Nucleic acid, pharmaceutical composition, conjugate, method of production and use

Abstract

FIELD: genetics.SUBSTANCE: invention discloses new miRNAs capable of inhibiting the blood coagulation factor XI (FXI) gene expression in plasma, as well as conjugates based on them. The invention can be used in the manufacture of a medicinal product used for the treatment or prevention of thrombotic events and ischemic strokes.EFFECT: improved gene expression.24 cl, 17 tbl, 8 ex

Description

[0001] FIELD OF TECHNOLOGY

[0002] The present invention relates to a nucleic acid capable of inhibiting the expression of the blood coagulation factor XI (FXI) gene, and a pharmaceutical composition and siRNA conjugate containing said nucleic acid. The present invention also relates to a method for producing and using such nucleic acids, pharmaceutical compositions, and siRNA conjugates.

[0003] STATE OF THE ART

[0004] Blood coagulation factor XI (hereinafter referred to as "FXI") is an important component of the intrinsic coagulation pathway, which promotes thrombin production, which in turn is an important component involved in fibrin formation and provides protection against fibrinolysis. High FXI levels are a risk factor for venous thrombosis. Inhibition of FXI gene expression allows for the prevention and treatment of thrombotic diseases (particularly venous thrombosis and ischemic stroke) at the cellular level.

[0005] Small interfering RNA (siRNA) can inhibit or block the expression of any target gene of interest in a sequence-specific manner based on the RNA interference (RNAi) mechanism, which will achieve the purpose of treating diseases.

[0006] The key to developing siRNA-based drugs that inhibit FXI gene expression and treat thrombotic diseases is to find suitable siRNA, its modification and an effective delivery system.

[0007] BRIEF DESCRIPTION OF THE INVENTION

[0008] The present inventors have unexpectedly discovered that the following siRNAs and modified sequences thereof provided by the present invention are capable of specifically inhibiting the expression of the FXI gene, and pharmaceutical compositions or siRNA conjugates containing such siRNAs are capable of specifically targeting the liver, thereby inhibiting the expression of the FXI gene in the liver to ensure the prevention or treatment of thrombotic diseases, thereby realizing the purpose of the present invention.

[0009] According to some embodiments of the present invention, there is provided a first siRNA capable of inhibiting the expression of the FXI gene, comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, which allows for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 1, and differs therefrom by no more than 3 nucleotides, and the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 2, and differs therefrom by no more than 3 nucleotides:

[0010] 5'-GGGGUAUUUCUUUCAAGCAAZ1-3' (SEQ ID NO: 1);

[0011] 5'-Z2UUGCUUGAAAGAAUACCC-3' (SEQ ID NO: 2),

[0012] where Z1 represents U, and Z2 represents A, and

[0013] nucleotide sequence I contains nucleotide Z3 at a position corresponding to Z1; nucleotide sequence II contains nucleotide Z4 at a position corresponding to Z2, where nucleotide Z4 is the first nucleotide from the 5' end of the antisense strand.

[0014] According to some embodiments of the present invention, there is provided a second siRNA capable of inhibiting the expression of the FXI gene, comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 61 and differs therefrom by no more than 3 nucleotides, and wherein the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 62 and differs therefrom by no more than 3 nucleotides:

[0015] 5'-GGCAUAAACUAUAACAGCZ5-3' (SEQ ID NO: 61);

[0016] 5'-Z6GCUGUUUAAUAAGUUUUUAUGCC-3' (SEQ ID NO: 62),

[0017] where Z5 represents U, and Z6 represents A, and

[0018] nucleotide sequence I contains nucleotide Z7 at a position corresponding to Z5; nucleotide sequence II contains nucleotide Z8 at a position corresponding to Z6, where nucleotide Z8 is the first nucleotide from the 5' end of the antisense strand.

[0019] According to some embodiments of the present invention, there is provided a third siRNA capable of inhibiting the expression of the FXI gene, comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 121 and differs therefrom by no more than 3 nucleotides, and wherein the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 122 and differs therefrom by no more than 3 nucleotides:

[0020] 5'-GCUCAAGAAUGCCAAGAZ9-3' (SEQ ID NO: 121);

[0021] 5'-Z 10 UUCUUGGCAUUUCUUGAGC-3' (SEQ ID NO: 122),

[0022] where Z9 represents A, and Z 10 represents U, and

[0023] nucleotide sequence I contains nucleotide Z 11 at the position corresponding to Z9; nucleotide sequence II contains the nucleotide Z 12 in the position corresponding to Z 10 , where the nucleotide is Z 12 represents the first nucleotide from the 5' end of the antisense strand.

[0024] According to some embodiments of the present invention, there is provided a fourth siRNA capable of inhibiting the expression of the FXI gene, comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 181 and differs therefrom by no more than 3 nucleotides, and wherein the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 182 and differs therefrom by no more than 3 nucleotides:

[0025] 5'-GCAACAAAGACAUUUUAUGZ 13 -3' (SEQ ID NO: 181);

[0026] 5'-Z 14 CAUAAAUGUCUUUGUGC-3' (SEQ ID NO: 182),

[0027] where Z 13 represents U, and Z 14 represents A, and

[0028] nucleotide sequence I contains nucleotide Z 15 in the position corresponding to Z 13 ; nucleotide sequence II contains nucleotide Z 16 in the position corresponding to Z 14 , where the nucleotide is Z 16 represents the first nucleotide from the 5' end of the antisense strand.

[0029] According to some embodiments of the present invention, there is provided a fifth siRNA capable of inhibiting the expression of the FXI gene, comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, which allows for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 241 and differs therefrom by no more than 3 nucleotides, and wherein the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 242 and differs therefrom by no more than 3 nucleotides:

[0030] 5'-GAAUCUCUCAAAGAAAUCUUZ17 -3' (SEQ ID NO: 241);

[0031] 5'-Z 18 AAGAUUUUCUUUUUGAGAUUC-3' (SEQ ID NO: 242),

[0032] where Z 17 represents U, and Z 18 represents A, and

[0033] nucleotide sequence I contains nucleotide Z 19 in the position corresponding to Z 17 ; nucleotide sequence II contains nucleotide Z 20 in the position corresponding to Z 18 , where the nucleotide is Z 20 represents the first nucleotide from the 5' end of the antisense strand.

[0034] According to some embodiments of the present invention, there is provided a sixth siRNA capable of inhibiting the expression of the FXI gene, comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, which allows for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 301, and differs therefrom by no more than 3 nucleotides, and the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 302, and differs therefrom by no more than 3 nucleotides:

[0035] 5'-GUACGUGGGACUGGAUUCUZ21 -3' (SEQ ID NO: 301);

[0036] 5'-Z 22 AGAAUCCAGUCCACGUAC-3' (SEQ ID NO: 302),

[0037] in which Z 21 represents G, and Z 22 represents C, and

[0038] nucleotide sequence I contains nucleotide Z 23 in the position corresponding to Z 21 ; nucleotide sequence II contains nucleotide Z 24 in the position corresponding to Z 22 , where the nucleotide is Z 24 represents the first nucleotide from the 5' end of the antisense strand.

[0039] According to some embodiments of the present invention, there is provided a seventh siRNA capable of inhibiting the expression of the FXI gene, comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, which allows for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 361, and differs therefrom by no more than 3 nucleotides, and the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 362, and differs therefrom by no more than 3 nucleotides:

[0040] 5'-AUUUCUGGGUAUUUCUUUCZ25 -3' (SEQ ID NO: 361);

[0041] 5'-Z 26 GAAAGAAUACCCAGAAAU-3' (SEQ ID NO: 362),

[0042] where Z 25 represents A and Z 26 represents U, and

[0043] nucleotide sequence I contains nucleotide Z 27 in the position corresponding to Z 25 ; nucleotide sequence II contains nucleotide Z 28 in the position corresponding to Z 26 , where the nucleotide is Z 28 represents the first nucleotide from the 5' end of the antisense strand.

[0044] According to some embodiments of the present invention, there is provided an eighth siRNA capable of inhibiting the expression of the FXI gene, comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, which allows for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 421, and differs therefrom by no more than 3 nucleotides, and the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 422, and differs therefrom by no more than 3 nucleotides:

[0045] 5'-CAUGAAGGGCAUAAACUAZ29 -3' (SEQ ID NO: 421);

[0046] 5'-Z 30 UAGUUUAUGCCCUUCAUG-3' (SEQ ID NO: 422),

[0047] where Z 29 represents U, and Z 30 represents A, and

[0048] nucleotide sequence I contains nucleotide Z 31 in the position corresponding to Z 29 ; nucleotide sequence II contains nucleotide Z 32 in the position corresponding to Z 30 , where the nucleotide is Z 32 represents the first nucleotide from the 5' end of the antisense strand.

[0049] According to some embodiments of the present invention, there is provided a ninth siRNA capable of inhibiting the expression of the FXI gene, comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, which allows for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 481, and differs therefrom by no more than 3 nucleotides, and the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 482, and differs therefrom by no more than 3 nucleotides:

[0050] 5'-GG AUUCU GG AG AAA ACUCZ 33 -3' (SEQ ID NO: 481);

[0051] 5'-Z 34 GAGUUUUUCUCCAGAAUCC-3' (SEQ ID NO: 482),

[0052] where Z 33 represents A and Z 34 represents U, and

[0053] nucleotide sequence I contains nucleotide Z 35 in the position corresponding to Z 33 ; nucleotide sequence II contains nucleotide Z 36 in the position corresponding to Z 34 , where the nucleotide is Z 36 represents the first nucleotide from the 5' end of the antisense strand.

[0054] According to some embodiments of the present invention, there is provided a pharmaceutical composition comprising an siRNA according to the present invention and a pharmaceutically acceptable carrier.

[0055] According to some embodiments of the present invention, there is provided an siRNA conjugate comprising an siRNA according to the present invention and a conjugating group conjugated to the siRNA.

[0056] According to some embodiments of the present invention, there is provided the use of siRNA and / or a pharmaceutical composition and / or siRNA conjugate according to the present invention in the manufacture of a medicament for the treatment and / or prevention of thrombotic diseases and / or ischemic stroke caused by increased expression of the FXI gene.

[0057] According to some embodiments of the present invention, a method for treating and / or preventing thrombotic diseases and / or ischemic stroke is provided, comprising administering an effective amount of siRNA and / or a pharmaceutical composition and / or siRNA conjugate according to the present invention to a subject suffering from thrombotic diseases and / or ischemic stroke.

[0058] According to some embodiments of the present invention, a method for inhibiting the expression of the FXI gene in hepatocytes is provided, comprising contacting an effective amount of siRNA and / or a pharmaceutical composition and / or siRNA conjugate according to the present invention with hepatocytes.

[0059] According to some embodiments of the present invention, a kit is provided comprising an siRNA and / or a pharmaceutical composition and / or an siRNA conjugate according to the present invention.

[0060] PREFERRED EFFECTS

[0061] The siRNA, pharmaceutical composition and siRNA conjugate according to the present invention exhibit high stability, have high inhibitory activity against FXI gene mRNA and low off-target effect and / or significantly treat or alleviate the symptoms of thrombotic diseases and / or ischemic stroke.

[0062] According to some embodiments of the present invention, the siRNA, pharmaceutical composition, or siRNA conjugate of the present invention exhibits excellent inhibitory activity against a target gene in in vitro cell assays. According to some embodiments of the present invention, the siRNA, pharmaceutical composition, or siRNA conjugate of the present invention exhibits a percentage of inhibition against target gene expression in hepatocytes of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. According to some embodiments of the present invention, the siRNA of the present invention exhibits inhibitory activity against FXI mRNA in the psiCHECK system, wherein the IC values 50for FXI mRNA are in the range from 0.013 nM to 0.119 nM. According to some embodiments of the present invention, the siRNA of the present invention exhibits high inhibitory activity in HepG2 cells, wherein the IC values 50 for FXI mRNA range from 1.49 nM to 11.1 nM. According to some embodiments of the present invention, the siRNA conjugate of the present invention exhibits high inhibitory activity in primary mouse hepatocytes, with IC values 50 for FXI mRNA range from 0.012 nM to 3.86 nM. According to some embodiments of the present invention, the siRNA of the present invention can inhibit the expression of FXI mRNA in HepG2 cells and exhibit an inhibition percentage of FXI mRNA of up to 86.9% at a concentration of 50 nM.

[0063] According to some embodiments of the present invention, the siRNA, siRNA pharmaceutical composition, or siRNA conjugate of the present invention can exhibit significantly higher stability and / or activity in vivo. According to some embodiments of the present invention, the siRNA, siRNA pharmaceutical composition, or siRNA conjugate of the present invention exhibits an inhibition percentage of the target gene expression in vivo of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. According to some embodiments of the present invention, the siRNA, siRNA pharmaceutical composition, or siRNA conjugate of the present invention exhibits an inhibition percentage of FXI gene expression in vivo of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.According to some embodiments of the present invention, the siRNA, siRNA pharmaceutical composition, or siRNA conjugate of the present invention exhibits a percentage of inhibition of FXI gene expression in the liver in vivo of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. According to some embodiments of the present invention, the siRNA, siRNA pharmaceutical composition, or siRNA conjugate of the present invention exhibits a percentage of inhibition of FXI gene expression in the liver in vivo in animal models of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. According to some embodiments of the present invention, the siRNA, pharmaceutical composition, or siRNA conjugate of the present invention exhibits an inhibition percentage of FXI gene expression in the liver in vivo in humans of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.In some embodiments, an siRNA conjugate of the invention exhibits a percentage inhibition of FXI mRNA expression in mice of up to 95.0% in vivo at an siRNA concentration of 5 mg / kg. In some embodiments, an siRNA conjugate of the invention exhibits a percentage inhibition of human FXI mRNA expression in humanized mice of up to 93.09% in vivo at an siRNA concentration of 3 mg / kg. Furthermore, the siRNA conjugate can significantly inhibit FXI protein concentration in plasma, exhibiting a percentage inhibition of up to about 99%. In some embodiments, an siRNA conjugate of the invention can significantly increase activated partial thromboplastin time (APTT) in plasma in CD57 mice in vivo, for example, by 64.9%.

[0064] According to some embodiments of the present invention, the siRNA, siRNA pharmaceutical composition, or siRNA conjugate of the present invention do not exhibit an obvious off-target effect. An off-target effect may be, for example, inhibition of the normal expression of a gene that is not the target gene. If the binding / inhibition of off-target gene expression is less than 50%, 40%, 30%, 20%, or 10% compared to the effect on the target gene, the off-target effect is considered to be insignificant.

[0065] Therefore, the siRNA, the siRNA pharmaceutical composition and the siRNA conjugate according to the present invention can inhibit the expression of the FXI gene, effectively treat and / or prevent thrombotic diseases and / or ischemic stroke caused by overexpression of the FXI gene, and thus their use is very promising.

[0066] Additional features and advantages of the present invention will be illustrated in detail in the following "Detailed Description of the Invention" section.

[0067] DETAILED DESCRIPTION OF THE INVENTION

[0068] Specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are intended only to illustrate and explain the present invention and are not intended to limit the present invention in any respect.

[0069] For the purposes of the present invention, FXI mRNA refers to mRNA with a nucleotide sequence disclosed under Genbank accession number NM 000128.3. Furthermore, unless otherwise specified, the term “target gene” used in the present invention refers to a gene that can transcribe the above-mentioned FXI mRNA; and the term “target mRNA” refers to the above-mentioned FXI mRNA.

[0070] Definitions

[0071] For the purposes of the present invention, unless otherwise specified, C, G, U, A, and T represent the base composition of nucleotides; the letter m represents that the nucleotide to the left of the letter m is a methoxy-modified nucleotide; f represents that the nucleotide to the left of the letter f is a fluorine-modified nucleotide; s represents that the two nucleotides on either side of the letter s are linked by a thiophosphorothioate bond; P1 denotes that the nucleotide adjacent to the right side of P1 is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analog, VP denotes that the nucleotide adjacent to the right side of VP is a nucleotide modified with vinyl phosphate (5''-(E)-vinyl phosphate, E-VP), Ps denotes that the nucleotide adjacent to the right side of Ps is a thiophosphate-modified nucleotide; and P denotes that the nucleotide adjacent to the right side of P is a 5'-phosphate nucleotide.

[0072] As used in the present invention, the term "fluorine-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl of the ribose group of a nucleotide with fluorine. The term "nucleotide with a non-fluorine modification" refers to a nucleotide formed by replacing the 2'-hydroxyl of the ribose group of a nucleotide with a group other than a fluorine group, or to a nucleotide analog. "Nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid, while it is structurally different from an adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide, such as an isonucleotide, a bridged nucleic acid (BNA) nucleotide, or an acyclic nucleotide. "Methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl of the ribose group with a methoxy group.

[0073] For the purposes of the present invention, the terms "complementary" and "reverse complementary" may be used interchangeably and have the generally understood meaning in the art, namely, bases in one strand are complementarily paired with bases in the other strand of a double-stranded nucleic acid molecule. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); and the purine base guanine (G) is always paired with the pyrimidine base cytosine (C). Each base pair contains a purine and a pyrimidine. If adenines in one strand are always paired with thymines (or uracils) in the other strand, and guanines are always paired with cytosines, the two strands are considered complementary; and the sequence of a strand can be deduced from the sequence of its complementary strand.Accordingly, "mispairing" means that in a double-stranded nucleic acid the bases at the corresponding positions are not presented as complementarily paired.

[0074] For the purposes of the present invention, unless otherwise specified, “substantially reverse complementary” means that there are no more than 3 mispaired bases between two nucleotide sequences. “Substantially reverse complementary” means that there are no more than 1 mispaired base between two nucleotide sequences. “Completely reverse complementary” means that there are no mispaired bases between two nucleotide sequences.

[0075] For purposes of the present invention, if a nucleotide sequence has a "nucleotide difference" from another nucleotide sequence, the nucleotide bases at the same position between them are changed. For example, if the nucleotide base in the second sequence is A, and the nucleotide base at the same position in the first sequence is U, C, G, or T, these two nucleotide sequences are considered to have a nucleotide difference at that position. According to some embodiments of the present invention, if a nucleotide at a certain position is substituted with an abasic nucleotide or nucleotide analog, a nucleotide difference is also considered to exist at that position.

[0076] With reference to the present invention, in particular in the description of the method for producing siRNA, siRNA composition or siRNA conjugate according to the present invention, unless otherwise specified, the term "nucleoside monomer" refers, according to the type and sequence of nucleotides in the siRNA or siRNA conjugate to be obtained, to unmodified or modified RNA phosphoamidites (RNA phosphoamidites are sometimes called nucleoside phosphoamidites) used in solid-phase phosphoamidite synthesis. Solid-phase phosphoamidite synthesis is a method for synthesizing RNA well known to those skilled in the art. All nucleoside monomers used in the present invention are commercially available.

[0077] For the purposes of the present invention, unless otherwise specified, "conjugation" refers to two or more chemical groups, each with a specific function, linked to each other via a covalent bond. Accordingly, "conjugate" refers to a compound formed by the covalent bonding of individual chemical groups. Furthermore, "miRNA conjugate" is a compound formed by the covalent attachment of miRNA and one or more chemical groups, each with a specific function. In the following text, the miRNA conjugate according to the present invention is sometimes abbreviated as "conjugate." According to the context of the present invention, siRNA conjugate should be understood as a general term for siRNA conjugates, a general term for siRNA conjugates represented by Formulas (305) and (307), or siRNA conjugates represented by Formulas (305), (307) or (308).For the purposes of the present invention, the term "conjugating molecule" shall be understood to mean a specific compound that can be conjugated to siRNA by reactions to ultimately yield the siRNA conjugate according to the present invention.

[0078] As used herein, the term "optional" or "optionally" means that the subsequently described event or condition may or may not occur, and that the description includes instances where the event or condition does or does not occur. For example, "optionally substituted alkyl" includes both "alkyl" and "substituted alkyl," as defined below. Those skilled in the art will understand, with respect to any group containing one or more substituents, that such groups are not intended to introduce any substitution or substitution patterns that are sterically impractical, synthetically unfeasible, and / or inherently unstable.

[0079] As used herein, the term "alkyl" refers to a straight chain and branched chain containing the specified number of carbon atoms, typically from 1 to 20 carbon atoms, such as from 1 to 10 carbon atoms, such as from 1 to 8 or from 1 to 6 carbon atoms. For example, C1-C6 alkyl includes both straight-chain and branched alkyl containing from 1 to 6 carbon atoms. When referring to an alkyl residue containing a specified number of carbon atoms, it is intended to include all branched-chain and straight-chain forms containing such a number of carbon atoms; thus, for example, "butyl" means n-butyl, sec-butyl, isobutyl and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subgroup of alkyl referring to the same residues as alkyl, but having two attachment positions.

[0080] As used herein, the term "alkenyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond that is derived by the appropriate removal of one hydrogen molecule from two adjacent carbon atoms of the parent alkyl. The group may be in the cis or trans configuration of the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl; propenyl such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl; butenyl such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl; and the like. According to certain embodiments of the present invention, the alkenyl group contains from 2 to 20 carbon atoms, and in other embodiments from 2 to 10, from 2 to 8, or from 2 to 6 carbon atoms.Alkenylene is a subgroup of alkenyl, referring to the same residues as alkenyl but having two attachment positions.

[0081] As used herein, the term "alkynyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond derived by the removal of two hydrogen molecules from two adjacent carbon atoms of the parent alkyl. 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. According to certain embodiments of the present invention, the alkynyl group contains from 2 to 20 carbon atoms, and in other embodiments from 2 to 10, from 2 to 8, or from 2 to 6 carbon atoms. Alkynylene is a subgroup of alkynyl, referring to the same residues as alkynyl but having two attachment positions.

[0082] In this document, the term "alkoxy" refers to an alkyl group with a specified number of carbon atoms bonded via an oxygen bridge, such as methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy, and the like. The alkoxy group usually contains 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms bonded via an oxygen bridge.

[0083] As used herein, the term "aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon, including 6 to 18 carbon atoms, with at least one ring in the ring system being completely unsaturated, i.e., it contains a cyclic delocalized (4n+2)π-electron system according to Hückel theory. Aryl groups include, but are not limited to, groups such as phenyl, fluorenyl, and naphthyl. Arylene is a subgroup of aryl, referring to the same residues as aryl, but having two attachment positions.

[0084] In this document, the term "halo substituent" or "halogen" refers to fluoro, chloro, bromo and iodo, and the term "halogen" includes fluorine, chlorine, bromine and iodine.

[0085] As used herein, the term "haloalkyl" refers to an alkyl group as defined above, with the specified number of carbon atoms, substituted with one or more halogen atoms, up to the maximum permissible number of halogen atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.

[0086] "Heterocyclyl" refers to a stable 3- to 18-membered non-aromatic ring radical that contains from 2 to 12 carbon atoms and from 1 to 6 heteroatoms selected from nitrogen, oxygen, or sulfur. Unless otherwise specified in the description, heterocyclyl is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may contain fused or bridged ring system(s). The heteroatom(s) in the heterocyclyl substituent may optionally be oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heterocyclyl substituent is partially or fully saturated. The heterocyclyl may be connected to the rest of the molecule through any atom of the ring(s).Examples of such heterocyclyl substituents include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxapyrimidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxathiomorpholinyl and 1,1-dioxathiomorpholyl.

[0087] “Heteroaryl” refers to a substituent derived from a 3- to 18-membered aromatic ring substituent that contains from 2 to 17 carbon atoms and from 1 to 6 heteroatoms selected from nitrogen, oxygen or sulfur. As used herein, the heteroaryl substituent may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., contains a cyclic delocalized (4n+2)ππ-electron system in accordance with Hückel theory. Heteroaryl includes fused or bridged ring system(s). The heteroatom(s) in the heteroaryl substituent are(are) optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. A heteroaryl is attached to the rest of the molecule through any atom of the ring(s). Examples of such heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl,benzindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzooxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl, benzothienyl[3,2-c1]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]-thieno[2,3-d]-pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothienyl, furanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocyclohepta[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl,naphthyridinonyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, 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, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl and thiophenyl / thienyl.,

[0088] Various hydroxyl-protecting groups can be used in the present invention. Generally, protecting groups render chemical functional groups inert to particular reaction conditions and can be attached to and removed from such functional groups in a molecule without significantly damaging the rest of the molecule. Typical hydroxyl-protecting groups are disclosed in Beaucage, et al., Tetrahedron 1992, 48, 2223-2311, and in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2nd ed., John Wiley & Sons, New York, 1991, each of which is hereby incorporated by reference in its entirety. According to some embodiments of the present invention, the protecting group is stable under basic conditions but can be removed under acidic conditions.In some embodiments of the present invention, non-exclusive examples of hydroxyl protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthen-9-yl (Mox). In some embodiments of the present invention, non-exclusive examples of hydroxyl protecting groups used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4''-trimethoxytrityl).

[0089] As used herein, the term "subject" refers to any animal, such as a mammal or marsupial. A subject according to the present invention includes, but is not limited to, a human, a non-human primate (e.g., a rhesus macaque or other macaque species), a mouse, a pig, a horse, a donkey, a cow, a sheep, a rat, and any species of poultry.

[0090] As used herein, the term "treatment" refers to a method for achieving a preferred or targeted result, including, but not limited to, a therapeutic benefit. "Therapeutic benefit" means eliminating or ameliorating the potential disorder being treated. Therapeutic benefit is also achieved by eliminating or reducing one or more physiological symptoms associated with the potential disorder, such that the subject experiences improvement, even though the subject may still be affected by the potential disorder.

[0091] As used herein, the term "prevention" refers to a method for achieving a preferred or targeted outcome, including, but not limited to, a prophylactic benefit. To achieve a "prophylactic benefit," siRNA, siRNA conjugates, or siRNA pharmaceutical compositions may be administered to a subject at risk of developing a specific disease or to a subject reporting one or more physiological symptoms of the disease, even if the disease has not been diagnosed.

[0092] In one aspect, the present invention provides first to ninth siRNAs capable of inhibiting the expression of the FXI gene. They will be described in detail sequentially below.

[0093] The miRNA according to the present invention contains nucleotide groups as its basic structural units. It is well known to those skilled in the art that a nucleotide group contains a phosphate group, a ribose group, and a base. Detailed illustrations of these groups are not provided herein.

[0094] The first miRNA

[0095] According to the present invention, the siRNA may be a first siRNA.

[0096] The first siRNA comprises a sense strand and an antisense strand; each nucleotide in the first siRNA is independently a modified or unmodified nucleotide; wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 1 and differs therefrom by no more than 3 nucleotides, and the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 2 and differs therefrom by no more than 3 nucleotides:

[0097] 5'-GGGGUAUUUCUUUCAAGCAAZ1-3' (SEQ ID NO: 1);

[0098] 5'-Z2UUGCUUGAAAGAAUACCC-3' (SEQ ID NO: 2),

[0099] where Z1 represents U, and Z2 represents A, and

[0100] nucleotide sequence I contains nucleotide Z3 at a position corresponding to Z1; nucleotide sequence II contains nucleotide Z4 at a position corresponding to Z2, where nucleotide Z4 is the first nucleotide from the 5' end of the antisense strand.

[0101] As used herein, the term "corresponding position" means being located at the same position in a nucleotide sequence when counted from the same end of the nucleotide sequence. For example, the first nucleotide at the 3' end of nucleotide sequence I is a nucleotide at a position corresponding to the first nucleotide at the 3' end of SEQ ID NO: 1.

[0102] According to some embodiments of the present invention, the sense strand comprises only the nucleotide sequence I, and the antisense strand comprises only the nucleotide sequence II.

[0103] According to some embodiments of the present invention, nucleotide sequence I contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 1, and / or nucleotide sequence II contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 2.

[0104] According to some embodiments of the present invention, the nucleotide difference between said nucleotide sequence II and said nucleotide sequence presented in SEQ ID NO: 2 comprises a difference at the position of Z4, wherein Z4 is selected from U, C or G. According to some embodiments of the present invention, the nucleotide difference is a difference at the position of Z4, wherein Z4 is selected from U, C or G. According to some embodiments of the present invention, Z3 is a nucleotide complementary to Z4. siRNAs containing the above nucleotide differences also have a high inhibitory ability towards the target mRNA, and such siRNAs containing nucleotide differences are also included within the scope of the present invention.

[0105] According to some embodiments of the present invention, the nucleotide sequence I is predominantly reverse complementary, substantially reverse complementary, or completely reverse complementary to the nucleotide sequence II; “predominantly reverse complementary” refers to no more than 3 mispaired bases in the two nucleotide sequences; “substantially reverse complementary” refers to no more than 1 mispaired base in the two nucleotide sequences; “completely reverse complementary” refers to the absence of mispaired bases in the two nucleotide sequences.

[0106] According to some embodiments of the present invention, nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 3, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 4:

[0107] 5'-GGGGUAUUUCUUUCAAGCAAZ3-3' (SEQ ID NO: 3);

[0108] 5'-Z4UUGCUUGAAAGAAUACCC-3' (SEQ ID NO: 4),

[0109] wherein Z4 is the first nucleotide from the 5' end of the antisense strand, Z3 is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3; in some embodiments, Z3 is U and Z4 is A.

[0110] In addition, the sense strand and the antisense strand have the same or different lengths, with the sense strand containing 19-23 nucleotides in length and the antisense strand containing 19-26 nucleotides in length. Thus, the ratio of the length of the sense strand to the length of the antisense strand in the siRNA according to the present invention may be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25, or 23 / 26. In some embodiments, the ratio of the length of the sense strand to the length of the antisense strand in the siRNA of the present invention may be 19 / 21, 21 / 23, or 23 / 25.

[0111] In some embodiments of the present invention, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV independently have a length of 1 to 4 nucleotides; the nucleotide sequence III and the nucleotide sequence IV have the same length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5'-end of the nucleotide sequence I; and the nucleotide sequence IV is connected to the 3'-end of the nucleotide sequence II.According to some embodiments of the present invention, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which refers to a nucleotide sequence that is connected to the 5' end of the nucleotide sequence of the target mRNA presented in SEQ ID NO: 1 in the target mRNA, and has the same length as the nucleotide sequence IV.

[0112] According to some embodiments of the present invention, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 20 / 20; or, the nucleotide sequence III and the nucleotide sequence IV are both 2 nucleotides in length, and, in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is CU, and the base composition of the nucleotide sequence IV is AG; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21;or, the nucleotide sequence III and the nucleotide sequence IV are both 3 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is UCU, and the base composition of the nucleotide sequence IV is AGA; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 22 / 22; or, the nucleotide sequence III and the nucleotide sequence IV are both 4 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is UUCU, and the base composition of the nucleotide sequence IV is AGAA;in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23 / 23. According to some embodiments of the present invention, the nucleotide sequence III and the nucleotide sequence IV have a length of 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is CU, and the base composition of the nucleotide sequence IV is AG; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21.

[0113] According to some embodiments of the present invention, the nucleotide sequence III is completely reverse complementary to the nucleotide sequence IV. Thus, if the base of the nucleotide sequence III is proposed, the base of the nucleotide sequence IV is also determined.

[0114] Second miRNA

[0115] According to the present invention, the siRNA may be a second siRNA.

[0116] The second siRNA comprises a sense strand and an antisense strand; each nucleotide in the second siRNA is independently a modified or unmodified nucleotide; wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 61 and differs therefrom by no more than 3 nucleotides, and the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 62 and differs therefrom by no more than 3 nucleotides:

[0117] 5'-GGCAUAAACUAUAACAGCZ5-3' (SEQ ID NO: 61);

[0118] 5'-Z6GCUGUUUAAUAAGUUUUUAUGCC-3' (SEQ ID NO: 62),

[0119] where Z5 represents U, and Z6 represents A, and

[0120] nucleotide sequence I contains nucleotide Z7 at a position corresponding to Z5; nucleotide sequence II contains nucleotide Z8 at a position corresponding to Z6, where nucleotide Z8 is the first nucleotide from the 5' end of the antisense strand.

[0121] According to some embodiments of the present invention, the sense strand comprises only the nucleotide sequence I, and the antisense strand comprises only the nucleotide sequence II.

[0122] According to some embodiments of the present invention, nucleotide sequence I contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 61, and / or nucleotide sequence II contains no more than 1 nucleotide different from the nucleotide sequence of SEQ ID NO: 62.

[0123] According to some embodiments of the present invention, the nucleotide difference between said nucleotide sequence II and said nucleotide sequence presented in SEQ ID NO: 62 comprises a difference at the position of Z8, wherein Z8 is selected from U, C or G. According to some embodiments of the present invention, the nucleotide difference is a difference at the position of Z8, wherein Z8 is selected from U, C or G. According to some embodiments of the present invention, Z7 is a nucleotide complementary to Z8. siRNAs containing the above nucleotide differences also have a high inhibitory ability towards the target mRNA, and such siRNAs containing nucleotide differences are also included within the scope of the present invention.

[0124] According to some embodiments of the present invention, the nucleotide sequence I is predominantly reverse complementary, substantially reverse complementary, or completely reverse complementary to the nucleotide sequence II.

[0125] According to some embodiments of the present invention, nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 63, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 64:

[0126] 5'-GGCAUAAACUAUAACAGCZ7-3' (SEQ ID NO: 63);

[0127] 5'-Z8GCUGUUAUAGUUUAUGCC-3' (SEQ ID NO: 64),

[0128] wherein Z8 is the first nucleotide from the 5' end of the antisense strand, Z7 is selected from A, U, G or C, and Z8 is a nucleotide complementary to Z7; in some embodiments, Z7 is U and Z8 is A.

[0129] In addition, the sense strand and the antisense strand have the same or different lengths, with the sense strand containing 19-23 nucleotides in length and the antisense strand containing 19-26 nucleotides in length.

[0130] In some embodiments of the present invention, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV independently have a length of 1 to 4 nucleotides; the nucleotide sequence III and the nucleotide sequence IV have the same length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II;the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which refers to a nucleotide sequence that is connected to the 5' end of the nucleotide sequence of the target mRNA presented in SEQ ID NO: 61 and has the same length as the nucleotide sequence IV.

[0131] According to some embodiments of the present invention, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 20 / 20; or, the nucleotide sequence III and the nucleotide sequence IV are both 2 nucleotides in length, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is AG, and the base composition of the nucleotide sequence IV is CU; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21;or, the nucleotide sequence III and the nucleotide sequence IV are both 3 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is AAG, and the base composition of the nucleotide sequence IV is CUU; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 22 / 22; or, the nucleotide sequence III and the nucleotide sequence IV are both 4 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is GAAG, and the base composition of the nucleotide sequence IV is CUUC;in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23 / 23. According to some embodiments of the present invention, the nucleotide sequence III and the nucleotide sequence IV have a length of 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is AG, and the base composition of the nucleotide sequence IV is CU; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21.

[0132] According to some embodiments of the present invention, the nucleotide sequence III is completely reverse complementary to the nucleotide sequence IV. Thus, if the base of the nucleotide sequence III is proposed, the base of the nucleotide sequence IV is also determined.

[0133] The third miRNA

[0134] According to the present invention, the siRNA may be a third siRNA.

[0135] The third siRNA comprises a sense strand and an antisense strand; each nucleotide in the third siRNA is independently a modified or unmodified nucleotide; wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 121 and differs therefrom by no more than 3 nucleotides, and the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 122 and differs therefrom by no more than 3 nucleotides:

[0136] 5'-GCUCAAGAAUGCCAAGAZ9-3' (SEQ ID NO: 121);

[0137] 5'-Z 10 UUCUUGGCAUUUCUUGAGC-3' (SEQ ID NO: 122),

[0138] where Z9 represents A, and Z 10 represents U, and

[0139] Nucleotide sequence I contains nucleotide Z 11 at the position corresponding to Z9; nucleotide sequence II contains the nucleotide Z 12 in the position corresponding to Z 10 , where the nucleotide is Z 12 represents the first nucleotide from the 5' end of the antisense strand.

[0140] According to some embodiments of the present invention, the sense strand comprises only the nucleotide sequence I, and the antisense strand comprises only the nucleotide sequence II.

[0141] According to some embodiments of the present invention, nucleotide sequence I contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 121, and / or nucleotide sequence II contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 122.

[0142] According to some embodiments of the present invention, the difference in nucleotides between said nucleotide sequence II and said nucleotide sequence presented in SEQ ID NO: 122 comprises a difference in the position of Z 12 , and Z 12 selected from A, C or G. In some embodiments of the present invention, the nucleotide difference is a difference in the position of Z 12 , and Z 12 selected from A, C or G. According to some embodiments of the present invention, Z 12 is a nucleotide complementary to Z 12 . The miRNAs containing the above-mentioned nucleotide differences also have a high inhibitory ability towards the target mRNA, and such miRNAs containing nucleotide differences are also included within the scope of the present invention.

[0143] According to some embodiments of the present invention, the nucleotide sequence I is predominantly reverse complementary, substantially reverse complementary, or completely reverse complementary to the nucleotide sequence II.

[0144] According to some embodiments of the present invention, nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 123, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 124:

[0145] 5'-GCUCAAGAAUGCCAAGAZ 11 -3' (SEQ ID NO: 123);

[0146] 5'-Z 12 UUCUUGGCAUUCUGAGC-3' (SEQ ID NO: 124),

[0147] in which Z 12 represents the first nucleotide from the 5' end of the antisense strand, Z 11 selected from A, U, G or C, and Z 12 is a nucleotide complementary to Z 11 ; in some embodiments of Z11 represents A and Z 12 represents U.

[0148] In addition, the sense strand and the antisense strand have the same or different lengths, with the sense strand containing 19-23 nucleotides in length and the antisense strand containing 19-26 nucleotides in length.

[0149] In some embodiments of the present invention, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV independently have a length of 1 to 4 nucleotides; the nucleotide sequence III and the nucleotide sequence IV have the same length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II;the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which refers to a nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence of the target mRNA presented in SEQ ID NO: 121 and has the same length as the nucleotide sequence IV.

[0150] According to some embodiments of the present invention, in the direction from the 5' end to the 3' end, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 20 / 20; or, the nucleotide sequence III and the nucleotide sequence IV are both 2 nucleotides in length, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is GU, and the base composition of the nucleotide sequence IV is AC; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21;or, the nucleotide sequence III and the nucleotide sequence IV are both 3 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is AGU, and the base composition of the nucleotide sequence IV is ACU; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 22 / 22; or, the nucleotide sequence III and the nucleotide sequence IV are both 4 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is GAGU, and the base composition of the nucleotide sequence IV is ACUC;in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23 / 23. According to some embodiments of the present invention, the nucleotide sequence III and the nucleotide sequence IV have a length of 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is GU, and the base composition of the nucleotide sequence IV is AC; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21.

[0151] According to some embodiments of the present invention, the nucleotide sequence III is completely reverse complementary to the nucleotide sequence IV. Thus, if the base of the nucleotide sequence III is proposed, the base of the nucleotide sequence IV is also determined.

[0152] The fourth miRNA

[0153] According to the present invention, the siRNA may be a fourth siRNA.

[0154] The fourth siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 181 and differs therefrom by no more than 3 nucleotides, and the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 182 and differs therefrom by no more than 3 nucleotides:

[0155] 5'-GCAACAAAGACAUUUUAUGZ 13 -3' (SEQ ID NO: 181);

[0156] 5'-Z 14CAUAAAUGUCUUUGUGC-3' (SEQ ID NO: 182),

[0157] where Z 13 represents U, and Z 14 represents A, and

[0158] nucleotide sequence I contains nucleotide Z 15 in the position corresponding to Z 13 ; nucleotide sequence II contains nucleotide Z 16 in the position corresponding to Z 14 , where the nucleotide is Z 16 represents the first nucleotide from the 5' end of the antisense strand.

[0159] According to some embodiments of the present invention, the sense strand comprises only the nucleotide sequence I, and the antisense strand comprises only the nucleotide sequence II.

[0160] According to some embodiments of the present invention, nucleotide sequence I contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 181, and / or nucleotide sequence II contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 182.

[0161] According to some embodiments of the present invention, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 182 comprises a difference in the position of Z 16 , and Z 16 selected from U, C or G. In some embodiments of the present invention, the nucleotide difference is a difference in the position of Z 16 , and Z 16 selected from U, C or G. According to some embodiments of the present invention, Z 15 is a nucleotide complementary to Z 16. The miRNAs containing the above-mentioned nucleotide differences also have a high inhibitory ability towards the target mRNA, and such miRNAs containing nucleotide differences are also included within the scope of the present invention.

[0162] According to some embodiments of the present invention, the nucleotide sequence I is predominantly reverse complementary, substantially reverse complementary, or completely reverse complementary to the nucleotide sequence II.

[0163] According to some embodiments of the present invention, nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 183, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 184:

[0164] 5'-GCAACAAAGACAUUUUUAUGZ 15 -3' (SEQ ID NO: 183);

[0165] 5'-Z 16 CAUAAAUGUCUUUGUGC-3' (SEQ ID NO: 184),

[0166] in which Z 16 represents the first nucleotide from the 5' end of the antisense strand, Z 15 selected from A, U, G or C, and Z 16 is a nucleotide complementary to Z 15 ; in some embodiments of Z 15 represents U, and Z 16 represents A.

[0167] In addition, the sense strand and the antisense strand have the same or different lengths, with the sense strand containing 19-23 nucleotides in length and the antisense strand containing 19-26 nucleotides in length.

[0168] According to some embodiments of the present invention, said sense strand further comprises a nucleotide sequence III, wherein said antisense strand further comprises a nucleotide sequence IV, and said nucleotide sequence III and said nucleotide sequence IV independently have a length of from 1 to 4 nucleotides; said nucleotide sequence III and said nucleotide sequence IV have the same length and are substantially reverse complementary or completely reverse complementary; said nucleotide sequence III is connected to the 5'-end of nucleotide sequence I, and said nucleotide sequence IV is connected to the 3'-end of nucleotide sequence II;said nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which refers to a nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence of the target mRNA presented in SEQ ID NO: 181 and has the same length as said nucleotide sequence IV.

[0169] According to some embodiments of the present invention, in the direction from the 5' end to the 3' end, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 20 / 20; or, the nucleotide sequence III and the nucleotide sequence IV are both 2 nucleotides in length, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is UU, and the base composition of the nucleotide sequence IV is AA; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21;or, the nucleotide sequence III and the nucleotide sequence IV are both 3 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is CUU, and the base composition of the nucleotide sequence IV is AAG; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 22 / 22; or, the nucleotide sequence III and the nucleotide sequence IV are both 4 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is GCUU, and the base composition of the nucleotide sequence IV is AAGC;in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23 / 23. According to some embodiments of the present invention, the nucleotide sequence III and the nucleotide sequence IV have a length of 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is UU, and the base composition of the nucleotide sequence IV is AA; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21.

[0170] According to some embodiments of the present invention, the nucleotide sequence III is completely reverse complementary to the nucleotide sequence IV. Thus, if the base of the nucleotide sequence III is proposed, the base of the nucleotide sequence IV is also determined.

[0171] The fifth miRNA

[0172] According to the present invention, the siRNA may be a fifth siRNA.

[0173] The fifth siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 241 and differs therefrom by no more than 3 nucleotides, and the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 242 and differs therefrom by no more than 3 nucleotides:

[0174] 5''-GAAUCUCUCAAAGAAAUCUUZ 17 -3' (SEQ ID NO: 241);

[0175] 5'-Z 18 AAGAUUUUCUUUUUGAGAUUC-3' (SEQ ID NO: 242),

[0176] where Z 17 represents U, and Z 18 represents A, and

[0177] nucleotide sequence I contains nucleotide Z 19 in the position corresponding to Z 17 ; nucleotide sequence II contains nucleotide Z 20 in the position corresponding to Z 18 , where the nucleotide is Z 20 represents the first nucleotide from the 5' end of the antisense strand.

[0178] According to some embodiments of the present invention, the sense strand comprises only the nucleotide sequence I, and the antisense strand comprises only the nucleotide sequence II.

[0179] According to some embodiments of the present invention, nucleotide sequence I contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 241, and / or nucleotide sequence II contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 242.

[0180] According to some embodiments of the present invention, the difference in nucleotides between said nucleotide sequence II and said nucleotide sequence according to SEQ ID NO: 242 comprises a difference in the Z position 20 , and Z 20 selected from U, C or G. In some embodiments of the present invention, the nucleotide difference is a difference in the position of Z 20 , and Z 20 selected from U, C or G. According to some embodiments of the present invention, Z 19 is a nucleotide complementary to Z20 . The miRNAs containing the above-mentioned nucleotide differences also have a high inhibitory ability towards the target mRNA, and such miRNAs containing nucleotide differences are also included within the scope of the present invention.

[0181] According to some embodiments of the present invention, the nucleotide sequence I is predominantly reverse complementary, substantially reverse complementary, or completely reverse complementary to the nucleotide sequence II.

[0182] According to some embodiments of the present invention, nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 243, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 244:

[0183] 5'-GAAUCUCUCAAAGAAAUCUUZ 19 -3' (SEQ ID NO: 243);

[0184] 5'-Z 20 AAGAUUUCUUUUGAGAUUC-3' (SEQ ID NO: 244),

[0185] in which Z 20 represents the first nucleotide from the 5' end of the antisense strand, Z 19 selected from A, U, G or C, and Z 20 is a nucleotide complementary to Z 19 ; in some embodiments of Z 19 represents U, and Z 20 represents A.

[0186] In addition, the sense strand and the antisense strand have the same or different lengths, with the sense strand containing 19-23 nucleotides in length and the antisense strand containing 19-26 nucleotides in length.

[0187] In some embodiments of the present invention, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV independently have a length of 1 to 4 nucleotides; the nucleotide sequence III and the nucleotide sequence IV have the same length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II;the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which refers to a nucleotide sequence that is connected to the 5' end of the nucleotide sequence of the target mRNA presented in SEQ ID NO: 241 and has the same length as the nucleotide sequence IV.

[0188] According to some embodiments of the present invention, in the direction from the 5' end to the 3' end, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is U; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 20 / 20; or, the nucleotide sequence III and the nucleotide sequence IV are both 2 nucleotides in length, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is AA, and the base composition of the nucleotide sequence IV is UU; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21;or, the nucleotide sequence III and the nucleotide sequence IV are both 3 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is AAA, and the base composition of the nucleotide sequence IV is UUU; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 22 / 22; or, the nucleotide sequence III and the nucleotide sequence IV are both 4 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is CAAA, and the base composition of the nucleotide sequence IV is UUUG;in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23 / 23. According to some embodiments of the present invention, the nucleotide sequence III and the nucleotide sequence IV have a length of 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is AA, and the base composition of the nucleotide sequence IV is UU; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21.

[0189] According to some embodiments of the present invention, the nucleotide sequence III is completely reverse complementary to the nucleotide sequence IV. Thus, if the base of the nucleotide sequence III is proposed, the base of the nucleotide sequence IV is also determined.

[0190] The sixth miRNA

[0191] According to the present invention, the siRNA may be the sixth siRNA.

[0192] The sixth siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 301 and differs therefrom by no more than 3 nucleotides, and the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 302 and differs therefrom by no more than 3 nucleotides:

[0193] 5'-GUACGUGGGACUGGAUUCUZ 21 -3' (SEQ ID NO: 301);

[0194] 5'-Z 22 AGAAUCCAGUCCACGUAC-3' (SEQ ID NO: 302),

[0195] in which Z 21 represents G, and Z 22 represents C, and

[0196] nucleotide sequence I contains nucleotide Z 23 in the position corresponding to Z 21 ; nucleotide sequence II contains nucleotide Z 24 in the position corresponding to Z 22 , where Z 24 represents the first nucleotide from the 5' end of the antisense strand.

[0197] According to some embodiments of the present invention, the sense strand comprises only the nucleotide sequence I, and the antisense strand comprises only the nucleotide sequence II.

[0198] According to some embodiments of the present invention, nucleotide sequence I contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 301, and / or nucleotide sequence II contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 302.

[0199] According to some embodiments of the present invention, the difference in nucleotides between said nucleotide sequence II and said nucleotide sequence according to SEQ ID NO: 302 comprises a difference in the Z position 24 , and Z 24 selected from U, G or A. In some embodiments of the present invention, the nucleotide difference is a difference in the position of Z 24 , and Z 24 selected from U, G or A. According to some embodiments of the present invention, Z 23 is a nucleotide complementary to Z24 . The miRNAs containing the above-mentioned nucleotide differences also have a high inhibitory ability towards the target mRNA, and such miRNAs containing nucleotide differences are also included within the scope of the present invention.

[0200] According to some embodiments of the present invention, the nucleotide sequence I is predominantly reverse complementary, substantially reverse complementary, or completely reverse complementary to the nucleotide sequence II.

[0201] According to some embodiments of the present invention, nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 303, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 304:

[0202] 5'-GUACGUGGGACUGGAUUCUZ 23 -3' (SEQ ID NO: 303);

[0203] 5'-Z 24 AGAAUCCAGUCCACGUAC-3' (SEQ ID NO: 304),

[0204] in which Z 24 represents the first nucleotide from the 5' end of the antisense strand, Z 23 selected from A, U, G or C, and Z 24 is a nucleotide complementary to Z 23 ; in some embodiments of the invention Z 23 represents G, and Z 24 represents C.

[0205] In addition, the sense strand and the antisense strand have the same or different lengths, with the sense strand containing 19-23 nucleotides in length and the antisense strand containing 19-26 nucleotides in length.

[0206] In some embodiments of the present invention, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV independently have a length of 1 to 4 nucleotides; the nucleotide sequence III and the nucleotide sequence IV have the same length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II;the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the second nucleotide sequence, which refers to a nucleotide sequence that is connected to the 5' end of the nucleotide sequence of the target mRNA presented in SEQ ID NO: 301 and has the same length as the nucleotide sequence IV.

[0207] According to some embodiments of the present invention, in the direction from the 5' end to the 3' end, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is U; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 20 / 20; or, the nucleotide sequence III and the nucleotide sequence IV are both 2 nucleotides in length, and, in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is GA, and the base composition of the nucleotide sequence IV is UC; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21;or, the nucleotide sequence III and the nucleotide sequence IV are both 3 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is CGA, and the base composition of the nucleotide sequence IV is UCG; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 22 / 22; or, the nucleotide sequence III and the nucleotide sequence IV are both 4 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is UCGA, and the base composition of the nucleotide sequence IV is UCGA;in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23 / 23. According to some embodiments of the present invention, the nucleotide sequence III and the nucleotide sequence IV have a length of 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is GA, and the base composition of the nucleotide sequence IV is UC; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21.

[0208] According to some embodiments of the present invention, the nucleotide sequence III is completely reverse complementary to the nucleotide sequence IV. Thus, if the base of the nucleotide sequence III is proposed, the base of the nucleotide sequence IV is also determined.

[0209] The seventh miRNA

[0210] According to the present invention, the siRNA may be the seventh siRNA.

[0211] The seventh siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; nucleotide sequence I and nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 361 and differs therefrom by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 362 and differs therefrom by no more than 3 nucleotides:

[0212] 5'-AUUUCUGGGUAUUUCUUUCZ 25 -3' (SEQ ID NO: 361);

[0213] 5'-Z 26 GAAAGAAUACCCAGAAAU-3' (SEQ ID NO: 362),

[0214] where Z 25 represents A and Z 26 represents U, and

[0215] Nucleotide sequence I contains nucleotide Z 27 in the position corresponding to Z 25 ; nucleotide sequence II contains nucleotide Z 28 in the position corresponding to Z 26 , where the nucleotide is Z 28 represents the first nucleotide from the 5' end of the antisense strand.

[0216] According to some embodiments of the present invention, the sense strand comprises only the nucleotide sequence I, and the antisense strand comprises only the nucleotide sequence II.

[0217] According to some embodiments of the present invention, nucleotide sequence I contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 361, and / or nucleotide sequence II contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 362.

[0218] According to some embodiments of the present invention, the nucleotide difference between said nucleotide sequence II and said nucleotide sequence presented in SEQ ID NO: 362 comprises a difference in the Z position 28 , and Z 28 selected from A, C or G. In some embodiments of the present invention, the nucleotide difference is a difference in the position of Z 28 , and Z 28 selected from A, C or G. According to some embodiments of the present invention, Z 27 is a nucleotide complementary to Z28 . The miRNAs containing the above-mentioned nucleotide differences also have a high inhibitory ability towards the target mRNA, and such miRNAs containing nucleotide differences are also included within the scope of the present invention.

[0219] According to some embodiments of the present invention, the nucleotide sequence I is predominantly reverse complementary, substantially reverse complementary, or completely reverse complementary to the nucleotide sequence II.

[0220] According to some embodiments of the present invention, nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 363, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 364:

[0221] 5'-AUUUCUGGGUAUUUCUUUCZ 27 -3' (SEQ ID NO: 363);

[0222] 5'-Z 28 GAAAGAAUACCCAGAAAU-3' (SEQ ID NO: 364),

[0223] in which Z 28 represents the first nucleotide from the 5' end of the antisense strand, Z 27 selected from A, U, G or C, and Z 28 is a nucleotide complementary to Z 27 ; in some embodiments of Z 27 represents A and Z 28 represents U.

[0224] In addition, the sense strand and the antisense strand have the same or different lengths, with the sense strand containing 19-23 nucleotides in length and the antisense strand containing 19-26 nucleotides in length.

[0225] In some embodiments of the present invention, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV independently have a length of 1 to 4 nucleotides; the nucleotide sequence III and the nucleotide sequence IV have the same length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I; and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II.According to some embodiments of the present invention, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which refers to a nucleotide sequence that is connected to the 5' end of the nucleotide sequence of the target mRNA presented in SEQ ID NO: 361 and has the same length as the nucleotide sequence IV.

[0226] According to some embodiments of the present invention, in the direction from the 5' end to the 3' end, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is G and the base of the nucleotide sequence IV is C; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 20 / 20; or, the nucleotide sequence III and the nucleotide sequence IV are both 2 nucleotides in length, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is CG and the base composition of the nucleotide sequence IV is CG; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21;or, the nucleotide sequence III and the nucleotide sequence IV are both 3 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is GCG, and the base composition of the nucleotide sequence IV is CGC; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 22 / 22; or, the nucleotide sequence III and the nucleotide sequence IV are both 4 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is AGCG, and the base composition of the nucleotide sequence IV is CGCU;in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23 / 23. According to some embodiments of the present invention, the nucleotide sequence III and the nucleotide sequence IV have a length of 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is CG, and the base composition of the nucleotide sequence IV is CG; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21.

[0227] According to some embodiments of the present invention, the nucleotide sequence III is completely reverse complementary to the nucleotide sequence IV. Thus, if the base of the nucleotide sequence III is proposed, the base of the nucleotide sequence IV is also determined.

[0228] The eighth miRNA

[0229] According to the present invention, the siRNA may be the eighth siRNA.

[0230] The eighth siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 421 and differs therefrom by no more than 3 nucleotides, and the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 422 and differs therefrom by no more than 3 nucleotides:

[0231] 5'-CAUGAAGGGCAUAAACUAZ 29 -3' (SEQ ID NO: 421);

[0232] 5'-Z 30 UAGUUUAUGCCCUUCAUG-3' (SEQ ID NO: 422),

[0233] where Z 29 represents U, and Z 30 represents A, and

[0234] nucleotide sequence I contains nucleotide Z 31 in the position corresponding to Z 29 ; nucleotide sequence II contains nucleotide Z 32 in the position corresponding to Z 30 , where the nucleotide is Z 32 represents the first nucleotide from the 5' end of the antisense strand.

[0235] According to some embodiments of the present invention, the sense strand comprises only the nucleotide sequence I, and the antisense strand comprises only the nucleotide sequence II.

[0236] According to some embodiments of the present invention, nucleotide sequence I contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 421, and / or nucleotide sequence II contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 422.

[0237] According to some embodiments of the present invention, the difference in nucleotides between said nucleotide sequence II and said nucleotide sequence according to SEQ ID NO: 422 comprises a difference in the Z position 32 , and Z 32 selected from U, C or G. In some embodiments of the present invention, the nucleotide difference is a difference in the position of Z 32 , and Z 32 selected from U, C or G. According to some embodiments of the present invention, Z 31 is a nucleotide complementary to Z32 . The miRNAs containing the above-mentioned nucleotide differences also have a high inhibitory ability towards the target mRNA, and such miRNAs containing nucleotide differences are also included within the scope of the present invention.

[0238] According to some embodiments of the present invention, the nucleotide sequence I is predominantly reverse complementary, substantially reverse complementary, or completely reverse complementary to the nucleotide sequence II.

[0239] According to some embodiments of the present invention, nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 423, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 424:

[0240] 5'-CAUGAAGGGGCAUAAACUAZ 31 -3' (SEQ ID NO: 423);

[0241] 5'-Z 32 UAGUUUUAUGCCCUUCAUG-3' (SEQ ID NO: 424),

[0242] in which Z 32 represents the first nucleotide from the 5' end of the antisense strand, Z 31 selected from A, U, G or C, and Z 32 is a nucleotide complementary to Z 31 ; in some embodiments of Z 31 represents U, and Z 32 represents A.

[0243] In addition, the sense strand and the antisense strand have the same or different lengths, with the sense strand containing 19-23 nucleotides in length and the antisense strand containing 19-26 nucleotides in length.

[0244] In some embodiments of the present invention, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV independently have a length of 1 to 4 nucleotides; the nucleotide sequence III and the nucleotide sequence IV have the same length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I; and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II.According to some embodiments of the present invention, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which refers to a nucleotide sequence that is connected to the 5' end of the nucleotide sequence of the target mRNA presented in SEQ ID NO: 421 and has the same length as the nucleotide sequence IV.

[0245] According to some embodiments of the present invention, in the direction from the 5' end to the 3' end, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is A and the base of the nucleotide sequence IV is U; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 20 / 20; or, the nucleotide sequence III and the nucleotide sequence IV are both 2 nucleotides in length, and, in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is GA and the base composition of the nucleotide sequence IV is UC; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21;or, the nucleotide sequence III and the nucleotide sequence IV are both 3 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is AGA, and the base composition of the nucleotide sequence IV is UCU; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 22 / 22; or, the nucleotide sequence III and the nucleotide sequence IV are both 4 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is UAGA, and the base composition of the nucleotide sequence IV is UCUA;in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23 / 23. According to some embodiments of the present invention, the nucleotide sequence III and the nucleotide sequence IV have a length of 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is GA, and the base composition of the nucleotide sequence IV is UC; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21.

[0246] According to some embodiments of the present invention, the nucleotide sequence III is completely reverse complementary to the nucleotide sequence IV. Thus, if the base of the nucleotide sequence III is proposed, the base of the nucleotide sequence IV is also determined.

[0247] The ninth miRNA

[0248] According to the present invention, the miRNA may be the ninth miRNA.

[0249] The ninth siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 481 and differs therefrom by no more than 3 nucleotides, and the nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 482 and differs therefrom by no more than 3 nucleotides:

[0250] 5'-GGAUUCUGGAGAAAACUCZ 33 -3' (SEQ ID NO: 481);

[0251] 5'-Z 34GAGUUUUUCUCCAGAAUCC-3' (SEQ ID NO: 482),

[0252] where Z 33 represents A and Z 34 represents U, and

[0253] nucleotide sequence I contains nucleotide Z 35 in the position corresponding to Z 33 ; nucleotide sequence II contains nucleotide Z 36 in the position corresponding to Z 34 , where the nucleotide is Z 36 represents the first nucleotide from the 5' end of the antisense strand.

[0254] According to some embodiments of the present invention, the sense strand comprises only the nucleotide sequence I, and the antisense strand comprises only the nucleotide sequence II.

[0255] According to some embodiments of the present invention, nucleotide sequence I contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 481, and / or nucleotide sequence II contains no more than 1 nucleotide different from the nucleotide sequence presented in SEQ ID NO: 482.

[0256] According to some embodiments of the present invention, the difference in nucleotides between said nucleotide sequence II and said nucleotide sequence presented in SEQ ID NO: 482 comprises a difference in the position of Z 36 , and Z 36 selected from A, C or G. In some embodiments of the present invention, the nucleotide difference is a difference in the position of Z 36 , and Z 36 selected from A, C or G. According to some embodiments of the present invention, Z 35 is a nucleotide complementary to Z36 . The miRNAs containing the above-mentioned nucleotide differences also have a high inhibitory ability towards the target mRNA, and such miRNAs containing nucleotide differences are also included within the scope of the present invention.

[0257] According to some embodiments of the present invention, the nucleotide sequence I is predominantly reverse complementary, substantially reverse complementary, or completely reverse complementary to the nucleotide sequence II.

[0258] According to some embodiments of the present invention, nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 483, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 484:

[0259] 5'-GGAUUCUGGAGAAAACUCZ 35 -3' (SEQ ID NO: 483);

[0260] 5'-Z 36 GAGUUUUUCUCCAGAAUCC-3' (SEQ ID NO: 484),

[0261] in which Z 36 represents the first nucleotide from the 5' end of the antisense strand, Z 35 selected from A, U, G or C, and Z 36 is a nucleotide complementary to Z 35 ; in some embodiments of Z 35 represents A and Z 36 represents U.

[0262] In addition, the sense strand and the antisense strand have the same or different lengths, with the sense strand containing 19-23 nucleotides in length and the antisense strand containing 19-26 nucleotides in length.

[0263] According to some embodiments of the present invention, said sense strand further comprises a nucleotide sequence III, wherein said antisense strand further comprises a nucleotide sequence IV, and said nucleotide sequence III and said nucleotide sequence IV independently have a length of from 1 to 4 nucleotides; said nucleotide sequence III and said nucleotide sequence IV have the same length and are substantially reverse complementary or completely reverse complementary; said nucleotide sequence III is connected to the 5'-end of said nucleotide sequence I, and said nucleotide sequence IV is connected to the 3'-end of said nucleotide sequence II.According to some embodiments of the present invention, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which refers to a nucleotide sequence that is connected to the 5' end of the nucleotide sequence of the target mRNA presented in SEQ ID NO: 481 and has the same length as the nucleotide sequence IV.

[0264] According to some embodiments of the present invention, in the direction from the 5' end to the 3' end, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 20 / 20; or, the nucleotide sequence III and the nucleotide sequence IV are both 2 nucleotides in length, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is CU, and the base composition of the nucleotide sequence IV is AG; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21;or, the nucleotide sequence III and the nucleotide sequence IV are both 3 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is ACU, and the base composition of the nucleotide sequence IV is AGU; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 22 / 22; or, the nucleotide sequence III and the nucleotide sequence IV are both 4 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is GACU, and the base composition of the nucleotide sequence IV is AGUC;in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23 / 23. According to some embodiments of the present invention, the nucleotide sequence III and the nucleotide sequence IV have a length of 2 nucleotides, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is CU, and the base composition of the nucleotide sequence IV is AG; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21 / 21.

[0265] According to some embodiments of the present invention, the nucleotide sequence III is completely reverse complementary to the nucleotide sequence IV. Thus, if the base of the nucleotide sequence III is proposed, the base of the nucleotide sequence IV is also determined.

[0266] The following description regarding the nucleotide sequence V, the nucleic acid sequence or the nucleotide modification, and the modified sequence of the siRNA applies to any of the above-mentioned first to ninth siRNAs. Specifically, unless otherwise specified, the following description of the siRNAs should be regarded as a description of the first, second, third, fourth, fifth, sixth, seventh, eighth, and ninth siRNAs in turn. For example, if a specific siRNA is not specified, the expression "the siRNA further comprises the nucleotide sequence V" means that "the first siRNA, the second siRNA, the third siRNA, the fourth siRNA, the fifth siRNA, the sixth siRNA, the seventh siRNA, the eighth siRNA, and the ninth siRNA further comprise the nucleotide sequence V."

[0267] According to some embodiments of the present invention, the antisense strand further comprises a nucleotide sequence V. The nucleotide sequence V has a length of 1 to 3 nucleotides and is connected to the 3' end of the antisense strand to form a 3' overhang of the antisense strand. In this case, the ratio of the length of the sense strand to the length of the antisense strand in the siRNA according to the present invention may 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. According to some embodiments of the present invention, the nucleotide sequence V comprises 2 nucleotides in length. In this case, the ratio of the length of the sense strand to the length of the antisense strand of the siRNA according to the present invention may be 19 / 21, 21 / 23, or 23 / 25.

[0268] Each nucleotide in the nucleotide sequence V can be any nucleotide. To facilitate and reduce the cost of synthesis, the nucleotide sequence V is 2 consecutive thymine deoxyribonucleotides (dTdT) or 2 consecutive uracil ribonucleotides (UU); or, to increase the affinity between the antisense strand of the siRNA and the target mRNA, the nucleotide sequence V is complementary to the nucleotides at the corresponding positions of the target mRNA. Thus, according to some embodiments of the present invention, the ratio of the length of the sense strand to the length of the antisense strand of the siRNA according to the present invention is 19 / 21 or 21 / 23. In this case, the siRNA according to the present invention exhibits better suppressive activity against the target mRNA.

[0269] The nucleotides at the corresponding positions of the target mRNA refer to the nucleotides or nucleotide sequence that are connected to the 5' end of the nucleotide sequence segment of the target mRNA. This nucleotide sequence segment of the target mRNA refers to a nucleotide sequence segment that is substantially reverse complementary or completely reverse complementary to nucleotide sequence II, or substantially reverse complementary or completely reverse complementary to a nucleotide sequence consisting of nucleotide sequence II and nucleotide sequence IV.

[0270] According to some embodiments of the present invention, with respect to the first siRNA, the sense strand of the siRNA comprises the nucleotide sequence presented in SEQ ID NO: 5, and the antisense strand of the siRNA comprises the nucleotide sequence presented in SEQ ID NO: 6:

[0271] 5'-GGGGUAUUUCUUUCAAGCAAZ3-3' (SEQ ID NO: 5);

[0272] 5'-Z4UUGCUUGAAAGAAUACCCAG-3' (SEQ ID NO: 6);

[0273] or the sense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 8:

[0274] 5'-CUGGGGUAUUUCUUUCAAGCAAZ3-3' (SEQ ID NO: 7);

[0275] 5'-Z4UUGCUUGAAAGAAUACCCAGAA-3' (SEQ ID NO: 8);

[0276] wherein Z4 is the first nucleotide from the 5'-terminus of said antisense strand; Z3 is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3.

[0277] According to some embodiments, with respect to the second siRNA, the sense strand of the siRNA comprises the nucleotide sequence presented in SEQ ID NO: 65, and the antisense strand of the siRNA comprises the nucleotide sequence presented in SEQ ID NO: 66:

[0278] 5'-GGCAUAAACUAUAACAGCZ7-3' (SEQ ID NO: 65);

[0279] 5'-Z8GCUGUUAUAGUUUAUGCCCU-3' (SEQ ID NO: 66),

[0280] or the sense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 67, and the antisense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 68:

[0281] 5'-AGGGCAUAAACUAUAACAGCZ7-3' (SEQ ID NO: 67);

[0282] 5'-Z8GCUGUUAUAGUUUAUGCCCUUC-3' (SEQ ID NO: 68),

[0283] wherein Z8 is the first nucleotide from the 5'-end of said antisense strand; Z7 is selected from A, U, G or C, and Z8 is a nucleotide complementary to Z7.

[0284] According to some embodiments of the present invention, with respect to the third siRNA, the sense strand of the siRNA comprises the nucleotide sequence presented in SEQ ID NO: 125, and the antisense strand of the siRNA comprises the nucleotide sequence presented in SEQ ID NO: 126:

[0285] 5'-GCUCAAGAAUGCCAAGAZ 11 -3' (SEQ ID NO: 125);

[0286] 5'-Z 12 UUCUUUGGCAUUUCUUUGAGCAC-3' (SEQ ID NO: 126),

[0287] or the sense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 127, and the antisense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 128:

[0288] 5'-GUGCUCAAGAAUGCCAAGAZn-3' (SEQ ID NO: 127); 5'-Zi2UUCUUGGCAUUCUUGAGCACUC-3' (SEQ ID NO: 128),

[0289] in which Z 12 represents the first nucleotide from the 5' end of the specified antisense strand; Z 11 selected from A, U, G or C, and Z 12 is a nucleotide complementary to Z 11 .

[0290] According to some embodiments of the present invention, with respect to the fourth miRNA, the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 185, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 186:

[0291] 5'-GCAACAAAGACAUUUUUAUGZ15 -3' (SEQ ID NO: 185);

[0292] 5'-Z 16 CAUAAAUGUCUUUUUGUUGCAA-3' (SEQ ID NO: 186),

[0293] or the sense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 187, and the antisense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 188:

[0294] 5'-UUGCAACAAAAGACAUUUUAUUGZ 15 -3' (SEQ ID NO: 187);

[0295] 5'-Z 16 CAUAAAUGUCUUUGUGCAAGC-3' (SEQ ID NO: 188),

[0296] in which Z 16 represents the first nucleotide from the 5' end of the specified antisense strand; Z 15 selected from A, U, G or C, and Z 16 is a nucleotide complementary to Z 15 .

[0297] According to some embodiments of the present invention, with respect to the fifth siRNA, the sense strand of the siRNA comprises the nucleotide sequence presented in SEQ ID NO: 245, and the antisense strand of the siRNA comprises the nucleotide sequence presented in SEQ ID NO: 246:

[0298] 5'-GAAUCUCUCAAAGAAAUCUUZ 19 -3' (SEQ ID NO: 245);

[0299] 5'-Z 20 AAGAUUUCUUUUGAGAUUCUU-3' (SEQ ID NO: 246),

[0300] or the sense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 247, and the antisense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 248:

[0301] 5'-AAGAAUCUCAAAGAAAUCUUZ 19 -3' (SEQ ID NO: 247);

[0302] 5'-Z 20 AAGAUUUUCUUUUUGAGAUUCUUUUG-3' (SEQ ID NO: 248),

[0303] in which Z 20 represents the first nucleotide from the 5' end of the specified antisense strand; Z 19 selected from A, U, G or C, and Z20 is a nucleotide complementary to Z 19 .

[0304] According to some embodiments of the present invention, with respect to the sixth miRNA, the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 305, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 306:

[0305] 5'-GUACGUGGGACUGGAUUCUZ 23 -3' (SEQ ID NO: 305);

[0306] 5'-Z 24 AGAAUCCAGUCCACGUACUC-3' (SEQ ID NO: 306),

[0307] or the sense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 307, and the antisense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 308:

[0308] 5'-GAGUACGUGGACUGGAUUCUZ 23 -3' (SEQ ID NO: 307);

[0309] 5'-Z 24 AGAAUCCAGUCCACGUACUCGA-3' (SEQ ID NO: 308),

[0310] in which Z 24represents the first nucleotide from the 5' end of the specified antisense strand; Z 23 selected from A, U, G or C, and Z 24 is a nucleotide complementary to Z 23 .

[0311] According to some embodiments of the present invention, with respect to the seventh miRNA, the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 365, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 366:

[0312] 5'-AUUUUCUGGGGUUAUUUUCUUUCZ 27 -3' (SEQ ID NO: 365);

[0313] 5'-Z 28 GAAAGAAUACCCAGAAAUCG-3' (SEQ ID NO: 366),

[0314] or the sense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 367, and the antisense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 368:

[0315] 5'-CGAUUUUCUGGGGGUAUUUUCUUUCZ 27 -3'' (SEQ ID NO: 367);

[0316] 5'-Z 28GAAAGAAUACCCAGAAAUCGCU-3' (SEQ ID NO: 368),

[0317] in which Z 28 represents the first nucleotide from the 5' end of the specified antisense strand; Z 27 selected from A, U, G or C, and Z 28 is a nucleotide complementary to Z 27 .

[0318] According to some embodiments of the present invention, with respect to the eighth siRNA, the sense strand of the siRNA comprises the nucleotide sequence presented in SEQ ID NO: 425, and the antisense strand of the siRNA comprises the nucleotide sequence presented in SEQ ID NO: 426:

[0319] 5'-CAUGAAGGGGCAUAAACUAZ 31 -3' (SEQ ID NO: 425);

[0320] 5'-Z 32 UAGUUUUAUGCCCUUCAUGUC-3' (SEQ ID NO: 426),

[0321] or the sense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 427, and the antisense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 428:

[0322] 5'-GACAUGAAGGGGCAUAAACUAZ 31 -3' (SEQ ID NO: 427);

[0323] 5'-Z 32 UAGUUUUAUGCCCUUCAUGUCUAUGUCUA-3' (SEQ ID NO: 428),

[0324] in which Z 32 represents the first nucleotide from the 5' end of the specified antisense strand; Z 31 selected from A, U, G or C, and Z 32 is a nucleotide complementary to Z 31 .

[0325] According to some embodiments of the present invention, with respect to the ninth miRNA, the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 485, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 486:

[0326] 5'-GGAUUCUGGAGAAAACUCZ 35 -3' (SEQ ID NO: 485);

[0327] 5'-Z 36 GAGUUUUUCUCCAGAAUCCAG-3' (SEQ ID NO: 486),

[0328] or the sense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 487, and the antisense strand of the miRNA comprises the nucleotide sequence shown in SEQ ID NO: 488:

[0329] 5'-CUGGAUUCUGGAGAAAACUCZ 35 -3' (SEQ ID NO: 487);

[0330] 5'-Z 36 GAGUUUUUCUCCAGAAUCCAGUC-3' (SEQ ID NO: 488),

[0331] in which Z 36 represents the first nucleotide from the 5' end of the specified antisense strand; Z 35 selected from A, U, G or C, and Z 36 is a nucleotide complementary to Z 35 .

[0332] According to some embodiments of the present invention, the siRNA of the present invention is siFXIa1, siFXIa2, siFXIb1, siFXIb2, siFXIc1, siFXIc2, siFXId1, siFXId2, siFXIe1, siFXIe2, siFXIf1, siFXIf2, siFXIg1, siFXIg2, siFXIh1, siFXIh2, siFXIi1 or siFXIi2, listed in Tables 1a-1i.

[0333] As mentioned above, in the siRNA of the present invention, each nucleotide independently represents a modified or unmodified nucleotide. In some embodiments of the present invention, a nucleotide in the siRNA of the present invention is an unmodified nucleotide; in some embodiments of the present invention, some or all nucleotides in the siRNA of the present invention are modified nucleotides. Such modifications on nucleotide groups will not cause a significant reduction or loss of the functions of the siRNA conjugate of the present invention for inhibiting the expression of the FXI gene.

[0334] According to some embodiments of the present invention, the siRNA of the present invention comprises at least 1 modified nucleotide. As used herein, the term “modified nucleotide” refers to a nucleotide formed by replacing the 2'-hydroxyl of the ribose group with other groups, or a nucleotide analog, or a nucleotide with a modified base. The modified nucleotide will not cause a significant reduction or loss of the functions of the siRNA in inhibiting gene expression. For example, the modified nucleotides disclosed in JK Watts, GF Deleavey and MJ Damha, Chemically Modified siRNA: tools and applications, DrugDiscov Today, 2008.13(19-20): p. 842-55 can be selected.

[0335] According to some embodiments of the present invention, at least one nucleotide in the sense or antisense strand of the siRNA according to the present invention is a modified nucleotide and / or at least one phosphate group is a phosphate group with a modified group(s). In other words, at least a portion of the phosphate and / or ribose groups in the phosphate-ribose backbone of at least one single strand in the sense strand and the antisense strand are phosphate and / or ribose groups with modified groups.

[0336] According to some embodiments of the present invention, all nucleotides in the sense strand and / or antisense strand are modified nucleotides. According to some embodiments of the present invention, each nucleotide in the sense strand and antisense strand of the siRNA according to the present invention is independently a fluorine-modified nucleotide or a nucleotide with a non-fluorine modification.

[0337] The present inventors unexpectedly found that the siRNAs of the present invention achieve a high balance between plasma stability and gene silencing efficiency in animal experiments.

[0338] According to some embodiments, said fluorine-modified nucleotides are located within nucleotide sequences I and II. Furthermore, in the 5'-to-3'-direction, at least the nucleotides at positions 7, 8, and 9 of nucleotide sequence I are fluorine-modified nucleotides; and, in the 5'-to-3'-direction, at least the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II are fluorine-modified nucleotides.

[0339] According to some embodiments of the present invention, fluorine-modified nucleotides are located within nucleotide sequences I and II; and no more than 5 fluorine-modified nucleotides are present in nucleotide sequence I. Furthermore, in the direction from the 5'-end to the 3'-end, at least nucleotides at positions 7, 8 and 9 of said nucleotide sequence I are fluorine-modified nucleotides; no more than 7 fluorine-modified nucleotides are present in nucleotide sequence II; and at least nucleotides at positions 2, 6, 14 and 16 of said nucleotide sequence II are fluorine-modified nucleotides.

[0340] According to some embodiments of the present invention, in the 5'-to-3'-direction, the nucleotides at positions 7, 8, and 9, or at positions 5, 7, 8, and 9, of the nucleotide sequence I in the sense strand are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are nucleotides with a non-fluorine modification; in the 5'-to-3'-direction, the nucleotides at positions 2, 6, 14, and 16, or at positions 2, 6, 8, 9, 14, and 16, of the nucleotide sequence II in the antisense strand are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are nucleotides with a non-fluorine modification.

[0341] As used in the present invention, the term "fluorine-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl of the ribose group of a nucleotide with a fluorine atom, which has a structure represented by the following Formula (7). "A nucleotide with a non-fluorine modification" refers to a nucleotide formed by replacing the 2'-hydroxyl of the ribose group of a nucleotide with a group other than fluorine, or a nucleotide analog. According to some embodiments of the present invention, each nucleotide with a non-fluorine modification is independently selected from a nucleotide formed by replacing the 2'-hydroxyl of its ribose group with a group other than fluorine, or a nucleotide analog.

[0342] Nucleotides formed by substituting the 2'-hydroxyl of a ribose group with a group other than fluorine are well known to those skilled in the art and can be selected from the group consisting of 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides and 2'-deoxynucleotides.

[0343] According to some embodiments of the present invention, the 2'-alkoxy-modified nucleotide is a 2'-methoxy (2'-OMe)-modified nucleotide represented by Formula (8). According to some embodiments of the present invention, the 2'-substituted alkoxy-modified nucleotide is, for example, a 2'-methoxyethyl (2'-MOE)-modified nucleotide represented by Formula (9). According to some embodiments of the present invention, the 2'-amino- (2'-NH2)-modified nucleotide is one represented by Formula (10). According to some embodiments of the present invention, the 2'-deoxynucleotide (DNA) is one represented by Formula (11).

[0344]

[0345] A nucleotide analog refers to a group that can replace a nucleotide in a nucleic acid, but is structurally different from an adenine ribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine deoxyribonucleotide. In some embodiments of the present invention, the nucleotide analog may be an isonucleotide, a bridged nucleic acid nucleotide, or an acyclic nucleotide.

[0346] Bridged nucleic acid (BNA) refers to a limited or inaccessible nucleotide. BNA may comprise a 5-, 6-membered, or 7-membered ring bridge structure with a wrinkle of the "fixed" C3'-endo sugar. The bridge is typically inserted into the 2'- and 4'-positions of ribose to form a 2',4'-BNA nucleotide. According to some embodiments of the present invention, BNA may be LNA, ENA, cET BNA, etc., which are represented by Formulas (12), (13), and (14), respectively:

[0347]

[0348] An acyclic nucleotide refers to a class of nucleotides in which the sugar ring is unclosed. According to some embodiments of the present invention, the acyclic nucleotide may be an unlocked nucleic acid (UNA) or a glycerol-based nucleic acid (GNA), which are represented by Formulas (15) and (16), respectively:

[0349]

[0350] In the above formulas (15) and (16), R is selected from H, OH or alkoxy (O-alkyl).

[0351] Isonucleotide is a compound formed by changing the position of a base in the ribose ring of a nucleotide. According to some embodiments of the present invention, an isonucleotide may be a compound in which the base is moved from the -1' position to the -2' or -3' position in the ribose ring, represented by Formula (17) or (18), respectively.

[0352]

[0353] In the above compounds of formulas (17) to (18), the "base" is a nucleic acid base such as A, U, G, C or T; R is selected from H, OH, F or the above group other than fluorine.

[0354] According to some embodiments of the present invention, the nucleotide analog is an analog selected from the group consisting of an isonucleotide, LNA, ENA, cET, UNA, and GNA. According to some embodiments of the present invention, each nucleotide with a non-fluorine modification is a methoxy-modified nucleotide. For the purposes of the present invention, a methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl of a ribose group with a methoxy group.

[0355] As used herein, the terms "fluorine-modified nucleotide", "2'-fluoro-modified nucleotide", "a nucleotide in which the 2'-hydroxyl of the ribose group is substituted with a fluorine atom", and "2'-fluororibosyl" have the same meaning when referring to a compound in which the 2'-hydroxyl of the nucleotide is substituted with a fluorine atom, having a structure represented by Formula (7). "Methoxy-modified nucleotide", "2'-methoxy-modified nucleotide", "a nucleotide in which the 2'-hydroxyl of the ribose group is substituted with methoxy", and "nucleotide with 2'-methoxyribosyl" have the same meaning when referring to a compound in which the 2'-hydroxyl of the ribose group in the nucleotide is substituted with methoxy, having a structure represented by Formula (8).

[0356] According to some embodiments of the present invention, the siRNA of the present invention is an siRNA with the following modifications: in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 or at positions 5, 7, 8 and 9 of the nucleotide sequence I in the sense strand are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 or at positions 2, 6, 8, 9, 14 and 16 of the nucleotide sequence II in the antisense strand are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are methoxy-modified nucleotides.

[0357] According to some embodiments of the present invention, the siRNA of the present invention is an siRNA with the following modifications: in the 5'-to-3'-direction, the nucleotides at positions 5, 7, 8, and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides; and in the 5'-to-3'-direction, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are methoxy-modified nucleotides;

[0358] or, in the direction from the 5'-end to the 3'-end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides; and in the direction from the 5'-end to the 3'-end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are methoxy-modified nucleotides;

[0359] or, in 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 fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides; and in the direction from the 5'-end to the 3'-end, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are methoxy-modified nucleotides.

[0360] Download the phone from the phone There is a lot of information about this item. siFXIa1-M1, siFXIa1-M2, siFXIa1-M3, siFXIa2-M1, siFXIa2-M2, siFXIa2-M3, siFXIb1-M1, siFXIb1-M2, siFXIb1-M3, siFXIb2-M1, siFXIb2-M2, siFXIb2-M3, siFXIc1-M1, siFXIc1-M2, siFXIc1-M3, siFXIc2-M1, siFXIc2-M2, siFXIc2-M3, siFXId1-M1, siFXId1-M2, siFXId1-M3, siFXId2-M1, siFXId2-M2, siFXId2-M3, siFXIe1-M1, siFXIe1-M2, siFXIe1-M3, siFXIe2-M1, siFXIe2-M2, siFXIe2-M3, siFXIf1-M1, siFXIf1-M2, siFXIf1-M3, siFXIf2-M1 siFXIf2-M2, siFXIf2-M3, siFXIg1-M1, siFXIg1-M2, siFXIg1-M3, siFXIg2-M1, siFXIg2-M2, siFXIg2-M3, siFXIh1-M1, siFXIh1-M2, siFXIh1-M3, siFXIh2-M1, siFXIh2-M2, siFXIh2-M3, siFXIi1-M1, siFXIi1-M2, siFXIi1-M3, siFXIi2-M1, siFXIi2-M2 and siFXIi2-M3 перечисленных в Таблицах 1a-1i.

[0361] MiRNAs with the above modifications can be produced not only at a lower cost but also reduce the tendency of ribonucleases to cleave nucleic acid in the blood, thereby increasing nucleic acid stability and ensuring greater resistance to nuclease hydrolysis. Furthermore, siRNAs with the above modifications exhibit higher inhibitory activity against the target mRNA.

[0362] According to some embodiments of the present invention, at least a portion of the phosphate groups in the phosphate-ribose backbone of at least one single strand in the sense strand and the antisense strand of the siRNA of the present invention are phosphate groups with modified groups. According to some embodiments of the present invention, the phosphate group with modified group(s) is a phosphorothioate group formed by replacing at least one oxygen atom in a phosphodiester bond in a phosphate group with a sulfur atom. According to some embodiments, the phosphate group with modified group(s) is a phosphorothioate group having a structure represented by Formula (1):

[0363]

[0364] This modification can stabilize the double-stranded structure of siRNA, which maintains high specificity and high affinity of base pairing.

[0365] According to some embodiments of the present invention, in the siRNA of the present invention, a phosphorothioate bond is present at at least one position selected from the group consisting of the following positions: a position between the first and second nucleotides at either end of the sense or antisense strand, a position between the second and third nucleotides at either end of the sense or antisense strand, or any combination thereof. According to some embodiments of the present invention, a phosphorothioate bond is present at all of the above positions except the 5' end of the sense strand. According to some embodiments of the present invention, a phosphorothioate bond is present at all of the above positions except the 3' end of the sense strand.According to some embodiments of the present invention, the phosphorothioate linkage is present at least in one of the following positions: a position between the first and second nucleotides at the 5'' end of the sense strand; a position between the second and third nucleotides at the 5'' end of the sense strand; a position between the first and second nucleotides at the 3'' end of the sense strand; a position between the second and third nucleotides at the 3'' end of the sense strand; a position between the first and second nucleotides at the 5'' end of the antisense strand; a position between the second and third nucleotides at the 5'' end of the antisense strand; a position between the first and second nucleotides at the 3'' end of the antisense strand; and a position between the second and third nucleotides at the 3'' end of the antisense strand.

[0366] In some embodiments, the siRNA of the present invention is any one of siFXIa1-M1S, siFXIa1-M2S, siFXIa1-M3S, siFXIa2-M1S, siFXIa2-M2S, siFXIa2-M3S, siFXIb1-M1S, siFXIb1-M2S, siFXIb1-M3S, siFXIb2-M1S, siFXIb2-M2S, siFXIb2-M3S, siFXIc1-M1S, siFXIc1-M2S, siFXIc1-M3S, siFXIc2-M1S, siFXIc2-M2S, siFXIc2-M3S, siFXId1-M1S, siFXId1-M2S, siFXId1-M3S, siFXId2-M1S, siFXId2-M2S, siFXId2-M3S, siFXIe1-M1S, siFXIe1-M2S, siFXIe1-M3S, siFXIe2-M1S, siFXIe2-M2S, siFXIe2-M3S, siFXIf1-M1S, siFXIf1-M2S, siFXIf1-M3S, siFXIf2-M1S, siFXIf2-M2S, siFXIf2-M3S, siFXIg1-M1S, siFXIg1-M2S, siFXIg1-M3S, siFXIg2-M1S, siFXIg2-M2S, siFXIg2-M3S, siFXIh1-M1S, siFXIh1-M2S, siFXIh1-M3S, siFXIh2-M1S, siFXIh2-M2S, siFXIh2-M3S, FXIi1-M1S, siFXIi1-M2S, siFXIi1-M3S, siFXIi2-M1S, siFXIi2-M2S and siFXIi2-M3S, listed in Tables 1a-1i.

[0367] According to some embodiments of the present invention, the 5'-terminal nucleotide in the antisense strand of the siRNA is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analog.

[0368] Commonly used 5'-phosphate nucleotides or nucleotides modified with a 5'-phosphate analog are well known to those skilled in the art. For example, 5'-phosphate nucleotides may have the following structure:

[0369]

[0370] As another example, disclosed in Anastasia Khvorova and Jonathan K. Watts, "The chemical evolution of oligonucleotide therapies of clinical utility." Nature Biotechnology, 2017, 35(3): 238–48, the following four nucleotides are given, modified with a 5'-phosphate analog:

[0371]

[0372] where R is selected from H, OH, methoxy and F;

[0373] “Base” is a nucleic acid base selected from A, U, C, G, or T.

[0374] According to some embodiments of the present invention, the 5'-phosphate nucleotide is a nucleotide with a 5'-phosphate modification represented by Formula (2); the nucleotide modified with a 5'-phosphate analog is a nucleotide with a vinylphosphonate modification represented by Formula (3), or a phosphorothioate-modified nucleotide represented by Formula (5).

[0375] According to some embodiments of the present invention, the siRNA of the present invention is any one of siFXIa1-M1P1, siFXIa1-M2P1, siFXIa1-M3P1, siFXIa2-M1P1, siFXIa2-M2P1, siFXIa2-M3P1, siFXIa1-M1SP1, siFXIa1-M2SP1, siFXIa1-M3SP1, siFXIa2-M1 SP1, siFXIa2-M2SP1, siFXIa2-M3SP1, siFXIb1-M1P1, siFXIb1-M2P1, siFXIb1-M3P1, siFXIb2-M1P1, siFXIb2-M2P1, siFXIb2-M3P1, siFXIb1-M1SP1, siFXIb1-M2SP1, siFXIb1-M3SP1, siFXIb2-M1SP1, siFXIb2-M2SP1, siFXIb2-M3SP1, siFXIcl-M1P1, siFXIcl-M2P1, siFXIcl-M3P1, siFXIc2-M1P1, siFXIc2-M2P1, siFXIc2-M3P1, siFXIcl-M1 SP1, siFXIcl-M2SP1, siFXIcl-M3SP1, siFXIc2-M1SP1, siFXIc2-M2SP1, siFXIc2-M3SP1, siFXId1-M1P1, siFXId1-M2P1, siFXId1-M3P1, siFXId2-M1P1, siFXId2-M2P1, siFXId2-M3P1, siFXId1-M1SP1, siFXId1-M2SP1, siFXId1-M3SP1, siFXId2-M1SP1, siFXId2-M2SP1, siFXId2-M3SP1, siFXIel-M1P1, siFXIel-M2P1, siFXIel-M3P1, siFXIe2-M1P1, siFXIe2-M2P1, siFXIe2-M3P1, siFXIel-M1SP1, siFXIel-M2SP1, siFXIel-M3SP1, siFXIe2-M1SP1, siFXIe2-M2SP1,siFXIe2-M3SP1, siFXIf1-M1P1, siFXIf1-M2P1, siFXIf1-M3P1, siFXIf2-M1P1, siFXIf2-M2P1, siFXIf2-M3P1, siFXIf1-M1SP1, siFXIf1-M2SP1, siFXIf1-M3SP1, siFXIf2-M1 SP1, siFXIf2-M2SP1, siFXIf2-M3SP1, siFXIg1-M1P1, siFXIg1-M2P1, siFXIg1-M3P1, siFXIg2-M1P1, siFXIg2-M2P1, siFXIg2-M3P1, siFXIg1-M1SP1, siFXIg1-M2SP1, siFXIg1-M3SP1, siFXIg2-M1SP1, siFXIg2-M2SP1, siFXIg2-M3SP1, siFXTn1-M1P1, siFXIh1-M2P1, siFXIh1-M3P1, siFXIh2-M1P1, siFXIn2-M2P1, siFXIh2-M3P1, siFXIh1-M1SP1, siFXIh1-M2SP1, siFXIh1-M3SP1, siFXIh2-M1SP1, siFXIh2-M2SP1, siFXHi2-M3SP1, FXIi1-M1P1, siFXIi1-M2P1, siFXIi1-M3P1, siFXIi2-M1P1, siFXIi2-M2P1, siFXIi2-M3P1, siFXIi1-M1SP1, siFXIi1-M2SP1, siFXIi1-M3SP1, siFXIi2-M1 SP1, siFXIi2-M2SP1 and siFXIi2-M3SP1, listed in Tables 1a-1i.,

[0376] The present inventors unexpectedly found that the siRNAs of the present invention have significantly increased stability in plasma and lysosomes, reduced off-target effects, while maintaining higher inhibitory activity against the target mRNA.

[0377] The siRNAs according to the present invention can be produced using conventional siRNA production methods in the art, such as solid-phase synthesis and liquid-phase synthesis. Customized services for solid-phase synthesis are already commercially available. Modified nucleotides can be introduced into the siRNAs according to the present invention using a nucleotide monomer having an appropriate modification. Methods for producing a nucleotide monomer having an appropriate modification and methods for introducing a modified nucleotide into siRNAs are also well known to those skilled in the art.

[0378] Pharmaceutical composition

[0379] According to the present invention, there is provided a pharmaceutical composition comprising the above-mentioned siRNA as an active ingredient and a pharmaceutically acceptable carrier.

[0380] The pharmaceutically acceptable carrier may be a carrier generally used in the field of siRNA administration, such as, but not limited to, one or more of magnetic nanoparticles (such as Fe3O4 and Fe2O3-based nanoparticles), carbon nanotubes, mesoporous silica, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamidoamine dendrimer (PAMAM), poly-L-lysine (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), D- and L-lactic acid-glycolic acid copolymer (PLGA), poly-2-aminoethylethylene phosphate (PPEEA), poly-2-dimethylaminoethyl methacrylate (PDMAEMA) and derivatives thereof.

[0381] There are no special requirements for the content of siRNA and a pharmaceutically acceptable carrier in the pharmaceutical composition according to the present invention. They can be present in any amount usually used for each component. According to some embodiments of the present invention, the weight ratio of siRNA to the pharmaceutically acceptable carrier can be 1: (1-500), and according to some embodiments of the present invention, the above-mentioned weight ratio is 1: (1-50).

[0382] According to some embodiments of the present invention, the pharmaceutical composition may also contain other pharmaceutically acceptable excipients, which may be one or more of various compositions or compounds commonly used in the art. For example, said other pharmaceutically acceptable excipients may comprise at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0383] The pH buffer may be a tris-hydroxymethylaminomethane hydrochloride buffer solution with a pH of 7.5 to 8.5 and / or a phosphate buffer solution with a pH of 5.5 to 8.5, such as a phosphate buffer solution with a pH of 5.5 to 8.5.

[0384] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The content of the protective agent may be from 0.01% by weight to 30% by weight based on the total weight of the pharmaceutical composition.

[0385] The osmotic pressure regulator can be sodium chloride and / or potassium chloride. The osmotic pressure regulator content ensures the osmotic pressure of the pharmaceutical composition is 200-700 mOsm / kg. Those skilled in the art can easily determine the osmotic pressure regulator content based on the target osmotic pressure.

[0386] According to some embodiments of the present invention, the pharmaceutical composition may be a liquid formulation, such as an injection solution; or a lyophilized powder for injection, which is mixed with a liquid excipient to obtain a liquid formulation for administration. The liquid formulation can be administered, but is not limited to, by subcutaneous, intramuscular, or intravenous injection, and can also be administered, but is not limited to, into the lungs by nebulization or other organs (such as the liver) through the lungs by nebulization. According to some embodiments of the present invention, the pharmaceutical composition is administered by intravenous injection.

[0387] According to some embodiments of the present invention, the pharmaceutical composition may be in the form of a liposomal formulation. According to some embodiments of the present invention, the pharmaceutically acceptable carrier used in the liposomal formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), an auxiliary lipid and / or a PEGylated lipid. Wherein the organic amine, the auxiliary lipid and the PEGylated lipid may be selected, respectively, from one or more of the amine-containing transfection compounds or pharmaceutically acceptable salts or derivatives thereof, auxiliary lipids and PEGylated lipids described in CN 103380113 A, which is fully incorporated herein by reference.

[0388] According to some embodiments of the present invention, the organic amine may be a compound represented by Formula (201) disclosed in CN 103380113 A, or a pharmaceutically acceptable salt thereof:

[0389]

[0390] where

[0391] X 101 their 102 independently of each other are selected from O, S, N-A or C-A, where A is hydrogen or C1-C 20 hydrocarbon chain;

[0392] Y 101 and Z 101 independently of one another, selected from C=O, C=S, S=O, CH-OH or SO2;

[0393] R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107are independently selected from hydrogen; a cyclic or acyclic, substituted or unsubstituted, branched or linear aliphatic group; a cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic group; a substituted or unsubstituted, branched or linear acyl group; a substituted or unsubstituted, branched or linear aryl group; and a substituted or unsubstituted, branched or linear heteroaryl group;

[0394] x represents an integer between 1 and 10;

[0395] n is an integer from 1 to 3, m is an integer from 0 to 20, p is 0 or 1, and if m=p=0, then R 102 is hydrogen; and

[0396] if at least one of n and m is 2, then R 103 and nitrogen in Formula (201) form a structure represented by Formula (202) or (203):

[0397]

[0398] in which g, e and f independently represent six integers of 1 to 6; "HCC" represents a hydrocarbon chain and each *N represents a nitrogen atom shown in Formula (201).

[0399] According to some embodiments of the present invention R 103 is a polyamine. According to other embodiments of the present invention, R 103 is a ketal. According to some embodiments of the present invention, R 101 and R 102 in Formula (201) independently of one another represent any substituted or unsubstituted, branched or linear alkyl or alkenyl, wherein said alkyl or alkenyl contains from 3 to about 20 carbon atoms (e.g., from 8 to about 18 carbon atoms) and 0-4 double bonds (e.g., 0-2 double bonds).

[0400] According to some embodiments of the present invention, if n and m are independently 1 or 3, R 103can be any of the following formulas (204)-(213):

[0401]

[0402] where in Formulas (204) to (213), g, e, and f independently represent an integer from 1 to 6, each “HCC” represents a hydrocarbon chain, and each * symbol represents a potential point of attachment of R 103 to the nitrogen atom in Formula (201), wherein each H in any * position can be substituted to effect attachment to the nitrogen atom in Formula (201).

[0403] The compound represented by Formula (201) can be produced according to the description of CN 103380113 A.

[0404] According to some embodiments of the present invention, the organic amine is an organic amine represented by Formula (214) and / or an organic amine represented by Formula (215):

[0405]

[0406] The accessory lipid is cholesterol, cholesterol analogs and / or cholesterol derivatives, and

[0407] The said PEGylated lipid is 1,2-dipalmitoylamine-sn-glycero-3-phosphatidylethanolamine-N-[methoxypolyethyleneglycol]-2000.

[0408] According to some embodiments of the present invention, the molar ratio between the organic amine, the auxiliary lipid and the PEGylated lipid in the pharmaceutical composition is (19.7-80): (19.7-80): (0.3-50), for example, the molar ratio may be (50-70): (20-40): (3-20).

[0409] According to some embodiments of the present invention, the particles of the pharmaceutical composition formed by the siRNA of the present invention and the above-mentioned amine-containing transfection reagents have an average diameter of from about 30 nm to about 200 nm, typically from about 40 nm to about 135 nm, and more typically the average diameter of the liposome particles is from about 50 nm to about 120 nm, from about 50 nm to about 100 nm, from about 60 nm to about 90 nm, or from 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 160 nm.

[0410] According to some embodiments of the present invention, in the pharmaceutical composition formed by the siRNA of the present invention and the above-mentioned amine-containing transfection agents, the weight ratio (weight / weight ratio) of the siRNA to total lipids, such as organic amines, auxiliary lipids and / or PEGylated lipids, ranges from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10. For example, the mass ratio of siRNA according to the present invention to total 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.

[0411] According to some embodiments of the present invention, the pharmaceutical composition may be sold with each component provided separately and may be used in the form of a liquid formulation. According to some embodiments of the present invention, the pharmaceutical composition formed by the siRNA of the present invention and the above-mentioned pharmaceutically acceptable carrier may be produced by various known methods, with the exception of replacing the existing siRNA with the siRNA of the present invention. According to some specific embodiments of the present invention, the pharmaceutical composition may be produced according to the following method:

[0412] Organic amines, auxiliary lipids and PEGylated lipids are suspended in alcohol at the molar ratio described above and mixed uniformly to obtain a lipid solution; the alcohol is used in such an amount that the resulting lipid solution is present at a total mass concentration of 2 to 25 mg / mL (for example, 8 to 18 mg / mL). The alcohol is a pharmaceutically acceptable alcohol, such as an alcohol that is in liquid form at about room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300 and polyethylene glycol 400, such as ethanol.

[0413] The siRNA according to the present invention is dissolved in a buffered saline solution to obtain an aqueous siRNA solution. The buffered saline solution has a concentration of 0.05 to 0.5 M, such as 0.1 to 0.2 M. The pH of the buffered saline solution is adjusted to 4.0 to 5.5, such as 5.0 to 5.2. The buffered saline solution is used in an amount such that the siRNA is present at a concentration of no more than 0.6 mg / mL, such as 0.2 to 0.4 mg / mL. The buffer salt may be one or more selected from the group consisting of soluble acetate and soluble citrate, such as sodium acetate and / or potassium acetate.

[0414] The lipid solution and the aqueous siRNA solution are mixed. The resulting product is incubated at 40 to 60°C for at least 2 minutes (e.g., 5 to 30 minutes) to obtain an incubated liposomal composition. The volume ratio of the lipid solution to the aqueous siRNA solution is 1:(2-5) (e.g., 1:4).

[0415] The incubated liposome formulation is concentrated or diluted and then subjected to impurity removal and sterilization to obtain a pharmaceutical composition according to the present invention, which has the following physicochemical parameters: pH of 6.5 to 8, encapsulation percentage of not less than 80%, particle size of 40 to 200 nm, polydispersity index of not more than 0.30, and osmotic pressure of 250 to 400 mOsm / kg. For example, the physicochemical parameters may be as follows: pH of 7.2 to 7.6, encapsulation percentage of not less than 90%, particle size of 60 to 100 nm, polydispersity index of not more than 0.20, and osmotic pressure of 300 to 400 mOsm / kg.

[0416] The concentration or dilution step may be performed before, after, or simultaneously with the impurity removal. The impurity removal method may be any of various existing methods, such as ultrafiltration using a 100 kDa hollow fiber column, phosphate-buffered saline (PBS) at pH 7.4 as the exchange solution for ultrafiltration, and a tangential flow system. The sterilization method may be any of various existing methods, such as sterilization by filtration on a filter with a pore size of 0.22 μm.

[0417] siRNA conjugate

[0418] According to the present invention, there is provided an siRNA conjugate that comprises the above-described siRNA and a conjugating group attached thereto.

[0419] Typically, the conjugating group comprises at least one pharmaceutically acceptable targeting group and an optional linker. In addition, the siRNA, the linker, and the targeting group are connected sequentially. According to some embodiments of the present invention, the number of targeting groups is from 1 to 6. According to some embodiments of the present invention, the number of targeting groups is from 2 to 4. The siRNA molecule can be non-covalently or covalently conjugated to the conjugating group, for example, the siRNA molecule can be covalently conjugated to the conjugating group. The conjugation position between the siRNA and the conjugating group can be at the 3'-end or 5'-end of the sense strand of the siRNA, or at the 5'-end of the antisense strand of the siRNA, or in the internal sequence of the siRNA. According to some embodiments of the present invention, the conjugation position between the siRNA and the conjugating group is at the 3' end of the sense strand of the siRNA.

[0420] According to some embodiments of the present invention, the conjugating group may be linked to a phosphate group, a 2'-hydroxy group, or a base of a nucleotide. According to some embodiments of the present invention, the conjugating group may also be linked to a 3'-hydroxy group if the nucleotides are linked via a 2'-5'-phosphodiester linkage. If the conjugating group is linked to the end of the siRNA chain, the conjugating group is typically linked to a phosphate group of a nucleotide; if the conjugating group is linked to an internal sequence of the siRNA, the conjugating group is typically linked to a ribose ring or a base. For various types of linkage, reference may be made to: Muthiah Manoharan et. al. siRNA conjugates carrying sequentially assembled triva1ent N-acetylgalactosamine linked through nucleosides detect robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7.

[0421] According to some embodiments of the present invention, the siRNA and the conjugating group can be linked via an acid-labile or reducible chemical bond, and these chemical bonds can be cleaved in the acidic environment of cellular endosomes, thereby releasing the siRNA. For non-cleavable conjugation types, the conjugating group can be linked to the sense strand of the siRNA, thereby minimizing the effect of conjugation on siRNA activity.

[0422] According to some embodiments of the present invention, the pharmaceutically acceptable targeting group may be a ligand commonly used in the field of siRNA administration, such as the various ligands described in WO 2009082607 A2, which is incorporated herein by reference in its entirety.

[0423] According to some embodiments of the present invention, the pharmaceutically acceptable targeting group may be selected from one or more ligands formed by the following targeting molecules or derivatives thereof: lipophilic molecules such as cholesterol, bile acids, vitamins (such as vitamin E), lipid molecules with different chain lengths; polymers such as polyethylene glycol; polypeptides such as a cell penetration peptide; aptamers; antibodies; quantum dots; saccharides such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid salts; or receptor ligands expressed in liver parenchymatous cells, such as asialoglycoprotein, asialo-sugar residue, lipoproteins (such as high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.

[0424] According to some embodiments of the present invention, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. According to some embodiments of the present invention, at least one ligand is a ligand capable of binding to a hepatocyte surface receptor. According to some embodiments of the present invention, at least one ligand is a ligand capable of binding to a mammalian hepatocyte surface receptor. According to some embodiments of the present invention, at least one ligand is a ligand capable of binding to a human hepatocyte surface receptor. According to some embodiments of the present invention, at least one ligand is a ligand capable of binding to the liver surface asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes.The types of these ligands are well known to those skilled in the art and typically function by binding to a specific receptor on the surface of a target cell, which mediates the delivery of ligand-bound miRNA to the target cell.

[0425] According to some embodiments of the present invention, the pharmaceutically acceptable targeting moiety can be any ligand that has affinity for asialoglycoprotein receptors (ASGP-R) on the surface of mammalian hepatocytes. In some embodiments of the present invention, each ligand is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF). In some embodiments of the present invention, the ligand is a saccharide or derivatives thereof.

[0426] According to some embodiments of the present invention, at least one ligand is a saccharide. In some embodiments of the present invention, each ligand is a saccharide. In some embodiments of the present invention, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a saccharide derivative. In some embodiments of the present invention, at least one ligand can be a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments of the present invention, at least one ligand is a modified saccharide. In some embodiments of the present invention, each ligand is a modified saccharide.According to some embodiments of the present invention, each ligand is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, and a monosaccharide derivative. According to some embodiments of the present invention, each ligand or at least one ligand is selected from the group consisting of glucose and its derivatives, mannose and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.

[0427] According to some embodiments of the present invention, each ligand can be independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucofuranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-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-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, methyl-2,3,4-tris-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-B-glucoheptopyranoside, 2,5-anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose, L-4-thioribose. Other ligand variants can be found, for example, in the disclosure of CN 105378082 A, which is incorporated herein by reference in its entirety.

[0428] According to some embodiments of the present invention, the pharmaceutically acceptable targeting group in the siRNA conjugate may be galactose or N-acetyl galactosamine, wherein the galactose or N-acetyl galactosamine molecules may be mono-, bi-, tri-, or tetravalent. It should be understood that the terms mono-, bi-, tri-, or tetravalent described herein, respectively, mean that the molar ratio of the siRNA molecule to the galactose or N-acetyl galactosamine molecule in the siRNA conjugate is 1:1, 1:2, 1:3, or 1:4, wherein the siRNA conjugate is formed from the siRNA molecule and a conjugating group containing a galactose or N-acetyl galactosamine molecule as a targeting group. According to some embodiments of the present invention, the pharmaceutically acceptable targeting moiety is N-acetylgalactosamine.According to some embodiments of the present invention, when the siRNA of the present invention is conjugated to a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. According to some embodiments of the present invention, when the siRNA of the present invention is conjugated to a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0429] The targeting group can be linked to the siRNA molecule via an appropriate linker, and the appropriate linker can be selected by those skilled in the art according to the specific type of targeting group. The types of these linkers and targeting groups, as well as the types of connection with siRNA, can be found in the disclosure of WO 2015006740 A2, which is incorporated herein by reference in its entirety.

[0430] According to some embodiments of the present invention, if the targeting group is N-acetylgalactosamine, a suitable linker may have the following structure represented by Formula (301):

[0431]

[0432] where

[0433] k represents an integer between 1 and 3;

[0434] L A is a chain group containing an amide bond, which has a structure represented by Formula (302), and each L A appropriately connected to the targeting group and the L group C by means of a simple etheric connection at its two ends:

[0435]

[0436] L B is a chain group containing N-acylpyrrolidine, which has a structure represented by Formula (303), wherein the chain group contains a carbonyl group at one end and is connected to the L group Cvia an amide bond, and also contains an hydroxy group at the other end and is connected to the specified miRNA via a complex phosphoester bond:

[0437]

[0438]

[0439] L C is a divalent or tetravalent linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or

[0440] trihydroxymethylaminomethane, and group L C connected to each of the L groups A via an ether bond through an oxygen atom and is connected to the L group B via an amide bond through the nitrogen atom.

[0441] According to some embodiments of the present invention, if n=3 and L C is a tetravalent trihydroxymethylaminomethane-based linking group, a miRNA conjugate formed by linking N-acetylgalactosamine molecules to a miRNA molecule via -(L A)3-trihydroxymethylaminomethane-L B - as a linker, has a structure represented by Formula (304):

[0442]

[0443] in which the double helix structure represents miRNA.

[0444] Similarly, the conjugation position between the siRNA and the conjugating group may be at the 3'-end or 5'-end of the sense strand of the siRNA, or at the 5'-end of the antisense strand, or in the internal sequence of the siRNA.

[0445] According to some embodiments of the present invention, the 3'-end of the sense strand of the siRNA of the present invention is covalently conjugated to three molecules of N-acetylgalactosamine (GalNAc) via a linker -(L A )3-trihydroxymethylaminomethane-L B- to obtain an siRNA conjugate in which the molar ratio of the siRNA molecule to the GalNAc molecule is 1:3 (hereinafter also referred to as (GalNAc)3-siRNA), and this siRNA conjugate has a structure represented by Formula (305):

[0446]

[0447] in which the double helix structure represents miRNA; and a linker is connected to the 3'-end of the sense strand of the miRNA.

[0448] According to some embodiments of the present invention, if the targeting group is N-acetylgalactosamine, a suitable linker may have a structure represented by Formula (306):

[0449]

[0450] where

[0451] 1 represents an integer between 0 and 3;

[0452] * represents a position on the specified linker connected to the specified targeting group via an ether bond; and

[0453] #represents a position on the specified linker that is connected to the specified miRNA via a complex phosphoester bond.

[0454] According to some embodiments of the present invention, if 1=2, the siRNA conjugate has a structure represented by Formula (307):

[0455]

[0456] in which the double helix structure denotes miRNA; and the linker is connected to the 3'-end of the sense strand of miRNA.

[0457] The above conjugates can be synthesized according to the method described in detail in the prior art. For example, WO 2015006740 A2 describes in detail the methods for producing various conjugates. The siRNA conjugate of the present invention can be produced by methods well known to those skilled in the art. For example, WO 2014025805 A1 describes a method for producing a conjugate having a structure represented by Formula (305). Rajeev et al, ChemBioChem 2015, 16, 903-908 described a method for producing a conjugate having a structure represented by Formula (307).

[0458] According to some embodiments of the present invention, the siRNA conjugate has a structure represented by Formula (308):

[0459]

[0460] where

[0461] n1 represents an integer from 1 to 3, and n3 represents an integer from 0 to 4;

[0462] m1, m2 and m3 independently represent integers from 2 to 10;

[0463] R 10 , R 11 , R 12 , R 13 , R 14 and R 15 independently of each other are H, or are selected from the group consisting of C1-C 10 alkyl, C1-C 10 haloalkyl and C1-C 10 alkoxy,

[0464] R3 is a group having a structure represented by Formula (A59):

[0465]

[0466] in which E1 is OH, SH or BHr; and Nu is the siRNA according to the present invention;

[0467] R2 is a linear alkylene of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally substituted with any one or more groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenylene, C2-C 10 alkynylene, C6-C 10 arylene, C3-C 18heterocyclylene and C5-C 10 heteroarylene, and wherein R2 optionally contains any one or more substituents selected from the group consisting of: C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C 10 haloalkyl, -OC1-C 10 alkyl, -OC1-C 10 alkylphenyl, -C1-C 10 alkyl-OH, -OC1-C 10 haloalkyl, -SC1-C 10 alkyl, -SC1-C 10 alkylphenyl, -C1-C 10 alkyl-SH, -SC1-C 10 haloalkyl, halogen, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 alkyl)(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C 10 alkyl)(C1-C 10 alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)0(C1-C 10 ) alkyl, -CON(C1-C 10 alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 alkyl), -N(C1-C 10alkyl)C(O)(phenyl), -C(O)C1-C 10 alkyl, -C(O)C1-C 10 alkylphenyl, -C(O)C1-C 10 haloalkyl, -OC(O)C1-C 10 alkyl, -SO2(C1-C 10 alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0468] each L1 is a linear alkylene of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally substituted with any one or more groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenylene, C2-C 10 alkynylene, C6-C 10 arylene, C3-C 18 heterocyclylene and C5-C 10 heteroarylene, and wherein L1 optionally contains any one or more substituents selected from the group consisting of: C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C 10 haloalkyl, -OC1-C 10alkyl, -OC1-C 10 alkylphenyl, -C1-C 10 alkyl-OH, -OC1-C 10 haloalkyl, -SC1-C 10 alkyl, -SC1-C 10 alkylphenyl, -C1-C 10 alkyl-SH, -SC1-C 10 haloalkyl, halogen, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 alkyl)(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C 10 alkyl)(C1-C 10 alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 alkyl), -CON(C1-C 10 alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 alkyl, -C(O)C1-C 10 alkylphenyl, -C(O)C1-C 10 haloalkyl, -OC(O)C1-C 10 alkyl, -SO2(C1-C 10 alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl).

[0469] According to some embodiments of the present invention, L1 may be selected from the group consisting of groups of formulas (A1) to (A26), or any combination thereof, wherein the structures and definitions of A1 to A26 are given below:

[0470]

[0471]

[0472] where j1 is an integer between 1 and 20;

[0473] j2 represents an integer between 1 and 20;

[0474] R' represents C1-C 10 alkyl;

[0475] Ra is selected from the group consisting of Formula groups (A27) to (A45) or any combination thereof:

[0476]

[0477]

[0478] Rb is C1-C 10 alkyl; and

[0479] represents the position at which the group is covalently attached.

[0480] Those skilled in the art will understand that, although L1 is defined as a linear alkyl for convenience, it cannot be a linear group or otherwise named, for example, amine or alkenyl, obtained by the above-mentioned substitution and / or replacement. For the purposes of the present invention, the length of L1 represents the number of atoms in the chain connecting the two attachment points. For this purpose, a ring obtained by substituting a carbon atom of a linear alkylene, such as heterocyclylene or heteroarylene, is counted as one atom.

[0481] M1 is a targeting group, the definitions and variants of which are similar to the definitions and variants of the above targeting groups. According to some embodiments of the present invention, each M1 is independently selected from ligands that have affinity for the asialoglycoprotein receptor on the surface of mammalian hepatocytes.

[0482] If M1 is a ligand that has affinity for the asialoglycoprotein receptor on the surface of a mammalian hepatocyte, according to some embodiments, n1 may be an integer from 1 to 3, and n3 may be an integer from 0 to 4, to ensure that the number of the M1 targeting group in the conjugate can be at least 2. According to some embodiments of the present invention, n1+n3≥2, so the number of the M1 targeting group is at least 3, which ensures easier binding of the M1 targeting group to the asialoglycoprotein receptor on the surface of hepatocytes, which can facilitate the endocytosis of the conjugate into cells. Experiments have shown that if the number of M1 targeting groups exceeds 3, the ease of binding of the M1 targeting groups to the asialoglycoprotein receptor on the surface of hepatocytes does not increase significantly.Thus, taking into account various aspects such as ease of synthesis, cost of the structure / process and delivery efficiency, according to some embodiments of the present invention, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1 and n1+n3=2 to 3.

[0483] According to some embodiments of the present invention, if m1, m2 and m3 independently of each other are an integer selected from 2-10, the steric positions among multiple targeting groups of M1 may be suitable for binding the targeting groups of M1 to the asialoglycoprotein receptor on the surface of hepatocytes. In order for the conjugate according to the present invention to have a simpler structure, easier synthesis and / or reduced cost, according to some embodiments of the present invention, m1, m2 and m3 independently of each other are integers 2-5, according to some embodiments of the present invention m1=m2=m3.

[0484] Those skilled in the art will understand that if R 10 , R 11 , R 12 , R 13 , R 14 or R 15 independently selected from H, C1-C 10 alkyl, C1-C 10 haloalkyl and C1-C 10 alkoxy, they will not change the properties of the conjugate according to the present invention and will all be able to achieve the purpose of the present invention. According to some embodiments of the present invention R 10 , R 11 , R 12 , R 13 , R 14 or R 15 independently selected from H, methyl, and ethyl. According to some embodiments of the present invention, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 represent N.

[0485] R3 is a group having a structure represented by Formula A59, wherein E1 is OH, SH or BH2, and in view of the high availability of starting materials, according to some embodiments of the present invention, E1 is OH or SH.

[0486] R2 is selected to provide a bond between the group represented by Formula A59 and the N atom on the nitrogen backbone. As used herein, the term "nitrogen backbone" refers to a chain structure in which the N atom is coaxially bonded to the carbon atoms to which R is attached. 10 , R 11 , R 12 , R 13 , R 14 and R 15. Thus, R2 can be any linking group capable of attaching a group represented by Formula (A59) to an N atom on the nitrogen backbone by suitable means. According to some embodiments of the present invention, in case the siRNA conjugate of Formula (308) is prepared by a solid phase synthesis method, the group R2 must have both a position bonding to the N atom on the nitrogen backbone and a position bonding to the P atom in R3. According to some embodiments of the present invention, in R2, the position bonding to the N atom on the nitrogen backbone forms an amide bond with the N atom, and the position bonding to the P atom in R3 forms a phosphoester bond with the P atom. According to some embodiments of the present invention, R2 can be B5, B6, B5, or B6:

[0487]

[0488] wherein q2 represents a position at which the group is covalently attached; q2 may be an integer from 1 to 10; according to some embodiments of the present invention, q2 is an integer from 1 to 5.

[0489] L1 is used to connect the targeting group M1 to the N atom on the nitrogen backbone, which provides the liver targeting function for the siRNA conjugate represented by Formula (308). According to some embodiments of the present invention, L1 is selected from connected combinations of one or more of the groups of Formulas (A1) to (A26). According to some embodiments of the present invention, L1 is selected from connected combinations of one or more of Formulas a1, A4, A5, A6, A8, A10, A11 and A13. According to some embodiments of the present invention, L1 is selected from connected combinations of at least two of Formulas (A1), (A4), (A8), (A10) and (A11). According to some embodiments of the present invention, L1 is selected from connected combinations of at least two of Formulas (A1), (A8) and (A10).

[0490] According to some embodiments of the present invention, the length of L1 may be from 3 to 25, from 3 to 20, from 4 to 15, or from 5 to 12 atoms. According to some embodiments of the present invention, L1 comprises 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, or 60 atoms in length.

[0491] According to some embodiments of the present invention, j1 is an integer from 2 to 10, and in some embodiments of the present invention, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3 to 5. R' is C1-C4 alkyl, and in some embodiments, R is methyl, ethyl, or isopropyl. Ra is one of Formulas (A27), (A28), (A29), (AZO), and (A31), and in some embodiments, Ra is Formula (A27) or (A28). Rb is C1-C5 alkyl, and in some embodiments, it is methyl, ethyl, isopropyl, or butyl.According to some embodiments of the present invention, j1, j2, R', Ra and Rb in Formulas (A1)-(A26), respectively, are selected to provide a connection between the M1 targeting groups and the N atom on the nitrogen backbone, and to create a steric position among the M1 targeting groups that is more favorable for binding of the M1 targeting groups to the asialoglycoprotein receptor on the surface of hepatocytes.

[0492] According to some embodiments of the present invention, the siRNA conjugate has a structure represented by Formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422):

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501] According to some embodiments of the present invention, the P atom in Formula (A59) may be linked to any possible position in the siRNA sequence. For example, the P atom in Formula (A59) may be linked to any nucleotide in the sense or antisense strand of the siRNA. According to some embodiments of the present invention, the P atom in Formula A59 is linked to any nucleotide in the sense strand of the siRNA. According to some embodiments of the present invention, the P atom in Formula (A59) may be linked to the terminal region of the sense or antisense strand of the siRNA. According to some embodiments of the present invention, the P atom in Formula (A59) is linked to the terminal region of the sense strand of the siRNA. The said terminal region refers to the first 4 nucleotides counted from one end of the sense or antisense strand. According to some embodiments of the present invention, the P atom in Formula A59 is linked to any end of the sense strand or the antisense strand of the siRNA.According to some embodiments of the present invention, the P atom in Formula A59 is linked to the 3'' end of the sense strand of the siRNA. If the P atom in Formula (A59) is linked to the above-mentioned position of the sense strand of the siRNA, after entering cells, the siRNA conjugate, as shown in Formula (308), can release a separate antisense strand of the siRNA during unwinding, which blocks the translation of FXI mRNA into protein and inhibits the expression of the FXI gene.

[0502] According to some embodiments of the present invention, the P atom in Formula (A59) can be linked to any possible position of a nucleotide in the siRNA, such as the 5' position, the 2' position, the 3' position, or a base of the nucleotide. According to some embodiments of the present invention, the P atom in Formula A59 can be linked to the 2'', 3'', or 5'' position of the nucleotide in the siRNA by forming a phosphodiester bond.According to some embodiments of the present invention, the P atom in Formula (A59) is bonded to an oxygen atom formed by deprotonation of the 3'-hydroxyl of a nucleotide at the 3'-end of the sense strand in the siRNA (in such a case, the P atom in Formula (A59) is also referred to as the P atom in the phosphate group of the siRNA), or the P atom in Formula (A59) is bonded to a nucleotide by substituting for a hydrogen atom in the 2'-hydroxyl of a nucleotide of the sense strand in the siRNA, or the P atom in Formula (A59) is bonded to a nucleotide by substituting for a hydrogen atom in the 5'-hydroxyl of a nucleotide at the 5'-end of the sense strand in the siRNA.

[0503] The present inventors unexpectedly discovered that the siRNA conjugate of the present invention exhibits significantly improved plasma stability and low off-target effect, and, in addition, exhibits higher suppressive activity against FXI mRNA. In some embodiments of the present invention, the siRNA of the present invention may be one of the siRNAs shown in Tables 1a-1i. The siRNA conjugates containing such siRNAs exhibit significantly higher suppressive activity against FXI mRNA.

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527] where C, G, U, and A represent the base compositions of nucleotides; m represents that the nucleotide to the left of m is a methoxy-modified nucleotide; f represents that the nucleotide to the left of f is a fluorine-modified nucleotide; s represents that the two nucleotides on either side of s are linked by a thiophosphorothioate bond;P1 denotes that the nucleotide adjacent to the right side of P1 is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analog, in some embodiments P1 is a specially modified nucleotide VP, Ps, or P, where VP denotes that the nucleotide adjacent to the right side of VP is a nucleotide modified with vinyl phosphate, Ps denotes that the nucleotide adjacent to the right side of Ps is a thiophosphate-modified nucleotide, and P denotes that the nucleotide adjacent to the right side of P is a 5'-phosphate nucleotide.

[0528] In the siRNA or siRNA conjugate according to the present invention, each pair of adjacent nucleotides is linked by a phosphodiester bond or a phosphothioates ester bond. The non-bridging oxygen or sulfur atom in the phosphodiester bond or phosphorothioates ester bond is negatively charged and may be present in the form of a hydroxyl or sulfhydryl. In addition, the hydrogen ion in the hydroxyl or sulfhydryl may be partially or completely replaced by a cation. The cation may be any cation, such as one of a metal cation, an ammonium cation NH4 + or an organic ammonium cation. In order to increase solubility, according to one embodiment of the present invention, the cation is selected from one or more of an alkali metal cation, an ammonium cation formed by a tertiary amine, and a quaternary ammonium cation. The alkali metal ion can be K + and / or Na +, and the cation formed by the tertiary amine may be an ammonium cation formed by triethylamine and / or an ammonium cation formed by N,N-diisopropylethylamine. Thus, the siRNA and the siRNA conjugate according to the present invention may be at least partially present in the form of a salt. According to one embodiment of the present invention, a non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphothiodiester bond is at least partially bound to a sodium ion, and thus the siRNA and the siRNA conjugate according to the present invention are present or partially present in the form of a sodium salt.

[0529] It is well known to those skilled in the art that a modified nucleotide moiety can be introduced into the siRNA of the present invention using a nucleoside monomer with an appropriate modification. Methods for producing a nucleoside monomer having an appropriate modification and methods for introducing a modified nucleotide moiety into siRNA are also well known to those skilled in the art. All modified nucleoside monomers can be commercially available or prepared using known methods.

[0530] Obtaining an siRNA conjugate represented by Formula (308)

[0531] The siRNA conjugate represented by Formula (308) can be produced by any appropriate synthetic routes.

[0532] According to some embodiments, the siRNA conjugate represented by Formula (308) can be obtained by the following method, including: sequentially connecting nucleoside monomers in the 3' to 5' direction according to the type and sequence of nucleotides in the sense strand and the antisense strand of the siRNA, respectively, under the condition of solid-phase phosphoamidite synthesis, wherein the step of connecting each nucleoside monomer comprises a four-step reaction of deprotection, coupling, capping and oxidation or sulfurization; isolating the sense strand and the antisense strand of the siRNA; and renaturation; wherein the siRNA is the above-mentioned siRNA according to the present invention.

[0533] Furthermore, the method further comprises: bringing the compound represented by Formula (321) into contact with a nucleoside monomer or a nucleotide sequence linked to a solid phase support under a coupling reaction condition and in the presence of a coupling agent, which results in the binding of the compound represented by Formula (321) to the nucleotide sequence by the coupling reaction. Hereinafter, the compound represented by Formula (321) is also referred to as a conjugating molecule.

[0534]

[0535] where

[0536] R4 is a group capable of binding to the siRNA represented by Nu in the compound represented by Formula (308). According to some embodiments of the present invention, R4 is a group capable of binding to the siRNA represented by Nu via a covalent bond. According to some embodiments of the present invention, Pa is a group that can be conjugated to any functional group of the siRNA represented by Nu via a phosphodiester bond by the reaction;

[0537] Each S1 independently represents a group formed by replacing all active hydroxyls in M1 with a YCOO- group, wherein each Y is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; in some embodiments of the invention, Y is methyl.

[0538] definitions and options n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L1 and M1, respectively, are described above.

[0539] R4 is selected to provide a connection to the N atom on the nitrogen backbone and to provide a suitable reaction site for the synthesis of the siRNA conjugate represented by Formula (308). According to some embodiments of the present invention, R4 comprises a linking group R2 or a protected linking group R2 and a functional group that can react with siRNA to form a structure represented by Formula (A59).

[0540] According to some embodiments of the present invention, R4 comprises a first functional group that can react with a group on the siRNA represented by Nu or a nucleoside monomer to form a phosphite ester, and a second functional group that can form a covalent bond with a hydroxy group or an amino group, or comprises a solid phase support attached via a covalent bond. According to some embodiments of the present invention, the first functional group is a phosphoamidite, hydroxyl, or protected hydroxyl. According to some embodiments of the present invention, the second functional group is a phosphoamidite, carboxyl, or carboxylate salt.According to some embodiments of the present invention, the second functional group is a solid phase support linked to the rest of the molecule via a covalent bond formed by a hydroxyl group or an amino group. According to some embodiments of the present invention, the solid phase support is linked via a phosphoester linkage, a carboxyester linkage, or an amide linkage. According to some embodiments of the present invention, the solid phase support is a resin.

[0541] According to some embodiments of the present invention, the first functional group comprises hydroxy, -OR k or a group represented by Formula (C3); the second functional group has a structure represented by Formula (C1), (C2), (C3), (C1') or (C3'):

[0542]

[0543] in which q1 is an integer from 1 to 4, X is O or NH, M+ is a cation, R k is the hydroxyl protecting group, SPS is the solid phase support and represents the position at which the group is covalently attached.

[0544] According to some embodiments of the present invention, the first functional group comprises a phosphoamidite group such as a group represented by Formula (C3). The phosphoamidite group can form a phosphite ester with a hydroxyl at any position on a nucleotide (such as 2'-hydroxy or 3'-hydroxy) through a coupling reaction, and the phosphite ester can form a phosphodiester bond or a phosphorothioester bond represented by Formula (A59) through oxidation or sulfurization, so as to conjugate the conjugating molecule with the siRNA. Herein, even if the second functional group does not exist, the compound represented by Formula (321) can still be conjugated with a nucleotide without affecting the production of the siRNA conjugate represented by Formula (308).Under such circumstances, after obtaining the sense or antisense strand of the siRNA by a method such as solid-phase phosphoamidite synthesis, the compound represented by Formula (321) is reacted with a hydroxyl at the terminal nucleotide of the nucleotide sequence and, through a subsequent oxidation or sulfurization process, a complex phosphodiester bond or a bond with a phosphorothioate compound is formed, resulting in conjugation of the compound represented by Formula (321) with the siRNA.

[0545] According to some embodiments of the present invention, the first functional group comprises a protected hydroxy group. According to some embodiments of the present invention, the second functional group comprises a group that can react with a solid phase support to provide a conjugate molecule comprising the solid phase support. According to some embodiments of the present invention, the second functional group comprises a carboxyl, a carboxylate salt, or a phosphoamidite, for example, a functional group represented by Formula (C1), (C2), or (C3). If the second functional group comprises a carboxyl or carboxylate salt, the compound represented by Formula (321) can react with a hydroxy or amino group on a solid phase support (such as a resin) via an esterification or amidation reaction to form a conjugate molecule comprising a solid phase support attached via a carboxylate ester linkage.If the second functional group contains a phosphoamidite functional group, the compound represented by Formula (321) can be linked to a hydroxy group on a universal solid-phase support (such as a resin) and, by oxidation, forms a conjugating molecule containing the solid-phase support attached via a phosphodiester bond. Then, starting from the above product coupled to the solid-phase support, nucleoside monomers are sequentially coupled using a solid-phase phosphoamidite synthesis method to obtain the sense strand or antisense strand of siRNA linked to the conjugating group. During the solid-phase phosphoamidite synthesis, the first functional group is deprotected and then coupled to the phosphoamidite group on the nucleoside monomer under coupling reaction conditions.

[0546] According to some embodiments of the present invention, the first functional group comprises a hydroxy group or a protected hydroxy group; the second functional group comprises a solid phase support attached via a carboxyl ester bond, an amide bond, or a phosphoester bond as represented by Formula (C1') or (C3'). Under such circumstances, starting from a compound represented by Formula (321), instead of a solid phase support, nucleoside monomers are sequentially coupled by a solid-phase phosphoamidite synthesis method to obtain a sense strand or an antisense strand of siRNA linked to a conjugating group.

[0547] According to some embodiments of the present invention, the carboxylate may be represented as -COO - M + , where M + is a cation such as selected from a metal cation, an ammonium cation NH4 +and an organic ammonium cation. According to one embodiment of the present invention, the metal cation may be an alkali metal cation, such as K + or Na + To enhance solubility and facilitate the reaction, according to some embodiments of the present invention, the organic ammonium cation is an ammonium cation formed by a tertiary amine or a quaternary ammonium cation, such as an ammonium cation formed by triethylamine or an ammonium cation formed by N,N-diisopropylethylamine. According to some embodiments of the present invention, the carboxylate is triethylamine carboxylate or N,N-diisopropylethylamine carboxylate.

[0548] According to some embodiments of the present invention, R4 comprises a structure represented by Formula (B9), (B10), (B9'), (B10'), (B11), (B12), (B11') or B(12'):

[0549]

[0550]

[0551] in which q1 is an integer from 1 to 4, q2 is an integer from 1 to 10, X is O or NH, M + is a cation, R k is a hydroxyl protecting group, SPS is a solid phase support and represents the position at which the group is covalently attached. In some embodiments of the present invention, q1 is 1 or 2. In some embodiments of the present invention, q2 is an integer from 1 to 5. In some embodiments of the present invention, R4 comprises a structure represented by Formula (B9) or (B10). In some embodiments of the present invention, R4 comprises a structure represented by Formula (B11) or (B12).

[0552] According to some embodiments of the present invention R kis one or more of Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4''-trimethoxytrityl). According to some embodiments of the present invention, R k may represent DMTr, i.e. 4,4''-dimethoxytrityl.

[0553] The definition of L1 is described above.

[0554] According to some embodiments of the present invention, L1 is used to connect the targeting group M1 to the N atom on the nitrogen backbone, which provides the liver targeting function for the siRNA conjugate represented by Formula (308). According to some embodiments of the present invention, L1 comprises any one of Formulas (A1) to (A26) or a combination thereof.

[0555] According to the above description, those skilled in the art will readily understand that, compared with the solid-phase phosphoamidite synthesis method well known in the art, the siRNA conjugate represented by Formula (308), in which the conjugating molecule is linked to any possible position of the nucleotide sequence, can be obtained using the above-mentioned first functional group and an optional second functional group. For example, the conjugating molecule is linked to the terminal region of the nucleotide sequence or to the end of the nucleotide sequence.Accordingly, unless otherwise stated, in the following description concerning the preparation of a conjugate and / or conjugating molecule, when referring to reactions such as "deprotection," "coupling," "capping," "oxidation," and "sulfurization," it should be understood that the reaction conditions and agents involved in the solid-phase phosphoamidite synthesis method, well known in the art, will also apply to these reactions. Exemplary reaction conditions and agents will be described in detail below.

[0556] According to some embodiments of the present invention, each S1 is independently M1. In some embodiments of the present invention, each S1 is independently a group formed by protecting at least one active hydroxyl group in M1 with a hydroxyl-protecting group. In some embodiments of the present invention, each S1 is independently a group formed by protecting all active hydroxyl groups in M1 with hydroxyl-protecting groups. In some embodiments of the present invention, any hydroxyl-protecting group known to one of skill in the art can be used to protect an active hydroxyl group in M1. In some embodiments of the present invention, the protected hydroxyl can be represented by the Formula YCOO-, wherein each Y is independently selected from the group consisting of C1-C 10 alkyl and C6-C10 aryl, which is optionally substituted with one or more substituents selected from the group consisting of halogen and C1-C6 alkyl. According to some embodiments of the present invention, each Y is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and C1-C6 alkylphenyl.

[0557] According to some embodiments of the present invention, each S1 is independently selected from the group consisting of Formulas (A46) to (A54):

[0558]

[0559] According to some embodiments of the present invention, S1 is A49 or A50.

[0560] According to some embodiments of the present invention, each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl. According to some embodiments of the present invention, Y is methyl.

[0561] As mentioned previously, the method for producing the siRNA conjugate represented by Formula (308) further comprises the following steps: synthesizing another strand of siRNA (for example, if the sense strand of siRNA linked to the conjugating molecule is synthesized in the above step, the method further comprises synthesizing the antisense strand of siRNA by a solid-phase synthesis method, and vice versa), isolating the sense strand and the antisense strand, and renaturing. Specifically, in the isolating step, the solid-phase support linked to the nucleotide sequence and / or the conjugating molecule is cleaved, and the necessary protecting group is removed (in this case, each S1 group in the compound of Formula (321) is converted into the corresponding targeting group M1) to obtain the sense strand (or antisense strand) of siRNA linked to the conjugating molecule and the corresponding antisense strand (or sense strand).The sense strand and antisense strand are annealed to form a double-stranded RNA structure, resulting in an siRNA conjugate represented by Formula (308).

[0562] According to some embodiments of the present invention, a method for producing an siRNA conjugate represented by Formula (308) comprises the following steps: bringing a compound represented by Formula (321) into contact with a first nucleoside monomer at the 3'-end of a sense strand or an antisense strand under a coupling reaction condition in the presence of a coupling agent, which results in the coupling of the compound represented by Formula (321) to the first nucleotide in the sequence;sequentially coupling nucleoside monomers in a 3' to 5' direction to synthesize a sense or antisense strand of siRNA according to a desired type and sequence of nucleotides of the sense or antisense strand under solid-phase phosphoramidite synthesis conditions, wherein the compound represented by Formula (321) is a compound in which R4 comprises a first functional group and a second functional group, wherein the first functional group comprises a protected hydroxyl, and the second functional group has a structure represented by Formula (C1') or (C3'), wherein the compound of Formula (321) is deprotected before coupling with the first nucleoside monomer; and coupling of each nucleoside monomer comprises a four-step reaction of deprotection, coupling, capping, and oxidation or sulfurization; thereby obtaining a sense or antisense strand of nucleic acid coupled to a conjugating group;sequentially coupling nucleoside monomers in a 3' to 5' direction to synthesize an antisense or sense strand of nucleic acid according to the type and sequence of nucleotides in the sense or antisense strand under solid-phase phosphoramidite synthesis conditions; wherein coupling of each nucleoside monomer comprises a four-step reaction of deprotection, coupling, capping, and oxidation or sulfurization; removal of the protecting group and cleavage of the solid-phase support; isolation and purification of the sense strand and the antisense strand of nucleic acid; and renaturation.

[0563] According to some embodiments of the present invention, a method for producing an siRNA conjugate represented by Formula (308) comprises the following steps: sequentially linking nucleoside monomers in a 3' to 5' direction to synthesize an antisense strand and a sense strand according to a type and sequence of nucleotides in a double-stranded siRNA; wherein the linking of each nucleoside monomer comprises a four-step reaction of deprotection, coupling, capping, and oxidation or sulfurization to produce a sense strand linked to a solid phase support and an antisense strand linked to a solid phase support;contacting a compound represented by Formula (321) with a sense strand coupled to a solid phase support or an antisense strand coupled to a solid phase support under a coupling reaction condition in the presence of a coupling agent, resulting in the coupling of the compound represented by Formula (321) with the sense strand or the antisense strand; wherein the compound represented by Formula (321) is a compound in which R4 comprises a first functional group which is a phosphoramidite group; removing the protecting group and cleaving the solid phase support; respectively, isolating and purifying the sense strand or the antisense strand of the siRNA; and renaturing, wherein the sense strand or the antisense strand of the siRNA is coupled to a conjugating group.

[0564] According to some embodiments of the present invention, the P atom in Formula (A59) is connected to the 3'-end of the sense strand of the siRNA, and the method for producing the siRNA conjugate represented by Formula (308) comprises:

[0565] (1) Removal of the hydroxyl protecting group P k in a compound represented by Formula (321), wherein the compound represented by Formula (321) is a compound in which R4 contains a first functional group containing a protected hydroxyl group OR k , and a second functional group that contains a structure represented by Formula (C1') or (C3'); contacting the deprotected product with a nucleoside monomer to obtain a nucleoside monomer linked to a solid phase support via a conjugating molecule under coupling reaction conditions;

[0566] (2) starting from a nucleoside monomer linked to a solid-phase support via a conjugating molecule, synthesizing a sense strand of siRNA in the 3' to 5' direction using a solid-phase phosphoamidite synthesis method;

[0567] (3) synthesizing the antisense strand of siRNA by a solid-phase phosphoamidite synthesis method; and

[0568] (4) isolating the sense strand and the antisense strand of the siRNA and renaturing them to obtain an siRNA conjugate represented by Formula (308).

[0569] At the same time, in step (1), the method of removing the protective group R kin a compound represented by Formula (321) comprises contacting the compound represented by Formula (321) with a deprotecting agent under deprotecting conditions. The deprotecting conditions include a temperature of 0-50°C and, in some embodiments, 15-35°C, and a reaction time of 30-300 seconds and, in some embodiments, 50-150 seconds. The deprotecting agent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid and monochloroacetic acid and, in some embodiments, the deprotecting agent is dichloroacetic acid. The molar ratio of the deprotecting agent to the compound represented by Formula (321) is from 10:1 to 1000:1 and, in some embodiments, from 50:1 to 500:1.

[0570] The coupling reaction condition and coupling agent may be any condition and agent suitable for the above coupling reaction. According to some embodiments of the present invention, the same conditions and agent as in the coupling reaction in the solid-phase synthesis method may be used.

[0571] According to some embodiments of the present invention, the coupling reaction condition comprises a reaction temperature of 0-50°C, and according to some embodiments of the present invention, 15-35°C. The molar ratio of the compound represented by Formula (321) to the nucleoside monomer is from 1:1 to 1:50, and according to some embodiments of the present invention, from 1:2 to 1:5. The molar ratio of the compound represented by Formula (321) to the coupling agent can be from 1:1 to 1:50, and according to some embodiments of the present invention, from 1:3 to 1:10. The reaction time is 200-3000 seconds, and according to some embodiments of the present invention, 500-1500 seconds. The binding agent is selected from one or more of 1H-tetrazole, 5-ethylthio-1H-tetrazole and 5-benzylthio-1H-tetrazole and, according to some embodiments of the present invention, is 5-ethylthio-1H-tetrazole.The coupling reaction can be performed in an organic solvent. The organic solvent is selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, and, according to some embodiments of the present invention, is anhydrous acetonitrile. Relative to the compound represented by Formula (321), the amount of organic solvent is 3-50 L / mol, and, according to some embodiments of the present invention, 5-20 L / mol.

[0572] In step (2), the sense strand of the SS siRNA conjugate is synthesized in the 3' to 5' direction using solid-phase phosphoamidite synthesis, starting from a nucleoside monomer linked to a solid-phase support via a conjugating molecule obtained in the preceding step. In this case, the conjugation group is attached to the 3' end of the resulting sense strand.

[0573] As other solid-phase synthesis conditions in steps (2) and (3), including the deprotection condition of the nucleoside monomer, the type and amount of the deprotection agent, the coupling reaction condition, the type and amount of the coupling agent, the capping reaction condition, the type and amount of the capping agent, the oxidation reaction condition, the type and amount of the oxidizing agent, the sulfurization reaction condition, and the type and amount of the sulfurization agent, various agents, amounts and conditions commonly used in the art can be used.

[0574] According to some embodiments of the present invention, for example, the solid phase synthesis in steps (2) and (3) can be carried out using the following conditions:

[0575] The deprotection condition for the nucleoside monomer includes a reaction temperature of 0-50°C and, according to some embodiments of the present invention, 15-35°C, and a reaction time of 30-300 seconds and, according to some embodiments of the present invention, 50-150 seconds. The deprotecting agent can be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid and monochloroacetic acid and, according to some embodiments of the present invention, is dichloroacetic acid. The molar ratio of the deprotecting agent to the 4,4'-dimethoxytrityl protecting group on the solid phase support is from 2:1 to 100:1 and, according to some embodiments, from 3:1 to 50:1.

[0576] The reaction condition for the coupling includes a reaction temperature of 0-50°C and, according to some embodiments of the invention, 15-35°C. The molar ratio of the nucleic acid sequence linked to the solid phase support to the nucleoside monomer is from 1:1 to 1:50 and, according to some embodiments, from 1:5 to 1:15. The molar ratio of the nucleic acid sequence linked to the solid phase support to the coupling agent is from 1:1 to 1:100 and, according to some embodiments of the present invention, is from 1:50 to 1:80. The selection of the reaction time and the coupling agent is the same as indicated above.

[0577] The capping reaction condition includes a reaction temperature of 0-50°C and, according to some embodiments of the present invention, 15-35°C, as well as a reaction time of 5-500 seconds and, according to some embodiments of the present invention, 10-100 seconds. The selection of the capping agent is the same as indicated above. The molar ratio of the total amount of the capping agent to the nucleic acid sequence linked to the solid phase support is from 1:100 to 100:1 and, according to some embodiments of the present invention, is from 1:10 to 10:1. In the case where an equimolar amount of acetic anhydride and N-methylimidazole is used as the capping agent, the molar ratio of acetic anhydride, N-methylimidazole and nucleic acid sequence linked to the solid phase support may be 1:1:10-10:10:1 and, according to some embodiments of the present invention, is 1:1:2:2:1.

[0578] The oxidation reaction condition includes a reaction temperature of 0-50°C and, according to some embodiments of the present invention, 15-35°C, and a reaction time of 1-100 seconds and, according to some embodiments of the present invention, 5-50 seconds. According to some embodiments of the present invention, the oxidizing agent is iodine (according to some embodiments of the present invention, provided in the form of a solution of iodine in water). The molar ratio of the oxidizing agent to the nucleic acid sequence linked to the solid phase support in the binding step can be from 1:1 to 100:1 and, according to some embodiments of the present invention, from 5:1 to 50:1. According to some embodiments of the present invention, the oxidation reaction is performed in a mixed solvent in which the ratio of tetrahydrofuran:water:pyridine is 3:1:1 to 1:1:3.The sulfurization reaction condition comprises a reaction temperature of 0-50°C and, according to some embodiments of the present invention, 15-35°C, and a reaction time of 50-2000 seconds and, according to some embodiments of the present invention, 100-1000 seconds. According to some embodiments of the present invention, the sulfurization agent is xanthan hydride. The molar ratio of the sulfurization agent to the nucleic acid sequence linked to the solid phase support in the linking step is from 10:1 to 1000:1 and, according to some embodiments of the present invention, from 10:1 to 500:1. According to some embodiments of the present invention, the sulfurization reaction is carried out in a mixed solvent in which the acetonitrile:pyridine ratio is 1:3-3:1.

[0579] The method further includes isolating the sense strand and antisense strand of the siRNA after coupling all nucleoside monomers and before renaturation. Isolation methods are well known to those skilled in the art and typically involve cleaving the synthesized nucleotide sequence from the solid-phase support, removing protecting groups from bases, phosphate groups, and ligands, purification, and desalting.

[0580] Generally accepted methods for cleavage and deprotection in the synthesis of siRNA can be used to cleave the synthesized nucleotide sequence from the solid phase support and remove the protecting groups on the bases, phosphate groups and ligands. For example, the obtained nucleotide sequence bound to the solid phase support is brought into contact with a concentrated aqueous ammonia solution; during deprotection, the YCOO- protecting group in the A46-A54 groups is converted into a hydroxyl group, and thus the S1 groups are converted into the corresponding M1 groups, which provides an siRNA conjugate represented by Formula (308); wherein the concentrated aqueous ammonia solution may be an aqueous ammonia solution with a concentration of 25-30 mass%. In relation to the target siRNA sequence, the amount of concentrated aqueous ammonia solution can range from 0.2 ml / μmol to 0.8 ml / μmol.

[0581] If the synthesized nucleotide sequence has at least one 2'-TBDMS protection, the method further comprises contacting the nucleotide sequence cleaved from the solid phase support with triethylamine trihydrofluoride to remove the 2'-TBDMS protection. Herein, the corresponding nucleoside in the obtained target siRNA sequence has a free 2'-hydroxyl. With respect to the target siRNA sequence, the amount of pure triethylamine trihydrofluoride can be 0.4 ml / μmol-1.0 ml / μmol. Thus, the siRNA conjugate represented by Formula (308) can be obtained.

[0582] Purification and desalting methods are well known to those skilled in the art. For example, nucleic acid purification can be accomplished using a preparative ion chromatography column with gradient elution using NaBr or NaCl; after product collection and pooling, desalting can be accomplished using a reversed-phase chromatography column.

[0583] The non-bridging oxygen or sulfur atom in the phosphodiester bond or the phosphothiodiester bond between nucleotides in the resulting siRNA conjugate of Formula (308) is substantially bound to a sodium ion, and the siRNA conjugate of Formula (308) is substantially present in the form of a sodium salt. Well-known ion exchange methods can be used in which the sodium ion can be replaced by a hydrogen ion and / or other cations, thereby providing other forms of the siRNA conjugates represented by Formula (308). The cations are those described above.

[0584] During synthesis, the purity and molecular weight of the nucleic acid sequence can be determined at any time to better control the quality of the synthesis. Such determination methods are well known to those skilled in the art. For example, nucleic acid purity can be determined using ion-exchange chromatography, and molecular weight can be determined using liquid chromatography-mass spectrometry (LC-MS).

[0585] Methods for renaturation are also well known to those skilled in the art. For example, the synthesized sense strand (S-strand) and antisense strand (AS-strand) can be simply mixed in water for injection at an equimolar ratio, heated to 70-95°C, and then cooled at room temperature to form a double-stranded structure through hydrogen bonding. Consequently, an siRNA conjugate represented by Formula (308) can be obtained.

[0586] After obtaining the conjugate, according to some embodiments of the present invention, the synthesized siRNA conjugate represented by Formula (308) can also be characterized by means such as determining the molecular weight using methods such as liquid chromatography-mass spectrometry to confirm that the synthesized siRNA conjugate is the siRNA conjugate represented by Formula (308) of interest, and the sequence of the synthesized siRNA is a desired siRNA sequence, for example, is one of the sequences listed in Table 1.

[0587] The compound represented by Formula (321) can be produced by the following production method, which comprises: bringing the compound represented by Formula (313) into contact with a cyclic anhydride in an organic solvent under esterification reaction condition in the presence of a base and an esterification catalyst; ion exchange and isolating the compound represented by Formula (321):

[0588]

[0589] in this case, definitions and options n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L1 and S1, respectively, are described above.

[0590] R6 is a group to provide R4 of Formula (321); in some embodiments of the invention, R6 has a structure represented by Formula (A61):

[0591]

[0592] where R irepresents any group capable of combining with an N atom on the nitrogen skeleton, combining with R k O and combine with a free hydroxy group; R k represents a hydroxyl-protecting group. In this case, a compound represented by Formula (321) is obtained, where R4 contains a first functional group as a hydroxyl-protecting group and a second functional group that contains a structure represented by Formula (C1) or (C2).

[0593] The esterification reaction condition comprises a reaction temperature of 0-100°C and a reaction time of 8-48 hours. According to some embodiments of the present invention, the esterification reaction condition comprises a reaction temperature of 10-40°C and a reaction time of 20-30 hours.

[0594] According to some embodiments of the present invention, the organic solvent comprises one or more of an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. According to some embodiments of the present invention, the epoxy solvent is dioxane and / or tetrahydrofuran, the ether solvent is diethyl ether and / or tert-butyl methyl ether, and the haloalkane solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. According to some embodiments of the present invention, the organic solvent is dichloromethane. The amount of the organic solvent is 3-50 L / mol, and, according to some embodiments of the present invention, 5-20 L / mol, relative to the compound represented by Formula (313).

[0595] According to some embodiments of the present invention, the cyclic anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride or pimelic anhydride, and according to some embodiments of the present invention, the cyclic anhydride is succinic anhydride. The molar ratio of the cyclic anhydride to the compound represented by Formula (313) is from 1:1 to 10:1, and, according to some embodiments of the present invention, from 2:1 to 5:1.

[0596] The esterification catalyst may be any catalyst capable of catalyzing esterification, such as 4-dimethylaminopyridine. The molar ratio of the catalyst to the compound represented by Formula (313) is from 1:1 to 10:1, and according to some embodiments of the present invention, is from 2:1 to 5:1.

[0597] According to some embodiments of the present invention, the base can be any inorganic base, organic base, or a combination thereof. Considering the solubility and stability of the product, the base can be, for example, a tertiary amine. According to some embodiments of the present invention, the tertiary amine is triethylamine or N,N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound represented by Formula (313) is from 1:1 to 20:1, and according to some embodiments of the present invention, from 3:1 to 10:1.

[0598] Ion exchange serves the function of converting the compound represented by Formula (321) into the target carboxylic acid or carboxylic acid salt form, and ion exchange methods are well known to those skilled in the art. The above-mentioned conjugating molecule in which the cation is M +, can be obtained using a suitable ion exchange solution and ion exchange conditions that are not specified herein. According to some embodiments of the present invention, a triethylamine phosphate solution is used in the ion exchange reaction, and the concentration of the triethylamine phosphate solution is 0.2-0.8 M. According to some embodiments of the present invention, the concentration of the triethylamine phosphate solution is 0.4-0.6 M, and the amount of the triethylamine phosphate solution is 3-6 L / mol, and in further embodiments 4-5 L / mol, relative to the compound represented by Formula (313).

[0599] The compound represented by Formula (321) can be isolated from the reaction mixture using any suitable isolation methods. According to some embodiments of the present invention, the compound represented by Formula (321) can be isolated by removing the solvent by evaporation followed by chromatography. For example, the following two chromatographic conditions can be used for isolation: (1) silica gel purification with a normal phase: silica gel filler with 200-300 mesh, gradient elution triethylamine in dichloromethane:methanol = 100:18-100:20; or (2) reverse phase purification: reverse phase filler C18 and C8, gradient elution methanol:acetonitrile = 0.1:1-1:0.1. According to some embodiments of the present invention, the solvent can be directly removed to obtain a crude product of the compound represented by Formula (321), which can be directly used in subsequent reactions.

[0600] According to some embodiments of the present invention, the method for producing a compound represented by Formula (321) further comprises: further bringing the product obtained as a result of the above ion exchange reaction into contact with a solid phase support with amino or hydroxy groups in an organic solvent under a condensation reaction condition in the presence of a condensing agent, a condensation catalyst and a tertiary amine. In this case, a compound represented by Formula (321) is obtained, wherein R4 comprises a first functional group containing a hydroxyl protecting group and a second functional group having a structure represented by Formula (C1').

[0601] The solid phase support is one of the supports used in the solid-phase synthesis of siRNA, some of which are well known to those skilled in the art. For example, the solid phase support can be selected from solid phase supports containing an active hydroxy or amino functional group(s), and in some embodiments, is an amino or hydroxy resin. In some embodiments, the amino or hydroxy resin has the following parameters: a particle size of 100-400 mesh and a surface content of amino groups or hydroxy groups of 0.2-0.5 mmol / g. The ratio of the compound represented by Formula (321) to the solid phase support is 10-400 μmol of the compound per gram of the solid phase support (μmol / g). According to some embodiments of the present invention, the ratio of the compound represented by Formula (321) to the solid phase support is from 50 μmol / g to 200 μmol / g.

[0602] The organic solvent may be any suitable solvent or mixed solvent known to those skilled in the art. According to some embodiments of the present invention, the organic solvent is one or more of acetonitrile, an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. According to some embodiments of the present invention, the epoxy solvent is dioxane and / or tetrahydrofuran; the ether solvent is diethyl ether and / or tert-butyl methyl ether; the haloalkane solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. According to some embodiments of the present invention, the organic solvent is acetonitrile.The amount of the organic solvent is 20-200 L / mol, and according to some embodiments of the present invention 50-100 L / mol, relative to the compound represented by Formula (321).

[0603] According to some embodiments of the present invention, the condensing agent may be benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 3-diethoxyphosphoryl-1,2,3-benzotrizin-4(3H)-one (DEPBT) and / or O-benzotriazoletetramethyluronium hexafluorophosphate. According to some embodiments of the present invention, the condensing agent is O-benzotriazoletetramethyluronium hexafluorophosphate. The molar ratio of the condensing agent to the compound represented by Formula (321) is from 1:1 to 20:1, and, according to some embodiments of the present invention, from 1:1 to 5:1.

[0604] According to some embodiments of the present invention, the tertiary amine is triethylamine and / or N,N-diisopropylethylamine, and, according to some embodiments of the present invention, N,N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound represented by Formula (321) is from 1:1 to 20:1, and, according to some embodiments of the present invention, from 1:1 to 5:1.

[0605] According to some embodiments of the present invention, the method for producing a compound represented by Formula (321) further comprises: bringing the obtained condensation product into contact with a capping agent and an acylation catalyst in an organic solvent under a capping reaction condition and isolating the compound represented by Formula (321). The capping reaction is used to remove any active functional group that does not react completely in order to avoid the formation of unnecessary by-products in subsequent reactions. The capping reaction condition includes a reaction temperature of 0-50°C and, according to some embodiments of the present invention, 15-35°C, as well as a reaction time of 1-10 hours and, according to some embodiments of the present invention, 3-6 hours. The capping agent may be a capping agent used in solid-phase siRNA synthesis, which is well known to those skilled in the art.

[0606] According to some embodiments of the present invention, the capping agent consists of a capping agent 1 (cap1) and a capping agent 2 (cap2). Cap1 is N-methylimidazole and, according to some embodiments of the present invention, is provided in the form of a mixed solution of N-methylimidazole in pyridine / acetonitrile, wherein the volume ratio of pyridine to acetonitrile is from 1:10 to 1:1, and, according to some embodiments of the present invention, from 1:3 to 1:1. According to some embodiments of the present invention, the ratio of the total volume of pyridine and acetonitrile to the volume of N-methylimidazole is from 1:1 to 10:1, and, according to some embodiments of the present invention, from 3:1 to 7:1.Sar2 is acetic anhydride and in some embodiments of the invention is provided in the form of a solution of acetic anhydride in acetonitrile, wherein the volume ratio of acetic anhydride to acetonitrile is from 1:1 to 1:10 and, in further embodiments of the invention, from 1:2 to 1:6.

[0607] According to some embodiments of the present invention, the volume ratio of the mixed solution of N-methylimidazole in pyridine / acetonitrile to the weight of the compound represented by Formula (321) is 5 ml / g-50 ml / g, and according to some embodiments of the present invention, 15 ml / g-30 ml / g. The volume ratio of the solution of acetic anhydride in acetonitrile to the weight of the compound represented by Formula (321) is 0.5 ml / g-10 ml / g, and according to some embodiments of the present invention, 1 ml / g-5 ml / g.

[0608] According to some embodiments of the present invention, the capping agent comprises an equimolar amount of acetic anhydride and N-methylimidazole. According to some embodiments of the present invention, the organic solvent is one or more of acetonitrile, an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. According to some embodiments of the present invention, the organic solvent is acetonitrile. The amount of the organic solvent is 10-50 L / mol and, according to some embodiments of the present invention, 5-30 L / mol relative to the compound represented by Formula (321).

[0609] In some embodiments of the invention, the acylation catalyst may be selected from any catalyst that can be used for condensation by esterification or amide condensation, such as alkali heterocyclic compounds. According to some embodiments of the present invention, the acylation catalyst is 4-dimethylaminopyridine. The weight ratio of the catalyst to the compound represented by Formula (321) is from 0.001:1 to 1:1, and, according to some embodiments of the present invention, from 0.01:1 to 0.1:1.

[0610] In some embodiments, the compound represented by Formula (321) can be isolated from the reaction mixture using any suitable separation methods. According to some embodiments of the present invention, the compound represented by Formula (321) can be obtained by thoroughly washing with an organic solvent and filtration to remove unreacted reagents, excess capping agent and other impurities, wherein the organic solvent is selected from acetonitrile, dichloromethane and methanol. According to some embodiments of the present invention, the organic solvent is acetonitrile.

[0611] According to some embodiments of the present invention, a method for producing a conjugate molecule represented by Formula (321) comprises contacting a compound represented by Formula (313) with a phosphodiamidite in an organic solvent under a coupling reaction condition in the presence of a coupling agent, and isolating the compound represented by Formula (321). In this case, a compound represented by Formula (321) is obtained, in which R4 comprises a first functional group containing a hydroxyl protecting group and a second functional group containing a structure represented by Formula (C3).

[0612] According to some embodiments of the present invention, the coupling reaction condition comprises: a reaction temperature of 0-50°C, such as 15-35°C; a molar ratio of the compound represented by Formula (313) to the phosphodiamidite of 1:1 to 1:50, such as 1:5 to 1:15; a molar ratio of the compound represented by Formula (313) to the coupling agent of 1:1 to 1:100, such as 1:50 to 1:80; and a reaction time of 200 to 3000 seconds, such as 500 to 1500 seconds. The phosphodiamidite can be, for example, bis(diisopropylamino)(2-cyanoethoxy)phosphine, which can be commercially available or synthesized according to methods well known in the art. The coupling agent is selected from one or more of 1H-tetrazole, 5-ethylthio-1H-tetrazole, and 5-benzylthio-1H-tetrazole, for example, 5-ethylthio-1H-tetrazole. The coupling reaction can be performed in an organic solvent.The organic solvent is selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, such as anhydrous acetonitrile. In some embodiments, the amount of organic solvent is 3-50 L / mol, such as 5-20 L / mol, relative to the compound represented by Formula (313). In the coupling reaction, the hydroxy group in the compound represented by Formula (313) reacts with the phosphodiamidite to form a phosphoamidite group. According to some embodiments of the present invention, the solvent can be directly removed to obtain a crude product of the compound represented by Formula (321), which can be directly used in subsequent reactions.

[0613] According to some embodiments, the method for producing a compound represented by Formula (321) further comprises the following steps: further contacting the isolated product with a solid phase support with hydroxyl groups in an organic solvent under a coupling reaction condition in the presence of a coupling agent, followed by capping, oxidation and isolation to obtain a compound represented by Formula (321), wherein R4 comprises a first functional group containing a protecting hydroxy group and a second functional group having a structure represented by Formula (C3').

[0614] According to some embodiments of the present invention, the solid phase support is a solid support well known in the art of solid phase nucleic acid synthesis, such as the commercially available universal deprotected solid phase support (NittoPhase oligonucleotide synthesis support). ®HL UnyLinker™ 300, Kinovate Life Sciences, represented by Formula B80):

[0615]

[0616] The deprotection reaction is well known to those skilled in the art. According to some embodiments of the present invention, the deprotection condition includes a temperature of 0-50°C, for example, 15-35°C, and a reaction time of 30-300 seconds, for example, 50-150 seconds. The deprotecting agent can be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid and monochloroacetic acid. According to some embodiments of the present invention, the deprotecting agent is dichloroacetic acid. The molar ratio of the deprotecting agent to the -DMTr (4,4'-dimethoxytrityl) protecting group on the solid phase support is from 2:1 to 100:1, for example, from 3:1 to 50:1. By means of such deprotection, reactive free hydroxyl groups are obtained on the surface of the solid phase support to facilitate the subsequent coupling reaction.

[0617] The coupling reaction conditions and coupling agent can be selected as described above. During the coupling reaction, the free hydroxyl groups formed during deprotection react with the phosphoamidite groups to form an ester phosphite bond.

[0618] According to some embodiments of the present invention, 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. The capping reaction is carried out in the presence of a capping agent. The selection and amount of the capping agent are described above.

[0619] The oxidation reaction condition includes a temperature of 0-50°C, for example, 15-35°C, and a reaction time of 1-100 seconds, for example, 5-50 seconds. The oxidizing agent may be, for example, iodine (according to some embodiments of the present invention, it is provided in the form of an iodine solution in water). According to some embodiments of the present invention, the molar ratio of the oxidizing agent to the nucleic acid sequence linked to the solid phase support is from 1:1 to 100:1, for example, it may be from 5:1 to 50:1. According to some embodiments of the present invention, the oxidation reaction is performed in a mixed solvent in which the ratio of tetrahydrofuran: water: pyridine = 3:1:1-1:1:3.

[0620] According to some embodiments of the present invention, R6 is one of the groups of Formula B7 or B8:

[0621]

[0622] where the definition of q2 is the one described above.

[0623] In this case, the compound represented by Formula (313) can be obtained by the following production method, including: bringing the compound represented by Formula (314) into contact with a compound represented by Formula (A-1) or (A-2) in an organic solvent under an amidation reaction condition in the presence of a condensing agent for an amidation reaction and a tertiary amine, followed by isolation:

[0624]

[0625] in this case, definitions and options n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L1, S1, q2 and R k , respectively, are described above.

[0626] The amidation reaction condition may include a reaction temperature of 0-100°C and a reaction time of 1-48 hours. According to some embodiments of the present invention, the amidation reaction condition includes a reaction temperature of 10-40°C and a reaction time of 2-16 hours.

[0627] According to some embodiments of the present invention, the organic solvent is one or more of an alcohol solvent, an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. According to some embodiments of the present invention, the alcohol solvent is one or more of methanol, ethanol, and propanol, and, in some embodiments, is ethanol. According to some embodiments of the present invention, the epoxy solvent is dioxane and / or tetrahydrofuran. According to some embodiments of the present invention, the ether solvent is diethyl ether and / or tert-butyl methyl ether.According to some embodiments of the present invention, the haloalkane solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. According to some embodiments of the present invention, the organic solvent is dichloromethane. The amount of the organic solvent is 3-50 L / mol and, according to some embodiments of the present invention, 3-20 L / mol relative to the compound represented by Formula (314).

[0628] In some embodiments, the condensing agent for the amidation reaction is benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-benzotrizin-4(3H)-one, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) or O-benzotriazoletetramethyluronium hexafluorophosphate, and in other embodiments, is 3-diethoxyphosphoryl-1,2,3-benzotrizin-4(3H)-one. The molar ratio of the condensing agent for the amidation reaction to the compound represented by Formula (314) can be from 1:1 to 10:1 and, according to some embodiments of the present invention, from 2.5:1 to 5:1.

[0629] According to some embodiments of the invention, the tertiary amine is triethylamine or N,N-diisopropylethylamine, and according to some embodiments of the invention is N,N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound represented by Formula (314) is from 3:1 to 20:1, and, according to some embodiments of the present invention, from 5:1 to 10:1.

[0630] The compounds represented by Formulas (A-1) and (A-2) can be prepared by any suitable methods. For example, if R kis a DMTr group, the compound represented by Formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl. Similarly, the compound represented by Formula (A-2) can be prepared by contacting 3-amino-1,2-propanediol with a cyclic anhydride and then reacting with DMTrCl, wherein the cyclic anhydride can contain 4-13 carbon atoms and, in some embodiments, 4-8 carbon atoms. Those skilled in the art will readily appreciate that sets of different cyclic anhydrides correspond to different q2 values ​​in the compound represented by Formula (A-2). For example, if the cyclic anhydride is succinic anhydride, q2=1; if the cyclic anhydride is glutaric anhydride, q2=2, and so on.

[0631] According to some embodiments of the present invention, the compound represented by Formula (313) can also be obtained by sequentially reacting the compound represented by Formula (314) with a cyclic anhydride, 3-amino-1,2-propanediol and DMTrCl. Those skilled in the art will readily understand that these variations will not affect the structure and function of the compound represented by Formula (313), and these variations are easily realized by those skilled in the art based on the above methods.

[0632] Similarly, the compound represented by Formula (313) can be isolated from the reaction mixture by any suitable isolation methods. According to some embodiments of the present invention, the compound represented by Formula (313) can be isolated by removing the solvent by evaporation, followed by chromatography. For example, the following two chromatographic conditions can be used for the isolation: (1) normal phase silica gel purification: 200-300 mesh silica gel packing, gradient elution of petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5-1:1:0.6; and (2) reversed phase purification: C18 and C8 reversed phase packings, gradient elution of methanol: acetonitrile = 0.1:1-1:0.1.According to some embodiments of the present invention, the solvent can be directly removed to yield a crude product of the compound represented by Formula (313), which can be directly used in subsequent reactions.

[0633] According to some embodiments of the present invention, a compound represented by Formula (314) can be obtained by the following production method, comprising: bringing a compound represented by Formula (320) into contact with a compound represented by Formula (316) in an organic solvent under a condensation reaction condition in the presence of a condensation agent for an amidation reaction and a tertiary amine, followed by isolation:

[0634]

[0635]

[0636]

[0637] in this case, definitions and options n1, n3, m1, m2, m3, R 10 , R11 , R 12 , R 13 , R 14 and R 15 , respectively, are described above.

[0638] The compound represented by Formula (316) may be, for example, a compound described in J. Am. Chem. Soc. 2014, 136, 16958-16961. According to another embodiment, the compounds represented by Formula (316) can be prepared by those skilled in the art using various methods. For example, some compounds represented by Formula (316) can be prepared according to the method described in Example 1 of U.S. Patent US 8,106,022 B2, which is fully incorporated into this application by reference.

[0639] According to some embodiments of the present invention, the condensation reaction condition includes a reaction temperature of 0-100°C and a reaction time of 0.1-24 hours, and, according to some embodiments of the present invention, a reaction temperature of 10-40°C and a reaction time of 0.5-16 hours.

[0640] Considering the structure of the target product compound represented by Formula (314), the molar ratio of the compound represented by Formula (316) to the compound represented by Formula (320) should be determined based on the sum of n1 and n3 in Formula (320). According to some embodiments of the present invention, for example, when n1+n3=3, in order to complete the reaction without leaving any excess reagent, the molar ratio of the compound represented by Formula (316) to the compound represented by Formula (320) may be from 3:1 to 3.5:1, and according to some embodiments of the present invention, from 3.01:1 to 3.15:1.

[0641] According to some embodiments of the present invention, the organic solvent is one or more of acetonitrile, an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. According to some embodiments of the present invention, the epoxy solvent is dioxane and / or tetrahydrofuran. According to some embodiments of the present invention, the ether solvent is diethyl ether and / or tert-butyl methyl ether. According to some embodiments of the present invention, the haloalkane solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. According to some embodiments of the present invention, the organic solvent is dichloromethane.The amount of organic solvent may be 3-50 L / mol and, according to some embodiments of the present invention, 5-20 L / mol relative to the compound represented by Formula (320).

[0642] According to some embodiments of the present invention, the condensing agent for the amidation reaction is one or more of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryloxy-1,2,3-benzotrizin-4(3H)-one (DEPBT), O-benzotriazoletetramethyluronium hexafluorophosphate, or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride or 1-hydroxybenzotriazole, and according to further embodiments of the present invention is a mixture of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate with 1-hydroxybenzotriazole, wherein benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate and 1-hydroxybenzotriazole are used in equimolar amounts. The molar ratio of the common condensing agent for the amide reaction to the compound represented by Formula (316) can be from 1:1 to 3:1 and, according to some embodiments of the present invention, from 1.05:1 to 1.5:1.

[0643] The tertiary amine may be N-methylmorpholine, triethylamine, or N,N-diisopropylethylamine, and, according to some embodiments of the present invention, N-methylmorpholine. The molar ratio of the tertiary amine to the compound represented by Formula (316) may be from 2:1 to 10:1, and, according to some embodiments of the present invention, from 2:1 to 5:1.

[0644] Similarly, the compound represented by Formula (314) can be isolated from the reaction mixture by any suitable isolation method. According to some embodiments of the present invention, the compound represented by Formula (314) can be isolated by removing the solvent by evaporation followed by chromatography, for example, using the following two chromatographic conditions for isolation: (1) normal phase silica gel purification: silica gel packing with 200-300 mesh, gradient elution of dichloromethane:methanol = 100:5-100:7; and (2) reverse phase purification: reverse phase packings C 18 and C8, gradient elution methanol:acetonitrile = 0.1:1-1:0.1. According to some embodiments of the present invention, the solvent can be directly removed to obtain a crude product of the compound represented by Formula (314), which can be directly used in subsequent reactions.

[0645] The compound represented by Formula (320) may be commercially available or prepared by those skilled in the art using known methods. For example, if m1=m2=m3=3, n1=1, n3=2 and R 10 , R 11 , R 12 , R 13 , R 14 and R 15 all represent H, the compound represented by Formula (320) can be commercially available from Alfa Aesar Inc.

[0646] The siRNA conjugate of the present invention can also be used in combination with other pharmaceutically acceptable excipients, which may be one or more of various formulations or compounds commonly used in the art. Detailed information is provided in the above description of the pharmaceutical compositions of the present invention.

[0647] Use of siRNA, pharmaceutical composition and conjugate containing siRNA according to the present invention

[0648] According to some embodiments of the present invention, there is provided the use of siRNA and / or a pharmaceutical composition and / or siRNA conjugate according to the present invention in the manufacture of a medicament for the treatment and / or prevention of thrombotic diseases and / or ischemic stroke.

[0649] According to some embodiments of the present invention, a method for preventing and / or treating thrombotic diseases and / or ischemic stroke is provided, comprising administering an effective amount of siRNA and / or a pharmaceutical composition and / or siRNA conjugate according to the present invention to a subject in need thereof.

[0650] The purpose of preventing and / or treating thrombotic diseases and / or ischemic stroke can be achieved based on the RNA interference mechanism by administering the siRNA according to the present invention as an active ingredient to a subject in need thereof. Therefore, the siRNA and / or pharmaceutical composition and / or siRNA conjugate according to the present invention can be used for preventing and / or treating thrombotic diseases and / or ischemic stroke or for producing a medicament for preventing and / or treating thrombotic diseases and / or ischemic stroke.

[0651] As used herein, the term "administering / administering" refers to the delivery of siRNA and / or a pharmaceutical composition and / or siRNA conjugate according to the present invention to a subject's body using a method or route that at least partially localizes the siRNA and / or pharmaceutical composition and / or siRNA conjugate according to the present invention to a target site to achieve a target effect. Suitable routes of administration for the methods according to the present invention include local administration and systemic administration. Typically, local administration results in the delivery of a larger amount of siRNA conjugate to a specific site compared to the subject's systemic circulation; whereas systemic administration results in the delivery of siRNA and / or a pharmaceutical composition and / or siRNA conjugate according to the present invention to the subject's main systemic circulation.Taking into account that the present invention is intended to provide means for the prevention and / or treatment of thrombotic diseases and / or ischemic stroke, according to some embodiments of the present invention, a method of administration is used that is capable of delivering a drug to the liver.

[0652] Administration to a subject can be carried out 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, transdermal administration, intratracheal administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including buccal administration and sublingual administration). The frequency of administration can be once or more times per day, weekly, once every two weeks, once every three weeks, monthly, or annually.

[0653] The dosage of siRNA or the pharmaceutical composition or siRNA conjugate used according to the present invention may be a conventional dosage in the art, which can be determined according to various parameters, in particular, the age, weight, and gender of the subject. Toxicity and efficacy can be determined in cell cultures or in experimental animals using standard pharmaceutical procedures, for example, by determining the LD 50 (lethal dose causing death of 50% of the population) and ED 50 (the dose that can elicit 50% of the maximal response intensity for a quantitative response and that results in 50% of experimental subjects having a positive response for a qualitative response). The dose range for human use can be derived from data obtained from cell culture analysis and animal studies.

[0654] When administering the siRNA, pharmaceutical composition and / or siRNA conjugate of the present invention to, for example, a male or female C57b1 / 6N mouse aged 6 to 12 weeks and weighing 18 to 25 g, depending on the amount of siRNA: (i) for the siRNA conjugate, the dose of siRNA may be from 0.001 to 100 mg / kg body weight, in some embodiments from 0.01 to 50 mg / kg body weight, in some embodiments from 0.05 to 20 mg / kg body weight, in further embodiments from 0.1 to 15 mg / kg body weight, and in further embodiments from 0.1 to 10 mg / kg body weight; (ii) for a pharmaceutical composition formed by siRNA and a pharmaceutically acceptable carrier, the dose of siRNA may be from 0.001 to 50 mg / kg body weight, in some embodiments from 0.01 to 10 mg / kg body weight, in some embodiments from 0.05 to 5 mg / kg body weight, and in some embodiments from 0.1 to 3 mg / kg body weight.

[0655] According to some embodiments of the present invention, there is provided a method for inhibiting the expression of the FXI gene in hepatocytes, comprising contacting an effective amount of siRNA and / or a pharmaceutical composition and / or siRNA conjugate according to the present invention with hepatocytes and administering the siRNA and / or the pharmaceutical composition and / or siRNA conjugate according to the present invention to the hepatocytes in order to inhibit the expression of the FXI gene in hepatocytes via an RNA interference mechanism. The hepatocytes can be selected from hepatoma cell lines (such as SMMC-7721, HepG2 and Huh7) or isolated primary liver cells. According to some embodiments of the present invention, the hepatocytes are HepG2 hepatoma cells.

[0656] When the expression of the FXI gene in a cell is inhibited using the method of the present invention, the amount of siRNA in the modified siRNA and / or pharmaceutical composition and / or siRNA conjugate of the present invention is generally such an amount that is sufficient to reduce the expression of the target gene and 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 on the surface of target cells. The amount required to achieve this local concentration will vary depending on various factors, including the delivery method, the delivery site, the number of cell layers between the delivery site and the target cells or tissues, the delivery route (local or systemic), etc. The concentration at the delivery site can be significantly higher than the concentration on the surface of the target cells or tissues.

[0657] Set

[0658] According to the present invention, there is provided a kit comprising an effective amount of at least one of a modified siRNA, a pharmaceutical composition and an siRNA conjugate according to the present invention.

[0659] According to some embodiments of the present invention, a kit according to the present invention may provide a modified siRNA in a container. According to some embodiments of the present invention, a kit according to the present invention may comprise a container containing a pharmaceutically acceptable excipient. According to some embodiments, said kit may further comprise other ingredients, such as stabilizers or preservatives. According to some embodiments of the present invention, a kit according to the present invention may comprise at least one additional therapeutic agent in a container different from the container for providing the modified siRNA according to the present invention.According to some embodiments of the present invention, the kit may contain instructions for mixing the modified siRNA with pharmaceutically acceptable carriers and / or excipients or other ingredients (if any).

[0660] In the kit according to the present invention, the modified siRNA and the pharmaceutically acceptable carrier and / or excipient, as well as the modified siRNA, the pharmaceutical composition, and / or the siRNA conjugate, and / or the conjugate, and / or the pharmaceutically acceptable excipient can be provided in any form, for example, in liquid form, dry form, or lyophilized form. According to some embodiments of the present invention, the modified siRNA and the pharmaceutically acceptable carrier and / or excipient, as well as the pharmaceutical composition and / or conjugate and the optional pharmaceutically acceptable excipient(s) are substantially pure and / or sterile. According to some embodiments of the present invention, the kit according to the present invention may contain sterile water.

[0661] Next, the present invention will be further described below using production examples and experimental examples, but is not limited thereto in any respect.

[0662] Examples

[0663] Unless otherwise stated, the agents and culture media used in the following examples are commercially available, and all procedures used, such as nucleic acid electrophoresis and real-time PCR, were performed in accordance with the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0664] When the siRNA or siRNA conjugate against the FXI gene synthesized in the present invention, or the siRNA or siRNA conjugate as a negative control, was used for cell transfection, Lipofectamine™2000 (Invitrogen) was used as the transfection reagent. Specific procedures may refer to the manufacturer's instructions.

[0665] C57b1 / 6N mice: 6-8 weeks old, purchased from Beijing Vital River Laboratory Anima1 Technology Co., Ltd., hereinafter referred to as C57 mice.

[0666] Heterozygous humanized mice: 6-8 weeks old, purchased from Cyagen Biosciences Inc.

[0667] Unless otherwise stated, all reagent ratios shown below are calculated as volume ratios (v / v).

[0668] Unless otherwise stated, all experimental data on in vivo / in vitro effects are expressed as and Graphpad Prism 5.0 statistical analysis software is used to analyze the data.

[0669] Production Example 1: Preparation of L10-siFXIf1M1S conjugate

[0670] In this preparation example, the L10-siFXIf1M1S conjugate was synthesized. This conjugate is an siRNA conjugate formed by conjugating the L-9 conjugating molecule with siRNA No. siFXIf1M1S. The sequence of the siRNA conjugated in this conjugate is presented in Table 3.

[0671] (1-1) Synthesis of compound L-10:

[0672] Compound L-10 was synthesized according to the following method:

[0673]

[0674] (1-1-1) Synthesis of the terminal segment of the GAL-5 conjugating molecule

[0675]

[0676] 100.0 g GaL-1 (N-acetyl-D-galactosamine hydrochloride, CAS No. 1772-03-8, purchased from Ning Bo hongxiang bio-chem Co., Ltd., 463.8 mmol) was dissolved in 1000 mL anhydrous pyridine, to which 540 mL acetic anhydride (purchased from Enox Inc., 5565.6 mmol) was added in an ice water bath for reaction with stirring at room temperature for 1.5 hours. The resulting reaction solution was poured into 10 L ice water and subjected to vacuum filtration under reduced pressure. The residue was washed with 2 L ice water, and then a mixed solvent of acetonitrile / toluene (v / v ratio of acetonitrile: toluene = 1:1) was added until completely dissolved. The solvent was evaporated to yield 130.0 g of the product GaL-2 as a white solid.

[0677] (1-1-1b) GaL-3 synthesis

[0678] GaL-2 (35.1 g, 90.0 mmol) obtained in step (1-1-1a) was dissolved in 213 mL of anhydrous 1,2-dichloroethane, to which 24.0 g of TMSOTf (CAS No. 27607-77-8, purchased from Macklin Inc., 108.0 mmol) was added in an ice water bath under a nitrogen atmosphere to react at room temperature overnight.

[0679] 400 mL of dichloromethane was added to the reaction solution for dilution, filtered through diatomaceous earth, and then 1 L of saturated aqueous sodium bicarbonate solution was added and stirred until homogeneous. The organic phase was isolated. The remaining aqueous phase was extracted twice, each time with 300 mL of dichloroethane. The organic phases were combined and washed with 300 mL of saturated aqueous sodium bicarbonate solution and 300 mL of saturated brine, respectively. The organic phase was isolated and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness under reduced pressure to obtain 26.9 g of the product GaL-3 as a light yellow viscous syrup.

[0680] (1-1-1c) GaL-4 Synthesis

[0681] GaL-3 (26.9 g, 81.7 mmol) obtained in step (1-1-1b) was dissolved in 136 mL of anhydrous 1,2-dichloroethane, 30 g of dry 4Å molecular sieve powder were added, followed by 9.0 g of 5-hexen-1-ol (CAS No. 821-41-0, purchased from Adamas-beta Inc., 89.9 mmol), and stirred at room temperature for 30 min. 9.08 g of TMSOTf (40.9 mmol) was added in an ice bath under a nitrogen atmosphere to react with stirring at room temperature overnight. The 4Å molecular sieve powder was removed by filtration. The filtrate was diluted with 300 mL of dichloroethane, filtered through diatomaceous earth, and then washed with 500 mL of saturated aqueous sodium bicarbonate solution. The mixture was stirred for 10 minutes to rinse. The organic phase was separated. The aqueous phase was extracted once with 300 mL of dichloroethane. The organic phases were combined and washed with 300 mL of saturated aqueous sodium bicarbonate solution and 300 mL of saturated brine, respectively.The organic phase was isolated and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness under reduced pressure, yielding 41.3 g of the product GaL-4 as a yellow syrup, which was used directly in the next oxidation reaction without purification.

[0682] (1-1-1d) GaL-5 Synthesis

[0683] GaL-4 (14.9 g, 34.7 mmol) obtained according to the method described in step (1-1-1c) was dissolved in a mixed solvent of 77 ml of dichloromethane and 77 ml of acetonitrile, 103 ml of deionized water and 29.7 g of sodium periodate (CAS No. 7790-28-5, purchased from Aladdin Inc., 138.8 mmol) were added, respectively, and stirred in an ice bath for 10 minutes. Ruthenium trichloride (CAS No. 14898-67-0, available from Energy Chemical, 238 mg, 1.145 mmol) was added to react at room temperature overnight. The resulting reaction solution was diluted by adding 300 ml of water while stirring and adjusted to a pH of approximately 7.5 with saturated sodium bicarbonate. The organic phase was separated and discarded. The aqueous phase was extracted three times, each time with 200 ml of dichloromethane, and the organic phase was discarded.The aqueous phase was adjusted to a pH of approximately 3 using dry citric acid, extracted three times, each time with 200 ml of dichloromethane, and the resulting organic phases were combined and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness under reduced pressure to yield 6.85 g of GaL-5 as a white foamy solid. 1 H NMR (400 MHz, DMSO) δ 12.01 (br, 1H), 7.83 (d, J=9.2 Hz, 1H), 5.21 (d, J=3.2 Hz, 1H), 4.96 (dd, J=11.2, 3.2 Hz, 1H), 4.49 (d, J=8.4 Hz, 1H), 4.07-3.95 (m, 3H), 3.92-3.85 (m, 1H), 3.74-3.67 (m, 1H), 3.48-3.39 (m, 1H), 2.20 (t, J=6.8 Hz, 2H), 2.11 (s, 3H), 2.00 (s, 3H), 1.90 (s, 3H), 1.77 (s, 3H), 1.55-1.45 (m, 4H).

[0684] (1-1-2) L-8 Synthesis:

[0685]

[0686] J-0 (9.886 g, 52.5 mmol, purchased from Alfa Aesar Inc.) and GaL-5 (72.819 g, 162.75 mmol, obtained by combining several batches of products) obtained in step (1-1-1) were dissolved in 525 mL of dichloromethane, and diisopropylethylamine (DIEA, 44.782 g, 346.50 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 90.158 g, 173.25 mmol), and hydroxybenzotriazole (HOBt, 23.410 g, 173.25 mmol) were added thereto for reaction at room temperature for 4 hours. The resulting reaction solution was washed by adding 20 ml of saturated sodium bicarbonate solution and 200 ml of saturated brine. The aqueous phase was extracted twice, each time with 100 ml of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The solvent was then evaporated to dryness under reduced pressure to yield the crude product.The crude product was purified using a normal-phase silica gel column (200-300 mesh). The column was loaded with 10 wt.% triethylamine to neutralize the acidity of the silica gel and then equilibrated. triethylamine and eluted with a gradient elution of dichloromethane:methanol = 100:25-100:40. The product eluate was collected and the solvent was evaporated to dryness under reduced pressure to yield 38.8 g of pure L-8. 1 H NMR (400 MHz, DMSO) δ 7.84 (d, J=9.0 Hz, 3H), 7.27-7.23 (m, 1H), 7.13-7.18 (m, 1H), 5.22 (d, J=3.1 Hz, 3H), 4.97 (dd, J=11.3, 3.1 Hz, 3H), 4.48 (d, J=8.4 Hz, 3H), 4.09-3.98 (m, 9H), 3.88 (dd, J=19.3, 9.3 Hz, 3H), 3.75-3.66 (m, 3H), 3.44 -3.38 (m, 3H), 3.17-3.30 (m, 4H), 3.10-2.97 (m, 4H), 2.35-2.20 (m, 6H), 2.15-2.08 (m, 9H), 2.07-1.98 (m, 13H), 1.94-1.87 (m, 9H), 1.81-1.74 (m, 9H), 1.65-1.42 (m, 18H). MS m / z: C 85 H 119 N7O 30 , [M+N] + , calculated: 1477.59, measured: 1477.23.

[0687] (1-1-3) L-7 Synthesis

[0688] (1-1-3a) Synthesis A-1

[0689]

[0690] DMTrCl (4,4'-dimethoxytrityl chloride, 101.65 g, 300 mmol) was dissolved in 1000 mL of anhydrous pyridine, and calcium DL-glycerate hydrate (28.63 g, 100 mmol) was added thereto for reaction at 45°C for 20 hours. The resulting reaction solution was filtered. The residue was washed with 200 mL of DCM, and the filtrate was concentrated to dryness under reduced pressure. The residue was redissolved in 500 mL of dichloromethane and washed twice, each time with 200 mL of 0.5 M triethylamine phosphate (pH = 7-8). The aqueous phase was extracted twice, each time with 200 mL of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated to dryness under reduced pressure, and the residue was purified using a normal phase silica gel column (200-300 mesh). The column was eluted with a gradient of petroleum ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.35-1:1:1:0.55.The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure. The residue was redissolved in 600 mL of dichloromethane and washed once with 200 mL of 0.5 M triethylamine phosphate. The aqueous phase was extracted once with 200 mL of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated to dryness under reduced pressure, and the residue was subjected to reduced pressure using a vacuum oil pump overnight to yield 50.7 g of product A-1 as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.46 (ddd, J=6.5, 2.3, 1.1 Hz, 1H), 7.40-7.28 (m, 7H), 6.89 -6.81 (m, 4H), 4.84 (d, J=5.0 Hz, 1H), 4.36-4.24 (m, 1H), 4.29 (s, 6H), 3.92 (dd, J=12.4, 7.0 Hz, 1H), 3.67 (dd, J=12.3, 7.0 Hz, 1H), 2.52 (q, J=6.3 Hz, 6H), 1.03 (t, J=6.3 Hz, 9H). MS m / z: C 24 N 23 O6, [M-N] - , calculated: 407.15, measured: 406.92.

[0691] (1-1-3b) Synthesis of L-7

[0692]

[0693] L-8 (40 g, 27.09 mmol, obtained by combining several batches of products) obtained in step (1-1-2) and A-1 (41.418 g, 81.27 mmol) obtained in step (1-1-3a) were mixed and dissolved in 271 ml of dichloromethane, 3-diethoxyphosphoryl-1,2,3-benzotrizin-4(3H)-one (DEPBT) (24.318 g, 81.37 mmol) was added, and then diisopropylethylamine (21.007 g, 162.54 mmol) was added to react with stirring at 25°C for 1.5 hours. The organic phase was washed with 800 ml of saturated sodium bicarbonate solution. The aqueous phase was extracted three times, each time with 50 ml of dichloromethane. The organic phase was washed with 150 ml of saturated brine, the aqueous phase was extracted once with 50 ml of dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, and filtered.The solvent was evaporated to dryness under reduced pressure, and the residue was dried in a foamed state using a vacuum oil pump overnight to obtain the crude product. The crude product was purified by column purification. The column was packed with 2 kg of normal phase silica gel (200-300 mesh), 200 ml of triethylamine was added to neutralize the acidity of the silica gel, equilibrated with petroleum ether containing 1 wt % triethylamine, and eluted with a gradient of petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:0.6. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 40.4 g of pure product L-7. 1H NMR (400 MHz, 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 (dd4, 5.5 J=11.2, 3.2 Hz, 3H), 4.48–4.30 (m, 4H), 4.02 (s, 9N), 3.93–3.84 (m, 3H), 3.76–3.66 (m, 9H), 3.45–3.35, (3, 3N), 3.45–3.35, (3, 3N), 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 N7O 35 , [M-DMTr] + , calculated: 1564.65, measured:

[0694] (1-1-4) Synthesis L-9

[0695]

[0696] L-7 (40 g, 21.4247 mmol) obtained in step (1-1-3b), succinic anhydride (4.288 g, 42.8494 mmol) and 4-dimethylaminopyridine (DMAP, 5.235 g, 42.8494 mmol) were mixed and dissolved in 215 ml of dichloromethane, then diisopropylethylamine (DIEA, 13.845 g, 107.1235 mmol) was added and stirred at 25 °C for 24 hours. The resulting reaction solution was washed with 800 ml of 0.5 M triethylamine phosphate. The aqueous phase was extracted three times, each time with 5 ml of dichloromethane. The organic phases were combined and evaporated to dryness under reduced pressure to yield the crude product. The crude product was purified on a column. The column was packed with 1 kg of normal phase silica gel (200-300 mesh), 1 wt % triethylamine was added to neutralize the acidity of the silica gel, equilibrated with dichloromethane, and eluted using a gradient elution of 1 wt % triethylamine-containing dichloromethane:methanol = 100:18-100:20.The product eluate was collected and the solvent was evaporated to dryness under reduced pressure to yield 31.0 g of pure L-9 conjugated molecule product. 1 H NMR (400 MHz, DMSO) δ 8.58 (d, J=4.2 Hz, 1H), 7.94-7.82 (m, 3H), 7.41-7.29 (m, 5H), 7.22 (d, J=8.1 Hz, 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=8.2 Hz, 3H), 4.02 (s, 9H), 3.88 (dd, J=19.4, 9.4 Hz, 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), 1.82-1.84 (m, 9H), 1.80-1.70 (m, 1.35-1.35 (m, 1.35-1H), 1.35 (m), 1.31-4.16 (m, 4H), 7.16 (t). MS m / z: C 94 H 132 N7O 38 , [M-DMTr] + , calculated: 1664.72, measured: 1665.03.

[0697] (1-1-5) Synthesis of compound L-10:

[0698]

[0699] In this step, compound L-10 was obtained by coupling the conjugating molecule L-9 to a solid phase support.

[0700] The conjugating molecule L-9 (22.751 g, 11 mmol) obtained in step (1-1-4), O-benzotriazoletetramethyluronium hexafluorophosphate (HBTU, 6.257 g, 16.5 mmol) and diisopropylethylamine (DIEA, 2.843 g, 22 mmol) were mixed and dissolved in 900 mL of acetonitrile and stirred at room temperature for 5 minutes. Aminomethyl resin (88 g, 100-200 mesh, amino group loading: 400 μmol / g, purchased from Tianjin Nankai HECHENG S&T Co., Ltd.) was added to the resulting reaction solution. The reaction was carried out on a shaker at 25°C and 150 rpm for 18 h, followed by filtration. The residue was washed twice (each time with 300 mL of DCM) and then three times (each time with 300 mL of acetonitrile) and dried using a vacuum oil pump for 18 h. The capping reaction was then performed by adding the starting materials (CapA, CarB, 4-dimethylaminopyridine (DMAP), and acetonitrile) according to the charge ratios shown in Table 2.The reaction was performed on a shaker at 25°C and 150 rpm for 5 hours. The reaction mixture was filtered. The residue was washed three times, each time with 300 ml of acetonitrile. The solvent was evaporated to dryness under reduced pressure, and the residue was dried under reduced pressure using a vacuum oil pump overnight to yield 102 g of compound L-10 (i.e., the conjugating molecule L-9 bound to the solid-phase support) with a loading of 90.8 μmol / g.

[0701]

[0702] In the table above, Cap A and Cap B are capping agent solutions. Cap A is a mixed solution of 20% by volume of N-methylimidazole in pyridine / acetonitrile, with the volume ratio of pyridine to acetonitrile being 3:5. Cap B is a 20% by volume solution of acetic anhydride in acetonitrile.

[0703] (1-2) Synthesis of the sense strand of the L10-siFXIf1M1S conjugate

[0704] The sense strand of siRNA Conjugate 1 in Table 3 was synthesized by coupling nucleoside monomers one by one in the 3' to 5' direction according to the nucleotide sequence of the sense strand using the solid-phase phosphoamidite synthesis method, starting from the compound L-10 obtained in the above step. The coupling of each nucleoside monomer involved a four-step reaction of deprotection, coupling, capping, and oxidation or sulfurization. Meanwhile, if two nucleotides are linked via a phosphoester bond, the four-step reaction of deprotection, coupling, capping, and oxidation was activated during the coupling of the subsequent nucleoside monomer; If two nucleotides are linked via a phosphorothioate bond, a four-step reaction of deprotection, coupling, capping, and sulfurization is initiated during the addition of the subsequent nucleoside monomer. The synthesis conditions are set as follows.

[0705] Nucleoside monomers are provided in a 0.1 M acetonitrile solution. The deprotection reaction conditions are identical for each step, i.e., a temperature of 25°C, a reaction time of 70 seconds, a solution of dichloroacetic acid in dichloromethane (3% v / v) as the deprotecting agent, and a molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on a solid-phase support of 5:1.

[0706] The coupling reaction condition is identical in each step, including a temperature of 25°C, a molar ratio of the nucleic acid sequence coupled to the solid phase support to the nucleoside monomers of 1:10, a molar ratio of the nucleic acid sequence coupled to the solid phase support to the coupling reagent of 1:65, a reaction time of 600 seconds, and a 0.5 M solution of 5-ethylthio-1H-tetrazole (ETT) in acetonitrile as the coupling agent.

[0707] The condition for the capping reaction is the same in each step, including a temperature of 25°C, a reaction time of 15 seconds, a mixed solution of CapA and CapB at a molar ratio of 1:1 is used as the capping agent solution, and the molar ratio of the capping agent to the nucleic acid sequence linked to the solid phase support is 1:1:1 (acetic anhydride: N-methylimidazole: nucleic acid sequence linked to the solid phase support).

[0708] The oxidation reaction condition is identical in each step, including a temperature of 25°C, a reaction time of 15 seconds, and 0.05 M iodine in water as the oxidizing agent; and the molar ratio of iodine to the nucleic acid sequence linked to the solid phase support in the binding step is 30:1. The reaction is carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3:1:1.

[0709] The condition for the sulfurization reaction is identical in each step, including a temperature of 25°C, a reaction time of 300 seconds, and xanthan hydride as the sulfurization agent; and the molar ratio of the sulfurization agent to the nucleic acid sequence linked to the solid phase support in the binding step is 120:1. The reaction is carried out in a mixed solvent of acetonitrile:pyridine=1:1.

[0710] After the binding of the last nucleoside monomer was completed, the nucleic acid sequence bound to the solid phase support was cleaved, deprotected, purified, desalted, and then lyophilized to obtain the sense strand, where:

[0711] The conditions for cleavage and removal of the protecting group are as follows: the synthesized nucleotide sequence linked to the support was added to a 25 wt% aqueous ammonia solution for reaction at 55°C for 16 hours, with the amount of aqueous ammonia solution being 0.5 ml / μmol. The liquid was removed by filtration, and the supernatant was concentrated to dryness under vacuum.

[0712] The purification and desalting conditions were as follows: nucleic acid purification was achieved by using a preparative ion chromatography column (Source 15Q) with a gradient elution of NaCl. Specifically, eluent A: 20 mM sodium phosphate (pH = 8.1), solvent: water / acetonitrile = 9:1 (v / v); eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), solvent: water / acetonitrile = 9:1 (v / v); the gradient elution was: the ratio of eluent A: eluent B = 100:0-50:50. The product eluate was collected, pooled, and desalted using a reversed-phase chromatography column. The specific condition includes: using a Sephadex column for desalting with Sephadex-C25 as a packing and elution with deionized water.

[0713] The detection method is as follows: the purity of the above-mentioned sense strand was determined by ion exchange chromatography (IC-HPLC); and the molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS), of which the calculated value was 7584.5 and the measured value was 7584.0. The fact that the measured value was consistent with the calculated value indicates that the SS sense strand whose 3'-terminal portion was conjugated with the conjugating molecule L-9 was synthesized.

[0714] (1-3) Synthesis of the antisense strand of the L10-siFXIf1M1S conjugate

[0715] The antisense strand of the L10-siFXIf1M1S conjugate was synthesized by solid-phase phosphoamidite synthesis, starting cycles from a universal solid-phase support (UnyLinker™-loaded NittoPhase solid supports ®HL, Kinovate Life Sciences Inc.). The reaction conditions for deprotection, coupling, capping, oxidation or sulfurization, cleavage and deprotection, as well as purification and desalting in the solid-phase synthesis method, were the same as those used for the synthesis of the sense strand. The AS antisense strand was prepared by lyophilization.

[0716] The purity of the antisense strand was determined using ion-exchange chromatography (IC-HPLC), and the molecular weight of the antisense strand was analyzed by liquid chromatography-mass spectrometry (LC-MS). The fact that the measured value was consistent with the calculated value indicates that the AS antisense strand with the target sequence was synthesized.

[0717] (1-4) Synthesis of L10-siFXFf1M1S conjugate

[0718] For the L10-siFXIf1M1S conjugate, the sense strand and antisense strand were respectively dissolved in water for injection to prepare a 40 mg / mL solution. They were mixed at an equimolar ratio, heated at 50°C for 15 minutes, cooled at room temperature to form a double-stranded structure, and then lyophilized to obtain a lyophilized powder. After the conjugate was diluted to a concentration of 0.2 mg / mL with ultrapure water (Milli-Q pure water instrument, resistivity 18.2 MΩ cm (25°C)), the molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS) (purchased from Waters Corp., model: LCT Premier). The fact that the measured value matched the calculated value indicates that the synthesized siRNA conjugate was the designed target double-stranded nucleic acid sequence with the conjugating molecule L-9. The siRNA conjugate has a structure represented by Formula (403).The miRNA has a sequence corresponding to the L10-siFXIf1M1S conjugate, as shown in Table 3.

[0719]

[0720]

[0721] where C, G, U, A, and T represent the base compositions of nucleotides; the letter m represents that the nucleotide to the left of m is a methoxy-modified nucleotide; f represents that the nucleotide to the left of f is a fluorine-modified nucleotide; s represents that the two nucleotides on either side of s are linked by a thiophosphorothioate bond; and P represents that the nucleotide adjacent to the right side of P is a 5'-phosphate nucleotide.

[0722] Production Examples 2-10: Synthesis of siRNA Conjugates According to the Present Invention

[0723] siRNA conjugates of the present invention: L10-siFXIelM1SP, L10-siFXIh1M1SP, L10-siFXIclM1SP, L10-siFXId1M1SP, L10-siFXIe1M1SP, L10-siFXIg1M1SP, L10-siFXIh1M1SP, L10-siFXIi1M1S and L10-siFXIi1M1SP conjugates (which had sequences corresponding to siFXIa1M1SP, siFXIb1M1SP, siFXIc1M1SP, siFXId1M1SP, siFXIg1M1SP, siFXIhIM1SP, siFXIm1S and siFXIiM1SP, as shown in Table 3, respectively) were further synthesized, respectively, by the same methods as in Example 1, except that (1) the sequences of the sense strand and antisense strand of LiF1M1S, siFXIb1M1S, and siFXIm1SP were the sequences of the sense strand and antisense strand as shown in Table 3; and (2) in the case of the conjugates L10-siFXIa1M1SP, L10-siFXIb1M1SP, L10-siFXIc1M1SP, L10-siFXId1M1SP, L10-siFXIe1M1SP, L10-siFXIg1M1SP, L10-siFXIh1M1SP, and L10-siFXIi1M1SP, the first nucleotide from the 5'-end of the antisense strands was a 5'-phosphate nucleotide;Accordingly, during the preparation of antisense strand sequences by the solid-phase phosphoamidite synthesis method, after the last nucleoside monomer was coupled, the monomer of Formula (CPR-I) (obtained from Suzhou GenePharma Inc., Cat#13-2601-XX) was coupled to the 5'-end of the antisense strand through a four-step reaction of deprotection, coupling, capping, and oxidation to form a 5'-phosphate nucleotide.

[0724] (CPR-I)

[0725] During coupling, deprotection, coupling, capping, and oxidation conditions were identical to those used for the synthesis of the sense strand. After complete coupling, the sequence was further cleaved, deprotected, purified, desalted, and finally lyophilized to yield the AS antisense strand.

[0726] After obtaining the conjugates, their molecular weights were determined in the same manner as in Preparation Example 1. The results showed that the measured values ​​were consistent with the calculated values, indicating that the synthesized siRNA conjugates were designed target double-stranded nucleic acid sequences with the L-9 conjugation molecule and had the structure represented by Formula (403). The miRNAs contained in these conjugates have sequences corresponding to the conjugates L10-siFXIa1M1SP, L10-siFXIb1M1SP, L10-siFXIc1M1SP, L10-siFXId1M1SP, L10-siFXIe1M1SP, L10-siFXIg1M1SP, L10-siFXIh1M1SP, L10-siFXIi1M1S or L10-siFXIi1M1SP, as shown in Table 3.

[0727] Production Examples 11-20: Synthesis of siRNA According to the Present Invention

[0728] The siRNA sequences listed in Table 4 were synthesized using the solid-phase synthesis method, respectively, and their molecular weights were determined. The sense strands and antisense strands, which were present at an equilibrium ratio and complementary to each other, as shown in Table 4, were dissolved in DEPC water and then annealed to obtain the siRNAs of the present invention: siFXIa1M1SP, siFXIb1M1SP, siFXIc1M1SP, siFXId1M1SP, siFXIelM1SP, siFXIf1M1SP, siFXIg1M1SP, siFXIhIM1SP, siFXIi1M1SP and siFXIel, as shown in Table 4.

[0729] During sequencing of siFXIe1, the target sequence contained an unmodified nucleotide. In this case, under cleavage and deprotection conditions, after treatment with aqueous ammonia, the product was dissolved in 0.4 mL / μmol N-methylpyrrolide, followed by the addition of 0.3 mL / μmol triethylamine and 0.6 mL / μmol triethylamine trihydrofluoride, based on the amount of single-stranded nucleic acid, which resulted in the removal of the 2'-TBDMS protecting group on ribose.

[0730] Furthermore, in the case where the first nucleotide from the 5'-terminal of the antisense strand in the target sequence was a 5'-phosphate nucleotide, during the preparation of the antisense strand according to the solid-phase phosphoramidite synthesis method, after the last nucleoside monomer in the antisense strand was coupled, the monomer of Formula (CPR-I) (purchased from Suzhou GenePharma Inc., catalog number 13-2601-XX) was coupled to the 5'-terminal of the antisense strand through a four-step reaction of deprotection, coupling, capping, and oxidation to form a 5'-phosphate nucleotide.

[0731] (CPR-I)

[0732] During coupling, deprotection, coupling, capping, and oxidation conditions were identical to those used for the synthesis of the sense strand. After complete coupling, the sequence was further cleaved, deprotected, purified, desalted, and finally lyophilized to yield the AS antisense strand.

[0733] Comparative Production Example 1: Synthesis of Comparative siRNA

[0734] The sense strand and antisense strand of siRNA, numbered as NC in Table 4, were synthesized using the solid-phase synthesis method, respectively, and their molecular weights were determined. The sense strand and antisense strand, which were present at an equilibrium ratio, were dissolved in DEPC water and then annealed to obtain the reference siRNA, numbered as NC.

[0735]

[0736]

[0737] where C, G, U, A, and T represent the base compositions of nucleotides; the letter m represents that the nucleotide to the left of m is a methoxy-modified nucleotide; f represents that the nucleotide to the left of f is a fluorine-modified nucleotide; s represents that the two nucleotides on either side of s are linked by a thiophosphorothioate bond; and P represents that the nucleotide adjacent to the right side of P is a 5'-phosphate nucleotide.

[0738] After the above-mentioned siRNAs or conjugates according to the present invention were completely prepared, they were lyophilized into a solid powder and stored until use. Upon use, they can be reconstituted with water for injection, normal saline (NS), phosphate buffer (PB), or phosphate-buffered saline (PBS) to obtain a solution of the required concentration.

[0739] Experimental Example 1: In vitro inhibitory activity of the siRNA according to the present invention

[0740] HEK293A cells (purchased from Nanjing Cobioer Biosciences Co., LTD) were cultured in DMEM complete medium (Hyclone) containing 10% fetal bovine serum (FBS, Hyclone) and 0.2% (v / v / v) penicillin-streptomycin (Gibco, Invitrogen) at 37°C in a 5% CO2 / 95% air incubator.

[0741] According to the method described by Kumico Ui-Tei et al., Functiona1 dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammary gene silencing with significantly reduced off-target effect. Nucleic Acids Research, 2008.36(7), 2136-2151, detection plasmids were constructed and co-transfected with the siRNA (siFXIe1) to be detected into HEK293A cells; and the expression levels of the dual luciferase reporter gene reflect the on-target activity of siRNA and the off-target effect of siRNA. The specific steps are described below:

[0742] [1] Construction of plasmid for detection

[0743] The detection plasmid was constructed using the psiCHECK™-2 plasmid (Promega™). This plasmid contains the target sequence, i.e., the target sequence of the miRNA. The miRNAs to be detected have the target sequence shown below. Specifically, siFXIe1 (obtained from Preparation Example 20) has the following target sequence:

[0744] GAATCTCAAAGAAATCTT (SEQ ID NO: 565).

[0745] The target sequence was inserted into the Xho I / Not I site of psiCHECK™-2 plasmid.

[0746] [2] Transfection

[0747] HEK293A cells were inoculated into a 96-well plate at 8×10 3 cells / well. After 16 hours, the cell growth density reached 70-80%. The complete H-DMEM media in the culture wells was aspirated. 80 µl of Opti-MEM medium (GIBCO) was added to each well, and the culture was continued for an additional 1.5 hours.

[0748] The above detection plasmid was diluted with DEPC-treated water to obtain 200 ng / μL detection plasmid working solution; siFXIe1 was prepared with DEPC-treated water into siRNA working solutions at concentrations of 10 nM and 3 nM (based on the amount of siRNA), respectively.

[0749] Solution 1A1 was prepared. Each portion of solution 1A1 contained 1 μl of 10 nM siRNA working solution, 0.05 μl of detection plasmid working solution (containing 10 ng of detection plasmid), and 10 μl of Opti-MEM medium.

[0750] Solution 1A2 was prepared. Each portion of solution 1A2 contained 1 μl of 3 nM siRNA working solution, 0.05 μl of detection plasmid working solution (containing 10 ng of detection plasmid), and 10 μl of Opti-MEM medium.

[0751] Solution 1B was prepared. Each portion of solution 1B contains 0.2 µl Lipofectamine™ 2000 and 10 µl Opti-MEM medium.

[0752] Solution 1C was prepared. Each portion of solution 1C contains 0.05 μl of working solution with detection plasmid (containing 10 ng of detection plasmid) and 10 μl of Opti-MEM medium.

[0753] One portion of solution 1B was mixed with one portion of solution 1A1 or one portion of solution 1A2, respectively. The mixed solution was incubated for 20 minutes at room temperature to form transfection complexes 1X1 and 1X2. One portion of solution 1B was mixed with one portion of solution 1C, and the mixed solution was incubated for 20 minutes at room temperature to form transfection complex 1X3.

[0754] The 1X1 transfection complex was added at 20 μL / well to three culture wells respectively and then mixed evenly to prepare a co-transfection mixture with a final siRNA concentration of 0.1 nM (recorded as test group 1).

[0755] The 1X2 transfection complex was added at 20 μL / well to three additional culture wells, respectively, and then mixed evenly to prepare a co-transfection mixture with a final siRNA concentration of 0.03 nM (recorded as test group 2).

[0756] The 1X3 transfection complex was added at 20 μL / well to three additional culture wells, respectively, to obtain a transfection mixture without siRNA (recorded as the control group).

[0757] After the co-transfection mixtures containing siRNA and the transfection mixture without siRNA were co-transfected in culture wells for 4 hours, each well was supplemented with 100 μl of complete H-DMEM medium containing 20% ​​FBS. The 96-well plate was placed in a CO2 incubator and further cultured for 24 hours.

[0758] [3] Definition

[0759] The media in the culture wells was aspirated. 150 μl of the mixed solution of Dual-glo reagent ® Luciferase and H-DMEM (in a 1:1 volume ratio) were added to each well and mixed thoroughly. After incubation for 10 minutes at room temperature, 120 µl of the mixed solution was transferred to a 96-well ELISA plate. The Firefly chemiluminescence (Fir) value in each well of the ELISA plate was read using a Synergy II multimode plate reader (BioTek). Then, 60 µl of Dual-glo reagent was added to each well of the ELISA plate. ® Stop & Glo ® and mixed thoroughly. After incubation at room temperature for 10 minutes, the Renilla chemiluminescence (REN) value in each well of the ELISA plate was read using a microplate reader according to the FIR reading procedure.

[0760] The luminescence ratio (Ratio = REN / FIR) of each well was calculated, and the luminescence ratio (Ratio (test) or Ratio (control)) of each test group or control group was the average value of the ratios of three culture wells. Using the luminescence ratio of the control group as the reference value, the luminescence ratio of each test group was normalized to obtain the ratio R of Ratio (test) / Ratio (control), which represents the expression level, i.e., residual activity, of the reporter gene. Renilla. The inhibition rate of siRNA was (1 - R) × 100%.

[0761] The inhibition results of siFXIe1 activity at different concentrations relative to the target sequence are shown in Table 5.

[0762] Comparative Experimental Example 1: In vitro inhibitory activity of comparative NC siRNA

[0763] The inhibitory activity of the comparative NC siRNA in the psiCHECK system was examined using the same method as described in Experimental Example 1, except that the test siRNA was replaced with the comparative NC siRNA. The results are shown in Table 5.

[0764]

[0765] The results showed that siFXIe1 exhibited good concentration-dependent inhibitory activity in vitro against the target sequence at the appropriate concentration. Specifically, the inhibition rate of siFXIe1 against the target sequence at an siRNA concentration of 0.1 nM was 72.43%, demonstrating a good inhibitory effect on FXI gene expression.

[0766] Experimental Example 2: IC Measurement 50 miRNA sequences against FXI mRNA in the psiCHECK system

[0767] This experimental example studied the IC values 50siFXIa1M1SP, siFXIb1M1SP, siFXIc1M1SP, siFXId1M1SP, siFXIelM1SP and siFXIi1M1SP in the psiCHECK in vitro system.

[0768] According to the method described by Kumico Ui-Tei et al., Functiona1 dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect Nucleic Acids Research, 2008.36 (7), 2136-2151, detection plasmids were constructed and co-transfected with the siRNA (siFXIe1) to be detected into HEK293A cells; and the expression levels of the dual luciferase reporter gene reflected the on-target activity of siRNA and the off-target effect of siRNA. The specific steps are described as follows:

[0769] [1] Construction of plasmid for detection

[0770] The detection plasmid was constructed using the psiCHECK™-2 plasmid (Promega™). This plasmid contains the target sequence, which was as shown under Genbank accession number NM 000128.3.

[0771] The target sequence was inserted into the Xho I / Not I site of psiCHECKTM-2 plasmid.

[0772] [2] Cell culture and transfection

[0773] HepG2 cells (purchased from GuangZhou Jennio Biotech Co., Ltd) were cultured in complete DMEM medium (Hyclone) containing 20% ​​fetal bovine serum (FBS, Hyclone) and 0.2% (v / v / v) penicillin-streptomycin (Gibco, hwitrogen) at 37°C in an incubator containing 5% CO2 / 95% air.

[0774] HepG2 cells were inoculated into a 96-well plate at 8×10 3cells / well. After 16 hours, the cell growth density reached 70-80%. The complete H-DMEM media in the culture wells was aspirated. 80 µl of Opti-MEM medium (GIBCO) was added to each well, and the culture was continued for an additional 1.5 hours.

[0775] The above detection plasmid was diluted with DEPC-treated water to prepare 200 ng / μL detection plasmid working solution; each of the following siRNAs was prepared with DEPC-treated water into siRNA working solutions at 10 different concentrations of 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, 1.23 nM, 4.12 nM, 0.137 nM, 0.0457 nM, 0.0152 nM, and 0.00508 nM, respectively. The siRNAs used were siFXIa1M1SP, siFXIb1M1SP, siFXIc1M1SP, siFXId1M1SP, siFXIe1M1SP, and siFXIi1M1SP, respectively.

[0776] Solutions 2A1 through 2A10 were prepared for each siRNA, respectively. Each portion of solutions 2A1 through 2A10 contained 1 μl of each of the siRNA working solutions at the 10 concentrations listed above, 0.05 μl of the working solution with the detection plasmid (containing 10 ng of the detection plasmid), and 10 μl of Opti-MEM medium.

[0777] One portion of solution 1B was mixed with one portion of the resulting solutions 2A1-2A10 for each siRNA, respectively. The mixed solution was incubated for 20 minutes at room temperature to form transfection complexes ranging from 2X1 to 2X10 for each siRNA.

[0778] 2X1 to 2X10 transfection complexes for each siRNA were added at 20 μL / well to the culture wells, respectively, and then uniformly mixed to obtain transfection complexes at final concentrations of about 1 nM, 0.333 nM, 0.111 nM, 0.0370 nM, 0.0123 nM, 0.00412 nM, 0.00137 nM, 0.000457 nM, 0.000152 nM, and 0.0000508 nM for each siRNA. Transfection complexes of 2x1-2x10 for each siRNA were transfected respectively in three culture cells to obtain co-transfection mixtures containing siRNA (recorded as test groups).

[0779] The 1X3 transfection complex was added at 20 μL / well to three additional culture wells, respectively, to prepare a co-transfection mixture without siRNA (recorded as the control group).

[0780] After the co-transfection mixtures containing siRNA and the co-transfection mixture without siRNA were transfected in the culture wells for 4 hours, each well was supplemented with 100 μl of complete H-DMEM medium containing 20% ​​FBS. The 96-well plate was placed in a CO2 incubator and further cultured for 24 hours.

[0781] [3] Definition

[0782] The media in the culture wells was aspirated. 150 μl of the mixed solution of Dual-glo reagent ® Luciferase and H-DMEM (in a 1:1 volume ratio) were added to each well and mixed thoroughly. After incubation for 10 minutes at room temperature, 120 µl of the mixed solution was transferred to a 96-well ELISA plate. The Firefly chemiluminescence (Fir) value in each well of the ELISA plate was read using a Synergy II multimode plate reader (BioTek). Then, 60 µl of Dual-glo reagent was added to each well of the ELISA plate. ® Stop & Glo ®and mixed thoroughly. After incubation at room temperature for 10 minutes, the Renilla chemiluminescence (REN) value in each well of the ELISA plate was read using a microplate reader according to the FIR reading procedure.

[0783] The luminescence ratio (Ratio=REN / FIR) of each well was calculated, and the luminescence ratio (Ratio (test) or Ratio (control)) of each test group or control group was the average value of the ratios of three culture wells. Using the luminescence ratio of the control group as the reference value, the luminescence ratio of each test group was normalized to obtain the ratio R of Ratio (test) / Ratio (control), which represents the expression level, i.e., residual activity, of the Renilla reporter gene. The inhibition rate of siRNA was (1 - R) × 100%.

[0784] Dose response curves were fitted using the log(inhibitor) versus response-variable slope function of Graphpad 5.0 software. IC values 50 The dose of siRNA targeting GSCM was calculated based on the dose-response curve. Specifically, the determined dose-response curves corresponded to the formula below:

[0785]

[0786] in which:

[0787] Y - ratio R, i.e. residual activity,

[0788] X represents the logarithm of the concentration of transfected siRNAs,

[0789] Bot represents the Y value at the bottom of the stationary stage,

[0790] Tor represents the value of Y at the top of the stationary stage,

[0791] X' represents the value of X obtained by fitting, with Y being the median value between the bottom and the top, and HillSlope is the slope of the curve by fitting on X'.

[0792] When Y=50%, the corresponding value of X 50 was determined based on the dose-response curve and the corresponding calculation formula. It was calculated that the IC value 50 each miRNA is 10^X 50 .

[0793] Specific IC values 50 are summarized in Table 6.

[0794]

[0795] As can be seen from the results of Table 6 above, the siRNAs of the present invention exhibit very high inhibitory activity against the target sequence 1 in vitro in HepG2 cells, and the IC value 50 is in the range from 0.013 to 0.119 nM.

[0796] Experimental Example 3: Measurement of IC 50 siRNA against FXI mRNA in HepG2 cells

[0797] HepG2 cells were inoculated into a 24-well plate at 7×10 4 cells / well. After 16 hours, the cell growth density reached 70-80%. The complete H-DMEM media in the culture wells was aspirated. 500 µl of Opti-MEM medium (GIBCO) was added to each well, and the culture was continued for an additional 1.5 hours.

[0798] Each of the following siRNAs was prepared with DEPC-treated water into siRNA working solutions at 7 different concentrations of 20 μM, 6.67 μM, 2.22 μM, 0.741 μM, 0.247 μM, 0.0823 μM, and 0.0274 μM, respectively. The siRNAs used were siFXIa1M1SP, siFXIb1M1SP, siFXIc1M1SP, or siFXId1M1SP, respectively.

[0799] For each siRNA, solutions 3A1 through 3A7 were prepared, respectively. Each portion of solutions 3A1-3A7 contains 3 µl of each of the siRNA working solutions at the seven concentrations listed above and 50 µl of Opti-MEM medium.

[0800] Solution 3B was prepared. Each portion of solution 3B contains 1 µl of Lipofectamine™2000 and 50 µl of Opti-MEM medium.

[0801] One portion of solution 3B was mixed with one portion of the resulting solutions 3A1-3A7 for each siRNA, respectively. The mixed solution was incubated for 20 minutes at room temperature to form transfection complexes from 3X1 to 3X7 for each siRNA.

[0802] One portion of solution 3B was mixed with 50 µl of Opti-MEM medium. The mixed solution was incubated for 20 min at room temperature to form a 3X8 transfection complex.

[0803] 3X1 to 3X7 transfection complexes for each siRNA were added at 100 μl / well to the culture wells, respectively, and then uniformly mixed to obtain transfection mixtures with final concentrations of about 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, 1.23 nM, 0.412 nM, and 0.137 nM for each siRNA. 3X1 to 3X7 transfection complexes for each siRNA were transfected in three culture cells, respectively, to obtain transfection mixtures containing siRNA (recorded as test groups).

[0804] The 3X8 transfection complex was added at 100 μL / well to three additional culture wells, respectively, to obtain a transfection mixture without siRNA (recorded as the control group).

[0805] After the transfection mixtures containing siRNA and the transfection mixture without siRNA were transfected in the culture wells for 4 hours, each well was supplemented with 1 ml of complete H-DMEM medium containing 20% ​​FBS. The 24-well plate was placed in a CO2 incubator and further cultured for 24 hours.

[0806] Subsequently, total RNA in the cells of each well was extracted using RNAVzol (purchased from Vigorous Biotechnology Beijing Co., Ltd., Cat. No. N002) according to the detailed steps described in the instructions.

[0807] For the cells in each well, 1 μg of total RNA and the reagent provided in the Goldenstar™ RT6 cDNA Synthesis Kit (purchased from Beijing Tsingke Biotechnology Co., Ltd., Cat. No. TSK301M) were used, in which Goldenstar™ Oligo (dT) was selected as the primer. 17. 20 μl of the reverse transcription reaction system were prepared according to the reverse transcription procedures in the instructions of the reverse transcription kit for total RNA of cells in each well. The reverse transcription conditions were as follows: each reverse transcription reaction system was placed and incubated at 50°C for 50 minutes, then incubated at 85°C for 5 minutes, and finally incubated at 4°C for 30 seconds; after completion of the reaction, 80 μl of DEPC water was added to each reverse transcription reaction system to obtain a cDNA-containing solution.

[0808] For each reverse transcription reaction system, 5 μL of the above-mentioned cDNA-containing solution and the reagent provided in the NovoStart kit were used as a template. ®SYBR qPCR SuperMix P1us (purchased from Novoprotein Scientific Co., Ltd., Cat. No. E096-01B) was used to prepare 20 μl qPCR reaction system, where the PCR primer sequences used to amplify the target gene FXI and the internal reference gene GAPDH were as shown in Table 7, and the final concentration of each primer was 0.25 μM. Each qPCR reaction system was loaded onto an ABI StepOneP1us real-time PCR instrument and amplified using a three-step method. The amplification procedures included pre-denaturation at 95°C for 10 min, followed by denaturation at 95°C for 30 s, renaturation at 60°C for 30 s, and extension at 72°C for 30 s. After repeating the above-mentioned denaturation, renaturation, and extension process 40 times, the W product containing the amplified target gene FXI and the internal reference gene GAPDH was obtained.Then, the W product was incubated at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The melting curves of the target gene FXI and the internal reference gene GAPDH in the W product were collected respectively using a fluorescent real-time qPCR instrument, and the Ct values ​​of the target gene FXI and the internal reference gene GAPDH were obtained.

[0809]

[0810] The relative expression levels of the FXI target gene in each of the test groups and the control group were quantitatively calculated using the Ct comparison method (ΔΔCt). The calculation method was described as follows:

[0811] ΔCt(test group) = Ct(target gene in test group) - Ct(internal reference gene in test group)

[0812] ΔCt(control group) = Ct(target gene in control group) - Ct(internal reference gene in control group)

[0813] ΔCt(test group) = ΔCt(test group) - ΔCt(average value in control group)

[0814] ΔCt(control group) = ΔCt(control group) - ΔCt(mean value in control group)

[0815] where ΔCt (mean value in the control group) is the arithmetic mean of the ΔCt (control group) values ​​of each of the three culture wells in the control group. Thus, each culture well in the test group or control group corresponds to one ΔΔCt value.

[0816] The FXI mRNA expression levels in the test groups were normalized based on the expression level in the control group, and the FXI mRNA expression level in the control group was defined as 100%;

[0817] The relative expression level of FXI mRNA in the test group = 2-ΔΔCt(test group) × 100%.

[0818] For the miRNAs in the same test group, the mean value of the relative expression levels of FXI mRNA in the test group at each concentration was the arithmetic mean of the relative expression levels of three culture wells at that concentration.

[0819] Dose response curves were fitted using the log(inhibitor) versus response-variable slope function of Graphpad 5.0 software. IC values 50 The affinity of each miRNA against FXI mRNA was calculated based on the dose-response curve. Specifically, the dose-response curves obtained by fitting were fit to the following formula:

[0820]

[0821] in which:

[0822] Y represents the relative expression level of FXI mRNA in each test group,

[0823] X represents the logarithm of the final concentration of miRNA used in the corresponding test group,

[0824] Bot represents the Y value at the bottom of the stationary stage,

[0825] Tor represents the value of Y at the top of the stationary stage,

[0826] X' represents the value of X obtained by fitting Y to which the median value between the minimum and maximum values ​​is given, and HillSlope represents the slope of the curve obtained by fitting on X.

[0827] When Y=50%, the corresponding value of X 50 was determined based on the dose-response curve and the corresponding calculation formula. It was calculated that the IC value 50 each miRNA is 10^X 50 (nM).

[0828] IC Values 50 The affinities of each miRNA against FXI mRNA are summarized in Table 8.

[0829]

[0830] As shown in Table 8, the siRNAs of the present invention exhibited very high inhibitory activity against FXI mRNA in vitro in HepG2 cell lines, and the IC value 50 is in the range from 1.49 to 11.1 nM.

[0831] Experimental Example 4: IC Measurement 50 miRNA against FXI mRNA in primary mouse hepatocytes

[0832] Primary mouse hepatocytes were extracted from fresh liver tissues of normal C57b1 / 6N mice. Hepatocytes of the appropriate density were inoculated into a glass, plastic, or tissue dish coated with type I collagen, cultured in RPM11460 medium containing 1x double antibody and 10% FBS, and further cultured in an incubator containing 5% CO2 / 95% air at 37°C for 30 minutes.

[0833] Inhibitory activity and IC value 50siRNA against FXI mRNA was measured by the same methods as described in Experimental Example 3, except that the siRNA to be detected was siFXHTM1SP; the cells used were mouse primary hepatocytes; and the final siRNA concentrations included a total of 8 concentrations (100 nM, 25 nM, 6.25 nM, 1.56 nM, 0.391 nM, 0.098 nM, 0.0244 nM, and 6.1×10 -3 nM), respectively. The results are presented in Table 9.

[0834]

[0835] As shown in Table 9, siFXIf1M1SP exhibited very high inhibitory activity against FXI mRNA in vitro in mouse primary hepatocytes, with the IC value 50 was 0.021 nM.

[0836] Experimental Example 5: Measurement of the efficiency of siRNA inhibition on FXI mRNA expression levels in HepG2 cells

[0837] The inhibition rates of siRNA against the expression levels of FXI mRNA were measured using the same method as described in Experimental Example 3, except that the siRNAs used were siFXIg1M1SP and siFXIh1M1SP; for each siRNA, the final siRNA concentrations included a total of 3 concentrations (50 nM, 5 nM, and 0.5 nM), respectively; and 2 culture wells were used at each concentration. The results are shown in Table 10.

[0838]

[0839] As can be seen from Table 10, the siRNAs of the present invention exhibit very high inhibitory activity in vitro in HepG2 cells; and an inhibition rate of up to 83% against FXI mRNA can be achieved at an siRNA concentration of 50 nM.

[0840] Experimental Example 6: Detection of the inhibitory efficacy of L10-siFXIf1M1S, L10-siFXIi1M1S and L10-siFXIi1M1SP conjugates on FXI mRNA expression levels in mice in vivo

[0841] C57b1 / 6N mice (all female) were randomly divided into groups (5 mice in each group) and numbered accordingly. The test conjugate (i.e., L10-siFXIf1M1S, L10-siFXIi1M1S, or L10-siFXIi1M1SP) was subcutaneously administered at two different doses of 5 mg / kg and 1 mg / kg (depending on the amount of siRNA) to the mice in each group, respectively. Each siRNA conjugate was administered at concentrations of 1 mg / mL and 0.2 mg / mL in the form of 0.9 wt % NaCl aqueous solution and an administration volume of 5 mL / kg.

[0842] One group of mice was administered 1×PBS at an injection volume of 5 ml / kg and registered as a control group.

[0843] Mice were sacrificed on day 7 post-injection. Liver tissue from each mouse was then collected and stored with RNA (Sigma Aldrich), and the liver tissue was homogenized using a tissue homogenizer. Total RNA was then extracted and prepared using Trizol according to the procedures described in the instructions.

[0844] FXI mRNA expression levels were measured by fluorescence qPCR, and the inhibition levels against FXI mRNA were calculated using the same methods as in Experimental Example 3, except that the extracted total RNA was reverse transcribed to obtain cDNA using the ImProm-IITM Reverse Transcription Kit (Promega Company) according to the instructions for preparing the cDNA solution. Then, the FXI mRNA expression level in liver tissue was measured using a fluorescence qPCR kit (Beijing ComWin Biotech Co., Ltd). In the fluorescence qPCR method, the mouse GAPDH gene (mGAPDH) was used as the internal reference gene, and mouse FXI and GAPDH were detected using primers for mouse FXI and GAPDH, respectively. The primer sequences for detection were as shown in Table 11.

[0845] To measure FXI mRNA expression levels and calculate the inhibition rate against FXI mRNA, mice in the control group of this experiment were injected with PBS; mice in the test groups were injected with different siRNA conjugates, respectively. The FXI mRNA ex...

Claims

1. An siRNA capable of inhibiting expression of the blood coagulation factor XI gene in plasma, comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II; wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein the nucleotide sequence I and the nucleotide sequence II are selected from any sequence presented in i) -ix): i) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 1 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 2 and differs from it by no more than 3 nucleotides: 5'-GGGUAUUCUUUCAAGCAAZ1-3' (SEQ ID NO: 1); 5'-Z2UUGCUUGAAAGAAUACCC-3' (SEQ ID NO: 2), where Z1 represents U, and Z2 represents A, and nucleotide sequence I contains nucleotide Z3 at a position corresponding to Z1; nucleotide sequence II contains nucleotide Z4 at a position corresponding to Z2, where Z4 is the first nucleotide from the 5' end of the antisense strand; ii) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 61 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 62 and differs from it by no more than 3 nucleotides: 5'-GGCAUAAACUAUAACAGCZ5-3' (SEQ ID NO: 61); 5'-Z6GCUGUUAUAGUUUAUGCC-3' (SEQ ID NO: 62), where Z5 represents U, and Z6 represents A, and nucleotide sequence I contains nucleotide Z7 at a position corresponding to Z5; nucleotide sequence II contains nucleotide Z8 at a position corresponding to Z6, where Z8 is the first nucleotide from the 5' end of the antisense strand; iii) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 121 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 122 and differs from it by no more than 3 nucleotides: 5'-GCUCAAGAAUGCCAAGAAZ9-3' (SEQ ID NO: 121); 5'-Z10UUCUUGGCAUUCUUGAGC-3 (SEQ ID NO: 122), where Z9 represents A, and Z 10 represents U, and nucleotide sequence I contains nucleotide Z 11 at the position corresponding to Z9; nucleotide sequence II contains the nucleotide Z 12 in the position corresponding to Z 10 , where Z 12 represents the first nucleotide from the 5' end of the antisense strand; iv) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 181 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 182 and differs from it by no more than 3 nucleotides: 5'-GCAACAAAGACAUUUAUGZ13-3' (SEQ ID NO: 181); 5'-Z14CAUAAAUGUCUUUGUUGC-3' (SEQ ID NO: 182), where Z 13 represents U, and Z 14 represents A, and nucleotide sequence I contains nucleotide Z 15 in the position corresponding to Z 13 ; nucleotide sequence II contains nucleotide Z 16 in the position corresponding to Z 14 , where Z 16 represents the first nucleotide from the 5' end of the antisense strand; v) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 241 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 242 and differs from it by no more than 3 nucleotides: 5'-GAAUCUCAAAGAAAUCUUZ17-3' (SEQ ID NO: 241); 5'-Z18AAGAUUUCUUUGAGAUUC-3' (SEQ ID NO: 242), where Z 17 represents U, and Z 18 represents A, and nucleotide sequence I contains nucleotide Z 19 in the position corresponding to Z 17 ; nucleotide sequence II contains nucleotide Z 20 in the position corresponding to Z 18 , where Z 20 represents the first nucleotide from the 5' end of the antisense strand; vi) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 301 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 302 and differs from it by no more than 3 nucleotides: 5'-GUACGUGGACUGGAUUCUZ21-3' (SEQ ID NO: 301); 5'-Z22AGAAUCCAGUCCACGUAC-3' (SEQ ID NO: 302), in which Z 21 represents G, and Z 22 represents C, and nucleotide sequence I contains nucleotide Z 23 in the position corresponding to Z 21 ; nucleotide sequence II contains nucleotide Z 24 in the position corresponding to Z 22 , where Z 24 represents the first nucleotide from the 5' end of the antisense strand; vii) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 361 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 362 and differs from it by no more than 3 nucleotides: 5'-AUUUCUGGGUAUUCUUUCZ25-3' (SEQ ID NO: 361); 5'-Z 26 GAAAGAAUACCCAGAAAU-3' (SEQ ID NO: 362), where Z 25 represents A, and Z 26 represents U, and nucleotide sequence I contains nucleotide Z 27 in the position corresponding to Z 25 ; nucleotide sequence II contains nucleotide Z 28 in the position corresponding to Z 26 , where Z 28 represents the first nucleotide from the 5' end of the antisense strand; viii) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 421 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 422 and differs from it by no more than 3 nucleotides: 5'-CAUGAAGGGCAUAAACUAZ 29 -3' (SEQ ID NO: 421); 5'-Z 30 UAGUUUAUGCCCUUCAUG-3' (SEQ ID NO: 422), where Z 29 represents U, and Z 30 represents A, and nucleotide sequence I contains nucleotide Z 31 in the position corresponding to Z 29 ; nucleotide sequence II contains nucleotide Z 32 in the position corresponding to Z 30 , where the nucleotide is Z 32 represents the first nucleotide from the 5' end of the antisense strand: ix) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 481 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 482 and differs from it by no more than 3 nucleotides: 5'-GGAUUCUGGAGAAAACUCZ 33 -3' (SEQ ID NO: 481); 5'-Z34GAGUUUUCUCCAGAAUCC-3' (SEQ ID NO: 482), where Z 33 represents A, and Z 34 represents U, and nucleotide sequence I contains nucleotide Z 35 in the position corresponding to Z 33 ; nucleotide sequence II contains nucleotide Z 36 in the position corresponding to Z 34 , where the nucleotide is Z 36 represents the first nucleotide from the 5' end of the antisense strand; wherein the sense strand and the antisense strand have the same or different lengths, wherein the sense strand contains 19-23 nucleotides in length and the antisense strand contains 19-26 nucleotides in length.

2. The miRNA according to claim 1, characterized in that the nucleotide sequence I differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 1, and / or the nucleotide sequence II differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 2; or the nucleotide sequence I differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 61, and / or the nucleotide sequence II differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 62; or the nucleotide sequence I differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 121, and / or the nucleotide sequence II differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 122; or the nucleotide sequence I differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 181, and / or the nucleotide sequence II differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 182; or the nucleotide sequence I differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 241, and / or the nucleotide sequence II differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 242; or the nucleotide sequence I differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 301, and / or the nucleotide sequence II differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 302; or the nucleotide sequence I differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 361, and / or the nucleotide sequence II differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 362; or the nucleotide sequence I differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 421, and / or the nucleotide sequence II differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 422; or the nucleotide sequence I differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO: 481, and / or the nucleotide sequence II differs by no more than 1 nucleotide from the nucleotide sequence presented in SEQ ID NO:

482.

3. The miRNA of claim 1 or 2, wherein the difference in nucleotides between the nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 2 includes a difference in the position of Z4, wherein Z4 is selected from U, C or G; or the nucleotide difference between the nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 62 comprises a difference in the position of Z8, wherein Z8 is selected from U, C or G; or the nucleotide difference between the nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 122 includes a difference in the position of Z 12 , and Z 12selected from A, C or G; or the nucleotide difference between the nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 182 includes a difference in the position of Z 16 , and Z 16 selected from U, C or G; or the nucleotide difference between the nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 242 includes a difference in the position of Z 20 , and Z 20 selected from U, C or G; or the nucleotide difference between the nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 302 includes a difference in the position of Z 24 , and Z 24 selected from A, U, or G; or the nucleotide difference between the nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 362 includes a difference in the position of Z 28 , and Z 28 selected from A, C or G; or the nucleotide difference between the nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 422 includes a difference in the position of Z 32 , and Z 32 selected from U, C or G; or the difference in nucleotides between the nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 482 includes a difference in the position of Z 36 , and Z 36 selected from A, C or G.

4. The miRNA according to any one of paragraphs 1-3, characterized in that Z3 is a nucleotide complementary to Z4; or Z7 is a nucleotide complementary to Z8; or Z 11 is a nucleotide complementary to Z 12 ; 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 ; or Z 27is a nucleotide complementary to Z 28 ; or Z 31 is a nucleotide complementary to Z 32 ; or Z 35 is a nucleotide complementary to Z 36 .

5. The miRNA according to any one of claims 1-4, characterized in that the nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 3, and the nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 4: 5'-GGGUAUUCUUUCAAGCAAZ3-3' (SEQ ID NO: 3); 5'-Z4UUGCUUGAAAGAAUACCC-3' (SEQ ID NO: 4), in which Z3 is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3; or nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 63, and nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 64: 5'-GGCAUAAACUAUAACAGCZ7-3' (SEQ ID NO: 63); 5'-Z8GCUGUUAUAGUUUAUGCC-3' (SEQ ID NO: 64), in which Z7 is selected from A, U, G or C, and Z8 is a nucleotide complementary to Z7; or nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 123, and nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 124: 5'-GCUCAAGAAUGCCAAGAAZ 11 -3' (SEQ ID NO: 123); 5'-Z 12 UUCUUGGCAUUCUGAGC-3' (SEQ ID NO: 124), in which Z 11 selected from A, U, G or C, and Z 12 is a nucleotide complementary to Z 11 ; or nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 183, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 184: 5'-GCAACAAAGACAUUUAUGZ15-3' (SEQ ID NO: 183); 5'-Z16CAUAAAUGUCUUUGUUGC-3' (SEQ ID NO: 184), in which Z 15 selected from A, U, G or C, and Z 16 is a nucleotide complementary to Z 15 ; or nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 243, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 244: 5'-GAAUCUCAAAGAAAUCUUZ19-3' (SEQ ID NO: 243); 5'-Z20AAGAUUUCUUUGAGAUUC-3' (SEQ ID NO: 244), in which Z 19 selected from A, U, G or C, and Z 20 is a nucleotide complementary to Z19 ; or nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 303, and nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 304: 5'-GUACGUGGACUGGAUUCUZ23-3' (SEQ ID NO: 303); 5'-Z 24 AGAAUCCAGUCCACGUAC-3' (SEQ ID NO: 304), in which Z 23 selected from A, U, G or C, and Z 24 is a nucleotide complementary to Z 23 ; or nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 363, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 364: 5'-AUUUCUGGGUAUUCUUUCZ27-3' (SEQ ID NO: 363); 5'-Z 28 GAAAGAAUACCCAGAAAU-3' (SEQ ID NO: 364), in which Z 27 selected from A, U, G or C, and Z 28is a nucleotide complementary to Z 27 ; or nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 423, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 424: 5'-CAUGAAGGGCAUAAACUAZ31-3' (SEQ ID NO: 423); 5'-Z32UAGUUUAUGCCCUUCAUG-3' (SEQ ID NO: 424), in which Z 31 selected from A, U, G or C, and Z 32 is a nucleotide complementary to Z 31 ; or nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 483, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 484: 5'-GGAUUCUGGAGAAAACUCZ 35 -3' (SEQ ID NO: 483); 5'-Z36GAGUUUUCUCCAGAAUCC-3' (SEQ ID NO: 484), in which Z 35selected from A, U, G or C, and Z 36 is a nucleotide complementary to Z 35 .

6. An siRNA capable of inhibiting expression of the blood coagulation factor XI gene in plasma, comprising a sense strand and an antisense strand, wherein each nucleotide in said siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II; nucleotide sequence I and nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein the sense strand further comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, and nucleotide sequence III and nucleotide sequence IV independently have a length of from 1 to 4 nucleotides; nucleotide sequence III is connected to the 5' end of nucleotide sequence I;and the nucleotide sequence IV is joined to the 3' end of the nucleotide sequence II; the nucleotide sequence III and the nucleotide sequence IV are of equal length and are substantially reverse complementary or completely reverse complementary to each other; where nucleotide sequence I, nucleotide sequence II, nucleotide sequence III and nucleotide sequence IV are selected from the sequences presented in any of i) to ix): i) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 1 and differs from it by no more than 3 nucleotides, and wherein nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 2 and differs from it by no more than 3 nucleotides 5'-GGGUAUUCUUUCAAGCAAZ1-3' (SEQ ID NO: 1); 5'-Z2UUGCUUGAAAGAAUACCC-3' (SEQ ID NO: 2), where Z1 is U and Z2 is A, and nucleotide sequence I contains nucleotide Z3 at a position corresponding to Z1; nucleotide sequence II contains nucleotide Z4 at a position corresponding to Z2, where Z4 is the first nucleotide from the 5' end of the antisense strand; nucleotide sequence III and nucleotide sequence IV are both 1 nucleotide in length, and the base of nucleotide sequence III is U; or nucleotide sequence III and nucleotide sequence IV are both 2 nucleotides in length, and, in the 5' to 3' direction, the base composition of nucleotide sequence III is CU; or nucleotide sequence III and nucleotide sequence IV are both 3 nucleotides in length, and, in the 5' to 3' direction, the base composition of nucleotide sequence III is UCU; or nucleotide sequence III and nucleotide sequence IV are both 4 nucleotides in length, and, in the 5' to 3' direction, the base composition of nucleotide sequence III is UUCU; or ii) wherein nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 61 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 62 and differs from it by no more than 3 nucleotides: 5'-GGCAUAAACUAUAACAGCZ5-3' (SEQ ID NO: 61); 5'-Z6GCUGUUAUAGUUUAUGCC-3' (SEQ ID NO: 62), where Z5 is U and Z6 is A, and nucleotide sequence I contains nucleotide Z7 at a position corresponding to Z5; nucleotide sequence II contains nucleotide Z8 at a position corresponding to Z6, where Z8 is the first nucleotide from the 5' end of the antisense strand; nucleotide sequence III and nucleotide sequence IV are both 1 nucleotide long, and the base of nucleotide sequence III is G; or nucleotide sequence III and nucleotide sequence IV are both 2 nucleotides long, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is AG; or nucleotide sequence III and nucleotide sequence IV are both 3 nucleotides long, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is AAG; or nucleotide sequence III and nucleotide sequence IV are both 4 nucleotides long, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is GAAG; or iii) wherein nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 121 and differs from it by no more than 3 nucleotides, and where nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 122 and differs from it by no more than 3 nucleotides: 5'-GCUCAAGAAUGCCAAGAAZ9-3' (SEQ ID NO: 121); 5'-Z 10 UUCUUGGCAUUCUGAGC-3' (SEQ ID NO: 122), where Z9 represents A and Z 10 represents U, and the nucleotide sequence I contains the nucleotide Z 11 at the position corresponding to Z9; nucleotide sequence II contains the nucleotide Z 12 in the position corresponding to Z 10 , where Z 12 represents the first nucleotide from the 5' end of the antisense strand; nucleotide sequence III and nucleotide sequence IV are both 1 nucleotide in length, and the base of nucleotide sequence III is U; or nucleotide sequence III and nucleotide sequence IV are both 2 nucleotides in length, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is GU; or nucleotide sequence III and nucleotide sequence IV are both 3 nucleotides long, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is AGU; or nucleotide sequence III and nucleotide sequence IV are both 4 nucleotides long, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is GAGU; or iv) wherein nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 181 and differs from it by no more than 3 nucleotides, and wherein nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 182 and differs from it by no more than 3 nucleotides: 5'-GCAACAAAGACAUUUAUGZ13-3' (SEQ ID NO: 181); 5'-Z14CAUAAAUGUCUUUGUUGC-3' (SEQ ID NO: 182), where Z 13 represents U, and Z 14 represents A, and the nucleotide sequence I contains the nucleotide Z 15 in the position corresponding to Z 13 ; nucleotide sequence II contains nucleotide Z 16 in the position corresponding to Z 14 , where Z 16 represents the first nucleotide from the 5' end of the antisense strand; nucleotide sequence III and nucleotide sequence IV are both 1 nucleotide long, and the base of nucleotide sequence III is U; or nucleotide sequence III and nucleotide sequence IV are both 2 nucleotides long, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is UU; or nucleotide sequence III and nucleotide sequence IV are both 3 nucleotides in length, and in the 5' to 3' direction, the base composition of nucleotide sequence III is CUU; or nucleotide sequence III and nucleotide sequence IV are both 4 nucleotides in length, and in the 5' to 3' direction, the base composition of nucleotide sequence III is GCUU; or v) wherein nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 241 and differs from it by no more than 3 nucleotides, and wherein nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 242 and differs from it by no more than 3 nucleotides: 5'-GAAUCUCAAAGAAAUCUUZ 17 -3' (SEQ ID NO: 241); 5'-Z 18 AAGAUUUCUUUGAGAUUC-3' (SEQ ID NO: 242), where Z 17 represents U, and Z 18 represents A, and the nucleotide sequence I contains the nucleotide Z 19 in the position corresponding to Z 17 ; nucleotide sequence II contains nucleotide Z 20 in the position corresponding to Z 18 , where Z 20 represents the first nucleotide from the 5' end of the antisense strand; nucleotide sequence III and nucleotide sequence IV are both 1 nucleotide in length, and the base of nucleotide sequence III is A; or nucleotide sequence III and nucleotide sequence IV are both 2 nucleotides in length, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is AA; or nucleotide sequence III and nucleotide sequence IV are both 3 nucleotides in length, and in the 5' to 3' direction, the base composition of nucleotide sequence III is AAA; or nucleotide sequence III and nucleotide sequence IV are both 4 nucleotides in length, and in the 5' to 3' direction, the base composition of nucleotide sequence III is CAAA; or vi) wherein nucleotide sequence I is the same length as the nucleotide sequence presented in SEQ ID NO: 301 and differs therefrom by no more than 3 nucleotides, and wherein nucleotide sequence II is the same length as the nucleotide sequence presented in SEQ ID NO: 302 and differs therefrom by no more than 3 nucleotides: 5'-GUACGUGGACUGGAUUCUZ 21 -3' (SEQ ID NO: 301); 5'-Z 22 AGAAUCCAGUCCACGUAC-3' (SEQ ID NO: 302), where Z 21represents G and Z 22 represents C, and the nucleotide sequence I contains the nucleotide Z 23 in the position corresponding to Z 21 ; nucleotide sequence II contains nucleotide Z24 at the position corresponding to Z 22 , where Z 24 represents the first nucleotide from the 5' end of the antisense strand; nucleotide sequence III and nucleotide sequence IV are both 1 nucleotide in length, and the base of nucleotide sequence III is A; or nucleotide sequence III and nucleotide sequence IV are both 2 nucleotides in length, and in the 5' to 3' direction, the base composition of nucleotide sequence III is GA; or nucleotide sequence III and nucleotide sequence IV are both 3 nucleotides in length, and in the 5' to 3' direction, the base composition of nucleotide sequence III is CGA; or nucleotide sequence III and nucleotide sequence IV are both 4 nucleotides long, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is UCGA; or vii) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 361 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 362 and differs from it by no more than 3 nucleotides: 5'-AUUUCUGGGUAUUCUUUCZ 25 -3' (SEQ ID NO: 361); 5'-Z 26 GAAAGAAUACCCAGAAAU-3' (SEQ ID NO: 362), where Z 25 represents A and Z 26 represents U, and the nucleotide sequence I contains the nucleotide Z 27 in the position corresponding to Z 25 ; nucleotide sequence II contains nucleotide Z28 in the position corresponding to Z 26 , where Z 28 represents the first nucleotide from the 5' end of the antisense strand; nucleotide sequence III and nucleotide sequence IV are both 1 nucleotide in length, and the base of nucleotide sequence III is G; or nucleotide sequence III and nucleotide sequence IV are both 2 nucleotides in length, and in the 5' to 3' direction, the base composition of nucleotide sequence III is CG; or nucleotide sequence III and nucleotide sequence IV are both 3 nucleotides in length, and in the 5' to 3' direction, the base composition of nucleotide sequence III is GCG; or nucleotide sequence III and nucleotide sequence IV are both 4 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is AGCG; or viii) wherein nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 421 and differs from it by no more than 3 nucleotides, and wherein nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 422 and differs from it by no more than 3 nucleotides: 5'-CAUGAAGGGCAUAAACUAZ 29 -3' (SEQ ID NO: 421); 5'-Z 30 UAGUUUAUGCCCUUCAUG-3' (SEQ ID NO: 422), where Z 29 represents U, and Z 30 represents A, and the nucleotide sequence I contains the nucleotide Z 31 in the position corresponding to Z 29 ; nucleotide sequence II contains nucleotide Z 32 in the position corresponding to Z 30 , where Z 32represents the first nucleotide from the 5' end of the antisense strand; nucleotide sequence III and nucleotide sequence IV are both 1 nucleotide long, and the base of nucleotide sequence III is A; or nucleotide sequence III and nucleotide sequence IV are both 2 nucleotides long, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is GA; or nucleotide sequence III and nucleotide sequence IV are both 3 nucleotides long, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is AGA; or nucleotide sequence III and nucleotide sequence IV are both 4 nucleotides long, and in the direction from the 5'-end to the 3'-end, the base composition of the nucleotide sequence III is UAGA; or ix) wherein nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 481 and differs from it by no more than 3 nucleotides, and wherein nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 482 and differs from it by no more than 3 nucleotides: 5'-GGAUUCUGGAGAAAACUCZ 33 -3' (SEQ ID NO: 481); 5'-Z 34 GAGUUUUCUCCAGAAUCC-3' (SEQ ID NO: 482), where Z 33 represents A and Z 34 represents U, and the nucleotide sequence I contains the nucleotide Z 35 in the position corresponding to Z 33 ; nucleotide sequence II contains nucleotide Z 36 in the position corresponding to Z 34 , Where Z 36 represents the first nucleotide from the 5' end of the antisense strand; where the sense strand and the antisense strand have the same or different lengths, where the sense strand has a length of 19-23 nucleotides, and the antisense strand has a length of 19-26 nucleotides; nucleotide sequence III and nucleotide sequence IV are both 1 nucleotide long, and the base of nucleotide sequence III is U; or nucleotide sequence III and nucleotide sequence IV are both 2 nucleotides long, and in the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is CU; or nucleotide sequence III and nucleotide sequence IV are both 3 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is ACU; or nucleotide sequence III and nucleotide sequence IV are both 4 nucleotides in length, and in the direction from the 5'-end to the 3'-end, the base composition of nucleotide sequence III is GACU.

7. An siRNA capable of inhibiting expression of the blood coagulation factor XI gene in plasma, comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II; nucleotide sequence I and nucleotide sequence II are at least partially reverse complementary, allowing for the formation of a double-stranded region; wherein nucleotide sequence I and nucleotide sequence II are selected from the sequences presented in any of i) to ix): i) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 1 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 2 and differs from it by no more than 3 nucleotides: 5'-GGGUAUUCUUUCAAGCAAZ1-3' (SEQ ID NO: 1); 5'-Z2UUGCUUGAAAGAAUACCC-3' (SEQ ID NO: 2), where Z1 represents U, and Z2 represents A, and nucleotide sequence I contains nucleotide Z3 at a position corresponding to Z1; nucleotide sequence II contains nucleotide Z4 at a position corresponding to Z2, where Z4 is the first nucleotide from the 5' end of the antisense strand; ii) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 61 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 62 and differs from it by no more than 3 nucleotides: 5'-GGCAUAAACUAUAACAGCZ5-3' (SEQ ID NO: 61); 5'-Z6GCUGUUAUAGUUUAUGCC-3' (SEQ ID NO: 62), where Z5 represents U, and Z6 represents A, and nucleotide sequence I contains nucleotide Z7 at a position corresponding to Z5; nucleotide sequence II contains nucleotide Z8 at a position corresponding to Z6, where Z8 is the first nucleotide from the 5' end of the antisense strand; iii) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 121 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 122 and differs from it by no more than 3 nucleotides: 5'-GCUCAAGAAUGCCAAGAAZ9-3' (SEQ ID NO: 121); 5'-Z 10 UUCUUGGCAUUCUGAGC-3' (SEQ ID NO: 122), where Z9 represents A and Z 10 represents U, and nucleotide sequence I contains nucleotide Z 11 at the position corresponding to Z9; nucleotide sequence II contains the nucleotide Z 12 in the position corresponding to Z 10 , where Z 12 represents the first nucleotide from the 5' end of the antisense strand; iv) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 181 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 182 and differs from it by no more than 3 nucleotides: 5'-GCAACAAAGACAUUUAUGZ 13 -3' (SEQ ID NO: 181); 5'-Z 14 CAUAAAUGUCUUUGUUGC-3' (SEQ ID NO: 182), where Z 13 represents U, and Z 14 represents A, and nucleotide sequence I contains nucleotide Z 15 in the position corresponding to Z 13 ; nucleotide sequence II contains nucleotide Z 16 in the position corresponding to Z 14 , where Z 16 represents the first nucleotide from the 5' end of the antisense strand; v) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 241 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 242 and differs from it by no more than 3 nucleotides: 5'-GAAUCUCAAAGAAAUCUUZ 17 -3' (SEQ ID NO: 241); 5'-Z 18 AAGAUUUCUUUGAGAUUC-3' (SEQ ID NO: 242), where Z 17 represents U, and Z 18 represents A, and nucleotide sequence I contains nucleotide Z 19 in the position corresponding to Z 17 ; nucleotide sequence II contains nucleotide Z 20 in the position corresponding to Z 18 , where Z 20 represents the first nucleotide from the 5' end of the antisense strand; vi) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 301 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 302 and differs from it by no more than 3 nucleotides: 5'-GUACGUGGACUGGAUUCUZ 21 -3' (SEQ ID NO: 301); 5'-Z 22 AGAAUCCAGUCCACGUAC-3' (SEQ ID NO: 302), where Z 21 represents G, and Z 22 represents C, and nucleotide sequence I contains nucleotide Z 23 in the position corresponding to Z 21 ; nucleotide sequence II contains nucleotide Z 24 in the position corresponding to Z 22 , where Z 24 represents the first nucleotide from the 5' end of the antisense strand; vii) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 361 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 362 and differs from it by no more than 3 nucleotides: 5'-AUUUCUGGGUAUUCUUUCZ 25 -3' (SEQ ID NO: 361); 5'-Z 26 GAAAGAAUACCCAGAAAU-3' (SEQ ID NO: 362), where Z 25 represents A and Z 26 represents U, and nucleotide sequence I contains nucleotide Z 27 in the position corresponding to Z 25 ; nucleotide sequence II contains nucleotide Z 28 in the position corresponding to Z 26 , where Z 28 represents the first nucleotide from the 5' end of the antisense strand; viii) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 421 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 422 and differs from it by no more than 3 nucleotides: 5'-CAUGAAGGGCAUAAACUAZ 29 -3' (SEQ ID NO: 421); 5'-Z 30 UAGUUUAUGCCCUUCAUG-3' (SEQ ID NO: 422), where Z 29 represents U, and Z 30 represents A, and nucleotide sequence I contains nucleotide Z 31 in the position corresponding to Z 29 ; nucleotide sequence II contains nucleotide Z 32 in the position corresponding to Z 30 , where Z 32 represents the first nucleotide from the 5' end of the antisense strand. ix) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 481 and differs from it by no more than 3 nucleotides, and nucleotide sequence II has the same length as the nucleotide sequence presented in SEQ ID NO: 482 and differs from it by no more than 3 nucleotides: 5'-GGAUUCUGGAGAAAACUCZ 33 -3' (SEQ ID NO: 481); 5'-Z 34 GAGUUUUCUCCAGAAUCC-3' (SEQ ID NO: 482), where Z 33 represents A, and Z 34 represents U, and nucleotide sequence I contains nucleotide Z 35 in the position corresponding to Z 33 ; nucleotide sequence II contains nucleotide Z 36 in the position corresponding to Z 34 , where Z 36 represents the first nucleotide from the 5' end of the antisense strand; where the sense strand and the antisense strand have the same or different lengths, wherein the sense strand has a length of 19-23 nucleotides, and the antisense strand has a length of 19-26 nucleotides; wherein the antisense strand further comprises a nucleotide sequence V; the nucleotide sequence V has a length of 1 to 3 nucleotides and is connected to the 3'-end of the antisense strand, forming a 3'-sticky end of the antisense strand; preferably, the nucleotide sequence V has a length of 2 nucleotides; preferably, the nucleotide sequence V is 2 consecutive thymine deoxyribonucleotides or 2 consecutive uracil ribonucleotides; or the nucleotide sequence V is complementary to the nucleotides at the corresponding positions of the target mRNA; more preferably, the sense strand of the siRNA comprises the nucleotide sequence presented in SEQ ID NO: 5, and the antisense strand of the siRNA comprises the nucleotide sequence presented in SEQ ID NO: 6: 5'-GGGUAUUCUUUCAAGCAAZ3-3' (SEQ ID NO: 5); 5'-Z4UUGCUUGAAAGAAUACCCAG-3' (SEQ ID NO: 6); or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 7, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 8: 5'-CUGGGUAUUCUUUCAAGCAAZ3-3' (SEQ ID NO: 7); 5'-Z4UUGCUUGAAAGAAUACCCAGAA-3' (SEQ ID NO: 8); where Z4 is the first nucleotide from the 5' end of the antisense strand; Z3 is selected from A, U, G, or C, and Z4 is a nucleotide complementary to Z3; or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 65, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 66: 5'-GGCAUAAACUAUAACAGCZ7-3' (SEQ ID NO: 65); 5'-Z8GCUGUUAUAGUUUAUGCCCU-3' (SEQ ID NO: 66); or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 67, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 68: 5'-AGGGCAUAAACUAUAACAGCZ7-3' (SEQ ID NO: 67); 5'-Z8GCUGUUAUAGUUUAUGCCCUUC-3' (SEQ ID NO: 68), where Z8 is the first nucleotide from the 5' end of the antisense strand; Z7 is selected from A, U, G, or C, and Z8 is a nucleotide complementary to Z7; or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 125, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 126: 5'-GCUCAAGAAUGCCAAGAA Z 11 -3' (SEQ ID NO: 125); 5'-Z 12 UUCUUGGCAUUCUUGAGCAC-3' (SEQ ID NO: 126), or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 127, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 128: 5'-GUGCUCAAGAAUGCCAAGAAZ 11 -3' (SEQ ID NO: 127); 5'-Z 12 UUCUUGGCAUUCUUGAGCACUC-3' (SEQ ID NO: 128), where Z 12 represents the first nucleotide from the 5' end of the antisense strand; Z 11 selected from A, U, G or C, and Z 12 is a nucleotide complementary to Z 11 ; or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 185, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 186: 5'-GCAACAAAGACAUUUAUGZ 15 -3' (SEQ ID NO: 185); 5'-Z 16 CAUAAAUGUCUUUGUUGCAA-3' (SEQ ID NO: 186), or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 187, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 188: 5'-UUGCAACAAAGACAUUUAUGZ 15 -3'(SEQ ID NO: 187); 5'-Z 16 CAUAAAUGUCUUUGUUGCAAGC-3'(SEQ ID NO: 188), where Z 16 represents the first nucleotide from the 5' end of the antisense strand; Z 15 selected from A, U, G or C, and Z 16 is a nucleotide complementary to Z 15 ; or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 245, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 246: 5'-GAAUCUCAAAGAAAUCUUZ 19 -3' (SEQ ID NO: 245); 5'-Z 20 AAGAUUUCUUUGAGAUUCUU-3' (SEQ ID NO: 246), or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 247, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 248: 5'-AAGAAUCUCAAAGAAAUCUUZ 19 -3' (SEQ ID NO: 247); 5'-Z 20 AAGAUUUCUUUGAGAUUCUUUG-3' (SEQ ID NO: 248), where Z 20 represents the first nucleotide from the 5' end of the antisense strand; Z 19 selected from A, U, G or C, and Z 20 is a nucleotide complementary to Z 19 ; or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 305, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 306: 5'-GUACGUGGACUGGAUUCUZ 23 -3' (SEQ ID NO: 305); 5'-Z 24 AGAAUCCAGUCCACGUACUC-3' (SEQ ID NO: 306), or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 307, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 308: 5'-GAGUACGUGGACUGGAUUCUZ 23 -3' (SEQ ID NO: 307); 5'-Z 24 AGAAUCCAGUCCACGUACUCGA-3' (SEQ ID NO: 308), where Z 24 represents the first nucleotide from the 5' end of the antisense strand; Z 23 selected from A, U, G or C, and Z 24 is a nucleotide complementary to Z 23 ; or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 365, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 366: 5'-AUUUCUGGGUAUUCUUUCZ 27 -3' (SEQ ID NO: 365); 5'-Z 28 GAAAGAAUACCCAGAAAUCG-3' (SEQ ID NO: 366); or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 367, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 368: 5'-CGAUUUCUGGGUAUUCUUUCZ 27 -3'(SEQ ID NO: 367); 5'-Z 28 GAAAGAAUACCCAGAAAUCGCU-3'(SEQ ID NO: 368); where Z 28 represents the first nucleotide from the 5' end of the antisense strand; Z 27 selected from A, U, G or C, and Z 28 is a nucleotide complementary to Z 27 ; or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 425, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 426: 5'-CAUGAAGGGCAUAAACUAZ 31 -3' (SEQ ID NO: 425); 5'-Z 32 UAGUUUAUGCCCUUCAUGUC-3' (SEQ ID NO: 426), or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 427, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 428: 5'-GACAUGAAGGGCAUAAACUAZ 31 -3'(SEQ ID NO: 427); 5'-Z 32 UAGUUUAUGCCCUUCAUGUCUA-3'(SEQ ID NO: 428), where Z 32 represents the first nucleotide from the 5' end of the antisense strand; Z 31 selected from A, U, G or C, and Z 32 is a nucleotide complementary to Z 31 ; or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 485, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 486: 5'-GGAUUCUGGAGAAAACUCZ 35 -3' (SEQ ID NO: 485); 5'-Z 36 GAGUUUUCUCCAGAAUCCAG-3' (SEQ ID NO: 486), or the sense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 487, and the antisense strand of the miRNA comprises the nucleotide sequence presented in SEQ ID NO: 488: 5'-CUGGAUUCUGGAGAAAACUCZ 35 -3'(SEQ ID NO: 487); 5'-Z 36 GAGUUUUCUCCAGAAUCCAGUC-3'(SEQ ID NO: 488), where Z 36 represents the first nucleotide from the 5' end of the antisense strand; Z 35 selected from A, U, G or C, and Z 36 is a nucleotide complementary to Z 35 ; even more preferably, the siRNA is any one of siFXIa1, siFXIa2, siFXIb1, siFXIb2, siFXIc1, siFXIc2, siFXId1, siFXId2, siFXIe1, siFXIe2, siFXIf1, siFXIf2, siFXIg1, siFXIg2, siFXIh1, siFXIh2, siFXIi1 and siFXIi2; the sense strand of siFXIa1 comprises the nucleotide sequence presented in SEQ ID NO: 9, and the antisense strand of siFXIa1 comprises the nucleotide sequence presented in SEQ ID NO: 10; or the sense strand of siFXIa2 comprises the nucleotide sequence presented in SEQ ID NO: 11, and the antisense strand of siFXIa2 comprises the nucleotide sequence presented in SEQ ID NO: 12; or the sense strand of siFXIb1 comprises the nucleotide sequence presented in SEQ ID NO: 69, and the antisense strand of siFXIb1 comprises the nucleotide sequence presented in SEQ ID NO: 70; or the sense strand of siFXIb2 comprises the nucleotide sequence presented in SEQ ID NO: 71, and the antisense strand of siFXIb2 comprises the nucleotide sequence presented in SEQ ID NO: 72; or the sense strand of siFXIc1 comprises the nucleotide sequence presented in SEQ ID NO: 129, and the antisense strand of siFXIc1 comprises the nucleotide sequence presented in SEQ ID NO: 130; or the sense strand of siFXIc2 comprises the nucleotide sequence presented in SEQ ID NO: 131, and the antisense strand of siFXIc2 comprises the nucleotide sequence presented in SEQ ID NO: 132; or the sense strand of siFXId1 comprises the nucleotide sequence presented in SEQ ID NO: 189, and the antisense strand of siFXId1 comprises the nucleotide sequence presented in SEQ ID NO: 190; or the sense strand of siFXId2 comprises the nucleotide sequence presented in SEQ ID NO: 191, and the antisense strand of siFXId2 comprises the nucleotide sequence presented in SEQ ID NO: 192; or the sense strand of siFXIe1 comprises the nucleotide sequence presented in SEQ ID NO: 249, and the antisense strand of siFXIe1 comprises the nucleotide sequence presented in SEQ ID NO: 250; or the sense strand of siFXIe2 comprises the nucleotide sequence presented in SEQ ID NO: 251, and the antisense strand of siFXIe2 comprises the nucleotide sequence presented in SEQ ID NO: 252; or the sense strand of siFXIf1 comprises the nucleotide sequence presented in SEQ ID NO: 309, and the antisense strand of siFXIf1 comprises the nucleotide sequence presented in SEQ ID NO: 310; or the sense strand of siFXIf2 comprises the nucleotide sequence presented in SEQ ID NO: 311, and the antisense strand of siFXIf2 comprises the nucleotide sequence presented in SEQ ID NO: 312; or the sense strand of siFXIg1 comprises the nucleotide sequence presented in SEQ ID NO: 369, and the antisense strand of siFXIg1 comprises the nucleotide sequence presented in SEQ ID NO: 370; or the sense strand of siFXIg2 comprises the nucleotide sequence presented in SEQ ID NO: 371, and the antisense strand of siFXIg2 comprises the nucleotide sequence presented in SEQ ID NO: 372; or the sense strand of siFXIh1 comprises the nucleotide sequence presented in SEQ ID NO: 429, and the antisense strand of siFXIh1 comprises the nucleotide sequence presented in SEQ ID NO: 430; or the sense strand of siFXIh2 comprises the nucleotide sequence presented in SEQ ID NO: 431, and the antisense strand of siFXIh2 comprises the nucleotide sequence presented in SEQ ID NO: 432; or the sense strand of siFXIi1 comprises the nucleotide sequence presented in SEQ ID NO: 489, and the antisense strand of siFXIi1 comprises the nucleotide sequence presented in SEQ ID NO: 490; or the sense strand of siFXIi2 comprises the nucleotide sequence presented in SEQ ID NO: 491, and the antisense strand of siFXIi2 comprises the nucleotide sequence presented in SEQ ID NO:

492.

8. The miRNA according to any one of claims 1 to 7, characterized in that at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide and / or at least one phosphate group is a phosphate group with modified group(s).

9. The miRNA of claim 8, wherein each nucleotide in the sense strand and the antisense strand is independently a fluorine-modified nucleotide or a nucleotide with a non-fluorine modification; preferably, the fluorine-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II; and, in the direction from the 5'-end to the 3'-end, at least the nucleotides in positions 7, 8 and 9 of nucleotide sequence I are fluorine-modified nucleotides; and, in the direction from the 5'-end to the 3'-end, at least the nucleotides in positions 2, 6, 14 and 16 of nucleotide sequence II are fluorine-modified nucleotides; more preferably, in the 5'-to-3'-direction, the nucleotides at positions 7, 8, and 9, or at positions 5, 7, 8, and 9, of the nucleotide sequence I in the sense strand are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are nucleotides with a non-fluorine modification; and, in the 5'-to-3'-direction, the nucleotides at positions 2, 6, 14, and 16, or at positions 2, 6, 8, 9, 14, and 16, of the nucleotide sequence II in the antisense strand are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are nucleotides with a non-fluorine modification; preferably, each nucleotide with a non-fluorine modification is independently selected from a nucleotide formed by replacing the 2'-hydroxyl of its ribose group with a group other than fluorine, or a nucleotide analogue; more preferably, the nucleotide formed by substituting the 2'-hydroxyl of the ribose group with a group other than fluorine is a nucleotide selected from the group consisting of 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides and 2'-deoxynucleotides; and the nucleotide analogue is an analogue selected from the group consisting of an isonucleotide, LNA, ENA, cET, UNA and GNA; Even more preferably, each nucleotide with a non-fluorine modification is a methoxy-modified nucleotide; and the methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl of a ribose group with a methoxy group.

10. The siRNA of any one of claims 1 to 9, wherein, in the 5' to 3' direction, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides; and, in the 5' to 3' direction, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are methoxy-modified nucleotides; or in the 5' to 3' direction, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides; and in the 5' to 3' direction, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are methoxy-modified nucleotides; or in 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 fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides; and in the direction from the 5'-end to the 3'-end, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are methoxy-modified nucleotides.

11. The miRNA according to claim 8, characterized in that the phosphate group with the modified group(s) is a phosphorothioate group formed by replacing at least one oxygen atom in a complex phosphodiester bond in the phosphate group with a sulfur atom; preferably, the phosphate group with the modified group(s) is a phosphorothioate group having a structure represented by Formula (1): Formula (1); more preferably, the phosphorothioate linkage is present in at least one of the groups consisting of the following positions: position between the first and second nucleotides at the 5' end of the sense strand; position between the second and third nucleotides at the 5' end of the sense strand; position between the first and second nucleotides at the 3' end of the sense strand; position between the second and third nucleotides at the 3' end of the sense strand; position between the first and second nucleotides at the 5' end of the antisense strand; position between the second and third nucleotides at the 5' end of the antisense strand; the position between the first and second nucleotides at the 3' end of the antisense strand; and position between the second and third nucleotides at the 3' end of the antisense strand.

12. The miRNA according to any one of paragraphs 1-11, characterized in that the nucleotide at the 5'-end of the antisense strand is a 5'-phosphate nucleotide and a nucleotide modified with a 5'-phosphate analogue; wherein preferably the 5'-phosphate nucleotide is a nucleotide having a structure represented by Formula (2), and the nucleotide modified with a 5'-phosphate analogue is a nucleotide having a structure represented by any one of Formulas (3) to (6): where R is selected from H, OH, methoxy or F; "base" is a base selected from A, U, C, G or T.

13. The miRNA according to any one of claims 1-12, characterized in that the siRNA is any of: siFXIa1-M1, siFXIa1-M2, siFXIa1-M3, siFXIa2-M1, siFXIa2-M2, siFXIa2-M3, siFXIb1-M1, siFXIb1-M2, siFXIb1-M3, siFXIb2-M1, siFXIb2-M2, siFXIb2-M3, siFXIc1-M1, siFXIc1-M2, siFXIc1-M3, siFXIc2-M1, siFXIc2-M2, siFXIc2-M3, siFXId1-M1, siFXId1-M2, siFXId1-M3, siFXId2-M1, siFXId2-M2, siFXId2-M3, siFXIe1-M1, siFXIe1-M2, siFXIe1-M3, siFXIe2-M1, siFXIe2-M2, siFXIe2-M3, siFXIf1-M1, siFXIf1-M2, siFXIf1-M3, siFXIf2-M1, siFXIf2-M2, siFXIf2-M3, siFXIg1-M1, siFXIg1-M2, siFXIg1-M3, siFXIg2-M1, siFXIg2-M2, siFXIg2-M3, siFXIh1-M1, siFXIh1-M2, siFXIh1-M3, siFXIh2-M1, siFXIh2-M2, siFXIh2-M3, siFXIi1-M1, siFXIi1-M2, siFXIi1-M3, siFXIi2-M1, siFXIi2-M2 and siFXIi2-M3; the sense strand of siFXIa1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 13, and the antisense strand of siFXIa1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 14; or the sense strand of siFXIa1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 15, and the antisense strand of siFXIa1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 16; or the sense strand of siFXIa1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 17, and the antisense strand of siFXIa1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 18; or the sense strand of siFXIa2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 19, and the antisense strand of siFXIa2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 20; or the sense strand of siFXIa2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 21, and the antisense strand of siFXIa2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 22; or the sense strand of siFXIa2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 23, and the antisense strand of siFXIa2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 24; or the sense strand of siFXIb1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 73, and the antisense strand of siFXIb1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 74; or the sense strand of siFXIb1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 75, and the antisense strand of siFXIb1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 76; or the sense strand of siFXIb1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 77, and the antisense strand of siFXIb1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 78; or the sense strand of siFXIb2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 79, and the antisense strand of siFXIb2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 80; or the sense strand of siFXIb2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 81, and the antisense strand of siFXIb2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 82; or the sense strand of siFXIb2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 83, and the antisense strand of siFXIb2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 84; or the sense strand of siFXIc1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 133, and the antisense strand of siFXIc1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 134; or the sense strand of siFXIc1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 135, and the antisense strand of siFXIc1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 136; or the sense strand of siFXIc1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 137, and the antisense strand of siFXIc1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 138; or the sense strand of siFXIc2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 139, and the antisense strand of siFXIc2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 140; or the sense strand of siFXIc2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 141, and the antisense strand of siFXIc2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 142; or the sense strand of siFXIc2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 143, and the antisense strand of siFXIc2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 144; or the sense strand of siFXId1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 193, and the antisense strand of siFXId1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 194; or the sense strand of siFXId1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 195, and the antisense strand of siFXId1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 196; or the sense strand of siFXId1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 197, and the antisense strand of siFXId1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 198; or the sense strand of siFXId2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 199, and the antisense strand of siFXId2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 200; or the sense strand of siFXId2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 201, and the antisense strand of siFXId2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 202; the sense strand of siFXId2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 203, and the antisense strand of siFXId2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 204; or the sense strand of siFXIe1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 253, and the antisense strand of siFXIe1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 254; or the sense strand of siFXIe1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 255, and the antisense strand of siFXIe1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 256; or the sense strand of siFXIe1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 257, and the antisense strand of siFXIe1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 258; or the sense strand of siFXIe2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 259, and the antisense strand of siFXIe2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 260; or the sense strand of siFXIe2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 261, and the antisense strand of siFXIe2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 262; or the sense strand of siFXIe2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 263, and the antisense strand of siFXIe2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 264; or the sense strand of siFXIf1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 313, and the antisense strand of siFXIf1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 314; or the sense strand of siFXIf1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 315, and the antisense strand of siFXIf1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 316; or the sense strand of siFXIf1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 317, and the antisense strand of siFXIf1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 318; or the sense strand of siFXIf2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 319, and the antisense strand of siFXIf2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 320; or the sense strand of siFXIf2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 321, and the antisense strand of siFXIf2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 322; or the sense strand of siFXIf2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 323, and the antisense strand of siFXIf2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 324; or the sense strand of siFXIg1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 373, and the antisense strand of siFXIg1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 374; or the sense strand of siFXIg1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 375, and the antisense strand of siFXIg1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 376; or the sense strand of siFXIg1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 377, and the antisense strand of siFXIg1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 378; or the sense strand of siFXIg2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 379, and the antisense strand of siFXIg2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 380; or the sense strand of siFXIg2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 381, and the antisense strand of siFXIg2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 382; or the sense strand of siFXIg2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 383, and the antisense strand of siFXIg2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 384; or the sense strand of siFXIh1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 433, and the antisense strand of siFXIh1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 434; or the sense strand of siFXIh1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 435, and the antisense strand of siFXIh1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 436; or the sense strand of siFXIh1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 437, and the antisense strand of siFXIh1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 438; or the sense strand of siFXIh2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 439, and the antisense strand of siFXIh2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 440; or the sense strand of siFXIh2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 441, and the antisense strand of siFXIh2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 442; or the sense strand of siFXIh2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 443, and the antisense strand of siFXIh2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 444; or the sense strand of siFXIi1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 493, and the antisense strand of siFXIi1-M1 comprises the nucleotide sequence presented in SEQ ID NO: 494; or the sense strand of siFXIi1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 495, and the antisense strand of siFXIi1-M2 comprises the nucleotide sequence presented in SEQ ID NO: 496; or the sense strand of siFXIi1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 497, and the antisense strand of siFXIi1-M3 comprises the nucleotide sequence presented in SEQ ID NO: 498; or the sense strand of siFXIi2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 499, and the antisense strand of siFXIi2-M1 comprises the nucleotide sequence presented in SEQ ID NO: 500; or the sense strand of siFXIi2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 501, and the antisense strand of siFXIi2-M2 comprises the nucleotide sequence presented in SEQ ID NO: 502; or the sense strand of siFXIi2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 503, and the antisense strand of siFXIi2-M3 comprises the nucleotide sequence presented in SEQ ID NO: 504; or the siRNA is any of: siFXIa1-M1S, siFXIa1-M2S, siFXIa1-M3S, siFXIa2-M1S, siFXIa2-M2S, siFXIa2-M3S, siFXIb1-M1S, siFXIb1-M2S, siFXIb1-M3S, siFXIb2-M1S, siFXIb2-M2S, siFXIb2-M3S, siFXIc1-M1S, siFXIc1-M2S, siFXIc1-M3S, siFXIc2-M1S, siFXIc2-M2S, siFXIc2-M3S, siFXId1-M1S, siFXId1-M2S, siFXId1-M3S, siFXId2-M1S, siFXId2-M2S, siFXId2-M3S, siFXIe1-M1S, siFXIe1-M2S, siFXIe1-M3S, siFXIe2-M1S, siFXIe2-M2S, siFXIe2-M3S, siFXIf1-M1S, siFXIf1-M2S, siFXIf1-M3S, siFXIf2-M1S, siFXIf2-M2S, siFXIf2-M3S, siFXIg1-M1S, siFXIg1-M2S, siFXIg1-M3S, siFXIg2-M1S, siFXIg2-M2S, siFXIg2-M3S, siFXIh1-M1S, siFXIh1-M2S, siFXIh1-M3S, siFXIh2-M1S, siFXIh2-M2S, siFXIh2-M3S, FXIi1-M1S, siFXIi1-M2S, siFXIi1-M3S, siFXIi2-M1S, siFXIi2-M2S and siFXIi2-M3S; the sense strand of siFXIa1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 25, and the antisense strand of siFXIa1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 26; or the sense strand of siFXIa1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 27, and the antisense strand of siFXIa1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 28; or the sense strand of siFXIa1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 29, and the antisense strand of siFXIa1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 30; or the sense strand of siFXIa2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 31, and the antisense strand of siFXIa2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 32; or the sense strand of siFXIa2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 33, and the antisense strand of siFXIa2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 34; or the sense strand of siFXIa2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 35, and the antisense strand of siFXIa2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 36; or the sense strand of siFXIb1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 85, and the antisense strand of siFXIb1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 86; or the sense strand of siFXIb1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 87, and the antisense strand of siFXIb1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 88; or the sense strand of siFXIb1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 89, and the antisense strand of siFXIb1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 90; or the sense strand of siFXIb2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 91, and the antisense strand of siFXIb2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 92; or the sense strand of siFXIb2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 93, and the antisense strand of siFXIb2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 94; or the sense strand of siFXIb2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 95, and the antisense strand of siFXIb2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 96; or the sense strand of siFXIc1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 145, and the antisense strand of siFXIc1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 146; or the sense strand of siFXIc1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 147, and the antisense strand of siFXIc1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 148; or the sense strand of siFXIc1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 149, and the antisense strand of siFXIc1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 150; or the sense strand of siFXIc2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 151, and the antisense strand of siFXIc2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 152; or the sense strand of siFXIc2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 153, and the antisense strand of siFXIc2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 154; or the sense strand of siFXIc2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 155, and the antisense strand of siFXIc2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 156; or the sense strand of siFXId1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 205, and the antisense strand of siFXId1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 206; or the sense strand of siFXId1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 207, and the antisense strand of siFXId1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 208; or the sense strand of siFXId1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 209, and the antisense strand of siFXId1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 210; or the sense strand of siFXId2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 211, and the antisense strand of siFXId2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 212; or the sense strand of siFXId2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 213, and the antisense strand of siFXId2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 214; or the sense strand of siFXId2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 215, and the antisense strand of siFXId2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 216; or the sense strand of siFXIe1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 265, and the antisense strand of siFXIe1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 266; or the sense strand of siFXIe1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 267, and the antisense strand of siFXIe1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 268; or the sense strand of siFXIe1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 269, and the antisense strand of siFXIe1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 270; or the sense strand of siFXIe2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 271, and the antisense strand of siFXIe2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 272; or the sense strand of siFXIe2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 273, and the antisense strand of siFXIe2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 274; or the sense strand of siFXIe2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 275, and the antisense strand of siFXIe2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 276; or the sense strand of siFXIf1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 325, and the antisense strand of siFXIf1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 326; or the sense strand of siFXIf1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 327, and the antisense strand of siFXIf1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 328; or the sense strand of siFXIf1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 329, and the antisense strand of siFXIf1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 330; or the sense strand of siFXIf2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 331, and the antisense strand of siFXIf2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 332; or the sense strand of siFXIf2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 333, and the antisense strand of siFXIf2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 334; or the sense strand of siFXIf2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 335, and the antisense strand of siFXIf2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 336; or the sense strand of siFXIg1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 385, and the antisense strand of siFXIg1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 386; or the sense strand of siFXIg1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 387, and the antisense strand of siFXIg1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 388; or the sense strand of siFXIg1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 389, and the antisense strand of siFXIg1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 390; or the sense strand of siFXIg2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 391, and the antisense strand of siFXIg2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 392; or the sense strand of siFXIg2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 393, and the antisense strand of siFXIg2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 394; or the sense strand of siFXIg2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 395, and the antisense strand of siFXIg2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 396; or the sense strand of siFXIh1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 445, and the antisense strand of siFXIh1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 446; or the sense strand of siFXIh1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 447, and the antisense strand of siFXIh1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 448; or the sense strand of siFXIh1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 449, and the antisense strand of siFXIh1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 450; or the sense strand of siFXIh2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 451, and the antisense strand of siFXIh2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 452; or the sense strand of siFXIh2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 453, and the antisense strand of siFXIh2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 454; or the sense strand of siFXIh2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 455, and the antisense strand of siFXIh2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 456; or the sense strand of FXIi1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 505, and the antisense strand of FXIi1-M1S comprises the nucleotide sequence presented in SEQ ID NO: 506; or the sense strand of siFXIi1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 507, and the antisense strand of siFXIi1-M2S comprises the nucleotide sequence presented in SEQ ID NO: 508; or the sense strand of siFXIi1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 509, and the antisense strand of siFXIi1-M3S comprises the nucleotide sequence presented in SEQ ID NO: 510; or the sense strand of siFXIi2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 511, and the antisense strand of siFXIi2-M1S comprises the nucleotide sequence presented in SEQ ID NO: 512; or the sense strand of siFXIi2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 513, and the antisense strand of siFXIi2-M2S comprises the nucleotide sequence presented in SEQ ID NO: 514; or the sense strand of siFXIi2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 515, and the antisense strand of siFXIi2-M3S comprises the nucleotide sequence presented in SEQ ID NO: 516; or the miRNA is any of: siFXIa1-M1P1, siFXIa1-M2P1, siFXIa1-M3P1, siFXIa2-M1P1, siFXIa2-M2P1, siFXIa2-M3P1, siFXIa1-M1SP1, siFXIa1-M2SP1, siFXIa1-M3SP1, siFXIa2-M1SP1, siFXIa2-M2SP1, siFXIa2-M3SP1, siFXIb1-M1P1, siFXIb1-M2P1, siFXIb1-M3P1, siFXIb2-M1P1, siFXIb2-M2P1, siFXIb2-M3P1, siFXIb1-M1SP1, siFXIb1-M2SP1, siFXIb1-M3SP1, siFXIb2-M1SP1, siFXIb2-M2SP1, siFXIb2-M3SP1, siFXIc1-M1P1, siFXIc1-M2P1, siFXIc1-M3P1, siFXIc2-M1P1, siFXIc2-M2P1, siFXIc2-M3P1, siFXIc1-M1SP1, siFXIc1-M2SP1, siFXIc1-M3SP1, siFXIc2-M1SP1, siFXIc2-M2SP1, siFXIc2-M3SP1, siFXId1-M1P1, siFXId1-M2P1, siFXId1-M3P1, siFXId2-M1P1, siFXId2-M2P1, siFXId2-M3P1, siFXId1-M1SP1, siFXId1-M2SP1, siFXId1-M3SP1, siFXId2-M1SP1, siFXId2-M2SP1, siFXId2-M3SP1, siFXIe1-M1P1, siFXIe1-M2P1, siFXIe1-M3P1, siFXIe2-M1P1, siFXIe2-M2P1, siFXIe2-M3P1, siFXIe1-M1SP1, siFXIe1-M2SP1, siFXIe1-M3SP1, siFXIe2-M1SP1, siFXIe2-M2SP1, siFXIe2-M3SP1, siFXIf1-M1P1, siFXIf1-M2P1, siFXIf1-M3P1, siFXIf2-M1P1, siFXIf2-M2P1, siFXIf2-M3P1,siFXIf1-M1SP1, siFXIf1-M2SP1, siFXIf1-M3SP1, siFXIf2-M1SP1, siFXIf2-M2SP1, siFXIf2-M3SP1, siFXIg1-M1P1, siFXIg1-M2P1, siFXIg1-M3P1, siFXIg2-M1P1, siFXIg2-M2P1, siFXIg2-M3P1, siFXIg1-M1SP1, siFXIg1-M2SP1, siFXIg1-M3SP1, siFXIg2-M1SP1, siFXIg2-M2SP1, siFXIg2-M3SP1, siFXIh1-M1P1, siFXIh1-M2P1, siFXIh1-M3P1, siFXIh2-M1P1, siFXIh2-M2P1, siFXIh2-M3P1, siFXIh1-M1SP1, siFXIh1-M2SP1, siFXIh1-M3SP1, siFXIh2-M1SP1, siFXIh2-M2SP1, siFXIh2-M3SP1, siFXIi1-M1P1, siFXIi1-M2P1, siFXIi1-M3P1, siFXIi2-M1P1, siFXIi2-M2P1, siFXIi2-M3P1, siFXIi1-M1SP1, siFXIi1-M2SP1, siFXIi1-M3SP1, siFXIi2-M1SP1, siFXIi2-M2SP1 and siFXIi2-M3SP1, the sense strand of siFXIa1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 37, and the antisense strand of siFXIa1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 38; or the sense strand of siFXIa1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 39, and the antisense strand of siFXIa1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 40; or the sense strand of siFXIa1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 41, and the antisense strand of siFXIa1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 42; or the sense strand of siFXIa2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 43, and the antisense strand of siFXIa2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 44; or the sense strand of siFXIa2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 45, and the antisense strand of siFXIa2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 46; or the sense strand of siFXIa2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 47, and the antisense strand of siFXIa2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 48; or the sense strand of siFXIa1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 49, and the antisense strand of siFXIa1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 50; or the sense strand of siFXIa1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 51, and the antisense strand of siFXIa1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 52; or the sense strand of siFXIa1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 53, and the antisense strand of siFXIa1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 54; or the sense strand of siFXIa2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 55, and the antisense strand of siFXIa2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 56; or the sense strand of siFXIa2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 57, and the antisense strand of siFXIa2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 58; or the sense strand of siFXIa2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 59, and the antisense strand of siFXIa2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 60; or the sense strand of siFXIb1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 97, and the antisense strand of siFXIb1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 98; or the sense strand of siFXIb1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 99, and the antisense strand of siFXIb1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 100; or the sense strand of siFXIb1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 101, and the antisense strand of siFXIb1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 102; or the sense strand of siFXIb2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 103, and the antisense strand of siFXIb2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 104; or the sense strand of siFXIb2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 105, and the antisense strand of siFXIb2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 106; or the sense strand of siFXIb2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 107, and the antisense strand of siFXIb2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 108; or the sense strand of siFXIb1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 109, and the antisense strand of siFXIb1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 110; or the sense strand of siFXIb1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 111, and the antisense strand of siFXIb1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 112; or the sense strand of siFXIb1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 113, and the antisense strand of siFXIb1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 114; or the sense strand of siFXIb2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 115, and the antisense strand of siFXIb2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 116; or the sense strand of siFXIb2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 117, and the antisense strand of siFXIb2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 118; or the sense strand of siFXIb2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 119, and the antisense strand of siFXIb2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 120; or the sense strand of siFXIc1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 157, and the antisense strand of siFXIc1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 158; or the sense strand of siFXIc1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 159, and the antisense strand of siFXIc1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 160; or the sense strand of siFXIc1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 161, and the antisense strand of siFXIc1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 162; or the sense strand of siFXIc2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 163, and the antisense strand of siFXIc2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 164; or the sense strand of siFXIc2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 165, and the antisense strand of siFXIc2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 166; or the sense strand of siFXIc2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 167, and the antisense strand of siFXIc2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 168; or the sense strand of siFXIc1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 169, and the antisense strand of siFXIc1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 170; or the sense strand of siFXIc1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 171, and the antisense strand of siFXIc1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 172; or the sense strand of siFXIc1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 173, and the antisense strand of siFXIc1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 174; or the sense strand of siFXIc2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 175, and the antisense strand of siFXIc2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 176; or the sense strand of siFXIc2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 177, and the antisense strand of siFXIc2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 178; or the sense strand of siFXIc2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 179, and the antisense strand of siFXIc2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 180; or the sense strand of siFXId1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 217, and the antisense strand of siFXId1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 218; or the sense strand of siFXId1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 219, and the antisense strand of siFXId1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 220; or the sense strand of siFXId1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 221, and the antisense strand of siFXId1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 222; or the sense strand of siFXId2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 223, and the antisense strand of siFXId2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 224; or the sense strand of siFXId2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 225, and the antisense strand of siFXId2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 226; or the sense strand of siFXId2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 227, and the antisense strand of siFXId2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 228; or the sense strand of siFXId1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 229, and the antisense strand of siFXId1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 230; or the sense strand of siFXId1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 231, and the antisense strand of siFXId1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 232; or the sense strand of siFXId1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 233, and the antisense strand of siFXId1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 234; or the sense strand of siFXId2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 235, and the antisense strand of siFXId2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 236; or the sense strand of siFXId2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 237, and the antisense strand of siFXId2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 238; or the sense strand of siFXId2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 239, and the antisense strand of siFXId2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 240; or the sense strand of siFXIe1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 277, and the antisense strand of siFXIe1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 278; or the sense strand of siFXIe1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 279, and the antisense strand of siFXIe1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 280; or the sense strand of siFXIe1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 281, and the antisense strand of siFXIe1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 282; or the sense strand of siFXIe2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 283, and the antisense strand of siFXIe2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 284; or the sense strand of siFXIe2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 285, and the antisense strand of siFXIe2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 286; or the sense strand of siFXIe2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 287, and the antisense strand of siFXIe2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 288; or the sense strand of siFXIe1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 289, and the antisense strand of siFXIe1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 290; or the sense strand of siFXIe1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 291, and the antisense strand of siFXIe1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 292; or the sense strand of siFXIe1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 293, and the antisense strand of siFXIe1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 294; or the sense strand of siFXIe2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 295, and the antisense strand of siFXIe2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 296; or the sense strand of siFXIe2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 297, and the antisense strand of siFXIe2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 298; or the sense strand of siFXIe2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 299, and the antisense strand of siFXIe2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 300; or the sense strand of siFXIf1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 337, and the antisense strand of siFXIf1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 338; or the sense strand of siFXIf1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 339, and the antisense strand of siFXIf1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 340; or the sense strand of siFXIf1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 341, and the antisense strand of siFXIf1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 342; or the sense strand of siFXIf2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 343, and the antisense strand of siFXIf2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 344; or the sense strand of siFXIf2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 345, and the antisense strand of siFXIf2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 346; or the sense strand of siFXIf2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 347, and the antisense strand of siFXIf2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 348; or the sense strand of siFXIf1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 349, and the antisense strand of siFXIf1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 350; or the sense strand of siFXIf1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 351, and the antisense strand of siFXIf1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 352; or the sense strand of siFXIf1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 353, and the antisense strand of siFXIf1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 354; or the sense strand of siFXIf2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 355, and the antisense strand of siFXIf2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 356; or the sense strand of siFXIf2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 357, and the antisense strand of siFXIf2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 358; or the sense strand of siFXIf2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 359, and the antisense strand of siFXIf2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 360; or the sense strand of siFXIg1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 397, and the antisense strand of siFXIg1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 398; or the sense strand of siFXIg1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 399, and the antisense strand of siFXIg1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 400; or the sense strand of siFXIg1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 401, and the antisense strand of siFXIg1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 402; or the sense strand of siFXIg2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 403, and the antisense strand of siFXIg2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 404; or the sense strand of siFXIg2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 405, and the antisense strand of siFXIg2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 406; or the sense strand of siFXIg2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 407, and the antisense strand of siFXIg2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 408; or the sense strand of siFXIg1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 409, and the antisense strand of siFXIg1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 410; or the sense strand of siFXIg1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 411, and the antisense strand of siFXIg1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 412; or the sense strand of siFXIg1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 413, and the antisense strand of siFXIg1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 414; or the sense strand of siFXIg2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 415, and the antisense strand of siFXIg2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 416; or the sense strand of siFXIg2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 417, and the antisense strand of siFXIg2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 418; or the sense strand of siFXIg2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 419, and the antisense strand of siFXIg2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 420; or the sense strand of siFXIh1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 457, and the antisense strand of siFXIh1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 458; or the sense strand of siFXIh1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 459, and the antisense strand of siFXIh1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 460; or the sense strand of siFXIh1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 461, and the antisense strand of siFXIh1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 462; or the sense strand of siFXIh2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 463, and the antisense strand of siFXIh2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 464; or the sense strand of siFXIh2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 465, and the antisense strand of siFXIh2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 466; or the sense strand of siFXIh2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 467, and the antisense strand of siFXIh2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 468; or the sense strand of siFXIh1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 469, and the antisense strand of siFXIh1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 470; or the sense strand of siFXIh1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 471, and the antisense strand of siFXIh1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 472; or the sense strand of siFXIh1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 473, and the antisense strand of siFXIh1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 474; or the sense strand of siFXIh2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 475, and the antisense strand of siFXIh2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 476; or the sense strand of siFXIh2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 477, and the antisense strand of siFXIh2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 478; or the sense strand of siFXIh2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 479, and the antisense strand of siFXIh2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 480; or the sense strand of siFXIi1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 517, and the antisense strand of siFXIi1-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 518; or the sense strand of siFXIi1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 519, and the antisense strand of siFXIi1-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 520; or the sense strand of siFXIi1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 521, and the antisense strand of siFXIi1-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 522; or the sense strand of siFXIi2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 523, and the antisense strand of siFXIi2-M1P1 comprises the nucleotide sequence presented in SEQ ID NO: 524; or the sense strand of siFXIi2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 525, and the antisense strand of siFXIi2-M2P1 comprises the nucleotide sequence presented in SEQ ID NO: 526; or the sense strand of siFXIi2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 527, and the antisense strand of siFXIi2-M3P1 comprises the nucleotide sequence presented in SEQ ID NO: 528; or the sense strand of siFXIi1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 529, and the antisense strand of siFXIi1-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 530; or the sense strand of siFXIi1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 531, and the antisense strand of siFXIi1-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 532; or the sense strand of siFXIi1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 533, and the antisense strand of siFXIi1-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 534; or the sense strand of siFXIi2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 535, and the antisense strand of siFXIi2-M1SP1 comprises the nucleotide sequence presented in SEQ ID NO: 536; or the sense strand of siFXIi2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 537, and the antisense strand of siFXIi2-M2SP1 comprises the nucleotide sequence presented in SEQ ID NO: 538; or the sense strand of siFXIi2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO: 539, and the antisense strand of siFXIi2-M3SP1 comprises the nucleotide sequence presented in SEQ ID NO:

540.

14. A pharmaceutical composition for the treatment or prevention of thrombotic diseases or ischemic stroke caused by increased expression of the FXI gene, characterized in that the pharmaceutical composition contains an effective amount of siRNA according to any one of claims 1-13 and a pharmaceutically acceptable carrier; Preferably, the mass ratio of siRNA to pharmaceutically acceptable carrier is 1:(1-500).

15. An siRNA conjugate for the treatment or prevention of thrombotic diseases or ischemic stroke caused by increased expression of the FXI gene, comprising the siRNA of any one of claims 1-13 and a conjugating group attached thereto, wherein the conjugating group comprises a pharmaceutically acceptable targeting group and a linker; and the siRNA, linker and targeting group are connected sequentially by covalent or non-covalent bonds; wherein the siRNA conjugate has a structure represented by Formula (308): , Formula (308) Where n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4; m1, m2 and m3 independently represent integers from 2 to 10; R 10 , R 11 , R 12 , R 13 , R 14 and R 15 independently of each other are H, or are selected from the group consisting of C1-C 10 alkyl, C1-C 10 haloalkyl and C1-C 10 alkoxy, R3 is a group having the structure represented by Formula (A59): , Formula (A59) where E1 is OH, SH, or BH2; and Nu is miRNA; R2 is a linear alkylene of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally substituted with any one or more groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10alkenylene, C2-C 10 alkynylene, C6-C 10 arylene, C3-C 18 heterocyclylene and C5-C 10 heteroarylene, wherein R2 optionally contains any one or more substituents selected from the group consisting of: C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C 10 haloalkyl, OC1-C 10 alkyl, OC1-C 10 alkylphenyl, -C1-C 10 alkylOH, OC1-C 10 haloalkyl, SC1-C 10 alkyl, SC1-C 10 alkylphenyl, -C1-C 10 alkylSH, SC1-C 10 haloalkyl, halogen, OH, -SH, NH2, C1-C 10 alkylNH2, N(C1-C 10 alkyl)(C1-C 10 alkyl), NH(C1-C 10 alkyl), N(C1-C 10 alkyl)(C1-C 10 alkylphenyl), NH(C1-C 10 alkylphenyl), cyano, nitro, CO2H, C(O)O(C1-C 10 alkyl), CON(C1-C 10 alkyl)(C1-C 10 alkyl), CONH(C1-C 10 alkyl), CONH2, NHC(O)(C1-C 10 alkyl), NHC(O)(phenyl), N(C1-C 10alkyl)C(O)(C1-C 10 alkyl), N(C1-C 10 alkyl)C(O)(phenyl), C(O)C1-C 10 alkyl, C(O)C1-C 10 alkylphenyl, C(O)C1-C 10 haloalkyl, OC(O)C1-C 10 alkyl, -SO2(C1-C 10 alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, SO2NH(C1-C 10 alkyl), SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and NHSO2(C1-C 10 haloalkyl); each L1 is a linear alkylene of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally substituted with any one or more groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenylene, C2-C 10 alkynylene, C6-C 10 arylene, C3-C 18 heterocyclylene and C5-C 10 heteroarylene, and wherein L1 optionally contains any one or more substituents selected from the group consisting of: C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C10 haloalkyl, OC1-C 10 alkyl, OC1-C 10 alkylphenyl, -C1-C 10 alkylOH, OC1-C 10 haloalkyl, SC1-C 10 alkyl, SC1-C 10 alkylphenyl, -C1-C 10 alkylSH, SC1-C 10 haloalkyl, halogen, OH, -SH, NH2, C1-C 10 alkylNH2, N(C1-C 10 alkyl)(C1-C 10 alkyl), NH(C1-C 10 alkyl), N(C1-C 10 alkyl)(C1-C 10 alkylphenyl), NH(C1-C 10 alkylphenyl), cyano, nitro, CO2H, C(O)O(C1-C 10 alkyl), CON(C1-C 10 alkyl)(C1-C 10 alkyl), CONH(C1-C 10 alkyl), CONH2, NHC(O)(C1-C 10 alkyl), NHC(O)(phenyl), N(C1-C 10 alkyl)C(O)(C1-C 10 alkyl), N(C1-C 10 alkyl)C(O)(phenyl), C(O)C1-C 10 alkyl, C(O)C1-C 10 alkylphenyl, C(O)C1-C 10 haloalkyl, OC(O)C1-C 10 alkyl, -SO2(C1-C 10 alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, SO2NH(C1-C 10alkyl), SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl) and NHSO2(C1-C 10 haloalkyl); represents the position at which the specified group is covalently attached; M1 is a targeting group.

16. The siRNA conjugate of claim 15, wherein each L1 is independently selected from the group consisting of the groups of Formulas (A1)-(A26) and any combination thereof: And where each j1 independently represents an integer from 1 to 20; each j2 independently represents an integer from 1 to 20; each R' independently represents C1-C 10 alkyl; each Ra is selected from the group consisting of formula groups (A27)-(A45) and any combination thereof: each Rb independently represents C1-C 10 alkyl; and represents the position at which the group is covalently attached; preferably, L1 is selected from the group consisting of the groups of formulas (A1), (A4), (A5), (A6), (A8), (A10), (A11) and (A13) and their combined combinations; more preferably, L1 is a connected combination of at least two groups of Formulas (A1), (A4), (A8), (A10) and (A11); even more preferably, L1 is a connected combination of at least two groups of Formulas (A1), (A8) and (A10); or preferably, j1 is an integer from 2 to 10; j2 is an integer from 2 to 10; R' is C1-C4 alkyl; Ra is one of formulas (A27), (A28), (A29), (A30) and (A31); and Rb is C1-C5 alkyl; more preferably, j1 is an integer from 3 to 5; j2 is an integer from 3 to 5; R' is one of methyl, ethyl, and isopropyl; Ra is the formula (A27) or (A28); Rb is one of methyl, ethyl, isopropyl, and butyl.

17. The siRNA conjugate according to any one of claims 15, 16, characterized in that L1 has a length of 3 to 25 atoms; preferably L1 has a length of 4 to 15 atoms; or preferably, n1 is an integer from 1 to 2; n3 is an integer from 0 to 1; and n1+n3 = 2 to 3; or preferably, each m1, m2 and m3 independently of one another represents an integer from 2 to 5; or preferably, m1 = m2 = m3; or preferably, each of the targeting groups independently represents a ligand that has affinity for asialoglycoprotein receptors on the surface of mammalian hepatocytes; more preferably, each of the targeting groups is independently an asialoglycoprotein or a saccharide; more preferably, each of the targeting groups is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucofuranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-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-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, methyl-2,3,4-tris-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranoside, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyranoside, 2,5-anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose, L-4-thioribose;, even more preferably, at least one or each of the targeting groups is galactose or N-acetylgalactosamine; preferably R 10 , R 11 , R 12 , R 13 , R 14 and R 15 independently of one another, selected from H, methyl and ethyl.

18. An siRNA conjugate according to any one of claims 15-17, characterized in that the R2 group contains both a position bonding to an N atom on the nitrogen backbone and a position bonding to a P atom in R3; preferably, in R2, the position bonding with the N atom on the nitrogen backbone forms an amide bond with the N atom, and the position bonding with the P atom in R3 forms a complex phosphoester bond with the P atom; more preferably, R2 is selected from B5, B6, B5' or B6': in which represents the position at which the group is covalently attached; q2 is an integer between 1 and 10; Preferably, q2 is an integer between 1 and 5.

19. The siRNA conjugate of any one of claims 15-18, wherein the siRNA conjugate has a structure represented by Formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422):

20. The siRNA conjugate of any one of claims 15 to 19, wherein the P atom in Formula (A59) is linked to a terminal region of the sense or antisense strand of the siRNA; and the terminal region refers to the first 4 nucleotides counted from one end of the sense or antisense strand; preferably, the P atom in Formula (A59) is connected to either end of the sense or antisense strand of the miRNA; more preferably, the P atom in Formula (A59) is connected to the 3' end of the sense strand of the miRNA; or preferably, the P atom in Formula A59 is linked to the 2', 3' or 5' position of the nucleotide in the siRNA by forming a phosphodiester bond.

21. The siRNA conjugate of claim 20, wherein the siRNA conjugate is L10-siFXIf1M1S, wherein L10 has the structure represented by Formula (403), and the sense strand of siFXIf1M1S has the nucleotide sequence represented in SEQ ID NO: 541, and the antisense strand of siFXIf1M1S has the nucleotide sequence represented in SEQ ID NO: 542: 5'-GmsUmsAmCmGmUmGfGfAfCmUmGmGmAmUmUmCmUmGm-3' (SEQ ID NO: 541) 5'-CmsAfsGmAmAmUfCmCmAmGmUmCmCmAfCmGfUmAmCmsUmsUm-3' (SEQ ID NO: 542).

22. The use of siRNA according to any one of claims 1-13 or a pharmaceutical composition according to claim 14 or an siRNA conjugate according to any one of claims 15-21 in the manufacture of a medicinal product for the treatment or prevention of thrombotic diseases or ischemic stroke caused by increased expression of the FXI gene.

23. A method for treating or preventing thrombotic diseases or ischemic stroke caused by increased expression of the FXI gene, comprising administering an effective amount of siRNA according to any one of claims 1-13 or a pharmaceutical composition according to claim 14 or an siRNA conjugate according to any one of claims 15-21 to a subject suffering from thrombotic diseases and / or ischemic stroke.

24. A method for inhibiting expression of the blood coagulation factor XI gene, comprising bringing into contact with cells an effective amount of siRNA according to any one of claims 1-13 or a pharmaceutical composition according to claim 14 or an siRNA conjugate according to any one of claims 15-21.

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