Double-stranded RNA targeting coagulation factor xi

Abstract

Provided are a double-stranded RNA targeting coagulation factor XI, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof。 Further provided are a pharmaceutical composition comprising the double-stranded RNA, the pharmaceutically acceptable salt thereof, or the ligand conjugate thereof, as well as a therapeutic use of the double-stranded RNA, the pharmaceutically acceptable salt thereof, or the ligand conjugate thereof.

Description

Double-stranded RNA targeting coagulation factor XI

[0001] Citation of Related Applications

[0002] This application claims priority and benefits from Chinese patent application No. 202311121470.9 filed with the State Intellectual Property Office of the People's Republic of China on August 31, 2023, the entire contents of which are hereby incorporated by reference into the text in their entirety. Technical Field

[0003] The present application belongs to the field of biomedicine and relates to a double-stranded ribonucleic acid (dsRNA), a pharmaceutically acceptable salt thereof, a ligand conjugate thereof, or a pharmaceutical composition, which can be used to inhibit coagulation factor XI. Background Art

[0004] Factor XI is a serine protease that is primarily synthesized in the liver and megakaryocytes. Factor XI participates in the intrinsic coagulation pathway. During the coagulation cascade, thrombin can activate factor XI. Activated factor XI, in turn, stimulates the production of thrombin, thereby amplifying the coagulation cascade. Therefore, drugs targeting factor XI can block the intrinsic pathway and inhibit the amplification of the coagulation cascade, thereby exerting antithrombotic effects without causing excessive bleeding.

[0005] Currently, relatively few factor XI inhibitors have been discovered, mainly including monoclonal antibodies, antisense oligonucleotides, small molecule inhibitors, peptide mimetics and peptide inhibitors. No factor XI inhibitor has been approved for marketing.

[0006] Small interfering RNA (siRNA) can inhibit or block the translation or transcription of target genes in a sequence-specific manner based on the RNA interference (RNAi) mechanism to inhibit target gene expression, exert an inhibitory effect at the mRNA level, and simultaneously reduce the level of coagulation factor XI, thereby achieving the purpose of treating diseases.

[0007] SUMMARY OF THE INVENTION

[0008] On the one hand, the present application provides a double-stranded ribonucleic acid (dsRNA), a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the dsRNA comprises a sense strand and an antisense strand, the sense strand containing a nucleotide sequence of no more than 23 nucleotides in length, the antisense strand containing a nucleotide sequence of no more than 25 nucleotides in length, the sense strand or the antisense strand being optionally modified, and the double-stranded ribonucleic acid (dsRNA), a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof targets coagulation factor XI mRNA.

[0009] On the other hand, the present application provides a double-stranded ribonucleic acid (dsRNA), a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25 or SEQ ID NO.27, and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID At least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.26 or SEQ ID NO.28, and the length of the antisense strand does not exceed 23 nucleotides:

[0010] Sense strand: 5'-GAUCUCCAACUAAAAUACU-3' (SEQ ID NO. 1),

[0011] Antisense strand: 5'-AGUAUUUUAGUUGGAGAUCCG-3' (SEQ ID NO. 2);

[0012] Sense strand: 5'-GGAUGAUUUUCUUAUAUCA-3' (SEQ ID NO. 3),

[0013] Antisense strand: 5'-UGAUAUAAGAAAAUCAUCCUG-3' (SEQ ID NO. 4);

[0014] Sense strand: 5'-GGUCUUUUCAGGAUGAUUU-3' (SEQ ID NO. 5),

[0015] Antisense strand: 5'-AAAUCAUCCUGAAAAGACCUU-3' (SEQ ID NO. 6);

[0016] Sense strand: 5'-GUCUUUUCAGGAUGAUUUU-3' (SEQ ID NO. 7),

[0017] Antisense strand: 5'-AAAAUCAUCCUGAAAAGACCU-3' (SEQ ID NO. 8);

[0018] Sense strand: 5'-CUUUUCAGGAUGAUUUUCU-3' (SEQ ID NO. 9),

[0019] Antisense strand: 5′-AGAAAAUCAUCCUGAAAAGAC-3′ (SEQ ID NO. 10);

[0020] Sense strand: 5'-UUUUCAGGAUGAUUUUCUU-3' (SEQ ID NO. 11),

[0021] Antisense strand: 5′-AAGAAAAUCAUCCUGAAAAGA-3′ (SEQ ID NO. 12);

[0022] Sense strand: 5'-UCAGGAUGAUUUUCUUAUA-3' (SEQ ID NO. 13),

[0023] Antisense strand: 5′-UAUAAGAAAAUCAUCCUGAAA-3′ (SEQ ID NO. 14);

[0024] Sense strand: 5'-CAGGAUGAUUUUCUUAUAU-3' (SEQ ID NO. 15),

[0025] Antisense strand: 5'-AUAUAAGAAAAUCAUCCUGAA-3' (SEQ ID NO. 16);

[0026] Sense strand: 5'-AGGAUGAUUUUCUUAUAUC-3' (SEQ ID NO. 17),

[0027] Antisense strand: 5'-GAUAUAAGAAAAUCAUCCUGA-3' (SEQ ID NO. 18);

[0028] Sense strand: 5'-GAUGAUUUUCUUAUAUCAA-3' (SEQ ID NO. 19),

[0029] Antisense strand: 5′-UUGAUAUAAGAAAAUCAUCCU-3′ (SEQ ID NO. 20);

[0030] Sense strand: 5'-AUGAUUUUCUUAUAUCAAG-3' (SEQ ID NO. 21),

[0031] Antisense strand: 5′-CUUGAUAUAAGAAAAUCAUCC-3′ (SEQ ID NO. 22);

[0032] Sense strand: 5'-UGAUUUUCUUAUAUCAAGU-3' (SEQ ID NO. 23),

[0033] Antisense strand: 5′-ACUUGAUAUAAGAAAAUCAUC-3′ (SEQ ID NO. 24);

[0034] Sense strand: 5'-CUUAUAUCAAGUGGUACAU-3' (SEQ ID NO. 25),

[0035] Antisense strand: 5'-AUGUACCACUUGAUAUAAGAA-3' (SEQ ID NO. 26);

[0036] Sense strand: 5'-AAACGGAUCUCCAACUAAA-3' (SEQ ID NO. 27),

[0037] Antisense strand: 5′-UUUAGUUGGAGAUCCGUUUGA-3′ (SEQ ID NO. 28);

[0038] The sense or antisense strand is optionally modified.

[0039] In another aspect, the present application provides a pharmaceutical composition comprising the dsRNA of the present application, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof. In another aspect, the present application provides a pharmaceutical composition comprising the dsRNA of the present application, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, and a pharmaceutically acceptable carrier or excipient.

[0040] On the other hand, the present application provides a kit for treating and / or preventing thromboembolism and its complications, comprising the dsRNA of the present application, a pharmaceutically acceptable salt thereof or a ligand conjugate thereof, or a pharmaceutical composition; and optional instructions for use.

[0041] On the other hand, the present application provides a method for treating and / or preventing thromboembolism and its complications, which comprises administering the dsRNA, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, or a pharmaceutical composition of the present application to a subject for treatment and / or prevention (hereinafter also referred to as a subject).

[0042] In some embodiments, the present application provides a method for treating and / or preventing thromboembolism and its complications, which comprises administering to a subject for treatment and / or prevention (hereinafter also referred to as a subject) a therapeutically / preventively effective amount of the dsRNA of the present application, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, or a pharmaceutical composition.

[0043] In another aspect, the present application provides use of the dsRNA, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, or a pharmaceutical composition of the present application in the preparation of a medicament for treating and / or preventing thromboembolism and its complications.

[0044] In another aspect, the present application provides use of the dsRNA, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, or a pharmaceutical composition of the present application in treating and / or preventing thromboembolism and its complications.

[0045] In another aspect, the present application provides the dsRNA, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, or a pharmaceutical composition of the present application for treating and / or preventing thromboembolism and its complications.

[0046] In some embodiments, the thromboembolism and its complications may include various types of thrombosis, embolism and thromboembolism, such as arterial embolism and venous embolism, including deep vein thrombosis, pulmonary embolism, myocardial infarction and stroke.

[0047] In some embodiments, the thromboembolism and its complications specifically include thromboembolism in patients with end-stage renal disease undergoing hemodialysis, venous thromboembolism in patients undergoing knee replacement surgery, stroke or systemic embolism in patients with atrial fibrillation, cancer-related venous thromboembolism, etc.

[0048] In some embodiments, the dsRNA, its pharmaceutically acceptable salt or ligand conjugate, or pharmaceutical composition of the present application is used as a single therapeutic agent for treating and / or preventing thromboembolism and its complications.

[0049] In some embodiments, the dsRNA, its pharmaceutically acceptable salt or ligand conjugate, or pharmaceutical composition of the present application is used in combination with other therapeutic agents for treating and / or preventing thromboembolism and its complications.

[0050] Detailed Description of the Invention

[0051] The exemplary embodiments of the present application will be described below, but those skilled in the art will understand that the scope of protection of the present application is not limited thereto. Instead, various modifications, changes or alterations may be made based on the spirit and concept of the present application, and the contents after such modifications, changes or alterations still fall within the scope of the present application.

[0052] The present application provides a double-stranded ribonucleic acid (dsRNA), a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleic acid sequence that differs from at least 15 (e.g., 15, 16, 17, 18, or 19) consecutive nucleotides in the nucleotide sequence shown in Table 1 by 0, 1, 2, or 3 nucleotides, and the length of the sense strand does not exceed 21 nucleotides; the antisense strand comprises a nucleic acid sequence that differs from at least 15 (e.g., 15, 16, 17, 18, or 19) consecutive nucleotides in the nucleotide sequence shown in Table 2 by 0, 1, 2, or 3 nucleotides, and the length of the antisense strand does not exceed 23 nucleotides.

[0053] The present application provides a double-stranded ribonucleic acid (dsRNA), a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides of a nucleotide sequence as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, or SEQ ID NO.27, and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID At least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.26 or SEQ ID NO.28, and the length of the antisense strand does not exceed 23 nucleotides:

[0054] Sense strand: 5'-GAUCUCCAACUAAAAUACU-3' (SEQ ID NO. 1),

[0055] Antisense strand: 5'-AGUAUUUUAGUUGGAGAUCCG-3' (SEQ ID NO. 2);

[0056] Sense strand: 5'-GGAUGAUUUUCUUAUAUCA-3' (SEQ ID NO. 3),

[0057] Antisense strand: 5'-UGAUAUAAGAAAAUCAUCCUG-3' (SEQ ID NO. 4);

[0058] Sense strand: 5'-GGUCUUUUCAGGAUGAUUU-3' (SEQ ID NO. 5),

[0059] Antisense strand: 5'-AAAUCAUCCUGAAAAGACCUU-3' (SEQ ID NO. 6);

[0060] Sense strand: 5'-GUCUUUUCAGGAUGAUUUU-3' (SEQ ID NO. 7),

[0061] Antisense strand: 5'-AAAAUCAUCCUGAAAAGACCU-3' (SEQ ID NO. 8);

[0062] Sense strand: 5'-CUUUUCAGGAUGAUUUUCU-3' (SEQ ID NO. 9),

[0063] Antisense strand: 5′-AGAAAAUCAUCCUGAAAAGAC-3′ (SEQ ID NO. 10);

[0064] Sense strand: 5'-UUUUCAGGAUGAUUUUCUU-3' (SEQ ID NO. 11),

[0065] Antisense strand: 5′-AAGAAAAUCAUCCUGAAAAGA-3′ (SEQ ID NO. 12);

[0066] Sense strand: 5'-UCAGGAUGAUUUUCUUAUA-3' (SEQ ID NO. 13),

[0067] Antisense strand: 5′-UAUAAGAAAAUCAUCCUGAAA-3′ (SEQ ID NO. 14);

[0068] Sense strand: 5'-CAGGAUGAUUUUCUUAUAU-3' (SEQ ID NO. 15),

[0069] Antisense strand: 5'-AUAUAAGAAAAUCAUCCUGAA-3' (SEQ ID NO. 16);

[0070] Sense strand: 5'-AGGAUGAUUUUCUUAUAUC-3' (SEQ ID NO. 17),

[0071] Antisense strand: 5'-GAUAUAAGAAAAUCAUCCUGA-3' (SEQ ID NO. 18);

[0072] Sense strand: 5'-GAUGAUUUUCUUAUAUCAA-3' (SEQ ID NO. 19),

[0073] Antisense strand: 5′-UUGAUAUAAGAAAAUCAUCCU-3′ (SEQ ID NO. 20);

[0074] Sense strand: 5'-AUGAUUUUCUUAUAUCAAG-3' (SEQ ID NO. 21),

[0075] Antisense strand: 5′-CUUGAUAUAAGAAAAUCAUCC-3′ (SEQ ID NO. 22);

[0076] Sense strand: 5'-UGAUUUUCUUAUAUCAAGU-3' (SEQ ID NO. 23),

[0077] Antisense strand: 5′-ACUUGAUAUAAGAAAAUCAUC-3′ (SEQ ID NO. 24);

[0078] Sense strand: 5'-CUUAUAUCAAGUGGUACAU-3' (SEQ ID NO. 25),

[0079] Antisense strand: 5'-AUGUACCACUUGAUAUAAGAA-3' (SEQ ID NO. 26);

[0080] Sense strand: 5'-AAACGGAUCUCCAACUAAA-3' (SEQ ID NO. 27),

[0081] Antisense strand: 5′-UUUAGUUGGAGAUCCGUUUGA-3′ (SEQ ID NO. 28);

[0082] The sense or antisense strand is optionally modified.

[0083] In some embodiments, the positive strand contains at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25 or SEQ ID NO.27, and the length of the positive strand does not exceed 21 nucleotides, wherein the at least 15 consecutive nucleotides can be selected from 15, 16, 17, 18 or 19 consecutive nucleotides.

[0084] In some embodiments, the antisense strand contains at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26 or SEQ ID NO.28, and the length of the antisense strand does not exceed 23 nucleotides, wherein the at least 15 consecutive nucleotides can be selected from 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides.

[0085] In some embodiments, the present application provides a double-stranded ribonucleic acid (dsRNA), a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, or SEQ ID NO.27, and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, or SEQ ID The nucleotide sequence shown in NO.28 and the length of the antisense strand does not exceed 23 nucleotides:

[0086] Sense strand: 5'-GAUCUCCAACUAAAAUACU-3' (SEQ ID NO. 1),

[0087] Antisense strand: 5'-AGUAUUUUAGUUGGAGAUCCG-3' (SEQ ID NO. 2);

[0088] Sense strand: 5'-GGAUGAUUUUCUUAUAUCA-3' (SEQ ID NO. 3),

[0089] Antisense strand: 5'-UGAUAUAAGAAAAUCAUCCUG-3' (SEQ ID NO. 4);

[0090] Sense strand: 5'-GGUCUUUUCAGGAUGAUUU-3' (SEQ ID NO. 5),

[0091] Antisense strand: 5'-AAAUCAUCCUGAAAAGACCUU-3' (SEQ ID NO. 6);

[0092] Sense strand: 5'-GUCUUUUCAGGAUGAUUUU-3' (SEQ ID NO. 7),

[0093] Antisense strand: 5'-AAAAUCAUCCUGAAAAGACCU-3' (SEQ ID NO. 8);

[0094] Sense strand: 5'-CUUUUCAGGAUGAUUUUCU-3' (SEQ ID NO. 9),

[0095] Antisense strand: 5′-AGAAAAUCAUCCUGAAAAGAC-3′ (SEQ ID NO. 10);

[0096] Sense strand: 5'-UUUUCAGGAUGAUUUUCUU-3' (SEQ ID NO. 11),

[0097] Antisense strand: 5′-AAGAAAAUCAUCCUGAAAAGA-3′ (SEQ ID NO. 12);

[0098] Sense strand: 5'-UCAGGAUGAUUUUCUUAUA-3' (SEQ ID NO. 13),

[0099] Antisense strand: 5′-UAUAAGAAAAUCAUCCUGAAA-3′ (SEQ ID NO. 14);

[0100] Sense strand: 5'-CAGGAUGAUUUUCUUAUAU-3' (SEQ ID NO. 15),

[0101] Antisense strand: 5'-AUAUAAGAAAAUCAUCCUGAA-3' (SEQ ID NO. 16);

[0102] Sense strand: 5'-AGGAUGAUUUUCUUAUAUC-3' (SEQ ID NO. 17),

[0103] Antisense strand: 5'-GAUAUAAGAAAAUCAUCCUGA-3' (SEQ ID NO. 18);

[0104] Sense strand: 5'-GAUGAUUUUCUUAUAUCAA-3' (SEQ ID NO. 19),

[0105] Antisense strand: 5′-UUGAUAUAAGAAAAUCAUCCU-3′ (SEQ ID NO. 20);

[0106] Sense strand: 5'-AUGAUUUUCUUAUAUCAAG-3' (SEQ ID NO. 21),

[0107] Antisense strand: 5′-CUUGAUAUAAGAAAAUCAUCC-3′ (SEQ ID NO. 22);

[0108] Sense strand: 5'-UGAUUUUCUUAUAUCAAGU-3' (SEQ ID NO. 23),

[0109] Antisense strand: 5′-ACUUGAUAUAAGAAAAUCAUC-3′ (SEQ ID NO. 24);

[0110] Sense strand: 5'-CUUAUAUCAAGUGGUACAU-3' (SEQ ID NO. 25),

[0111] Antisense strand: 5'-AUGUACCACUUGAUAUAAGAA-3' (SEQ ID NO. 26);

[0112] Sense strand: 5'-AAACGGAUCUCCAACUAAA-3' (SEQ ID NO. 27),

[0113] Antisense strand: 5′-UUUAGUUGGAGAUCCGUUUGA-3′ (SEQ ID NO. 28);

[0114] The sense or antisense strand is optionally modified.

[0115] In some embodiments, the present application provides a double-stranded ribonucleic acid (dsRNA), a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand is a nucleotide sequence as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, or SEQ ID NO.27, and the antisense strand is a nucleotide sequence as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, or SEQ ID NO.28:

[0116] Sense strand: 5'-GAUCUCCAACUAAAAUACU-3' (SEQ ID NO. 1),

[0117] Antisense strand: 5'-AGUAUUUUAGUUGGAGAUCCG-3' (SEQ ID NO. 2);

[0118] Sense strand: 5'-GGAUGAUUUUCUUAUAUCA-3' (SEQ ID NO. 3),

[0119] Antisense strand: 5'-UGAUAUAAGAAAAUCAUCCUG-3' (SEQ ID NO. 4);

[0120] Sense strand: 5'-GGUCUUUUCAGGAUGAUUU-3' (SEQ ID NO. 5),

[0121] Antisense strand: 5'-AAAUCAUCCUGAAAAGACCUU-3' (SEQ ID NO. 6);

[0122] Sense strand: 5'-GUCUUUUCAGGAUGAUUUU-3' (SEQ ID NO. 7),

[0123] Antisense strand: 5'-AAAAUCAUCCUGAAAAGACCU-3' (SEQ ID NO. 8);

[0124] Sense strand: 5'-CUUUUCAGGAUGAUUUUCU-3' (SEQ ID NO. 9),

[0125] Antisense strand: 5′-AGAAAAUCAUCCUGAAAAGAC-3′ (SEQ ID NO. 10);

[0126] Sense strand: 5'-UUUUCAGGAUGAUUUUCUU-3' (SEQ ID NO. 11),

[0127] Antisense strand: 5′-AAGAAAAUCAUCCUGAAAAGA-3′ (SEQ ID NO. 12);

[0128] Sense strand: 5'-UCAGGAUGAUUUUCUUAUA-3' (SEQ ID NO. 13),

[0129] Antisense strand: 5′-UAUAAGAAAAUCAUCCUGAAA-3′ (SEQ ID NO. 14);

[0130] Sense strand: 5'-CAGGAUGAUUUUCUUAUAU-3' (SEQ ID NO. 15),

[0131] Antisense strand: 5'-AUAUAAGAAAAUCAUCCUGAA-3' (SEQ ID NO. 16);

[0132] Sense strand: 5'-AGGAUGAUUUUCUUAUAUC-3' (SEQ ID NO. 17),

[0133] Antisense strand: 5'-GAUAUAAGAAAAUCAUCCUGA-3' (SEQ ID NO. 18);

[0134] Sense strand: 5'-GAUGAUUUUCUUAUAUCAA-3' (SEQ ID NO. 19),

[0135] Antisense strand: 5′-UUGAUAUAAGAAAAUCAUCCU-3′ (SEQ ID NO. 20);

[0136] Sense strand: 5'-AUGAUUUUCUUAUAUCAAG-3' (SEQ ID NO. 21),

[0137] Antisense strand: 5′-CUUGAUAUAAGAAAAUCAUCC-3′ (SEQ ID NO. 22);

[0138] Sense strand: 5'-UGAUUUUCUUAUAUCAAGU-3' (SEQ ID NO. 23),

[0139] Antisense strand: 5′-ACUUGAUAUAAGAAAAUCAUC-3′ (SEQ ID NO. 24);

[0140] Sense strand: 5'-CUUAUAUCAAGUGGUACAU-3' (SEQ ID NO. 25),

[0141] Antisense strand: 5'-AUGUACCACUUGAUAUAAGAA-3' (SEQ ID NO. 26);

[0142] Sense strand: 5'-AAACGGAUCUCCAACUAAA-3' (SEQ ID NO. 27),

[0143] Antisense strand: 5′-UUUAGUUGGAGAUCCGUUUGA-3′ (SEQ ID NO. 28);

[0144] The sense or antisense strand is optionally modified.

[0145] In some embodiments, the present application provides a double-stranded ribonucleic acid (dsRNA), a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.1 and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.2 and the length of the antisense strand does not exceed 23 nucleotides; the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.3 and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.4 and the length of the antisense strand does not exceed 23 nucleotides; the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.5 and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.6 and the length of the antisense strand does not exceed 23 nucleotides; the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. NO.7, wherein the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.7 and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.8 and the length of the antisense strand does not exceed 23 nucleotides; the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.9 and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.10 and the length of the antisense strand does not exceed 23 nucleotides; the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.11 and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.12 and the length of the antisense strand does not exceed 23 nucleotides; the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.13 and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO. NO.14, wherein the antisense strand contains at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.14 and the length of the antisense strand does not exceed 23 nucleotides; the sense strand contains at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.15 and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.16 and the length of the antisense strand does not exceed 23 nucleotides; the sense strand contains the nucleotide sequence shown in SEQ ID NO.17 and the sense strand is no longer than 21 nucleotides, the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.18 and the antisense strand is no longer than 23 nucleotides; the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.19 and the sense strand is no longer than 21 nucleotides, the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.20 and the antisense strand is no longer than 23 nucleotides; the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.21 and the sense strand is no longer than 21 nucleotides, the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.22 and the antisense strand is no longer than 23 nucleotides; the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.23 and the sense strand is no longer than 21 nucleotides, the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO. The sense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence shown in SEQ ID NO. 24, and the antisense strand is no longer than 23 nucleotides; the sense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence shown in SEQ ID NO. 25, and the sense strand is no longer than 21 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence shown in SEQ ID NO. 26, and the antisense strand is no longer than 23 nucleotides; or the sense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence shown in SEQ ID NO. 27, and the sense strand is no longer than 21 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence shown in SEQ ID NO. 28, and the antisense strand is no longer than 23 nucleotides, and the sense strand or antisense strand is optionally modified.

[0146] In some embodiments, the present application provides a double-stranded ribonucleic acid (dsRNA), a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence as shown in SEQ ID NO.1 and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO.2 and the length of the antisense strand does not exceed 23 nucleotides; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO.3 and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO.4 and the length of the antisense strand does not exceed 23 nucleotides; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO.5 and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO.6 and the length of the antisense strand does not exceed 23 nucleotides; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO.7 and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO.8 and the length of the antisense strand does not exceed 23 nucleotides; the sense strand comprises a nucleotide sequence as shown in SEQ ID NO. NO.9, and the sense strand is no longer than 21 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.10, and the antisense strand is no longer than 23 nucleotides; the sense strand contains the nucleotide sequence shown in SEQ ID NO.11, and the sense strand is no longer than 21 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.12, and the antisense strand is no longer than 23 nucleotides; the sense strand contains the nucleotide sequence shown in SEQ ID NO.13, and the sense strand is no longer than 21 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.14, and the antisense strand is no longer than 23 nucleotides; the sense strand contains the nucleotide sequence shown in SEQ ID NO.15, and the sense strand is no longer than 21 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.16, and the antisense strand is no longer than 23 nucleotides; the sense strand contains the nucleotide sequence shown in SEQ ID NO.17, and the sense strand is no longer than 21 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. NO.18 and the antisense strand is no longer than 23 nucleotides; the sense strand contains the nucleotide sequence shown in SEQ ID NO.19 and the sense strand is no longer than 21 nucleotides, the antisense strand contains the nucleotide sequence shown in SEQ ID NO.20 and the antisense strand is no longer than 23 nucleotides; the sense strand contains the nucleotide sequence shown in SEQ ID NO.The antisense strand contains the nucleotide sequence shown in SEQ ID NO. 21 and the sense strand is no longer than 21 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 22 and the antisense strand is no longer than 23 nucleotides; the sense strand contains the nucleotide sequence shown in SEQ ID NO. 23 and the sense strand is no longer than 21 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 24 and the antisense strand is no longer than 23 nucleotides; the sense strand contains the nucleotide sequence shown in SEQ ID NO. 25 and the sense strand is no longer than 21 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 26 and the antisense strand is no longer than 23 nucleotides; or the sense strand contains the nucleotide sequence shown in SEQ ID NO. 27 and the sense strand is no longer than 21 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. 28 and the antisense strand is no longer than 23 nucleotides, and the sense strand or antisense strand is optionally modified.

[0147] In some embodiments, the present application provides a double-stranded ribonucleic acid (dsRNA), a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand is the nucleotide sequence shown in SEQ ID NO.1, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.2; the sense strand is the nucleotide sequence shown in SEQ ID NO.3, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.4; the sense strand is the nucleotide sequence shown in SEQ ID NO.5, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.6; the sense strand is the nucleotide sequence shown in SEQ ID NO.7, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.8; the sense strand is the nucleotide sequence shown in SEQ ID NO.9, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.10; the sense strand is the nucleotide sequence shown in SEQ ID NO.11, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.12; the sense strand is the nucleotide sequence shown in SEQ ID NO.13, and the antisense strand is the nucleotide sequence shown in SEQ ID NO. NO.14; the sense strand is the nucleotide sequence shown in SEQ ID NO.15, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.16; the sense strand is the nucleotide sequence shown in SEQ ID NO.17, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.18; the sense strand is the nucleotide sequence shown in SEQ ID NO.19, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.20; the sense strand is the nucleotide sequence shown in SEQ ID NO.21, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.22; the sense strand is the nucleotide sequence shown in SEQ ID NO.23, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.24; the sense strand is the nucleotide sequence shown in SEQ ID NO.25, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.26; or, the sense strand is the nucleotide sequence shown in SEQ ID NO.27, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.28, and the sense strand or antisense strand is optionally modified.

[0148] The present application also provides a dsRNA, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the dsRNA comprises a sense strand and an antisense strand, and the sense strand and the antisense strand have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the full length of the nucleotide sequence of the sense strand and the antisense strand, respectively.

[0149] In some embodiments, the dsRNA, pharmaceutically acceptable salt thereof, or ligand conjugate thereof of the present application is an RNAi drug.

[0150] In some embodiments, the dsRNA, its pharmaceutically acceptable salt, or its ligand conjugate of the present application is siRNA or its ligand conjugate, or its pharmaceutically acceptable salt.

[0151] In a specific embodiment, the dsRNA, its pharmaceutically acceptable salt or its ligand conjugate has a double-stranded region consisting of the sense strand and the antisense strand complementarily, and optionally an overhang located at the 5' and / or 3' end of the sense strand and / or antisense strand. In a preferred embodiment, the dsRNA, its pharmaceutically acceptable salt or its ligand conjugate has a double-stranded region consisting of the sense strand and the antisense strand complementarily, and an overhang located at the 5' and / or 3' end of the sense strand and / or antisense strand. In a preferred embodiment, the dsRNA, its pharmaceutically acceptable salt or its ligand conjugate has a double-stranded region consisting of the sense strand and the antisense strand complementarily, and an overhang located at the 3' end of the antisense strand. In a preferred embodiment, the dsRNA, its pharmaceutically acceptable salt or its ligand conjugate has a double-stranded region consisting of the sense strand and the antisense strand complementarily, and an overhang located at the 3' end of the sense strand.

[0152] In some embodiments, the sense strand contains a nucleic acid sequence that differs from the nucleotide sequence in the nucleotide sequence shown in Table 1 by 0, 1, 2 or 3 nucleotides, and the length of the sense strand does not exceed 21 nucleotides; the antisense strand contains a nucleic acid sequence that differs from the nucleotide sequence in the nucleotide sequence shown in Table 2 by 0, 1, 2 or 3 nucleotides, and the length of the antisense strand does not exceed 23 nucleotides.

[0153] In some embodiments, the positive strand is selected from a nucleic acid sequence that differs from the nucleotide sequence in the nucleotide sequence shown in Table 1 by 0, 1, 2 or 3 nucleotides; the antisense strand is selected from a nucleotide sequence that differs from the nucleotide sequence in the nucleotide sequence shown in Table 2 by 0, 1, 2 or 3 nucleotides.

[0154] In some embodiments, the present application provides a double-stranded ribonucleic acid (dsRNA)-ligand conjugate, which comprises the double-stranded ribonucleic acid (dsRNA) described herein, a pharmaceutically acceptable salt thereof, and a ligand.

[0155] Double-stranded region

[0156] In some embodiments, the double-stranded region of the dsRNA has a length of 15-23 nucleotide pairs, for example, the double-stranded region of the dsRNA has a length of 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide pairs.

[0157] In some embodiments, the double-stranded region of the dsRNA has a length of 19-23 nucleotide pairs, for example, the double-stranded region of the dsRNA has a length of 19, 20, 21, 22 or 23 nucleotide pairs.

[0158] protrusion

[0159] In some embodiments, the sense strand or antisense strand optionally includes an overhang at the 5' end and / or the 3' end.

[0160] In some embodiments, the overhang contains 1, 2, 3, 4, or 5 nucleotides. In some embodiments, the overhang contains 1 or 2 nucleotides.

[0161] In some embodiments, the sense strand optionally includes an overhang at the 5' end and / or the 3' end. In some embodiments, the sense strand optionally includes an overhang of 1, 2, 3, 4, or 5 nucleotides at the 5' end and / or the 3' end. In some embodiments, the sense strand optionally includes an overhang of 1 or 2 nucleotides at the 5' end and / or the 3' end.

[0162] In some embodiments, the antisense strand optionally includes an overhang at the 5' end and / or the 3' end. In some embodiments, the antisense strand optionally includes an overhang of 1, 2, 3, 4, or 5 nucleotides at the 5' end and / or the 3' end. In some embodiments, the antisense strand optionally includes an overhang of 1 or 2 nucleotides at the 5' end and / or the 3' end.

[0163] In some embodiments, the overhang is selected from unmodified or modified A, G, C, U, or T.

[0164] In a specific embodiment, the overhang may be 1, 2, 3, 4 or 5 nucleotides at the 5' end and / or 3' end of the sense strand or antisense strand, preferably 1 or 2 nucleotides. In a specific embodiment, the dsRNA, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof comprises an additional sequence as an overhang, and the additional sequence may comprise 1, 2, 3, 4 or 5 nucleotides, preferably 1 or 2 nucleotides, attached to the 5' end and / or 3' end of the sense strand or antisense strand.

[0165] As an example, in the case where the ribonucleotides at the 5' and / or 3' ends of the sense strand or antisense strand serve as overhangs, when the sense strand is 21 nucleotides and the antisense strand is 23 nucleotides, when positions 1-21 of the sense strand are complementary to positions 1-21 of the antisense strand (in the direction from the 5' end to the 3' end), then nucleotides 22-23 of the antisense strand constitute an overhang located at the 3' end of the antisense strand, that is, the 3' end of the sense strand is blunt or blunt; when the sense strand is 21 nucleotides and the antisense strand is 21 nucleotides, when positions 1-21 of the sense strand are complementary to positions 1-21 of the antisense strand (in the direction from the 5' end to the 3' end), then nucleotides 22-23 of the antisense strand constitute an overhang located at the 3' end of the antisense strand, that is, the 3' end of the sense strand is blunt or blunt; -20 positions are complementary to positions 1-20 of the antisense strand (in the direction from 5' end to 3' end), then the 21st nucleotides of the sense strand and the antisense strand respectively constitute an overhang located at the 3' end of the sense strand and the antisense strand; when the sense strand is 21 nucleotides and the antisense strand is 23 nucleotides, when positions 1-21 of the sense strand are complementary to positions 3-23 of the antisense strand (in the direction from 5' end to 3' end), then nucleotides 1-2 of the antisense strand constitute an overhang located at the 5' end of the antisense strand, that is, the 5' end of the sense strand is blunt or blunt.

[0166] In some embodiments, when the nucleotides at the 5' and / or 3' ends of the sense strand or antisense strand serve as overhangs, the antisense strand optionally includes an overhang at the 5' and / or 3' ends, wherein the overhang is selected from an unmodified or modified ribonucleotide sequence of CG, AA, GA, CC, CA, AC, GC, AA, UG, GG, CU, UU, or UC. In some embodiments, the antisense strand optionally includes an overhang at the 3' end, wherein the overhang is selected from an unmodified or modified ribonucleotide sequence of CG, AA, GA, CC, CA, AC, GC, AA, UG, GG, CU, UU, or UC.

[0167] In a specific embodiment, the dsRNA, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof comprises an additional sequence at the 5' end and / or 3' end of the antisense strand or sense strand as an overhang or part of an overhang, and the additional sequence may comprise 1, 2, 3, 4, or 5 nucleosides, preferably 1 or 2 nucleotides, at the 5' end and / or 3' end. In some embodiments, the overhang consists of the nucleotides at the 5' end and / or 3' end of the antisense strand or sense strand and the additional sequence, or the overhang may consist solely of the additional sequence.

[0168] In some embodiments, the overhang is an additional sequence optionally located at the 5' and / or 3' end of the antisense strand, and the overhang is selected from unmodified or modified CG, AA, GA, CC, CA, AC, GC, AA, UG, GG, CU, UU, or UC. In some embodiments, the overhang is an additional sequence located at the 3' end of the antisense strand, and the overhang is selected from unmodified or modified CG, AA, GA, CC, CA, AC, GC, AA, UG, GG, CU, UU, or UC.

[0169] In some embodiments, the additional sequence in the overhang can be selected from unmodified or modified U or T, for example, can be selected from uu or dTdT.

[0170] In some embodiments, when the overhang is one nucleotide, the additional sequence serving as the overhang can be selected from unmodified or modified U or T. In some embodiments, when the overhang is one nucleotide, the additional sequence serving as the overhang can be selected from 2'-O-methyl-modified U (hereinafter also referred to as u) or deoxythymidine nucleotide (hereinafter also referred to as dT).

[0171] In some embodiments, when the overhang is 2 nucleotides, the additional sequence as the overhang can be selected from unmodified or modified UU or TT. In some embodiments, when the overhang is 2 nucleotides, the additional sequence as the overhang can be selected from uu or dTdT.

[0172] In some embodiments, the sense strand optionally includes an overhang at the 5' end and / or the 3' end, wherein the overhang is selected from uu or dTdT.

[0173] In some embodiments, the antisense strand optionally includes an overhang at the 5' end and / or the 3' end, wherein the overhang is selected from uu or dTdT.

[0174] In some embodiments, the overhang is linked to the adjacent nucleotide via a phosphate group or a phosphorothioate group.

[0175] In some embodiments, one or more nucleotides in the overhang are linked by phosphate or phosphorothioate groups.

[0176] Modification

[0177] In some embodiments, the sense strand or the antisense strand is optionally modified.

[0178] In some embodiments, one or more nucleotides of the sense strand or antisense strand are modified.

[0179] In some embodiments, each nucleotide can be modified with the same or different modifications, which can include, but are not limited to, one or more changes in one or both of the non-linked phosphate oxygens and / or one or more of the linked phosphate oxygens; changes in the composition of the ribose sugar (e.g., the 2' hydroxyl group on the ribose sugar); complete replacement of the phosphate moiety with a "dephospho" linker; modification of the bases; and modification of the ribose-phosphate backbone.

[0180] In some embodiments, before each nucleotide is modified, the base is selected from naturally occurring bases, such as A, U, C, G and T. When the modification occurs on the nucleotide base, it refers to chemical modification based on the backbone of A, U, C, G and T.

[0181] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides of the sense strand are modified; or a range of nucleotides within any of the foregoing values ​​is modified, e.g., 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, or 1-10 nucleotides are modified.

[0182] In some embodiments, more than one, more than two, more than three, more than four, more than five, more than six, more than seven, more than eight, more than nine, more than ten, more than eleven, more than twelve, more than thirteen, more than fourteen, more than fifteen, more than sixteen, more than seventeen, more than eighteen, more than nineteen, more than twenty, or more than twenty-one nucleotides of the sense strand are modified; typically, more than ten, more than eleven, more than twelve, more than thirteen, more than fourteen, more than fifteen, more than sixteen, more than seventeen, more than eighteen, more than nineteen, more than twenty, or more than twenty-one nucleotides of the sense strand are modified.

[0183] In some embodiments, all nucleotides in the sense strand are modified.

[0184] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides of the antisense strand are modified; or a range of nucleotides within any of the foregoing values ​​is modified, e.g., 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, or 1-10 nucleotides are modified.

[0185] In some embodiments, more than one, more than two, more than three, more than four, more than five, more than six, more than seven, more than eight, more than nine, more than ten, more than eleven, more than twelve, more than thirteen, more than fourteen, more than fifteen, more than sixteen, more than seventeen, more than eighteen, more than nineteen, more than twenty-one, more than twenty-two, or more than twenty-three nucleotides of the antisense strand are modified; typically, more than ten, more than eleven, more than twelve, more than thirteen, more than fourteen, more than fifteen, more than sixteen, more than seventeen, more than eighteen, more than nineteen, more than twenty-one, more than twenty-two, or more than twenty-three nucleotides of the antisense strand are modified.

[0186] In some embodiments, all nucleotides in the antisense strand are modified.

[0187] In some embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified.

[0188] In some embodiments, the modification is selected from the group consisting of nucleotide sugar modification, base modification, internucleotide linkage modification, and terminal modification. In some embodiments, the modification is selected from the group consisting of nucleotide sugar modification, internucleotide linkage modification, and terminal modification.

[0189] In some embodiments, the sugar modification of the nucleotide is selected from dehydroxylation, fluorination, amination, alkylation, hydroxyalkylation or hydroxyalkenylation.

[0190] In some embodiments, the sugar modification of the nucleotide occurs at the 2' position of the sugar group.

[0191] In some embodiments, the glycosyl modification is selected from 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-alkyl, 2'-O-alkyl, 2'-O-ether, 2'-O-alkenyl. In some embodiments, the glycosyl modification is selected from 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-methyl, 2'-ethyl, 2'-methyl-O-methyl, 2'-ethyl-O-methyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-ethyl-O-methyl, or 2'-O-allyl.

[0192] In some embodiments, the internucleotide linkage modification is selected from phosphorothioate (PS), phosphorodithioate (PS2), methylphosphonate (MP), methoxypropylphosphonate (MOP) or aminophosphonate. In some embodiments, the internucleotide linkage modification is selected from phosphorothioate (PS). In some embodiments, the nucleotides are connected by phosphorothioate diester bonds.

[0193] In some embodiments, the terminal modification is selected from a modification on a phosphate group or a hydroxyl group at the terminal. In some embodiments, the terminal modification is selected from a 5'-terminal modification or a 3'-terminal modification. In some embodiments, the terminal modification is selected from a 5'-phosphate, a 5'-methylphosphonate (5'-MP), a 5'-thiophosphate (5'-PS) or a 5'-(E)-vinylphosphonate (5'-(E)-VP) on the ribose group of the terminal nucleotide. In some embodiments, the terminal modification is selected from a 5'-phosphate or a 5'-(E)-vinylphosphonate on the ribose group of the terminal nucleotide.

[0194] In some embodiments, the modification is selected from 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-methyl, 2'-ethyl, 2'-methyl-O-methyl, 2'-ethyl-O-methyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-allyl, phosphorothioate, methylphosphonate, aminophosphonate, 5'-phosphate, 3'-phosphate, 5'-(E)-vinyl phosphate, or 3'-(E)-vinyl phosphate.

[0195] In some embodiments, the modification is selected from 2'-dehydroxylation, 2'-fluoro, 2'-O-methyl, 2'-O-ethyl, 2'-O-ethyl-O-methyl, 2'-O-allyl, phosphorothioate, or 5'-(E)-vinyl phosphate.

[0196] In some embodiments, the modification is selected from 2'-fluoro, 2'-O-methyl, phosphorothioate, or 5'-(E)-vinyl phosphate.

[0197] As will be described in detail below, when referring to the nth nucleotide of the sense strand and the antisense strand, counting starts from the 5' end unless otherwise specified.

[0198] In some embodiments, phosphorothioate modification is used between the first two nucleotides and / or between the first three nucleotides at either or both ends of the two nucleic acid strands of the dsRNA, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof. For example, taking the sense strand as an example, phosphorothioate modification is used between nucleotides 1 and 2 and / or between nucleotides 2 and 3 of the sense strand.

[0199] In some embodiments, when there is no additional sequence as an overhang, the space between the 1st and 2nd nucleotides and / or the space between the 2nd and 3rd nucleotides of the sense strand is modified with a phosphorothioate group.

[0200] In some embodiments, when there is no additional sequence as an overhang, the antisense strand is modified between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 1 and 2 at the 3' end of the antisense strand, and / or between nucleotides 2 and 3 at the 3' end of the antisense strand. In some embodiments, when there is no additional sequence as an overhang, the antisense strand is modified between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 19 and 20, and / or between nucleotides 20 and 21.

[0201] In some embodiments, when there is an additional sequence as an overhang, the phosphorothioate modification may be located between nucleotides in the additional sequence.

[0202] In some embodiments, the 5'-terminal nucleotide of the antisense strand has a 5'-(E)-vinyl phosphate modification. In some embodiments, when there is an additional sequence as an overhang, the 5'-terminal modification may be present on the nucleotide of the additional sequence.

[0203] In some embodiments, the sense strand has 2'-fluoro modifications at 2, 3, 4, or 5 nucleotides. In some embodiments, the sense strand has 14, 15, 16, or 17 nucleotides with 2'-O-methyl modifications. In some embodiments, the sense strand has 3 or 4 nucleotides with 2'-fluoro modifications and 15, 16, 17, or 18 nucleotides with 2'-O-methyl modifications.

[0204] In some embodiments, 0, 1, or 2 nucleotides from position 1 to 6 of the sense strand are modified with 2'-fluoro.

[0205] In some embodiments, 4, 5, or 6 nucleotides from positions 1 to 6 of the sense strand are modified with 2'-O-methyl.

[0206] In some embodiments, two or three nucleotides from position 7 to 9 of the sense strand are modified with 2'-fluoro.

[0207] In some embodiments, 0 or 1 nucleotides from position 7 to 9 of the sense strand are modified with 2'-O-methyl.

[0208] In some embodiments, 0 or 1 nucleotides from position 10 to 19 of the sense strand are 2'-fluoro-modified.

[0209] In some embodiments, 8, 9, or 10 nucleotides from positions 10 to 19 of the sense strand are modified with 2'-O-methyl.

[0210] In some embodiments, the 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro-modified, and 0, 1 or 2 nucleotides in nucleotides 1 to 6 and / or 10 to 19 of the sense strand are also 2'-fluoro-modified. In some embodiments, the 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro-modified, and 0, 1 or 2 nucleotides in nucleotides 1 to 6 and 10 to 19 of the sense strand are also 2'-fluoro-modified. In some embodiments, the 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro-modified, and 0 or 1 nucleotide in nucleotides 1 to 6 and 10 to 19 of the sense strand are also 2'-fluoro-modified.

[0211] In some embodiments, the antisense strand has 2, 3, 4, 5, 6, or 7 nucleotides modified with 2'-fluoro.

[0212] In some embodiments, the antisense strand has 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides modified with 2'-O-methyl.

[0213] In some embodiments, three, four, five, or six nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-fluoro-modified. In some embodiments, four nucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-fluoro-modified. Six nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-fluoro-modified.

[0214] In some embodiments, nucleotides 2, 6, 14, and 16 of the antisense strand are 2'-fluoro-modified, and any one or two of nucleotides 1, 3 to 5, 7 to 13, 15, and / or 17 to 21 of the antisense strand are also 2'-fluoro-modified. In some embodiments, nucleotides 2, 6, 14, and 16 of the antisense strand are 2'-fluoro-modified, and any one or two of nucleotides 1, 3 to 5, 7 to 13, 15, and 17 to 21 of the antisense strand are also 2'-fluoro-modified.

[0215] In some embodiments, 14, 15, 16, 17, 18 or 19 nucleotides among nucleotides 1, 3 to 5, 7 to 13, 15 and / or 17 to 21 of the antisense strand are modified with 2'-O-methyl.

[0216] In a specific embodiment, the 5th, 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro-modified, and the other ribonucleotides of the sense strand are 2'-O-methyl-modified, and the 2nd, 6th, 8th, 9th, 14th and 16th nucleotides of the antisense strand are 2'-fluoro-modified, and the other ribonucleotides of the antisense strand are 2'-O-methyl-modified.

[0217] In a specific embodiment, the 5th, 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro-modified, and the other ribonucleotides of the sense strand are 2'-O-methyl-modified, and the 2nd, 6th, 14th and 16th nucleotides of the antisense strand are 2'-fluoro-modified, and the other ribonucleotides of the antisense strand are 2'-O-methyl-modified.

[0218] In a specific embodiment, nucleotides 7, 8 and 9 of the sense strand are 2'-fluoro-modified, and the other ribonucleotides of the sense strand are 2'-O-methyl-modified, and nucleotides 2, 6, 14 and 16 of the antisense strand are 2'-fluoro-modified, and the other ribonucleotides of the antisense strand are 2'-O-methyl-modified.

[0219] In a specific embodiment, the 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro-modified, the other ribonucleotides of the sense strand are 2'-O-methyl-modified, and the 2nd, 6th, 8th, 9th, 14th and 16th nucleotides of the antisense strand are 2'-fluoro-modified, and the other ribonucleotides of the antisense strand are 2'-O-methyl-modified. In a specific embodiment, the 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro-modified, the other ribonucleotides of the sense strand are 2'-O-methyl-modified, and the 2nd, 6th, 14th and 16th nucleotides of the antisense strand are 2'-fluoro-modified, and the other ribonucleotides of the antisense strand are 2'-O-methyl-modified. In a specific embodiment, the 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro-modified, the other ribonucleotides of the sense strand are 2'-O-methyl-modified, and the antisense strand includes 2'-fluoro (e.g., position 14, etc.) and 2'-O-methyl modifications, optionally further including one or more other types of modifications.

[0220] In a specific embodiment, the 5th, 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro modified, the other ribonucleotides of the sense strand are 2'-O-methyl modified, and the 2nd, 6th, 8th, 9th, 14th and 16th nucleotides of the antisense strand are 2'-fluoro modified, the other ribonucleotides of the antisense strand are 2'-O-methyl modified, and between the 1st and 2nd nucleotides of the sense strand, between the 2nd and 3rd nucleotides of the sense strand, between the 1st and 2nd nucleotides of the antisense strand, between the 2nd and 3rd nucleotides of the antisense strand, between the 19th and 20th nucleotides of the antisense strand and / or between the 20th and 21st nucleotides of the antisense strand are phosphorothioate modified. Optionally, the 5'-terminus of the antisense strand is modified with 5'-(E)-vinyl phosphate, for example, the 5'-terminal ribonucleotide of the antisense strand has a 5'-(E)-vinyl phosphate modification.

[0221] In a specific embodiment, the 5th, 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro modified, the other ribonucleotides of the sense strand are 2'-O-methyl modified, and the 2nd, 6th, 14th and 16th nucleotides of the antisense strand are 2'-fluoro modified, the other ribonucleotides of the antisense strand are 2'-O-methyl modified, and between the 1st and 2nd nucleotides of the sense strand, between the 2nd and 3rd nucleotides of the sense strand, between the 1st and 2nd nucleotides of the antisense strand, between the 2nd and 3rd nucleotides of the antisense strand, between the 19th and 20th nucleotides of the antisense strand and / or between the 20th and 21st nucleotides of the antisense strand are phosphorothioate modified. Optionally, the 5'-terminus of the antisense strand is modified with 5'-(E)-vinyl phosphate, for example, the 5'-terminal ribonucleotide of the antisense strand has a 5'-(E)-vinyl phosphate modification.

[0222] In a specific embodiment, the 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro modified, the other ribonucleotides of the sense strand are 2'-O-methyl modified, the 2nd, 6th, 14th and 16th nucleotides of the antisense strand are 2'-fluoro modified, the other ribonucleotides of the antisense strand are 2'-O-methyl modified, and the 1st and 2nd nucleotides of the sense strand, the 2nd and 3rd nucleotides of the sense strand, the 1st and 2nd nucleotides of the antisense strand, the 2nd and 3rd nucleotides of the antisense strand, the 19th and 20th nucleotides of the antisense strand and / or the 20th and 21st nucleotides of the antisense strand are phosphorothioate modified. Optionally, the 5'-terminus of the antisense strand is modified with 5'-(E)-vinyl phosphate, for example, the 5'-terminal ribonucleotide of the antisense strand has a 5'-(E)-vinyl phosphate modification.

[0223] In a specific embodiment, the 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro modified, the other ribonucleotides of the sense strand are 2'-O-methyl modified, the 2nd, 6th, 8th, 9th, 14th and 16th nucleotides of the antisense strand are 2'-fluoro modified, the other ribonucleotides of the antisense strand are 2'-O-methyl modified, and between the 1st and 2nd nucleotides of the sense strand, between the 2nd and 3rd nucleotides of the sense strand, between the 1st and 2nd nucleotides of the antisense strand, between the 2nd and 3rd nucleotides of the antisense strand, between the 19th and 20th nucleotides of the antisense strand and / or between the 20th and 21st nucleotides of the antisense strand are phosphorothioate modified. Optionally, the 5'-terminus of the antisense strand is modified with 5'-(E)-vinyl phosphate, for example, the 5'-terminal ribonucleotide of the antisense strand has a 5'-(E)-vinyl phosphate modification.

[0224] ligand

[0225] In some embodiments, the ligand includes a branching group and a linking group. In some embodiments, the branching group may include a targeting group.

[0226] In some embodiments, the ligand is linked to the dsRNA via a linker. Herein, the dsRNA linked to the ligand is referred to as a dsRNA ligand conjugate.

[0227] In some embodiments, the ligand is connected to one or more targeting groups via a branching group. In some embodiments, the branching group comprises a targeting group.

[0228] In some embodiments, the ligand contains at least one targeting group. In some embodiments, the ligand contains one, two, three, four, or five targeting groups. In some embodiments, the ligand contains two, three, or four targeting groups. In some embodiments, the ligand contains three targeting groups.

[0229] In some embodiments, the targeting group is selected from a GalNAc group.

[0230] In some embodiments, the ligand contains one, two, three, four, or five GalNAc groups. In some embodiments, the ligand contains two, three, or four GalNAc groups. In some embodiments, the ligand contains three GalNAc groups.

[0231] In some embodiments, the branching group is selected from:

[0232] In some embodiments, the linking group is selected from:

[0233] In some embodiments, the ligand is selected from:

[0234] In some embodiments, the ligand is attached to the sense strand or the antisense strand. In some embodiments, the ligand is attached to the 5' or 3' end of the sense strand or the antisense strand. In some embodiments, the ligand is attached to the 5' or 3' end of the sense strand. In some embodiments, the ligand is attached to the 3' end of the sense strand.

[0235] In some embodiments, the part is connected with the sense strand or the antisense strand of dsRNA through a phosphate group or a thiophosphate group. In some embodiments, the part is connected with the sense strand through a phosphate group or a thiophosphate group. In some embodiments, the part is connected with the 3 ' end of the sense strand through a phosphate group or a thiophosphate group.

[0236] In some embodiments, the dsRNA ligand conjugate is as follows:

[0237] In some embodiments, the dsRNA ligand conjugate is selected from:

[0238] In some embodiments, the dsRNA ligand conjugate is selected from:

[0239] In some embodiments, X is OH or O - .

[0240] In some embodiments, the unmodified sense strand of the dsRNA is selected from:

[0241] Table 1. Unmodified sense strand

[0242] In some embodiments, the unmodified antisense strand of the dsRNA is selected from:

[0243] Table 2. Unmodified antisense strand

[0244] In some embodiments, the modified sense strand of the dsRNA is selected from:

[0245] Table 3. Modified sense strands

[0246] In some embodiments, the modified antisense strand of the dsRNA is selected from:

[0247] Table 4. Modified antisense strands

[0248] The dsRNA of the present application can be formed by including any one of the above-mentioned sense strands and any one of the above-mentioned antisense strands.

[0249] For example, the dsRNA comprises the sense strand of any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 and

[0250] The dsRNA includes any one of the following antisense strands: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84.

[0251] In some embodiments, the dsRNA (unmodified) is selected from:

[0252] Table 5. dsRNA (unmodified)

[0253] In some embodiments, the dsRNA (modified) is selected from:

[0254] Table 6. dsRNA (modified)

[0255] In some embodiments, the dsRNA ligand conjugate (siRNA ligand conjugate) is selected from:

[0256] Table 7. dsRNA ligand conjugates

[0257] Wherein, L represents a ligand, and the ligand is as described above. In some embodiments, the ligand is selected from L01 ligand or L02 ligand. The sequence ID number of the sense strand refers to the sequence ID number of the nucleic acid sequence in the sense strand. The L at the 3' end of the sense strand sequence in the dsRNA ligand conjugate serial number indicates that a ligand is attached to the 3' end of the sense strand sequence in the dsRNA ligand conjugate.

[0258] Pharmaceutically acceptable salts

[0259] In some embodiments, the salt described above is selected from base addition salts, acid addition salts, and combinations thereof.

[0260] In some embodiments, the base addition salt is selected from sodium, potassium, calcium, ammonium, organic amine, magnesium salts and combinations thereof, and the acid addition salt is selected from inorganic acid salts, organic acid salts and combinations thereof.

[0261] In some embodiments, the inorganic acid is selected from hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and combinations thereof, and the organic acid is selected from acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and combinations thereof.

[0262] In some embodiments, the pharmaceutically acceptable salt is selected from sodium salt. Beneficial effects

[0263] The double-stranded RNA, its pharmaceutically acceptable salt, or its ligand conjugate of the present application has good inhibitory activity against coagulation factor XI. The double-stranded RNA, its pharmaceutically acceptable salt, or its ligand conjugate of the present application has good stability and can continue to function in vivo for a long time. The double-stranded RNA, its pharmaceutically acceptable salt, or its ligand conjugate of the present application has low off-target effects, minimal toxic side effects, and high safety. Therefore, it has good prospects for drug development.

[0264] Definition and Description

[0265] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. The absence of a specific definition for a particular term or phrase should not be construed as undefined or ambiguous, but rather should be understood as meaning understood by one of ordinary skill in the art. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0266] In this application, unless otherwise stated, the terms "comprise, include and contain" or equivalents are open-ended expressions, which mean that in addition to the listed elements, components or steps, other unspecified elements, components or steps may also be included.

[0267] When any variable occurs more than once in a compound, nucleotide, or single-stranded or double-stranded structure, its definition in each instance is independent. For example, modifications described herein occur independently, meaning that, unless otherwise indicated, modifications to the sense strand do not affect modifications to the antisense strand, modifications to one nucleotide do not affect modifications to another nucleotide, and modifications to the sugar group of one nucleotide do not affect modifications to another sugar group of the same nucleotide. Such effects include whether a modification occurs and what type of modification is used.

[0268] The coagulation factor XI gene sequence of the present application can be obtained from a publicly available database. siRNAs were designed using the human coagulation factor XI gene transcript NM_000128.4 and the cynomolgus monkey coagulation factor XI gene transcript XM_005556483.2 as target genes. All siRNA sequences had 100% identity or 1-3 mismatches with the human transcript, and 100% identity or 1 mismatch with the cynomolgus monkey transcript.

[0269] As known in the art, the term "interfering RNA" or "RNAi" or "interfering RNA sequence" refers to a single-stranded RNA (e.g., a mature miRNA) or double-stranded RNA (e.g., a duplex RNA such as siRNA, aiRNA, or pre-miRNA) that can reduce or inhibit the expression of a target gene or sequence when the interfering RNA and the target gene or sequence are in the same cell (e.g., by mediating the degradation of an mRNA complementary to the interfering RNA sequence or inhibiting the translation or transcription of an mRNA complementary to the interfering RNA sequence). The interfering RNA can have substantial or complete identity to the target gene or sequence, or can include mismatch regions (i.e., mismatch sequences).

[0270] The double-stranded RNA of the present application functions as an interfering RNA. Hereinafter, the double-stranded RNA of the present application may also be referred to as siRNA.

[0271] As known in the art, the term "mismatch region" or "mismatch sequence" refers to a portion of an interfering RNA (e.g., siRNA, aiRNA, miRNA) sequence that does not have 100% complementarity with its target sequence. An interfering RNA (e.g., siRNA, aiRNA, miRNA) can have at least 1, 2, 3, 4, 5, 6 or more mismatch regions. Mismatch regions can be continuous or can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides. Mismatch regions can include a single nucleotide or can include 2, 3, 4, 5, 6 or more nucleotides.

[0272] The term "identity" refers to the similarity between two nucleotide sequences or between two amino acid sequences. The identity of a sequence preferably relates to the percentage of nucleotides or amino acids in the same position in two or more sequences of the same length. Specifically, the "% identity" of two amino acid sequences or two nucleotide sequences can be determined as follows: compare sequences for the purpose of optimal comparison (for example, a room can be introduced in either sequence to optimally compare with another sequence) and compare the amino acids or nucleotides in the corresponding positions. Rooms are generally considered to be non-identical positions, regardless of their actual position in the comparison. "Optimal comparison" is generally the comparison of the two sequences that results in the highest percentage identity. Percent identity is determined by the number of identical nucleotides in the compared sequences (that is, % identity = number of identical positions / total number of positions × 100). The application's sequence identity is at least 80%, 85%, 90% or 95%, preferably at least 90%, and non-limiting examples include: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. Mathematical algorithm well known to those skilled in the art can be utilized to complete the determination of identity percentage between two sequences.

[0273] As known in the art, interfering RNA includes "small interfering RNA (siRNA)", which has a length of, for example, about 15-60, 15-50, 15-40, 15-30, 15-25, 19-25, 19-23, or 19-21 nucleotides. The double-stranded RNA of the present application can function as such siRNA and can have a corresponding length.

[0274] As used herein, the nucleotide positions of the sense strand or antisense strand are counted from the 5' end. For a dsRNA with a sense strand of 19 nucleotides and an antisense strand of 21 nucleotides, for example, position 1 of 5'-GAUCUCCAACUAAAAUACU-3' (SEQ ID NO: 1) is G and position 19 is U; and position 1 of 5'-AGUAUUUUAGUUGGAGAUCCG-3' (SEQ ID NO: 2) is A and position 21 is G. For a dsRNA with a sense strand of more than 19 nucleotides and an antisense strand of more than 21 nucleotides, for example, if the sense strand has several nucleotides added to the terminal based on SEQ ID NO: 1 and the antisense strand has several nucleotides added to the terminal based on SEQ ID NO: 2, the counting method of the nucleotide positions of the sense strand or antisense strand will not change accordingly, that is, position 1 of the sense strand will still be position 1 of SEQ ID NO: 1, and position 1 of the antisense strand will still be position 1 of SEQ ID NO: 2.

[0275] As is known in the art, and unless otherwise indicated, the term "complementary" when used to describe the relationship between a first nucleic acid sequence and a second nucleic acid sequence refers to the ability of an oligonucleotide or polynucleotide containing the first nucleic acid sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleic acid sequence and form a double-stranded structure under specific conditions. As used herein, "complementary" sequences may also include a double-stranded structure formed by base pairing formed by non-Watson-Crick base pairing and / or non-natural or modified nucleotides, or may be a double-stranded structure formed entirely by base pairing formed by non-Watson-Crick base pairing and / or non-natural or modified nucleotides, as long as the above requirements with respect to their ability to hybridize are met.

[0276] As known in the art, a "fully complementary" sequence includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleic acid sequence with an oligonucleotide or polynucleotide comprising a second nucleic acid sequence over the entire length of the first nucleic acid sequence and the second nucleic acid sequence.

[0277] As known in the art, "substantially complementary" means that two nucleic acid sequences are completely complementary or at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the overlapping nucleotides are complementary.

[0278] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in terms of base pairing between the sense and antisense strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as can be understood from the context in which they are used.

[0279] In the art, "G", "C", "A", "T" and "U" generally represent the bases guanine, cytosine, adenine, thymine and uracil, respectively. However, it is also generally known in the art that "G", "C", "A", "T" and "U" each generally represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases, respectively. This is a common way to represent deoxyribonucleic acid sequences and / or ribonucleic acid sequences. Therefore, in the context of the present disclosure, the meanings represented by "G", "C", "A", "T" and "U" include the above-mentioned various possible situations. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide (as further described in other parts of this document) or having an alternative replacement part. It is recognized by those skilled in the art that guanine, cytosine, adenine and uracil can be replaced by other parts without substantially changing the base pairing properties of an oligonucleotide (including a nucleotide having such a replacement part). For example, without limitation, nucleotides comprising inosine as their bases can base pair with nucleotides comprising adenine, cytosine or uracil. Therefore, nucleotides containing uracil, guanine or adenine can be replaced by nucleotides containing, for example, inosine in the nucleotide sequence of the dsRNA characterized by the application. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced by guanine and uracil, respectively, to form a GU wobble base pairing with the target mRNA. Sequences containing this type of replacement portion are suitable for the compositions and methods characterized by the present disclosure.

[0280] As known in the art, "double-stranded ribonucleic acid," "double-stranded RNA," or "dsRNA" are used interchangeably. The term "dsRNA" comprises two antiparallel, complementary nucleic acid strands that are oriented in either a "sense" or "antisense" direction relative to a target RNA (e.g., a factor XI gene). In embodiments of the present application, dsRNA can degrade a target RNA (e.g., mRNA) via an RNA interference (RNAi) mechanism.

[0281] The double-stranded RNA of the present application contains a sense strand and an antisense strand. The term "sense strand" refers to a single strand in the dsRNA double strand that is substantially complementary to a region of the antisense strand. The term "antisense strand" refers to a single strand in the dsRNA double strand that is substantially complementary to a region of the target sequence. If the sense strand is not fully complementary to the antisense strand, mismatches may occur within the molecule or in the terminal regions. Generally, the most tolerable mismatches are within the terminal regions. If the antisense strand is not fully complementary to the target sequence, mismatches may occur within the molecule or in the terminal regions. Generally, the most tolerable mismatches are within the terminal regions.

[0282] As is known in the art, the double strands of dsRNA can have the same or different numbers of nucleotides. The length of the double stranded region formed by complementation can be any length that allows degradation of the target RNA, and possible lengths are in the range of about 9 to 36 pairs of nucleotides, such as 15 to 30 pairs, 16 to 28 pairs, 19 to 21 pairs, etc.

[0283] As is known in the art, outside the double-stranded region, dsRNA may include one or more nucleotide overhangs, which refer to at least one unpaired nucleotide / nucleoside analog. For example, when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand (or vice versa), an overhang is present. The nucleotides at the "overhang" may include 0-5 nucleotides, where "0" indicates no "overhang," and "5" indicates that there are 5 additional (i.e., not paired with the other single strand) nucleotides on the single strand of the dsRNA double strand. These optional "overhangs" may be located at the 5' and / or 3' end of any single strand in the two strands of the dsRNA. In some embodiments, the "overhang" includes 0-5 nucleotides. In some embodiments, the "overhang" includes 0-2 nucleotides. In some embodiments, the "overhang" at the 3' and / or 5' end of the sense strand of the dsRNA has 0-2 nucleotides. In some embodiments, the "overhang" at the 3' and / or 5' end of the antisense strand of the dsRNA has 0-2 nucleotides. The nucleotides forming the "overhang" can be A, G, C, U, or T, or modified structures thereof. The nucleotides forming the "overhang" can be U, T, or dT, or modified structures thereof. In some embodiments, the "overhang" includes, but is not limited to, "TT," "dTdT," "UU," or their corresponding modified structures, such as UU modified with a 2' methoxy group, i.e., uu. In some embodiments, the "overhang" at the 3' and / or 5' end of the antisense strand of the dsRNA is substantially complementary to the target RNA. In some embodiments, the "overhang" at the 3' and / or 5' end of the antisense strand of the dsRNA is completely complementary to the target RNA. In some embodiments, the "overhang" at the 3' end of the antisense strand of the dsRNA is completely complementary to the target RNA. The term "blunt end" or "blunt end" refers to the absence of unpaired nucleotides at the terminus of the dsRNA, i.e., the absence of nucleotide overhangs. A dsRNA with "blunt ends" or "blunt ends" at both ends is a dsRNA that is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule.

[0284] In this application, the terms "phosphorothioate linkage", "phosphorothiodiester linkage" and "phosphorothioate group" are used interchangeably.

[0285] In the present application, the dsRNA or any single strand thereof is optionally modified. Whether unmodified or modified, the dsRNA is within the scope of protection of this application. The modification does not significantly impair or abolish the dsRNA's ability to inhibit factor XI gene expression. The modification of the dsRNA or any single strand thereof may be at the 5' and / or 3' termini, at nucleotides, or at internucleotide linkages. The dsRNA or any single strand thereof may be synthesized or modified using methods known in the art.

[0286] In the present application, “the sense strand or antisense strand is optionally modified” means that any nucleotide of the sense strand or antisense strand is optionally modified.

[0287] In the present application, the modification of the nucleotide includes but is not limited to occurring on the sugar group of the nucleotide, including one or more substituted or removed sugar moieties, such as removing the hydroxyl group on the carbonyl, or fluoro, amination, alkylation, hydroxyalkylation or hydroxyalkenylation. The modification on the sugar group can occur at various positions on the sugar ring. Exemplarily, the modification on the sugar group of the nucleotide includes but is not limited to 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-methyl, 2'-ethyl, 2'-methyl-O-methyl, 2'-ethyl-O-methyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-ethyl-O-methyl or 2'-O-allyl, and modification and modified nucleoside structure can be as follows:

[0288] Wherein Base represents base.

[0289] In the present application, the modification of the internucleotide linkage includes substitution or replacement of the atom or functional group of the phosphate group, such as phosphorothioate (PS), phosphorodithioate (PS2), methylphosphonate (MP), methoxypropylphosphonate (MOP) or aminophosphonate.

[0290] In this application, the capital letters G, C, A, U, or T each generally represent a nucleotide containing guanine, cytosine, adenine, uracil, or thymine as a base, respectively, wherein the letter combination dT represents a deoxyribonucleotide whose base is thymine. The lowercase letters g, c, a, and u respectively represent that the ribose group of the nucleotide represented by the corresponding capital letter is modified by 2'-methoxy, i.e., g, c, a, and u represent 2'-O-methyl G, 2'-O-methyl C, 2'-O-methyl A, and 2'-O-methyl U, respectively. A capital letter plus a lowercase letter f to the right represents that the ribose group of the nucleotide represented by the corresponding capital letter is modified by 2'-fluorine, i.e., Gf, Cf, Af, and Uf represent 2'-fluorine G, 2'-fluorine C, 2'-fluorine A, and 2'-fluorine U, respectively. A lowercase letter "s" indicates that the two nucleotide residues adjacent to the "s" are linked by a phosphorothioate group. For example, "csu" indicates that the C and U residues are linked by a phosphorothioate group. VP- indicates that the nucleotide to the right of the hyphen is a (E)-vinyl phosphate-modified nucleotide. For example, "VP-u" indicates a (E)-vinyl phosphate-modified 2'-O-methyl U.

[0291] In this application, the 5'-end and / or 3'-end modifications refer to modifications occurring at the 5'-end and / or 3'-end of dsRNA or any single strand thereof, such as phosphorylation, conjugation, or reverse linkage. Taking the 5'-end as an example, including but not limited to 5'-phosphate, 5'-methylphosphonate (5'-MP), 5'-thiophosphate (5'-PS), or 5'-(E)-vinylphosphonate (5'-(E)-VP), the modification and the structure of the modified nucleotide can be shown as follows:

[0292] wherein Base represents a base, and X is selected from hydrogen, hydroxyl, or a 2' modification on a sugar group.

[0293] In the present application, the ligand is a group connected to the dsRNA, and the ligand includes a branched group and a linker, and the dsRNA, the linker and the branched group are connected in sequence (for example, as shown in Formula 104). The branched group contains at least one (for example, one, two, three, four or five) pharmaceutically acceptable targeting group, which targets the dsRNA to a specific tissue or enhances cellular absorption. The targeting group is, for example, but not limited to, a GalNAc (N-acetylgalactosamine, N-Acetylgalactosamine, for example, as shown in Formula 105) group. A plurality of the targeting groups are connected in series or in parallel through a branched group. Wherein, the GalNAc group can be monovalent, divalent, trivalent or tetravalent. The monovalent, divalent, trivalent, and tetravalent terms used herein refer to the molar ratio of dsRNA to GalNAc molecules in the dsRNA ligand conjugate formed by the dsRNA molecule and the ligand containing GalNAc as a targeting group being 1:1, 1:2, 1:3, and 1:4, respectively. In some embodiments, when the dsRNA of the present application is conjugated to a ligand containing GalNAc, the GalNAc molecule is trivalent or tetravalent. In some embodiments, when the dsRNA of the present application is conjugated to a ligand containing GalNAc, the GalNAc molecule is trivalent.

[0294] In the present application, the ligand can be attached to the phosphate group, 2'-hydroxyl group, 3'-hydroxyl group, or base of a nucleotide. The ligand can be attached to any nucleotide of the dsRNA, including but not limited to the 5' or 3' terminal nucleotide of the sense or antisense strand or a non-terminal intermediate nucleotide. When the ligand is attached to the end of the dsRNA strand, the ligand can be attached to the phosphate group of the nucleotide; when the ligand is attached to the intermediate nucleotide of the dsRNA, the ligand can be attached to the sugar ring or base of the nucleotide.

[0295] In this application, the types and preparation methods of the ligands described herein can refer to methods known in the art, including but not limited to the ligands and preparation methods described in WO2009082607, WO2014025805, WO2015006740, and WO2021249484, the entire disclosures of which are incorporated herein by reference. Exemplary ligands include but are not limited to L01 or L02 described above. The L01 ligand described herein is the same as the L96 ligand known in the prior art.

[0296] In this application, unless otherwise specified, "conjugation" refers to the connection of two or more chemical moieties, each with a specific function, to each other via non-covalent or covalent bonding. Accordingly, "conjugate" refers to a compound formed by non-covalent or covalent bonding of these chemical moieties. In this application, conjugates that are covalently bonded are preferred.

[0297] In the present application, the ligand is linked to the 5' or 3' end of the sense strand or antisense strand. Preferably, the ligand is linked to the 5' or 3' end of the sense strand. More preferably, the ligand is linked to the 3' end of the sense strand. Exemplarily, the dsRNA ligand conjugate formed by linking the dsRNA to the ligand is shown in Formula 102 or Formula 103 below:

[0298] The compounds of the present application (e.g., the double-stranded RNA, pharmaceutically acceptable salts thereof, and ligand conjugates thereof) may exist in specific geometric or stereoisomeric forms, all of which are within the scope of the present application. The present application contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and racemic mixtures and other mixtures thereof, such as enantiomer- or diastereomer-enriched mixtures, all of which are within the scope of the present application. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present application.

[0299] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0300] Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

[0301] Unless otherwise specified, use a solid wedge key. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond and straight dashed key Indicate the relative configuration of stereocenters with a wavy line Indicates a wedge-shaped solid key or dotted wedge key Or use a wavy line Indicates a straight solid bond and / or straight dashed key

[0302] Unless otherwise indicated, the terms "enriched in one isomer", "isomerically enriched", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100%, and the content of that isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

[0303] Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

[0304] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary groups are cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art and then recovered to obtain the pure enantiomer. In addition, the separation of enantiomers and diastereoisomers is typically accomplished by using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate formation from an amine). The compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that constitute the compound. For example, compounds can be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I) or C-14( 14 C). For example, deuterated drugs can be formed by replacing hydrogen with heavy hydrogen. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and extended drug biological half-life. All isotopic variations of the compounds of this application, whether or not radioactive, are included within the scope of this application.

[0305] The term "treatment" means administering the compound or preparation described herein (e.g., the double-stranded RNA, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present invention) to improve or eliminate a disease or one or more symptoms associated with the disease, and includes: (i) inhibiting the disease or disease state, i.e., curbing its development; (ii) alleviating the disease or disease state, i.e., causing the disease or disease state to regress.

[0306] The term "prevention" means administering the compound or preparation described herein (e.g., the double-stranded RNA, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present invention) to prevent a disease or one or more symptoms associated with the disease, and includes preventing the occurrence of a disease or disease state in a subject, particularly when such subject is susceptible to the disease state but has not yet been diagnosed as having the disease state.

[0307] The terms "subject," "patient," or "subject" are used interchangeably herein to refer to an animal that has been the subject of treatment, observation, or experiment. In some embodiments, the subject is a mammal, preferably a primate, and more preferably a human.

[0308] The term "therapeutically effective amount" means an amount of a compound of the present application (e.g., a double-stranded RNA, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof) that (i) treats or prevents a specific disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. The amount of a compound of the present application that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the route of administration, and the age of the subject to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and the disclosure of this application.

[0309] The therapeutic dose of the compound of the present application may be determined based on, for example, the specific use of the treatment, the mode of administering the compound, the health and condition of the patient, and the judgment of the prescribing physician. The ratio or concentration of the compound of the present application in the pharmaceutical composition may not be fixed and depends on a variety of factors, including dosage, chemical properties (e.g., hydrophobicity), and route of administration. For example, the compound of the present application may be provided in a physiologically buffered saline solution containing about 0.1 to 10% w / v of the compound for parenteral administration. Some typical dosage ranges are about 1 μg / kg to about 1 g / kg body weight / day. In certain embodiments, the dosage range is about 0.01 mg / kg to about 100 mg / kg body weight / day. The dosage is likely to depend on such variables as the type and extent of the disease or condition, the general health status of the specific patient, the relative biological efficacy of the selected compound, the excipient formulation, and its route of administration. The effective dose can be obtained by extrapolation of a dose-response curve derived from an in vitro or animal model test system.

[0310] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and other animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0311] As the pharmaceutically acceptable salt, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids and the like can be mentioned.

[0312] As used herein, the pharmaceutically acceptable salts include pharmaceutically acceptable salts of double-stranded ribonucleic acid and pharmaceutically acceptable salts of double-stranded ribonucleic acid ligand conjugates.

[0313] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application (e.g., double-stranded RNA, pharmaceutically acceptable salts thereof, or ligand conjugates thereof) and pharmaceutically acceptable carriers, excipients, or vehicles. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism. In this article, the terms "pharmaceutical composition" and "preparation" have the same meaning and are used interchangeably. The carriers, excipients, or vehicles used herein include any and all solvents, diluents, or other liquid excipients, dispersants, or suspending agents, surfactants, isotonic agents, thickeners, or emulsifiers, preservatives, solid binders, lubricants, and the like that are suitable for the desired specific dosage form. Various carriers, excipients, or vehicles used to formulate pharmaceutically acceptable compositions and methods for their preparation can employ known techniques. Except for any conventional carrier medium that is incompatible with the compounds of the present application (e.g., produces any adverse biological effects or otherwise interacts in a harmful manner with any other component of the pharmaceutically acceptable composition), its use is encompassed within the scope of this application. In some specific embodiments, the carriers, adjuvants or excipients used herein are conventionally used in the field of dsRNA administration.

[0314] The pharmaceutical compositions of the present application can be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable carriers, excipients or vehicles, and can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.

[0315] Typical routes of administration of the compounds of the present application or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0316] The pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing methods, dissolution methods, granulation methods, sugar-coated pill making methods, grinding methods, emulsification methods, freeze-drying methods, etc.

[0317] The kit of the present application includes the double-stranded ribonucleic acid, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present application; and optionally, instructions for using the double-stranded ribonucleic acid, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present application to treat and / or prevent thromboembolism and its complications.

[0318] The solvents used in this application are commercially available.

[0319] Unless otherwise specified, the solvent ratios used in column chromatography and preparative thin-layer silica gel chromatography in this application are all volume ratios.

[0320] It is known in the art that the modified nucleotide groups can be introduced into the dsRNA described herein by using nucleoside monomers with corresponding modifications. Those skilled in the art can obtain methods for preparing corresponding modified nucleoside monomers and methods for introducing modified nucleotide groups into dsRNA from the prior art. All modified nucleoside monomers can be obtained from commercial sources or prepared using known methods.

[0321] It is known in the art that the desired ribonucleic acid can be obtained by conventional ribonucleic acid preparation methods (such as solid phase synthesis and liquid phase synthesis) in the art, for example, the desired ribonucleic acid can be synthesized by phosphoramidite solid phase synthesis technology. The preparation method of the double-stranded ribonucleic acid of the present application, its pharmaceutically acceptable salt or its ligand conjugate comprises the following steps: according to the nucleotide type or order of the sense strand or antisense strand of the double-stranded ribonucleic acid, nucleotide monomers are sequentially connected to synthesize sense strand and antisense strand in a 3' to 5' direction. The connection of each nucleotide monomer includes a four-step reaction of deprotection, coupling, capping, oxidation or sulfurization. Those skilled in the art can adopt conventional reaction conditions, the type and dosage of reagents, or adjust according to the test situation to realize the deprotection, coupling, capping, oxidation or sulfurization reaction.

[0322] In some embodiments, for the synthesis of ribonucleic acid containing a ligand, the ligand can be connected to the ribonucleic acid through a coupling reaction during or after the synthesis of the oligoribonucleotide, or the ligand can be first connected to a solid phase support, and then the nucleoside monomer and the ligand-solid phase support can be connected in sequence in the 3' to 5' direction.

[0323] Methods for purification and desalting are well known to those skilled in the art. For example, RNA purification can be accomplished by preparative ion chromatography. For another example, RNA desalting can be accomplished by reverse phase chromatography purification or ultrafiltration centrifugation.

[0324] Annealing methods are well known to those skilled in the art. For example, the sense strand and the antisense strand can be mixed at a molar ratio of 1:1, heated to 70-95° C., and then cooled to room temperature to form a double-stranded structure.

[0325] During the synthesis process, the RNA concentration can be detected by, for example, ion exchange chromatography, or the molecular weight can be determined by liquid chromatography-mass spectrometry, or the concentration can be determined by microspectrophotometry to control the synthesis quality. Such detection methods are well known to those skilled in the art.

[0326] Unless otherwise specifically stated, singular terms encompass plural terms and plural terms encompass the singular. Unless otherwise specifically stated, the words "a" or "an" mean "at least one" or "at least one." Unless otherwise specified, the use of "or" means "and / or."

[0327] For the purposes of description and disclosure, all patents, patent applications, and other identified publications are expressly incorporated herein by reference. These publications are provided solely for their disclosure prior to the filing date of the present application. All statements regarding the dates of these documents or the representations of their contents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any citation of these publications herein does not constitute an admission that such publications become part of the common general knowledge in the art in any country. DETAILED DESCRIPTION

[0328] The present invention is described in detail below by way of examples, but is not intended to constitute any adverse restriction to the present invention. The compound of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by the combination thereof with other chemical synthesis methods and equivalent replacement modes well known to those skilled in the art, and preferred embodiments include but are not limited to the embodiments of the present invention. It will be apparent to those skilled in the art that various changes and improvements will be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0329] The prepared double-stranded RNA, its pharmaceutically acceptable salt or its ligand conjugate can be confirmed to be the target product by detection methods such as mass spectrometry.

[0330] Reagents and materials

[0331] Commercially available protected monomers used in the synthesis

[0332] Reagents used in synthesis

[0333] This application uses the following abbreviations:

[0334] DCM stands for dichloromethane; ACN stands for acetonitrile; DIC stands for diisopropylcarbodiimide; DMAP stands for dimethylaminopyridine; DMT stands for di-p-methoxytrityl; and MEM stands for 2-methoxyethoxymethyl.

[0335] siRNA sequence design and specificity analysis

[0336] siRNAs were designed targeting the human factor XI gene transcript NM_000128.4 and the cynomolgus monkey factor XI gene transcript XM_005556483.2. All siRNA sequences were 100% identical to the human transcript and 100% identical to the cynomolgus monkey transcript or had one mismatch.

[0337] Preparation Example 1: Connecting a GalNAc ligand (a ligand containing a GalNAc group as a targeting group) to a solid support

[0338] Weigh 1.0 g of amino-CPG (amino loading: 30-40 μmol / g) and add 5 mL of the decapping agent DBK. Let react for 1 minute, drain the reagent, and repeat this cycle four times. Based on a loading of 35 μmol, add 2 eq of a GalNAc ligand / acetonitrile solution, followed by a condensation reagent consisting of 2.5 eq of DIC and 2.5 eq of DMAP. Incubate at 25-30°C for 24 hours. After completion, filter out the solvent, rinse with anhydrous acetonitrile, then add the GalNAc ligand and condensation reagent in the aforementioned amounts and let react for an additional 24 hours. After the reaction, rinse the solid support with anhydrous acetonitrile, add 4 mL of a 1:1 mixture of Capping A and Capping B, let react for 2 minutes, drain, and then add 4 mL of the mixture. Let react for 2 minutes, drain, wash with anhydrous acetonitrile, and dry under vacuum at 30°C for 1 hour before use. In this example, the GalNAc ligand used is L01 (or L02).

[0339] Preparation Example 2 Preparation of double-stranded ribonucleic acid

[0340] 2.1.1 Synthesis of GalNAc ligand-ssRNA (sense strand)

[0341] On a K&A nucleic acid synthesizer, the GalNAc ligand-solid phase support was placed into a synthesis column and ssRNA was synthesized using standard phosphoramidite technology. The specific steps included:

[0342] 1) DMT removal (deprotection): Wash the resin twice with acetonitrile, remove DMT from the resin with 3% trichloroacetic acid / DCM, and wash with acetonitrile 4 to 5 times.

[0343] 2) Condensation (coupling): Add monomers to the synthesis column, add condensation reagents (2.5 eq DIC, 2.5 eq DMAP), and condense at room temperature for 10 minutes. Wash with acetonitrile 4-5 times.

[0344] 3) Blocking (capping): Block unreacted hydroxyl groups with Capping A first, then Capping B. Wash with acetonitrile 4-5 times.

[0345] 4) Oxidation: Add an oxidizing agent for 2 minutes, then wash with acetonitrile 4 to 5 times.

[0346] Repeat steps 1) to 4) until all sequences are synthesized. Finally, remove DMT with decapping agent DBK and wash with acetonitrile 4 to 5 times.

[0347] Formation of phosphorothioate bonds: Replace the oxidizing reagent with a thio reagent and set the thio time to 10 minutes to complete the formation of the phosphorothioate bond.

[0348] 2.1.2 Synthesis of ssRNA (ligand-free sense strand)

[0349] On a K&A nucleic acid synthesizer, a universal CPG solid phase carrier was placed into a synthesis column and ssRNA was synthesized using standard phosphoramidite technology. The specific steps include:

[0350] 1) DMT removal (deprotection): The resin was washed twice with acetonitrile, and then the DMT on the resin was removed with 3% trichloroacetic acid / DCM, and then washed with acetonitrile 4 to 5 times.

[0351] 2) Condensation (coupling): Add monomers to the synthesis column, add condensation reagents, and condense at room temperature for 10 minutes. Wash with acetonitrile 4-5 times.

[0352] 3) Blocking (capping): Block unreacted hydroxyl groups with Capping A first, then Capping B. Wash with acetonitrile 4-5 times.

[0353] 4) Oxidation: Add an oxidizing agent for 2 minutes, then wash with acetonitrile 4 to 5 times.

[0354] Repeat steps 1) to 4) until all sequences are synthesized. Finally, remove DMT with decapping agent DBK and wash with acetonitrile 4 to 5 times.

[0355] Formation of phosphorothioate bonds: Replace the oxidizing reagent with a thio reagent and set the thio time to 10 minutes to complete the formation of the phosphorothioate bond.

[0356] Synthesis of 2.2asRNA (ligand-free antisense strand)

[0357] On a K&A nucleic acid synthesizer, place the CPG solid phase carrier into a synthesis column and perform synthesis according to the method described in 2.1.1 or 2.1.2.

[0358] 2.3 Separation of ssRNA or asRNA from solid phase carrier

[0359] The lysis reagent is a 3:1 ratio of ammonia to ethanol (v:v, with an ammonia concentration of 25-28%). 5 mL of the lysis reagent is added to every 100 mg of the solid support and the reaction is stirred at 65-70°C for 3 hours. After the reaction is complete, the mixture is cooled to room temperature, precipitated with icy n-butanol, and placed in a -20°C refrigerator for 30 minutes. The precipitate is centrifuged and then washed with n-butanol twice. Finally, the precipitate is washed with acetone, centrifuged, dried under vacuum, and the crude product is analyzed by mass spectrometry.

[0360] LC-MS (negative ion mode) conditions are as follows:

[0361] Chromatographic column: HILIC column

[0362] Mobile phase A: 20 mM ammonium formate, pH 6.2

[0363] Mobile phase B: 20 ​​mm ammonium formate + 95% ACN

[0364] Column temperature: 45°C

[0365] Flow rate: 1 mL / min.

[0366] 2.4 Annealing to form siRNA (dsRNA)

[0367] Dissolve the crude single-stranded RNA obtained in step 2.3 in water and filter through a membrane to remove the carrier. Measure the concentrations of the sense and antisense strands using a microspectrophotometer. Add the sense and antisense strands to a 1:1 molar ratio and mix. Denature at 94°C for 4 minutes. Anneal naturally and cool to room temperature.

[0368] 2.5 Purification of siRNA (dsRNA)

[0369] Purification was performed using a DNAPac RP 10*150mm 4μm column. Mobile phase A was 0.1 mol / L triethylamine, pH 8.0, and mobile phase B was 0.1 mol / L triethylamine, pH 8.0 + 50% ACN. Detection wavelengths were 215 nm and 260 nm. The main peak was collected and the solvent was removed by rotary evaporation to obtain the desired product.

[0370] 2.6 Desalination and content calibration

[0371] Use 3K ultrafiltration centrifuge tubes, 12000×g, centrifugation time 12min, ultrafiltration concentration and desalination; after the rotary evaporation liquid is concentrated, add water for replacement, and repeat more than 5 times.

[0372] Concentration determination: After concentration, the sample was measured using a micro-spectrophotometer.

[0373] The double-stranded RNA or its ligand conjugate is shown below:

[0374] Table 8. Double-stranded RNA or its ligand conjugates

[0375] Among them, the capital letters G, C, A, or U respectively represent nucleotides containing guanine, cytosine, adenine, and uracil as bases; the lowercase letters g, c, a, and u respectively represent that the ribose group of the nucleotide represented by the corresponding capital letter is modified by 2'-methoxy, that is, g, c, a, and u represent 2'-O-methyl G, 2'-O-methyl C, 2'-O-methyl A, and 2'-O-methyl U respectively; the capital letter plus the lowercase letter f to the right represents that the ribose group of the nucleotide represented by the corresponding capital letter is modified by 2'-fluorine, that is, Gf, Cf, Af, and Uf represent 2'-fluorine G, 2'-fluorine C, 2'-fluorine A, and 2'-fluorine U respectively; the lowercase letter s indicates that the two nucleotide residues adjacent to s are connected by a thiophosphate group, for example, "csu" indicates that the c and u residues are connected by a thiophosphate group, and L represents a ligand. For example, the structures of the L01 ligand and the L02 ligand are shown below:

[0376] The above-mentioned double-stranded RNA, its pharmaceutically acceptable salt or its ligand conjugate (the ligand L is selected from L01 ligand or L02 ligand) can be prepared according to the method described in this application or methods known in the art.

[0377] The mass spectrometry detection data of the double-stranded RNA, its pharmaceutically acceptable salt or its ligand conjugate of the present application are consistent with expectations, and it can be confirmed that the target product has been obtained.

[0378] Test Example 1 In vitro screening

[0379] Cell culture and plasmid / transfection

[0380] Cos7 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in DMEM complete medium (Hyclone, Catalog No. SH30243.01) containing 10% fetal bovine serum (Gibco, Catalog No. 10099-141) at 37°C in an incubator containing 5% CO2. When the cells grew to near confluence, they were treated with trypsin and released from the culture flask. Cos7 cells were plated at 2×10 4Cells were seeded per well in a 96-well plate and cultured overnight. When the cell density reached 70-80%, Cos7 cells were transfected with the pmirGLO-FXI plasmid containing the FXI gene sequence (GenBank accession #NM_000128.4). Transfection was performed using 0.3 μL of Lipofectamine 3000 transfection reagent (Invitrogen, Cat. No. L3000015) at a concentration of 50 ng of plasmid per well, as described by the manufacturer. Six to eight hours after plasmid transfection, the culture medium was removed. siRNA was then transfected into the Cos7 cells transfected with the pmirGLO-FXI plasmid. The siRNA transfection method was to add 0.5 μL of siRNA to 5 μL of OPTI-MEM medium, and then add 0.3 μL of Lipofectamine RNAiMAX transfection reagent (Invitrogen, Cat. No. 13778150) to 5 μL of OPTI-MEM medium; the two were pipetted to mix thoroughly and allowed to stand at room temperature for 5 minutes; the complex was added to the culture wells, and 90 μL of complete DMEM medium was added to each well, and the culture was continued for 48 hours. Three concentrations of each siRNA were set, with two replicates, and single-dose experiments were performed with final dsRNA concentrations of 10 nM, 1 nM, and 0.1 nM.

[0381] Detection

[0382] Remove the culture medium from the wells and add 150 μL of A mixture of Luciferase reagent (Promega) and DMEM complete medium (volume ratio = 1:1) was mixed thoroughly and incubated at room temperature for 10 minutes. The chemiluminescence value (Fir) was read using a Spark multifunctional microplate reader (Tecan). 75 μL of Reagent (Promega), mix thoroughly, incubate at room temperature for 10 min, and read the chemiluminescence value (Ren) using a Spark multifunctional microplate reader.

[0383] The luminescence ratio for each well was calculated as Ratio = Fir / Ren. The luminescence ratio (Ratio(test)) or (Ratio(control)) for each test or control group was the average of the Ratios for each group. The luminescence ratios of each test group were normalized using the luminescence ratio of the control group as a benchmark to obtain the ratio (Ratio(test)) / Ratio(control), which represents the expression level of the firefly reporter gene. The inhibition rate (%) of the siRNA was (1-R) ​​× 100%. The results are shown in Table 9.

[0384] Table 9 Silencing efficiency

[0385] The test results show that the double-stranded RNA of the present application has good in vitro silencing efficiency.

[0386] Experimental Example 2 Dual luciferase assay

[0387] Cell culture and transfection

[0388] Cos7 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in DMEM complete medium (Hyclone, Catalog No. SH30243.01) containing 10% fetal bovine serum (Gibco, Catalog No. 10099-141) at 37°C in an incubator containing 5% CO2. When the cells grew to near confluence, they were treated with trypsin and released from the culture flask. Cos7 cells were plated at 2×10 4 Cells were seeded per well in a 96-well plate and cultured overnight. When the cell density reached 70-80%, Cos7 cells were transfected with the pmirGLO-FXI plasmid containing the FXI gene sequence (GenBank accession #NM_000128.4). Transfection was performed using 0.3 μL of Lipofectamine 3000 transfection reagent (Invitrogen, Cat. No. L3000015) at a concentration of 50 ng of plasmid per well, as described by the manufacturer. Six to eight hours after plasmid transfection, the culture medium was removed. siRNA was then transfected into the Cos7 cells transfected with the pmirGLO-FXI plasmid. The siRNA transfection method is to dilute the siRNA 5-fold with enzyme-free water to a concentration range of 2-0.0000256 μM; take 0.5 μL siRNA and add it to 5 μL OPTI-MEM medium, and take 0.3 μL Lipofectamine RNAiMAX transfection reagent (Invitrogen, catalog number 13778150) and add it to 5 μL OPTI-MEM medium; mix the two by pipetting and let it stand at room temperature for 5 minutes; add the complex to the culture well, add 90 μL DMEM complete medium to each well, and continue to culture for 48 hours; set 8 concentrations for each siRNA.

[0389] Detection

[0390] Remove the culture medium from the wells and add 150 μL of A mixture of Luciferase reagent (Promega) and DMEM complete medium (volume ratio = 1:1) was mixed thoroughly and incubated at room temperature for 10 minutes. The chemiluminescence value (Fir) was read using a Spark multifunctional microplate reader (Tecan). 75 μL of Reagent (Promega), mix thoroughly, incubate at room temperature for 10 min, and read the chemiluminescence value (Ren) using a Spark multifunctional microplate reader.

[0391] Calculate the luminescence ratio of each well: Ratio = Fir / Ren. The luminescence ratio of each test group or control group, Ratio (test) or Ratio (control), is the average of the Ratios of each group. The luminescence ratio of each test group is normalized to the luminescence ratio of the control group to obtain the ratio R of Ratio (test) / Ratio (control), which is used to express the expression level of the firefly reporter gene. The inhibition rate (%) of siRNA is (1-R) ​​× 100%. The concentration-inhibition rate (%) data are processed using EXCEL software, and the EC value of siRNA is calculated using a four-parameter nonlinear regression model. 50 , the results are shown in Table 10.

[0392] Table 10 EC of dual luciferase assay 50

[0393] The in vitro screening results showed that the double-stranded RNA of the present application had good in vitro silencing activity.

[0394] Experimental Example 3 Evaluation of in vivo activity of human coagulation factor XI model mice

[0395] The humanized mice used in this test case were purchased from Saiye Biotechnology Co., Ltd. The humanized mice were randomly divided into groups of 4, and each group of mice was given compounds and PBS controls (pH = 7.2). The first day of administration was set as day 0 of the experiment. According to the weight of the animals, a single subcutaneous injection of the corresponding compound solution was given at a dose of 1 and 5 mg / kg. Each PBS control mouse was given PBS at a volume of 10 mL / kg body weight. Before administration and on days 7, 14, 28, 42, and 63 after administration, orbital blood was collected, plasma was separated, and the content of coagulation factor XI protein in plasma was detected at each time point.

[0396] Approximately 165 μL of blood was collected from the orbital vein for plasma collection. Whole blood was treated with 3.8% trisodium citrate anticoagulant at a volume ratio of 1:9 (v / v) and centrifuged at 3000 rpm at 20°C for 15 minutes to collect plasma. Plasma obtained from each group of mice (compound-administered test group and PBS control group) was assayed for Factor XI protein levels using a Human Coagulation Factor XI ELISA kit (Sigma, Cat. No. RAB1385-1KT).

[0397] The factor XI protein inhibition rate was calculated according to the following equation:

[0398] Coagulation factor XI protein inhibition rate = (1-test group protein content / control group protein content) × 100%.

[0399] Table 11 shows that humanized homozygous mice were subcutaneously administered single doses of 1 and 5 mg / kg of the siRNA ligand conjugates (TD01M01L01 and TD02M01L01), and the FXI protein levels in the animals' plasma were measured before administration and on days 7, 14, 28, 42, and 63 after administration. As shown in Table 12, the results demonstrate that a single dose of the dsRNA ligand conjugates of the present application exhibited excellent inhibitory effects on human factor XI protein expression.

[0400] Table 11 In vivo experimental design

[0401] Table 12 Inhibition of Factor XI protein expression in mouse plasma

[0402] Experimental Example 4 Evaluation of in vivo activity of human coagulation factor XI model mice

[0403] The humanized mice used in this test case were purchased from Saiye Biotechnology Co., Ltd. Female humanized homozygous mice were randomly divided into groups of 4 per group, and each group of mice was given compounds and PBS controls (pH = 7.2). The first day of administration was set as day 0 of the experiment. According to the body weight of the animals, a single subcutaneous injection of the corresponding compound solution was performed at a dose of 3 mg / kg, and each PBS control mouse was given PBS with a dosing volume of 10 mL / kg body weight. Before and after administration, on days 3, 7, 14, 21, 28, 42, 56, and 70, orbital blood was collected, serum was separated, and the content of coagulation factor XI protein in serum at each time point was tested using Human Coagulation Factor XI ELISA kit (Thermo company, batch number 2350072722, item number EH118RB).

[0404] The factor XI protein inhibition rate was calculated according to the following equation:

[0405] Coagulation factor XI protein inhibition rate = (1-test group protein content / control group protein content) × 100%.

[0406] Table 13 shows that humanized homozygous mice were subcutaneously administered a single dose of 3 mg / kg of the siRNA-ligand conjugate, and the plasma levels of factor XI protein were measured before administration and on days 3, 7, 14, 21, 28, 42, 56, and 70 after administration. As shown in Table 14, the results demonstrate that a single dose of the siRNA-ligand conjugate exhibited excellent inhibitory effects on human factor XI protein expression.

[0407] Table 13 In vivo experimental design

[0408] Table 14 Inhibition of Factor XI protein expression in mouse plasma

[0409] Experimental Example 5 Evaluation of in vivo activity in AAV-FXI model mice

[0410] 4-6 week old C5BL / 6 male mice were injected with 1×10 11 vg AAV8-FXI (Weizhen Biopharmaceuticals). On the 28th day after modeling, mice with an hFXI protein content greater than 100 ng / mL were selected. The mice were randomly divided into groups according to body weight and injected with 3 mg / kg of the dsRNA ligand conjugate of the present application on day 0 (the dosage is calculated only as siRNA). On days 7, 14, 35, and 56, blood was collected from the eye sockets, and the serum was separated. The coagulation factor XI protein content in the serum at each time point was tested using a Human Coagulation Factor XI ELISA kit (Thermo, catalog number EH118RB).

[0411] The factor XI protein inhibition rate was calculated according to the following equation:

[0412] Coagulation factor XI protein inhibition rate = (1-test group protein content / control group protein content) × 100%.

[0413] AAV-FXI mice were subcutaneously administered a single dose of 3 mg / kg of the dsRNA ligand conjugate of the present application, and the levels of factor XI protein in the animal serum were measured before administration and on days 7, 14, 35, and 56 after administration (see Table 15). As shown in Table 16, the results confirmed that a single dose of the dsRNA ligand conjugate of the present application exhibited excellent inhibition of human factor XI protein expression.

[0414] Table 15 In vivo experimental design

[0415] Table 16 Inhibition of Factor XI protein expression in mouse serum

[0416] Test Example 6 Evaluation of in vivo activity in cynomolgus monkeys

[0417] On the 7th day before administration, 2-4 kg female cynomolgus monkeys were fasted overnight, and ~1 mL of blood was collected from the femoral vein and centrifuged to collect plasma. FXI protein (Human Coagulation Factor XI ELISA kit, Thermo Fisher Scientific, catalog number EH118RB), FXI activity (Coagulation Factor XI Assay Kit, Stago, catalog number 00723), and APTT (Activated Partial Thromboplastin Time Assay Kit, Stago, catalog number 00595) were measured. The monkeys were grouped according to their indicators and injected with the dsRNA ligand conjugate of the present application (TD02M02L01, etc.) on day 0. Plasma was collected on days 0, 3, 7, 14, 21, 28, 37, 54, and 68 to measure plasma FXI protein, FXI activity, and activated partial thromboplastin time (APTT).

[0418] The factor XI protein inhibition rate was calculated according to the following equation:

[0419] Coagulation factor XI protein inhibition rate = (protein content after administration / protein content before administration-1) × 100%.

[0420] The inhibition rate of coagulation factor XI activity was calculated according to the following equation:

[0421] Coagulation factor XI activity inhibition rate = (activity level after administration / activity level before administration - 1) x 100%.

[0422] The activated partial thromboplastin time prolongation rate was calculated according to the following equation:

[0423] Prolongation rate of activated partial thromboplastin time = (APTT value after administration / APTT value before administration - 1) × 100%.

[0424] Cynomolgus monkeys were subcutaneously administered 4.2 and 14 mg / kg of the dsRNA ligand conjugate of the present application, and the plasma factor XI protein content, FXI activity, and activated partial thromboplastin time (APTT) were measured before administration and on days 0, 3, 7, 14, 21, 28, 37, 54, and 68 after administration (Table 17). As shown in Tables 18 to 20, the results confirmed that the dsRNA ligand conjugate of the present application exhibited excellent inhibitory effects on the expression and activity of human factor XI protein and significantly prolonged the activated partial thromboplastin time (APTT).

[0425] Table 17 In vivo experimental design

[0426] Table 18 Inhibition of Factor XI Protein Expression in Cynomolgus Monkey Plasma

[0427] Table 19 Inhibition of Factor XI Activity in Cynomolgus Monkey Plasma

[0428] Table 20 Prolongation of activated partial thromboplastin time in cynomolgus monkey plasma

[0429] Test Example 7 Evaluation of in vitro liver homogenate stability

[0430] 1) For every 200 mg of liver tissue (C57BL / 6 mice), add 1 mL of tissue lysis buffer (100 mM Tris-HCl, 1 mM MgCl2, pH 6.0) and grind in a tissue grinder at -30°C. Collect the grinding solution, i.e., the blank liver homogenate, and store at -80°C until use.

[0431] 2) Pipette 17 μL of the stock solution of the dsRNA ligand conjugate to be tested (concentration is 4.1 mg / mL) and add 680 μL of enzyme-free water to dilute the stock solution to 100 μg / mL for use. The final concentration of the test compound in the system is 10 μg / mL (1:10).

[0432] 3) Obtain blank liver homogenate from mice and incubate at 37°C for 5 min, 18 h, 21 h, and 24 h.

[0433] 4) Pipette 540 μL of blank liver homogenate after 24 h incubation into a 1.5 mL Eppendorf tube, add 60 μL of the test compound stock solution (100 μg / mL), and vortex to mix. This is the 0 h sample.

[0434] 5) Pipette 540 μL of blank liver homogenate after 21 h of incubation into a 1.5 mL Eppendorf tube, add 60 μL of the test compound stock solution (100 μg / mL), vortex mix, and continue to place on a shaker (37°C, 300 rpm) for 3 h to prepare the 3 h sample.

[0435] 6) Pipette 540 μL of the blank liver homogenate after 18 h of incubation into a 1.5 mL Eppendorf tube, add 60 μL of the test compound stock solution (100 μg / mL), vortex mix, and continue to place on a shaker (37°C, 300 rpm) for another 6 h to prepare the 6 h sample.

[0436] 7) Pipette 540 μL of the blank liver homogenate after 5 min incubation into a 1.5 mL Eppendorf tube, add 60 μL of the test compound stock solution (100 μg / mL), vortex mix, and place on a shaker (37°C, 300 rpm) for 24 h to prepare the 24 h sample.

[0437] 8) Sample pretreatment:

[0438] (1) Vortex all the above samples to mix thoroughly. Pipette 50 μL of the sample to be tested and add 150 μL of Clarity Lysis Buffer (phenomenex, catalog number: AL0-8579). Vortex for 5 minutes and centrifuge at 15,000 rpm for 10 minutes.

[0439] (2) 1 mL of methanol and 1 mL of equilibration buffer (50 mM ammonium acetate (pH = 5.5) + 0.5% Triton_X100) were added to a Clarity OTX SPE plate (phenomenex, catalog number: 8E-S103-EGA) in sequence.

[0440] (3) 185 μL of the supernatant after centrifugation in step 1) was aspirated and loaded onto the SPE kit.

[0441] (4) Wash three times with 1 mL of equilibration buffer (50 mM ammonium acetate (pH = 5.5)), and then wash three times with 1 mL of buffer (50 mM ammonium acetate (pH = 5.5) + 50% acetonitrile).

[0442] (5) Elution was performed twice with 1 mL of an eluent (100 mM ammonium bicarbonate (pH = 9.5) + 40% acetonitrile + 10% tetrahydrofuran).

[0443] (6) Finally, the tube was dried with nitrogen at 65°C, and 100 μL of TE buffer (10 mM Tris-HCl + 1 mM EDTA (pH = 8.0)) was added for re-dissolution. The tube was vortexed for 5 min and centrifuged at 15,000 rpm for 10 min. The supernatant was collected and injected for analysis.

[0444] 9) LC-MS / MS was used to detect sample concentration, with zero point as the reference, and the analyte peak area was used to calculate the remaining percentage and analyze the data.

[0445] According to the test results, it was found that the dsRNA ligand conjugate of the present application had good stability. The remaining amount of the sense chain after 24 hours of incubation was more than 50%, and the remaining amount of the antisense chain after 24 hours of incubation was more than 80%.

[0446] Experimental Example 8 In vitro on-target and off-target evaluation

[0447] Cell culture and transfection

[0448] Cos7 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in DMEM complete medium (Hyclone, catalog number SH30243.01) containing 10% fetal bovine serum (Gibco, catalog number 10099-141) at 37°C in an incubator containing 5% CO2. Cos7 cells were plated at 2×10 4Cells were seeded / well in a 96-well plate and cultured overnight. Cos7 cells were transfected with reporter gene plasmids (on-target and off-target plasmids) using 0.3 μL of Lipofectamine 3000 transfection reagent (Invitrogen, Cat. No. L3000015). Six to eight hours after plasmid transfection, the culture medium was removed and various concentrations of siRNA were transfected into Cos7 cells containing the reporter gene plasmid using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Cat. No. 13778150). The on-target plasmid was administered at concentrations ranging from 10 nM to 0.00457 nM, while the off-target plasmid was administered at concentrations ranging from 40 nM to 0.0183 nM. The cells were incubated at 37°C, 5% CO2 for 48 hours.

[0449] Detection

[0450] Detection was performed using a dual-luciferase assay kit (Promega, Cat. No. E2940). The luminescence ratio for each well was calculated as Ratio = Fir / Ren. The luminescence ratio of each test group was normalized to the luminescence ratio of the control group. The ratio R (test) / Ratio (control) was used to represent the expression level of the firefly reporter gene. The inhibition rate (%) of the siRNA was calculated as (1-R) ​​× 100%.

[0451] The on-target plasmid GSCM is constructed by inserting a sequence complementary to the antisense strand into the 3'-untranslated region of the firefly luciferase in the pmirGLO plasmid. The off-target plasmid GSSM is constructed by inserting a sequence that perfectly matches positions 1-8 of the 5' end of the antisense strand into the 3'-untranslated region of the firefly luciferase in the pmirGLO plasmid, with no other matches. To improve sensitivity, GSSM-X5 was constructed, consisting of five identical GSSM sequences connected together. The off-target plasmid PSCM is constructed by inserting a sequence complementary to the sense strand into the 3'-untranslated region of the firefly luciferase in the pmirGLO plasmid. The off-target plasmid PSSM is constructed by inserting a sequence that perfectly matches positions 1-8 of the 5' end of the sense strand into the 3'-untranslated region of the firefly luciferase in the pmirGLO plasmid, with no other matches.

[0452] The test results show that the double-stranded ribonucleic acid (dsRNA) of the present application has no obvious off-target effect.

Claims

1. A double-stranded ribonucleic acid (dsRNA), a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides of a nucleotide sequence as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25 or SEQ ID NO.27, and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID At least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.26 or SEQ ID NO.28 and the length of the antisense strand does not exceed 23 nucleotides: Sense strand: 5'-GAUCUCCAACUAAAAUACU-3' (SEQ ID NO. 1), Antisense strand: 5'-AGUAUUUUAGUUGGAGAUCCG-3' (SEQ ID NO. 2); Sense strand: 5'-GGAUGAUUUUCUUAUAUCA-3' (SEQ ID NO. 3), Antisense strand: 5'-UGAUAUAAGAAAAUCAUCCUG-3' (SEQ ID NO. 4); Sense strand: 5'-GGUCUUUUCAGGAUGAUUU-3' (SEQ ID NO. 5), Antisense strand: 5'-AAAUCAUCCUGAAAAGACCUU-3' (SEQ ID NO. 6); Sense strand: 5'-GUCUUUUCAGGAUGAUUUU-3' (SEQ ID NO. 7), Antisense strand: 5'-AAAAUCAUCCUGAAAAGACCU-3' (SEQ ID NO. 8); Sense strand: 5'-CUUUUCAGGAUGAUUUUCU-3' (SEQ ID NO. 9), Antisense strand: 5'-AGAAAAUCAUCCUGAAAAGAC-3' (SEQ ID NO. 10); Sense strand: 5'-UUUUCAGGAUGAUUUUCUU-3' (SEQ ID NO. 11), Antisense strand: 5'-AAGAAAAUCAUCCUGAAAAGA-3' (SEQ ID NO. 12); Sense strand: 5'-UCAGGAUGAUUUUCUUAUA-3' (SEQ ID NO. 13), Antisense strand: 5'-UAUAAGAAAAUCAUCCUGAAA-3' (SEQ ID NO. 14); Sense strand: 5'-CAGGAUGAUUUUCUUAUAU-3' (SEQ ID NO. 15), Antisense strand: 5'-AUAUAAGAAAAUCAUCCUGAA-3' (SEQ ID NO. 16); Sense strand: 5'-AGGAUGAUUUUCUUAUAUC-3' (SEQ ID NO. 17), Antisense strand: 5'-GAUAUAAGAAAAUCAUCCUGA-3' (SEQ ID NO. 18); Sense strand: 5'-GAUGAUUUUCUUAUAUCAA-3' (SEQ ID NO. 19), Antisense strand: 5'-UUGAUAUAAGAAAAUCAUCCU-3' (SEQ ID NO. 20); Sense strand: 5'-AUGAUUUUCUUAUAUCAAG-3' (SEQ ID NO. 21), Antisense strand: 5'-CUUGAUAUAAGAAAAUCAUCC-3' (SEQ ID NO. 22); Sense strand: 5'-UGAUUUUCUUAUAUCAAGU-3' (SEQ ID NO. 23), Antisense strand: 5'-ACUUGAUAUAAGAAAAUCAUC-3' (SEQ ID NO. 24); Sense strand: 5'-CUUAUAUCAAGUGGUACAU-3' (SEQ ID NO. 25), Antisense strand: 5'-AUGUACCACUUGAUAUAAGAA-3' (SEQ ID NO. 26); Sense strand: 5'-AAACGGAUCUCCAACUAAA-3' (SEQ ID NO. 27), Antisense strand: 5'-UUUAGUUGGAGAUCCGUUUGA-3' (SEQ ID NO. 28); The sense strand or antisense strand is optionally modified.

2. The double-stranded ribonucleic acid, its pharmaceutically acceptable salt or its ligand conjugate according to claim 1, wherein: The sense strand contains a nucleotide sequence as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25 or SEQ ID NO.27, and the length of the sense strand does not exceed 21 nucleotides; the antisense strand contains a nucleotide sequence as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26 or SEQ ID NO.28, and the length of the antisense strand does not exceed 23 nucleotides.

3. The double-stranded ribonucleic acid, its pharmaceutically acceptable salt or its ligand conjugate according to claim 1, wherein: The positive strand is a nucleotide sequence as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25 or SEQ ID NO.27, and the antisense strand is a nucleotide sequence as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26 or SEQ ID NO.

28.

4. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 3, wherein: The sense strand or antisense strand includes an overhang at the 5' end and / or the 3' end.

5. The double-stranded ribonucleic acid, its pharmaceutically acceptable salt or its ligand conjugate according to claim 4, wherein: The overhang contains 1, 2, 3, 4 or 5 nucleotides; or, the overhang contains 1 or 2 nucleotides; or, the overhang is 1, 2, 3, 4 or 5 nucleotides at the 5' end and / or 3' end of the sense strand or the antisense strand; or, the dsRNA, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof comprises an additional sequence as an overhang.

6. The double-stranded ribonucleic acid, its pharmaceutically acceptable salt or its ligand conjugate according to claim 4 or 5, wherein: The additional sequence in the overhang is selected from unmodified or modified A, G, C, U or T; or selected from unmodified or modified U or T; or when the overhang is 1 nucleotide, the overhang is selected from u or dT; or when the overhang is 2 nucleotides, the overhang is selected from uu or dTdT.

7. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 4 to 6, wherein: The additional sequence as the overhang is connected to the adjacent nucleotide via a phosphate group or a phosphorothioate group; or, one or more nucleotides in the additional sequence as the overhang are connected via a phosphate group or a phosphorothioate group.

8. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 7, wherein: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides of the sense strand are modified, or nucleotides within a range of any of the foregoing values ​​are modified; or 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-1 2, 1-11 or 1-10 nucleotides are modified; or more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 16, more than 17, more than 18, more than 19, more than 20 or more 21 nucleotides of the sense strand are modified; or all nucleotides of the sense strand are modified; and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides of the antisense strand are modified, or nucleotides within a range of any of the foregoing values ​​are modified; or 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13 of the antisense strand are modified. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides of the antisense strand are modified; or all nucleotides of the antisense strand are modified.

9. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 8, wherein: The modification is selected from the group consisting of nucleotide sugar modification, base modification, internucleotide linkage modification, and terminal modification.

10. The double-stranded ribonucleic acid, its pharmaceutically acceptable salt or its ligand conjugate according to claim 9, wherein: The sugar modification of the nucleotide is selected from 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-alkyl, 2'-O-alkyl, 2'-O-ether, 2'-O-alkenyl; or, the sugar modification of the nucleotide is selected from 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-methyl, 2'-ethyl, 2'-methyl-O-methyl, 2'-ethyl-O-methyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-ethyl-O-methyl or 2'-O-allyl; The internucleotide linkage modification is selected from phosphorothioate (PS), phosphorodithioate (PS2), methylphosphonate (MP), methoxypropylphosphonate (MOP) or aminophosphonate; or, the internucleotide linkage modification is selected from phosphorothioate (PS); and / or The terminal modification is selected from 5'-terminal modification or 3'-terminal modification; or, the terminal modification is selected from 5'-phosphate, 5'-methylphosphonate (5'-MP), 5'-thiophosphate (5'-PS) or 5'-(E)-vinylphosphonate (5'-(E)-VP).

11. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 10, wherein: The first and second nucleotides of the sense chain and / or the second and third nucleotides of the sense chain are modified with a thiophosphate group, and the first and second nucleotides of the antisense chain, the second and third nucleotides of the antisense chain, the 19th and 20th nucleotides of the antisense chain and / or the 20th and 21st nucleotides of the antisense chain are modified with a thiophosphate group.

12. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 11, wherein: The sense strand has 2, 3, 4 or 5 nucleotides modified with 2'-fluoro, and the sense strand has 14, 15, 16 or 17 nucleotides modified with 2'-O-methyl; and / or The antisense strand has 2, 3, 4, 5, 6 or 7 nucleotides modified with 2'-fluoro, and the antisense strand has 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides modified with 2'-O-methyl.

13. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 12, wherein: 0, 1 or 2 nucleotides in the 1st to 6th nucleotides of the sense strand are 2'-fluoro-modified, 4, 5 or 6 nucleotides in the 1st to 6th nucleotides of the sense strand are 2'-O-methyl-modified, 2 or 3 nucleotides in the 7th to 9th nucleotides of the sense strand are 2'-fluoro-modified, 0 or 1 nucleotide in the 7th to 9th nucleotides of the sense strand are 2'-O-methyl-modified, 0 or 1 nucleotide in the 10th to 19th nucleotides of the sense strand are 2'-fluoro-modified, 8, 9 or 10 nucleotides among the 10th to 19th nucleotides are modified with 2'-O-methyl; or the 7th, 8th and 9th nucleotides of the sense chain are modified with 2'-fluoro, and 0, 1 or 2 nucleotides among the 1st to 6th and / or 10th to 19th nucleotides of the sense chain are also modified with 2'-fluoro; or the 7th, 8th and 9th nucleotides of the sense chain are modified with 2'-fluoro, and 0, 1 or 2 nucleotides among the 1st to 6th and / or 10th to 19th nucleotides of the sense chain are also modified with 2'-fluoro.

14. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 13, wherein: Three or four nucleotides among the 2nd, 6th, 14th and 16th nucleotides of the antisense chain are modified with 2'-fluorine; or three, four, five or six nucleotides among the 2nd, 6th, 8th, 9th, 14th and 16th nucleotides of the antisense chain are modified with 2'-fluorine; or the 2nd, 6th, 14th and 16th nucleotides of the antisense chain are modified with 2'-fluorine, and any one or two of the 1st, 3 to 5th, 7 to 13th, 15th, 17 to 21st and / or 17 to 23rd nucleotides of the antisense chain are also modified with 2'-fluorine; or 14, 15, 16, 17, 18 or 19 nucleotides among the 1st, 3 to 5th, 7 to 13th, 15th, 17 to 21st, and / or 17 to 23rd nucleotides of the antisense chain are modified with 2'-O-methyl.

15. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 14, wherein: The ligand contains at least one targeting group; or the ligand contains one, two, three, four or five targeting groups.

16. The double-stranded ribonucleic acid, its pharmaceutically acceptable salt or its ligand conjugate according to claim 15, wherein: The targeting group is a GalNAc group.

17. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 16, wherein: The ligand includes the following branching groups:

18. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 17, wherein: The ligand is selected from:

19. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 18, wherein: The ligand is linked to the 5' or 3' end of the sense strand or the antisense strand; or, the ligand is linked to the 5' or 3' end of the sense strand; or, the ligand is linked to the 3' end of the sense strand.

20. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 19, wherein: The double-stranded ribonucleic acid comprises any one of the following sense strands: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 and The double-stranded ribonucleic acid comprises any one of the following antisense strands: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75 NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:

84.

21. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 20, wherein: The double-stranded ribonucleic acid contains the following sequence pairs of a sense strand and an antisense strand: SEQ ID NO:1 / SEQ ID NO:2, SEQ ID NO:3 / SEQ ID NO:4, SEQ ID NO:5 / SEQ ID NO:6, SEQ ID NO:7 / SEQ ID NO:8, SEQ ID NO:9 / SEQ ID NO:10, SEQ ID NO:11 / SEQ ID NO:12, SEQ ID NO:13 / SEQ ID NO:14, SEQ ID NO:15 / SEQ ID NO:16, SEQ ID NO:17 / SEQ ID NO:18, SEQ ID NO:19 / SEQ ID NO:20, SEQ ID NO:21 / SEQ ID NO:22, SEQ ID NO:23 / SEQ ID NO:24, SEQ ID NO:25 / SEQ ID NO:26, SEQ ID NO:27 / SEQ ID NO:28, SEQ ID NO:29 / SEQ ID NO:30, SEQ ID NO:31 / SEQ ID NO:32, SEQ ID NO:33 / SEQ ID NO:34, SEQ ID NO:35 / SEQ ID NO:36, SEQ ID NO:37 / SEQ ID NO:38, SEQ ID NO:39 / SEQ ID NO:40, SEQ ID NO:41 / SEQ ID NO:42, SEQ ID NO:43 / SEQ ID NO:44, SEQ ID NO:45 / SEQ ID NO:46, SEQ ID NO:47 / SEQ ID NO:48, SEQ ID NO:49 / SEQ ID NO:50, SEQ ID NO:51 / SEQ ID NO:52, SEQ ID NO:53 / SEQ ID NO:54, SEQ ID NO:55 / SEQ ID NO:56, SEQ ID NO:57 / SEQ ID NO:58, SEQ ID NO:59 / SEQ ID NO:60, SEQ ID NO:61 / SEQ ID NO:62, SEQ ID NO:63 / SEQ ID NO:64, SEQ ID NO:65 / SEQ ID NO:66, SEQ ID NO:67 / SEQ ID NO:68, SEQ ID NO:69 / SEQ ID NO:70, SEQ ID NO:71 / SEQ ID NO:72, SEQ ID NO:73 / SEQ ID NO:74, SEQ ID NO:75 / SEQ IDNO:76、SEQ ID NO:77 / SEQ ID NO:78、SEQ ID NO:79 / SEQ ID NO:80、SEQ ID NO:81 / SEQ ID NO:82、SEQ ID NO:83 / SEQ ID NO:84。 22. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 21, wherein: The sense strand comprises 5'-gsasucUfcCfAfAfcuaaaauacuL-3' (SEQ ID NO: 29), and the antisense strand comprises 5'-asGfsuauUfuUfAfguugGfaGfaucscsg-3' (SEQ ID NO: 30); The sense strand comprises 5'-gsasucucCfAfAfcuaaaauacuL-3' (SEQ ID NO: 31), and the antisense strand comprises 5'-asGfsuauUfuuaguugGfaGfaucscsg-3' (SEQ ID NO: 32); The sense strand comprises 5'-gsgsauGfaUfUfUfucuuauaucaL-3' (SEQ ID NO: 33), and the antisense strand comprises 5'-usGfsauaUfaAfGfaaaaUfcAfuccsusg-3' (SEQ ID NO: 34); The sense strand comprises 5'-gsgsaugaUfUfUfucuuauaucaL-3' (SEQ ID NO: 35), and the antisense strand comprises 5'-usGfsauaUfaagaaaaUfcAfuccsusg-3' (SEQ ID NO: 36); The sense strand comprises 5'-gsgsucUfuUfUfCfaggaugauuuL-3' (SEQ ID NO: 37), and the antisense strand comprises 5'-asAfsaucAfuCfCfugaaAfaGfaccsusu-3' (SEQ ID NO: 38); The sense strand comprises 5'-gsgsucuuUfUfCfaggaugauuuL-3' (SEQ ID NO: 39), and the antisense strand comprises 5'-asAfsaucAfuccugaaAfaGfaccsusu-3' (SEQ ID NO: 40); The sense strand comprises 5'-gsuscuUfuUfCfAfggaugauuuuL-3' (SEQ ID NO: 41), and the antisense strand comprises 5'-asAfsaauCfaUfCfcugaAfaAfgacscsu-3' (SEQ ID NO: 42); The sense strand comprises 5'-gsuscuuuUfCfAfggaugauuuuL-3' (SEQ ID NO: 43), and the antisense strand comprises 5'-asAfsaauCfauccugaAfaAfgacscsu-3' (SEQ ID NO: 44); The sense strand comprises 5'-csusuuUfcAfGfGfaugauuuucuL-3' (SEQ ID NO: 45), and the antisense strand comprises 5'-asGfsaaaAfuCfAfuccuGfaAfaagsasc-3' (SEQ ID NO: 46); The sense strand comprises 5'-csusuuucAfGfGfaugauuuucuL-3' (SEQ ID NO: 47), and the antisense strand comprises 5'-asGfsaaaAfucauccuGfaAfaagsasc-3' (SEQ ID NO: 48); The sense strand comprises 5'-ususuuCfaGfGfAfugauuuucuuL-3' (SEQ ID NO: 49), and the antisense strand comprises 5'-asAfsgaaAfaUfCfauccUfgAfaaasgsa-3' (SEQ ID NO: 50); The sense strand comprises 5'-ususuucaGfGfAfugauuuucuuL-3' (SEQ ID NO: 51), and the antisense strand comprises 5'-asAfsgaaAfaucauccUfgAfaaasgsa-3' (SEQ ID NO: 52); The sense strand comprises 5'-uscsagGfaUfGfAfuuuucuuauaL-3' (SEQ ID NO: 53), and the antisense strand comprises 5'-usAfsuaaGfaAfAfaucaUfcCfugasasa-3' (SEQ ID NO: 54); The sense strand comprises 5'-uscsaggaUfGfAfuuuucuuauaL-3' (SEQ ID NO: 55), and the antisense strand comprises 5'-usAfsuaaGfaaaaucaUfcCfugasasa-3' (SEQ ID NO: 56); The sense strand comprises 5'-csasggAfuGfAfUfuuucuuauauL-3' (SEQ ID NO: 57), and the antisense strand comprises 5'-asUfsauaAfgAfAfaaucAfuCfcugsasa-3' (SEQ ID NO: 58); The sense strand comprises 5'-csasggauGfAfUfuuucuuauauL-3' (SEQ ID NO: 59), and the antisense strand comprises 5'-asUfsauaAfgaaaaucAfuCfcugsasa-3' (SEQ ID NO: 60); The sense strand comprises 5'-asgsgaUfgAfUfUfuucuuauaucL-3' (SEQ ID NO: 61), and the antisense strand comprises 5'-gsAfsuauAfaGfAfaaauCfaUfccusgsa-3' (SEQ ID NO: 62); The sense strand comprises 5'-asgsgaugAfUfUfuucuuauaucL-3' (SEQ ID NO: 63), and the antisense strand comprises 5'-gsAfsuauAfagaaaauCfaUfccusgsa-3' (SEQ ID NO: 64); The sense strand comprises 5'-gsasugAfuUfUfUfcuuauaucaaL-3' (SEQ ID NO: 65), and the antisense strand comprises 5'-usUfsgauAfuAfAfgaaaAfuCfaucscsu-3' (SEQ ID NO: 66); The sense strand comprises 5'-gsasugauUfUfUfcuuauaucaaL-3' (SEQ ID NO: 67), and the antisense strand comprises 5'-usUfsgauAfuaagaaaAfuCfaucscsu-3' (SEQ ID NO: 68); The sense strand comprises 5'-asusgaUfuUfUfCfuuauaucaagL-3' (SEQ ID NO: 69), and the antisense strand comprises 5'-csUfsugaUfaUfAfagaaAfaUfcauscsc-3' (SEQ ID NO: 70); The sense strand comprises 5'-asusgauuUfUfCfuuauaucaagL-3' (SEQ ID NO: 71), and the antisense strand comprises 5'-csUfsugaUfauaagaaAfaUfcauscsc-3' (SEQ ID NO: 72); The sense strand comprises 5'-usgsauUfuUfCfUfuauaucaaguL-3' (SEQ ID NO: 73), and the antisense strand comprises 5'-asCfsuugAfuAfUfaagaAfaAfucasusc-3' (SEQ ID NO: 74); The sense strand comprises 5'-usgsauuuUfCfUfuauaucaaguL-3' (SEQ ID NO: 75), and the antisense strand comprises 5'-asCfsuugAfuauaagaAfaAfucasusc-3' (SEQ ID NO: 76); The sense strand comprises 5'-csusuaUfaUfCfAfagugguacauL-3' (SEQ ID NO: 77), and the antisense strand comprises 5'-asUfsguaCfcAfCfuugaUfaUfaagsasa-3' (SEQ ID NO: 78); The sense strand comprises 5'-csusuauaUfCfAfagugguacauL-3' (SEQ ID NO: 79), and the antisense strand comprises 5'-asUfsguaCfcacuugaUfaUfaagsasa-3' (SEQ ID NO: 80); The sense strand comprises 5'-asasacGfgAfUfCfuccaacuaaaL-3' (SEQ ID NO: 81), and the antisense strand comprises 5'-usUfsuagUfuGfGfagauCfcGfuuusgsa-3' (SEQ ID NO: 82); The sense strand comprises 5'-asasacggAfUfCfuccaacuaaaL-3' (SEQ ID NO: 83), and the antisense strand comprises 5'-usUfsuagUfuggagauCfcGfuuusgsa-3' (SEQ ID NO: 84); The L is L01 or L02.

23. A pharmaceutical composition comprising the double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 22, optionally comprising a pharmaceutically acceptable carrier or excipient.

24. Use of the double-stranded RNA according to any one of claims 1 to 22, its pharmaceutically acceptable salt or its ligand conjugate, or the pharmaceutical composition according to claim 23 in the preparation of a medicament for treating and / or preventing thromboembolism and its complications.

25. The use according to claim 24, wherein The double-stranded RNA, its pharmaceutically acceptable salt or ligand conjugate, or pharmaceutical composition is used together with other therapeutic agents.

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