WO2020147847A1 - Nucleic acid, composition and conjugate containing nucleic acid, preparation method, and use - Google Patents

Abstract

An siRNA for inhibiting expression of factor XII (FXII) gene, and a pharmaceutical composition and conjugate containing the siRNA. Nucleotides in the siRNA are each independently modified or unmodified nucleotides. The siRNA contains a sense strand and an antisense strand. The sense strand contains a nucleotide sequence I having a length equal to that of the nucleotide sequence as shown in SEQ ID NO: 1 and having no more than three nucleotides different from those of the nucleotide sequence as shown in SEQ ID NO: 1. The antisense strand contains a nucleotide sequence II having a length equal to that of the nucleotide sequence as shown in SEQ ID NO: 2 and having no more than three nucleotides different from those of the nucleotide sequence as shown in SEQ ID NO: 2. The siRNA and the pharmaceutical composition and conjugate thereof can treat and/or prevent hereditary angioedema (HAE) and/or thrombosis.

Description

Nucleic acid, composition and conjugate containing the nucleic acid, preparation method and application Technical field

The present disclosure relates to a nucleic acid capable of inhibiting the expression of factor XII gene and a composition and conjugate containing the nucleic acid. The present disclosure also relates to preparation methods and uses of these nucleic acids, compositions and conjugates.

Background technique

Hereditary angioedema (HAE) is a rare disease characterized by repeated episodes of severe swelling. The most common swollen areas of the body are the limbs, face, intestines, and airways. The onset may be spontaneous, or it may be caused by physical trauma or stress. Laryngeal (airway) edema can be life-threatening because it can lead to death from suffocation.

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

Factor XII is one of the key targets for the treatment of HAE. By inhibiting the expression of factor XII, the occurrence of HAE can be effectively inhibited. Therefore, if gene expression can be silenced at the gene level and the production of factor XII can be blocked, it will undoubtedly be the most ideal treatment. Small interfering RNA (siRNA) can be based on the mechanism of RNA interference (RNAi) to inhibit or block the expression of any target gene of interest in a sequence-specific manner, thereby achieving the purpose of treating diseases.

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

Summary of the invention

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

among them,

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 2-10;

Each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R _{15 is} independently H, or is selected from the group consisting of C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ haloalkyl and C ₁ -C ₁₀ alkoxy;

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

Wherein, E ₁ is OH, SH or BH ₂ ;

Nu is siRNA, the siRNA has a sense strand and an antisense strand, each nucleotide in the siRNA is independently a modified or unmodified nucleotide, and the sense strand contains a nucleotide sequence I, The antisense strand contains 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 and the nucleotide sequence The nucleotide sequence II is selected from the following group i)-v):

i) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length and have no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 2 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₃ whose position corresponds to Z ₁ , and the nucleotide sequence II Comprising the nucleotide Z ₄ at the position corresponding to Z ₂ , where Z ₄ is the first nucleotide at the 5'end of the antisense strand;

ii) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 62 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₇ whose position corresponds to Z ₅ , and the nucleotide sequence II Comprising the nucleotide Z ₈ at the position corresponding to Z ₆ , said Z ₈ being the first nucleotide at the 5'end of the antisense strand;

iii) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 122 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₁₅ whose position corresponds to Z ₁₃ , and the nucleotide sequence II Comprising the nucleotide Z ₁₆ at the position corresponding to Z ₁₄ , said Z ₁₆ being the first nucleotide at the 5'end of the antisense strand;

iv) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 182 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₁₉ at the position corresponding to Z ₁₇ , and the nucleotide sequence II Comprising the nucleotide Z ₂₀ at the position corresponding to Z ₁₈ , said Z ₂₀ being the first nucleotide at the 5'end of the antisense strand;

v) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 242 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₂₃ corresponding to the position Z ₂₁ , and the nucleotide sequence II _Comprising the nucleotide Z ₂₄ at the position corresponding to Z ₂₂ , said Z ₂₄ being the first nucleotide at the 5'end of the antisense strand;

R ₂ is a linear alkylene group having a length of 1-20 carbon atoms, wherein one or more carbon atoms is optionally replaced by one or more selected from the group consisting of: C( O), NH, O, S, CH=N, S(O) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C ₃ -C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene; and wherein R ₂ may optionally have any one or more substituents from the group consisting of: C ₁ -C ₁₀ alkane Group, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl -OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkyl phenyl, -C ₁ -C ₁₀ alkyl -SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl -NH ₂ , -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ Alkyl), -NH (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl phenyl), -NH (C ₁ -C ₁₀ alkyl benzene) Group), cyano, nitro, -CO ₂ H, -C(O)O (C ₁ -C ₁₀ alkyl), -CON (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl) , -CONH (C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O) (C ₁ -C ₁₀ alkyl), -NHC(O) (phenyl), -N(C ₁ -C ₁₀ Alkyl) C (O) (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ alkyl, -C(O)C ₁ -C ₁₀ alkylphenyl, -C(O)C ₁ -C ₁₀ haloalkyl, -OC(O)C ₁ -C ₁₀ alkyl, -SO ₂ (C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl), -SO ₂ NH _{(phenyl), - NHSO 2 (C 1} -C 10 alkyl), - NHSO ₂ (phenyl), and -NHSO ₂ (C ₁ -C ₁₀ haloalkyl);

Each L _{1 is} independently a linear alkylene group with a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally selected from any one or more of the following groups Replaced: C(O), NH, O, S, CH=N, S(O) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene , C ₃ -C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene; and wherein, L ₁ may optionally have any one or more substituents in the group consisting of the following groups: C ₁ -C ₁₀ alkyl, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkyl Phenyl, -C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkane Group -SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl -NH ₂ , -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl), -NH (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl phenyl), -NH (C ₁ -C ₁₀ alkyl phenyl), cyano, nitro, -CO ₂ H, -C (O) O (C ₁ -C ₁₀ alkyl), -CON (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl), -CONH (C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O) (C ₁ -C ₁₀ alkyl), -NHC(O) (phenyl), -N (C ₁ -C ₁₀ alkyl) C (O) (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ alkyl, -C (O) C ₁ -C ₁₀ alkyl phenyl, -C (O) C ₁ -C ₁₀ haloalkyl, -OC (O) C ₁ -C ₁₀ alkyl, -SO ₂ (C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl) ), - SO ₂ NH _{(phenyl), - NHSO 2 (C 1} -C 10 alkyl), - NHSO ₂ (phenyl), and -NHSO ₂ (C ₁ -C ₁₀ haloalkyl);

表示基团共价连接的位点；

Indicates the site where the group is covalently attached;

M ₁ represents a targeting group.

In some embodiments, the present disclosure provides a siRNA comprising a sense strand and an antisense strand, and each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified core Nucleotides or non-fluorinated modified nucleotides; the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide Sequence II is at least partially reverse complementary to form a double-stranded region. The fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and, in the direction from the 5'end to the 3'end, In the sense strand, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorinated modified nucleotides According to the direction from the 5'end to the 3'end, in the antisense strand, the nucleotides at 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-fluorinated modified nucleotides, and,

i) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length and have no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 2 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₃ whose position corresponds to Z ₁ , and the nucleotide sequence II The inclusion position corresponds to the nucleotide Z _{4 of} Z ₂ , where Z ₄ is the first nucleotide at the 5'end of the antisense strand; or,

ii) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 62 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₇ whose position corresponds to Z ₅ , and the nucleotide sequence II The inclusion position corresponds to the nucleotide Z _{8 of} Z ₆ , where Z ₈ is the first nucleotide at the 5'end of the antisense strand; or,

iii) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 122 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₁₅ whose position corresponds to Z ₁₃ , and the nucleotide sequence II The inclusion position corresponds to the nucleotide Z _{16 of} Z ₁₄ , where Z ₁₆ is the first nucleotide at the 5'end of the antisense strand; or,

iv) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 182 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₁₉ at the position corresponding to Z ₁₇ , and the nucleotide sequence II The inclusion position corresponds to the nucleotide Z _{20 of} Z ₁₈ , said Z ₂₀ being the first nucleotide at the 5'end of the antisense strand; or,

v) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 242 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₂₃ corresponding to the position Z ₂₁ , and the nucleotide sequence II comprising a position corresponding to nucleotide Z ₂₂ Z _24, Z ₂₄ is the antisense strand of the 5 'end of the first nucleotide.

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

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

In some embodiments, the present disclosure provides that the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure are used in the preparation of a medicine for the treatment and/or prevention of hereditary angioedema HAE and/or thrombosis the use of.

In some embodiments, the present disclosure provides a method for treating and/or preventing HAE and/or thrombosis, the method comprising conjugating an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA of the present disclosure The drug is administered to subjects with HAE.

In some embodiments, the present disclosure provides a method for inhibiting FXII gene expression in liver cells, the method comprising combining an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure with the liver. Cell contact.

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

Incorporate by reference

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually incorporated by reference into this document The same degree.

Beneficial effect

The siRNA, the composition containing the siRNA, and the siRNA conjugate provided in the present disclosure have good stability, high gene suppression activity, and/or can significantly treat or relieve HAE symptoms.

In some embodiments, the siRNA, the composition containing the siRNA, or the siRNA conjugate provided in the present disclosure show excellent target gene inhibitory activity in in vitro cell experiments. In some embodiments, at a dose of 50 nM, the siRNA provided in the present disclosure shows an inhibition rate of FXII mRNA expression as high as 78.70% in human liver primary cells. In some embodiments, at a dose of 50 nM, the siRNA provided in the present disclosure shows an inhibition rate of up to 70.09% of FXII mRNA expression in C57 mouse liver primary cells.

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

In some embodiments, the siRNA, composition containing the siRNA, or siRNA conjugate provided in the present disclosure does not show obvious off-target effects. Off-target effects can be, for example, suppression of normal expression of genes other than target genes. It is believed that if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20%, or 10% compared to the target gene effect, the off-target effect is not significant.

This shows that the siRNA, pharmaceutical composition and siRNA conjugate provided in the present disclosure can inhibit the expression of FXII gene, effectively treat and/or prevent HAE symptoms, and have good application prospects.

Other features and advantages of the present disclosure will be described in detail in the following specific embodiments.

BRIEF DESCRIPTION

Fig. 1 and Fig. 2 are scatter plots of FXII mRNA expression in liver tissue of C57 mice (relative value with GAPDH as internal reference) after administration of PBS and different doses of each conjugate, respectively.

detailed description

Specific embodiments of the present disclosure will be described in detail below. It should be understood that the specific embodiments described here are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.

In this disclosure, FXII mRNA refers to the sequence shown in Genbank registration number NM_000505.3. Further, unless otherwise specified, the term "target gene" used in the present disclosure refers to a gene that expresses the aforementioned FXII mRNA, and the term "target mRNA" refers to the aforementioned FXII mRNA.

definition

In the above and in the following, unless otherwise specified, capital letters C, G, U, A represent the base composition of nucleotides; lowercase letter m represents that the adjacent nucleotide to the left of the letter m is a methoxy group Modified nucleotides; a lowercase letter f indicates that the adjacent nucleotide to the left of the letter f is a fluorinated modified nucleotide; a lowercase letter s indicates that between the two adjacent nucleotides to the left and right of the letter s It is a phosphorothioate group connection; P1 indicates that the adjacent nucleotide to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide. In some embodiments, P1 represents a specifically modified VP, Ps, or P, where the letter combination VP indicates that the adjacent nucleotide on the right side of the letter combination VP is a nucleotide modified by vinyl phosphate. Ps indicates that the adjacent nucleotide to the right of the letter combination Ps is a phosphorothioate modified nucleotide, and the capital letter P indicates that the adjacent nucleotide 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 formed by replacing the hydroxyl group at the 2'position of the ribose group of a nucleotide with fluorine, and the “non-fluoro-modified nucleotide” refers to Nucleotides or nucleotide analogs formed by substituting a non-fluorine group for the hydroxyl group at the 2'position of the ribose group of a nucleotide. "Nucleotide analogue" refers to a nucleic acid that can replace nucleotides, but has a structure different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymus The group of pyrimidine deoxyribonucleotides. Such as heteronucleotides, bridged nucleotides (BNA for short) or acyclic nucleotides. The "methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.

In the context of this article, the expressions "complementary" or "reverse complement" can be used interchangeably, and have the meaning well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand and the other strand The bases are paired in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base Cytosine (G) matches. Each base pair includes a purine and a pyrimidine. When adenine on one chain always pairs with thymine (or uracil) on the other chain, and guanine always pairs with cytosine, the two chains are considered to be complementary to each other, and the sequence from their complementary chain The sequence of the chain can be inferred from. Correspondingly, "mismatch" in the art means that in a double-stranded nucleic acid, bases at corresponding positions are not paired in a complementary manner.

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

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

In the above and below, especially when describing the preparation method of the siRNA, siRNA-containing composition or siRNA conjugate of the present disclosure, unless otherwise specified, the nucleoside monomer refers to the preparation method according to the The type and sequence of nucleotides in the siRNA or siRNA conjugate, the modified or unmodified nucleoside phosphoramidite monomers used in the solid-phase synthesis of phosphoramidites (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also called Nucleoside phosphoramidites). Phosphoramidite solid-phase synthesis is a method used in RNA synthesis well known to those skilled in the art. The nucleoside monomers used in the present disclosure are all commercially available.

In the context of the present disclosure, unless otherwise specified, "conjugate" means that two or more chemical moieties each having a specific function are connected to each other in a covalent manner; correspondingly, "conjugate" is Refers to the compound formed by covalent linkage between the various chemical parts. Further, "siRNA conjugate" refers to a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA. Hereinafter, sometimes the siRNA conjugate of the present disclosure is also referred to simply as "conjugate". According to the context, siRNA conjugate should be understood as the general term of siRNA conjugate, the general term of siRNA conjugate represented by formula (305) and formula (307), or formula (305), formula (307), formula (308) SiRNA conjugate shown. In the context of the present disclosure, "conjugated molecule" should be understood as a specific compound that can be conjugated to siRNA through a reaction, and ultimately form the siRNA conjugate of the present disclosure.

As used herein, a dash ("-") that is not between two letters or between two symbols is used to indicate the point of attachment of substituents. For example: the leftmost dash in the structural formula "-C ₁ -C ₁₀ alkyl-NH ₂ "means that 66 is connected through a C ₁ -C ₁₀ alkyl group.

As used herein, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and the description includes situations where the event or condition occurs and when it does not occur. For example, "optionally substituted" "alkyl" includes "alkyl" and "substituted alkyl" defined below. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution patterns that are sterically impractical, synthetically impractical, and/or inherently unstable. .

As used herein, "alkyl" refers to straight and branched chains having a specified number of carbon atoms, the number is usually 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, C ₁ -C ₆ alkyl groups include straight and branched chain alkyl groups of 1 to 6 carbon atoms. When referring to alkyl residues having a specific number of carbons, it is intended to cover all branched and straight chain forms having that number of carbons; therefore, for example, "butyl" means including n-butyl, sec-butyl , Isobutyl and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to residues that are the same as alkyl but have two points of attachment.

As used herein, "alkenyl" refers to an unsaturated branched or unbranched alkyl group having at least one carbon-carbon double bond, which is obtained from adjacent carbon atoms of the parent alkyl group. Obtained by removing one molecule of hydrogen. The group can 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 Group), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-ene-1- But-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl, etc. In certain embodiments, alkenyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to the same residue as alkenyl but with two points of attachment.

As used herein, "alkynyl" refers to an unsaturated branched or unbranched alkyl group having at least one carbon-carbon triple bond, the carbon-carbon triple bond is derived from adjacent carbon atoms of the parent alkyl group Obtained by removing two molecules of hydrogen. 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, the alkynyl group has 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to residues that are the same as alkynyl but have two points of attachment.

As used herein, "alkoxy" refers to an alkyl group with a specified number of carbon atoms connected through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, S-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methyl Pentoxy and so on. Alkoxy groups generally have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by oxygen bridges.

As used herein, "aryl" refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing hydrogen atoms from ring carbon atoms. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, wherein at least one ring in the ring system is completely unsaturated, that is, contains a ring according to Hückel theory , Delocalized (4n+2)π-electron system. Aryl groups include but are not limited to groups such as phenyl, fluorenyl and naphthyl. Arylene is a subset of aryl and refers to residues that are the same as aryl but have two points of attachment.

As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring, usually having 3 to 7 ring carbon atoms. The ring can 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.

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

"Heterocyclyl" refers to a stable 3- to 18-membered non-aromatic cyclic group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless otherwise stated in the specification, a heterocyclic group is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, and may include a fused ring or a bridged ring system. The heteroatoms in the heterocyclic group may be optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heterocyclic group is partially saturated or fully saturated. The heterocyclic group can be connected to the rest of the molecule through any ring atom. Examples of such heterocyclic groups include but are not limited to: dioxanyl, thienyl[1,3]dithianyl (thienyl[1,3]dithianyl), decahydroisoquinolinyl, imidazolinyl, imidazolidine Group, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxa Pyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinone, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trisulfanyl (trithianyl) ), tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl and 1,1-dioxothio Morpholinyl (1,1-dioxo-thiomorpholinyl).

"Heteroaryl" refers to a group derived from a 3- to 18-membered aromatic ring radical, containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, a heteroaryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, that is, contains a cyclic delocalization according to Hückel's theory (4n +2) π-electron system. 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. The heteroaryl group is attached to the rest of the molecule through any ring atom. Examples of heteroaryl groups include, but are not limited to: azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodiaxazolyl, benzofuranyl, benzene Oxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl (benzo[b][1,4]dioxepinyl), benzo[ b][1,4]oxazinyl (benzo[b][1,4]oxazinyl), 1,4-benzodioxanyl (1,4-benzodioxanyl), benzonaphthofuranyl, benzo Oxazolyl, benzodioxolyl (benzodioxolyl), benzodioxinyl (benzodioxinyl), benzopyranyl, benzopyranone, benzofuranyl, benzofuranone , Benzothienyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazolyl, pyridyl Cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6- Dihydrobenzo[h]quinazolinyl (5,6-dihydrobenzo[h]quinazolinyl), 5,6-dihydrobenzo[h]cinnolinyl (5,6dihydrobenzo[h]cinnolinyl), 6,7 -Dihydro-5H-benzo[6,7]cycloheptano[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothienyl, furanyl, furanonyl, furo[ 3,2-c]pyridyl, 5,6,7,8,9,10-hexahydrocyclooctano[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocyclooctane And[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocyclooctano[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, Isoindolyl, indolinyl, isoindolinyl, isoquinolinyl, indolizinyl, isoxazolyl, 5,8-methanol-5,6,7,8- Tetrahydroquinazolinyl (5,8-methano-5,6,7,8-tetrahydroquinazolinyl), naphthyridinyl, 1,6-naphthyridinonyl, oxadiazole Group, 2-oxoazepinyl (2-oxoazepinyl), oxazolyl, oxiranyl (oxiranyl), 5,6,6a,7,8,9,10,10a-octahydrobenzo[ H)quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl , Pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridyl, pyrido[3,2-d]pyridine Iridinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinoline Group, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7, 8,9-tetrahydro-5H-cycloheptano[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridine Azinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[ 2,3-c]pyridyl (thieno[2,3-c]pridinyl) and thiophenyl/thienyl.

Various hydroxyl protecting groups can be used in this disclosure. Generally speaking, the protective group makes 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 rest of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d, John Wiley & Sons, New York, 1991, as cited In this way, the above-mentioned documents are incorporated into this article as a whole. In some embodiments, the protecting group is stable under basic conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthene-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that can be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxy Trityl) and TMTr (4,4',4"-trimethoxytrityl).

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

As used herein, "treat", "relief" or "improve" can be used interchangeably herein. These terms refer to methods for obtaining beneficial or desired results, including but not limited to therapeutic benefits. "Therapeutic benefit" means eradicating or improving the underlying barrier to be treated. In addition, therapeutic benefits are obtained by eradicating or improving one or more physiological symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be afflicted by the underlying disorder.

As used herein, "prevent" and "prevent" are used interchangeably. These terms refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits. In order to obtain a "preventive benefit", the conjugate or composition can be administered to a subject who is at risk of suffering from a particular disease, or to a subject who reports one or more physiological symptoms of the disease, even though the diagnosis of the disease Not yet made.

The siRNA of the present disclosure contains a nucleotide group as a basic structural unit, and those skilled in the art know that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be repeated here.

The first siRNA

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

The first siRNA contains a sense strand and an antisense strand, and each nucleotide in the first siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains 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 Sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length and have no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 2 The length is equal, and no more than 3 nucleotide differences:

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

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

Among them, Z ₁ is U, Z ₂ is A,

In addition, the nucleotide sequence I contains the nucleotide Z ₃ whose position corresponds to Z ₁ , and the nucleotide sequence II contains the nucleotide Z ₄ whose position corresponds to Z ₂ , and the Z ₄ is the The first nucleotide at the 5'end of the antisense strand.

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

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

In some embodiments, there is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1, and/or the nucleotide sequence II and SEQ ID NO: No more than one nucleotide difference between the nucleotide sequences shown in 2.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 includes the 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 the Z ₄ position, and Z _{4 is} selected from U, C, or G. In some embodiments, Z ₃ is a nucleotide that is complementary to Z ₄ . These nucleotide differences do not significantly reduce the target gene suppression ability of the siRNA conjugate, and siRNA conjugates containing these nucleotide differences are also within the protection scope of the present disclosure.

In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary. In the context of the present disclosure, the term "substantially reverse complementary" refers to the presence of no more than 3 base mismatches between two nucleotide sequences; "substantially reverse complementary" refers to two nucleotides There is no more than one base mismatch between the sequences; "complete reverse complementarity" means that there is no base mismatch between two nucleotide sequences.

In some embodiments, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 3, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 4:

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

5'-Z ₄ AGCACUUUAUUGAGUUCC-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 Where Z ₃ is U, Z ₄ is A;

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

In some embodiments, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV. The length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores. Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5 of the nucleotide sequence I 'End, 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 completely reverse-complementary to the second nucleotide sequence, and the second nucleotide sequence refers to the sequence ID of the target mRNA and NO: 1 represents a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence and has the same length as the nucleotide sequence IV.

In some embodiments, according to the 5'-3' direction, the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, The base of the nucleotide sequence IV is U; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is CA, and the base composition of nucleotide sequence IV is UG; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is GCA, and the base composition of nucleotide sequence IV is UGC; At this time, the length ratio of the sense strand and the antisense strand is 22/22; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, according to the direction from the 5'end to the 3'end, the nuclear The base composition of nucleotide sequence III is CGCA, and the base composition of nucleotide sequence IV is UGCG; at this time, the length ratio of the sense strand and the antisense strand is 23/23. In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, in the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is CA , The base composition of nucleotide sequence IV is UG; at this time, the length ratio of the sense strand and the antisense strand is 21/21.

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

The second siRNA

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

The second siRNA contains a sense strand and an antisense strand, each nucleotide in the second siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains 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 Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 61 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 62 The length is equal, and no more than 3 nucleotide differences:

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

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

Among them, Z ₅ is A and Z ₆ is U;

In addition, the nucleotide sequence I contains the nucleotide Z ₇ whose position corresponds to Z ₅ , and the nucleotide sequence II contains the nucleotide Z ₈ whose position corresponds to Z ₆ , and the Z ₈ is the The first nucleotide at the 5'end of the antisense strand.

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

In some embodiments, there is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61, and/or the nucleotide sequence II and SEQ ID NO: No more than one nucleotide difference between the nucleotide sequences shown in 62.

In some embodiments, the II and the nucleotide sequence SEQ ID NO: nucleotide differences between the nucleotide sequence shown at 62 include differences in location Z _8, and Z ₈ is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at the Z ₈ position, and Z _{8 is} selected from A, C, or G. In some embodiments, Z ₇ is a nucleotide that is complementary to Z ₈ . These nucleotide differences do not significantly reduce the target gene suppression ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the protection scope of the present disclosure.

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

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

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

5'-Z ₈ UCAAAGCACUUUAUUGAG-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 Where Z ₇ is A, Z ₈ is U;

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

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

In some embodiments, according to the 5'-3' direction, the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, The base of the nucleotide sequence IV is U; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is AA, and the base composition of nucleotide sequence IV is UU; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is GAA, and the base composition of nucleotide sequence IV is UUC; At this time, the length ratio of the sense strand and the antisense strand is 22/22; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, according to the direction from the 5'end to the 3'end, the nuclear The base composition of nucleotide sequence III is GGAA, and the base composition of nucleotide sequence IV is UUCC; at this time, the length ratio of the sense strand and the antisense strand is 23/23. In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, in the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is AA , The base composition of nucleotide sequence IV is UU; at this time, the length ratio of the sense strand and the antisense strand is 21/21.

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

The third siRNA

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

The third siRNA contains a sense strand and an antisense strand, and each nucleotide in the third siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains 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 Sequence I and the nucleotide sequence shown in SEQ ID NO: 121 are equal in length and have no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 122 The length is equal, and no more than 3 nucleotide differences:

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

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

Among them, Z ₁₃ is A, Z ₁₄ is U;

In addition, the nucleotide sequence I includes the nucleotide Z ₁₅ whose position corresponds to Z ₁₃ , and the nucleotide sequence II includes the nucleotide Z ₁₆ whose position corresponds to Z ₁₄ , and the Z ₁₆ is the The first nucleotide at the 5'end of the antisense strand.

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

In some embodiments, there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121, and/or the nucleotide sequence II and SEQ ID NO: 122 has no more than one nucleotide difference between the nucleotide sequences shown.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 122 includes the 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 the Z ₁₆ position, and Z _{16 is} selected from A, C, or G. In some embodiments, Z ₁₅ is a nucleotide that is complementary to Z ₁₆ . These nucleotide differences do not significantly reduce the target gene suppression ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the protection scope of the present disclosure.

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

In some embodiments, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 123, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 124:

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

5'-Z ₁₆ UUCACUUUCUUGGGCUCC-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 Where Z ₁₅ is A, Z ₁₆ is U;

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

In some embodiments, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV. The length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores. Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence, The second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5'end of the nucleotide sequence represented by SEQ ID NO: 121 in the target mRNA and has the same length as the nucleotide sequence IV .

In some embodiments, according to the 5'-3' direction, the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is U, The base of nucleotide sequence IV is A; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of nucleotide sequences III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is UU, and the base composition of nucleotide sequence IV is AA; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is UUU, and the base composition of nucleotide sequence IV is AAA; At this time, the length ratio of the sense strand and the antisense strand is 22/22; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, in the direction from the 5'end to the 3'end, the nuclear The base composition of nucleotide sequence III is GUUU, and the base composition of nucleotide sequence IV is AAAC; at this time, the length ratio of the sense strand and the antisense strand is 23/23. In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, in the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is AA , The base composition of nucleotide sequence IV is UU; at this time, the length ratio of the sense strand and the antisense strand is 21/21.

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

Fourth siRNA

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

The fifth siRNA contains a sense strand and an antisense strand, each nucleotide in the fifth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains 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 Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 181 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 182 The length is equal, and no more than 3 nucleotide differences:

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

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

Among them, Z ₁₇ is A, Z ₁₈ is U,

In addition, the nucleotide sequence I includes the nucleotide Z ₁₉ at a position corresponding to Z ₁₇ and the nucleotide sequence II includes the nucleotide Z ₂₀ at the position corresponding to Z ₁₈ , and the Z ₂₀ is the The first nucleotide at the 5'end of the antisense strand.

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

In some embodiments, there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181, and/or the nucleotide sequence II and SEQ ID NO: No more than 1 nucleotide difference between the nucleotide sequences shown in 182.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 182 includes the difference at position Z ₂₀ , and Z _{20 is} selected from A, C or G. In some embodiments, the difference is a difference between the nucleotide at position ₂₀ Z, 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 suppression ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the protection scope of the present disclosure.

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

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

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

5'-Z ₂₀ CUUUCACUUUCUUGGGCU-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 Where Z ₁₉ is A and Z ₂₀ is U;

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

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

In some embodiments, according to the 5'-3' direction, the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is G, and the nuclear The base of nucleotide sequence IV is C; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is GG, and the base composition of nucleotide sequence IV is CC; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is UGG, and the base composition of nucleotide sequence IV is CCA; At this time, the length ratio of the sense strand and the antisense strand is 22/22; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, according to the direction from the 5'end to the 3'end, the core The base composition of nucleotide sequence III is UUGG, and the base composition of nucleotide sequence IV is CCAA; at this time, the length ratio of the sense strand and the antisense strand is 23/23. In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, in the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is GG , The base composition of nucleotide sequence IV is CC; at this time, the length ratio of the sense strand and the antisense strand is 21/21.

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

The fifth siRNA

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

The fifth siRNA contains a sense strand and an antisense strand, each nucleotide in the fifth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleoside Acid sequence I, the antisense strand contains 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 Sequence I is the same length as the nucleotide sequence shown in SEQ ID NO: 241 and has no more than 3 nucleotide differences, and the nucleotide sequence II is the same as the nucleotide sequence shown in SEQ ID NO: 242 The length is equal, and no more than 3 nucleotide differences:

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

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

Among them, Z ₂₁ is A and Z ₂₂ is U;

The nucleotide sequence I contains the nucleotide Z ₂₃ whose position corresponds to Z ₂₁ , the nucleotide sequence II contains the nucleotide Z ₂₄ whose position corresponds to Z ₂₂ , and the Z ₂₄ is the reverse The first nucleotide at the 5'end of the sense strand.

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

In some embodiments, there is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241, and/or the nucleotide sequence II and SEQ No more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:242.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 242 includes the difference at position Z ₂₄ , and Z _{24 is} selected from A, C or G. In some embodiments, the difference is a difference between the nucleotide at position ₂₄ Z, 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 suppression ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the protection scope of the present disclosure.

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

In some embodiments, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 303, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 304:

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

5'-Z ₂₄ GGUCUUUCACUUUCUUGG-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 Where Z ₂₃ is A, Z ₂₄ is U;

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

In some embodiments, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV. The length of the nucleotide sequence III and the nucleotide sequence IV are each 1-4 cores. Nucleotide; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5 of the nucleotide sequence I 'End, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence, This second nucleotide sequence refers to a nucleotide sequence that is 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, according to the 5'-3' direction, the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is C, The base of nucleotide sequence IV is G; at this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end To the 3'end, the base composition of nucleotide sequence III is GC, and the base composition of nucleotide sequence IV is GC; at this time, the length ratio of the sense strand and the antisense strand is 21/21; or, The length of nucleotide sequence III and IV are both 3 nucleotides, according to the direction from 5'end to 3'end, the base composition of nucleotide sequence III is AGC, and the base composition of nucleotide sequence IV is GCU; At this time, the length ratio of the sense strand and the antisense strand is 22/22; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, in the direction from the 5'end to the 3'end, the nuclear The base composition of nucleotide sequence III is GAGC, and the base composition of nucleotide sequence IV is GCUC; at this time, the length ratio of the sense strand and the antisense strand is 23/23. In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is GC , The base composition of nucleotide sequence IV is GC; at this time, the length ratio of the sense strand and the antisense strand is 21/21.

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

Overhanging ends and modifications of siRNA

In the following, the description of nucleotide sequence V, nucleic acid sequence, nucleotide modification in siRNA, and modification sequence is applicable to any one of the above-mentioned first siRNA to fifth siRNA. That is, if there is no specific indication, the following description of siRNA should be regarded as describing the first siRNA, the second siRNA, the third siRNA, the fourth siRNA and the fifth siRNA one by one. For example, if no specific siRNA is specified, "the siRNA also contains the nucleotide sequence V" means "the first siRNA, the second siRNA, the third siRNA, the fourth siRNA or the fifth siRNA It also contains the nucleotide sequence V".

In some embodiments, the length of the sense strand and the antisense strand are different, and the antisense strand also contains a nucleotide sequence V. The length of the nucleotide sequence V is 1 to 3 nucleotides. The 3'end of the antisense strand constitutes the 3'overhang of the antisense strand. Thus, the length ratio of the siRNA sense strand and antisense strand 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 length of the nucleotide sequence V is 2 nucleotides. Therefore, the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure may be 19/21, 21/23, or 23. /25.

Each nucleotide in the nucleotide sequence V can be any nucleotide. In order to facilitate synthesis and save synthesis cost, the nucleotide sequence V is two consecutive thymine deoxyribonucleotides ( dTdT) or two consecutive uracil ribonucleotides (UU); or, in order to increase the affinity of the siRNA antisense strand with the target mRNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. Therefore, in some embodiments, the ratio of the length of the sense strand and the antisense strand of the siRNA of the present disclosure is 19/21 or 21/23. At this time, the siRNA of the present disclosure has better mRNA silencing activity.

The nucleotide at the corresponding position of the target mRNA refers to the nucleotide or nucleotide sequence adjacent to a nucleotide sequence of the target mRNA at the 5'end. The nucleotide sequence of the target mRNA is the same as the nucleotide sequence Sequence II is substantially reverse complementary or completely reverse complementary, or a nucleotide sequence that is substantially reverse complementary or completely reverse complementary to the nucleotide sequence composed of nucleotide sequence II and nucleotide sequence IV.

In some embodiments, for the first siRNA, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 5, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 6. The nucleotide sequence shown:

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

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

Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 8:

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

5'-Z ₄ AGCACUUUAUUGAGUUCCUGCG-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 contains the nucleotide sequence shown in SEQ ID NO: 65, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 66. The nucleotide sequence shown:

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

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

Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 67, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 68:

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

5'-Z ₈ UCAAAGCACUUUAUUGAGUUCC-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 contains the nucleotide sequence shown in SEQ ID NO: 125, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 126. The nucleotide sequence shown:

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

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

Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 127, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 128:

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

5'-Z ₁₆ UUCACUUUCUUGGGCUCCAAAC-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 contains the nucleotide sequence shown in SEQ ID NO: 185, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 186. The nucleotide sequence shown:

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

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

Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 187, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 188:

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

5'-Z ₂₀ CUUUCACUUUCUUGGGCUCCAA-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 the fifth siRNA, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 245, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 246. The nucleotide sequence shown:

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

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

Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 247, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 248:

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

5'-Z ₂₄ GGUCUUUCACUUUCUUGGGCUC-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 described in the present disclosure is siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1, or siFXIIf2:

siFXIIa1

Sense chain: 5'-GGAACUCAAUAAAGUGCUU-3' (SEQ ID NO: 9)

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

siFXIIa2

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

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

siFXIIb1

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

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

siFXIIb2

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

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

siFXIId1

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

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

siFXIId2

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

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

siFXIIe1

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

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

siFXIIe2

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

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

siFXIIf1

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

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

siFXIIf2

Sense chain: 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 (ie, a nucleic acid base sequence) shown in siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1 or siFXIIf2.

As mentioned above, the nucleotides in the siRNA 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. These modifications on the nucleoside group will not cause the siRNA conjugate of the present disclosure to significantly weaken or lose the function of inhibiting FXII gene expression.

In some embodiments, the siRNA of the present disclosure contains at least one modified nucleotide. In the context of the present disclosure, the term "modified nucleotides" used refers to nucleotides or nucleotide analogs formed by replacing the 2'hydroxyl group of the ribose group of nucleotides with other groups, or nucleosides The base on the acid is the nucleotide of the modified base. The modified nucleotides will not cause significant weakening or loss of the function of siRNA to inhibit gene expression. For example, 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 in the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate ester with a modified group In other words, at least a part of the phosphate group and/or ribose group in the phosphate-sugar backbone of at least one single chain in the sense strand and the antisense strand is a phosphate group with a modified group and/ Or a ribose group with a modified group.

In some embodiments, all 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 in the present disclosure is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.

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

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

In some embodiments, the fluoro-modified nucleotides are located in the nucleotide sequence I and the nucleotide sequence II, and there are no more than 5 fluoro-modified nucleotides in the nucleotide sequence I, In addition, according to the direction from the 5'end to the 3'end, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluorinated modified nucleotides; the fluorine in the nucleotide sequence II There are no more than 7 modified nucleotides, and the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides.

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

In the context of the present disclosure, "fluoromodified nucleotides" refer to nucleotides in which the hydroxyl group at the 2'position of the ribose group of the nucleotide is substituted with fluorine, and has a structure represented by the following formula (7). "Non-fluorinated modified nucleotides" refer to nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'position of the ribose group of a nucleotide with a non-fluorine group. In some embodiments, each non-fluorinated modified nucleotide is independently selected from among nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorine group. One kind.

The nucleotides formed by replacing the hydroxyl group at the 2'position of these ribose groups with non-fluorine groups are well known to those skilled in the art, and these nucleotides can be selected from 2'-alkoxy modified nucleotides, 2'- Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'- One of substituted amino-modified nucleotides and 2'-deoxynucleotides.

In some embodiments, the 2'-alkoxy modified nucleotides are methoxy modified nucleotides (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 ) Shown. 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 nucleotides that can replace nucleotides in nucleic acids, but the structure is different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxynucleotides The group of ribonucleotides. In some embodiments, the nucleotide analogs can be isonucleotides, bridged nucleotides (BNA), or acyclic nucleotides.

BNA refers to nucleotides that are constrained or inaccessible. BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endosaccharide condensed bridge structure. The bridge is usually incorporated into 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., where LNA is shown in formula (12), ENA is shown in formula (13), and cET BNA is shown in formula (14):

Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of nucleotides. In some embodiments, acyclic nucleotides can be unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA), where 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).

Isonucleotide refers to a compound formed by changing the position of the base in the nucleotide on the ribose ring. In some embodiments, the heteronucleotide may be a compound formed by moving a base from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formula (17) or (18):

In the compounds of formula (7) to formula (18) above, Base represents a nucleic acid base, such as 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 heteronucleotides, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluorinated modified nucleotide is a methoxy modified nucleotide. In the above and below, the methoxy modified nucleotide refers to the 2'of the ribose group. -Nucleotides formed by the substitution of hydroxy groups with methoxy groups.

In the above and below, "fluoro-modified nucleotides", "2'-fluoro-modified nucleotides", "nucleotides in which the 2'-hydroxyl group of the ribose group is substituted with fluorine" and "having 2 The meaning of'-fluororibose nucleotides' have the same meaning, and both refer to the compound having the structure shown in formula (7) formed by replacing the 2'-hydroxyl of the nucleotide with fluorine; "Methoxy modified core Nucleotides", "2'-methoxy-modified nucleotides", "nucleotides in which the 2'-hydroxyl group of a ribose group is substituted by a methoxy group" and "nuclei with 2'-methoxyribose groups The meaning of "nucleotide" has the same meaning, and both refer to the compound having the structure shown in formula (8) formed by replacing the 2'-hydroxyl group of the nucleotide ribose group with a methoxy group.

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

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

Alternatively, according to the direction from the 5'end to the 3'end, the 5th, 7th, 8th and 9th nucleotides of the nucleotide sequence I in the sense strand of the siRNA are fluorinated modified nucleotides, and the sense of siRNA The nucleotides at the remaining positions of the chain are methoxy-modified nucleotides, and in the direction from the 5'end to the 3'end, the second, sixth, and 14th nucleotide sequence II in the antisense strand of the siRNA The nucleotides at and 16 are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of siRNA are methoxy-modified nucleotides;

Alternatively, according to the direction from the 5'end to the 3'end, the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are -fluoro-modified nucleotides, and the sense strand of the siRNA The nucleotides at the remaining positions of the siRNA are methoxy-modified nucleotides, and in the direction from the 5'end to the 3'end, the second, sixth, 14th, and fourth nucleotide sequence II in the antisense strand of the siRNA The nucleotide at position 16 is a fluoro-modified nucleotide, and the nucleotide at the remaining position of the antisense strand of the siRNA is a methoxy-modified nucleotide.

In some embodiments, the siRNA provided in the present disclosure is 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 Any one of siFXIIe2-M2, siFXIIe2-M3, siFXIIf1-M1, siFXIIf1-M2, siFXIIf1-M3, siFXIIf2-M1, siFXIIf2-M2 and siFXIIf2-M3:

siFXIIa1-M1

Justice chain:

5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 13)

Antisense strand:

5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO: 14)

siFXIIa1-M2

Justice chain:

5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 15)

Antisense strand:

5'-AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO: 16)

siFXIIa1-M3

Justice chain:

5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 17)

Antisense strand:

5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO: 18)

siFXIIa2-M1

Justice chain:

5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 19)

Antisense strand:

5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO: 20)

siFXIIa2-M2

Justice chain:

5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 21)

Antisense strand:

5'-AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO: 22)

siFXIIa2-M3

Justice chain:

5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 23)

Antisense strand:

5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO: 24)

siFXIIb1-M1

Justice chain:

5'-CmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 73)

Antisense strand:

5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO: 74)

siFXIIb1-M2

Justice chain:

5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 75)

Antisense strand:

5'-UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO: 76)

siFXIIb1-M3

Justice chain:

5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 77)

Antisense strand:

5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO: 78)

siFXIIb2-M1

Justice chain:

5'-AmAmCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 79)

Antisense strand:

5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'

(SEQ ID NO: 80)

siFXIIb2-M2

Justice chain:

5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 81)

Antisense strand:

5'-UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'

(SEQ ID NO: 82)

siFXIIb2-M3

Justice chain:

5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 83)

Antisense strand:

5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'

(SEQ ID NO: 84)

siFXIId1-M1

Justice chain:

5'-GmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 133)

Antisense strand:

5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO: 134)

siFXIId1-M2

Justice chain:

5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 135)

Antisense strand:

5'-UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO: 136)

siFXIId1-M3

Justice chain:

5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 137)

Antisense strand:

5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO: 138)

siFXIId2-M1

Justice chain:

5'-UmUmGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 139)

Antisense strand:

5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'

(SEQ ID NO: 140)

siFXIId2-M2

Justice chain:

5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 141)

Antisense strand:

5'-UmUfUmCmAmCfUmUfUfCmUmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'

(SEQ ID NO: 142)

siFXIId2-M3

Justice chain:

5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 143)

Antisense strand:

5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'

(SEQ ID NO: 144)

siFXIIe1-M1

Justice chain:

5'-AmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 193)

Antisense strand:

5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO: 194)

siFXIIe1-M2

Justice chain:

5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 195)

Antisense strand:

5'-UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO: 196)

siFXIIe1-M3

Justice chain:

5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 197)

Antisense strand:

5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO: 198)

siFXIIe2-M1

Justice chain:

5'-GmGmAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 199)

Antisense strand:

5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'

(SEQ ID NO: 200)

siFXIIe2-M2

Justice chain:

5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 201)

Antisense strand:

5'-UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'

(SEQ ID NO: 202)

siFXIIe2-M3

Justice chain:

5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 203)

Antisense strand:

5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'

(SEQ ID NO: 204)

siFXIIf1-M1

Justice chain:

5'-CmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO:253)

Antisense strand:

5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO:254)

siFXIIf1-M2

Justice chain:

5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 255)

Antisense strand:

5'-UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO: 256)

siFXIIf1-M3

Justice chain:

5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 257)

Antisense strand:

5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO: 258)

siFXIIf2-M1

Justice chain:

5'-GmCmCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 259)

Antisense strand:

5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'

(SEQ ID NO: 260)

siFXIIf2-M2

Justice chain:

5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 261)

Antisense strand:

5'-UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'

(SEQ ID NO: 262)

siFXIIf2-M3

Justice chain:

5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 263)

Antisense strand:

5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'

(SEQ ID NO:264)

Among them, capital letters C, G, U, A indicate the base composition of nucleotides; lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates The adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide.

The modified siRNA not only has low cost, but also makes it difficult for ribonuclease in blood to cleave nucleic acid, thereby increasing the stability of nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis. At the same time, the above modifications did not significantly reduce the inhibitory performance of siRNA.

In some embodiments, at least a part of the phosphate groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand of the siRNA provided by the present disclosure is a phosphate group with a modification group. In some embodiments, the phosphate group with a modification group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, the The phosphate group with a modified group is a phosphorothioate group having a structure shown in formula (1):

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

In some embodiments, in the siRNA provided by the present disclosure, the phosphorothioate group linkage exists in at least one of the following positions: the first and second cores of either end of the sense strand or the antisense strand Between nucleotides; 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 the above positions except the 5'end of the sense chain. In some embodiments, the phosphorothioate group linkages are present at all the aforementioned positions except the 3'end of the sense chain. In some embodiments, the phosphorothioate group linkage is present in at least one of the following positions:

Between the first nucleotide and the second nucleotide of the 5'end of the sense strand;

Between the second nucleotide and the third nucleotide of the 5'end of the sense strand;

Between the first nucleotide and the second nucleotide of the 3'end of the sense strand;

Between the second nucleotide and the third nucleotide of the 3'end of the sense strand;

Between the first nucleotide and the second nucleotide of the 5'end of the antisense strand;

Between the second nucleotide and the third nucleotide of the 5'end of the antisense strand;

Between the first nucleotide and the second nucleotide of the 3'end of the antisense strand; and

Between the second nucleotide and the third nucleotide of the 3'end of the antisense strand.

In some embodiments, the siRNA provided by the present disclosure is siFXIIa1-M1S, siFXIIa1-M2S, siFXIIa1-M3S, siFXIIa2-M1S, siFXIIa2-M2S, siFXIIa2-M3S, siFXIIb1-M1S, siFXIIb1-M2S, siFXIIb1-M3S, siFXIIb1-M3S, M1S, siFXIIb2-M2S, siFXIIb2-M3S, siFXIId1-M1S, siFXIId1-M2S, siFXIId1-M3S, siFXIId2-M1S, siFXIId2-M2S, siFXIId2-M3S, siFXIIe1-M1S, siFXIIe1-e1-M1S, siFXIIe1-e1-M2S, siFXIIS Any one of siFXIIe2-M2S, siFXIIe2-M3S, siFXIIf1-M1S, siFXIIf1-M2S, siFXIIf1-M3S, siFXIIf2-M1S, siFXIIf2-M2S, and siFXIIf2-M3S:

siFXIIa1-M1S

Justice chain:

5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 25)

Antisense strand:

5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO: 26)

siFXIIa1-M2S

Justice chain:

5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 27)

Antisense strand:

5'-AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO: 28)

siFXIIa1-M3S

Justice chain:

5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 29)

Antisense strand:

5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO: 30)

siFXIIa2-M1S

Justice chain:

5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 31)

Antisense strand:

5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO: 32)

siFXIIa2-M2S

Justice chain:

5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 33)

Antisense strand:

5'-AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO: 34)

siFXIIa2-M3S

Justice chain:

5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 35)

Antisense strand:

5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO: 36)

siFXIIb1-M1S

Justice chain:

5'-CmsUmsCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 85)

Antisense strand:

5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO: 86)

siFXIIb1-M2S

Justice chain:

5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 87)

Antisense strand:

5'-UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO: 88)

siFXIIb1-M3S

Justice chain:

5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 89)

Antisense strand:

5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO: 90)

siFXIIb2-M1S

Justice chain:

5'-AmsAmsCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 91)

Antisense strand:

5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'

(SEQ ID NO: 92)

siFXIIb2-M2S

Justice chain:

5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 93)

Antisense strand:

5'-UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'

(SEQ ID NO: 94)

siFXIIb2-M3S

Justice chain:

5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 95)

Antisense strand:

5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'

(SEQ ID NO: 96)

siFXIId1-M1S

Justice chain:

5'-GmsGmsAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 145)

Antisense strand:

5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'

(SEQ ID NO: 146)

siFXIId1-M2S

Justice chain:

5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 147)

Antisense strand:

5'-UmsUfsUmCmAmCfUmUfUfCmUmUmUmGmGfGmCfUmCmCmsAmsAm-3'

(SEQ ID NO: 148)

siFXIId1-M3S

Justice chain:

5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 149)

Antisense strand:

5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'

(SEQ ID NO: 150)

siFXIId2-M1S

Justice chain:

5'-UmsUmsGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 151)

Antisense strand:

5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'

(SEQ ID NO: 152)

siFXIId2-M2S

Justice chain:

5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 153)

Antisense strand:

5'-UmsUfsUmCmAmCfUmUfUfCmUmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'

(SEQ ID NO: 154)

siFXIId2-M3S

Justice chain:

5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 155)

Antisense strand:

5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'

(SEQ ID NO: 156)

siFXIIe1-M1S

Justice chain:

5'-AmsGmsCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 205)

Antisense strand:

5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO: 206)

siFXIIe1-M2S

Justice chain:

5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 207)

Antisense strand:

5'-UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO: 208)

siFXIIe1-M3S

Justice chain:

5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 209)

Antisense strand:

5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO: 210)

siFXIIe2-M1S

Justice chain:

5'-GmsGmsAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 211)

Antisense strand:

5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'

(SEQ ID NO: 212)

siFXIIe2-M2S

Justice chain:

5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 213)

Antisense strand:

5'-UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'

(SEQ ID NO: 214)

siFXIIe2-M3S

Justice chain:

5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 215)

Antisense strand:

5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'

(SEQ ID NO: 216)

siFXIIf1-M1S

Justice chain:

5'-CmsCmsAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 265)

Antisense strand:

5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO: 266)

siFXIIf1-M2S

Justice chain:

5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 267)

Antisense strand:

5'-UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO: 268)

siFXIIf1-M3S

Justice chain:

5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 269)

Antisense strand:

5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO: 270)

siFXIIf2-M1S

Justice chain:

5'-GmsCmsCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 271)

Antisense strand:

5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'

(SEQ ID NO: 272)

siFXIIf2-M2S

Justice chain:

5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 273)

Antisense strand:

5'-UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'

(SEQ ID NO: 274)

siFXIIf2-M3S

Justice chain:

5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 275)

Antisense strand:

5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'

(SEQ ID NO: 276)

Among them, capital letters C, G, U, A indicate the base composition of nucleotides; lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates The adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; the lowercase letter s means that the two nucleotides on the left and right of the letter are connected by phosphorothioate groups.

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

The commonly used 5'-phosphate nucleotides or 5'-phosphate analog modified nucleotides are well known to those skilled in the art, for example, 5'-phosphate nucleotides can have the following formula (2) structure:

For another example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 discloses the following 4 kinds of 5'-phosphate analog modified nucleosides acid:

Wherein, R is selected from H, OH, methoxy, fluorine; Base represents a nucleic acid base, selected from A, U, C, G or T.

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

In some embodiments, the siRNA provided in the present disclosure is siFXIIa1-M1P1, siFXIIa1-M2P1, siFXIIa1-M3P1, siFXIIa2-M1P1, siFXIIa2-M2P1, siFXIIa2-M3P1, siFXIIa1-M1SP1, siFXIIa1-M2SP1, siFXIIa1-M3SP1, siFXIIa1-M3SP1 M1SP1, siFXIIa2-M2SP1, siFXIIa2-M3SP1, siFXIIa1U-M1P1, siFXIIa1U-M2P1, siFXIIa1U-M3P1, siFXIIa2U-M1P1, siFXIIa2U-M2P1, siFXIIa2U-M3P1, siFXIIa2U-M3PII, siFXIIa1, Msi1USPII siFXIIa2U-M2SP1, siFXIIa2U-M3SP1, siFXIIb1-M1P1, siFXIIb1-M2P1, siFXIIb1-M3P1, siFXIIb2-M1P1, siFXIIb2-M2P1, siFXIIb2-M3P1, siFXIIb1-M1b2-M3P1, siFXIIb1-M1SP1, siFXIIb1, siFXIIb1-M1SP1 M2SP1, siFXIIb2-M3SP1, siFXIId1-M1P1, siFXIId1-M2P1, siFXIId1-M3P1, siFXIId2-M1P1, siFXIId2-M2P1, siFXIId2-M3P1, siFXIId1-M1SP1, siFXIId1-M2SP1, siFXIId1-M3SP1, siFXIId1-M3SP1 siFXIId2-M3SP1, siFXIIe1-M1P1, siFXIIe1-M2P1, siFXIIe1-M3P1, siFXIIe2-M1P1, siFXIIe2-M2P1, siFXIIe2-M3P1, siFXIIe1-M1SP1, siFXIIe1-M2SP1, siFXIIe1-M3SP1, siFXIISP1, siFXIIe1-M3SP1, siFXIIe2-FX M3SP1, siFXIIf1-M1P1, siFXIIf1-M2P1, siFXIIf1-M3P1, siFXIIf2-M1P1, siFXIIf2-M2P1, siFXIIf2-M3P1, siFXIIf1-M1SP 1. Any one of siFXIIf1-M2SP1, siFXIIf1-M3SP1, siFXIIf2-M1SP1, siFXIIf2-M2SP1 and siFXIIf2-M3SP1:

siFXIIa1-M1P1

Justice chain:

5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 37)

Antisense strand:

5'-P1AmAfGmCmAmCfUmUmUmAmUmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO: 38)

siFXIIa1-M2P1

Justice chain:

5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 39)

Antisense strand:

5'-P1AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO: 40)

siFXIIa1-M3P1

Justice chain:

5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 41)

Antisense strand:

5'-P1AmAfGmCmAmCfUmUmUmAmUmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO: 42)

siFXIIa2-M1P1

Justice chain:

5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 43)

Antisense strand:

5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO: 44)

siFXIIa2-M2P1

Justice chain:

5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 45)

Antisense strand:

5'-P1AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO: 46)

siFXIIa2-M3P1

Justice chain:

5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 47)

Antisense strand:

5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO: 48)

siFXIIa1-M1SP1

Justice chain:

5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 49)

Antisense strand:

5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO: 50)

siFXIIa1-M2SP1

Justice chain:

5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 51)

Antisense strand:

5'-P1AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO: 52)

siFXIIa1-M3SP1

Justice chain:

5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 53)

Antisense strand:

5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO: 54)

siFXIIa2-M1SP1

Justice chain:

5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 55)

Antisense strand:

5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO: 56)

siFXIIa2-M2SP1

Justice chain:

5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 57)

Antisense strand:

5'-P1AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO: 58)

siFXIIa2-M3SP1

Justice chain:

5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'

(SEQ ID NO: 59)

Antisense strand:

5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO: 60)

siFXIIa1U-M1P1

Justice chain:

5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO: 335)

Antisense strand:

5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO: 336)

siFXIIa1U-M2P1

Justice chain:

5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO: 337)

Antisense strand:

5'-P1UmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO: 338)

siFXIIa1U-M3P1

Justice chain:

5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO: 339)

Antisense strand:

5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'

(SEQ ID NO: 340)

siFXIIa2U-M1P1

Justice chain:

5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO: 341)

Antisense strand:

5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO: 342)

siFXIIa2U-M2P1

Justice chain:

5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO: 343)

Antisense strand:

5'-P1UmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO: 344)

siFXIIa2U-M3P1

Justice chain:

5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO: 345)

Antisense strand:

5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'

(SEQ ID NO: 346)

siFXIIa1U-M1SP1

Justice chain:

5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO: 347)

Antisense strand:

5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO: 348)

siFXIIa1U-M2SP1

Justice chain:

5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO: 349)

Antisense strand:

5'-P1UmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO: 350)

siFXIIa1U-M3SP1

Justice chain:

5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO: 351)

Antisense strand:

5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'

(SEQ ID NO: 352)

siFXIIa2U-M1SP1

Justice chain:

5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO: 353)

Antisense strand:

5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO: 354)

siFXIIa2U-M2SP1

Justice chain:

5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO: 355)

Antisense strand:

5'-P1UmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO: 356)

siFXIIa2U-M3SP1

Justice chain:

5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'

(SEQ ID NO: 357)

Antisense strand:

5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'

(SEQ ID NO: 358)

siFXIIb1-M1P1

Justice chain:

5'-CmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 97)

Antisense strand:

5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO: 98)

siFXIIb1-M2P1

Justice chain:

5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 99)

Antisense strand:

5'-P1UmUfCmAmAmAfGmCfAfCmUmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO: 100)

siFXIIb1-M3P1

Justice chain:

5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 101)

Antisense strand:

5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmUmAfUmUfGmAmGmUmUm-3'

(SEQ ID NO: 102)

siFXIIb2-M1P1

Justice chain:

5'-AmAmCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 103)

Antisense strand:

5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'

(SEQ ID NO: 104)

siFXIIb2-M2P1

Justice chain:

5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 105)

Antisense strand:

5'-P1UmUfCmAmAmAfGmCfAfCmUmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'

(SEQ ID NO: 106)

siFXIIb2-M3P1

Justice chain:

5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 107)

Antisense strand:

5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'

(SEQ ID NO: 108)

siFXIIb1-M1SP1

Justice chain:

5'-CmsUmsCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 109)

Antisense strand:

5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO: 110)

siFXIIb1-M2SP1

Justice chain:

5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 111)

Antisense strand:

5'-P1UmsUfsCmAmAmAfGmCfAfCmUmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO: 112)

siFXIIb1-M3SP1

Justice chain:

5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 113)

Antisense strand:

5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'

(SEQ ID NO: 114)

siFXIIb2-M1SP1

Justice chain:

5'-AmsAmsCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 115)

Antisense strand:

5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'

(SEQ ID NO: 116)

SiFXIIb2-M2SP1

Justice chain:

5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 117)

Antisense strand:

5'-P1UmsUfsCmAmAmAfGmCfAfCmUmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'

(SEQ ID NO: 118)

SiFXIIb2-M3SP1

Justice chain:

5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'

(SEQ ID NO: 119)

Antisense strand:

5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'

(SEQ ID NO: 120)

siFXIId1-M1P1

Justice chain:

5'-GmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 157)

Antisense strand:

5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO: 158)

siFXIId1-M2P1

Justice chain:

5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 159)

Antisense strand:

5'-P1UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO: 160)

siFXIId1-M3P1

Justice chain:

5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 161)

Antisense strand:

5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'

(SEQ ID NO: 162)

siFXIId2-M1P1

Justice chain:

5'-UmUmGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 163)

Antisense strand:

5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'

siFXIId2-M2P1

Justice chain:

5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 165)

Antisense strand:

5'-P1UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'

(SEQ ID NO: 166)

siFXIId2-M3P1

Justice chain:

5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 167)

Antisense strand:

5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'

(SEQ ID NO: 168)

siFXIId1-M1SP1

Justice chain:

5'-GmsGmsAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 169)

Antisense strand:

5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'

(SEQ ID NO: 170)

siFXIId1-M2SP1

Justice chain:

5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 171)

Antisense strand:

5'-P1UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmsAmsAm-3'

(SEQ ID NO: 172)

siFXIId1-M3SP1

Justice chain:

5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 173)

Antisense strand:

5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'

(SEQ ID NO: 174)

siFXIId2-M1SP1

Justice chain:

5'-UmsUmsGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 175)

Antisense strand:

5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'

(SEQ ID NO: 176)

siFXIId2-M2SP1

Justice chain:

5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 177)

Antisense strand:

5'-P1UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'

(SEQ ID NO: 178)

siFXIId2-M3SP1

Justice chain:

5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'

(SEQ ID NO: 179)

Antisense strand:

5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'

(SEQ ID NO: 180)

siFXIIe1-M1P1

Justice chain:

5'-AmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 217)

Antisense strand:

5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO: 218)

siFXIIe1-M2P1

Justice chain:

5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 219)

Antisense strand:

5'-P1UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO: 220)

siFXIIe1-M3P1

Justice chain:

5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 221)

Antisense strand:

5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'

(SEQ ID NO: 222)

siFXIIe2-M1P1

Justice chain:

5'-GmGmAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 223)

Antisense strand:

5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'

(SEQ ID NO: 224)

siFXIIe2-M2P1

Justice chain:

5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 225)

Antisense strand:

5'-P1UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'

(SEQ ID NO: 226)

siFXIIe2-M3P1

Justice chain:

5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 227)

Antisense strand:

5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'

(SEQ ID NO: 228)

siFXIIe1-M1SP1

Justice chain:

5'-AmsGmsCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 229)

Antisense strand:

5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO: 230)

siFXIIe1-M2SP1

Justice chain:

5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 231)

Antisense strand:

5'-P1UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO: 232)

siFXIIe1-M3SP1

Justice chain:

5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 233)

Antisense strand:

5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'

(SEQ ID NO: 234)

siFXIIe2-M1SP1

Justice chain:

5'-GmsGmsAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 235)

Antisense strand:

5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'

(SEQ ID NO: 236)

siFXIIe2-M2SP1

Justice chain:

5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 237)

Antisense strand:

5'-P1UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'

(SEQ ID NO: 238)

siFXIIe2-M3SP1

Justice chain:

5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'

(SEQ ID NO: 239)

Antisense strand:

5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'

(SEQ ID NO: 240)

siFXIIf1-M1P1

Justice chain:

5'-CmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 277)

Antisense strand:

5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO: 278)

siFXIIf1-M2P1

Justice chain:

5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 279)

Antisense strand:

5'-P1UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO: 280)

siFXIIf1-M3P1

Justice chain:

5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 281)

Antisense strand:

5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'

(SEQ ID NO: 282)

siFXIIf2-M1P1

Justice chain:

5'-GmCmCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 283)

Antisense strand:

5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'

(SEQ ID NO: 284)

siFXIIf2-M2P1

Justice chain:

5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 285)

Antisense strand:

5'-P1UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'

(SEQ ID NO: 286)

siFXIIf2-M3P1

Justice chain:

5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 287)

Antisense strand:

5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'

(SEQ ID NO: 288)

siFXIIf1-M1SP1

Justice chain:

5'-CmsCmsAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 289)

Antisense strand:

5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO: 290)

siFXIIf1-M2SP1

Justice chain:

5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 291)

Antisense strand:

5'-P1UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO: 292)

siFXIIf1-M3SP1

Justice chain:

5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 293)

Antisense strand:

5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'

(SEQ ID NO: 294)

siFXIIf2-M1SP1

Justice chain:

5'-GmsCmsCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 295)

Antisense strand:

5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'

(SEQ ID NO: 296)

siFXIIf2-M2SP1

Justice chain:

5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 297)

Antisense strand:

5'-P1UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'

(SEQ ID NO: 298)

siFXIIf2-M3SP1

Justice chain:

5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'

(SEQ ID NO: 299)

Antisense strand:

5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'

(SEQ ID NO: 300)

Among them, capital letters C, G, U, A indicate the base composition of nucleotides; lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates The adjacent nucleotide on the left side of the letter f is a fluoro-modified nucleotide; the lowercase letter s means that the two nucleotides on the left and right sides of the letter are connected by phosphorothioate groups; The adjacent nucleotide is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide. In some embodiments, P1 represents a specifically modified VP, Ps or P, where the letter combination VP means that the adjacent nucleotide to the right of the letter combination VP is vinyl phosphate (5'-(E)- vinylphosphonate, E-VP) modified nucleotides, the letter combination Ps indicates that the adjacent nucleotide on the right side of the letter combination Ps is a phosphorothioate modified nucleotide, and the capital letter P indicates the right side of the letter P The adjacent nucleotide is a 5'-phosphate nucleotide.

The inventors of the present disclosure unexpectedly discovered that the siRNA provided in the present disclosure not only has significantly enhanced plasma and lysosome stability, but also retains high gene suppression activity.

The siRNA provided in the present disclosure can be obtained by conventional siRNA preparation methods in the art (for example, solid-phase synthesis and liquid-phase synthesis). Among them, solid-phase synthesis already has commercial customized services. The modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications, the method for preparing nucleoside monomers with corresponding modifications and the introduction of modified nucleotide groups The method of siRNA is also well known to those skilled in the art.

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 field of siRNA administration, such as but not limited to magnetic nanoparticles (magnetic nanoparticles, such as Fe ₃ O ₄ or Fe ₂ O _3- based nanoparticles), carbon nanotubes ( carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, polylysine Acid (poly(L-lysine), PLL), chitosan (chitosan), 1,2-dioleoyl-3-trimethylammonium-propane (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP), poly D Type or L type lactic acid/glycolic acid copolymer (poly(D&L-lactic/glycolic acid) copolymer, PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA) and poly( One or more of poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) and their derivatives.

In some embodiments, there is no special requirement for the content of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition. In some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier may be 1:( 1-500). In some embodiments, the weight ratio is 1:(1-50).

In some embodiments, the pharmaceutical composition may also include other pharmaceutically acceptable auxiliary materials, which may be one or more of various formulations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable auxiliary materials may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

The pH buffer may be a tris hydrochloride buffer with a pH of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, for example, it may be a phosphate with a pH of 5.5-8.5 Buffer.

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

The osmotic pressure regulator may be sodium chloride and/or potassium chloride. The content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 millimoles per kilogram (mOsm/kg). According to the required osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.

In some embodiments, the pharmaceutical composition may be a liquid preparation, such as an injection, or a freeze-dried powder injection, which is mixed with liquid excipients during administration to prepare a liquid preparation. The liquid formulation can be, but is not limited to, administered by subcutaneous, intramuscular or intravenous injection, and can also be, but not limited to, administered to the lungs by spraying, or administered to other organs and tissues (such as liver) by spraying through the lungs. In some embodiments, the pharmaceutical composition is for intravenous administration.

In some embodiments, the pharmaceutical composition may be in the form of a liposome formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome formulation contains an amine-containing transfection compound (hereinafter may also be referred to as an organic amine), auxiliary lipid and/or PEGylation Lipid. Wherein, the organic amine, auxiliary lipid and pegylated lipid can be selected from the amine-containing transfection compound described in CN103380113A (incorporated in its entirety by reference) or its pharmaceutically acceptable One or more of the accepted salt or derivative, auxiliary lipid, and pegylated lipid.

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

among them:

Each of X ₁₀₁ and X _{102 is} independently O, S, NA or CA, where A is hydrogen or a C1-C20 hydrocarbon chain;

Each of Y ₁₀₁ and Z _{101 is} independently C=O, C=S, S=O, CH-OH or SO ₂ ;

Each of R ₁₀₁ , R ₁₀₂ , R ₁₀₃ , R ₁₀₄ , R ₁₀₅ , R ₁₀₆ and R _{107 is} independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight chain lipid Group group, cyclic or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic group, substituted or unsubstituted, branched or straight chain acyl group, substituted Or unsubstituted, branched or straight chain aryl, substituted or unsubstituted, branched or straight chain heteroaryl;

x is an integer of 1-10;

n is an integer of 1-3, m is an integer of 0-20, and p is 0 or 1; where, if m=p=0, 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 from 1 to 6, "HCC" represents a hydrocarbon chain, and each *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. The radical or alkenyl group has 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 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):

Among them, in formulas (204)-(213), g, e and f are each independently an integer from 1 to 6, each "HCC" represents a hydrocarbon chain, and each * shows that R ₁₀₃ and the formula (201) The possible attachment points of the nitrogen atom in where each H at any * position can be replaced to achieve the attachment to the nitrogen atom in formula (201).

Among them, the compound represented by formula (201) can be prepared according to the description in CN103380113A.

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

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

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

In some embodiments, in the pharmaceutical composition, the molar ratio between the organic amine, the auxiliary lipid and the PEGylated lipid 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 by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, usually about 40 nm to about 135 nm, and more generally, the liposome The average diameter of the particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, for example, 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, in the pharmaceutical composition formed by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent, the weight of siRNA and all lipids (for example, organic amines, auxiliary lipids and/or PEGylated lipids) The ratio (weight/weight ratio) is 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: In the range of 6 to about 1:10, for example, the weight ratio of the siRNA of the present disclosure to all lipids 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, each component of the pharmaceutical composition may exist independently when sold, and may exist in the form of a liquid preparation when used. In some embodiments, the pharmaceutical composition formed by the siRNA provided in the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods, except that the siRNA provided in the present disclosure can replace the existing siRNA; in some In the embodiment, it can be prepared as follows:

The organic amine, auxiliary lipid and PEGylated lipid are suspended in alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol is such that the total mass concentration of the lipid solution is 2-25 mg/mL, For example, it can be 8-18 mg/mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400 One or more of, for example, ethanol.

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

The lipid solution and the siRNA aqueous solution are mixed, and the mixed product is incubated at 40-60° C. for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome preparation. The volume ratio of lipid solution and siRNA aqueous solution is 1: (2-5).

The incubated liposome preparation is concentrated or diluted to remove impurities and sterilize to obtain the pharmaceutical composition provided by the present disclosure. Its physical and chemical parameters are pH 6.5-8, encapsulation rate not less than 80%, and particle size 40-200nm, the polydispersity index is not higher than 0.30, the osmotic pressure is 250-400mOsm/kg; for example, the physical and chemical parameters can be pH 7.2-7.6, the encapsulation rate is not less than 90%, and the particle size is 60-100nm. The dispersion index is not higher than 0.20, and the osmotic pressure is 300-400mOsm/kg.

Among them, the concentration or dilution can be performed before, after or at the same time as removing impurities. Various existing methods can be used to remove impurities, such as a tangential flow system, a hollow fiber column, ultrafiltration under 100K Da conditions, and the ultrafiltration exchange solution is phosphate buffered saline (PBS) with pH 7.4. The method of sterilization can adopt various existing methods, for example, filter sterilization on a 0.22 μm filter.

siRNA conjugate

The present disclosure provides an siRNA conjugate containing the above-mentioned siRNA and a conjugating group conjugated to the siRNA.

Generally, the conjugating group includes at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA, the linker and the targeting group are connected in sequence. In some embodiments, the targeting groups are 1-6. In some embodiments, there are 2-4 targeting groups. The siRNA molecule may be non-covalently or covalently conjugated to the conjugating group, for example, may be covalently conjugated to the conjugating group. The conjugation site of the siRNA and the conjugating group can be at the 3'end or 5'end of the siRNA sense strand, or 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 and the conjugating group is at the 3'end of the sense strand of the siRNA.

In some embodiments, the conjugating group may be attached to the phosphate group, the 2'-position hydroxyl group or the base of the nucleotide. In some embodiments, the conjugating group can also be connected to the 3'-position hydroxyl group, in which case the nucleotides are connected by 2'-5' phosphodiester bonds. When the conjugating group is attached to the end of the siRNA chain, the conjugating group is usually attached to the phosphate group of the nucleotide; when the conjugating group is attached to the internal sequence of the siRNA, the conjugating group is Usually attached to the ribose ring or base. Various connection methods can refer to the literature: Muthiah Manoharan et al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silence in vivo in hepatocytes. ACS Chemical biology, 2015, 10 (5)

In some embodiments, the siRNA and the conjugating group may be connected by acid-labile or reducible chemical bonds. Under the acidic environment of cell endosomes, these chemical bonds can be degraded, so that the siRNA becomes a free state. For non-degradable conjugation methods, the conjugating group can be attached to the sense strand of siRNA, thereby minimizing the effect of conjugation on siRNA activity.

In some embodiments, the pharmaceutically acceptable targeting group may be a ligand commonly used in the field of siRNA administration, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated by reference This article.

In some embodiments, the pharmaceutically acceptable targeting group may be selected from one or more of the following targeting molecules or ligands formed by their derivatives: lipophilic molecules, such as cholesterol, bile acids, Vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, and mannose Sugar, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by liver parenchymal cells, such as asialoglycoprotein, asialoglycoside residues, lipoproteins (such as high-density Lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitter (such as adrenaline), growth factor, transferrin, etc.

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

In some embodiments, the pharmaceutically acceptable targeting group may be any ligand that binds to the asialoglycoprotein receptor (ASGPR) on the surface of mammalian liver cells. In some embodiments, each ligand is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF). In some embodiments, the ligand is a sugar or sugar derivative.

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, polysaccharide, modified monosaccharide, modified polysaccharide, or sugar derivative. In some embodiments, at least one of the ligands can 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 polysaccharides, modified polysaccharides, monosaccharides, modified monosaccharides, polysaccharide derivatives, or monosaccharide derivatives. In some embodiments, each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives Food, 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 D-mannanose, L-mannanose, D-arabinose, D-xylofuranose, L-xylofuranose, D- Glucose, L-glucose, D-galactose, L-galactose, α-D-mannanose, β-D-mannanose, α-D-mannanose, β-D-mannanose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-frutofuranose, α-D-fructose pyranose, α-D-pyranose Galactopyrose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-tris Fluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl ]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O -Methyl-D-mannanose, 2-deoxy-2-sulfoamino-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-pyran half Lactose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyrano-heptanoside ethyl ester, 2,5-anhydro-D-A Loose nitrile, ribose, D-ribose, D-4-thioribose, L-ribose or L-4-thioribose. For other selections of the ligands, see, for example, the description 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 , Second price, three price, four price. It should be understood that the monovalent, bivalent, trivalent, and tetravalent mentioned herein refer to siRNA molecules and conjugate molecules containing galactose or N-acetylgalactosamine groups as targeting groups to form siRNA conjugates. After compounding, the molar ratio of the siRNA group to the galactose or N-acetylgalactosamine group in the siRNA conjugate is 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 with a N-acetylgalactosamine-containing conjugating group, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described in the present disclosure is conjugated with a N-acetylgalactosamine-containing conjugating group, the N-acetylgalactosamine molecule is trivalent.

The targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group. For the types of linkers, targeting groups, and connection methods with siRNA, please refer to the disclosure of WO2015006740A2, and the entire content is incorporated herein by reference.

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

among them,

k is an integer of 1-3;

L ^A is a chain-like part containing an amide bond having a structure as shown in formula (302), and each of the L ^{A is} connected to one of the targeting groups and the L ^C moiety through ether bonds at its two ends. connection:

L ^B having the formula (303) comprises a pyrrolidine N- acyl chain portion shown structure, the linear portion having a carbonyl group at one end thereof and connected with the L ^C moiety through an amide bond, at the other end It has an oxygen group and is connected to the siRNA through a phosphate bond:

L ^C based aminomethane, two monovalent 2-4 aminomethane or tris (hydroxymethyl) aminomethane linking group, via an oxygen atom of the L ^C L ^A portion of each of the phases by ether linkages connection, and connected by an amide bond via a nitrogen atom and L ^B of the portion.

In some embodiments, when n = 3, L ^C, based on 4 Tris divalent linking group by a linker - (L ^A) ₃ Tris -L ^B - connector The structure of the siRNA conjugate formed by N-acetylgalactosamine molecule and siRNA molecule is shown in the following formula (304):

In the formula, the double helix structure represents siRNA.

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

In some embodiments, the present disclosure of the siRNA sense strand 3 'end by the linker - (L ^A) ₃ Tris -L ^B - with three N- acetylgalactosamine (GalNAc) Covalent Conjugation to obtain an siRNA conjugate with a molar ratio of siRNA molecule to GalNAc molecule of 1:3, which may also be referred to as (GalNAc) ₃ -siRNA below, and its structure is shown in the following formula (305):

Wherein, the double helix structure represents the siRNA, and the linker is connected 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 have a structure as shown in formula (306):

among them,

l is an integer of 0-3;

^* Indicates the point on the linker connected to the targeting group through an ether bond;

^# Indicates the site on the linker that is connected to the siRNA via a phosphate bond.

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

Wherein, the double helix structure represents the siRNA, and the linker is connected to the 3'end of the sense strand of the siRNA.

The above-mentioned conjugate can be synthesized by a method that has been described in detail in the prior art. For example, WO2015006740A2 describes the preparation methods of various conjugates in detail. The siRNA conjugate of the present disclosure is obtained by a method well known to those skilled in the art. For example, WO2014025805A1 describes the preparation method of the structure represented by formula (305), Rajeev et al. described the preparation method of the structure represented by formula (307) in ChemBioChem 2015, 16, 903-908.

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

among them:

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 2-10;

Each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R _{15 is} independently H, or is selected from the group consisting of C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ haloalkyl and C ₁ -C ₁₀ alkoxy;

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

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

R ₂ is a linear alkylene group having a length of 1-20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C (O), NH, O, S, CH=N, S(O) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C _3- C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene; and wherein R ₂ may optionally have any one or more substituents in the group consisting of: C ₁ -C ₁₀ alkyl, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkylphenyl,- C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl -NH ₂ , -N (C ₁ -C ₁₀ alkyl) (C _1- C ₁₀ alkyl), -NH (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl phenyl), -NH (C ₁ -C ₁₀ alkyl) Phenyl), cyano, nitro, -CO ₂ H, -C(O)O (C ₁ -C ₁₀ alkyl), -CON (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkane基), -CONH (C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O) (C ₁ -C ₁₀ alkyl), -NHC(O) (phenyl), -N(C _1- C ₁₀ alkyl) C (O) (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ alkane Group, -C(O)C ₁ -C ₁₀ alkylphenyl, -C(O)C ₁ -C ₁₀ haloalkyl, -OC(O)C ₁ -C ₁₀ alkyl, -SO ₂ (C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl), -SO ₂ NH _{(phenyl), - NHSO 2 (C 1} -C 10 alkyl), - NHSO ₂ (phenyl), and -NHSO ₂ (C ₁ -C ₁₀ haloalkyl);

Each L ₁ is a linear alkylene group with a length of 1-70 carbon atoms, wherein one or more carbon atoms is optionally replaced by any one or more selected from the group consisting of the following groups : C(O), NH, O, S, CH=N, S(O) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C _3- C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene; and wherein, L ₁ may optionally have any one or more substituents in the group consisting of: C ₁ -C ₁₀ alkyl, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkylphenyl , -C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl- SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl -NH ₂ , -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl), -NH (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl phenyl), -NH (C ₁ -C ₁₀ alkyl phenyl), cyano, nitro, -CO ₂ H, -C(O)O (C ₁ -C ₁₀ alkyl), -CON (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl), -CONH (C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O)(C ₁ -C ₁₀ alkyl), -NHC(O)(phenyl), -N(C ₁ -C ₁₀ alkyl) C (O) (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ alkyl, -C(O)C ₁ -C ₁₀ alkylphenyl, -C(O)C ₁ -C ₁₀ haloalkyl, -OC(O)C ₁ -C ₁₀ alkyl, -SO ₂ ( C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl), -SO ₂ NH _{(phenyl), - NHSO 2 (C 1} -C 10 alkyl), - NHSO ₂ (phenyl), and -NHSO ₂ (C ₁ -C ₁₀ haloalkyl).

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

Among them, j1 is an integer of 1-20; j2 is an integer of 1-20;

R'is C ₁ -C ₁₀ alkyl;

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

表示基团共价连接的位点。 Rb is C ₁ -C ₁₀ alkyl;

Represents the site where the group is covalently attached.

The skilled person will understand that although L ₁ is defined as a linear alkylene group for convenience, it may not be a linear group or have a different name, such as an amine or alkenyl group due to the above substitutions and/or substitutions. For the purposes of this disclosure, the length of L ₁ is the number of atoms in the chain connecting the two connection points. For this purpose, a ring (such as a heterocyclylene or heteroarylene) obtained by replacing the carbon atom of the linear alkylene group is counted as one atom.

M ₁ represents a targeting group, and its definition and selectable range are the same as the above-mentioned targeting group. In some embodiments, each M _{1 is} independently selected from one of the ligands having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.

When M ₁ is a ligand that has affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, in some embodiments, n1 may be an integer of 1-3, and n3 may be an integer of 0-4 , To ensure that the number of M ₁ targeting groups in the conjugate is at least 2; in some embodiments, n1+n3≥2, so that the number of M ₁ targeting groups can be at least 3, thereby This makes it easier for the M ₁ targeting group to bind to the asialoglycoprotein receptor on the liver surface, thereby promoting the conjugate to enter the cell through endocytosis. Experiments have shown that when the number of M ₁ targeting groups is greater than 3, the ease of binding of the M ₁ targeting group to the asialoglycoprotein receptor on the liver surface does not increase significantly. Therefore, from the ease of synthesis, Considering multiple aspects such as structure/process cost and delivery efficiency, in some embodiments, n1 is an integer of 1-2, n3 is an integer of 0-1, and n1+n3=2-3.

In some embodiments, when each m1, m2, and m3 are independently selected from an integer of 2-10, the spatial position between the multiple M ₁ targeting groups can be adapted to the M ₁ targeting group and the liver surface. The binding of sialoglycoprotein receptors, in order to make the conjugates provided in the present disclosure simpler, easier to synthesize and/or reduce costs, in some embodiments, each of m1, m2, and m3 is independently 2- An integer of 5. In some embodiments, m1=m2=m3.

Those skilled in the art can understand that when each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R _{15 is} independently selected from H, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ haloalkyl, As well as one of C ₁ -C ₁₀ alkoxy groups, the properties of the conjugate of the present disclosure will not be changed, and the objectives of the present disclosure can be achieved. In some embodiments, each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ are each independently selected from H, methyl, and ethyl. In some embodiments, each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R _14, and R ₁₅ is H.

R ₃ is a group represented by the formula A59, where E ₁ is OH, SH or BH _2. Based on the availability of raw materials for preparation, in some embodiments, E ₁ is OH or SH.

The choice of R ₂ is to realize the connection with the N atom on the nitrogen-containing skeleton and A59. In the context of the present disclosure, "nitrogen-containing skeleton" refers to a chain structure in which carbon atoms to which R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ are connected and N atoms are connected to each other. Therefore, R ₂ may be any linking group capable of linking the A59 group to the N atom on the nitrogen-containing skeleton in an appropriate manner. In some embodiments, when the siRNA conjugate represented by formula (308) is prepared by a solid-phase synthesis process, the R ₂ group needs to also contain a connection site to the N atom on the nitrogen-containing backbone. And the connection site connected to the P atom in R ₃ . In some embodiments, the site connected to the N atom on the nitrogen-containing skeleton in R ₂ forms an amide bond with N, and the site connected to the P atom on R ₃ forms a phosphate bond with the P atom; In some embodiments, R ₂ can be B5, B6, B5' or B6':

表示基团共价键连接的位点。 among them,

Represents the site where the groups are covalently bonded.

The value range of q ₂ can be an integer of 1-10. In some embodiments, q ₂ is an integer of 1-5.

The role of L ₁ is to connect the M ₁ targeting group to the N atom on the nitrogen-containing backbone to provide the liver targeting function for the siRNA conjugate represented by formula (308). In some embodiments, L _{1 is} selected from a linked combination of one or more of the groups of formula A1-A26. In some embodiments, L _{1 is} selected from the connection combination of one or more of A1, A4, A5, A6, A8, A10, A11, and A13. In some embodiments, L _{1 is} selected from a connection combination of at least two of A1, A4, A8, A10, and A11. In some embodiments, L _{1 is} selected from a connection combination of at least two of A1, A8, and A10.

In some embodiments, the length of L ₁ can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms. In some embodiments, the length of 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 atom.

In some embodiments, j1 is an integer of 2-10, and in some embodiments, j1 is an integer of 3-5. In some embodiments, j2 is an integer of 2-10, and in some embodiments, j2 is an integer of 3-5. R'is a C ₁ -C ₄ alkyl group, and in some embodiments, R'is one of methyl, ethyl, and isopropyl. Ra is one of A27, A28, A29, A30, and A31. In some embodiments, Ra is A27 or A28. Rb is a C ₁ -C ₅ alkyl group. In some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, each of j1, j2, R', Ra, Rb is selected in the formulas A1-A26, so that the M ₁ targeting group is connected to the N on the nitrogen-containing backbone, and M _{1 is} targeted The spatial position between the groups is more suitable for the binding of the M ₁ targeting group to the asialoglycoprotein receptor on the liver surface.

In some embodiments, the conjugate has formulas (403), (404), (405), (406), (407), (408), (409), (410), (411), (412 ), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422):

In some embodiments, the P atom in formula A59 can be attached to any possible position in the siRNA sequence. For example, the P atom in formula A59 can be attached to any nucleotide in the sense strand or antisense strand of the siRNA; In some embodiments, the P atom in formula A59 is connected to any nucleotide in the sense strand of the siRNA. In some embodiments, the P atom in formula A59 is connected to the end of the siRNA sense strand or antisense strand; in some embodiments, the P atom in formula A59 is connected to the end of the siRNA sense strand. 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 connected to the end of the siRNA sense strand or antisense strand; in some embodiments, the P atom in formula A59 is connected to the 3'end of the siRNA sense strand. In the case of being connected to the above position of the sense strand of siRNA, after the siRNA conjugate represented by formula (308) enters the cell, when unwinding, a separate siRNA antisense strand can be released to block FXII mRNA translation The protein process inhibits the expression of factor XII (FXII) gene.

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 of the nucleotide. 'Position or nucleotide base. In some embodiments, the P atom in formula A59 can be connected to the 2'position, 3'position or 5'position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments, the P atom in formula A59 is connected to the oxygen atom formed after the 3'hydroxyl of the 3'terminal nucleotide of the siRNA sense strand is dehydrogenated (at this time, the P atom in A59 can also be regarded as siRNA The P atom in the phosphate group contained in A59), or the P atom in formula A59 is connected to the nucleotide by substituting the hydrogen in the 2'-hydroxyl group of one nucleotide in the sense strand of siRNA, or the P in formula A59 The atom is connected to the nucleotide by replacing the hydrogen in the 5'hydroxyl group of the 5'terminal nucleotide of the siRNA sense strand.

The inventors of the present disclosure unexpectedly discovered that the siRNA conjugates of the present disclosure have significantly improved plasma stability and low off-target effects, while also showing FXII mRNA silencing activity that is not significantly reduced. Therefore, in some embodiments, the siRNA in the siRNA conjugate of the present disclosure may be any one of the siRNAs shown in Tables 1a, 1b, 1d, 1e, and 1f.

Table 1a The first siRNA sequence in the conjugate of the present disclosure

Table 1b The second siRNA sequence in the conjugate of the present disclosure

Table 1d The third siRNA sequence in the conjugate of the present disclosure

Table 1e The fourth siRNA sequence in the conjugate of the present disclosure

Table 1f The fifth siRNA sequence in the conjugate of the present disclosure

In the siRNA or siRNA conjugate described in the present disclosure, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate bond, and the phosphodiester bond or phosphorothioate bond is not bridged The oxygen atom or sulfur atom has a negative charge, and it can exist in the form of a hydroxyl group or a mercapto group. The hydrogen ions in the hydroxyl group or mercapto group can also be partially or completely replaced by cations. The cation may be any cation, such as one of metal cation, ammonium ion NH ₄ ⁺ , and organic ammonium cation. In consideration of improving solubility, in one embodiment, the cation is selected from one or more of alkali metal ions, ammonium cations formed by tertiary amines, and quaternary ammonium cations. The alkali metal ion may be K ⁺ and/or Na ⁺ , and the cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N,N-diisopropylethylamine. Therefore, the siRNA or siRNA conjugate described in the present disclosure may exist at least partially in the form of a salt. In one mode, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate bond is at least partially combined with sodium ions, and the siRNA or siRNA conjugate described in the present disclosure is sodium salt or partial sodium salt. The form exists.

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

Preparation of siRNA conjugate represented by formula (308)

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

In some embodiments, the siRNA conjugate represented by formula (308) can be prepared by the following method, which includes under the conditions of phosphoramidite solid phase synthesis, according to the nucleotides of the sense strand and antisense strand of the siRNA, respectively. Type and sequence, according to the 3'to 5'direction, connect the nucleoside monomers in turn. The connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or vulcanization; isolate the sense strand of siRNA And the antisense strand, annealing, wherein the siRNA is the above-mentioned siRNA of the present disclosure;

In addition, the method also includes contacting the compound represented by formula (321) with a nucleoside monomer or a nucleotide sequence linked to a solid support in the presence of coupling reaction conditions and a coupling reagent, so that the formula (321) The compound shown in) is linked to the nucleotide sequence through a coupling reaction. Hereinafter, the compound represented by formula (321) is also referred to as a conjugate molecule.

among them:

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

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

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

The choice of R ₄ is to realize the connection with the N atom on the nitrogen-containing backbone and to provide a suitable reaction site for the synthesis of the siRNA conjugate represented by formula (308). In some embodiments, R ₄ includes an R ₂ linking group or a protected R ₂ linking group, and a functional group that can react with siRNA to form the structure shown in A59.

In some embodiments, R ₄ contains a first functional group that can form a phosphite with a group on the siRNA or nucleoside monomer represented by Nu and a second functional group that can react with a hydroxyl or amino group to form a covalent bond, or contains The solid phase carrier connected by the covalent bond. In some embodiments, the first functional group is phosphoramidite, hydroxyl or protected hydroxyl. In some embodiments, the second functional group is phosphoramidite, carboxyl or carboxylate. In some embodiments, the second functional group is a solid support connected to other parts of the molecule via a covalent bond, and the covalent bond is formed by a hydroxyl group or an amino group. In some embodiments, the solid phase support is connected via a phosphate bond, a carboxylate bond or an amide bond. In some embodiments, the solid phase carrier is a resin.

In some embodiments, the first functional group contains a hydroxyl group, -OR _k, or a group represented by formula (C3); the second functional group contains formula (C1), (C2), (C3), (C1' ) Or (C3'):

表示基团共价连接的位点。 In the formula, q ₁ is an integer from ₁ to 4, X is O or NH, M ⁺ is a cation, R _k is a hydroxyl protecting group, SPS is a solid phase carrier,

Represents the site where the group is covalently attached.

In some embodiments, the first functional group contains a phosphoramidite group, as shown in formula (C3), the phosphoramidite group can be combined with a hydroxyl group at any position on the nucleotide, such as the 2'hydroxyl group or The 3'hydroxyl group undergoes a coupling reaction to form a phosphite, which is oxidized or vulcanized to form a phosphodiester bond or a phosphorothioate bond represented by formula A59, and the conjugation molecule is conjugated to the siRNA. At this time, even if the second functional group does not exist, the compound of formula (321) can be conjugated to nucleotides without affecting the obtaining of the siRNA conjugate represented by formula (308). In this case, after obtaining the sense strand or antisense strand of the siRNA by methods such as phosphoramidite solid-phase synthesis, the compound of formula (321) is reacted with the hydroxyl group on the terminal nucleotide in the nucleotide sequence, and subsequently During the oxidation or vulcanization process, a phosphodiester linkage or phosphorothioate linkage is formed, and the compound of formula (321) is conjugated to the siRNA.

In some embodiments, the first functional group contains a protected hydroxyl group. In some embodiments, the second functional group includes a group that can react with a solid-phase support, and the reaction provides a conjugate molecule including the solid-phase support. In some embodiments, the second functional group contains a carboxyl group, carboxylate or phosphoramidite, as shown in formula (C1), (C2) or (C3), when the second functional group contains a carboxyl group or carboxylate At this time, the compound of formula (321) undergoes an esterification reaction or amidation reaction with a solid support, such as a hydroxyl group or an amino group on the resin, to form a conjugated molecule containing the solid support connected via a carboxylate bond. When the second functional group contains a phosphoramidite functional group, the compound of formula (321) undergoes a coupling reaction with a general solid support, such as a hydroxyl group on a resin, and is oxidized to form a solid support containing a phosphodiester bond. Conjugation molecule. Subsequently, starting from the product after connecting the solid phase carrier, the nucleoside monomers are sequentially connected according to the phosphoramidite solid phase synthesis method to obtain the sense strand or antisense strand of the siRNA with the conjugation group attached. In the process of phosphoramidite solid-phase synthesis, the first functional group is deprotected and then coupled with the phosphoramidite group on the nucleoside monomer under coupling reaction conditions.

In some embodiments, the first functional group contains a hydroxyl group or a protected hydroxyl group; the second functional group contains a solid-phase carrier connected via a carboxylate bond or a solid-phase carrier connected via an amide bond, or a phosphate bond The connected solid phase carrier is represented by formula (C1') or (C3'). At this time, starting with the compound of formula (321) instead of the solid phase carrier, nucleoside monomers are sequentially connected according to the phosphoramidite solid phase synthesis method to obtain the sense strand or antisense strand of the siRNA with the conjugation group attached.

In some embodiments, the carboxylate may be expressed as -COO ^- M ⁺ , where M ⁺ is a cation, for example, one selected from the group consisting of metal cations, ammonium cations NH ₄ ⁺ , and organic ammonium cations. In one embodiment, the metal ion is selected from one of alkali metal ions, such as K ⁺ or Na ⁺ . In order to improve the solubility and make the reaction proceed smoothly, in some embodiments, the organic ammonium ion is an ammonium cation formed by a tertiary amine or a quaternary ammonium cation, such as an ammonium ion formed by triethylamine or N,N-diamine Ammonium ion formed by isopropylethylamine. In some embodiments, the carboxylate is triethylamine carboxylate or N,N-diisopropylethylamine carboxylate.

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

表示基团共价连接的位点。在一些实施方式中，q ₁为1或2。在一些实施方式中，q ₂为1-5的整数。在一些实施方式中，R ₄含有式(B9)或(B10)所示的结构。在一些实施方式中，R ₄含有式(B11)或(B12)所示的结构。 Wherein, q ₁ is an integer of 1-4, q ₂ is an integer of 1-10, X is O or NH, M ⁺ is a cation, R _k is a hydroxyl protecting group, and SPS is a solid phase carrier,

Represents the site where the group is covalently attached. In some embodiments, q ₁ is 1 or 2. In some embodiments, q ₂ is an integer of 1-5. 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 Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-bismethoxytrityl), TMTr (4 ,4',4'-trimethoxytrityl) one or more. In some embodiments, R _k may be DMTr, that is, 4,4'-dimethoxytrityl (4,4'-dimethoxytrityl).

The definition of L ₁ is as described above.

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

According to the above description, those skilled in the art can easily understand that, compared with the phosphoramidite solid-phase synthesis method known in the art, the first functional group and the optional second functional group can be used to connect the conjugated molecules. The siRNA conjugate represented by formula (308) to any possible position of the nucleotide sequence, for example, the conjugate molecule is connected to the end of the nucleotide sequence, and the conjugate molecule is connected to the end of the nucleotide sequence. Correspondingly, unless otherwise stated, in the following descriptions concerning the preparation of conjugates and/or conjugated molecules, when referring to "deprotection", "coupling", "cap", "oxidation", "vulcanization", etc. During the reaction, it should be understood that the reaction conditions and reagents involved in the phosphoramidite nucleic acid solid-phase synthesis method known in the art are also 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 by protecting at least one active hydroxyl group in M ₁ by a hydroxyl protecting group. In some embodiments, each S _{1 is} independently a group formed by protecting all active hydroxyl groups present in M ₁ by a hydroxyl protecting group. In some embodiments, any hydroxyl protecting group known to those skilled in the art can be used to protect the active hydroxyl group in M ₁ . In some embodiments, the protected hydroxyl group can be represented by the formula YCOO-, wherein each Y is independently selected from the group consisting of C ₁ -C ₁₀ alkyl and C ₆ -C ₁₀ aryl, and the C _{The 1-} C ₁₀ alkyl group and the C ₆ -C ₁₀ aryl group are optionally substituted with one or more substituents selected from the group consisting of halogen and C ₁ -C 6 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 -C ₆ alkylphenyl.

In some embodiments, each S _{1 is} independently selected from the group consisting of formula A46-A54:

In some embodiments, S ₁ is formula A49 or A50.

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

As mentioned above, the preparation method of the siRNA conjugate represented by formula (308) also includes the following steps: synthesizing another strand of siRNA (for example, when the above step synthesizes the siRNA sense strand connected with the conjugate molecule, also Including the synthesis of the antisense strand of siRNA according to the solid-phase synthesis method, and vice versa), separation of the sense strand and antisense strand, and annealing. Specifically, in the separation step, the solid-phase carrier connected to the nucleotide sequence and/or the conjugate molecule is cleaved, and the necessary protective group is removed (at this time, each S in the compound of formula (321) _{The 1} group is converted into the corresponding M ₁ targeting group), the siRNA sense strand (or antisense strand) and the corresponding antisense strand (or sense strand) connected with the conjugate molecule are obtained, and the sense strand and antisense strand are annealed A double-stranded RNA structure is formed, and the siRNA conjugate represented by formula (308) is obtained.

In some embodiments, the preparation method of the siRNA conjugate represented by formula (308) comprises the following steps: in the presence of coupling reaction conditions and coupling reagents, the compound represented by formula (321) is combined with the sense strand or reverse The first nucleoside monomer at the 3'end of the sense strand is contacted, and the compound represented by formula (321) is connected to the first nucleotide in the sequence. Under the conditions of phosphoramidite solid-phase synthesis, according to the desired sense Chain or antisense strand nucleotide type and sequence, nucleoside monomers are sequentially connected in the 3'to 5'direction to synthesize the sense strand or antisense strand of siRNA; wherein the compound represented by formula (321) is R ₄ A compound containing a first functional group and a second functional group, the first functional group contains a protected hydroxyl group, and the second functional group has the structure shown in formula (C1') or (C3'), before being connected to the first nucleoside monomer , The compound of formula (321) is deprotected; the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; the sense strand or antisense strand of the nucleic acid connected with the conjugation group is obtained ; Under the conditions of phosphoramidite solid-phase synthesis, according to the type and sequence of the antisense strand or sense strand nucleotides, the nucleoside monomers are sequentially connected in the 3'to 5'direction to synthesize the antisense strand or sense strand of the nucleic acid Chain; the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or vulcanization; removing the protecting group and cutting with the solid phase carrier, separating and purifying to obtain the sense strand and antisense strand, and annealing.

In some embodiments, the preparation method of the siRNA conjugate represented by formula (308) includes the following steps: according to the nucleotide type and sequence of the sense strand or the antisense strand in the double-stranded siRNA, according to 3'to 5' The nucleoside monomers are connected in sequence to synthesize the sense strand and the antisense strand. The connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or vulcanization to obtain the solid-phase carrier. The sense strand and the antisense strand attached to the solid phase carrier; in the presence of coupling reaction conditions and coupling reagents, the compound represented by formula (321) and the sense strand attached to the solid phase carrier or connected to the solid phase The antisense strand on the carrier is contacted to connect the compound of formula (321) to the sense strand or the antisense strand, wherein the compound of formula (321) is a formula in which R ₄ contains the first functional group and the first functional group is a phosphoramidite group (321) compound; remove the protective group and cut with the solid phase carrier, separate and purify separately, obtain the sense strand or antisense strand of siRNA, and anneal, wherein the sense strand or antisense strand of the siRNA is connected with a conjugating group group.

In some embodiments, the P atom in formula A59 is connected to the 3'end of the sense strand in the siRNA, and the preparation method of the siRNA conjugate represented by formula (308) includes:

(1) Removal of the compound of formula (321) (wherein, the compound of formula (321) is that R ₄ contains a first functional group and a second functional group, the first functional group contains a protected hydroxyl group OR _k , and the second functional group has the formula (C1 ') or (C3') the hydroxyl protecting group R _k in the compound of the structure shown in (C3'); in the presence of coupling reaction conditions and coupling reagents, the product obtained by deprotection is contacted with a nucleoside monomer to obtain a The nucleoside monomer connected to the solid phase carrier;

(2) Starting with the nucleoside monomer connected to the solid-phase carrier by the conjugation molecule, synthesize the sense strand of siRNA by phosphoramidite solid-phase synthesis in the 3'-5' direction;

(3) Synthesize the antisense strand of siRNA by solid phase synthesis of phosphoramidite;

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

Wherein, in step (1), the method for removing the protective group R _k in the compound of formula (321) includes contacting the compound of formula (321) with a deprotection reagent under deprotection conditions. The deprotection conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 30-300 seconds, and in some embodiments 50-150 seconds, the deprotection reagent may be selected from trifluoroacetic acid One or more of trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid, in some embodiments, dichloroacetic acid. The molar ratio of the deprotection reagent to the compound of formula (321) is 10:1 to 1000:1, and in some embodiments, 50:1 to 500:1.

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

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

In step (2), by the method of phosphoramidite nucleic acid solid-phase synthesis, starting from the nucleoside monomer prepared by the above-mentioned steps that is connected to the solid-phase carrier by the conjugation molecule, the third is synthesized according to the 3'-5' direction. The sense strand S of two siRNA conjugates. At this time, the conjugating group is attached to the 3'end of the resulting sense chain.

Other conditions for solid-phase synthesis described in steps (2) and (3) include deprotection conditions of nucleoside monomers, types and amounts of deprotection reagents, coupling reaction conditions, types and amounts of coupling reagents, and capping reaction conditions. The conditions, the type and amount of the capping reagent, the oxidation reaction conditions, the type and amount of the oxidizing reagent, the vulcanization reaction conditions, and the type and amount of the vulcanizing reagent adopt various reagents, amounts and conditions conventionally used in the art.

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

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

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

The capping reaction conditions include a temperature of 0-50°C, 15-35°C in some embodiments, a reaction time of 5-500 seconds, and 10-100 seconds in some embodiments, and the selection of capping reagents is the same as described above. The molar ratio of the total amount of capping reagent to the nucleic acid sequence linked on the solid phase carrier is 1:100-100:1, and in some embodiments, 1:10-10:1. In the case where the capping reagent uses equimolar amounts of acetic anhydride and N-methylimidazole, the molar ratio of acetic anhydride, N-methylimidazole, and the nucleic acid sequence linked to the solid support can be 1:1:10-10: 10:1, in some embodiments 1:1:2-2:2:1.

The oxidation reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 1-100 seconds, and in some embodiments 5-50 seconds, the oxidizing reagent is iodine in some embodiments (In some embodiments, it is provided in the form of iodine water). The molar ratio of the oxidizing reagent to the nucleic acid sequence linked to the solid support in the coupling step can be 1:1-100:1, and in some embodiments, 5:1-50:1. In some embodiments, the oxidation reaction is performed in a mixed solvent of tetrahydrofuran:water:pyridine=3:1:1-1:1:3. The vulcanization reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 50-2000 seconds, and in some embodiments 100-1000 seconds, the vulcanization reagent is hydrogenation in some embodiments. Xanthan. The molar ratio of the vulcanizing reagent to the nucleic acid sequence linked to the solid support in the coupling step is 10:1 to 1000:1, and in some embodiments, 10:1 to 500:1. In some embodiments, the vulcanization reaction is performed in a mixed solvent of acetonitrile:pyridine=1:3-3:1.

After all nucleoside monomers are connected and before annealing, the method also includes separating the sense strand and antisense strand of the siRNA. The method of separation is well-known to those skilled in the art, and generally includes cutting the synthesized nucleotide sequence from the solid support, removing the protective groups on the base, phosphate and ligand, purification and desalting .

Cleaving the synthesized nucleotide sequence from the solid-phase carrier and removing the protective groups on the base, phosphate and ligand can be carried out according to the conventional cutting and deprotection methods in siRNA synthesis. For example, the obtained nucleotide sequence connected with the solid phase carrier is contacted with concentrated ammonia; in the process of deprotection, the protecting group YCOO- of the A46-A54 group is converted into a hydroxyl group, and the S ₁ group is converted into the corresponding The M ₁ group produces the conjugate represented by formula (308). Wherein, the concentrated ammonia water may be 25-30% by weight of ammonia water, and the amount of concentrated ammonia water may be 0.2ml/μmol-0.8ml/μmol compared with the target siRNA sequence.

When there is at least one 2'-TBDMS protection on the synthesized nucleotide sequence, the method further comprises contacting the nucleotide sequence removed from the solid phase carrier with triethylamine trihydrofluoride to remove the 2'-TBDMS protection. At this time, the corresponding nucleotide in the obtained target siRNA sequence has a free 2'-hydroxyl group. Compared with the target siRNA sequence, the amount of pure triethylamine trihydrofluoride can be 0.4ml/μmol-1.0ml/μmol. In this way, the siRNA conjugate represented by formula (308) can be obtained.

Methods of purification and desalination are well known to those skilled in the art. For example, a preparative ion chromatography purification column can be used to complete the purification of nucleic acid through gradient elution of NaBr or NaCl; after the product is collected and combined, a reverse phase chromatography purification column can be used for desalting.

In the siRNA conjugate represented by the formula (308) thus obtained, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate bond between the nucleotides is basically bonded to the sodium ion, and the formula ( The siRNA conjugate shown in 308) basically exists in the form of sodium salt. A well-known ion exchange method can be used to replace the sodium ions with hydrogen ions and/or other cations to obtain other forms of siRNA conjugates represented by formula (308). The cation is as described above.

During the synthesis process, the purity and molecular weight of the nucleic acid sequence can be tested at any time to better control the synthesis quality. Such detection methods are well known to those skilled in the art. For example, nucleic acid purity can be detected by ion exchange chromatography, and molecular weight can be determined by liquid mass spectrometry (LC-MS).

The annealing method is also well known to those skilled in the art. For example, the synthesized sense strand (S chain) and antisense strand (AS chain) can be simply mixed in an equimolar ratio, heated to 70-95°C in water for injection, and then cooled at room temperature to form a double bond through hydrogen bonding. Chain structure. In this way, the siRNA conjugate represented by formula (308) can be obtained.

After the conjugate is obtained, in some embodiments, the synthesized siRNA conjugate represented by formula (308) can also be characterized by methods such as liquid-mass spectrometry chromatography, etc., by means of molecular weight detection. It is determined that the synthesized siRNA conjugate is the siRNA conjugate represented by formula (308) of the target design, and the sequence of the synthesized siRNA is the sequence of the desired siRNA, for example, as shown in Tables 1a, 1b, 1d, 1e and 1f One of the sequences listed in.

The compound represented by formula (321) can be obtained by the following preparation method: the method includes combining the compound represented by formula (313) with a cyclic compound in an organic solvent, under esterification reaction conditions, and in the presence of a base and an esterification catalyst. Contact with acid anhydride, ion exchange, and separation to obtain the compound represented by formula (321):

Wherein, n1, n3, m1, m2, m3, R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , L _{1, and} S ₁ are defined and selectable ranges 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 (A61):

Among them, R _i is any group that can be connected to the N atom on the nitrogen-containing skeleton, connected to R _k O and connected to a free hydroxyl group, and R _k is a hydroxyl protecting group. At this time, what is obtained is that R ₄ contains a first functional group and a second functional group as a hydroxyl protecting group, and the second functional group contains a compound of formula (321) having a structure represented by formula (C1) or (C2).

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

In some embodiments, the organic solvent includes epoxy-based solvents, ether-based solvents, halogenated alkane-based solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine One or more of. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran, the ether solvent is diethyl ether and/or methyl tert-butyl ether, and the halogenated alkane solvent is dichloromethane, trihydrofuran One or more of methyl chloride and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. Relative to the compound represented by formula (313), the amount of the organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol.

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

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 represented by formula (313) is 1:1-10:1, and in some embodiments, 2:1-5:1.

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

The ion exchange effect is to convert the compound of formula (321) into the desired form of carboxylic acid or carboxylate. The method of ion exchange is well known to those skilled in the art. Suitable ion exchange solutions and exchange conditions can be used to obtain The conjugate molecule of M ⁺ cation will not be described in detail here. In some embodiments, the ion exchange reaction is performed using a triethylamine phosphate solution, and the concentration of the triethylamine phosphate solution is 0.2-0.8M. In some embodiments, the triethylamine phosphate solution The concentration of triethylamine phosphate is 0.4-0.6M, relative to the compound of formula (313), the amount of the triethylamine phosphate solution is 3-6L/mol, in a further embodiment 4-5L/mol.

Any suitable separation method can be used to separate the compound of formula (321) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation, and then the compound of formula (321) can be separated by chromatographic methods. For example, the following two chromatographic conditions can be used for separation: (1) normal phase purified silica gel: 200-300 mesh silica gel filler, Use dichloromethane containing 1wt‰ of triethylamine: methanol = 100:18-100:20 gradient elution; or (2) reverse phase purification: C18, C8 reverse phase packing, use methanol: acetonitrile = 0.1:1-1 :0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be directly used in subsequent reactions.

In some embodiments, the preparation method of the compound of formula (321) further comprises further comprising, under condensation reaction conditions, in an organic solvent, in the presence of a condensing agent, a condensation catalyst and a tertiary amine, the product obtained by the above ion exchange reaction is further Contact with a solid phase carrier containing amino groups or hydroxyl groups. At this time, what is obtained is that R ₄ contains a first functional group and a second functional group, the first functional group contains a hydroxyl protecting group, and the second functional group contains a compound of formula (321) having a structure represented by formula (C1′).

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

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 acetonitrile, epoxy solvent, ether solvent, halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl. One or more of ethylamine. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran, the ether solvent is diethyl ether and/or methyl tert-butyl ether, and the halogenated alkane solvent is dichloromethane, trihydrofuran One or more of methyl chloride and 1,2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. Relative to the compound of formula (321), the amount of the organic solvent is 20-200 L/mol, in some embodiments 50-100 L/mol.

In some embodiments, the condensing agent may be benzotriazol-1-yl-oxytripyrrolidino phosphonium hexafluorophosphate (PyBop), 3- Diethoxy phosphoryl-1,2,3-benzoxazole 4(3H)-one and/or O-benzotriazole-tetramethylurea hexafluorophosphate. In some embodiments, the condensing agent It is O-benzotriazole-tetramethylurea hexafluorophosphate. The molar ratio of the condensing agent to the compound represented by formula (321) is 1:1-20:1, and in some embodiments, 1:1-5:1.

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

In some embodiments, the preparation method of the compound of formula (321) may also include contacting the obtained condensation product with a capping reagent and an acylation catalyst in an organic solvent under capping reaction conditions to obtain the formula (321) Compound. The function of the capping reaction is to remove any reactive functional groups that have not been completely reacted, so as to avoid unnecessary by-products in subsequent reactions. The conditions of the capping reaction include a reaction temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 1-10h, and in some embodiments 3-6h. The capping reagent can be the capping reagent used in siRNA solid-phase synthesis, and the capping reagent used in siRNA solid-phase synthesis is well known to those skilled in the art.

In some embodiments, the capping reagent consists of capping reagent 1 (cap1) and capping reagent 2 (cap2), wherein capping reagent 1 is N-methylimidazole, and in some embodiments, it is N-methylimidazole The pyridine/acetonitrile mixed solution form is provided, wherein the volume ratio of pyridine to acetonitrile is 1:10-1:1, in some embodiments, 1:3-1:1, and the total volume of pyridine and acetonitrile is compared with N-form The volume ratio of base imidazole is 1:1-10:1, and 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 volume of acetic anhydride and acetonitrile is 1:1-1:10, and in a further embodiment, 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-50ml/g, in some embodiments 15ml/g- 30ml/g. The ratio of the volume of the acetonitrile solution of acetic anhydride to the mass of the compound of formula (321) is 0.5ml/g-10ml/g, and in some embodiments is 1ml/g-5ml/g.

In some embodiments, the capping reagent uses equimolar amounts of acetic anhydride and N-methylimidazole. In some embodiments, the organic solvent is acetonitrile, epoxy solvent, ether solvent, halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl. One or more of ethylamine. In some embodiments, the organic solvent is acetonitrile. Relative to the compound of formula (321), the amount of the organic solvent is 10-50 L/mol, and in some embodiments, 5-30 L/mol.

In some embodiments, the acylation catalyst may be selected from any catalyst that can be used 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 represented by formula (321) is 0.001:1 to 1:1, in some embodiments 0.01:1 to 0.1:1.

In some embodiments, any suitable separation method may be used to separate the compound of formula (321) from the reaction mixture. In some embodiments, the compound of formula (321) can be obtained by fully washing with an organic solvent and filtering to remove unreacted reactants, excess capping reagents and other impurities. The organic solvent is selected from acetonitrile and dichloromethane. , Methanol, in some embodiments acetonitrile.

In some embodiments, the preparation method of the conjugate molecule represented by formula (321) includes combining the compound represented by formula (313) with phosphorous in an organic solvent, under coupling reaction conditions, and in the presence of a coupling reagent. The diamide is contacted and separated to obtain a compound represented by formula (321). At this time, what is obtained is that R ₄ contains a first functional group and a second functional group, the first functional group contains a hydroxyl protecting group, and the second functional group contains a compound of formula (321) having a structure represented by formula (C3).

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

In some embodiments, the preparation method of the compound of formula (321) further includes the following steps: under coupling reaction conditions, in an organic solvent, and in the presence of a coupling reagent, the separated product is further combined with hydroxyl-containing The solid support is brought into contact. Subsequently, through capping reaction and oxidation reaction, the compound of formula (321) is isolated. In this case, obtained is R ₄ contains a first functional group and a second functional group, the first functional group containing a hydroxyl protective group, the second functional group having a compound of the formula (C3 ') of formula (321) structure shown in FIG.

HL UnyLinker ^TM 300Oligonucleotide Synthesis Support，Kinovate Life Sciences公司，结构如式B80所示)： In some embodiments, the solid phase carrier is a solid phase carrier known in the art that can be used for nucleic acid solid phase synthesis, for example, it may be a commercially available universal solid phase carrier after deprotection reaction (

HL UnyLinker ^TM 300 Oligonucleotide Synthesis Support, Kinovate Life Sciences company, structure shown in formula B80):

The deprotection reaction is well known to those skilled in the art. In some embodiments, the deprotection conditions include a temperature of 0-50°C, such as 15-35°C, and a reaction time of 30-300 seconds, such as 50-150 seconds. The deprotection reagent can be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid. In some embodiments, the deprotection reagent is dichloroacetic acid. The molar ratio of the deprotection reagent to the -DMTr (4,4'-dimethoxytrityl) protecting group on the stationary phase is 2:1-100:1, for example, 3:1-50:1. By performing the deprotection, free hydroxyl groups with reactivity are obtained on the surface of the solid support, which facilitates subsequent coupling reactions.

The coupling reaction conditions and the selection of coupling reagents can be as described above. Through the coupling reaction, the free hydroxyl group formed in the deprotection reaction reacts with the phosphoramidite group to form a phosphite linkage.

In some embodiments, the capping reaction conditions include a temperature of 0-50°C, such as 15-35°C, and a reaction time of 5-500 seconds, such as 10-100 seconds, and the capping reaction is performed in the presence of a capping reagent. The selection and amount of capping reagent can be as described above.

The oxidation reaction conditions include a temperature of 0-50°C, such as 15-35°C, a reaction time of 1-100 seconds, such as 5-50 seconds, and an oxidizing reagent such as iodine (in some embodiments, iodine Provided in the form of water). In some embodiments, the molar ratio of the oxidizing reagent to the nucleic acid sequence linked to the solid phase carrier is 1:1-100:1, for example, it may be 5:1-50:1. In some embodiments, the oxidation reaction is performed in a mixed solvent of tetrahydrofuran:water:pyridine=3:1:1-1:1:3.

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

The definition of q ₂ is as mentioned before,

At this time, the compound represented by formula (313) can be obtained by the following preparation method: in an organic solvent, under amidation reaction conditions, and in the presence of a condensing agent and tertiary amine for amidation reaction, the formula (314) The compound is contacted with the compound represented by formula (A-1) or the compound represented by formula (A-2), and then separated:

Among them, n1, n3, m1, m2, m3, R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , L ₁ , S ₁ , q ₂ and R _k are each defined and selectable ranges as As mentioned earlier.

The amidation reaction conditions may include a reaction temperature of 0-100°C and a reaction time of 1-48 hours. In some embodiments, the amidation reaction conditions may include a reaction temperature of 10-40°C and a reaction time of 2- 16 hours.

In some embodiments, the organic solvent is alcohol solvent, epoxy solvent, ether solvent, halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diiso One or more of propylethylamine. The alcohol solvent is one or more of methanol, ethanol, and propanol in some embodiments, and ethanol in some embodiments. The epoxy solvent is dioxane and/or tetrahydrofuran in some embodiments. The ether solvent is diethyl ether and/or methyl tert-butyl ether in some embodiments. In some embodiments, the halogenated alkane solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. Relative to the compound of formula (314), the amount of organic solvent is 3-50 L/mol, and in a further embodiment, 3-20 L/mol.

In some embodiments, the amidation reaction condensing agent is hexafluorophosphate benzotriazol-1-yl-oxytripyrrolidinyl phosphorus, 3-diethoxyphosphoryl-1,2,3-benzene Azole 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 a further embodiment is 3-diethoxyphosphoryl-1,2,3- Benzazole 4(3H)-one. The molar ratio of the amidation reaction condensing agent to the compound represented by formula (314) may be 1:1-10:1, and in some embodiments, 2.5:1-5:1.

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

In some embodiments, the compounds of formula (A-1) and formula (A-2) can be prepared by any suitable means. For example, when R _k is a DMTr group, the compound of formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl; similarly, 3-amino-1,2-propanediol can be contacted with cyclic anhydride first, and then The compound of formula (A-2) is prepared by reacting with DMTrCl. The cyclic anhydride may be a cyclic anhydride having 4-13 carbon atoms, and in some embodiments, 4-8. It is easily understood by those skilled in the art that the selection of the cyclic anhydride corresponds to different values of q ₂ in the compound (A-2). For example, when the cyclic anhydride is succinic anhydride, q ₂ =1, When the cyclic anhydride is glutaric anhydride, q ₂ =2, and so on.

In some variations, the compound of formula (313) can also be prepared by sequentially reacting the compound represented by formula (314) with the cyclic anhydride, 3-amino-1,2-propanediol and DMTrCl. It is easily understood by those skilled in the art that these modifications will not affect the structure and function of the compound of formula (313), and these modifications are easily implemented by those skilled in the art on the basis of the above methods.

Similar to the above, any suitable separation method can be used to separate the compound of formula (313) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation, and then the compound of formula (313) can be separated by chromatographic methods. For example, the following two chromatographic conditions can be used for separation: (1) normal phase purification silica gel: 200-300 mesh silica gel filler, Use petroleum ether: ethyl acetate: dichloromethane: N, N-dimethylformamide=1:1:1:0.5-1:1:1:1:0.6 gradient elution; and (2) reverse phase purification: C18 , C8 reverse phase packing, using methanol: acetonitrile = 0.1:1-1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (313), which can be directly used in subsequent reactions.

In some embodiments, the compound represented by formula (314) can be obtained by the following preparation method: the method includes in an organic solvent, in the presence of a condensing agent for amidation reaction and a tertiary amine, under condensation reaction conditions, the formula ( The compound represented by 320) is contacted with the compound represented by formula (316), and then separated:

Wherein, n1, n3, m1, m2, m3, R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , and R _{15 have} the same definitions and selectable ranges as described above.

For the compound of formula (316), for example, the compounds disclosed in J. Am. Chem. Soc. 2014, 136, 16958-16961 can be used, or the compound of formula (316) can be prepared by various methods by those skilled in the art, for example, Some compounds of formula (316) were prepared with reference to the method disclosed in Example 1 of US Patent No. 8,106,022 B2, and the entire contents of the above documents are incorporated herein by reference in their entirety.

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

Considering the structure of the compound of formula (314) as the desired product, 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, in order to ensure that the reaction is complete and not excessive, the molar ratio of the compound represented by formula (316) to the compound represented by formula (320) may be 3:1- 3.5:1, in some embodiments 3.01:1-3.15:1.

In some embodiments, the organic solvent is acetonitrile, epoxy solvent, ether solvent, halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl. One or more of ethylamine, the epoxy solvent is dioxane and/or tetrahydrofuran in some embodiments, and the ether solvent is diethyl ether and/or methyl tert-butyl in some embodiments In some embodiments, the haloalkane solvent is one or more of dichloromethane, chloroform, and 1,2-dichloroethane. In some embodiments, the organic solvent is two Methyl chloride. Relative to the compound of formula (320), the amount of the organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol.

In some embodiments, the amidation reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2, 3-Benzazole 4(3H)-one (DEPBT), O-benzotriazole-tetramethylurea hexafluorophosphate/ester, 4-(4,6-dimethoxytriazin-2-yl) One or more of -4-methylmorpholine hydrochloride or 1-hydroxybenzotriazole, in a further embodiment benzotriazol-1-yl-oxytripyrrolidinyl phosphonium A mixture of fluorophosphate and 1-hydroxybenzotriazole, wherein benzotriazol-1-yl-oxytripyrrolidinyl phosphonium hexafluorophosphate and 1-hydroxybenzotriazole are equimolar Dosage. The molar ratio of the total amidation reaction condensing agent to the compound represented by formula (316) may be 1:1-3:1, and in some embodiments, 1.05:1-1.5:1.

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

Similar to the above, any suitable separation method can be used to separate the compound of formula (314) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation, and then the compound of formula (314) can be separated by chromatography. For example, the following two chromatographic conditions can be used for separation: (1) Normal phase purification silica gel: 200-300 mesh silica gel filler, using Dichloromethane: methanol = 100:5-100:7 gradient elution; and (2) reverse phase purification: C18, C8 reverse phase packing, using methanol: acetonitrile = 0.1:1 to 1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (314), which can be directly used in subsequent reactions.

The compound of formula (320) is commercially available or can be obtained by a person skilled in the art using a known method. For example, when m1=m2=m3=3, n1=1, n3=2, and each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₅ is H, the compound of formula (320) can Commercially purchased from Alfa Aesar.

The siRNA conjugate of the present disclosure can also be used in combination with other pharmaceutically acceptable excipients, which can be one or more of various formulations or compounds conventionally used in the art. For details, please refer to the above about the present disclosure Description of the pharmaceutical composition.

Application of siRNA, pharmaceutical composition containing the siRNA and conjugate of the present disclosure

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

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

By administering the siRNA active ingredients of the present disclosure to subjects in need, the purpose of preventing and/or treating HAE and/or thrombosis can be achieved through the mechanism of RNA interference. Therefore, the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure can be used to prevent and/or treat HAE and/or thrombosis, or be used in preparation for preventing and/or treating HAE and/or thrombosis medicine.

The term "administration/administration" as used herein refers to a method or approach that allows the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure to be at least partially positioned at a desired site to produce a desired effect. The siRNA, pharmaceutical composition, and/or siRNA conjugate of the present disclosure are placed in a subject. Suitable routes of administration for the method of the present disclosure include local administration and systemic administration. Generally speaking, local administration results in the delivery of more siRNA conjugates to a specific site than in the subject's systemic circulation; while systemic administration results in the delivery of the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure Basic systemic circulation to the subject. Considering that the present disclosure aims to provide a means to prevent and/or treat HAE, in some embodiments, an administration method capable of delivering drugs to the liver is adopted.

The subject can be administered to the subject by any suitable route known in the art, including but not limited to: oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, and transdermal administration. Medicine, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration and topical administration (including buccal administration and sublingual administration). The frequency of administration may be once or more daily, weekly, every two weeks, every three weeks, every month, every two months, every quarter, every six months, or every year.

The dosage of the siRNA, pharmaceutical composition or siRNA conjugate described in the present disclosure can be a conventional dosage in the art, and the dosage can be determined according to various parameters, especially the age, weight and sex of the subject. May be in cell cultures or experimental animals Determination Toxicity and therapeutic efficacy by standard pharmaceutical procedures, e.g. assays and ED ₅₀ dose (the amount of the reaction finger to cause 50% of the maximum intensity of the reaction of the LD ₅₀ (so that dose lethal to 50% of the population died), In the qualitative response, it refers to the dose that causes a positive response in 50% of the test subjects). The range of human doses can be derived based on data obtained from cell culture analysis and animal studies.

When administering the siRNA, pharmaceutical composition, and/or siRNA conjugate described in the present disclosure, for example, for male or female, 6-12 weeks old, 18-25g of C57BL/6J or 30-45g of ob/ ob mice, based on the amount of siRNA: (i) For siRNA conjugates, the amount of siRNA can be 0.001-100 mg/kg body weight, in some embodiments 0.01-50 mg/kg body weight, in some embodiments 0.05-20 mg/kg body weight, in some embodiments 0.1-15 mg/kg body weight, and in other embodiments 0.1-10 mg/kg body weight; (ii) for a pharmaceutical composition formed by siRNA and a pharmaceutically acceptable carrier The siRNA dosage can be 0.001-50mg/kg body weight, in some embodiments 0.01-10mg/kg body weight, in some embodiments 0.05-5mg/kg body weight, in some embodiments 0.1-3mg/kg body weight.

In some embodiments, the present disclosure provides a method for inhibiting FXII gene expression in liver cells, the method comprising combining an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure with the liver. Cell contact, the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure are introduced into the liver cell, and the purpose of inhibiting the expression of the FXII gene in the liver cell is achieved through the mechanism of RNA interference. The liver cells may be selected from liver cancer cell lines such as SMMC-7721, HepG2, Huh7, or isolated primary liver cells. In some embodiments, the cell is a human liver primary cell.

Using the method provided by the present disclosure to inhibit the expression of FXII gene in cells, the amount of siRNA in the provided modified siRNA, pharmaceutical composition and/or siRNA conjugate is generally such an amount that it is sufficient to reduce the expression of the target gene, and This results in an extracellular concentration of 1 pM to 1 μM, or 0.01 nM to 100 nM, or 0.05 nM to 50 nM, or 0.05 nM to about 5 nM at the surface of the target cell. The amount required to achieve this local concentration will vary with various factors, including the delivery method, the delivery site, the number of cell layers between the delivery site and the target cell or tissue, the delivery route (local or systemic), etc. . The concentration at the delivery site can be significantly higher than the concentration at the surface of the target cell or tissue.

Reagent test kit

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

In some embodiments, the kits described herein can provide modified siRNA in one container. In some embodiments, the kits described herein may include a container that provides pharmaceutically acceptable excipients. In some embodiments, the kit may also contain other ingredients, such as stabilizers or preservatives. In some embodiments, the kit described herein may contain at least one other therapeutic agent in a container other than the container that provides the modified siRNA described herein. In some embodiments, the kit may include instructions for mixing the modified siRNA with pharmaceutically acceptable carriers and/or excipients or other ingredients (if any).

In the kit of the present disclosure, the modified siRNA and a pharmaceutically acceptable carrier and/or adjuvant and the modified siRNA, pharmaceutical composition and/or siRNA conjugate and/or conjugate, and/ Or pharmaceutically acceptable excipients can be provided in any form, such as liquid form, dried form or lyophilized form. In some embodiments, the modified siRNA and pharmaceutically acceptable carriers and/or adjuvants, and the pharmaceutical composition and/or conjugate and optional pharmaceutically acceptable adjuvants are substantially pure and/or Sterile. In some embodiments, sterile water can be provided in the kit of the present disclosure.

The following examples will further illustrate the present disclosure, but the present disclosure is not limited in any way.

Examples

Unless otherwise specified, the reagents and culture media used in the following examples are all commercially available products. The nucleic acid electrophoresis, real-time PCR and other operations used are described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)) Method to proceed.

Human liver primary cells were provided by the Nucleic Acid Technology Laboratory of the Institute of Molecular Medicine, Peking University. Before use, they were thawed in a 37°C water bath, and then they were seeded on type I collagen-coated glass or plastic coverslips or tissue culture dishes. The cells were cultured in RPMI 1460 medium containing 1× double antibody and 10% FBS, and cultured in an incubator containing 5% CO ₂ /95% air at 37°C.

C57 mouse liver primary cells were provided by the Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking University, and were obtained from C57 mice (purchased from Zhaoyan Company). Inoculate the appropriate density of cells in type I collagen-coated glass or plastic coverslips or tissue culture dishes, and culture the cells in RPMI 1460 medium containing 1× double antibody and 10% FBS. Cultivate the cells in a CO ₂ /95% air incubator for 15-30 minutes.

The C57 mice used were 6-8 week old mice, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

Lipofectamine ^TM 2000 (Invitrogen) is used as the transfection reagent when transfecting cells with siRNA, siRNA conjugate or siRNA and siRNA conjugate for FXII gene synthesized in the present disclosure, and the specific operation refers to the instructions provided by the manufacturer .

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

表示，数据分析采用Graphpad prism统计分析软件。 The experimental data are

Said that the data analysis uses Graphpad prism statistical analysis software.

Preparation Example 1 Preparation of Conjugate 1

In this preparation example, conjugate 1 was synthesized. The conjugate is a conjugate formed by conjugating an L-9 conjugate molecule with the siFXIIa1M1SP siRNA 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 GAL-5

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

Dissolve 100.0g of GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS number: 1772-03-8, purchased from Ningbo Hongxiang Biochemical Company, 463.8mmol) in 1000ml of anhydrous pyridine, under ice water bath 540ml of acetic anhydride (purchased from Enox Company, 5565.6mmol) was added, and the reaction was stirred at room temperature for 1.5 hours. The reaction solution was poured into 10L ice water, filtered under reduced pressure, the filter cake was washed with 2L ice water, and acetonitrile/toluene mixed solvent (volume ratio acetonitrile:toluene = 1:1) was added to completely dissolve, and the solvent was evaporated to dryness and white The solid product GAL-2 130.0g.

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

Dissolve the GAL-2 (35.1g, 90.0mmol) obtained in step (1-1-1a) in 213ml of anhydrous 1,2-dichloroethane, add 24.0g TMSOTf in an ice water bath and under nitrogen protection (CAS number: 27607-77-8, purchased from Macleans Company, 108.0 mmol), react at room temperature overnight.

Add 400ml of dichloromethane to the reaction solution to dilute, filter with diatomaceous earth, then add 1L of saturated sodium bicarbonate aqueous solution, stir evenly, separate the organic phase, and extract the aqueous phase with dichloroethane twice, 300ml each time, and combine The organic phase was washed with 300 ml of saturated sodium bicarbonate aqueous solution and 300 ml of saturated brine respectively, the organic phase was separated, dried with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 26.9 g of light yellow viscous sugar thin product GAL-3.

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

分子筛粉末30g，再加入9.0g 5-己烯-1-醇(CAS号：821-41-0，购自Adamas-beta公司，89.9mmol)，室温下搅拌30分钟，冰浴和氮气保护下加入9.08g TMSOTf(40.9mmol)，室温下搅拌反应过夜。过滤除去

分子筛粉末，滤液中加入300ml二氯甲烷稀释，以硅藻土过滤，再加入500ml饱和碳酸氢钠水溶液搅拌10分钟洗涤，分出有机相，水相用300ml二氯乙烷萃取一次，合并有机相并分别用300ml饱和碳酸氢钠水溶液和300ml饱和食盐水洗涤，分出有机相，无水硫酸钠干燥，减压蒸干溶剂，得到黄色糖稀状产品GAL-4 41.3g，不进行纯化直接进行下一步氧化反应。 The GAL-3 (26.9g, 81.7mmol) obtained in step (1-1-1b) was dissolved in 136ml of anhydrous 1,2-dichloroethane, and the dried

30g molecular sieve powder, then add 9.0g 5-hexen-1-ol (CAS number: 821-41-0, purchased from Adamas-beta company, 89.9mmol), stir at room temperature for 30 minutes, add under ice bath and nitrogen protection 9.08g TMSOTf (40.9mmol), stirred and reacted overnight at room temperature. Filter to remove

Molecular sieve powder, the filtrate is diluted with 300ml dichloromethane, filtered with diatomaceous earth, and then 500ml saturated sodium bicarbonate aqueous solution is added and stirred for 10 minutes to wash, the organic phase is separated, the aqueous phase is extracted once with 300ml dichloroethane, and the organic phases are combined And washed with 300ml saturated sodium bicarbonate aqueous solution and 300ml saturated brine respectively, separated the organic phase, dried with anhydrous sodium sulfate, and evaporated the solvent under reduced pressure to obtain 41.3g of yellow sugar thin product GAL-4, proceed directly without purification. One-step oxidation reaction.

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

GAL-4 (14.9g, 34.7mmol,) obtained according to the method described in step (1-1-1c) was dissolved in a mixed solvent of 77ml dichloromethane and 77ml acetonitrile, and 103ml deionized water and 29.7g were added respectively Sodium periodate (CAS number: 7790-28-5, purchased from Aladdin Company, 138.8mmol), stirred under ice water bath for 10 minutes, adding ruthenium trichloride (CAS number: 14898-67-0, purchased from An Nai Kyrgyzstan, 238mg, 1.145mmol), react at room temperature overnight. The reaction solution was diluted with 300 ml of water and stirred, and 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 with dichloromethane three times with 200 ml each time, and the organic phase was discarded. Adjust the pH of the aqueous phase to about 3 with citric acid solid, extract three times with 200 ml of dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, and evaporate the solvent under reduced pressure to obtain 6.85g of white foamy solid product GAL-5 . ¹ H 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=11.2,3.2 Hz,1H), 4.49(d,J=8.4Hz,1H), 4.07–3.95(m,3H), 3.92–3.85(m,1H), 3.74–3.67(m,1H), 3.48– 3.39(m, 1H), 2.20 (t, J = 6.8Hz, 2H), 2.11 (s, 3H), 2.00 (s, 3H), 1.90 (s, 3H), 1.77 (s, 3H), 1.55-1.45 (m, 4H) ).

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

J-0 (9.886 g, 52.5 mmol, commercially available from Alfa Esha Company) and GAL-5 (72.819 g, 162.75 mmol) obtained in step (1-1-1), obtained by combining multiple batches of products ) Was dissolved in 525ml of dichloromethane, and diisopropylethylamine (DIEA, 44.782g, 346.50mmol), benzotriazol-1-yl-oxytripyrrolidinyl phosphonium hexafluorophosphate (PyBOP, 90.158g, 173.25mmol) and hydroxybenzotriazole (HOBt, 23.410g, 173.25mmol), react for 4h at room temperature, add 20ml saturated sodium bicarbonate and 200ml saturated brine for washing, and extract the aqueous phase twice with dichloromethane , 100ml each time, combine the organic phases, dry with anhydrous sodium sulfate, filter and evaporate the solvent under reduced pressure to obtain a crude product. Purification uses 200-300 mesh normal phase silica gel, neutralizes the acidity of silica gel with 10wt% triethylamine, equilibrates the column with 1wt‰ triethylamine, and eluates with a gradient of dichloromethane:methanol=100:25-100:40. Collect the product wash The liquid was removed, and the solvent was evaporated under reduced pressure to obtain 38.8 g of pure L-8. ¹ H NMR(400MHz,DMSO)δ7.84(d,J=9.0Hz,3H), 7.27–7.23(m,1H), 7.13–7.18(m,1H), 5.22(d,J=3.1Hz,3H ), 4.97 (dd, J = 11.3, 3.1 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: C ₈₅ H ₁₁₉ N ₇ O ₃₀ , [M+H] ⁺ , theory: 1477.59, actual measurement: 1477.23.

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

Dissolve DMTrCl (4,4'-bismethoxytrityl chloride, 101.65g, 300mmol) in 1000ml of anhydrous pyridine, add DL-glycerate calcium hydrate (28.63g, 100mmol), and react at 45℃ After 20h, the reaction solution was filtered, the filter cake was rinsed with 200ml DCM, the filtrate was concentrated to dryness under reduced pressure, the residue was redissolved with 500ml dichloromethane, and washed with 0.5M triethylamine phosphate (pH=7-8) twice, 200ml each time, the aqueous phase was extracted twice with dichloromethane, 200ml each time, the organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Purified by 200-300 mesh normal phase silica gel column and purified with petroleum ether : Ethyl acetate: dichloromethane: methanol=1:1:1:0.35-1:1:1:0.55 gradient elution, collect the product eluent, evaporate the solvent under reduced pressure, and re-dissolve in 600ml dichloromethane to Wash with 200ml 0.5M triethylamine phosphate once, extract the aqueous phase with 200ml dichloromethane once, combine the organic phases, dry with anhydrous sodium sulfate, filter, evaporate the solvent under reduced pressure, vacuum oil pump overnight under reduced pressure to obtain white The solid product A-1 50.7g. ¹ H NMR(400MHz,DMSO-d6)δ7.46(ddd,J=6.5,2.3,1.1Hz,1H), 7.40–7.28(m,7H), 6.89–6.81(m,4H), 4.84(d, J = 5.0Hz, 1H), 4.36–4.24 (m, 1H), 4.29 (s, 6H), 3.92 (dd, J = 12.4, 7.0 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: C ₂₄ H ₂₃ O ₆ , [MH] ^- , theory: 407.15, measured: 406.92 .

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

The L-8 (40g, 27.09mmol, obtained by combining multiple batches) obtained in step (1-1-2) and A-1 (41.418g, 81.27) obtained in step (1-1-3a) mmol), dissolve in 271ml of dichloromethane, add 3-diethoxyphosphoryl-1,2,3-benzoxazole 4(3H)-one (DEPBT) (24.318g, 81.37mmol), then add diiso Propylethylamine (21.007g, 162.54mmol), stirred for 1.5h at 25°C, washed the organic phase with 800ml saturated sodium bicarbonate, and extracted the aqueous phase with dichloromethane three times, each time with 50ml, and washed with 150ml saturated brine The organic phase and the aqueous phase were extracted once with 50 ml of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure, and the vacuum oil pump foamed and dried overnight to obtain a crude product. Use 2kg 200-300 mesh normal phase silica gel for column purification, neutralize the silica gel acidity with 200ml triethylamine, equilibrate the column with petroleum ether containing 1wt% triethylamine, and use petroleum ether: ethyl acetate: methylene chloride: N, N -Dimethylformamide=1:1:1:0.5-1:1:1:0.6 gradient elution, collect the product eluent, evaporate the solvent under reduced pressure to obtain 40.4g of pure product L-7. ¹ H NMR (400MHz, DMSO) δ 7.90--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, 3.2 Hz, 3H), 4.48–4.30 (m, 4H), 4.02 (s, 9H), 3.93–3.84 (m, 3H), 3.76–3.66 (m, 9H), 3.45–3.35(m, 3H), 3.24–2.98(m, 10H), 2.30–2.20(m, 2H), 2.11–1.88(m, 31H), 1.80–1.40(m, 28H).MS m /z: C ₉₀ H ₁₂₈ N ₇ O ₃₅ , [M-DMTr] ⁺ , theory: 1564.65, actual measurement: 1564.88.

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

The L-7 (40g, 21.4247mmol) obtained in step (1-1-3b), succinic anhydride (4.288g, 42.8494mmol) and 4-dimethylaminopyridine (DMAP, 5.235g, 42.8494mmol) were mixed and dissolved To 215ml of dichloromethane, add diisopropylethylamine (DIEA, 13.845g, 107.1235mmol), stir for 24h at 25°C, wash the reaction solution with 800ml of 0.5M triethylamine phosphate, and extract the aqueous phase with dichloromethane. 5ml each time, combine the organic phases and evaporate to dryness under reduced pressure to obtain a crude product. Column purification uses 1kg 200-300 mesh normal phase silica gel, neutralizes the acidity of silica gel with 1wt% triethylamine, equilibrates the column with dichloromethane, and uses dichloromethane containing 1wt‰ of triethylamine: methanol=100:18-100:20 Gradient elution, collecting the product eluate, evaporating the solvent under reduced pressure to obtain 31.0 g of pure L-9 conjugated molecule. ¹ H 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 = 8.3 Hz, 4H), 5.49-5.37 (m, 1H), 5.21 (d, J = 3.0 Hz, 3H), 4.97 (d, J = 11.1 Hz, 3H), 4.49 (d ,J=8.2Hz,3H),4.02(s,9H),3.88(dd,J=19.4,9.4Hz,3H),3.77–3.65(m,9H),3.50–3.39(m,6H),3.11– 2.90(m,5H), 2.61-2.54(m,4H), 2.47-2.41(m,2H), 2.26-2.17(m,2H), 2.15-1.95(m,22H),1.92-1.84(m,9H) ),1.80–1.70(m,10H),1.65–1.35(m,17H),1.31–1.19(m,4H),0.96(t,J=7.1Hz,9H).MS m/z: C ₉₄ H ₁₃₂ N ₇ O ₃₈ , [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 conjugated molecule to the solid support.

The L-9 conjugate molecule (22.751g, 11mmol) obtained in step (1-1-4), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 6.257g, 16.5mmol) ) And diisopropylethylamine (DIEA, 2.843g, 22mmol), dissolved in 900ml of acetonitrile, stirred at room temperature for 5 minutes, added aminomethyl resin (88g, 100-200 mesh, amino group loading 400μmol/ g, purchased from Nankai Hecheng Company), shaker reaction at 25℃, rotating speed 150 rpm, after 18h reaction, filter, filter cake with DCM rinsed twice, 300ml each time, acetonitrile rinsed 3 times, each time 300ml, vacuum oil pump drying for 18h, and then add the raw materials (CapA, CapB, 4-dimethylaminopyridine (DMAP) and acetonitrile) according to the feeding ratio shown in Table 2 for capping reaction. Placed on a shaker at 25°C, rotating speed 150 rpm, reacting for 5 hours, the reaction solution is filtered, the filter cake is rinsed with acetonitrile 3 times, 300 ml each time, the solvent is evaporated under reduced pressure to dryness, and dried under vacuum oil pump overnight to obtain The L-10 compound (ie, the L-9 conjugated molecule connected to the solid phase carrier) was 102 g, and the loading was 90.8 μmol/g.

Table 2 Feeding ratio of cap reaction

raw material Dosage specification batch number Manufacturer CapA 1980ml —— —— —— CapB 220ml —— —— —— DMAP 1.100g Analytically pure I1422139 Aladdin Acetonitrile 220ml Spectral purity O15161001 Shanghai Xingke

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

(1-2) Synthesis of the sense chain of Conjugate 1

Through the solid-phase phosphoramidite method, the L-10 compound prepared by the above steps starts the cycle, and connects the nucleoside monomers one by one from the 3'-5' direction according to the sequence of the sense strand nucleotides. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or vulcanization. Among them, when two nucleotides are connected by phosphate ester, when the next nucleoside monomer is connected, four steps of deprotection, coupling, capping and oxidation are included. When two nucleotides are connected by phosphorothioate, when the next nucleoside monomer is connected, there are four steps of protection, coupling, capping and vulcanization. The synthesis conditions are given as follows:

The nucleoside monomer is provided in 0.1M acetonitrile solution. The deprotection reaction conditions of each step are the same, that is, the temperature is 25℃, the reaction time is 70 seconds, and the deprotection reagent is dichloroacetic acid in dichloromethane (3% v/v), the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support is 5:1.

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

The capping conditions for each step are the same, including a temperature of 25°C 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: the nucleic acid sequence connected to the solid phase carrier =1:1:1.

The oxidation reaction conditions for each step are the same, including a temperature of 25°C, a reaction time of 15 seconds, and an oxidizing reagent of 0.05M iodine water. The molar ratio of iodine to the nucleic acid sequence connected to the solid phase carrier in the coupling step is 30:1. The reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine=3:1:1.

The conditions of each vulcanization reaction are the same, including the temperature is 25°C, the reaction time is 300 seconds, and the vulcanization reagent is hydroxanthin. The molar ratio of the vulcanizing reagent to the nucleic acid sequence connected to the solid support in the coupling step is 120:1. The reaction was carried out in a mixed solvent of acetonitrile:pyridine=1:1.

The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence linked to the carrier is added to 25wt% ammonia water, the amount of ammonia water is 0.5ml/μmol, reacted at 55℃ for 16h, the liquid is removed, and the residue is concentrated in vacuo to dry.

Purification and desalting: Use a preparative ion chromatography purification column (Source 15Q) to purify nucleic acid through NaCl gradient elution. Specifically: eluent A: 20mM sodium phosphate (pH 8.1), solvent is water/acetonitrile = 9:1 (volume ratio); eluent B: 1.5M sodium chloride, 20mM sodium phosphate (pH 8.1) , The solvent is water/acetonitrile=9:1 (volume ratio); elution gradient: eluent A: eluent B=100:0-50:50 gradient elution. After collecting the product eluates, they are combined and desalted using a reversed-phase chromatography purification column. The specific conditions include using Sephadex G25 (Sephadex G25) as the filler and eluting with deionized water.

Detection: Use ion exchange chromatography (IEX-HPLC) to detect purity, and use liquid mass spectrometry (LC-MS) to analyze molecular weight. The measured value is consistent with the theoretical value, indicating that the synthesized is the sense strand S conjugated with an L-9 conjugate molecule at the 3'end.

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

HL Solid Supports，Kinovate Life Sciences公司)起始循环，合成缀合物1的反义链AS。固相合成方法中的脱保护、偶联、盖帽、氧化或硫化反应条件，切割和脱保护，纯化与脱盐条件与合成正义链相同。 Through the solid phase phosphoramidite method, the universal solid phase carrier (UnyLinker ^TM loaded

HL Solid Supports, Kinovate Life Sciences Company) initiated the cycle to synthesize the antisense strand AS of Conjugate 1. The deprotection, coupling, capping, oxidation or vulcanization reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid phase synthesis method are the same as the synthetic sense strand.

When the target sequence has a 5-P modification at the first nucleotide at the 5'-end of the antisense strand, in the process of preparing the antisense strand according to the solid-phase phosphoramidite method, the last nucleoside single After the body, the CPR-I monomer (Suzhou Gima, Cat#13-2601-XX) was connected to the 5'end of the antisense strand through a four-step reaction of deprotection, coupling, capping, and oxidation to form a 5'- Phosphate modification.

In this connection, the general solid phase carrier used, deprotection, coupling, capping, oxidation or vulcanization reaction conditions, cleavage and deprotection, purification and desalting conditions are the same as the synthetic sense strand.

Detection: ion exchange chromatography (IEX-HPLC) was used to detect purity; liquid mass spectrometry (LC-MS) was used to analyze the molecular weight of the obtained product. As a result, the measured value agrees with the theoretical value, indicating that the synthesized antisense strand AS has the target sequence.

(1-4) Synthesis of Conjugate 1

For Conjugate 1, the S chain and AS chain were separately dissolved in water for injection to obtain a 40 mg/mL solution, mixed in an equimolar ratio, heated at 50°C for 15 min, and cooled at room temperature to obtain the annealed product, which was freeze-dried. A lyophilized powder is obtained. Use ultrapure water (Milli-Q ultrapure water meter, resistivity 18.2MΩ*cm(25℃)) to dilute the conjugate to a concentration of 0.2mg/mL, then use liquid mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, Model: LCT Premier) for molecular weight detection. The measured value is consistent with the theoretical value, indicating that the synthesized conjugate 1 is a target designed double-stranded nucleic acid sequence with an L-9 conjugate molecule. Its structure is shown in formula (403).

Preparation Example 2 Preparation of Conjugate 2-6

Using the same method as Preparation Example 1, Conjugate 2, Conjugate 3, Conjugate 4, Conjugate 5, and Conjugate 6 were synthesized respectively. The difference is that the siRNA in the conjugate was Having the sense strand and antisense strand corresponding to Conjugate 2, Conjugate 3, Conjugate 4, Conjugate 5, and Conjugate 6 shown in Table 3, in order to synthesize these conjugates, When preparing the sense strand and antisense strand, according to the siRNA sense strand and antisense strand sequence shown in Table 3, the corresponding sense strand and antisense strand were synthesized. The molecular weights of the obtained conjugates 2-6 were detected by LC-MS, and the measured values were consistent with the theoretical values, indicating that the synthesized conjugates were the target-designed double-stranded nucleic acid sequences with L-9 conjugate molecules. The structure of conjugate 2-6 is shown in formula (403).

Table 3 siRNA conjugates

Among them, capital letters C, G, U, A indicate the base composition of nucleotides; lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates The adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; a lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by phosphorothioate groups; the uppercase letter P indicates the letter The adjacent nucleotide to the right of P is a 5'-phosphate nucleotide modified nucleotide.

In the above conjugate, between the siRNA in conjugate 3 and conjugate 1, the sense strand and antisense strand each contain a nucleotide base difference (5'-3' direction, the second core of the sense strand) The difference of GA at the nucleotide position and the difference of CU at the 18th nucleotide of the antisense strand), the siRNA antisense strand of conjugate 1 is completely reverse complementary to human FXII mRNA, while the siRNA antisense strand of conjugate 3 is Mouse FXII mRNA is fully reverse complementary. The siRNA antisense strands of the remaining conjugates 2, 4, 5, and 6 are completely reverse complementary to human FXII mRNA and mouse FXII mRNA.

Preparation Example 3 Synthesis of siRNA sequence

The siRNA sense and antisense strands listed in Table 4 were obtained by solid-phase synthesis, and the equimolar mixture of sense and antisense strands was dissolved in DEPC water, and then annealed to form siRNA duplexes, lyophilized, and freeze-dried powder The siRNA listed in Table 4 were obtained in the form.

Table 4 siRNA sequence

Among them, capital letters C, G, U, A indicate the base composition of nucleotides; lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates The adjacent nucleotide to the left of the letter f is a fluoro-modified nucleotide; the lowercase letter s means that the two nucleotides on the left and right of the letter s are connected by phosphorothioate groups; dT means thymine deoxynucleus Glycidyl.

In the preparation process of the above sequence, when the target sequence contains unmodified nucleotides, in the cutting and deprotection conditions, after ammonia treatment, 0.4ml/μmol N-methyl is used relative to the amount of single-stranded nucleic acid. Pyrrolidone dissolves the product, and then 0.3ml/μmol triethylamine and 0.6ml/μmol triethylamine trihydrofluoride are added to remove the 2'-TBDMS protection on the ribose.

Experimental Example 4 Detection of the inhibitory efficiency of siRNA on FXII mRNA expression in human liver primary cells.

According to the instructions provided by the supplier, use Lipofectamine ^TM 2000 to combine the test siRNA (siRNA7, 8, 10, 11 and 12 and each solution of the comparison sequence NC, where NC is a negative control sequence with no obvious sequence correlation with FXII mRNA. The solution refers to the aqueous solution formed by re-dissolving siRNA at the required concentration with DEPC water before the experiment) was transfected into primary human liver cells. The final concentration of each siRNA was 50 nM, and each concentration was 2 replicates. hole.

The expression of FXII mRNA in human liver primary cells transfected with each siRNA was detected by Quantitative Real-Time PCR (Quantitative Real-Time PCR). The specific steps are: After culturing the transfected cells for 24 hours, use Trizol (Thermo Fisher) to extract the total RNA from the cells according to the standard operating procedures for total RNA extraction; take 1 μg of total RNA and use the reverse transcription kit (Promega) , Item No. A3500) Reverse transcription to obtain cDNA according to the operating method of its manual. Use the 2×Ultra SYBR Mixture (with ROX) (Beijing Kangwei Century Biotechnology Co., Ltd., catalog number CW0956) kit, and use cDNA as a template to detect FXII mRNA expression according to the instructions. Among them, the PCR primers used to amplify FXII and GAPDH as an internal reference gene are shown in Table 5.

Table 5 Primer information

FXII mRNA expression is calculated according to the following equation: FXII mRNA expression = (FXII mRNA expression of the test group/GAPDH mRNA expression of the test group)/(FXII mRNA expression of the control group/GAPDH mRNA expression of the control group) ×100%.

mRNA inhibition rate=(1-FXII mRNA expression level)×100%. Among them, each test group is human liver primary cells treated with various concentrations of siRNA, and the control group is a comparison sequence NC-treated cells. The results are shown in Table 6.

Table 6 Inhibition of FXII mRNA in human liver primary cells

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

It can be seen from the results in Table 6 that the modified siRNA provided in the present disclosure shows high FXII mRNA inhibitory activity in human liver primary cells, and the 50 nM siRNA12 can inhibit FXII mRNA expression by 78.70%.

Experimental Example 5 Detection of the inhibitory efficiency of siRNA on FXII mRNA expression in C57 mouse liver primary cells.

The detection was performed according to the same method 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, and at the same time used for amplification shown in Table 7 The PCR primers of FXII and GAPDH as the internal reference gene replace the primers shown in Table 5.

Table 7 Primer information

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

Table 8 Inhibition of FXII mRNA in C57 mouse liver primary cells

siRNA Numbering 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

It can be seen from the results in Table 8 that the siRNA provided by the present disclosure shows high FXII mRNA inhibitory activity in C57 mouse liver primary cells, and the 50 nM siRNA12 can inhibit FXII mRNA expression by 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 conjugate 1-6 on FXII mRNA in liver tissue in C57 mice was investigated through two experiments.

(6-A) Inhibition rate of conjugates 1, 2 and 3 on FXII mRNA in liver tissue in C57 mice

Randomly divide 6-8 weeks old C57 mice into 7 groups, each with 5 mice, and each group of mice were given conjugate 1, 2 or 3 (each conjugate corresponds to 2 different dose groups) or The solution of the comparative sequence NC (group 1) (the solution refers to the solution formed by re-dissolving the siRNA conjugate to the required concentration with 1×PBS (pH 7.4) buffer before the experiment), and PBS Blank control group (15 mice, given 1×PBS). All animals calculate the dose based on body weight, and use subcutaneous injection for a single dose. The dose of siRNA conjugate (based on the amount of siRNA) is 5 mg/kg and 1 mg/kg, respectively, and the dose is 10 mL. /kg, according to the dose and volume of administration, the concentration of each siRNA conjugate should be calculated.

The mice were sacrificed 7 days after the administration, and the liver was collected and preserved with RNA later (Sigma Aldrich); then the liver tissue was homogenized with a tissue homogenizer, and then extracted with Trizol (Thermo Fisher) according to the standard procedure of total RNA extraction Get total RNA from liver tissue.

Real-time fluorescent quantitative PCR was used to detect the expression level of FXII mRNA in liver tissue, specifically: reverse transcription using a reverse transcription kit (Promega company, catalog number A3500) according to the operating method of its manual to obtain cDNA. Using the SYBR Select Master Mix (Applied Biosystem, Catalog No. 4472897) kit, use the cDNA as a template to detect the FXII mRNA expression according to the steps of the instructions, and calculate the inhibition rate of the siRNA conjugate on the FXII mRNA expression. Among them, the PCR primers used to amplify FXII and GAPDH as an internal reference gene are shown in Table 7.

FXII mRNA expression, that is, the remaining expression, calculated according to the following equation: FXII mRNA expression = (FXII mRNA expression of the test group/GAPDH mRNA expression of the test group)/(FXII mRNA expression of the control group/Control group GAPDH mRNA expression level)×100%.

The inhibitory rate of the siRNA conjugate on the FXII mRNA expression is (1-FXII mRNA expression)×100%. Among them, the control group is a control group of mice administered with PBS in the experiment, and each test group is a administration group of mice administered with different siRNA conjugates.

Figure 1 is a scatter plot of FXII mRNA expression in liver tissue of C57 mice (relative value with GAPDH as internal reference) after administration of PBS or different doses of conjugate 1, 2 or 3 in C57 mice. It can be seen from Figure 1 that on the 7th day after administration, the comparison sequence NC did not show any inhibitory effect; at the same time, siRNA conjugate 2 at a dose of 5 mg/kg inhibited FXII mRNA expression The rate is as high as 94.9%. siRNA conjugate 2 at a dose of 1 mg/kg also shows a high inhibition rate of 74.8% on FXII mRNA expression; for siRNA conjugate 3, it shows 96.7% at a dose of 1 mg/kg The high inhibition rate of FXII mRNA expression at a dose of 5 mg/kg is as high as 98.7%; in addition, although siRNA conjugate 1 does not show a high inhibition rate of FXII mRNA expression in mice, Considering that the siRNA contained in the conjugate is the siRNA corresponding to human FXII mRNA, and the siRNA conjugate 3 having the same target mRNA site as it shows unexpectedly high inhibitory activity, it can be expected that it is also extremely effective in humans. It may show a high inhibition rate of FXII mRNA expression.

(6-B) Inhibition rate of conjugates 1, 3, 4, 5 and 6 on FXII mRNA expression in liver tissues in C57 mice

In this experiment, using the same experimental conditions and steps as (6-A), the inhibition rate of conjugates 1, 3, 4, 5, and 6 on the expression of FXII mRNA in liver tissues in C57 mice was measured. The difference is that the siRNA conjugates used are conjugates 1, 3, 4, 5 and 6, and the PBS group uses 10 mice.

Figure 2 is a scatter diagram of FXII mRNA expression in liver tissue of C57 mice (with GAPDH as the relative value of the internal control) after administration of PBS or different doses of conjugates 1, 3, 4, 5 and 6. It can be seen from the results in Figure 2 that Conjugate 1 and Conjugate 3 showed similar inhibitory effects to (6-A), especially Conjugate 3 showed 95.4% and 95.4% and 5 mg/kg at doses of 1 mg/kg and 5 mg/kg, respectively. 97.4% high FXII mRNA expression inhibition rate; Conjugates 4, 5 and 6 showed 54.9%, 61.8% and 41.2% FXII mRNA expression inhibition rates at a dose of 1 mg/kg, respectively, at a dose of 3 mg/kg It also showed high FXII mRNA expression inhibition rates of 83.9%, 87.0% and 83.8%, respectively.

From the results of the above experimental examples 4, 5, (6-A) and (6-B), it can be seen that the siRNA and siRNA conjugates of the present disclosure are used in in vitro human/mouse liver primary cell experiments and in vivo experiments in mice. Both show excellent FXII mRNA inhibitory activity.

Some embodiments of the present disclosure are described in detail above. However, the present disclosure is not limited to the specific details in the above-mentioned embodiments. Within the scope of the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure. These simple modifications All belong to the protection scope of the present disclosure.

In addition, it should be noted that the various specific technical features described in some of the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure provides various possible The combination method is not explained separately.

In addition, various different embodiments of the present disclosure can also be combined arbitrarily, as long as they do not violate the idea of the present disclosure, they should also be regarded as the content disclosed in the present disclosure.

Claims (57)

A siRNA conjugate having the structure shown in formula (308):

among them, 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 2-10; Each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R _{15 is} independently H, or is selected from the group consisting of C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ haloalkyl and C ₁ -C ₁₀ alkoxy; R ₃ is a group of the structure shown in formula A59:

Wherein, E ₁ is OH, SH or BH ₂ ; Nu is siRNA, the siRNA has a sense strand and an antisense strand, each nucleotide in the siRNA is independently a modified or unmodified nucleotide, and the sense strand contains a nucleotide sequence I, The antisense strand contains 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 and the nucleotide sequence The nucleotide sequence II is selected from the following group i)-v), i) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length and have no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 2 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₃ whose position corresponds to Z ₁ , and the nucleotide sequence II Comprising the nucleotide Z ₄ at the position corresponding to Z ₂ , where Z ₄ is the first nucleotide at the 5'end of the antisense strand; ii) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 62 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₇ whose position corresponds to Z ₅ , and the nucleotide sequence II Comprising the nucleotide Z ₈ at the position corresponding to Z ₆ , said Z ₈ being the first nucleotide at the 5'end of the antisense strand; iii) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 122 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₁₅ whose position corresponds to Z ₁₃ , and the nucleotide sequence II Comprising the nucleotide Z ₁₆ at the position corresponding to Z ₁₄ , said Z ₁₆ being the first nucleotide at the 5'end of the antisense strand; iv) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 182 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₁₉ at the position corresponding to Z ₁₇ , and the nucleotide sequence II Comprising the nucleotide Z ₂₀ at the position corresponding to Z ₁₈ , said Z ₂₀ being the first nucleotide at the 5'end of the antisense strand; v) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 242 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₂₃ corresponding to the position Z ₂₁ , and the nucleotide sequence II _Comprising the nucleotide Z ₂₄ at the position corresponding to Z ₂₂ , said Z ₂₄ being the first nucleotide at the 5'end of the antisense strand; R ₂ is a linear alkylene group having a length of 1-20 carbon atoms, wherein one or more carbon atoms is optionally replaced by one or more selected from the group consisting of: C( O), NH, O, S, CH=N, S(O) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C ₃ -C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene; and wherein R ₂ may optionally have any one or more substituents from the group consisting of: C ₁ -C ₁₀ alkane Group, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl -OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkyl phenyl, -C ₁ -C ₁₀ alkyl -SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl -NH ₂ , -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ Alkyl), -NH (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl phenyl), -NH (C ₁ -C ₁₀ alkyl benzene) Group), cyano, nitro, -CO ₂ H, -C(O)O (C ₁ -C ₁₀ alkyl), -CON (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl) , -CONH (C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O) (C ₁ -C ₁₀ alkyl), -NHC(O) (phenyl), -N(C ₁ -C ₁₀ Alkyl) C (O) (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ alkyl, -C(O)C ₁ -C ₁₀ alkylphenyl, -C(O)C ₁ -C ₁₀ haloalkyl, -OC(O)C ₁ -C ₁₀ alkyl, -SO ₂ (C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl), -SO ₂ NH _{(phenyl), - NHSO 2 (C 1} -C 10 alkyl), - NHSO ₂ (phenyl), and -NHSO ₂ (C ₁ -C ₁₀ haloalkyl); Each L _{1 is} independently a linear alkylene group with a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally selected from any one or more of the following groups Replaced: C(O), NH, O, S, CH=N, S(O) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene , C ₃ -C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene; and wherein, L ₁ may optionally have any one or more substituents in the group consisting of the following groups: C ₁ -C ₁₀ alkyl, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkyl Phenyl, -C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkane Group -SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl -NH ₂ , -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl), -NH (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl phenyl), -NH (C ₁ -C ₁₀ alkyl phenyl), cyano, nitro, -CO ₂ H, -C (O) O (C ₁ -C ₁₀ alkyl), -CON (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl), -CONH (C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O) (C ₁ -C ₁₀ alkyl), -NHC(O) (phenyl), -N (C ₁ -C ₁₀ alkyl) C (O) (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ alkyl, -C (O) C ₁ -C ₁₀ alkyl phenyl, -C (O) C ₁ -C ₁₀ haloalkyl, -OC (O) C ₁ -C ₁₀ alkyl, -SO ₂ (C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl) ), - SO ₂ NH _{(phenyl), - NHSO 2 (C 1} -C 10 alkyl), - NHSO ₂ (phenyl), and -NHSO ₂ (C ₁ -C ₁₀ haloalkyl);

Indicates the site where the group is covalently attached; M ₁ represents a targeting group. The siRNA conjugate of claim 1, wherein each L _{1 is} independently selected from the group consisting of the groups represented by formulas (A1)-(A26) and any combination thereof:

Wherein, each j1 is independently an integer of 1-20; each j2 is independently an integer of 1-20; Each R'is independently a C1-C10 alkyl group; Each Ra is selected from the group consisting of the groups represented by formulas (A27)-(A45) and any combination thereof:

Each Rb is independently C ₁ -C ₁₀ alkyl; Or, L ₁ is a connection combination of one or more of the groups A1, A4, A5, A6, A8, A10, A11, and A13; Alternatively, L ₁ is a linked combination of at least two of the groups A1, A4, A8, A10 and A11; Or, L ₁ is a linked combination of at least two of the groups A1, A8, and A10; Or, the length of L ₁ is 3-25 atoms; Alternatively, the length of L ₁ is 4-15 atoms. The siRNA conjugate according to claim 2, wherein j1 is an integer from 2 to 10, j2 is an integer from 2 to 10, R'is a C ₁ -C ₄ alkyl group, and Ra is A27, A28, A29, A30 And one of A31, Rb is C ₁ -C ₅ alkyl; Alternatively, j1 is an integer of 3-5, j2 is an integer of 3-5, R'is one of methyl, ethyl and isopropyl, and Ra is a group represented by formula (A27) or formula (A28 In the group shown in ), Rb is one of methyl, ethyl, isopropyl and butyl. The siRNA conjugate according to any one of claims 1-3, wherein n1 is an integer of 1-2, n3 is an integer of 0-1, and n1+n3=2-3. The siRNA conjugate according to any one of claims 1 to 4, wherein each of m1, m2 and m3 is independently an integer of 2-5, and/or m1=m2=m3. The siRNA conjugate according to any one of claims 1 to 5, wherein each of the targeting groups is independently a ligand that has affinity with the asialoglycoprotein receptor on the surface of mammalian liver cells ； Or each of the targeting groups is independently asialoglycoprotein or sugar; Or each of the targeting groups is independently selected from D-mannanose, L-mannanose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L- Glucose, D-galactose, L-galactose, α-D-mannfuranose, β-D-mannanose, α-D-mannanose, β-D-mannanose, α-D- Glucopyranose, β-D-glucopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-frutofuranose, α-D-fructose pyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactosamine, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactose Amine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2- Deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl- D-mannanose, 2-deoxy-2-sulfoamino-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-galactopyranoside, 3, 4,6,7-Tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyrano-heptanoside ethyl ester, 2,5-anhydro-D-allosenitrile, One of ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose; Alternatively, at least one or each of the targeting groups is galactose or N-acetylgalactosamine. The siRNA conjugate according to any one of claims 1 to 6, wherein each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R _{15 is} independently H, methyl or ethyl; Alternatively, each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ is H. The siRNA conjugate according to any one of claims 1-7, wherein R ₂ contains a linking site connected to the N atom on the nitrogen-containing backbone and a linking site connected to the P atom in R ₃ point; Alternatively, the site on R ₂ connected to the N atom on the nitrogen-containing skeleton forms an amide bond with the N atom, and the site on R ₃ connected to the P atom forms a phosphate bond with the P atom; Alternatively, R _{2 is} selected from the group represented by the formula (B5), (B6), (B5') or (B6'). The siRNA conjugate of any one of claims 1-8, wherein the conjugate has the formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421 ) Or (422). The siRNA conjugate according to any one of claims 1-9, wherein the P atom in formula A59 is connected to the end of the siRNA sense strand or antisense strand, and the end refers to the sense strand or antisense strand. The first 4 nucleotides from one end of the sense strand; Alternatively, the P atom in formula A59 is connected to the end of the siRNA sense strand or antisense strand; or the P atom in formula A59 is connected to the 3'end of the siRNA sense strand; Alternatively, the P atom in formula A59 is connected to the 2'position, 3'position or 5'position of the nucleotide in the siRNA by forming a phosphodiester bond. The siRNA conjugate according to claim 1, wherein i) there is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1, and/ Or there is no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2; or ii), the nucleotide sequence I and SEQ ID NO: 61 There is no more than 1 nucleotide difference between the nucleotide sequences shown, and/or no more than 1 nucleus between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 62 Nucleotide sequence difference; or, iii) no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121, and/or the nucleotide sequence II There is no more than 1 nucleotide difference between the nucleotide sequence shown in SEQ ID NO: 122; or, iv) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 No more than 1 nucleotide difference, and/or no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 182; or, v) There is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241, and/or the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 242 No more than one nucleotide difference between nucleotide sequences. The siRNA conjugate according to claim 1, wherein i) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 includes the difference at the Z ₄ position , And Z _{4 is} selected from A, C or G; or ii) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 62 includes the difference at the Z ₈ position, And Z _{8 is} selected from A, C or G; or iii) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 122 includes the difference at the Z ₁₆ position, and Z _{16 is} selected from A, C or G; or iv) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 182 includes the difference at the Z ₂₀ position, and Z _{20 is} selected from A, C or G; or v) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 242 includes the difference at position Z ₂₄ , and Z ₂₄ Selected from A, C or G. The siRNA conjugate of claim 12, 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 nucleoside complementary to Z ₁₆ Acid; or Z ₁₉ is a nucleotide complementary to Z ₂₀ ; or Z ₂₃ is a nucleotide complementary to Z ₂₄ . The siRNA conjugate according to any one of claims 11-13, wherein the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse Complementarity; the substantially reverse complementation refers to the presence of no more than 3 base mismatches between two nucleotide sequences; the substantially reverse complementation refers to the presence of no more than 3 base mismatches between the two nucleotide sequences More than 1 base mismatch; the complete reverse complementation means that there is no mismatch between two nucleotide sequences. The siRNA conjugate according to any one of claims 11-14, wherein the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV and a nucleotide sequence III And the length of the nucleotide sequence IV are each independently 1-4 nucleotides, the nucleotide sequence III is connected to the 5'end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the nucleotide sequence At the 3'end of II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the substantially reverse complement refers to two nucleotide sequences There is no more than 1 base mismatch between them; the complete reverse complementation means that there is no mismatch between two nucleotide sequences. The siRNA conjugate according to claim 15, wherein the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 are equal in length and have no more than 3 nucleotide differences, and, The length of the nucleotide sequence III and IV are both 1 nucleotide, and the base of the nucleotide sequence III is A; or the length of the nucleotide sequence III and IV are both 2 nuclei According to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is CA; or, the length of the nucleotide sequence III and IV are both 3 nucleotides, according to 5' From the end to the 3'end, the base composition of the nucleotide sequence III is GCA; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, according to the 5'end to the 3'end Direction, the base composition of nucleotide sequence III is CGCA; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 have the same length and no more than 3 nucleotide differences, and the lengths of the nucleotide sequences III and IV are both The length of the nucleotide sequence III is 1 nucleotide, and the base of the nucleotide sequence III is A; or the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end to the 3'end Direction, the base composition of the nucleotide sequence III is AA; or, the length of the nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the nucleotide sequence The base composition of III is GAA; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is GGAA; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 have the same length and no more than 3 nucleotide differences, and the lengths of the nucleotide sequences III and IV are both Is 1 nucleotide, the base of the nucleotide sequence III is U; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end to the 3'end Direction, the base composition of the nucleotide sequence III is UU; or, the length of the nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the nucleotide sequence The base composition of III is UUU; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is GUUU; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 have the same length and no more than 3 nucleotide differences, and the lengths of the nucleotide sequences III and IV are both The length of the nucleotide sequence III is 1 nucleotide, and the base of the nucleotide sequence III is G; or the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end to the 3'end Direction, the base composition of the nucleotide sequence III is GG; or, the length of the nucleotide sequence III and IV is 3 nucleotides, according to the direction from the 5'end to the 3'end, the nucleotide sequence The base composition of III is UGG; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is UUGG; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 have the same length and no more than 3 nucleotide differences, and the lengths of the nucleotide sequence III and IV are both The length of the nucleotide sequence III is 1 nucleotide, and the base of the nucleotide sequence III is C; or the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end to the 3'end Direction, the base composition of the nucleotide sequence III is GC; or, the length of the nucleotide sequence III and IV is 3 nucleotides, according to the direction from the 5'end to the 3'end, the nucleotide sequence The base composition of III is AGC; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is GAGC. The siRNA conjugate of claim 16, wherein the nucleotide sequences III and IV are completely reverse complementary. The siRNA conjugate according to any one of claims 11-17, wherein the antisense strand further contains a nucleotide sequence V, and the length of the nucleotide sequence V is 1 to 3 nucleotides. At the 3'end of the antisense strand, constitute the 3'hanging end of the antisense strand; or the length of the nucleotide sequence V is 2 nucleotides; or the nucleotide sequence V is continuous Two thymine deoxyribonucleotides or two consecutive uracil ribonucleotides; or the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. The siRNA conjugate according to any one of claims 11-18, wherein the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 5, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 5. The nucleotide sequence shown in NO: 6; or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand contains the nucleus sequence shown in SEQ ID NO: 8 The nucleotide sequence; or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 65, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 66; or, The sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 67, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 68; or, the sense strand of the siRNA It contains the nucleotide sequence shown in SEQ ID NO: 125, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 126; or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: : 127, the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 128; or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 185 Nucleotide sequence, the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 186; or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 187, The antisense strand contains the nucleotide sequence shown in SEQ ID NO: 188; or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 245, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 245. NO: 246; alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 247, and the antisense strand contains the nucleus sequence shown in SEQ ID NO: 248 Nucleotide sequence. The siRNA conjugate according to any one of claims 11-19, wherein the siRNA is siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1 or siFXIIf2. The siRNA conjugate according to any one of claims 11-20, wherein at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and/or at least one phosphate The ester group is a phosphate group having a modification group. The siRNA conjugate of claim 21, wherein each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide ； Or the fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and in the direction from the 5'end to the 3'end, the seventh, eighth, and eighth nucleotides of the nucleotide sequence I The nucleotides at position 9 are fluorinated modified nucleotides; 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 are fluorinated. Modified nucleotides; Or according to the direction from the 5'end to the 3'end, in the sense strand, the nucleotides at positions 7, 8, 9 or 5, 7, 8, and 9 of the nucleotide sequence I are fluoro Modified nucleotides, the nucleotides at the remaining positions in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5'end to the 3'end, in the antisense strand, the nucleoside The nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, and 16 of acid sequence II are fluoro-modified nucleotides, and the nucleosides at the remaining positions in the antisense strand The acid is a non-fluorinated modified nucleotide. The siRNA conjugate according to claim 21 or 22, wherein each non-fluorinated modified nucleotide is independently selected from the core formed by the substitution of a non-fluorinated group for the hydroxyl group at the 2'position of the ribose group of the nucleotide One of nucleotide or nucleotide analogues; Or the nucleotide formed by replacing the hydroxyl group at the 2'position of the ribose group with a non-fluorine group is selected from 2'-alkoxy-modified nucleotides and 2'-substituted alkoxy-modified nucleosides Acid, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, One of 2'-deoxynucleotides; the nucleotide analog is selected from one of heteronucleotides, LNA, ENA, cET, UNA and GNA; Or each non-fluoro-modified nucleotide is a methoxy-modified nucleotide, and the methoxy-modified nucleotide refers to a nucleoside formed by replacing the 2'-hydroxyl group of a ribose group with a methoxy group acid. The siRNA conjugate according to any one of claims 21-23, wherein, in the direction from the 5'end to the 3'end, the 5th, 7th, 8th, and 8th of the nucleotide sequence I in the sense strand of the siRNA The nucleotides at position 9 are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides, and in the direction from the 5'end to the 3'end, so The nucleotides at positions 2, 6, 8, 9, 14 and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are Methoxy modified nucleotides; Alternatively, according to the direction from the 5'end to the 3'end, the 5th, 7th, 8th and 9th nucleotides of the nucleotide sequence I in the sense strand of the siRNA are fluorinated modified nucleotides, and the sense of siRNA The nucleotides at the remaining positions of the chain are methoxy-modified nucleotides, and in the direction from the 5'end to the 3'end, the second, sixth, and 14th nucleotide sequence II in the antisense strand of the siRNA The nucleotides at and 16 are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of siRNA are methoxy-modified nucleotides; Alternatively, according to the direction from the 5'end to the 3'end, the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are -fluoro-modified nucleotides, and the sense strand of the siRNA The nucleotides at the remaining positions of the siRNA are methoxy-modified nucleotides, and in the direction from the 5'end to the 3'end, the second, sixth, 14th, and fourth nucleotide sequence II in the antisense strand of the siRNA The nucleotide at position 16 is a fluoro-modified nucleotide, and the nucleotide at the remaining position of the antisense strand of the siRNA is a methoxy-modified nucleotide. The siRNA conjugate according to any one of claims 11-24, wherein the siRNA is 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 Any one of siFXIIe1-M2, siFXIIe1-M3, siFXIIe2-M1, siFXIIe2-M2, siFXIIe2-M3, siFXIIf1-M1, siFXIIf1-M2, siFXIIf1-M3, siFXIIf2-M1, siFXIIf2-M2, siFXIIf2-M3. The siRNA conjugate according to any one of claims 11-25, wherein the phosphate group having a modification group is that at least one oxygen atom in the phosphodiester bond in the phosphate group is substituted by a sulfur atom And the phosphorothioate group formed; or the phosphorothioate group having a modified group is a phosphorothioate group having a structure as shown in formula (1):

The siRNA conjugate according to claim 26, wherein, in the siRNA, the phosphorothioate group linkage exists in at least one of the following positions: Between the first nucleotide and the second nucleotide of the 5'end of the sense strand; Between the second nucleotide and the third nucleotide of the 5'end of the sense strand; Between the first nucleotide and the second nucleotide of the 3'end of the sense strand; Between the second nucleotide and the third nucleotide of the 3'end of the sense strand; Between the first nucleotide and the second nucleotide of the 5'end of the antisense strand; Between the second nucleotide and the third nucleotide of the 5'end of the antisense strand; Between the first nucleotide and the second nucleotide of the 3'end of the antisense strand; and Between the second nucleotide and the third nucleotide of the 3'end of the antisense strand. The siRNA conjugate according to any one of claims 11-27, wherein the siRNA is 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-M2S1-M1S, siFXIId2-M2S, siFXII Any one of siFXIIe1-M2S, siFXIIe1-M3S, siFXIIe2-M1S, siFXIIe2-M2S, siFXIIe2-M3S, siFXIIf1-M1S, siFXIIf1-M2S, siFXIIf1-M3S, siFXIIf2-M1S, siFXIIf2-M2S, siFXIIf2-. The siRNA conjugate according to any one of claims 11-28, wherein the 5'terminal nucleotide of the siRNA antisense strand is modified with 5'-phosphate nucleotides or 5'-phosphate analogues Nucleotide Alternatively, the 5'-phosphate nucleotides are nucleotides having the structure shown in formula (2), and the nucleotides modified by the 5'-phosphate analogues are selected from the structures shown in formula (3)-formula ( 6) any one of the nucleotides shown in,

Wherein, R is selected from H, OH, methoxy or fluorine; Base represents a nucleic acid base, selected from A, U, C, G or T. The siRNA conjugate according to any one of claims 11-29, wherein the siRNA is siFXIIa1-M1P1, siFXIIa1-M2P1, siFXIIa1-M3P1, siFXIIa2-M1P1, siFXIIa2-M2P1, siFXIIa2-M3P1, siFXIIa1- M1SP1, siFXIIa1-M2SP1, siFXIIa1-M3SP1, siFXIIa2-M1SP1, siFXIIa2-M2SP1, siFXIIa2-M3SP1, siFXIIa1U-M1P1, siFXIIa1U-M2P1, siFXIIa1U-M3P1, siFXIIa1U-M3P1, siFXIIa2U-M2P1, siFXIIa2U-M2P1, siFXIIa1 siFXIIa1U-M2SP1, siFXIIa1U-M3SP1, siFXIIa2U-M1SP1, siFXIIa2U-M2SP1, siFXIIa2U-M3SP1, siFXIIb1-M1P1, siFXIIb1-M2P1, siFXIIb1-M3P1, siFXIIb2-M1M21-M3P1, siFXBIIsiII2-M2B1-M3P1, siFXIIb2 M2SP1, siFXIIb1-M3SP1, siFXIIb2-M1SP1, siFXIIb2-M2SP1, siFXIIb2-M3SP1, siFXIId1-M1P1, siFXIId1-M2P1, siFXIId1-M3P1, siFXIId2-M1P1, siFXIId2-M2P1, siFXIId2-M3P1-M2P1, siFXIId2-M3P1 siFXIId1-M3SP1, siFXIId2-M1SP1, siFXIId2-M2SP1, siFXIId2-M3SP1, siFXIIe1-M1P1, siFXIIe1-M2P1, siFXIIe1-M3P1, siFXIIe2-M1P1, siFXIIe2-M2P1, siFXIIe2-M3IIP1, siFXIISP1, siFXIIe1-M3SP1 M3SP1, siFXIIe2-M1SP1, siFXIIe2-M2SP1, siFXIIe2-M3SP1, siFXIIf1-M1P1, siFXIIf1-M2P1, siFXIIf1-M3P1, siFXIIf2-M1P1, siFXIIf2-M2P1, siFXIIf2-M Any of 3P1, siFXIIf1-M1SP1, siFXIIf1-M2SP1, siFXIIf1-M3SP1, siFXIIf2-M1SP1, siFXIIf2-M2SP1, siFXIIf2-M3SP1. An siRNA comprising a sense strand and an antisense strand, each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleoside Acid; the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double In the chain region, the fluoro-modified nucleotides are located in the nucleotide sequence I and the nucleotide sequence II, and, in the direction from the 5'end to the 3'end, in the sense strand, the nucleoside The nucleotides at positions 7, 8, and 9 of acid sequence I are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorinated modified nucleotides; according to the 5'end to In the direction of the 3'end, in the antisense strand, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides, and in the antisense strand The nucleotides at the remaining positions are non-fluorinated modified nucleotides, and, i) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length and have no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 2 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₃ whose position corresponds to Z ₁ , and the nucleotide sequence II The inclusion position corresponds to the nucleotide Z _{4 of} Z ₂ , where Z ₄ is the first nucleotide at the 5'end of the antisense strand; or, ii) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 62 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₇ whose position corresponds to Z ₅ , and the nucleotide sequence II The inclusion position corresponds to the nucleotide Z _{8 of} Z ₆ , where Z ₈ is the first nucleotide at the 5'end of the antisense strand; or, iii) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 122 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₁₅ whose position corresponds to Z ₁₃ , and the nucleotide sequence II The inclusion position corresponds to the nucleotide Z _{16 of} Z ₁₄ , where Z ₁₆ is the first nucleotide at the 5'end of the antisense strand; or, iv) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 182 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₁₉ at the position corresponding to Z ₁₇ , and the nucleotide sequence II The inclusion position corresponds to the nucleotide Z _{20 of} Z ₁₈ , said Z ₂₀ being the first nucleotide at the 5'end of the antisense strand; or, v) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 have the same length and no more than 3 nucleotide differences, and the nucleotide sequence II is the same as SEQ ID NO: 242 The nucleotide sequences shown are equal in length and have no more than 3 nucleotide differences. The nucleotide sequence I includes the nucleotide Z ₂₃ corresponding to the position Z ₂₁ , and the nucleotide sequence II comprising a position corresponding to nucleotide Z ₂₂ Z _24, Z ₂₄ is the antisense strand of the 5 'end of the first nucleotide. The siRNA of claim 31, wherein each non-fluorinated modified nucleotide is independently selected from nucleotides or nucleosides formed by substituting a non-fluorinated group for the hydroxyl group at the 2'position of the ribosyl group of the nucleotide One of the acid analogs. The siRNA according to claim 32, wherein the nucleotide formed by substituting the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorine group is selected from 2'-alkoxy modified nucleotides, 2'- Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'- One of substituted amino-modified nucleotides and 2'-deoxynucleotides; the nucleotide analog is selected from one of heteronucleotides, LNA, ENA, cET, UNA and GNA. The siRNA of any one of claims 31-33, wherein each non-fluorinated modified nucleotide is a methoxy modified nucleotide, and the methoxy modified nucleotide refers to ribose A nucleotide in which the 2'-hydroxy group of the group is replaced by a methoxy group. The siRNA according to any one of claims 31-34, wherein i) there is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 , And/or no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2; or ii), the nucleotide sequence I and SEQ ID There is no more than one nucleotide difference between the nucleotide sequence shown in NO: 61, and/or the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 62 are not more than 1 nucleotide difference; or, iii) no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121, and/or the nucleoside No more than one nucleotide difference between the acid sequence II and the nucleotide sequence shown in SEQ ID NO: 122; or, iv) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 No more than 1 nucleotide difference between the sequences, and/or no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 182; or, v) No more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241, and/or the nucleotide sequence II and SEQ ID NO: 242 There is no more than one nucleotide difference between the nucleotide sequences shown. The siRNA according to any one of claims 31-35, wherein i) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 includes the Z ₄ position And Z _{4 is} selected from A, C or G; or ii) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 62 includes the Z ₈ position And Z _{8 is} selected from A, C or G; or iii) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 122 includes the Z ₁₆ position Difference, and Z _{16 is} selected from A, C or G; or iv) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 182 includes the difference at position Z ₂₀ , And Z _{20 is} selected from A, C or G; or v) the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 242 includes the difference at position Z ₂₄ , And Z _{24 is} selected from A, C or G. The siRNA of claim 36, 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 . The siRNA according to any one of claims 31-37, wherein the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary; The "substantially reverse complementarity" means that there are no more than 3 base mismatches between two nucleotide sequences; the "substantially reverse complementarity" refers to the presence of no more than 1 base mismatch between the two nucleotide sequences. A base mismatch; the complete reverse complementation means that there is no mismatch between two nucleotide sequences. The siRNA according to any one of claims 31-38, wherein the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV, a nucleotide sequence III and a nucleoside The length of the acid sequence IV is each independently 1-4 nucleotides, 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 of the nucleotide sequence II. 'At the end, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the substantially reverse complement refers to the existence between two nucleotide sequences No more than 1 base mismatch; the complete reverse complementation means that there is no mismatch between two nucleotide sequences. The siRNA according to any one of claims 31-39, wherein the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 are equal in length and have no more than 3 nucleotide differences And, the length of the nucleotide sequence III and IV are both 1 nucleotide, and the base of the nucleotide sequence III is A; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is CA; or, the length of the nucleotide sequence III and IV are both 3 nucleotides, According to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is GCA; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, according to the 5'end to the 3' The direction of the end, the base composition of nucleotide sequence III is CGCA; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 have the same length and no more than 3 nucleotide differences, and the lengths of the nucleotide sequences III and IV are both The length of the nucleotide sequence III is 1 nucleotide, and the base of the nucleotide sequence III is A; or the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end to the 3'end Direction, the base composition of the nucleotide sequence III is AA; or, the length of the nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the nucleotide sequence The base composition of III is GAA; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is GGAA; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 have the same length and no more than 3 nucleotide differences, and the lengths of the nucleotide sequences III and IV are both Is 1 nucleotide, the base of the nucleotide sequence III is U; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end to the 3'end Direction, the base composition of the nucleotide sequence III is UU; or, the length of the nucleotide sequence III and IV are both 3 nucleotides, according to the direction from the 5'end to the 3'end, the nucleotide sequence The base composition of III is UUU; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is GUUU; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 have the same length and no more than 3 nucleotide differences, and the lengths of the nucleotide sequences III and IV are both The length of the nucleotide sequence III is 1 nucleotide, and the base of the nucleotide sequence III is G; or the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end to the 3'end Direction, the base composition of the nucleotide sequence III is GG; or, the length of the nucleotide sequence III and IV is 3 nucleotides, according to the direction from the 5'end to the 3'end, the nucleotide sequence The base composition of III is UGG; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is UUGG; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 have the same length and no more than 3 nucleotide differences, and the lengths of the nucleotide sequence III and IV are both The length of the nucleotide sequence III is 1 nucleotide, and the base of the nucleotide sequence III is C; or the length of the nucleotide sequence III and IV are both 2 nucleotides, according to the 5'end to the 3'end Direction, the base composition of the nucleotide sequence III is GC; or, the length of the nucleotide sequence III and IV is 3 nucleotides, according to the direction from the 5'end to the 3'end, the nucleotide sequence The base composition of III is AGC; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, according to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is GAGC. The siRNA of claim 40, wherein the nucleotide sequences III and IV are completely reverse complementary. The siRNA according to any one of claims 31-40, wherein the antisense strand further contains a nucleotide sequence V, the length of the nucleotide sequence V is 1 to 3 nucleotides, and is connected to the The 3'end of the antisense strand constitutes the 3'hanging end of the antisense strand; or the length of the nucleotide sequence V is 2 nucleotides; or the nucleotide sequence V is two consecutive thymus glands Pyrimidine deoxyribonucleotides or two consecutive uracil ribonucleotides; or the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. The siRNA according to any one of claims 31-42, wherein the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 5, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 6 Or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 8 Or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 65, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 66; or, the siRNA The sense strand contains the nucleotide sequence shown in SEQ ID NO: 67, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 68; or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 68. ID NO: 125, the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 126; or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 127 The antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 128; or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 185 Sequence, the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 186; or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 187, the antisense strand It contains the nucleotide sequence shown in SEQ ID NO: 188; or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 245, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 246. Or, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 247, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 248 . The siRNA according to any one of claims 31-43, wherein the siRNA has a nucleotide sequence of siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1 or siFXIIf2. The siRNA of any one of claims 31-44, wherein the siRNA is siFXIIa1-M1, siFXIIa2-M1, siFXIIb1-M1, siFXIIb2-M1, siFXIId1-M1, siFXIId2-M1, siFXIIe1-M1, siFXIIe2 -Any one of M1, siFXIIf1-M1, siFXIIf2-M1. The siRNA according to any one of claims 31-45, wherein at least one phosphate group in the sense strand or the antisense strand is a phosphate group with a modification group. The siRNA according to claim 46, wherein the phosphate group having a modification group is a phosphorothioate group formed by replacing at least one oxygen atom in a phosphodiester bond in the phosphate group with a sulfur atom; Or the phosphate group with a modified group is a phosphorothioate group having a structure as shown in formula (1):

The siRNA according to claim 47, wherein, in the siRNA, the phosphorothioate group linkage exists in at least one of the following positions: Between the first nucleotide and the second nucleotide of the 5'end of the sense strand; Between the second nucleotide and the third nucleotide of the 5'end of the sense strand; Between the first nucleotide and the second nucleotide of the 3'end of the sense strand; Between the second nucleotide and the third nucleotide of the 3'end of the sense strand; Between the first nucleotide and the second nucleotide of the 5'end of the antisense strand; Between the second nucleotide and the third nucleotide of the 5'end of the antisense strand; Between the first nucleotide and the second nucleotide of the 3'end of the antisense strand; and Between the second nucleotide and the third nucleotide of the 3'end of the antisense strand. The siRNA of any one of claims 31-48, wherein the siRNA is siFXIIa1-M1S, siFXIIa2-M1S, siFXIIb1-M1S, siFXIIb2-M1S, siFXIId1-M1S, siFXIId2-M1S, siFXIIe1-M1S, siFXIIe2 -Any one of M1S, siFXIIf1-M1S, siFXIIf2-M1S. The siRNA of any one of claims 31-49, wherein the 5'terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide ； Alternatively, the 5'-phosphate nucleotides are nucleotides having the structure shown in formula (2), and the nucleotides modified by the 5'-phosphate analogues are selected from the structures shown in formula (3)-formula ( 6) any one of the nucleotides shown in,

Wherein, R is selected from H, OH, methoxy or fluorine; Base represents a nucleic acid base, selected from A, U, C, G or T. The siRNA of any one of claims 31-50, wherein the siRNA is siFXIIa1-M1P1, siFXIIa2-M1P1, siFXIIa1U-M1P1, siFXIIa2U-M1P1, siFXIIa1U-M1SP1, siFXIIa2U-M1SP1, siFXIIb1-M1P1, siFXIIb2 -M1P1, siFXIId1-M1P1, siFXIId2-M1P1, siFXIIe1-M1P1, siFXIIe2-M1P1, siFXIIf1-M1P1, siFXIIf2-M1P1, siFXIIa1-M1SP1, siFXIIa2-M1SP1, siFXIIb1-M1SP1, siFXIIb2-M1FXII-M1SP1 , Any one of siFXIIe1-M1SP1, siFXIIe2-M1SP1, siFXIIf1-M1SP1, siFXIIf2-M1SP1. A pharmaceutical composition, characterized in that it contains the siRNA according to any one of claims 31-51 and a pharmaceutically acceptable carrier. An siRNA conjugate containing the siRNA according to any one of claims 31-51 and a conjugate group conjugated to the siRNA. The siRNA conjugate of any one of claims 1-30 and 53, the siRNA of any one of claims 31-51, and/or the pharmaceutical composition of claim 52 are in preparation for treatment and /Or use in medicines for preventing hereditary angioedema HAE and/or thrombosis. A method for treating and/or preventing hereditary angioedema HAE and/or thrombosis, wherein the method comprises adding an effective amount of the siRNA conjugate according to any one of claims 1-30 and 53, The siRNA according to any one of claims 31-51 and/or the pharmaceutical composition according to claim 52 is administered to a subject suffering from hereditary angioedema HAE. A method for inhibiting FXII gene expression in hepatocytes, the method comprising adding an effective amount of the siRNA conjugate of any one of claims 1-30 and 53, and any one of claims 31-51 The siRNA and/or the pharmaceutical composition of claim 52 are contacted with the hepatocytes. A kit, wherein the kit contains the siRNA conjugate according to any one of claims 1-30 and 53, the siRNA according to any one of claims 31-51 and/or the siRNA according to claim 52 The pharmaceutical composition.

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