Sirna for inhibiting FXI gene expression, and conjugate, pharmaceutical composition and use thereof

Abstract

The present invention relates to an siRNA for inhibiting FXI gene expression, an siRNA conjugate, a pharmaceutical composition containing same, and the use thereof. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, and the siRNA contains a sense strand and an antisense strand. The siRNA and the conjugate and pharmaceutical composition thereof can effectively treat and / or prevent diseases associated with FXI gene overexpression.

Description

siRNA, conjugates and pharmaceutical compositions thereof for inhibiting expression of FXI gene and uses thereof TECHNICAL FIELD

[0001] The present application relates to siRNA, siRNA conjugates, pharmaceutical compositions comprising the same, methods of making and uses thereof for inhibiting expression of FXI gene. BACKGROUND

[0002] The coagulation cascade is a series of activation of coagulation factors, which ultimately leads to the formation of fibrin. The coagulation cascade is initiated by the intrinsic pathway (also known as the contact activation pathway) and the extrinsic pathway (also known as the tissue factor pathway) to generate FXa, and then through the common pathway to generate thrombin (FIIa), which ultimately forms fibrin. Coagulation factor XI (in this document, also abbreviated as FXI or F11) is essential for maintaining the intrinsic pathway and plays a key role in the amplification of the coagulation cascade. In the coagulation cascade, thrombin can activate FXI, and activated FXI promotes the production of a large amount of thrombin, thereby amplifying the coagulation cascade. Therefore, drugs targeting FXI can block the intrinsic pathway and inhibit the amplification of the coagulation cascade, thereby having an antithrombotic effect. At the same time, a large amount of thrombin generated by FXIa can activate thrombin-activated fibrinolysis inhibitor (TAFI) to down-regulate the fibrinolysis system. In recent years, clinical data on human FXI deficiency or elevated FXI levels, as well as animal experiments on FXI deficiency or inhibition of antithrombotic agents, have shown that FXI is a new target for the prevention and treatment of antithrombotic agents with low risk of bleeding.

[0003] Thromboembolism can lead to conditions such as deep vein thrombosis, pulmonary embolism, myocardial infarction and stroke. Anticoagulants can reduce the risk of thromboembolism, and current anticoagulants such as warfarin, heparin and low molecular weight heparin (LMWH), factor X inhibitors, etc. all have significant drawbacks. Warfarin can interact with vitamin K-dependent coagulation factors including factors II, VII, IX and X. Warfarin also inhibits anticoagulant proteins C and S. Due to the difficulty in predicting treatment with warfarin, patients must be carefully monitored to detect any signs of abnormal bleeding. Heparin works by activating antithrombin, which inhibits thrombin and factor X (Bjork I, Lindahl U. Mol Cell Biochem. 1982 48: 161-182). Antithrombotic therapy with heparin also produces side effects known as heparin-induced thrombocytopenia (HIT), which causes increased bleeding and requires patient monitoring, and long-term treatment with heparin can also cause osteoporosis. Low molecular weight heparin can inhibit factor II, but to a lesser extent than unfractionated heparin (UFH). Thus, some current treatments for antithrombotic agents lack predictability and specificity, so patients need to be carefully monitored to prevent adverse side effects such as bleeding complications.

[0004] There is no anticoagulant nucleic acid drug targeting only endogenous pathway or exogenous pathway on the market at present, so developing drug targeting FXI target has important value. The present application aims to provide siRNA composition which can effectively cut RNA induced silencing complex (RISC) of RNA transcript of FXI gene, so as to selectively and effectively inhibit the expression of FXI gene and achieve the purpose of disease treatment. SUMMARY

[0005] The present application provides a siRNA for inhibiting the expression of FXI gene, which 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 contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein,

[0006] (1) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 1, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 2; or

[0007] (2) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 31, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 2.

[0008] In some embodiments, after the sense strand and the antisense strand are complementary to form a double-stranded region, the sense strand, the antisense strand or a combination thereof has a protruding nucleotide extending out of the double-stranded region. The number of protruding nucleotides can be one or more, for example, one or two. In addition, the protruding 1-2 nucleotides can be located at the 5' end, the 3' end or both ends of any antisense strand or sense strand, and each protruding nucleotide can be any type of nucleotide.

[0009] In some embodiments, the sense strand and the antisense strand are complementary to form the double-stranded region, the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the sense strand forms a blunt end.

[0010] In some embodiments, the sense strand and the antisense strand are complementary to form the double-stranded region, the 3' end of the sense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and / or the 3' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region.

[0011] In some embodiments, 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 a modification; preferably, the phosphate group with a modification is a phosphorothioate group in which one of the oxygen atoms of the phosphodiester bond is replaced by a sulfur atom.

[0012] In some embodiments, the siRNA comprises a sense strand that does not comprise 3' overhang nucleotides.

[0013] In some embodiments, the modified nucleotide is selected from a 2'-fluoro-modified nucleotide, a 2'-alkoxy-modified nucleotide, a 2'-substituted alkoxy-modified nucleotide, a 2'-alkyl-modified nucleotide, a 2'-substituted alkyl-modified nucleotide, a 2'-deoxy-modified nucleotide, a 2'-amino-modified nucleotide, a 2'-substituted amino-modified nucleotide, a nucleotide analogue, or a combination of any two or more thereof.

[0014] In some embodiments, the modified nucleotide is selected from a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a 2'-O-CH2-CH2-O-CH3-modified nucleotide, a 2'-O-CH2-CH=CH2-modified nucleotide, a 2'-CH2-CH2-CH=CH2-modified nucleotide, a 2'-deoxy-modified nucleotide, a nucleotide analogue, or a combination of any two or more thereof.

[0015] In some preferred embodiments, the nucleotide analogue is selected from an iso-nucleotide, a LNA, an ENA, a cET-BNA, a UNA, or a GNA.

[0016] In some embodiments, the overhang at the 3' end of the antisense strand is 2-3 bp in length, preferably, the overhang is 2 bp in length. Each nucleotide on the overhang is complementary or mismatched to the mRNA of the FXI gene.

[0017] In some embodiments, when the overhang at the 3' end of the antisense strand is 2 bp in length, the overhang is UC; when the overhang at the 3' end of the antisense strand is 3 bp in length, the overhang is UCU.

[0018] In some embodiments, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

[0019] In some embodiments, the modification of the sense strand comprises any one of the following:

[0020] In some embodiments, the nucleotides in positions 7, 9, 10, and 11 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0021] In some embodiments, the nucleotides in positions 5, 7, 8, and 9 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0022] In some embodiments, the nucleotides in positions 9, 10, and 11 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0023] In some embodiments, the nucleotides in positions 8, 9, and 10 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0024] In some embodiments, the nucleotides in positions 8, 9, 10, and 11 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0025] In some embodiments, the nucleotides in positions 7 and 9 in the sense strand are 2’-F modified nucleotides, the nucleotide in position 11 in the sense strand is a 2’-deoxy modified nucleotide, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0026] In some embodiments, the nucleotides in positions 7, 9, and 11 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0027] In some embodiments, the nucleotides in positions 7, 11, 13, and 15 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0028] In some embodiments, the nucleotides in positions 8, 9, 10, and 12 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0029] In some embodiments, the nucleotides in positions 7, 9, 11, 13, and 15 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0030] In some embodiments, the nucleotides in positions 9, 11, and 13 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0031] In some embodiments, the nucleotides in positions 7, 9, 11, and 13 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0032] In some embodiments, the nucleotides in positions 3, 7, 8, and 9 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0033] In some embodiments, the nucleotides in positions 3, 9, 11, and 13 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0034] In some embodiments, the nucleotides in positions 3, 7, 9, and 11 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0035] In some embodiments, the nucleotides in positions 7, 11, and 13 in the sense strand are 2’-F modified nucleotides, and the nucleotides in the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0036] In some embodiments, the nucleotides at positions 3, 7, 11, and 16 in the sense strand are 2’-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0037] In some embodiments, the nucleotides at positions 3, 7, 11, and 16 in the sense strand are 2’-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0038] In some embodiments, the nucleotides at positions 3, 7, 11, and 16 in the sense strand are 2’-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0039] In some embodiments, the nucleotides at positions 3, 7, 11, and 16 in the sense strand are 2’-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides, in the 5’ end to 3’ end direction.

[0040] In some embodiments, neither the 5’ end nor the 3’ end of the sense strand is attached to an inverted abasic deoxyribose residue (invAb).

[0041] In some embodiments, only the 5’ end of the sense strand is attached to an inverted abasic deoxyribose residue.

[0042] In some embodiments, only the 3’ end of the sense strand is attached to an inverted abasic deoxyribose residue.

[0043] In some embodiments, both the 5’ end and the 3’ end of the sense strand are attached to an inverted abasic deoxyribose residue.

[0044] In one embodiment, the inverted abasic deoxyribose residue is attached to the 3’ end nucleotide and / or the 5’ end nucleotide of the sense strand via a phosphodiester linkage, a phosphorothioate group, or other internucleoside linkage.

[0045] In one embodiment, the 5’ end nucleotide of the antisense strand is attached to a 5’ phosphate group or a 5’ phosphate derivative group.

[0046] In some embodiments, the sense strand comprises a phosphorothioate group at the following indicated positions, in the 5’ end to 3’ end direction:

[0047] between the 1st and 2nd nucleotides from the 5’ end of the sense strand;

[0048] between the 2ndand 3rdnucleotides from the 5' terminus of the sense strand;

[0049] between the 1stand 2ndnucleotides from the 3' terminus of the sense strand;

[0050] between the 2ndand 3rdnucleotides from the 3' terminus of the sense strand;

[0051] or,

[0052] the sense strand comprises a phosphorothioate group at a position selected from:

[0053] between the 1stand 2ndnucleotides from the 5' terminus of the sense strand;

[0054] between the 2ndand 3rdnucleotides from the 5' terminus of the sense strand;

[0055] or,

[0056] the sense strand comprises a phosphorothioate group at a position selected from:

[0057] between the 1stand 2ndnucleotides from the 5' terminus of the sense strand;

[0058] between the 2ndand 3rdnucleotides from the 5' terminus of the sense strand;

[0059] between the inverted deoxyabasic ribose residue and the 1stnucleotide from the 3' terminus of the sense strand;

[0060] or,

[0061] the sense strand comprises a phosphorothioate group at a position selected from:

[0062] between the inverted deoxyabasic ribose residue and the 1stnucleotide from the 5' terminus of the sense strand;

[0063] between the 1stand 2ndnucleotides from the 5' terminus of the sense strand;

[0064] between the inverted deoxyabasic ribose residue and the 1stnucleotide from the 3' terminus of the sense strand.

[0065] In some embodiments, the modifications of the antisense strand comprise any one of the following:

[0066] In some embodiments, the nucleotides in the 2nd, 6th, and 14th positions of the antisense strand, counting from the 5' end to the 3' end, are 2'-F modified nucleotides, and the nucleotides in the remaining positions of the antisense strand are 2'-0-CH3 modified nucleotides.

[0067] In some embodiments, the nucleotides in the 2nd, 6th, 14th, and 16th positions of the antisense strand, counting from the 5' end to the 3' end, are 2'-F modified nucleotides, and the nucleotides in the remaining positions of the antisense strand are 2'-0-CH3 modified nucleotides.

[0068] In some embodiments, the nucleotides in the 2nd, 3rd, 5th, 8th, 10th, 14th, 16th, and 18th positions of the antisense strand, counting from the 5' end to the 3' end, are 2'-F modified nucleotides, and the nucleotides in the remaining positions of the antisense strand are 2'-0-CH3 modified nucleotides.

[0069] In some embodiments, the nucleotides in the 2nd, 3rd, 4th, 5th, 7th, 10th, and 14th positions of the antisense strand, counting from the 5' end to the 3' end, are 2'-F modified nucleotides, and the nucleotides in the remaining positions of the antisense strand are 2'-0-CH3 modified nucleotides.

[0070] In some embodiments, the nucleotides in the 2nd, 4th, 6th, 12th, 14th, 16th, 18th, and 20th positions of the antisense strand, counting from the 5' end to the 3' end, are 2'-F modified nucleotides, and the nucleotides in the remaining positions of the antisense strand are 2'-0-CH3 modified nucleotides.

[0071] In some embodiments, the nucleotides in the 2nd and 14th positions of the antisense strand, counting from the 5' end to the 3' end, are 2'-F modified nucleotides, and the nucleotides in the remaining positions of the antisense strand are 2'-0-CH3 modified nucleotides.

[0072] In some embodiments, the nucleotides in the 2nd, 4th, 5th, 6th, 8th, 10th, 12th, 14th, 16th, 18th, and 20th positions of the antisense strand, counting from the 5' end to the 3' end, are 2'-F modified nucleotides, and the nucleotides in the remaining positions of the antisense strand are 2'-0-CH3 modified nucleotides.

[0073] In some embodiments, the nucleotides in positions 2, 3, 4, 5, 7, 10, and 14, counting from the 5' end to the 3' end, of the antisense strand are 2'-F modified nucleotides, the nucleotide in position 6 of the antisense strand is a GNA modified nucleotide, and the nucleotides in the remaining positions of the antisense strand are 2'-0-CH3 modified nucleotides.

[0074] In some embodiments, the nucleotides in positions 2, 7, 10, and 14, counting from the 5' end to the 3' end, of the antisense strand are 2'-F modified nucleotides, and the nucleotides in the remaining positions of the antisense strand are 2'-0-CH3 modified nucleotides.

[0075] In some embodiments, the nucleotides in positions 2, 7, and 14, counting from the 5' end to the 3' end, of the antisense strand are 2'-F modified nucleotides, the nucleotides in positions 5 and 12 of the antisense strand are 2'-deoxy modified nucleotides, and the nucleotides in the remaining positions of the antisense strand are 2'-0-CH3 modified nucleotides.

[0076] In some embodiments, the nucleotides in positions 2, 3, 7, 10, 12, 14, and 22, counting from the 5' end to the 3' end, of the antisense strand are 2'-F modified nucleotides, the nucleotide in position 5 of the antisense strand is a 2'-deoxy modified nucleotide, the nucleotide in position 6 of the antisense strand is a GNA modified nucleotide, and the nucleotides in the remaining positions of the antisense strand are 2'-0-CH3 modified nucleotides.

[0077] In some embodiments, the nucleotides in positions 2, 3, 7, 10, 12, 14, and 16, counting from the 5' end to the 3' end, of the antisense strand are 2'-F modified nucleotides, the nucleotide in position 5 of the antisense strand is a 2'-deoxy modified nucleotide, the nucleotide in position 6 of the antisense strand is a GNA modified nucleotide, and the nucleotides in the remaining positions of the antisense strand are 2'-0-CH3 modified nucleotides.

[0078] In some embodiments, the nucleotide at the 5' end of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group. Preferably, the 5' phosphate derivative group is a 5'-trans vinyl phosphonate.

[0079] In some embodiments, the modification method of the siRNA is a combination of any of the modifications of the sense strand described above and any of the modifications of the antisense strand described above, which can be obtained by a person skilled in the art according to the description of the modifications of the sense strand and the antisense strand above. Preferably, the modification method of the siRNA can be obtained according to the description of the modifications of the sense strand and the antisense strand above in any combination of the siRNA sequences listed in Table 1.

[0080] For example, in some embodiments, the nucleotides at positions 3, 7, 8, and 9 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides, in the 5' end to 3' end direction; the nucleotides at positions 2, 7, 10, and 14 in the antisense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are 2'-O-CH3 modified nucleotides, in the 5' end to 3' end direction. In some embodiments, the nucleotides at positions 3, 7, 8, and 9 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides, in the 5' end to 3' end direction; the nucleotides at positions 2, 7, and 14 in the antisense strand are 2'-F modified nucleotides, the nucleotides at positions 5 and 12 in the antisense strand are 2'-deoxy modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are 2'-O-CH3 modified nucleotides, in the 5' end to 3' end direction.

[0081] In some embodiments, the nucleotides at positions 3, 7, 8, and 9 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides, in the 5' end to 3' end direction; the nucleotides at positions 2, 7, 10, and 14 in the antisense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are 2'-O-CH3 modified nucleotides, in the 5' end to 3' end direction.

[0082] The antisense strand comprises phosphorothioate groups at positions as shown below:

[0083] between the 1st and 2nd nucleotides from the 5' end of the antisense strand;

[0084] between the 2nd and 3rd nucleotides from the 5' end of the antisense strand;

[0085] between the 1st and 2nd nucleotides from the 3' end of the antisense strand;

[0086] between the 2nd and 3rd nucleotides from the 3' end of the antisense strand;

[0087] or,

[0088] The antisense strand comprises phosphorothioate groups at positions as shown below:

[0089] between the 1st and 2nd nucleotides from the 5' end of the antisense strand;

[0090] between the 2ndand 3rdnucleotides from the 5' terminus of the antisense strand;

[0091] between the 1stand 2ndnucleotides from the 3' terminus of the antisense strand;

[0092] between the 2ndand 3rdnucleotides from the 3' terminus of the antisense strand;

[0093] between the 3rdand 4thnucleotides from the 3' terminus of the antisense strand.

[0094] In one embodiment, the application provides an siRNA selected from Table 1 ; preferably, the siRNA is selected from N-ER-FY005291D2, N-ER-FY005291M12, N-ER-FY005291M13, N-ER-FY005291M15, N-ER-FY005291M20, N-ER-FY005291M21, N-ER-FY005291M22, N-ER-FY005291M24, N-ER-FY005291M25, N-ER-FY005291M26, N-ER-FY005291M28, N-ER-FY005291M29, N-ER-FY005291M32, N-ER-FY005291M33, N-ER-FY005291M35, N-ER-FY005291M11D2, N-ER-FY005291M12D2, N-ER-FY005291M13D2, N-ER-FY005291M15D2, N-ER-FY005291M21D2, N-ER-FY005291M22D2, N-ER-FY005291M24D2, N-ER-FY005291M25D2, N-ER-FY005291M26D2, N-ER-FY005291M29D2, N-ER-FY005291M31D2, N-ER-FY005291M35D2, N-ER-FY005291M44, N-ER-FY005291M46, N-ER-FY005291M47, N-ER-FY005291M48, N-ER-FY005291M49, N-ER-FY005291M50.

[0095] The application also provides an siRNA conjugate comprising an siRNA of the application and a conjugate group conjugated to the siRNA (as shown below, the double helix structure represents the siRNA and the conjugate group is attached to the 3' terminus of the sense strand of the siRNA):

[0096] X in the above conjugate structure can be selected from O or S, in one embodiment, X is O.

[0097] In one embodiment, in the siRNA conjugate, the sense strand of the siRNA is complementary to the antisense strand to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-3 overhanging nucleotides extending out of the double-stranded region.

[0098] In one embodiment, the conjugate group (wherein, in some conjugate groups, a phosphoester bond or a phosphorothioate moiety is exemplarily shown) is selected from:

[0099] In one specific embodiment, the siRNA conjugate is selected from the siRNA conjugates of Table 2, preferably, the siRNA conjugate is selected from N-ER-FY005291M12L96, N-ER-FY005291M13L96, N-ER-FY005291M15L96, N-ER-FY005291M20L96, N-ER-FY005291M21L96, N-ER-FY005291M22L96, N-ER-FY005291M24L96, N-ER-FY005291M25L96, N-ER-FY005291M26L96, N-ER-FY005291M28L96, N-ER-FY005291M29L96, N-ER-FY005291M32L96, N-ER-FY005291M33L96, N-ER-FY005291M35L96, N-ER-FY005291M44L96, N-ER-FY005291M46L96, N-ER-FY005291M47L96, N-ER-FY005291M48L96, N-ER-FY005291M49L96, N-ER-FY005291M50L96.

[0100] The present application also provides a pharmaceutical composition comprising the siRNA of the present application, or the siRNA conjugate of the present application, and a pharmaceutically acceptable carrier.

[0101] The present application also provides a kit comprising the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application.

[0102] The present application also provides use of the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application for the preparation of a medicament for inhibiting the expression of FXI gene.

[0103] The present application also provides use of the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application for the preparation of a medicament for preventing and / or treating a disease associated with overexpression of FXI gene.

[0104] In specific embodiments, the disease is deep vein thrombosis, venous or arterial thrombosis, ischemic stroke, pulmonary embolism, myocardial infarction, stroke, Alzheimer's disease, thrombosis associated with chronic kidney disease or end-stage kidney disease.

[0105] The present application also provides a method for inhibiting the expression of FXI gene, comprising contacting a cell expressing FXI with a therapeutically effective amount of the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application, or administering to a subject in need thereof.

[0106] The present application also provides a method for treating and / or preventing a disease associated with overexpression of FXI gene, comprising administering to a subject in need thereof a therapeutically effective amount of the siRNA of the present application, or the siRNA conjugate of the present application, or the pharmaceutical composition of the present application.

[0107] In specific embodiments, the disease is deep vein thrombosis, venous or arterial thrombosis, ischemic stroke, pulmonary embolism, myocardial infarction, stroke, Alzheimer's disease, thrombosis associated with chronic kidney disease or end-stage kidney disease. Advantages

[0108] The siRNA, siRNA conjugate and pharmaceutical composition containing the same provided by the present application show excellent FXI gene expression inhibition activity in in vitro cell experiments, and have good potential for treating diseases associated with overexpression of FXI gene. For example, the siRNA and conjugate thereof disclosed in the present application can reduce the expression of FXI mRNA in the liver, have low toxic side effects, good plasma stability, and have good clinical application prospects.

[0109] The siRNA conjugate provided by the present application has high FXI gene inhibition activity in HepG2 cells. DETAILED DESCRIPTION

[0110] Definitions

[0111] Throughout the specification, unless specifically stated otherwise, "G", "C", "A", "T" and "U" generally represent the bases of guanine, cytosine, adenine, thymine, uracil, respectively, in the art, but it is also generally known in the art that "G", "C", "A", "T" and "U" each generally also represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as the base, respectively, which is a common way of expressing deoxyribonucleic acid sequences and / or ribonucleic acid sequences, and thus in the context of the present disclosure, the meaning of "G", "C", "A", "T", "U" includes the various possibilities described above, and "nucleotide" and "ribonucleotide" are used interchangeably herein, and "deoxyribonucleotide" and "2'-deoxyribonucleotide" are used interchangeably herein. mA, mG, mC, mU: represent 2'-methoxy modified nucleotides; Af, Gf, Cf, Uf: represent 2'-fluoro modified nucleotides; dA, dG, dC, dT: represent 2'-deoxy modified ribonucleotides at the corresponding positions in the motif sequence; the lower case letter s represents a phosphorothioate linkage between the two nucleotides adjacent to the letter s on the left and right; EVP: represents that the nucleotide adjacent to the right of the EVP is a 5'-trans vinylphosphonate nucleotide; [GNA]: represents that the nucleotide adjacent to the left of the [GNA] is a nucleotide with GNA modification; "(invAb)": represents an inverted abasic deoxyribose residue.

[0112] In the foregoing and hereinafter, the "2'-fluoro modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a fluorine. The "non-fluoro modified nucleotide" refers to a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorine group. In some embodiments, each of the non-fluoro modified nucleotides is independently selected from one of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorine group. These nucleotides in which the hydroxyl group at the 2' position of the ribose group is replaced by a non-fluorine group are well known to those skilled in the art, and these nucleotides can be selected from one of a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, a 2'-deoxy modified nucleotide.

[0113] "Alkyl" includes straight chain, branched chain, or cyclic saturated alkyl groups. For example, alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, iso-butyl, sec-butyl, t-butyl, cyclobutyl, n-pentyl, cyclohexyl, and the like. By way of example, "C 1-6 " in "alkyl" refers to a group having 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight chain, branched chain, or cyclic form. 1-6 " in "alkyl" refers to a group having 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight chain, branched chain, or cyclic form.

[0114] "Alkoxy" refers herein to an alkyl group attached to the remainder of the molecule through an oxygen atom (-O-alkyl), wherein the alkyl group is as defined herein. Non-limiting examples of alkoxy groups include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, i-propoxy, n-butoxy, t-butoxy, n-pentoxy, and the like.

[0115] "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. Such as an iso-nucleotide, a bridged nucleotide (BNA), or an acyclic nucleotide.

[0116] BNA refers to a constrained or inaccessible nucleotide. A BNA can contain a five-, six-, or seven-membered ring with a "fixed" C3'-endo sugar conformation bridged structure. The bridge is typically incorporated at the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET BNA, and the like, wherein LNA is shown in formula (1), ENA is shown in formula (2), and cET BNA is shown in formula (3):

[0117] Acyclic nucleotides are a class of nucleotides in which the sugar ring of the nucleotide is opened, such as unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA), wherein UNA is shown in formula (4) and GNA is shown in formula (5):

[0118] In the above formula (4) and formula (5), R is selected from H, OH, or alkoxy (O-alkyl).

[0119] Iso-nucleotides refer to compounds in which the position of the base on the ribose ring is changed, such as compounds in which the base is moved from the 1'-position to the 2'-position or 3'-position of the ribose ring, such as shown in formula (6) or (7):

[0120] In the above formula (6)-(7) compounds, Base represents a base, such as A, U, G, C, or T; and R is selected from H, OH, F, or a non-fluorine group as described above.

[0121] In some embodiments, the nucleotide analog is selected from one of an iso-nucleotide, LNA, ENA, cET BNA, UNA, and GNA. In some embodiments, each non-fluoro-modified nucleotide is a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, or a combination of any two or more thereof. In some preferred embodiments, each non-fluoro-modified nucleotide is a 2'-methoxy-modified nucleotide, which, as used hereinabove and hereinbelow, refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is replaced with a methoxy group.

[0122] The "2'-methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is replaced with a methoxy group. The "phosphorothioate group" refers to a phosphorothioate group in which one of the oxygen atoms in the phosphodiester linkage of the phosphate group is replaced with a sulfur atom.

[0123] The "phosphorothioate group" refers to a group of the following formula:

[0124] The "5'-phosphonucleotide" refers to a structure of the following formula:

[0125] In the context of the present specification, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meaning well known to persons skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand each pair with a base on the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or, in RNA, uracil (U)); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair includes one purine and one pyrimidine. When the adenine on one strand always pairs with the thymine (or uracil) on the other strand, and the guanine always pairs with the cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" in the art means that the bases at the corresponding positions in a double-stranded nucleic acid do not pair in a complementary manner.

[0126] In the context of the present specification, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meaning well known to persons skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand each pair with a base on the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or, in RNA, uracil (U)); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair includes one purine and one pyrimidine. When the adenine on one strand always pairs with the thymine (or uracil) on the other strand, and the guanine always pairs with the cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" in the art means that the bases at the corresponding positions in a double-stranded nucleic acid do not pair in a complementary manner.

[0127] In the foregoing and hereafter, a nucleotide sequence has a "nucleotide difference" from another nucleotide sequence if the base type of a nucleotide at a corresponding position in the former is different from that in the latter, e.g., if a nucleotide base is A in the latter, a corresponding nucleotide base is U, C, G, or T at the same position in the former, the nucleotide difference is recognized between the two nucleotide sequences at that position. In some embodiments, a nucleotide difference is also recognized at a position where an abasic nucleotide or its equivalent is substituted for a nucleotide at the position.

[0128] In the foregoing and hereafter, a "overhang" refers to one or more unpaired nucleotides that protrude from a duplex structure of an siRNA when a 3' end of one strand of the siRNA extends beyond a 5' end of the other strand, or vice versa. A "blunt end" or "blunt" means that there are no unpaired nucleotides at that end of the siRNA, i.e., there is no nucleotide overhang. A "blunt-ended" siRNA is one that is double-stranded over its entire length, i.e., there is no nucleotide overhang at either end of the molecule.

[0129] In the present application, "5'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 5' carbon of the pentose sugar, which is the predominant type of nucleotide found free in living organisms. "3'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 3' carbon of the pentose sugar, which can include, for example, adenosine-3'-phosphate, guanosine-3'-phosphate, cytidine-3'-phosphate, uridine-3'-phosphate, 2'-deoxythymidine-3'-phosphate, 2'-O-methyladenosine-3'-phosphate, 2'-O-methyladenosine-3'-phosphorothioate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroadenosine-3'-phosphorothioate, 2'-O-methylguanosine-3'-phosphate, 2'-O-methylguanosine-3'-phosphorothioate, 2'-fluoroguanosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphorothioate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylcytidine-3'-phosphorothioate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphorothioate, 2'-O-methyluridine-3'-phosphate, 2'-O-methyluridine-3'-phosphorothioate, 2'-fluorouridine-3'-phosphate, 2'-fluorouridine-3'-phosphorothioate, 2'-deoxythymidine-3'-phosphorothioate. The definition can apply to modified or unmodified nucleoside phosphoramidite monomers. The terms "iRNA," "RNAi agent," "iRNA agent," "RNA interference agent" used in the context of the present disclosure are used interchangeably herein to refer to the term as defined herein to include siRNA and to mediate the targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. iRNAs direct the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs modulate, e.g., inhibit, the expression of a target gene in a cell, such as a cell of a subject, e.g., a mammalian subject.

[0130] In the present application specification above and below, particularly in describing the preparation of siRNAs, pharmaceutical compositions or siRNA conjugates of the present application, the nucleoside monomers refer, unless otherwise specified, to modified or unmodified nucleoside phosphoramidite monomers used in the solid phase phosphoramidite synthesis according to the kind and order of nucleotides in the siRNA or siRNA conjugate to be prepared. Solid phase phosphoramidite synthesis is a method well known to those skilled in the art for use in RNA synthesis. All nucleoside monomers used in the present application are commercially available.

[0131] In the context of the present application, unless otherwise specified, "conjugation" refers to the linkage between two or more chemical moieties each having a specific function in a manner of covalent linkage; correspondingly, "conjugate" refers to the compound formed by the covalent linkage between the respective chemical moieties. Further, "siRNA conjugate" refers to the compound formed by the covalent linkage of one or more chemical moieties each having a specific function to siRNA. The siRNA conjugate should be understood as the general term for a plurality of siRNA conjugates or the siRNA conjugate represented by a certain chemical formula, depending on the context. In the context of the present specification, "conjugating molecule" should be understood as a specific compound that can be conjugated to siRNA through a reaction to ultimately form the siRNA conjugate of the present application.

[0132] As used in the present specification, "2'-deoxy-modified nucleotide" or "2'-deoxy-modification" refers to the replacement of ribonucleotides A, U, C, G with the corresponding deoxyribonucleotides dA, dT, dC, dG in the process of modification. For example, in the direction of 5' to 3', the replacement of the 5th nucleotide of the antisense strand of N-ER-FY005291D2 (SEQ ID NO: 2) with a deoxyribonucleotide means that the 5th ribonucleotide "A" is replaced with "dA"; similarly, the replacement of uracil ribonucleotide (U) with the corresponding deoxyribonucleotide (dT); the replacement of cytosine ribonucleotide (C) with the corresponding cytosine deoxyribonucleotide (dC); and the replacement of guanine ribonucleotide (G) with the corresponding guanine deoxyribonucleotide (dG). In the present specification, "2'-deoxy-modified nucleotide" or "2'-deoxy-modification" is also referred to as "replacement of nucleotide with deoxyribonucleotide".

[0133] The term "disease associated with abnormal expression of the FXI gene" is a disease or disorder associated with the involvement of the FXI gene. The term "disease associated with abnormal expression of the FXI gene" includes diseases, disorders or conditions that would benefit from a reduction in FXI (i.e. "FXI-associated diseases") expression. Such diseases are typically closely associated with thrombosis and thromboembolism.Non-limiting examples of diseases associated with abnormal expression of FXI include: stroke and venous thrombosis, ischemic stroke, venous thromboembolism, arterial thrombosis, pulmonary embolism, myocardial infarction, chronic kidney disease or end-stage renal disease (ESRD), Alzheimer’s disease, etc. (see, e.g., Steven P Grover et al., Intrinsic Pathway of Coagulation and Thrombosis, Arterioscler Thromb Vasc Biol. 2019 Mar; 39(3):331-338; Stefan Willmann et al., Stefan Willmann, CPT Pharmacometrics Syst Pharmacol. 2021 Aug; 10(8):890-901; C Zhong et al., Coagulation factor XI vaccination: an alternative strategy to prevent thrombosis, J Thromb Haemost. 2017 Jan; 15(1): 122-130; Mayken Visser et al., Role of Factor XIa and Plasma Kallikrein in Arterial and Venous Thrombosis, Thromb Haemost. 2020 Jun; 120(6):883-993; Karsten Engseth Kluge et al., Coagulation factors XI and XII as possible targets for anticoagulant therapy, Thromb Res. 2022 Jun; 214:53-62; Zu-Lin Chen et al., Anti-HK antibody inhibits the plasma contact system by blocking prekallikrein and factor XI activation in vivo, Blood Adv. 2022 Nov 21;. Tomasz et al., Active FXI Can Independently Predict Ischemic Stroke in Anticoagulated Atrial Fibrillation Patients: A Cohort Study, Thromb Haemost. 2022 Aug; 122(8): 1397-1406.

[0134] Various hydroxyl protecting groups can be used in the present application. Generally, a protecting group renders a chemical functional group insensitive to particular reaction conditions, and can be added to and removed from that functional group in a molecule without substantially damaging the rest of the molecule. In some embodiments, the protecting group is stable under basic conditions, but can be removed under acidic conditions. In some embodiments, nonexclusive examples of hydroxyl protecting groups that can be used in the present application include monomethoxytrityl, 9-phenylxanthine-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthine-9-yl (Mox). In some embodiments, nonexclusive examples of hydroxyl protecting groups that can be used in the present application include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4’-dimethoxytrityl), and TMTr (4,4’,4”-trimethoxytrityl).

[0135] As used in the present specification, “optional” or “optionally” means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0136] The term “subject,” as used herein, refers to any animal, such as a mammal or a marsupial. Subjects of the present application include, but are not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, rabbits, or any species of poultry.

[0137] As used herein, “treatment” refers to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit. “Therapeutic benefit” means eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with regard to a disorder being treated when one or more symptoms associated with the disorder are reduced, even if the disorder is not entirely eliminated.

[0138] As used herein, "prevention" refers to a method of obtaining a beneficial or desired result, including but not limited to prophylactic benefit. To obtain "prophylactic benefit," the siRNA, siRNA conjugate, or pharmaceutical composition can be administered to a subject at risk of suffering from a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of this disease can not have been made.

[0139] siRNA

[0140] The present application relates to an siRNA capable of inhibiting the expression of FXI gene. The siRNA of the present application contains nucleotide groups as basic structural units, which are well known to those skilled in the art, containing phosphate groups, ribose groups, and bases. The siRNA generally active, i.e., functional, is about 12-40 nucleotides in length, and in some embodiments, about 15-30 nucleotides in length.

[0141] The siRNA of the present application contains a sense strand and an antisense strand, each of the nucleotides in the siRNA is independently modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. In some embodiments, the length of the double-stranded region is 15-30 nucleotide pairs. In other embodiments, the length of the double-stranded region is 17-23 nucleotide pairs. In other embodiments, the length of the double-stranded region is 19-21 nucleotide pairs. In yet other embodiments, the length of the double-stranded region is 19 or 21 nucleotide pairs.

[0142] The length of the sense strand and the antisense strand provided in the present application is the same or different, in some embodiments, the sense strand or the antisense strand has 15-30 nucleotides. In other embodiments, the sense strand or the antisense strand has 19-25 nucleotides. In other embodiments, the sense strand or the antisense strand has 19-23 nucleotides. The length ratio of the siRNA sense strand and the antisense strand provided in the present application can be 15 / 15, 16 / 16, 17 / 17, 18 / 18, 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 20 / 19, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 21 / 19, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 22 / 19, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 23 / 19, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 24 / 24, 25 / 25, 26 / 26, 27 / 27, 28 / 28, 29 / 29, 30 / 30, 22 / 24, 22 / 25, 22 / 26, 23 / 24, 23 / 25, or 23 / 26, etc. In some embodiments, the length ratio of the siRNA sense strand and the antisense strand is 19 / 19, 21 / 21, 19 / 21, 21 / 23, or 23 / 23, at this time, the siRNA of the present disclosure has better cell mRNA silencing activity.

[0143] It is found that different modification strategies have completely different effects on the stability, biological activity and cytotoxicity of siRNA. For example, CN102140458B studies various chemical modification strategies of siRNA, and confirms that seven effective modification methods can improve the stability of siRNA in blood, and at the same time, maintain the inhibitory activity of siRNA which is basically the same as that of unmodified siRNA.

[0144] The nucleotides in the siRNA of the present application are each independently modified or unmodified nucleotides. In some embodiments, each nucleotide in the siRNA of the present application is an unmodified nucleotide; in some embodiments, part or all of the nucleotides in the siRNA of the present application are modified nucleotides, and these modifications on the nucleotide groups do not cause the function of the siRNA of the present application to significantly weaken or lose the ability to inhibit the expression of FXI gene.

[0145] In some embodiments, the siRNA of the present application contains at least one modified nucleotide. In the context of the present application, the term "modified nucleotide" as used herein refers to a nucleotide or a nucleotide analogue in which the ribosyl 2' position hydroxyl group is replaced with another group, or a nucleotide with a modified base. The modified nucleotide does not cause a significant reduction or loss of the function of the siRNA in inhibiting gene expression. For example, the modified nucleotide disclosed in J. K. Watts, G. F. Deleavey, and M. J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20):842-55 can be selected.

[0146] In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present application is a modified nucleotide, and / or at least one phosphate group is a phosphate group with a modification group; in other words, at least one of the phosphate groups in the phosphate-sugar backbone and / or at least one of the ribosyl groups in the sense strand and the antisense strand of the siRNA is a phosphate group with a modification group and / or a ribosyl group with a modification group. In some embodiments, the phosphate group with a modification group is a phosphorothioate group in which one of the oxygen atoms in the phosphodiester bond is replaced with a sulfur atom.

[0147] In some embodiments, the siRNA comprises a sense strand that does not comprise 3' overhanging nucleotides; that is, the sense strand of the siRNA can have 3' overhanging nucleotides, and the 3' overhanging nucleotides of the sense strand are excluded to form a blunt end. In some embodiments, the siRNA comprises a modification in which the 3' end of the sense strand in Table 1 is modified to be a blunt end, optionally by excluding the overhanging end of the 3' end of the sense strand in Table 1 that extends beyond the double-stranded region.

[0148] In some embodiments, when the nucleotide sequence of the sense strand and the antisense strand are complementary to form a double-stranded region, the 3' end of the sense strand has overhanging nucleotides that extend beyond the double-stranded region, and the overhanging nucleotides at the 3' end of the sense strand are excluded as the nucleotide sequence of the sense strand, and accordingly, the sense strand of the siRNA forms a blunt end.

[0149] In some embodiments, the 5' end nucleotide of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group, preferably, the 5' end nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate.

[0150] After the 5' end nucleotide of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group, the following structure is formed:

[0151] wherein, formula (8) shows the structure of 5' terminal nucleotide connecting 5' phosphate group, formula (9) shows the structure of 5' terminal nucleotide connecting 5' phosphate derivative group (EVP), formula (10) shows the structure of 5' terminal nucleotide connecting 5' phosphate derivative group (5' methylene phosphate group), wherein Base represents a base, such as A, U, G, C or T. R' is hydroxyl or hydrogen or various groups known to those skilled in the art, for example, the modified nucleotide after substitution can be 2'-fluoro (2'-F) modified nucleotide, 2'-alkoxy modified nucleotide, 2'-substituted alkoxy modified nucleotide, 2'-alkyl modified nucleotide, 2'-substituted alkyl modified nucleotide, 2'-amino modified nucleotide, 2'-substituted amino modified nucleotide, 2'-deoxy modified nucleotide.

[0152] In some embodiments, the 5' terminal nucleotide of the sense strand or the antisense strand is not connected to 5' phosphate group, 5' phosphate derivative group or inverted abasic deoxyribose residue (invAb) (i.e. the ribose group of the 5' terminal nucleotide of the sense strand or the antisense strand is 5' hydroxyl), which has the structure as shown below:

[0153] wherein, Base represents a base, such as A, U, G, C or T. R is hydroxyl or hydrogen or various groups known to those skilled in the art, for example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino.

[0154] Exemplary modified nucleotides have the structure as shown below:

[0155] wherein, Base represents a base, such as A, U, G, C or T. The hydroxyl group at 2' position of the ribose group is substituted by R. The hydroxyl group at 2' position of the ribose group can be substituted by various groups known to those skilled in the art, for example, the modified nucleotide after substitution can be 2'-fluoro (2'-F) modified nucleotide, 2'-alkoxy modified nucleotide, 2'-substituted alkoxy modified nucleotide, 2'-alkyl modified nucleotide, 2'-substituted alkyl modified nucleotide, 2'-amino modified nucleotide, 2'-substituted amino modified nucleotide, 2'-deoxy modified nucleotide.

[0156] In some embodiments, the sense strand can comprise one or more capping residues or moieties, sometimes referred to in the art as "caps," "terminal caps," or "capping residues." As used herein, a "capping residue" is a non-nucleotide compound or other moiety that can be incorporated at one or more of the terminal ends of the nucleotide sequence of an siRNA disclosed herein. In some cases, a capping residue can provide certain beneficial properties to the siRNA, such as protection against exonuclease degradation. In some embodiments, an inverted abasic deoxyribose residue (invAb) is added as a capping residue. In some embodiments, the capping residue occurs at the 5' terminal end, the 3' terminal end, or both the 5' and 3' terminal ends of the sense strand.

[0157] In some embodiments, one or more inverted abasic deoxyribose residues (invAb) are added to the 3' terminal end of the sense strand. In some embodiments, one or more inverted abasic deoxyribose residues (invAb) are added to the 5' terminal end of the sense strand. In some embodiments, one or more inverted abasic deoxyribose residues (invAb) are added to the 5' terminal end of the sense strand and the 3' terminal end of the sense strand. The inverted abasic deoxyribose residue can be linked via a phosphoester linkage, a phosphorothioate group, or other internucleoside linkage. In describing the modification site in a method of modification, the (invAb) is not counted as the 1st site of the sequence. In some embodiments, an inverted abasic deoxyribose residue (invAb), also referred to in the art as an "inverted abasic site," can be added. Among others, the (invAb) can have the structure shown below:

[0158] Formula B when the (invAb) is at the 3' terminal end of the siRNA; and Formula C when the (invAb) is at the 5' terminal end of the siRNA:

[0159] In some embodiments, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe, 2'-O-CH3) modified nucleotide, etc.

[0160] In some embodiments, the 2'-substituted alkoxy modified nucleotide is a 2'- methoxyethoxy (2'-O-CH2-CH2-O-CH3) modified nucleotide, a 2'-O-CH2-CH=CH2 modified nucleotide, etc.

[0161] In some embodiments, the 2'-substituted alkyl modified nucleotide is a 2'-CH2-CH2-CH=CH2 modified nucleotide, etc.

[0162] siRNA conjugates

[0163] The present application relates to an siRNA conjugate, which contains an siRNA and a conjugate group conjugated to the siRNA.

[0164] In the present application, the sense strand of the siRNA conjugate forms a double-stranded region of the siRNA conjugate with the antisense strand, and the 3' end of the sense strand of the siRNA conjugate forms a blunt end. In some embodiments, the 3' end of the sense strand of the siRNA conjugate forms a blunt end, and the 3' end of the antisense strand of the siRNA conjugate has 1-3 overhanging nucleotides extending out of the double-stranded region.

[0165] In some preferred embodiments, the siRNA conjugate is obtained by conjugating an siRNA with a conjugate group. In which, the sense strand of the siRNA is complementary to the antisense strand to form a double-stranded region of the siRNA, and the 3' end of the sense strand of the siRNA has a blunt end, the conjugate group is conjugated to the 3' end of the sense strand with a blunt end to form the siRNA conjugate.

[0166] For example, the siRNA with the sequence as shown in N-ER-FY005291M11D2, the 3' end of the sense strand of the siRNA has a blunt end, the sequence of the 3' end blunt end in the sense strand is used as the nucleotide sequence for connecting the L96 conjugate group, thus, the sense strand of the siRNA conjugate is mAsmAsmAmUCfmUUfUfGfmUmCmUmCmCmUmUmAmAmAL96, and the antisense strand is EVPmUsUfsmUmAmAGfmGmAmGmAmCmAmAAfmGAfmUmUmUsmCsmU.

[0167] In some preferred embodiments, the 3' end of the sense strand of the siRNA has overhanging nucleotides extending out of the double-stranded region, the sequence with a 3' blunt end formed by excluding the overhanging nucleotides at the 3' end of the sense strand is used as the nucleotide sequence for connecting the conjugate group, and the conjugate group is connected to the 3' blunt end of the sense strand to form the siRNA conjugate.

[0168] In some more preferred embodiments, when the 3' end of the sense strand has overhanging nucleotides extending out of the double-stranded region after the nucleotide sequence of the sense strand is complementary to the antisense strand to form a double-stranded region, the sequence with a 3' blunt end formed by excluding the overhanging nucleotides at the 3' end of the sense strand is used as the nucleotide sequence for connecting the conjugate group, and the conjugate group is connected to the 3' blunt end of the sense strand to form the siRNA conjugate.

[0169] Exemplarily, the siRNA has a sequence as shown in N-ER-FY005291M11, the 3' end of the sense strand of the siRNA has a protruding nucleotide extending out of the double-stranded region, and the flat end sequence mAsmAsmAmUCfmUUfUfGfmUmCmUmCmCmUmUmAmAmA formed by excluding the protruding -smAsmC nucleotide at the 3' end of the sense strand is used as the nucleotide sequence for linking the L96 conjugating group, thus the sense strand of the siRNA conjugate is mAsmAsmAmUCfmUUfUfGfmUmCmUmCmCmUmUmAmAmAL96, and the antisense strand is EVPmUsUfsmUmAmAGfmGmAmGmAmCmAmAAfmGAfmUmUmUsmCsmU.

[0170] Generally, the conjugating group comprises at least one pharmaceutically acceptable targeting group, or further comprises a linker, and the siRNA, the linker and the targeting group are sequentially connected. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The siRNA molecule can be non-covalently or covalently conjugated to the conjugating group, for example, can be covalently conjugated to the conjugating group. The conjugation site of the siRNA and the conjugating group can be at the 3' end or the 5' end of the sense strand of the siRNA, can also be at the 5' end of the antisense strand, and can also be in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and the conjugating group is at the 3' end of the sense strand of the siRNA.

[0171] In some embodiments, the conjugate group can be attached to the phosphate group, 2'-position hydroxyl group, or base of the nucleotide. In some embodiments, the conjugate group can also be attached to the 3'-position hydroxyl group, in which case the nucleotides are connected by 2'-5' phosphodiester bonds. When the conjugate group is attached to the end of the siRNA strand, the conjugate group is usually attached to the phosphate group of the nucleotide; when the conjugate group is attached to the internal sequence of the siRNA, the conjugate group is usually attached to the ribose sugar ring or base. Various attachment methods can be referred to in the literature: Muthiah Manoharan et. al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7.

[0172] In some embodiments, the siRNA and the conjugate group can be connected by acid-labile or reducible chemical bonds, which can be degraded in the acidic environment of the intracellular endosome, thereby allowing the siRNA to become free. For non-degradable conjugation, the conjugate group can be attached to the sense strand of the siRNA, thereby minimizing the impact of conjugation on the activity of the siRNA.

[0173] In some embodiments, the pharmaceutically acceptable targeting group can be a ligand commonly used in the field of siRNA administration, such as various ligands described in WO2009082607A2, which is incorporated herein by reference in its entirety.

[0174] In some embodiments, the pharmaceutically acceptable targeting group can be selected from one or more of the following ligands formed by the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots; sugars, such as lactose, poly-lactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folate; receptor ligands expressed by liver parenchymal cells, such as asialoglycoproteins, asialo sugar residues, lipoproteins (such as high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.

[0175] In some embodiments, each ligand is independently selected from a ligand that binds to a cell surface receptor. In some embodiments, at least one ligand is a ligand that binds to a liver cell surface receptor. In some embodiments, at least one ligand is a ligand that binds to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand that binds to a human liver cell surface receptor. In some embodiments, at least one ligand is a ligand that binds to a liver surface asialoglycoprotein receptor (ASGPR). The classes of such ligands are known to those skilled in the art and generally function to bind to a specific receptor on the surface of a target cell, mediating delivery of the siRNA linked to the ligand to the target cell.

[0176] In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to an asialoglycoprotein receptor (ASGPR) on the surface of a mammalian liver cell. In some embodiments, each ligand is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF). In some embodiments, the ligand is a sugar or a derivative of a sugar.

[0177] In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some embodiments, at least one of the ligands can be a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. In some embodiments, each ligand is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or a monosaccharide derivative. In some embodiments, each or at least one ligand is selected from the group consisting of glucose and derivatives thereof, mannans and derivatives thereof, galactose and derivatives thereof, xylose and derivatives thereof, ribose and derivatives thereof, fucose and derivatives thereof, lactose and derivatives thereof, maltose and derivatives thereof, arabinose and derivatives thereof, fructose and derivatives thereof, and sialic acid.

[0178] In some embodiments, each of the ligands can be independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, a-D-mannofuranose, β-D-mannofuranose, a-D-mannopyranose, β-D-mannopyranose, a-D-glucopyranose, β-D-glucopyranose, a-D-glucoruranose, β-D-glucoruranose, a-D-fructofuranose, a-D-fructopyranose, a-D-galactopyranose, β-D-galactopyranose, a-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-0-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L- glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-0-methyl-D-mannopyranose, 2-deoxy-2- sulfonamido-D-glucopyranose, N-glycolyl-a-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-tri-0-acetyl-1-thio-6-0-trityl-a-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-0-acetyl-2-deoxy-1,5-dithio-a-D-glucoheptoside ethyl ester, 2,5-anhydro-D-allosonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, or L-4-thioribose. Other choices of the ligands can be found, for example, in CN105378082A, which is incorporated by reference in its entirety.

[0179] In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that monovalent, divalent, trivalent, and tetravalent as used herein refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate formed by conjugation of the siRNA molecule to the conjugation group containing galactose or N-acetylgalactosamine as the targeting group is 1 : 1, 1:2, 1:3, or 1:4, respectively. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described herein is conjugated to the conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described herein is conjugated to the conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0180] The targeting group can be linked to the siRNA molecule via a suitable linker, which can be selected by a person skilled in the art according to the specific type of the targeting group. The linkers, the types of the targeting groups and the ways of connecting to the siRNA can be found in the disclosure of WO2015006740A2, which is incorporated herein by reference in its entirety.

[0181] Synthesis method of siRNA

[0182] The nucleotide monomers are connected one by one in the order of nucleotide arrangement from 3'-5' direction by the conventional solid-phase phosphoramidite method in the art. Each connection of a nucleotide monomer includes four steps of deprotection, coupling, oxidation or sulfurization, and capping. When the phosphate linkage is used between two nucleotides, the connection of the next nucleotide monomer includes four steps of deprotection, coupling, oxidation, and capping. When the phosphorothioate linkage is used between two nucleotides, the connection of the next nucleotide monomer includes four steps of deprotection, coupling, sulfurization, and capping. The nucleotide monomers are selected according to the target sequence in the present application, and the selected nucleotide monomers are the nucleotide monomers commonly used by a person skilled in the art, for example, the nucleotide monomers for synthesizing A can be but are not limited to selecting adenosine-3-phosphate. It should be understood that these monomers are connected to each other by 5'-3' phosphodiester bonds or 5'-3' phosphorothioate groups when they exist in an oligonucleotide, and when the last nucleotide at the 3' position is hydroxyl, for example, in the 5' to 3' direction, it is realized according to the conventional means in the art.

[0183] For example, the synthesis conditions of the siRNA of the present application can be as follows:

[0184] The deprotection conditions include: the reaction temperature is 25°C, the reaction time is 70 seconds, and the deprotection reagent is selected from dichloromethane solution (3% V / V) of dichloroacetic acid, and the molar ratio of the deprotection reagent to the protecting group on the solid support is 5:1.

[0185] The coupling reaction conditions include: the reaction temperature is 25°C, the reaction time is 600 seconds, the coupling reagent is selected from 0.25M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT), and the molar ratio of the nucleic acid sequence connected on the solid support to the nucleotide monomer is 1:10.

[0186] The oxidation reaction conditions include: the reaction temperature is 25°C, the reaction time is 15 seconds, the oxidation reagent is selected from 0.05M iodo-tetrahydrofuran solution, and the molar ratio of the oxidation reagent to the nucleic acid sequence connected on the solid support in the coupling step is 30:1.

[0187] The sulfurization reaction conditions include: the reaction temperature is 25°C, the reaction time is 300 seconds, the sulfurization reagent is selected from hydrogenated xanthine, and the molar ratio of the sulfurization reagent to the nucleic acid sequence connected on the solid support in the coupling step is 120:1.

[0188] The capping reaction conditions include: the reaction temperature is 25℃, the reaction time is 15 seconds, the capping reagent is selected from a mixed solution of CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methyl imidazole pyridine / acetonitrile solution) with a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence connected to the solid support is 1:1:1.

[0189] After all the nucleotide monomers are connected, the nucleic acid sequence connected to the solid support is subjected to amination, purification, desalination in sequence to obtain the siRNA sense strand and antisense strand, and finally the two strands are heated to anneal to obtain the product.

[0190] The methods of amination, purification, desalination and annealing are well known in the art. For example, amination is performed by contacting the nucleotide sequence connected to the solid support with concentrated ammonia water; purification is performed by chromatography; desalination is performed by reverse phase chromatography; and annealing is performed by gradually cooling the equimolar mixture of the sense strand and the antisense strand under different stringent conditions.

[0191] The synthesized siRNA is shown in Table 1.

[0192] siRNA conjugate synthesis method

[0193] Take the synthesis of L96 as an example:

[0194] In the first step, compound L96-A is obtained by reacting DMTr-L96 and succinic anhydride:

[0195] Preparation process: DMTr-L96, succinic anhydride, 4-dimethylaminopyridine and diisopropylethylamine are added to dichloromethane, stirred at 25℃ for 24 hours, then the reaction solution is washed with 0.5M triethylamine phosphate, the aqueous phase is washed with dichloromethane three times, the organic phases are combined and evaporated under reduced pressure to obtain the crude product. Then column chromatography is used to purify the pure product L96-A.

[0196] In the second step, L96-A is reacted with NH2-SPS to obtain L96-B:

[0197] Preparation process: L96-A, O-benzotriazol-tetramethylurea hexafluorophosphate (HBTU) and diisopropylethylamine (DIPEA) are mixed in acetonitrile, stirred at room temperature for 5 minutes to obtain a uniform solution, and then the amino methyl resin (NH2-SPS, 100-200 mesh) is added to the reaction solution, and the shaking table reaction is started at 25℃. After 18 hours of reaction, filtration is performed, and the filter cake is washed with dichloromethane and acetonitrile in sequence to obtain the filter cake. The filter cake is subjected to capping reaction with a CapA / CapB mixed solution to obtain L96-B, which is a solid support containing a conjugated molecule.

[0198] Third step, preparation of siRNA conjugate

[0199] L96-B was used as a solid support to synthesize siRNA conjugate according to the siRNA synthesis method described above, and the siRNA antisense strand was synthesized according to the siRNA synthesis method described above, and annealing generated the siRNA conjugate of the present application.

[0200] The synthesized siRNA conjugate is shown in Table 2.

[0201] Pharmaceutical composition

[0202] The present application provides a pharmaceutical composition containing siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.

[0203] The pharmaceutically acceptable carrier can be one or more of the carriers commonly used in the field of siRNA administration, such as, but not limited to, lipid nanoparticles (Lipid Nanoparticle, LNP), magnetic nanoparticles (such as nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (polyethylenimine, PEI), polyamidoamine (polyamidoamine (PAMAM) dendrimer), poly(L-lysine) (poly(L-lysine), PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP), poly(D&L-lactic / glycolic acid) copolymer (poly(D&L-lactic / glycolic acid) copolymer, PLGA), poly(2-aminoethyl ethylene phosphate) (poly(2-aminoethyl ethylene phosphate), PPEEA), and poly(2-dimethylaminoethyl methacrylate) (poly(2-dimethylaminoethyl methacrylate), PDMAEMA), and their derivatives.

[0204] The content of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly limited and can be the conventional content of each component.

[0205] In some embodiments, the pharmaceutical composition can further include a pharmaceutically acceptable other excipient, which can be one or more of various preparations or compounds conventionally used in the art. For example, the pharmaceutically acceptable other excipient can include at least one of a pH buffer, a protective agent, and an osmotic pressure adjusting agent.

[0206] The pH buffer can be a tris-hydroxymethyl aminomethane hydrochloride buffer having a pH of 7.5 to 8.5 and / or a phosphate buffer having a pH of 5.5 to 8.5, for example, a phosphate buffer having a pH of 5.5 to 8.5.

[0207] The protective agent can be at least one of myo-inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The content of the protective agent can be 0.01 to 30% by weight, based on the total weight of the pharmaceutical composition.

[0208] The osmotic pressure adjusting agent can be sodium chloride and / or potassium chloride. The content of the osmotic pressure adjusting agent can be such that the osmotic pressure of the pharmaceutical composition is 200 to 700 milliosmoles per kilogram (mOsm / kg). The content of the osmotic pressure adjusting agent can be easily determined by one skilled in the art according to the desired osmotic pressure.

[0209] In some embodiments, the pharmaceutical composition can be a liquid preparation, for example, an injection solution, or a lyophilized powder preparation that is mixed with a liquid excipient to prepare a liquid preparation when administered. The liquid preparation can be, but is not limited to, used for subcutaneous, intramuscular, or intravenous injection administration, or can be, but is not limited to, administered to the lung by spraying or to other organ tissues (e.g., the liver) by transpulmonary spraying. In some embodiments, the pharmaceutical composition is used for intravenous injection administration.

[0210] In some embodiments, the pharmaceutical composition can be in the form of a liposome preparation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome preparation includes an amine-containing transfection compound (hereinafter, also referred to as an organic amine), a helper lipid, and / or a pegylated lipid.

[0211] The following examples are intended to further illustrate the present application, but are not to be construed as limiting the same.

[0212] Example

[0213] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description, but it is to be understood that both the detailed description and the specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

[0214] The experimental techniques and experimental methods used in this example are conventional techniques and methods, and if no special instructions are given, the experimental methods not specifically mentioned in the following examples are usually carried out according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples, if no special instructions are given, can be obtained through regular commercial channels.

[0215] Example 1 Preparation of siRNA

[0216] The siRNA molecules with the following sequences were synthesized by Tianlin Biotech (Shanghai) Co., Ltd.

[0217] Table 1 siRNA and its sequence

[0218] Among them, the capital letters "G", "C", "A" and "U" each usually represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases, respectively; mA, mG, mC, mU: represent 2'-methoxy modified nucleotides; Af, Gf, Cf, Uf: represent 2'-fluorine modified nucleotides; dA, dG, dC, dT: represent 2'-deoxy modified ribonucleotides at the corresponding positions in the motif sequence; The lower case letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate group; EVP: indicates that the nucleotide adjacent to the right of the EVP is a 5'-trans-vinyl phosphonate nucleotide; [GNA]: indicates that the ribonucleotide adjacent to the left is a GNA modified ribonucleotide; "(invAb)": indicates the reverse dealkylated deoxyribose residue.

[0219] The siRNA conjugates with the following sequences were synthesized by Tianlin Biotech (Shanghai) Co., Ltd.:

[0220] Table 2 siRNA conjugates and their sequences:

[0221] Table 3 siRNA conjugates and their sequences:

[0222] Among them, L96 is connected to the 3' end of the sense strand or the blunt end formed by the 3' end of the sense strand in Table 1 or Table 3 through a phosphodiester bond, and L96 is:

[0223] In Tables 1-3, the 5' position of the ribose group of the 5' terminal nucleotide of the sense strand, the modified sense strand and the modified sense strand conjugated with the linking moiety is hydroxyl if not labeled EVP or (invAb), which structure is shown as Formula X:

[0224] wherein Base represents a base, such as A, U, G, C or T; R is hydroxyl or hydrogen or substituted by various groups known to those skilled in the art, for example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino, 2'-deoxy modified nucleotide.

[0225] In Tables 1-3, the 5' position of the ribose group of the 5' terminal nucleotide of the antisense strand and the modified antisense strand is hydroxyl if not labeled EVP, which structure is also shown as Formula X.

[0226] In Tables 1-3, the 3' position of the 3' terminal nucleotide of the sense strand and the modified sense strand is hydroxyl when the 3' terminal of the sense strand and the modified sense strand is not linked (invAb); the 3' position of the 3' terminal nucleotide of the antisense strand and the modified antisense strand is hydroxyl in Tables 1-3.

[0227] Example 2: Inhibition of FXI gene expression by siRNA and its modifications

[0228] 2.1 Experimental materials:

[0229] HepG2 cells were purchased from ATCC, item number: HB-8065;

[0230] RNA extraction kit, 96Kit, purchased from QIAGEN, item number: 74182;

[0231] RNAiMAX transfection reagent, purchased from Invitrogen, item number 13778-150;

[0232] RMPI1640 medium, purchased from Gibco, item number: 22400-089;

[0233] Fastking RT Kit(with gDNase), purchased from TianGen, item number: KR116-02;

[0234] FastStart Universal Probe Master, purchased from Roche, item number: 04914058001;

[0235] Opti-medium: purchased from Gibco, item number: 31985-070;

[0236] Target FXI primer and probe set, purchased from Thermo, item number: ID-HS01038035_m1;

[0237] TaqMan Gene Expression Assay (GAPDH), purchased from Thermo, item number: ID-Hs02786624_g1;

[0238] 2.2 Experimental method:

[0239] 2.2.1 Incubate HepG2 cells in fresh Opti-medium culture medium in a 96-well plate for 24 hours, resuspend the cultured cells in PS (penicillin-streptomycin mixture) free RMPI1640 culture medium to prepare a cell suspension with a density of 5.55x10 4 cells / mL, and add 90 μL of the cell suspension to each well of the 96-well plate, i.e. 5000 cells per well.

[0240] 2.2.2 Centrifuge the dry powder of the siRNA and siRNA modifiers (for convenience of description, the siRNA and siRNA modifiers are collectively referred to as siRNA in the description of the experimental process of this embodiment) at low temperature and high speed, then dissolve them with UltraPure Distilled Water to prepare a 100 μM stock solution.

[0241] 2.2.3 Prepare 20nM siRNA dilution Z and 2nM siRNA dilution W

[0242] (1) Preparation of 1 μM siRNA stock solution Q and 0.1 μM siRNA stock solution X:

[0243] a) Take 2 μL of the 100 μM siRNA stock solution prepared in step 2.2.2 above, add 18 μL of UltraPure Distilled Water to obtain a 10 μM siRNA dilution;

[0244] b) Take 2 μL of the 10 μM siRNA dilution prepared in step a), add 18 μL of UltraPure Distilled Water to obtain a 1 μM siRNA stock solution Q;

[0245] c) Take 2 μL of the 1 μM siRNA stock solution Q prepared in step b), add 18 μL of UltraPure Distilled Water to obtain a 0.1 μM siRNA stock solution X;

[0246] (2) Take 2 μL of each of the above-prepared siRNA stock solution Q and siRNA stock solution X, and add 98 μL of Opti-medium to obtain 20 nM siRNA dilution Z and 2 nM siRNA dilution W respectively.

[0247] 2.2.4 Transfection of HepG2 cells

[0248] (1) Take 3 μL of RNAiMAX transfection reagent was added to 97 μL of Opti-medium to obtain... RNAiMAX transfection reagent dilution solution; The RNAiMAX transfection reagent diluent and the 20 nM siRNA diluent Z prepared in step 2.2.3 were mixed at a volume ratio of 1:1 to prepare a transfection mixture. After standing for 5 minutes, 10 μL of the transfection mixture was added to a 96-well plate to transfect the HepG2 cells cultured in step 2.2.1 (final volume 100 μL, the concentration of siRNA in this system is 1 nM).

[0249] (2) Take 3 μL of RNAiMAX transfection reagent was added to 97 μL of Opti-medium to obtain... RNAiMAX transfection reagent dilution solution; The RNAiMAX transfection reagent diluent and the 2nM siRNA diluent W prepared in step 2.2.3 were mixed at a 1:1 volume ratio to prepare a transfection mixture. After standing for 5 minutes, 10 μL of the transfection mixture was added to a 96-well plate to transfect the HepG2 cells cultured in step 2.2.1 (final volume 100 μL, the concentration of siRNA in this system is 0.1 nM).

[0250] The cells were cultured for 24 hours after transfection; two replicates were set up for each concentration (1 nM and 0.1 nM).

[0251] 2.2.5 Utilization According to the 96Kit instructions, extract total RNA from HepG2 cells obtained in section 2.2.4.

[0252] 2.2.6 The extracted total RNA was reverse transcribed into cDNA using the Fastking RT Kit (with gDNase), following these steps:

[0253] a) Remove gDNA using gDNAase according to the table below;

[0254] 42℃, 3 min; 4℃, let stand.

[0255] b) Add each reagent as described below to the system obtained in step a) and perform reverse transcription;

[0256] 42°C, 15 min; 95°C, 3 min.

[0257] c) Store the reverse transcription product obtained in step b) at -20°C for real-time PCR analysis.

[0258] 2.2.7 Real-time PCR analysis using FastStart universal probe master

[0259] a) Prepare the qPCR reaction mixture as shown in the table below, and during the entire operation, all reagents are placed on ice;

[0260] Among them, the target special gene TaqMan probe primer (FXI) is purchased from Thermo, item ID-HS01038035_m1; the target special gene TaqMan probe primer (GAPDH) is purchased from Thermo, item 4326317E (VIC).

[0261] b) Perform the qPCR program as described below

[0262] 95°C, 10 minutes;

[0263] 95°C, 15 seconds, 60°C, 1 minute (40 cycles of this operation).

[0264] 2.2.8 Result analysis

[0265] a) Use Quant Studio 7 software to automatically calculate the Ct value with default settings;

[0266] b) Calculate the relative expression amount of the gene using the following formula:

[0267] ΔCt = Ct (FXI gene) - Ct (GAPDH)

[0268] ΔΔCt = ΔCt (detection sample group) - ΔCt (Mock group);

[0269] The expression amount of mRNA relative to the Mock group = 2 -ΔΔCt .

[0270] Mock group means: the group without adding siRNA compared with the detection sample group.

[0271] Inhibition rate (%) = (Mock group mRNA relative expression - test sample group mRNA relative expression) / Mock group mRNA relative expression x 100%

[0272] 2.3 Inhibition rate determination results

[0273] Select concentrations of 1 nM and 0.1 nM for testing, and the results are shown in Table 4.

[0274] Table 4 Note: “--” indicates that the results are not shown.

[0275] As can be seen from Table 4, the siRNA provided in the application has high FXI gene inhibition activity in HepG2 cells.

[0276] Example 3: siRNA and its conjugate inhibit FXI gene expression

[0277] 3.1 Experimental materials:

[0278] HepG2 cells, purchased from ATCC, item number: HB-8065;

[0279] RNA extraction kit, 96Kit, purchased from QIAGEN, item number: 74182;

[0280] RNAiMAX transfection reagent, purchased from Invitrogen, item number 13778-150;

[0281] RMPI1640 medium, purchased from Gibco, item number: 22400-089;

[0282] Fastking RT Kit(with gDNase), purchased from TianGen, item number: KR116-02;

[0283] FastStart Universal Probe Master, purchased from Roche, item number: 04914058001;

[0284] Opti-medium: purchased from Gibco, item number: 31985-070;

[0285] Target FXI primer and probe set, purchased from Thermo, item number: ID-HS01038035_m1;

[0286] TaqMan Gene Expression Assay (GAPDH), purchased from Thermo, item number: ID-Hs02786624_g1;

[0287] 3.2 Experimental method:

[0288] 3.2.1 Incubate HepG2 cells in fresh Opti-medium culture medium in a 96-well plate for 24 hours, resuspend the incubated cells in RMPI1640 culture medium without PS (penicillin streptomycin mixture) to prepare a cell suspension with a density of 5.55x10 4 cells / mL, and add 90 μL of the cell suspension to each well of the 96-well plate, i.e. 5000 cells per well.

[0289] 3.2.2 Centrifuge the dry powder of the siRNA to be tested (for the convenience of description, the siRNA and the siRNA conjugate are uniformly described as siRNA) at low temperature and high speed, then dissolve it with ultra-pure distilled water (ULtraPure Distilled Water) to prepare a 100 μM stock solution.

[0290] 3.2.3 Preparation of 200 nM siRNA dilution Z and 2 nM siRNA dilution W

[0291] (1) Preparation of 10 μM siRNA stock solution Q and 0.1 μM siRNA stock solution X:

[0292] a) Take 2 μL of the 100 μM siRNA stock solution prepared in step 3.2.2 above, add 18 μL of ultra-pure distilled water to obtain a 10 μM siRNA stock solution Q;

[0293] b) Take 2 μL of the 10 μM siRNA stock solution Q prepared in step a), add 18 μL of ultra-pure distilled water to obtain a 1 μM siRNA stock solution L;

[0294] c) Take 2 μL of the 1 μM siRNA stock solution L prepared in step b), add 18 μL of ultra-pure distilled water to obtain a 0.1 μM siRNA stock solution X;

[0295] (2) Take 2 μL of the prepared siRNA stock solution Q and siRNA stock solution X respectively, add 98 μL of Opti-medium respectively to obtain 200 nM siRNA dilution Z and 2 nM siRNA dilution W respectively.

[0296] 3.2.4 Transfection of HepG2 cells

[0297] (1) Take RNAiMAX transfection reagent 3 μL, added to 97 μL Opti-medium, to obtain RNAiMAX transfection reagent dilution; to RNAiMAX transfection reagent dilution was mixed with the 200 nM siRNA dilution Z prepared in step 3.2.3 in a 1:1 volume ratio to prepare a transfection mixture, which was allowed to stand for 5 minutes, and 10 μL of the transfection mixture was added to the HepG2 cells cultured in step 3.2.1 in a 96-well plate (final volume 100 μL, the concentration of siRNA in the system was 10 nM);

[0298] (2) Take RNAiMAX transfection reagent 3 μL, added to 97 μL Opti-medium, to obtain RNAiMAX transfection reagent dilution; to RNAiMAX transfection reagent dilution was mixed with the 2 nM siRNA dilution W prepared in step 3.2.3 in a 1:1 volume ratio to prepare a transfection mixture, which was allowed to stand for 5 minutes, and 10 μL of the transfection mixture was added to the HepG2 cells cultured in step 3.2.1 in a 96-well plate (final volume 100 μL, the concentration of siRNA in the system was 0.1 nM).

[0299] The above transfection was cultured for 48 hours; 2 repeats were set for each concentration (10 nM and 0.1 nM).

[0300] 3.2.5 Utilize 96 Kit instructions to extract total RNA from the HepG2 cells obtained in 3.2.4.

[0301] 3.2.6 Reverse transcription of the extracted total RNA to cDNA using the Fastking RT Kit (with gDNase) kit, according to the following steps:

[0302] a) Remove gDNA according to the following table using gDNA enzyme;

[0303] 42°C, 3 min; 4°C, stand

[0304] b) Add the following reagents to the system obtained in step a) and perform reverse transcription;

[0305] 42°C, 15 min; 95°C, 3 min.

[0306] c) Store the reverse transcription product obtained in step b) at -20°C for real-time PCR analysis.

[0307] 3.2.7 Real-time PCR analysis using FastStart universal probe master

[0308] a) Prepare the qPCR reaction mixture as shown in the table below, and keep all reagents on ice during the entire procedure;

[0309] b) Perform the qPCR program as described below

[0310] 95°C, 10 minutes;

[0311] 95°C, 15 seconds, 60°C, 1 minute (40 cycles of this operation).

[0312] 3.2.8 Result analysis

[0313] a) Use the Quant Studio 7 software to automatically calculate the Ct value using the default settings;

[0314] b) Calculate the relative expression of the gene using the following formula:

[0315] ΔCt = Ct (FXI gene) - Ct (GAPDH)

[0316] ΔΔCt = ΔCt (test sample group) - ΔCt (Mock group);

[0317] Expression of mRNA relative to the Mock group = 2 -ΔΔCt .

[0318] Mock group means: a group without the addition of siRNA compared with the test sample group.

[0319] Inhibition rate (%) = (Mock group mRNA relative expression - test sample group mRNA relative expression) / Mock group mRNA relative expression x 100%

[0320] 3.3 Inhibition rate determination results

[0321] Select 10 nM and 0.1 nM for testing, and the results are shown in Table 5.

[0322] Table 5

[0323] As can be seen from Table 5, the siRNA conjugate provided in the application has high FXI gene inhibition activity in HepG2 cells.

[0324] Example 4

[0325] This example is basically the same as Example 2, except that the modifier is N-ER-FY005291M44, N-ER-FY005291M46, N-ER-FY005291M47, N-ER-FY005291M48, N-ER-FY005291M49, N-ER-FY005291M50. The inhibition rate results are shown in Table 6.

[0326] Table 6

[0327] As can be seen from Table 6, the siRNA modifier provided by the present application has high FXI gene inhibition activity in HepG2 cells.

[0328] Example 5: Inhibition of human FXI (hFXI) gene expression in humanized mice by siRNA conjugates

[0329] 6-8 week old C57BL / 6-hC5 mice (provided by Shanghai Southern Model Organism Technology Co., Ltd.) were introduced into the feeding facility and adaptively fed for 7 days, then the mice were subcutaneously administered with N-ER-FY005291M44L96, N-ER-FY005291M46L96, N-ER-FY005291M50L96 (5 mice per group) at a single dose of 3 mg / kg. The expression of hFXI protein in serum was detected every 7 days after administration, and the inhibition rate of siRNA conjugates on hFXI protein expression was obtained.

[0330] From this experiment, it can be concluded that the siRNA conjugates of the present disclosure have high inhibition activity on hFXI gene expression in vivo, and can reduce the level of hFXI protein for a long time. The inhibition rate result on the 7th day of the experiment is as high as more than 90%, indicating that the designed compound can better inhibit the generation of hFXI protein. On the 35th day of the experiment, it still has a high inhibition rate, as high as more than 75%, and the in vivo inhibition effect has long-acting.

Claims

1. An siRNA for inhibiting the expression of FXI gene, the siRNA comprising a sense strand and an antisense strand, wherein each of the nucleotides in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I and the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein, (1) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 1, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 2; or (2) the nucleotide sequence I comprises the nucleotide sequence set forth in SEQ ID NO: 31, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:

2.

2. The siRNA of claim 1, wherein, the sense strand and the antisense strand are complementary to form the double-stranded region, the 3’-terminal end of the antisense strand has 1-2 overhanging nucleotides extending out of the double-stranded region, and the 3’-terminal end of the sense strand forms a blunt end; or, the sense strand and the antisense strand are complementary to form the double-stranded region, the 3’-terminal end of the sense strand has 1-2 overhanging nucleotides extending out of the double-stranded region, and / or the 3’-terminal end of the antisense strand has 1-3 overhanging nucleotides extending out of the double-stranded region.

3. The siRNA of claim 1 or 2, wherein at least one of the nucleotides in the sense strand or the antisense strand is a modified nucleotide, and / or at least one of the phosphate groups is a phosphate group with a modification group; preferably, the phosphate group with a modification group is a phosphorothioate group in which one of the oxygen atoms in the phosphodiester bond is replaced by a sulfur atom; and / or, the siRNA comprises a sense strand that does not comprise 3’ overhanging nucleotides. neither the 5’-terminal end nor the 3’-terminal end of the sense strand is connected to an inverted abasic deoxyribose residue, or only the 5’-terminal end of the sense strand is connected to an inverted abasic deoxyribose residue, or only the 3’-terminal end of the sense strand is connected to an inverted abasic deoxyribose residue, or the 5’-terminal end and the 3’-terminal end of the sense strand are respectively connected to an inverted abasic deoxyribose residue; preferably, the inverted abasic deoxyribose residue is connected to the 3’-terminal nucleotide and / or the 5’-terminal nucleotide of the sense strand by a phosphodiester bond, a phosphorothioate group or other internucleoside linkage. in the 5’-terminal end to 3’-terminal end direction, the 7th, 9th, 10th and 11th nucleotides in the sense strand are 2’-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides; or, in the 5’-terminal end to 3’-terminal end direction, the 5th, 7th, 8th and 9th nucleotides in the sense strand are 2’-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2’-O-CH3 modified nucleotides. ​ ​ ​ ​ 4. The siRNA according to any one of claims 1 to 3, wherein, ​ ​ 5. The siRNA according to any one of claims 1 to 4, wherein, ​ ​ or, in the direction of the 5' end to the 3' end, the nucleotides at positions 9, 10, and 11 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, in the direction of the 5' end to the 3' end, the nucleotides at positions 8, 9, and 10 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, in the direction of the 5' end to the 3' end, the nucleotides at positions 8, 9, 10, and 11 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, in the direction of the 5' end to the 3' end, the nucleotides at positions 7 and 9 in the sense strand are 2'-F modified nucleotides, the nucleotide at position 11 in the sense strand is a 2'-deoxy modified nucleotide, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, in the direction of the 5' end to the 3' end, the nucleotides at positions 7, 9, and 11 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, in the direction of the 5' end to the 3' end, the nucleotides at positions 7, 11, 13, and 15 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, in the direction of the 5' end to the 3' end, the nucleotides at positions 8, 9, 10, and 12 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, in the direction of the 5' end to the 3' end, the nucleotides at positions 7, 9, 11, 13, and 15 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, in the direction of the 5' end to the 3' end, the nucleotides at positions 9, 11, and 13 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, in the direction of the 5' end to the 3' end, the nucleotides at positions 7, 9, 11, and 13 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, in the direction of the 5' end to the 3' end, the nucleotides at positions 3, 7, 8, and 9 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, along the 5' end to the 3' end direction, the nucleotides at positions 3, 9, 11 and 13 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, along the 5' end to the 3' end direction, the nucleotides at positions 3, 7, 9 and 11 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, along the 5' end to the 3' end direction, the nucleotides at positions 7, 11 and 13 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, along the 5' end to the 3' end direction, the nucleotides at positions 7, 11, 12 and 13 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, along the 5' end to the 3' end direction, the nucleotides at positions 3, 7, 11 and 16 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, along the 5' end to the 3' end direction, the nucleotides at positions 3, 7, 11 and 17 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides; or, along the 5' end to the 3' end direction, the nucleotides at positions 3, 7, 11, 16 and 17 in the sense strand are 2'-F modified nucleotides, and the nucleotides at the remaining positions in the sense strand are 2'-O-CH3 modified nucleotides.

6. The siRNA according to any one of claims 1 to 5, wherein, along the 5' end to the 3' end direction, the sense strand comprises a phosphorothioate group at a position selected from: between the 1st and 2nd nucleotides from the 5' end of the sense strand; between the 2nd and 3rd nucleotides from the 5' end of the sense strand; between the 1st and 2nd nucleotides from the 3' end of the sense strand; between the 2nd and 3rd nucleotides from the 3' end of the sense strand; or, along the 5' end to the 3' end direction, the sense strand comprises a phosphorothioate group at a position selected from: between the 1st and 2nd nucleotides from the 5' end of the sense strand; between the 2nd and 3rd nucleotides from the 5' end of the sense strand; or, along the 5' end to the 3' end direction, the sense strand comprises a phosphorothioate group at a position selected from: between the 1st and 2nd nucleotides from the 5' end of the sense strand; between the 2nd and 3rd nucleotides from the 5' end of the sense strand; between the reverse abasic deoxyribosyl residue and the 1st nucleotide from the 3' end of the sense strand; or, along the 5' end to the 3' end direction, the sense strand comprises a phosphorothioate group at a position selected from: a reverse deoxyribonucleotide residue between the 5' terminal start and the 1st nucleotide of the sense strand; a reverse deoxyribonucleotide residue between the 5' terminal start and the 1st nucleotide of the sense strand; a reverse deoxyribonucleotide residue between the 5' terminal start and the 1st nucleotide of the sense strand.

7. The siRNA according to any one of claims 1 to 6, wherein, the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand are 2'-F modified nucleotides, and the remaining nucleotides in the antisense strand are 2'-O-CH3 modified nucleotides, in the direction from the 5' terminal end to the 3' terminal end; the 2nd, 6th and 14th nucleotides in the antisense strand or, in the 5' end to 3' end direction of the antisense strand, the nucleotides at positions 2, 7, and 14 are 2'-F modified nucleotides, the nucleotide at position 5 is a 2'-deoxy modified nucleotide, and the nucleotides at the remaining positions are 2'-0-CH3 modified nucleotides; or, in the 5' end to 3' end direction of the antisense strand, the nucleotides at positions 2, 3, 7, 10, 12, 14, and 22 are 2'-F modified nucleotides, the nucleotide at position 5 is a 2'-deoxy modified nucleotide, the nucleotide at position 6 is a GNA modified nucleotide, and the nucleotides at the remaining positions are 2'-0-CH3 modified nucleotides; or, in the 5' end to 3' end direction of the antisense strand, the nucleotides at positions 2, 3, 7, 10, 12, 14, and 16 are 2'-F modified nucleotides, the nucleotide at position 5 is a 2'-deoxy modified nucleotide, the nucleotide at position 6 is a GNA modified nucleotide, and the nucleotides at the remaining positions are 2'-0-CH3 modified nucleotides.

8. The siRNA according to any one of claims 1 to 7, wherein, in the 5' end to 3' end direction of the antisense strand, the nucleotide at the 5' end is linked to a 5' phosphate group or a 5' phosphate derivative group; preferably, the 5' phosphate derivative group is a 5'-trans vinyl phosphonate.

9. The siRNA according to any one of claims 1 to 8, wherein, in the 5' end to 3' end direction of the antisense strand, the antisense strand comprises a phosphorothioate group at a position selected from: between the 1st and 2nd nucleotides from the 5' end of the antisense strand; between the 2nd and 3rd nucleotides from the 5' end of the antisense strand; between the 1st and 2nd nucleotides from the 3' end of the antisense strand; between the 2nd and 3rd nucleotides from the 3' end of the antisense strand; or, in the 5' end to 3' end direction of the antisense strand, the antisense strand comprises a phosphorothioate group at a position selected from: between the 1st and 2nd nucleotides from the 5' end of the antisense strand; between the 2nd and 3rd nucleotides from the 5' end of the antisense strand; between the 1st and 2nd nucleotides from the 3' end of the antisense strand; between the 2nd and 3rd nucleotides from the 3' end of the antisense strand; between the 3rd and 4th nucleotides from the 3' end of the antisense strand.

10. The siRNA according to any one of claims 1 to 9, which is selected from the siRNAs of Table 1; preferably, the siRNA is selected from N-ER-FY005291D2, N-ER-FY005291M12, N-ER-FY005291M13, N-ER-FY005291M15, N-ER-FY005291M20, N-ER-FY005291M21, N-ER-FY005291M22, N-ER-FY005291M24, N-ER-FY005291M25, N-ER-FY005291M26, N-ER-FY005291M28, N-ER-FY005291M29, N-ER-FY005291M32, N-ER-FY005291M33, N-ER-FY005291M35, N-ER-FY005291M11D2, N-ER-FY005291M12D2, N-ER-FY005291M13D2, N-ER-FY005291M15D2, N-ER-FY005291M21D2, N-ER-FY005291M22D2, N-ER-FY005291M24D2, N-ER-FY005291M25D2, N-ER-FY005291M26D2, N-ER-FY005291M29D2, N-ER-FY005291M31D2, N-ER-FY005291M35D2, N-ER-FY005291M44, N-ER-FY005291M46, N-ER-FY005291M47, N-ER-FY005291M48, N-ER-FY005291M49, N-ER-FY005291M50.

11. An siRNA conjugate, which contains an siRNA according to any one of claims 1 to 10 and a conjugation group conjugated to the siRNA.

12. The siRNA conjugate of claim 11, wherein, The sense strand of the siRNA is complementary to the antisense strand to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1 to 3 overhanging nucleotides extending out of the double-stranded region.

13. The siRNA conjugate of claim 11 or 12, wherein the conjugate group is selected from:

14. The siRNA conjugate according to any one of claims 11-13, wherein the siRNA conjugate is selected from the group consisting of siRNA conjugates of Table 2, preferably the siRNA conjugate is selected from the group consisting of N-ER-FY005291M12L96, N-ER-FY005291M13L96, N-ER-FY005291M15L96, N-ER-FY005291M20L96, N-ER-FY005291M21L96, N-ER-FY005291M22L96, N-ER-FY005291M24L96, N-ER-FY005291M25L96, N-ER-FY005291M26L96, N-ER-FY005291M28L96, N-ER-FY005291M29L96, N-ER-FY005291M32L96, N-ER-FY005291M33L96, N-ER-FY005291M35L96, N-ER-FY005291M44L96, N-ER-FY005291M46L96, N-ER-FY005291M47L96, N-ER-FY005291M48L96, N-ER-FY005291M49L96, N-ER-FY005291M50L96.

15. A pharmaceutical composition comprising the siRNA according to any one of claims 1-10, or the siRNA conjugate according to any one of claims 11-14, and a pharmaceutically acceptable carrier.

16. A kit comprising the siRNA according to any one of claims 1-10, or the siRNA conjugate according to any one of claims 11-14, or the pharmaceutical composition according to claim 15.

17. Use of the siRNA according to any one of claims 1-10, or the siRNA conjugate according to any one of claims 11-14, or the pharmaceutical composition according to claim 15 for the manufacture of a medicament for inhibiting the expression of a FXI gene.

18. Use of the siRNA according to any one of claims 1-10, or the siRNA conjugate according to any one of claims 11-14, or the pharmaceutical composition according to claim 15 for the manufacture of a medicament for preventing and / or treating a disease associated with overexpression of a FXI gene.

19. The use according to claim 18, wherein the disease comprises deep vein thrombosis, venous or arterial thrombosis, ischemic stroke, pulmonary embolism, myocardial infarction, stroke, Alzheimer’s disease, thrombosis associated with chronic kidney disease or end-stage kidney disease.

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