFXIIa2U-M1 SP1、siFXIIb1-M1P1、siFXIIb2-M1P1、siFXIId1-M1P1、siiFXIId2-M1P1、siFXIIe1-M1P1、siFXIIe2-M1P1、siFXIIf1-M1P1、siFXIIf2-M1P1、siFXIIa1-M1 SP1、siFXIIa2-M1SP1、siFXIIb1-M1SP1、siFXIIb2-M1 SP1、siFXIId1-M1SP1、siFXIId2-M1SP1、siFXIIe1-M1 SP1、siFXIIe2-M1SP1、siFXIIf1-M1SP1、siFXIIf2-M1SP1:

siFXIIa1-M1P1

Sense strand:

5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:37)

antisense strand:

5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO:38)；

siFXIIa2-M1P1

Sense strand:

5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:43)

antisense strand:

5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO:44)；

siFXIIa1-M1SP1

Sense strand:

5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:49)

antisense strand:

5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO:50)；

siFXIIa2-M1SP1

Sense strand:

5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO:55)

antisense strand:

5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO:56)；

siFXIIa1U-M1P1

Sense strand:

5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:335)

antisense strand:

5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO:336)；

siFXIIa2U-M1P1

Sense strand:

5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:341)

antisense strand:

5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO:342)；

siFXIIa1U-M1SP1

Sense strand:

5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:347)

antisense strand:

5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO:348)；

siFXIIa2U-M1SP1

Sense strand:

5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO:353)

antisense strand:

5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO:354)；

siFXIIb1-M1P1

Sense strand:

5'-CmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:97)

antisense strand:

5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO:98)；

siFXIIb2-M1P1

Sense strand:

5'-AmAmCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:103)

antisense strand:

5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'

(SEQ ID NO:104)；

siFXIIb1-M1SP1

Sense strand:

5'-CmsUmsCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:109)

antisense strand:

5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO:110)；

siFXIIb2-M1SP1

Sense strand:

5'-AmsAmsCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO:115)

antisense strand:

5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm

-3'

(SEQ ID NO:116)；siFXIId1-M1P1

Sense strand:

5'-GmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 157) antisense strand:

5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO:158)；siFXIId2-M1P1

Sense strand:

5'-UmUmGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 163) antisense strand:

5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'(SEQ ID NO:164)；siFXIId1-M1SP1

Sense strand:

5'-GmsGmsAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 169) antisense strand:

5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'(SEQ ID NO:170)；siFXIId2-M1SP1

Sense strand:

5'-UmsUmsGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 175) antisense strand:

5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAms Cm-3'

(SEQ ID NO:176)；siFXIIe1-M1P1

Sense strand:

5'-AmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 217) antisense strand:

5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO:218)；siFXIIe2-M1P1

Sense strand:

5'-GmGmAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 223) antisense strand:

5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'(SEQ ID NO:224)；siFXIIe1-M1SP1

Sense strand:

5'-AmsGmsCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:229)

antisense strand:

5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO:230)；

siFXIIe2-M1SP1

Sense strand:

5'-GmsGmsAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO:235)

antisense strand:

5'-

P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm

-3'

(SEQ ID NO:236)；

siFXIIf1-M1P1

Sense strand:

5'-CmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:277)

antisense strand:

5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO:278)；

siFXIIf2-M1P1

Sense strand:

5'-GmCmCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:283)

antisense strand:

5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'

(SEQ ID NO:284)；

siFXIIf1-M1SP1

Sense strand:

5'-CmsCmsAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:289)

antisense strand:

5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO:290)；

siFXIIf2-M1SP1

Sense strand:

5'-GmsCmsCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:295)

antisense strand:

5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUms

Cm-3'

(SEQ ID NO:296)

Wherein, the capital letter C, G, U, A represents the base composition of the nucleotide; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; lowercase letters s represent phosphorothioate linkages between the left and right nucleotides of the letter; the capital letter P1 indicates that the nucleotide adjacent to the right of the letter is a nucleotide modified with a 5 '-phosphonucleotide or 5' -phosphoanalog.

18. A pharmaceutical composition comprising the siRNA of any one of claims 1-17 and a pharmaceutically acceptable carrier.

19. An siRNA conjugate comprising the siRNA of any one of claims 1-17 and a conjugate group conjugated to the siRNA.

20. The siRNA conjugate of claim 19, wherein the conjugate has a structure represented by formula (403):

The Nu is siRNA.

21. The siRNA conjugate of claim 20, wherein the P atom is attached to a terminus of the sense strand or the antisense strand of the siRNA, said terminus referring to the first 4 nucleotides in said sense strand or antisense strand from one end thereof.

22. The siRNA conjugate of claim 21, wherein P is attached to the 3' end of the siRNA sense strand.

23. The siRNA conjugate of claim 20 or 21, wherein P is linked to the 2' position, 3' position or 5' position of a nucleotide in the siRNA by formation of a phosphodiester linkage.

24. A method of inhibiting FXII gene expression in a hepatocyte in vitro, the method comprising contacting an effective amount of the siRNA of any one of claims 1-17, the pharmaceutical composition of claim 18 and/or the siRNA conjugate of any one of claims 19-23 with the hepatocyte.

25. A kit comprising the siRNA of any one of claims 1-17, the pharmaceutical composition of claim 18, and/or the siRNA conjugate of any one of claims 19-23.