PD-L1-targeting double-stranded ribonucleic acid

Abstract

The invention provides a PD-L1 targeting small interfering nucleotide, a pharmaceutically acceptable salt thereof or a ligand conjugate thereof, and also provides a pharmaceutical composition containing the small interfering nucleotide, the pharmaceutically acceptable salt thereof or the ligand conjugate thereof, and a therapeutic use of the small interfering nucleotide, the pharmaceutically acceptable salt thereof or the ligand conjugate thereof.

Description

Double-stranded RNA targeting PD-L1

[0001] Citation of Related Applications

[0002] This application claims the priority and benefits of Chinese Patent Application No. 202310982666.0 filed with the State Intellectual Property Office of the People's Republic of China on August 4, 2023, Chinese Patent Application No. 202311654515.9 filed with the State Intellectual Property Office of the People's Republic of China on December 4, 2023, and Chinese Patent Application No. 202410114631.X filed with the State Intellectual Property Office of the People's Republic of China on January 26, 2024, 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 PD-L1. Background Art

[0004] Hepatitis B, also known as HBV, is a disease caused by infection with the hepatitis B virus (HBV). HBV is a hepatotropic virus that primarily resides in and damages liver cells, causing inflammation, necrosis, and fibrosis. Hepatitis B can be classified as acute or chronic. Acute HBV resolves spontaneously in most adults through the body's own immune system. However, chronic HBV (CHB) has become a significant global healthcare challenge and a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) (Edward JG, et al., The oral toll-like receptor-7 agonist GS-9620 in patients with chronic hepatitis B virus infection. Journal of Hepatology (2015); 63:320-328).

[0005] Currently approved anti-HBV drugs are primarily immunomodulators (interferon-α and pegylated interferon-α-2a, among others) and antiviral drugs (lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, and clavulanate, among others). These antiviral drugs are nucleoside or nucleotide drugs, whose mechanism of action is to inhibit HBV DNA synthesis and do not directly reduce HBsAg levels. As with extended therapy, nucleoside or nucleotide drugs have demonstrated HBsAg clearance rates similar to those observed naturally (Janssen et al. Lancet (2005), 365, 123-129; Marcellin et al. N. Engl. J. Med. (2004), 351, 1206-1217; Buster et al. Hepatology (2007), 46, 388-394).

[0006] Immune checkpoint inhibitors have been a subject of extensive research in recent years, primarily targeting the PD-1 / PD-L1 pathway. PD-L1 is a 40kDa transmembrane protein that binds to PD-1 to inhibit T cell activation, thereby maintaining immune homeostasis. Under normal circumstances, PD-L1 expression is induced on most lymphocytes, such as epithelial cells, endothelial cells, and some tumor cells. In normal liver tissue, PD-L1 is primarily expressed in the portal vein region, with low expression also observed on hepatocytes and sinusoidal endothelial cells. Viral infection significantly increases PD-L1 expression on hepatocytes. During HBV infection, PD-L1 expression on the hepatocyte surface is upregulated, interacting with the PD-1 receptor on T cells, leading to T cell exhaustion. Therefore, inhibiting PD-L1 expression can enhance T cell function, help restore immune responses, and trigger immune-mediated clearance of HBV-infected hepatocytes, which is crucial for the cure of chronic hepatitis B.

[0007] 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 and exert an inhibitory effect at the mRNA level, thereby achieving the purpose of treating diseases.

[0008] SUMMARY OF THE INVENTION

[0009] 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 PD-L1 mRNA.

[0010] 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 of the nucleotide sequence shown in SEQ ID NO.9 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.10 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.11 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.12 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of 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 comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. NO.14, wherein the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.15, wherein the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.16, wherein the antisense strand does not exceed 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.17, wherein the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.18, wherein the antisense strand does not exceed 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.19, wherein the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.20, wherein the antisense strand does not exceed 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. NO.21 and the sense strand is no longer than 23 nucleotides, and 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 25 nucleotides; or, 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 23 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.24, and the antisense strand is no longer than 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 25, and the sense strand is no longer than 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 26, and the antisense strand is no longer than 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 27, and the sense strand is no longer than 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 28, and the antisense strand is no longer than 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 29, and the sense strand is no longer than 21 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 30, and the antisense strand is no longer than 23 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. The antisense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence shown in SEQ ID NO. 53 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence shown in SEQ ID NO. 54 and the length of the antisense strand does not exceed 25 nucleotides.

[0011] Sense strand: 5'-AGCAAUAUGACAAUUGAAUGA-3' (SEQ ID NO. 9),

[0012] Antisense strand: 5′-UCAUUCAAUUGUCAUAUUGCUAC-3′ (SEQ ID NO. 10);

[0013] Sense strand: 5'-AUUUACUGUCACGGUUCCCAA-3' (SEQ ID NO. 11),

[0014] Antisense strand: 5′-UUGGGAACCGUGACAGUAAUAGC-3′ (SEQ ID NO. 12);

[0015] Sense strand: 5'-CUAUUUAUUUUGAGUCUGU-3' (SEQ ID NO. 13),

[0016] Antisense strand: 5'-ACAGACUCAAAAUAAAUAGGA-3' (SEQ ID NO. 14);

[0017] Sense strand: 5'-UGAAGAUAUAUUGUAGUAGAU-3' (SEQ ID NO. 15),

[0018] Antisense strand: 5′-AUCUACUACAAUAUAUCUUCAAA-3′ (SEQ ID NO. 16);

[0019] Sense strand: 5'-UUGUCUCAUGUUUCAUCGUAA-3' (SEQ ID NO. 17),

[0020] Antisense strand: 5′-UUACGAUGAAACAUGAGACAAAA-3′ (SEQ ID NO. 18);

[0021] Sense strand: 5'-CUGUGCAGUAUCUGUUCCAUU-3' (SEQ ID NO. 19),

[0022] Antisense strand: 5'-AAUGGAACAGAUACUGCACAGAC-3' (SEQ ID NO. 20);

[0023] Sense strand: 5'-AAUGAAAGGACUCACUUGGUA-3' (SEQ ID NO. 21),

[0024] Antisense strand: 5′-UACCAAGUGAGUCCUUUCAUUUG-3′ (SEQ ID NO. 22);

[0025] Sense strand: 5'-GCUGCAUGAUCAGCUAUGGUA-3' (SEQ ID NO. 23),

[0026] Antisense strand: 5′-UACCAUAGCUGAUCAUGCAGCGG-3′ (SEQ ID NO. 24);

[0027] Sense strand: 5'-GGUGCUUGGUCUCCUCUAUAA-3' (SEQ ID NO. 25),

[0028] Antisense strand: 5′-UUAUAGAGGAGACCAAGCACCUU-3′ (SEQ ID NO. 26);

[0029] Sense strand: 5'-AUUGGUCAUCCCAGAACUACA-3' (SEQ ID NO. 27),

[0030] Antisense strand: 5′-UGUAGUUCUGGGAUGACCAAUUC-3′ (SEQ ID NO. 28);

[0031] Sense strand: 5'-UAUUUAUUUUGAGUCUGUG-3' (SEQ ID NO. 29),

[0032] Antisense strand: 5'-CACAGACUCAAAAUAAAUAGG-3' (SEQ ID NO. 30);

[0033] Sense strand: 5'-AUUUACUGUCACGGUUCCCAA-3' (SEQ ID NO. 53),

[0034] Antisense strand: 5′-UUGGGAACCGUGACAGUAAAUGC-3′ (SEQ ID NO. 54);

[0035] The sense or antisense strand is optionally modified.

[0036] On the other hand, 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.

[0037] On the other hand, the present application provides a kit for treating and / or preventing hepatitis B virus infection, comprising the dsRNA, a pharmaceutically acceptable salt thereof, a ligand conjugate thereof, or a pharmaceutical composition of the present application; and optional instructions for use.

[0038] On the other hand, the present application provides a method for treating and / or preventing hepatitis B virus infection, which comprises administering the dsRNA, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present application to a subject for treatment and / or prevention (hereinafter also referred to as a subject).

[0039] In some embodiments, the present application provides a method for treating and / or preventing hepatitis B virus infection, which comprises administering to the subject of treatment and / or prevention a therapeutically / preventively effective amount of the dsRNA described herein, a pharmaceutically acceptable salt thereof, a ligand conjugate thereof, or a pharmaceutical composition.

[0040] In another aspect, the present application provides use of the dsRNA, pharmaceutically acceptable salts thereof, ligand conjugates thereof, or pharmaceutical compositions of the present application in the preparation of a medicament for treating and / or preventing hepatitis B virus infection.

[0041] In another aspect, the present application provides use of the dsRNA, pharmaceutically acceptable salt thereof, ligand conjugate thereof, or pharmaceutical composition of the present application in treating and / or preventing hepatitis B virus infection.

[0042] In another aspect, the present application provides the dsRNA, pharmaceutically acceptable salt thereof, ligand conjugate thereof, or pharmaceutical composition of the present application for treating and / or preventing hepatitis B virus infection.

[0043] In some embodiments, the hepatitis B virus infection can be at any stage of the disease, such as acute hepatitis B or chronic hepatitis B, or liver diseases caused by hepatitis B virus infection include hepatitis, liver fibrosis, cirrhosis, liver failure or liver cancer. In some embodiments, the hepatitis B virus infection is chronic hepatitis B.

[0044] In some embodiments, the dsRNA, pharmaceutically acceptable salts thereof, ligand conjugates thereof, or pharmaceutical compositions of the present application are used as a single therapeutic agent for treating and / or preventing hepatitis B virus infection.

[0045] In some embodiments, the dsRNA, pharmaceutically acceptable salts thereof, ligand conjugates thereof, or pharmaceutical compositions of the present application are used in combination with other therapeutic agents for treating and / or preventing hepatitis B virus infection.

[0046] Detailed Description of the Invention

[0047] 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.

[0048] 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.9 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.10 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.11 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO.12 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of 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 comprises 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; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. The antisense strand comprises 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 23 nucleotides, and the antisense strand comprises 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 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 17 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 18 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 19 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 20 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 21 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. NO.22 and the antisense strand is no longer than 25 nucleotides; or, 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 23 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.24, and the antisense strand is no longer than 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 25, and the sense strand is no longer than 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 26, and the antisense strand is no longer than 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 27, and the sense strand is no longer than 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 28, and the antisense strand is no longer than 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 29, and the sense strand is no longer than 21 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 30, and the antisense strand is no longer than 23 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. The antisense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence shown in SEQ ID NO. 53 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence shown in SEQ ID NO. 54 and the length of the antisense strand does not exceed 25 nucleotides.

[0049] Sense strand: 5'-AGCAAUAUGACAAUUGAAUGA-3' (SEQ ID NO. 9),

[0050] Antisense strand: 5′-UCAUUCAAUUGUCAUAUUGCUAC-3′ (SEQ ID NO. 10);

[0051] Sense strand: 5'-AUUUACUGUCACGGUUCCCAA-3' (SEQ ID NO. 11),

[0052] Antisense strand: 5′-UUGGGAACCGUGACAGUAAUAGC-3′ (SEQ ID NO. 12);

[0053] Sense strand: 5'-CUAUUUAUUUUGAGUCUGU-3' (SEQ ID NO. 13),

[0054] Antisense strand: 5'-ACAGACUCAAAAUAAAUAGGA-3' (SEQ ID NO. 14);

[0055] Sense strand: 5'-UGAAGAUAUAUUGUAGUAGAU-3' (SEQ ID NO. 15),

[0056] Antisense strand: 5′-AUCUACUACAAUAUAUCUUCAAA-3′ (SEQ ID NO. 16);

[0057] Sense strand: 5'-UUGUCUCAUGUUUCAUCGUAA-3' (SEQ ID NO. 17),

[0058] Antisense strand: 5′-UUACGAUGAAACAUGAGACAAAA-3′ (SEQ ID NO. 18);

[0059] Sense strand: 5'-CUGUGCAGUAUCUGUUCCAUU-3' (SEQ ID NO. 19),

[0060] Antisense strand: 5'-AAUGGAACAGAUACUGCACAGAC-3' (SEQ ID NO. 20);

[0061] Sense strand: 5'-AAUGAAAGGACUCACUUGGUA-3' (SEQ ID NO. 21),

[0062] Antisense strand: 5′-UACCAAGUGAGUCCUUUCAUUUG-3′ (SEQ ID NO. 22);

[0063] Sense strand: 5'-GCUGCAUGAUCAGCUAUGGUA-3' (SEQ ID NO. 23),

[0064] Antisense strand: 5′-UACCAUAGCUGAUCAUGCAGCGG-3′ (SEQ ID NO. 24);

[0065] Sense strand: 5'-GGUGCUUGGUCUCCUCUAUAA-3' (SEQ ID NO. 25),

[0066] Antisense strand: 5′-UUAUAGAGGAGACCAAGCACCUU-3′ (SEQ ID NO. 26);

[0067] Sense strand: 5'-AUUGGUCAUCCCAGAACUACA-3' (SEQ ID NO. 27),

[0068] Antisense strand: 5′-UGUAGUUCUGGGAUGACCAAUUC-3′ (SEQ ID NO. 28);

[0069] Sense strand: 5'-UAUUUAUUUUGAGUCUGUG-3' (SEQ ID NO. 29),

[0070] Antisense strand: 5'-CACAGACUCAAAAUAAAUAGG-3' (SEQ ID NO. 30);

[0071] Sense strand: 5'-AUUUACUGUCACGGUUCCCAA-3' (SEQ ID NO. 53),

[0072] Antisense strand: 5′-UUGGGAACCGUGACAGUAAAUGC-3′ (SEQ ID NO. 54);

[0073] The sense or antisense strand is optionally modified.

[0074] In some embodiments, the sense strand contains at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 9, 11, 15, 17, 19, 21, 23, 25, 27 or 53 and the length of the sense strand does not exceed 23 nucleotides, wherein the at least 15 consecutive nucleotides can be selected from 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides.

[0075] In some embodiments, the antisense strand contains at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 10, 12, 16, 18, 20, 22, 24, 26, 28 or 54, and the length of the antisense strand does not exceed 25 nucleotides, wherein the at least 15 consecutive nucleotides can be selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides.

[0076] In some embodiments, the sense strand contains at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 13 or 29 and the length of the sense strand does not exceed 21 nucleotides, wherein the at least 15 consecutive nucleotides can be selected from 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides.

[0077] In some embodiments, the antisense strand contains at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO. 14 or 30 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, 21, 22 or 23 consecutive nucleotides.

[0078] 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 the nucleotide sequence shown in SEQ ID NO.9 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.10 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.11 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.12 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand comprises 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 comprises the nucleotide sequence shown in SEQ ID NO.14 and the length of the antisense strand does not exceed 23 nucleotides; or, the sense strand comprises the nucleotide sequence shown in SEQ ID NO.15 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO. NO.16, and the antisense strand is no longer than 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.17, and the sense strand is no longer than 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.18, and the antisense strand is no longer than 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.19, and the sense strand is no longer than 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.20, and the antisense strand is no longer than 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.21, and the sense strand is no longer than 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.22, and the antisense strand is no longer than 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.23, and the sense strand is no longer than 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.24, and the antisense strand is no longer than 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO. NO.25 and the sense strand is no longer than 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.26 and the antisense strand is no longer than 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.27 and the sense strand is no longer than 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.28, and the antisense strand is no longer than 25 nucleotides; or, the sense strand comprises the nucleotide sequence shown in SEQ ID NO. 29, and the sense strand is no longer than 21 nucleotides, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO. 30, and the antisense strand is no longer than 23 nucleotides; or, the sense strand comprises the nucleotide sequence shown in SEQ ID NO. 53, and the sense strand is no longer than 23 nucleotides, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO. 54, and the antisense strand is no longer than 25 nucleotides.

[0079] Sense strand: 5'-AGCAAUAUGACAAUUGAAUGA-3' (SEQ ID NO. 9),

[0080] Antisense strand: 5′-UCAUUCAAUUGUCAUAUUGCUAC-3′ (SEQ ID NO. 10);

[0081] Sense strand: 5'-AUUUACUGUCACGGUUCCCAA-3' (SEQ ID NO. 11),

[0082] Antisense strand: 5′-UUGGGAACCGUGACAGUAAUAGC-3′ (SEQ ID NO. 12);

[0083] Sense strand: 5'-CUAUUUAUUUUGAGUCUGU-3' (SEQ ID NO. 13),

[0084] Antisense strand: 5'-ACAGACUCAAAAUAAAUAGGA-3' (SEQ ID NO. 14);

[0085] Sense strand: 5'-UGAAGAUAUAUUGUAGUAGAU-3' (SEQ ID NO. 15),

[0086] Antisense strand: 5′-AUCUACUACAAUAUAUCUUCAAA-3′ (SEQ ID NO. 16);

[0087] Sense strand: 5'-UUGUCUCAUGUUUCAUCGUAA-3' (SEQ ID NO. 17),

[0088] Antisense strand: 5′-UUACGAUGAAACAUGAGACAAAA-3′ (SEQ ID NO. 18);

[0089] Sense strand: 5'-CUGUGCAGUAUCUGUUCCAUU-3' (SEQ ID NO. 19),

[0090] Antisense strand: 5'-AAUGGAACAGAUACUGCACAGAC-3' (SEQ ID NO. 20);

[0091] Sense strand: 5'-AAUGAAAGGACUCACUUGGUA-3' (SEQ ID NO. 21),

[0092] Antisense strand: 5′-UACCAAGUGAGUCCUUUCAUUUG-3′ (SEQ ID NO. 22);

[0093] Sense strand: 5'-GCUGCAUGAUCAGCUAUGGUA-3' (SEQ ID NO. 23),

[0094] Antisense strand: 5′-UACCAUAGCUGAUCAUGCAGCGG-3′ (SEQ ID NO. 24);

[0095] Sense strand: 5'-GGUGCUUGGUCUCCUCUAUAA-3' (SEQ ID NO. 25),

[0096] Antisense strand: 5′-UUAUAGAGGAGACCAAGCACCUU-3′ (SEQ ID NO. 26);

[0097] Sense strand: 5'-AUUGGUCAUCCCAGAACUACA-3' (SEQ ID NO. 27),

[0098] Antisense strand: 5′-UGUAGUUCUGGGAUGACCAAUUC-3′ (SEQ ID NO. 28);

[0099] Sense strand: 5'-UAUUUAUUUUGAGUCUGUG-3' (SEQ ID NO. 29),

[0100] Antisense strand: 5'-CACAGACUCAAAAUAAAUAGG-3' (SEQ ID NO. 30);

[0101] Sense strand: 5'-AUUUACUGUCACGGUUCCCAA-3' (SEQ ID NO. 53),

[0102] Antisense strand: 5′-UUGGGAACCGUGACAGUAAAUGC-3′ (SEQ ID NO. 54);

[0103] The sense or antisense strand is optionally modified.

[0104] 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.9, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.10; or, 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; or, 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.14; or, 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; or, 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; or, 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; or, the sense strand is the nucleotide sequence shown in SEQ ID NO. NO.21, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.22; or, 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; or, 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; or, the sense strand is the nucleotide sequence shown in SEQ ID NO.29, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.30; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.53, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.54:

[0105] Sense strand: 5'-AGCAAUAUGACAAUUGAAUGA-3' (SEQ ID NO. 9),

[0106] Antisense strand: 5′-UCAUUCAAUUGUCAUAUUGCUAC-3′ (SEQ ID NO. 10);

[0107] Sense strand: 5'-AUUUACUGUCACGGUUCCCAA-3' (SEQ ID NO. 11),

[0108] Antisense strand: 5′-UUGGGAACCGUGACAGUAAUAGC-3′ (SEQ ID NO. 12);

[0109] Sense strand: 5'-CUAUUUAUUUUGAGUCUGU-3' (SEQ ID NO. 13),

[0110] Antisense strand: 5'-ACAGACUCAAAAUAAAUAGGA-3' (SEQ ID NO. 14);

[0111] Sense strand: 5'-UGAAGAUAUAUUGUAGUAGAU-3' (SEQ ID NO. 15),

[0112] Antisense strand: 5′-AUCUACUACAAUAUAUCUUCAAA-3′ (SEQ ID NO. 16);

[0113] Sense strand: 5'-UUGUCUCAUGUUUCAUCGUAA-3' (SEQ ID NO. 17),

[0114] Antisense strand: 5′-UUACGAUGAAACAUGAGACAAAA-3′ (SEQ ID NO. 18);

[0115] Sense strand: 5'-CUGUGCAGUAUCUGUUCCAUU-3' (SEQ ID NO. 19),

[0116] Antisense strand: 5'-AAUGGAACAGAUACUGCACAGAC-3' (SEQ ID NO. 20);

[0117] Sense strand: 5'-AAUGAAAGGACUCACUUGGUA-3' (SEQ ID NO. 21),

[0118] Antisense strand: 5′-UACCAAGUGAGUCCUUUCAUUUG-3′ (SEQ ID NO. 22);

[0119] Sense strand: 5'-GCUGCAUGAUCAGCUAUGGUA-3' (SEQ ID NO. 23),

[0120] Antisense strand: 5′-UACCAUAGCUGAUCAUGCAGCGG-3′ (SEQ ID NO. 24);

[0121] Sense strand: 5'-GGUGCUUGGUCUCCUCUAUAA-3' (SEQ ID NO. 25),

[0122] Antisense strand: 5′-UUAUAGAGGAGACCAAGCACCUU-3′ (SEQ ID NO. 26);

[0123] Sense strand: 5'-AUUGGUCAUCCCAGAACUACA-3' (SEQ ID NO. 27),

[0124] Antisense strand: 5′-UGUAGUUCUGGGAUGACCAAUUC-3′ (SEQ ID NO. 28);

[0125] Sense strand: 5'-UAUUUAUUUUGAGUCUGUG-3' (SEQ ID NO. 29),

[0126] Antisense strand: 5'-CACAGACUCAAAAUAAAUAGG-3' (SEQ ID NO. 30);

[0127] Sense strand: 5'-AUUUACUGUCACGGUUCCCAA-3' (SEQ ID NO. 53),

[0128] Antisense strand: 5′-UUGGGAACCGUGACAGUAAAUGC-3′ (SEQ ID NO. 54);

[0129] The sense or antisense strand is optionally modified.

[0130] 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.

[0131] In some embodiments, the dsRNA, pharmaceutically acceptable salt thereof, or ligand conjugate thereof of the present application is an RNAi drug.

[0132] In some embodiments, the dsRNA, its pharmaceutically acceptable salt, or its ligand conjugate of the present application is siRNA or its ligand conjugate.

[0133] 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.

[0134] Double-stranded region

[0135] 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.

[0136] protrusion

[0137] In some embodiments, the sense strand or antisense strand optionally includes an overhang at the 5' end and / or the 3' end.

[0138] In some embodiments, the overhang contains 1, 2, 3, 4, or 5 nucleotides. In some embodiments, the overhang contains 1 or 2 nucleotides.

[0139] 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.

[0140] 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.

[0141] In some embodiments, the overhang is selected from unmodified or modified A, G, C, U, or T.

[0142] 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.

[0143] 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, then nucleotides 22-23 of the antisense strand constitute an overhang located at the 3' end of the antisense strand, i.e., 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, the ribonucleotides at positions 22-23 of the antisense strand constitute an overhang located at the 3' end of the antisense strand. When positions 1-20 of the sense strand are complementary to positions 1-20 of the antisense strand, 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, the 1st-2nd nucleotides 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.

[0144] 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 GA, CA, AC, GC, AA, UG, GG, 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 GA, CA, AC, GC, AA, UG, GG, UU, or UC.

[0145] In a specific embodiment, the dsRNA, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof comprises an additional sequence as an overhang at the 5' end and / or 3' end of the antisense strand or the sense strand, 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.

[0146] In some embodiments, the overhang is an additional sequence optionally located at the 5' end and / or 3' end of the antisense strand, and the overhang is selected from unmodified or modified GA, CA, AC, GC, AA, UG, GG, 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 GA, CA, AC, GC, AA, UG, GG, UU, or UC.

[0147] In some embodiments, the additional sequence serving as the overhang comprises unmodified or modified U or T.

[0148] In some embodiments, when the additional sequence as the overhang is one nucleotide, the overhang is selected from unmodified or modified U or T. In some embodiments, when the additional sequence as the overhang is one nucleotide, the overhang is selected from 2'-O-methyl-modified U (hereinafter also referred to as u) or deoxythymidine nucleotide (hereinafter also referred to as dT).

[0149] In some embodiments, when the additional sequence as the overhang is 2 nucleotides, the overhang is selected from unmodified or modified UU or TT. In some embodiments, when the additional sequence as the overhang is 2 nucleotides, the overhang is selected from uu or dTdT.

[0150] In some embodiments, the overhang is an additional sequence optionally located at the 5' end and / or the 3' end of the sense strand, and the overhang is selected from uu or dTdT.

[0151] In some embodiments, the overhang is an additional sequence optionally located at the 5' end and / or the 3' end of the antisense strand, and the overhang is selected from uu or dTdT.

[0152] In some embodiments, the overhang is linked to the adjacent nucleotide via a phosphate group or a phosphorothioate group.

[0153] In some embodiments, one or more nucleotides in the overhang are linked by phosphate or phosphorothioate groups.

[0154] Modification

[0155] In some embodiments, the sense strand or the antisense strand is optionally modified.

[0156] In some embodiments, one or more nucleotides of the sense strand or antisense strand are modified.

[0157] In some embodiments, each nucleotide can be modified with the same or different modifications, which can include 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 naturally occurring bases; and modification of the ribose-phosphate backbone.

[0158] 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.

[0159] 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 sense 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.

[0160] 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 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, more than 21, more than 22, or more 23 nucleotides of the sense strand are modified; typically, 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 than 21 nucleotides of the sense strand are modified.

[0161] In some embodiments, all nucleotides in the sense strand are modified.

[0162] 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, 23, 24, or 25 nucleotides of the antisense strand are modified; or a range of nucleotides within any of the foregoing values ​​is modified, e.g., 1-25, 1-24, 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.

[0163] 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 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, more than 21, more than 22, more than 23, more than 24, or more 25 nucleotides of the antisense strand are modified; typically, 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, more than 21, more than 22, or more than 23 nucleotides of the antisense strand are modified.

[0164] In some embodiments, all nucleotides in the antisense strand are modified.

[0165] In some embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified.

[0166] 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.

[0167] In some embodiments, the sugar modification of the nucleotide is selected from dehydroxylation, fluorination, amination, alkylation, hydroxyalkylation or hydroxyalkenylation.

[0168] In some embodiments, the sugar modification of the nucleotide occurs at the 2' position of the sugar group.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] In some embodiments, the modification is selected from 2'-fluoro, 2'-O-methyl, phosphorothioate, or 5'-(E)-vinyl phosphate.

[0175] 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.

[0176] In some embodiments, phosphorothioate groups are used to modify the first two nucleotides and / or the first three nucleotides of one or both 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 groups are used to modify the first and second nucleotides of the sense strand and / or the second and third nucleotides of the sense strand and / or the first and second nucleotides and the second and third nucleotides of the sense strand.

[0177] 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.

[0178] In some embodiments, for the case where there is no additional sequence as an overhang, the antisense strand is modified between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 1st and 2nd nucleotides at the 3' end of the antisense strand, and / or between the 2nd and 3rd nucleotides at the 3' end of the antisense strand using a thiophosphate group.

[0179] In some embodiments, when there is an additional sequence as an overhang, the phosphorothioate modification may be located between nucleotides in the additional sequence.

[0180] In some embodiments, the 5'-terminal nucleotide of the antisense strand has a 5'-(E)-vinyl phosphate modification.

[0181] 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, 17, 18, or 19 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.

[0182] In some embodiments, 0, 1, or 2 nucleotides in positions 1 to 6 or 1 to 8 of the sense strand are 2'-fluoro-modified.

[0183] In some embodiments, 4, 5, 6, 7 or 8 nucleotides in positions 1 to 6 or 1 to 8 of the sense strand are modified with 2'-O-methyl.

[0184] In some embodiments, two or three nucleotides among nucleotides 7 to 9 or 9 to 11 of the sense strand are 2'-fluoro-modified.

[0185] In some embodiments, 0 or 1 nucleotides in positions 7 to 9 or positions 9 to 11 of the sense strand are modified with 2'-O-methyl.

[0186] In some embodiments, 0 or 1 nucleotide among nucleotides 10 to 19 or 12 to 21 of the sense strand is 2'-fluoro-modified.

[0187] In some embodiments, 8, 9, 10, 11 or 12 nucleotides from positions 10 to 19 or from positions 12 to 21 of the sense strand are modified with 2'-O-methyl.

[0188] 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 nucleotides in nucleotides 1 to 6 and 10 to 19 of the sense strand are also 2'-fluoro-modified.

[0189] In some embodiments, the 9th, 10th and 11th nucleotides of the sense strand are 2'-fluoro-modified, and 0, 1 or 2 nucleotides of nucleotides 1 to 8 and / or 12 to 21 of the sense strand are also 2'-fluoro-modified. In some embodiments, the 9th, 10th and 11th nucleotides of the sense strand are 2'-fluoro-modified, and 0, 1 or 2 nucleotides of nucleotides 1 to 8 and 12 to 21 of the sense strand are also 2'-fluoro-modified. In some embodiments, the 9th, 10th and 11th nucleotides of the sense strand are 2'-fluoro-modified, and 0 nucleotides of nucleotides 1 to 8 and 12 to 21 of the sense strand are also 2'-fluoro-modified.

[0190] In some embodiments, the antisense strand has 2, 3, 4, 5, 6, or 7 nucleotides modified with 2'-fluoro.

[0191] In some embodiments, the antisense strand has 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides modified with 2'-O-methyl.

[0192] In some embodiments, three or four nucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-fluorinated. In some embodiments, four nucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-fluorinated.

[0193] 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, three or four nucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-fluoro-modified. In some embodiments, four or six nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-fluoro-modified.

[0194] 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, 17 to 21, and / or 17 to 23 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, 17 to 21, and / or 17 to 23 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 no nucleotides 1, 3 to 5, 7 to 13, 15, 17 to 21, and / or 17 to 23 of the antisense strand are 2'-fluoro-modified.

[0195] In some embodiments, 14, 15, 16, 17, 18 or 19 nucleotides among nucleotides 1, 3 to 5, 7 to 13, 15, 17 to 21 and / or 17 to 23 of the antisense strand are modified with 2'-O-methyl.

[0196] In some embodiments, nucleotides 2, 6, 14, and 16 of the antisense strand are 2'-fluoro-modified, and 0 nucleotides among nucleotides 1, 3 to 5, 7 to 13, 15, 17 to 21, and / or 17 to 23 of the antisense strand are 2'-fluoro-modified, and 14, 15, 16, 17, 18, or 19 nucleotides among nucleotides 1, 3 to 5, 7 to 13, 15, 17 to 21, and / or 17 to 23 of the antisense strand are 2'-O-methyl-modified.

[0197] In some embodiments, the sense strand has 2, 3, 4, or 5 nucleotides modified with 2'-fluoro, and the sense strand has 14, 15, 16, 17, 18, or 19 nucleotides modified with 2'-O-methyl; and / or;

[0198] 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.

[0199] In a specific embodiment, the 7th, 8th and 9th or 9th, 10th and 11th nucleotides of the sense strand are modified with 2'-fluoro, and the other nucleotides of the sense strand are modified with 2'-O-methyl, and the 2nd, 6th, 14th and 16th nucleotides of the antisense strand are modified with 2'-fluoro, and the other nucleotides of the antisense strand are modified with 2'-O-methyl.

[0200] In a specific embodiment, the 7th, 8th and 9th or 9th, 10th and 11th nucleotides of the sense strand are modified with 2'-fluoro, the other nucleotides of the sense strand are modified with 2'-O-methyl, and the 2nd, 6th, 14th and 16th nucleotides of the antisense strand are modified with 2'-fluoro, the other nucleotides of the antisense strand are modified with 2'-O-methyl, 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 1st and 2nd nucleotides of the 3' end of the antisense strand and / or between the 3rd and 4th nucleotides of the antisense strand are modified with phosphorothioate groups. Optionally, the 5'-terminal nucleotide of the antisense strand has a 5'-(E)-vinyl phosphate modification.

[0201] ligand

[0202] In some embodiments, the ligand includes a branching group and a linking group. In some embodiments, the branching group may include a targeting group.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] In some embodiments, the targeting group is selected from a GalNAc group.

[0207] 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.

[0208] In some embodiments, the branching group is selected from:

[0209] In some embodiments, the linking group is selected from:

[0210] In some embodiments, the ligand is selected from:

[0211] 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.

[0212] 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.

[0213] In some embodiments, the dsRNA ligand conjugate is as follows:

[0214] In some embodiments, the dsRNA ligand conjugate is selected from:

[0215] In some embodiments, the dsRNA ligand conjugate is selected from:

[0216] In some embodiments, the unmodified sense strand of the dsRNA is selected from:

[0217] Table 1. Unmodified sense strand

[0218] In some embodiments, the unmodified antisense strand of the dsRNA is selected from:

[0219] Table 2. Unmodified antisense strand

[0220] In some embodiments, the modified sense strand of the dsRNA is selected from:

[0221] Table 3. Modified sense strands

[0222] In some embodiments, the modified antisense strand of the dsRNA is selected from:

[0223] Table 4. Modified antisense strands

[0224] 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.

[0225] For example, the dsRNA comprises the sense strand of any one of 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: 53, 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, or SEQ ID NO: 55; and

[0226] The dsRNA comprises the antisense strand of any one of 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: 54, 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, or SEQ ID NO: 56.

[0227] In some embodiments, the dsRNA (or siRNA, unmodified) is selected from:

[0228] Table 5. siRNA (unmodified)

[0229] In some embodiments, the dsRNA (modified) is selected from:

[0230] Table 6. dsRNA (modified)

[0231] In some embodiments, the siRNA (or dsRNA) ligand conjugate is selected from:

[0232] Table 7. dsRNA ligand conjugates

[0233] 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.

[0234] Pharmaceutically acceptable salts

[0235] In some embodiments, the salt described above is selected from base addition salts, acid addition salts, and combinations thereof.

[0236] 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.

[0237] 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.

[0238] In some embodiments, the pharmaceutically acceptable salt is selected from sodium salt. Beneficial effects

[0239] The double-stranded RNA, its pharmaceutically acceptable salt or its ligand conjugate of the present application have good PD-L1 inhibitory activity, in vitro silencing activity, and in vivo (human PD-L1 model mice or AAV-hPD-L1 mice) inhibition or reduction of PD-L1 mRNA expression. The double-stranded RNA, its pharmaceutically acceptable salt or its ligand conjugate of the present application have good stability and can continue to play a role in the body for a long time. The double-stranded RNA, its pharmaceutically acceptable salt or its ligand conjugate of the present application have low off-target effects, small toxic side effects and high safety. Therefore, it has good prospects for drug development.

[0240] Definition and Description

[0241] 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.

[0242] 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.

[0243] 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.

[0244] The PD-L1 gene sequence of the present application can be obtained through a publicly available database, such as Genbank, etc., such as the human CD274 reference sequence (NM_014143.4).

[0245] 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).

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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 21 nucleotides and an antisense strand of 23 nucleotides, for example, position 1 of 5'-AGCAAUAUGACAAUUGAAUGA-3' (SEQ ID NO: 9) is A and position 21 is A; and position 1 of 5'-UCAUUCAAUUGUCAUAUUGCUAC-3' (SEQ ID NO: 10) is U and position 23 is C. For a dsRNA with a sense strand of more than 21 nucleotides and an antisense strand of more than 23 nucleotides, for example, if the sense strand has several nucleotides added to the terminal based on SEQ ID NO: 9 and the antisense strand has several nucleotides added to the terminal based on SEQ ID NO: 10, 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: 9, and position 1 of the antisense strand will still be position 1 of SEQ ID NO: 10.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] As known in the art, the terms "double-stranded ribonucleic acid," "double-stranded RNA," or "dsRNA" are used interchangeably. The term "dsRNA" comprises two antiparallel and complementary nucleic acid strands that are oriented "sense" or "antisense" relative to a target RNA (e.g., a PD-L1 gene). In embodiments of the present application, dsRNA can degrade target RNA (e.g., mRNA) through the RNA interference (RNAi) mechanism.

[0257] The double-stranded RNA of the present application contains a sense strand and an antisense strand. The term "sense strand" or "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.

[0258] 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.

[0259] 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. In some embodiments, the "overhang" at the 3' end of the antisense strand of the dsRNA is selected from unmodified or modified GA. The term "blunt end" or "blunt end" means that there are no unpaired nucleotides at the end of the dsRNA, i.e., there are no nucleotide overhangs. A dsRNA with "blunt" or "blunt" ends at both ends is a dsRNA that is double-stranded throughout its entire length, ie, has no nucleotide overhangs at either end of the molecule.

[0260] In this application, the terms "phosphorothioate linkage" or "phosphorothioate group" are used interchangeably.

[0261] In the present application, the dsRNA or any single strand thereof is optionally modified, and both unmodified and modified ribonucleic acids are within the scope of protection of the present application. The modification does not significantly weaken or lose the function of the dsRNA in inhibiting PD-L1 gene expression. The modification of the dsRNA or any single strand thereof may be located at the 5' and / or 3' termini, nucleotides, or internucleotide linkages. Synthesis or modification can be performed using methods known in the art.

[0262] In the present application, the sense strand or antisense strand is optionally modified, which means that any nucleotide of the sense strand or antisense strand is optionally modified.

[0263] 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:

[0264] Wherein Base represents base.

[0265] 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.

[0266] 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.

[0267] 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:

[0268] wherein Base represents a base, and X is selected from a hydroxyl group or a 2' modification on a sugar group.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] In this application, unless otherwise specified, "conjugation" refers to the non-covalent or covalent attachment of two or more chemical moieties, each with a specific function, to one another; accordingly, "conjugate" refers to a compound formed by non-covalent or covalent attachment of these chemical moieties. In this application, conjugates attached to one another by covalent attachment are preferred.

[0273] 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:

[0274] 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.

[0275] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0276] 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.

[0277] 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 bond 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

[0278] 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%.

[0279] 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%.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] 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.

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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.

[0288] 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.

[0289] 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.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] 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 hepatitis B virus infection.

[0294] The solvents used in this application are commercially available.

[0295] Unless otherwise specified, the solvent ratios used in column chromatography and preparative thin-layer silica gel chromatography in this application are all volume ratios.

[0296] 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.

[0297] 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.

[0298] 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.

[0299] 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.

[0300] 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.

[0301] 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.

[0302] 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."

[0303] 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

[0304] 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.

[0305] 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.

[0306] Reagents and materials

[0307] Commercially available protected monomers used in the synthesis

[0308] Reagents used in synthesis

[0309] This application uses the following abbreviations:

[0310] 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.

[0311] Preparation Example 1: Connecting a GalNAc ligand (a ligand containing a GalNAc group as a targeting group) to a solid support

[0312] 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 2.5 eq of a condensation reagent consisting of DIC and 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 amounts indicated above 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 above mixture. Let react for 2 minutes, drain, rinse with anhydrous acetonitrile, and dry under vacuum at 30°C for 1 hour before use. In this example, the GalNAc ligand L01 can be used.

[0313] Preparation Example 2 Preparation of double-stranded ribonucleic acid

[0314] 2.1.1 Synthesis of GalNAc ligand-ssRNA (sense strand)

[0315] 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:

[0316] 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.

[0317] 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.

[0318] 3) Blocking (capping): Block unreacted hydroxyl groups with Capping A first, then Capping B. Wash with acetonitrile 4-5 times.

[0319] 4) Oxidation: Add an oxidizing agent for 2 minutes, then wash with acetonitrile 4 to 5 times.

[0320] 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.

[0321] 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 phosphorothioate bonds.

[0322] 2.1.2 Synthesis of ssRNA (ligand-free sense strand)

[0323] 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:

[0324] 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.

[0325] 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.

[0326] 3) Blocking (capping): Block unreacted hydroxyl groups with Capping A first, then Capping B. Wash with acetonitrile 4-5 times.

[0327] 4) Oxidation: Add an oxidizing agent for 2 minutes, then wash with acetonitrile 4 to 5 times.

[0328] 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.

[0329] 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 phosphorothioate bonds.

[0330] 2.2 Synthesis of asRNA (ligand-free antisense strand)

[0331] 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.

[0332] 2.3 Separation of ssRNA or asRNA from solid phase carrier

[0333] The lysis reagent was a 3:1 ratio of ammonia to ethanol. 5 mL of lysis reagent was added per 100 mg of solid support and the reaction was stirred at 65-70°C for 3 h. After the reaction, the mixture was cooled to room temperature and precipitated with icy n-butanol. The mixture was then placed in a -20°C refrigerator for 30 min. The precipitate was centrifuged and washed with n-butanol twice. Finally, the precipitate was washed with acetone, centrifuged, and dried under vacuum to obtain the crude product for mass spectrometry analysis.

[0334] LC-MS (negative ion mode) conditions are as follows:

[0335] Chromatographic column: HILIC column

[0336] Mobile phase A: 20 mM ammonium formate, pH 6.2

[0337] Mobile phase B: 20 ​​mm ammonium formate + 95% ACN

[0338] Column temperature: 45°C

[0339] Flow rate: 1 mL / min.

[0340] 2.4 Annealing to form siRNA (dsRNA)

[0341] Dissolve the crude single-stranded RNA in water and remove the carrier by membrane filtration. Measure the concentrations of the sense and antisense strands using a microspectrophotometer. Add the sense and antisense strands at a molar ratio of 1:1 and mix. Denature at 94°C for 4 minutes. Anneal naturally and cool to room temperature.

[0342] 2.5 Purification of siRNA (dsRNA)

[0343] 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.

[0344] 2.6 Desalination and content calibration

[0345] 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.

[0346] Concentration determination: After concentration, the sample was measured using a micro-spectrophotometer.

[0347] Preparation Example 3

[0348] Solid phase synthesis: (antisense strand)

[0349] The monomer was weighed and a calculated amount of anhydrous acetonitrile was added to prepare a 0.2 M monomer solution.

[0350] 1) Weigh the Unylinker Support and pack it into the Fineline70P synthetic column according to the SOP column packing requirements.

[0351] 2) Referring to the standard operating procedures of the synthesis instrument, connect the monomer solution and reagent solution to the synthesis instrument, open the nitrogen pressure valve and run the solid phase synthesis command.

[0352] 3) After the solid-phase synthesis is completed, remove the pipeline at the upper end of the column, connect the nitrogen pipeline, and empty the solvent in the column under positive pressure for 5 minutes.

[0353] 4) Close the lower opening, open the plunger cavity valve, press in nitrogen, push the plunger rod to the opening, release the pressure, remove the screw and valve, and pour the wet Oligo-Solid-support into a clean crystallizing dish.

[0354] 5) Vacuum dry until constant weight is reached, the net weight of the dry product is 123.6 g.

[0355] 2. Cracking

[0356] 1) 123 g of the intermediate prepared above and 1230 mL of 28 wt % concentrated ammonia water were added to a 3 L stainless steel reactor, and the stirring speed was set to 100 rpm.

[0357] 2) Control the external temperature of heating to 60-85°C and the internal temperature to 50-60°C, and heat the reaction for 12 hours.

[0358] 3) After the reaction was completed, the feed solution was poured into a Fineline 70P chromatography column and filtered under positive pressure to obtain a filtrate. The filter cake was rinsed with purified water and filtered again. All filtrates were combined to obtain 2353.4 g of lysate with a total OD value of 1375420.4.

[0359] 3. Purification

[0360] 1) Column efficiency test.

[0361] 2) Disinfect the column.

[0362] 3) Prepare the sample, eluent, and sample collection bottle. Record the weight and total OD of the sample to be purified. Connect the prepared eluent to the appropriate line of the instrument and open the appropriate valve.

[0363] 4) Manually run the program. Ensure that both pumps A and B are on, flush and equilibrate the column, and adjust the eluent percentage during the elution process based on the crude product purity and bench-test results.

[0364] 5) After the run, flush the column.

[0365] 6) Based on the in-process control results, combine the purified collected solutions and store them in a refrigerator at 2-8°C.

[0366] 4. Ultrafiltration

[0367] 5. Synthesize the sense chain according to the method of synthesizing the antisense chain.

[0368] 6. Annealing

[0369] 1) Pour 1402.5 g of the antisense strand solution and 1356.2 g of the sense strand solution into the bottle, rinse the glass bottle containing the solution with 200 g of purified water, and then pour them into the reaction bottle.

[0370] 2) Start stirring and heating at 110°C; raise the reaction system temperature to 87-91°C and maintain for 30 minutes. Turn off heating and lower the reaction system temperature to approximately 25°C; sample and determine double-strand purity; the double-strand content is 94.44%.

[0371] 3) Collect the feed liquid into a sterilized 5L PP material barrel, rinse the reaction bottle with a small amount of purified water, and combine it with the feed liquid. The net weight of the feed liquid is 2978.7 g.

[0372] 7. Freeze-drying to obtain 71.6 g of freeze-dried solid.

[0373] Double-stranded RNA looks like this:

[0374] Table 8. Double-stranded RNA

[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 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 A

[0379] 1. Cell Culture and Plasmid / Transfection for Dual-Luciferase Assay

[0380] Cos7 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in DMEM (Viva Cell, catalog number C3113-0500) containing 10% fetal bovine serum (Gibco, catalog number 10099-141) in a complete culture medium at 37°C in an incubator containing 5% CO2 / 95% air. When the cells grew to near confluence, they were treated with trypsin and released from the culture flask. Cos7 cells were plated at 1.5×10 4 Cells / well were seeded in 96-well plates and cultured overnight. The complete human CD274 reference sequence (NM_014143.4) was cloned into the dual-luciferase pmirGLO vector (Promega, cat. no. E1330) (Nanjing GenScript Biotechnology Co., Ltd., C3872GD150) using a construct with an insert length of approximately 3634 bp. The pmirGLO plasmid and siRNA were transfected into 1.5×10 4 in a cell. The transfection method is to take 0.3 μL LipofectamineTM 3000 transfection reagent and add it to 5 μL OPTI-MEM medium (Thermo, product number 11058021), and mix it by pipetting; then add 50 ng / well pmirGLO plasmid and 0.2 μL / well P3000 to 5 μL OPTI-MEM medium (Thermo, product number 11058021), and mix it by pipetting; the diluted plasmid (at a ratio of 1:1) is added to the OPTI-MEM medium containing the transfection reagent, mix it by pipetting, and let it stand at room temperature for 5 minutes; the complex (a mixture of diluted transfection reagent Lipofectamine 3000 and diluted plasmid and p3000) is added to the culture well, and 90 μL DMEM complete medium is added to each well, and the culture is continued for 8 hours; the medium is changed; siRNA is diluted to 2 μM and 0.2 μM with enzyme-free water; 0.3 μL Lipofectamine TM RNAiMAX transfection reagent was added to 10 μL of OPTI-MEM medium and pipetted to mix. Then 0.5 μL of siRNA was added to the OPTI-MEM medium containing transfection reagent, pipetted to mix, and allowed to stand at room temperature for 5 minutes. The complex was added to the culture wells, and 90 μL of DMEM complete medium was added to each well. The culture was continued for 48 hours, and then the dual luciferase detection kit was used. Luciferase was measured using a luciferase assay system (Promega, Cat. No. E2940); single-dose experiments were performed at final duplex concentrations of 10 nM and 1 nM. siRNAs used for transfection were the sequences shown below: AD01, AD02, AD03, AD04, AD05, AD06, AD07, AD08, AD09, AD10, AD11, and AD12.

[0381] 2. Luciferase Assay

[0382] After 48h of siRNA transfection, firefly (transfection control) luciferase and Renilla (fused to PD-L1 target sequence) luciferase were measured. First, the culture medium was removed from the cells. Firefly luciferase activity was then measured by adding 75 μL of Dual-luciferase reagent equivalent to the volume of PBS (pH 7.4) to each well and mixing. The mixture was incubated at room temperature for 10min, and then the luminescence (500nm) was measured on a multifunctional microplate reader (TECAN) to detect the firefly luciferase signal. Renilla luciferase activity was measured by adding 75 μL of room temperature Stop&Glo reagent to each well, and the plate was incubated for 10min, and then the luminescence was measured again to determine the signal of Renilla luciferase. Stop&Glo reagent quenches the firefly luciferase signal and causes the Renilla luciferase reaction to continue luminescence. siRNA activity was determined by benchmarking the firefly (control) signal to the Renilla (PD-L1) signal in each well. The magnitude of siRNA activity was then assessed compared to cells transfected with the same vector but without siRNA treatment or with non-targeting siRNA. All transfections were performed in triplicate.

[0383] 3. Cell culture and transfection for QuantiGene method:

[0384] HCC4006 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in RPMI 1640 (Bio-Channel, catalog number BC-M-017-500 mL) containing 10% fetal bovine serum at 37°C in an incubator containing 5% CO2 / 95% air. When the cells grew to near confluence, they were treated with trypsin and released from the culture flask. HCC4006 cells were plated at 2×10 5Cells were seeded / well in a 96-well plate and cultured overnight. siRNA transfection was performed by diluting siRNA to 2 μM and 0.02 μM using enzyme-free water. 0.3 μL of Lipofectamine RNAiMAX transfection reagent was added to 10 μL of OPTI-MEM medium and mixed by pipetting. 0.5 μL of siRNA was then added to the OPTI-MEM medium containing the transfection reagent, mixed by pipetting, and allowed to stand at room temperature for 5 minutes. The complex was then added to the wells, and 90 μL of complete RPMI 1640 medium was added to each well. Cultures were continued for 48 hours. Single-dose experiments were performed at final duplex concentrations of 10 nM and 0.1 nM. For dose-response experiments, eight 3-fold serial dilutions were performed, starting at 10 nM. The siRNAs used for transfection were the sequences shown in M01, M02, M03, M04, M05, M06, M07, M08, M09, M10, M11 and M12.

[0385] 4.QuantiGene Detection

[0386] iGene assay

[0387] 48 h after siRNA transfection, the TM Sample processing kit QuantiGene TM Sample Processing Kit (Thermo, catalog number QS0103), QuantiGene TM QuantiGene probe TM Probe Set (Thermo, catalog number QGS-1000) and QuantiGene TM Signal detection kit QuantiGene TM PD-L1 mRNA expression levels in cells were detected using the Signalplex Assay Kit (Thermo, Cat. No. QS0384). The procedure was performed according to the manufacturer's instructions. Chemiluminescence (RLU) values ​​were measured using a multi-functional plate reader (TECAN). Inhibition rate (%) = [1 - siRNA group (RLU) / control group (RLU)] × 100. Data were processed using Excel software to calculate the concentration-inhibition rate (%) data, and the EC50 for siRNA inhibition of PD-L1 was calculated using a four-parameter nonlinear regression model. All transfections were performed in duplicate.

[0388] The results are shown in Tables 9, 10 and 11.

[0389] Table 9 Silencing efficiency of dual luciferase method (%)

[0390] Table 10 Silencing efficiency of single-dose experiment by QuantiGene method (%)

[0391] Table 11 EC of QuantiGene method 50

[0392] The results of in vitro screening showed that the siRNA of the present application had good in vitro silencing activity.

[0393] Test Example 2 In vitro screening B

[0394] HCC4006 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in RPMI 1640 complete medium (Bio-Channel, catalog number BC-M-017-500 mL) containing 10% fetal bovine serum at 37°C in an incubator containing 5% CO2 / 95% air. When the cells grew to near confluence, they were treated with trypsin and released from the culture flask. HCC4006 cells were plated at 5×10 4 Cells / well were seeded in a 48-well plate and cultured overnight. The siRNA transfection method was to dilute siRNA (20 μM) to 2 μM, 0.4 μM, 0.08 μM, 0.016 μM, 0.0032 μM, and 0.00064 μM using enzyme-free water; 1.25 μL of Lipofectamine RNAiMAX (Invitrogen, catalog number 13778150) transfection reagent was added to 25 μL of OPTI-MEM medium (Thermo, catalog number 11058021) and mixed by pipetting; then 1.25 μL of the siRNA dilutions of different concentrations was added to 25 μL of OPTI-MEM medium containing the transfection reagent, pipetting and mixing, and standing at room temperature for 5 minutes; the complex was added to the culture wells, and 225 μL of RPMI 1640 complete medium was added to each well, and the culture was continued for 48 hours. Dose response experiments were performed using 6 x 5-fold serial dilutions starting at a final duplex concentration of 10 nM. siRNAs used for transfection were the sequences shown for M03 and M12, and all transfections were performed in duplicate.

[0395] 1. qRT-PCR Detection

[0396] 48 h after siRNA transfection, total cellular RNA was extracted using the RNA extraction kit TaKaRa MiniBEST Universal RNA Extraction Kit (TAKARA, catalog number 9767) according to the total RNA extraction procedure in the instruction manual.

[0397] For the extracted total cell RNA, 500 ng of total RNA was used as a template for reverse transcription using the PrimeScript reverse transcription kit. TM Using the reagents provided by RT Master Mix (Takara, Cat. No. RR036A), prepare 10 μL of the reverse transcription reaction system according to the reverse transcription protocol in the kit instructions and reverse transcribe total cellular RNA. Reverse transcription conditions are as follows: incubate the reaction system at 37°C for 15 minutes, followed by incubation at 85°C for 5 seconds. After completion of the reaction, a solution containing cDNA is obtained.

[0398] For each reverse transcription reaction system, the cDNA-containing solution was used as a template for qPCR. A 20 μL qPCR reaction system was prepared using the reagents provided in the ChamQ SYBR qPCR Master Mix kit (Novozymes Biotech, Cat. No. Q331). The PCR primer sequences for amplifying the target gene PD-L1 and the internal reference gene GAPDH are shown in Table 12, with a final concentration of 10 μM for each primer. The qPCR reaction system was placed on a LightCycler 480 fluorescence quantitative PCR instrument and amplified using a three-step method. The amplification program was pre-denaturation at 95°C for 3 minutes, followed by denaturation at 95°C for 10 seconds, and annealing at 60°C for 30 seconds. This denaturation and annealing process was repeated 40 times to obtain a PCR amplification product containing the target gene PD-L1 and the internal reference gene GAPDH. The PCR amplification products were then incubated at 95°C for 5s, 65°C for 1min, and 97°C for 15s. The melting curves of the target gene PD-L1 and the internal reference gene GAPDH in the PCR amplification products were collected by real-time fluorescence quantitative PCR instrument to obtain the Ct values ​​of the target gene PD-L1 and the internal reference gene GAPDH.

[0399] Table 12 Primer sequences

[0400] The relative quantitative method, namely the ΔΔCt value method, was used to calculate the expression level of PD-L1 mRNA.

[0401] The calculation method is as follows:

[0402] ΔCt(siRNA)=Ct(PD-L1)–Ct(GAPDH)

[0403] ΔCt(NC)=Ct(PD-L1)–Ct(GAPDH)

[0404] ΔΔCt(siRNA)=ΔCt(siRNA)-ΔCt(average NC)

[0405] ΔΔCt(NC)=ΔCt(NC)-ΔCt(average NC)

[0406] PD-L1 mRNA relative expression level = 2^(-ΔΔCt(siRNA))×100%,

[0407] siRNA inhibition rate of PD-L1 mRNA = (1-2^(-ΔΔCt(siRNA))) × 100%;

[0408] The concentration-inhibition rate (%) data were processed using EXCEL software, and the EC value of siRNA inhibition of PD-L1 was calculated using a four-parameter nonlinear regression model. 50 .

[0409] The results are shown in Table 13.

[0410] Table 13 EC of qRT-PCR method 50

[0411] Experimental Example 3 Evaluation of in vivo activity of human PD-L1 model mice A

[0412] 6-8 week old male human PD-L1 mice (purchased from Shanghai Nanmo Biotechnology Co., Ltd.) were randomly divided into groups according to body weight, with 3 mice in each group, respectively designated as PBS group and each sample group (dsRNA ligand conjugate of the present application).

[0413] Each sample group (dsRNA ligand conjugate of the present application) was prepared into a 1 mg / mL solution using 1×PBS (pH 7.4).

[0414] Each mouse in the PBS group was subcutaneously injected with 10 mL / kg body weight of 1×PBS (pH 7.4), and each mouse in each sample group (dsRNA ligand conjugate of the present application) was subcutaneously injected with the corresponding above-prepared solution. The dosage volume for each animal was 10 mL / kg body weight, and the dosage was 10 mg / kg body weight. The day of the first administration was set as day 0. All mice were killed 14 days after administration, and the livers were collected and stored in 1.5 mL sterile EP tubes containing 1 mL of animal tissue RNA stabilization solution (Shanghai Biyuntian Biotechnology Co., Ltd., article number R0118). After being placed in a 4°C refrigerator for 24 hours, it was transferred to -80°C for storage. The liver tissue of each mouse was then homogenized with a tissue homogenizer, and the total RNA of the liver tissue of each mouse was extracted separately using the RNA extraction kit TaKaRa MiniBEST Universal RNA Extraction Kit (TAKARA, article number 9767) according to the operating steps of the total RNA extraction in the instruction manual.

[0415] For each mouse liver tissue, 1 μg of total RNA was used as a template for reverse transcription. Using the reagents provided in the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Novozymes Biotech, Cat. No. R312), 20 μL of the reverse transcription reaction system was prepared according to the reverse transcription protocol in the kit's instructions. Reverse transcription was performed on the obtained liver total RNA. The reverse transcription conditions were as follows: incubate the reaction system at 37°C for 15 minutes, followed by incubation at 85°C for 5 seconds. After completion of the reaction, a solution containing cDNA was obtained.

[0416] For each reverse transcription reaction system, the cDNA-containing solution was used as a template for qPCR. 20 μL of the qPCR reaction system was prepared using the reagents provided in the ChamQ SYBR qPCR Master Mix kit (Novozymes Biotech, Cat. No. Q311). The PCR primer sequences for amplifying the target genes PD-L1 and GAPDH are shown in Table 14, with a final concentration of 0.2 μM for each primer. The qPCR reaction system was placed on a LightCycler 480 fluorescence quantitative PCR instrument and amplified using a three-step method. The amplification program was pre-denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 10 seconds. This denaturation, annealing, and extension process was repeated 40 times to obtain a PCR amplification product containing the target gene PD-L1 and the internal reference gene GAPDH. The PCR amplification products were then incubated at 95°C for 5s, 65°C for 1min, and 97°C for 15s. The melting curves of the target gene PD-L1 and the internal reference gene GAPDH in the PCR amplification products were collected by real-time fluorescence quantitative PCR instrument to obtain the Ct values ​​of the target gene PD-L1 and the internal reference gene GAPDH.

[0417] Table 14 Primer sequences

[0418] The comparative Ct (ΔΔCt) method was used to calculate the inhibition rate of the dsRNA ligand conjugate of the present application on PD-L1 mRNA. The calculation method is as follows:

[0419] ΔCt(test group) = Ct(test group target gene) – Ct(test group reference gene)

[0420] ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene)

[0421] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average)

[0422] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0423] Wherein, the test group is each sample group (dsRNA ligand conjugate of the present application), the control group is the PBS group, and ΔCt (control group average) is the arithmetic mean of the ΔCt (control group) of each animal in the PBS group. Thus, each animal in each sample group (dsRNA ligand conjugate of the present application) corresponds to a ΔΔCt (test group), and each animal in the PBS group corresponds to a ΔΔCt (control group).

[0424] The inhibition rate of dsRNA ligand conjugate on PD-L1 mRNA of each animal in the test group = (1-2^(-ΔΔCt(test group)) / 2^(-ΔΔCt(control group)))×100%,

[0425] The inhibition rate of each dsRNA ligand conjugate on PD-L1 mRNA in liver tissue is shown in Table 15.

[0426] Table 15 Inhibition rate of dsRNA ligand conjugates on PD-L1 mRNA

[0427] Experimental Example 4 Evaluation of in vivo activity of human PD-L1 model mice B

[0428] 6-8 week old male human PD-L1 mice (purchased from Shanghai Nanmo Biotechnology Co., Ltd.) were randomly divided into groups according to body weight, with 3 mice in each group, respectively designated as PBS group and each sample group (dsRNA ligand conjugate of the present application).

[0429] Each sample group (the dsRNA ligand conjugate of the present application) was prepared into a 1 mg / mL solution using 1×PBS (pH 7.4).

[0430] Each mouse in the PBS group was subcutaneously injected with 10 mL / kg body weight of 1×PBS (pH 7.4), and each mouse in each sample group (dsRNA ligand conjugate of the present application) was subcutaneously injected with the corresponding above-prepared solution. The dosage volume for each animal was 10 mL / kg body weight, and the dosage was 10 mg / kg body weight. The day of the first administration was set as day 0. Liver tissue was taken from the body 14 days and 28 days after administration and stored in 1.5 mL sterile EP tubes containing 1 mL animal tissue RNA stabilization solution (Shanghai Biyuntian Biotechnology Co., Ltd., article number R0118). After being placed in a 4°C refrigerator for 24 hours, it was transferred to -80°C for storage. The liver tissue of each mouse was then homogenized with a tissue homogenizer, and the total RNA of the liver tissue of each mouse was extracted according to the operating steps of the total RNA extraction in the instruction manual using the RNA extraction kit TaKaRa MiniBEST Universal RNA Extraction Kit (TAKARA, article number 9767).

[0431] The reverse transcription synthesis of cDNA, fluorescence quantitative PCR operation steps and primers and inhibition rate calculation are shown in Experimental Example 3.

[0432] The inhibition rate of each dsRNA ligand conjugate on PD-L1 mRNA in liver tissue is shown in Table 16.

[0433] Table 16 Inhibition rate of dsRNA ligand conjugates on PD-L1 mRNA

[0434] Experimental Example 5 Evaluation of AAV-hPD-L1 Activity in Mice

[0435] AAV-CTM-PDL1 (human full-length PD-L1 gene, titer 6.25×10 13 vg / mL, purchased from Shandong Weizhen Biotechnology Co., Ltd.) was prepared with sterile PBS to a concentration of 5×10 11 vg / mL solution. Male C57BL / 6 mice aged 4-6 weeks (purchased from Shanghai Bikeway Biotechnology Co., Ltd.) were injected with 200 μL of the above-prepared solution with a concentration of 5×10 11 vg / mL AAV-CTM-PDL1. 14 days after infection, livers of 2-5 mice were harvested to detect PDL1 mRNA expression levels. A Ct value of 24±2 was observed, indicating successful model establishment. Mice were randomly divided into groups of 4 according to body weight.

[0436] Each sample group (dsRNA ligand conjugate of the present application) was prepared into a 1 mg / mL solution using 1×PBS (pH 7.4).

[0437] Each mouse in the PBS group was subcutaneously injected with 10 mL / kg body weight of 1×PBS (pH 7.4), and each mouse in each sample group was subcutaneously injected with the corresponding solution prepared above. The dosage volume for each animal was 10 mL / kg body weight, and the dosage was 10 mg / kg body weight. The day of the first administration was set as day 0. Liver tissues were taken from the body 14 days and 28 days after administration and stored in 1.5 mL sterile EP tubes containing 1 mL of animal tissue RNA stabilization solution (Shanghai Biyuntian Biotechnology Co., Ltd., product number R0118). After being placed in a 4°C refrigerator for 24 hours, they were transferred to -80°C for storage. Subsequently, the liver tissues of each mouse were homogenized with a tissue homogenizer, and then the total RNA of each mouse liver tissue was extracted using an automatic nucleic acid extractor (Aosheng, model AUTO-PURE96) and the RNA extraction kit Universal RNA Extraction CZ Kit (H) (Onrew, product number RNC643-03H) according to the total RNA extraction steps in the instruction manual.

[0438] For each mouse liver tissue, 500 ng of total RNA was used as a template for reverse transcription. Using the reagents provided in the PrimeScript™ RT Master Mix (Perfect Real Time) Reverse Transcription Kit (TaKaRa, Cat. No. RR036A), 10 μL of the reverse transcription reaction system was prepared according to the reverse transcription protocol in the kit instructions. Reverse transcription was performed on the obtained liver total RNA. The reverse transcription conditions were as follows: incubate the reaction system at 37°C for 15 minutes, followed by incubation at 85°C for 5 seconds. After completion of the reaction, a solution containing cDNA was obtained.

[0439] The fluorescence quantitative PCR operation steps, primers and inhibition rate calculation are shown in Experimental Example 3.

[0440] According to the test results, the dsRNA ligand conjugate of the present application has a good inhibitory effect on the inhibition rate of PD-L1 mRNA in liver tissue, and the PD-L1 expression inhibition rate 14 days or 28 days after administration is greater than 50%, preferably greater than 60% or more preferably greater than 70%.

[0441] Test Example 6 Evaluation of in vitro liver homogenate stability

[0442] 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.

[0443] 2) Pipette 40 μL of the stock solution of the dsRNA ligand conjugate to be tested (concentration is 1 mg / mL) and add 360 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).

[0444] 3) Obtain blank liver homogenate from mice and incubate at 37°C for 5 minutes, 24 hours, 42 hours, and 48 hours.

[0445] 4) Pipette 720 μL of blank liver homogenate after 48 h incubation into a 1.5 mL Eppendorf tube, add 80 μL of the test compound stock solution (100 μg / mL), and vortex to mix. This is the 0 h sample.

[0446] 5) Pipette 720 μL of blank liver homogenate after 5 min incubation into a 1.5 mL Eppendorf tube, add 80 μL of the test compound stock solution (100 μg / mL), vortex mix, and place on a shaker (37°C, 300 rpm) for 48 h to prepare the 48 h sample.

[0447] 6) Sample pretreatment:

[0448] (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.

[0449] (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.

[0450] (3) 185 μL of the supernatant after centrifugation in step 1) was aspirated and loaded onto the SPE kit.

[0451] (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).

[0452] (5) Elution was performed twice with 1 mL of an eluent (100 mM ammonium bicarbonate (pH = 9.5) + 40% acetonitrile + 10% tetrahydrofuran).

[0453] (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.

[0454] 7) LC-MS / MS was used to detect the sample concentration, with the zero point as the reference, and the peak area of ​​the analyte internal standard was used to calculate the remaining percentage and analyze the data.

[0455] According to the test results, the dsRNA ligand conjugate of the present application has good stability. The remaining amount of the sense chain after incubation for 48 hours is more than 30% or 40%, and the remaining amount of the antisense chain after incubation for 48 hours is more than 60% or 80%.

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 in the nucleotide sequence as shown in SEQ ID NO.9 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides in the nucleotide sequence as shown in SEQ ID NO.10 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides in the nucleotide sequence as shown in SEQ ID NO.11 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides in the nucleotide sequence as shown in SEQ ID NO.12 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand comprises at least 15 consecutive nucleotides in the nucleotide sequence as shown in SEQ ID NO.13 and the length of the sense strand does not exceed 21 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides in the nucleotide sequence as shown in SEQ ID NO. NO.14 contains at least 15 consecutive nucleotides and the antisense strand is no longer than 23 nucleotides; or, the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.15 and the sense strand is no longer than 23 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.16 and the antisense strand is no longer than 25 nucleotides; or, the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.17 and the sense strand is no longer than 23 nucleotides, and 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 25 nucleotides; or, 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 23 nucleotides, and 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 25 nucleotides; or, the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO. NO.21, and the length of the sense strand is no more than 23 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.22, and the length of the antisense strand is no more than 25 nucleotides; or, the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.23, and the length of the sense strand is no more than 23 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.24, and the length of the antisense strand is no more than 25 nucleotides; or, the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.25, and the length of the sense strand is no more than 23 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.26, and the length of the antisense strand is no more than 25 nucleotides; or, the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.27, and the length of the sense strand is no more than 23 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.28, and the length of the antisense strand is no more than 25 nucleotides; or, the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.29, and the length of the sense strand is no more than 21 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.30, and the length of the antisense strand is no more than 23 nucleotides; or, the sense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO. At least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.53 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO.54 and the length of the antisense strand does not exceed 25 nucleotides:. Sense strand: 5'-AGCAAUAUGACAAUUGAAUGA-3' (SEQ ID NO. 9), Antisense strand: 5'-UCAUUCAAUUGUCAUAUUGCUAC-3' (SEQ ID NO. 10); Sense strand: 5'-AUUUACUGUCACGGUUCCCAA-3' (SEQ ID NO.11), Antisense strand: 5'-UUGGGAACCGUGACAGUAAUAGC-3' (SEQ ID NO. 12); Sense strand: 5'-CUAUUUAUUUUGAGUCUGU-3' (SEQ ID NO. 13), Antisense strand: 5'-ACAGACUCAAAAUAAAUAGGA-3' (SEQ ID NO. 14); Sense strand: 5'UGAAGAUAUAUUGUAGUAGAU-3' (SEQ ID NO. 15), Antisense strand: 5'-AUCUACUACAAUAUAUCUUCAAA-3' (SEQ ID NO. 16); Sense strand: 5'-UUGUCUCAUGUUUCAUCGUAA-3' (SEQ ID NO. 17), Antisense strand: 5'-UUACGAUGAAACAUGAGACAAAA-3' (SEQ ID NO. 18); Sense strand: 5'-CUGUGCAGUAUCUGUUCCAUU-3' (SEQ ID NO. 19), Antisense strand: 5'-AAUGGAACAGAUACUGCACAGAC-3' (SEQ ID NO. 20); Sense strand: 5'-AAUGAAAGGACUCACUUGGUA-3' (SEQ ID NO. 21), Antisense strand: 5'-UACCAAGUGAGUCCUUUCAUUUG-3' (SEQ ID NO. 22); Sense strand: 5'-GCUGCAUGAUCAGCUAUGGUA-3' (SEQ ID NO. 23), Antisense strand: 5'-UACCAUAGCUGAUCAUGCAGCGG-3' (SEQ ID NO. 24); Sense strand: 5'-GGUGCUUGGUCUCCUCUAUAA-3' (SEQ ID NO. 25), Antisense strand: 5'-UUAUAGAGGAGACCAAGCACCUU-3' (SEQ ID NO. 26); Sense strand: 5'-AUUGGUCAUCCCAGAACUACA-3' (SEQ ID NO. 27), Antisense strand: 5'-UGUAGUUCUGGGAUGACCAAUUC-3' (SEQ ID NO. 28); Sense strand: 5'-UAUUUAUUUUGAGUCUGUG-3' (SEQ ID NO. 29), Antisense strand: 5'-CACAGACUCAAAAUAAAUAGG-3' (SEQ ID NO. 30); Sense strand: 5'-AUUUACUGUCACGGUUCCCAA-3' (SEQ ID NO.53), Antisense strand: 5'-UUGGGAACCGUGACAGUAAAUGC-3' (SEQ ID NO. 54); 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 the nucleotide sequence shown in SEQ ID NO.9, and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.10, and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.11, and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.12, and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand contains 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 the nucleotide sequence shown in SEQ ID NO.14, and the length of the antisense strand does not exceed 23 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.15, and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.16, and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.17, and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO. NO.18 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.19 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.20 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.21 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.22 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.23 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.24 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.25 and the length of the sense strand does not exceed 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.26 and the length of the antisense strand does not exceed 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO. NO.27 and the length of the sense strand is no more than 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.28 and the length of the antisense strand is no more than 25 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.29 and the length of the sense strand is no more than 21 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.30 and the length of the antisense strand is no more than 23 nucleotides; or, the sense strand contains the nucleotide sequence shown in SEQ ID NO.53 and the length of the sense strand is no more than 23 nucleotides, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.54 and the length of the antisense strand is no more than 25 nucleotides.

3. The double-stranded ribonucleic acid, its pharmaceutically acceptable salt or its ligand conjugate according to claim 1, wherein: The sense strand is a nucleotide sequence as shown in SEQ ID NO.9, and the antisense strand is a nucleotide sequence as shown in SEQ ID NO.10; or, the sense strand is a nucleotide sequence as shown in SEQ ID NO.11, and the antisense strand is a nucleotide sequence as shown in SEQ ID NO.12; or, the sense strand is a nucleotide sequence as shown in SEQ ID NO.13, and the antisense strand is a nucleotide sequence as shown in SEQ ID NO.14; or, the sense strand is a nucleotide sequence as shown in SEQ ID NO.15, and the antisense strand is a nucleotide sequence as shown in SEQ ID NO.16; or, the sense strand is a nucleotide sequence as shown in SEQ ID NO.17, and the antisense strand is a nucleotide sequence as shown in SEQ ID NO.18; or, the sense strand is a nucleotide sequence as shown in SEQ ID NO.19, and the antisense strand is a nucleotide sequence as shown in SEQ ID NO.20; or, the sense strand is a nucleotide sequence as shown in SEQ ID NO.21, and the antisense strand is a nucleotide sequence as shown in SEQ ID NO.22; or, the sense strand is a nucleotide sequence as shown in SEQ ID NO. The nucleotide sequence of SEQ ID NO.23 is as shown in SEQ ID NO.23, and the antisense strand is the nucleotide sequence as shown in SEQ ID NO.24; or, the sense strand is the nucleotide sequence as shown in SEQ ID NO.25, and the antisense strand is the nucleotide sequence as shown in SEQ ID NO.26; or, the sense strand is the nucleotide sequence as shown in SEQ ID NO.27, and the antisense strand is the nucleotide sequence as shown in SEQ ID NO.28; or, the sense strand is the nucleotide sequence as shown in SEQ ID NO.29, and the antisense strand is the nucleotide sequence as shown in SEQ ID NO.30; or, the sense strand is the nucleotide sequence as shown in SEQ ID NO.53, and the antisense strand is the nucleotide sequence as shown in SEQ ID NO.

54.

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 double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof optionally 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 as 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 additional sequence as the overhang is selected from u or dT; or when the overhang is 2 nucleotides, the additional sequence as the overhang is selected from uu or dTdT; or when the overhang is 2 nucleotides, the additional sequence as the overhang is selected from unmodified or modified GA.

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, 21, 22 or 23 nucleotides of the sense strand are modified, or the nucleotides within the range formed by any of the foregoing values ​​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, more than 21, more than 22 or more 23 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, 23, 24 or 25 nucleotides of the antisense strand are modified, or nucleotides within the range formed by any of the foregoing values ​​are modified; or more than 1, more than 2, more than 3, more than 4, more than 5 nucleotides 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, 23, 24, or 25 nucleotides 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 8, 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 / or the first and second nucleotides of the antisense chain, the second and third nucleotides of the antisense chain, the first and second nucleotides at the 3' end of the antisense chain and / or the second and third 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, 17, 18 or 19 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 among nucleotides 1 to 6 or 1 to 8 of the sense strand are 2'-fluoro-modified, 4, 5, 6, 7 or 8 nucleotides among nucleotides 1 to 6 or 1 to 8 of the sense strand are 2'-O-methyl-modified, 2 or 3 nucleotides among nucleotides 7 to 9 or 9 to 11 of the sense strand are 2'-fluoro-modified, 0 or 1 nucleotide among nucleotides 7 to 9 or 9 to 11 of the sense strand are 2'-O-methyl-modified, 0 or 1 nucleotide among nucleotides 10 to 19 or 12 to 21 of the sense strand are 2'-fluoro-modified, and 8, 9, 10, 11 or 12 nucleotides among nucleotides 10 to 19 or 12 to 21 of the sense strand are 2'-O-methyl-modified; 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 9th, 10th and 11th nucleotides of the sense chain are modified with 2'-fluoro, and 0, 1 or 2 nucleotides among the 1st to 8th and / or 12th to 21st 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 16, 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: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:53, 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, or SEQ ID NO:55; and The double-stranded ribonucleic acid comprises any one of the following antisense strands: 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:54, 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 or SEQ ID NO:

56.

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 RNA comprises the following sequence pairs of sense strand and antisense strand: 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:53 / SEQ ID NO:54, 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 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:55 / SEQ ID NO:

56.

22. 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: a) the sense strand comprises 5'-asgscaauauGfAfCfaauugaaugaL01-3' (SEQ ID NO: 31), and the antisense strand comprises 5'-usCfsauuCfaauugucAfuAfuugcusasc-3' (SEQ ID NO: 32); b) the sense strand comprises 5'-asgscaauauGfAfCfaauugaaugaL02-3' (SEQ ID NO: 31), and the antisense strand comprises 5'-usCfsauuCfaauugucAfuAfuugcusasc-3' (SEQ ID NO: 32); c) the sense strand comprises 5'-asusuuacugUfCfAfcgguucccaaL01-3' (SEQ ID NO: 33), and the antisense strand comprises 5'-usUfsgggAfaccgugaCfaGfuaauasgsc-3' (SEQ ID NO: 34); d) the sense strand comprises 5'-asusuuacugUfCfAfcgguucccaaL02-3' (SEQ ID NO: 33), and the antisense strand comprises 5'-usUfsgggAfaccgugaCfaGfuaauasgsc-3' (SEQ ID NO: 34); e) the sense strand comprises 5'-csusauuuAfUfUfuugagucuguL01-3' (SEQ ID NO: 35), and the antisense strand comprises 5'-asCfsagaCfucaaaauAfaAfuagsgsa-3' (SEQ ID NO: 36); f) the sense strand comprises 5'-csusauuuAfUfUfuugagucuguL02-3' (SEQ ID NO: 35), and the antisense strand comprises 5'-asCfsagaCfucaaaauAfaAfuagsgsa-3' (SEQ ID NO: 36); g) the sense strand comprises 5'-usgsaagauaUfAfUfuguaguagauL01-3' (SEQ ID NO: 37), and the antisense strand comprises 5'-asUfscuaCfuacaauaUfaUfcuucasasa-3' (SEQ ID NO: 38); h) the sense strand comprises 5'-usgsaagauaUfAfUfuguaguagauL02-3' (SEQ ID NO: 37), and the antisense strand comprises 5'-asUfscuaCfuacaauaUfaUfcuucasasa-3' (SEQ ID NO: 38); i) the sense strand comprises 5'-ususgucucaUfGfUfuucaucguaaL01-3' (SEQ ID NO: 39), and the antisense strand comprises 5'-usUfsacgAfugaaacaUfgAfgacaasasa-3' (SEQ ID NO: 40); j) the sense strand comprises 5'-ususgucucaUfGfUfuucaucguaaL02-3' (SEQ ID NO: 39), and the antisense strand comprises 5'-usUfsacgAfugaaacaUfgAfgacaasasa-3' (SEQ ID NO: 40); k) the sense strand comprises 5'-csusgugcagUfAfUfcuguuccauuL01-3' (SEQ ID NO: 41), and the antisense strand comprises 5'-asAfsuggAfacagauaCfuGfcacagsasc-3' (SEQ ID NO: 42); 1) the sense strand comprises 5'-csusgugcagUfAfUfcuguuccauuL02-3' (SEQ ID NO: 41), and the antisense strand comprises 5'-asAfsuggAfacagauaCfuGfcacagsasc-3' (SEQ ID NO: 42); m) the sense strand comprises 5'-asasugaaagGfAfCfucacuugguaL01-3' (SEQ ID NO: 43), and the antisense strand comprises 5'-usAfsccaAfgugagucCfuUfucauususg-3' (SEQ ID NO: 44); n) the sense strand comprises 5'-asasugaaagGfAfCfucacuugguaL02-3' (SEQ ID NO: 43), and the antisense strand comprises 5'-usAfsccaAfgugagucCfuUfucauususg-3' (SEQ ID NO: 44); o) the sense strand comprises 5'-gscsugcaugAfUfCfagcuaugguaL01-3' (SEQ ID NO: 45), and the antisense strand comprises 5'-usAfsccaUfagcugauCfaUfgcagcsgsg-3' (SEQ ID NO: 46); p) the sense strand comprises 5'-gscsugcaugAfUfCfagcuaugguaL02-3' (SEQ ID NO: 45), and the antisense strand comprises 5'-usAfsccaUfagcugauCfaUfgcagcsgsg-3' (SEQ ID NO: 46); q) the sense strand comprises 5'-gsgsugcuugGfUfCfuccucuauaaL01-3' (SEQ ID NO: 47), and the antisense strand comprises 5'-usUfsauaGfaggagacCfaAfgcaccsusu-3' (SEQ ID NO: 48); r) the sense strand comprises 5'-gsgsugcuugGfUfCfuccucuauaaL02-3' (SEQ ID NO: 47), and the antisense strand comprises 5'-usUfsauaGfaggagacCfaAfgcaccsusu-3' (SEQ ID NO: 48); s) the sense strand comprises 5'-asusuggucaUfCfCfcagaacuacaL01-3' (SEQ ID NO: 49), and the antisense strand comprises 5'-usGfsuagUfucugggaUfgAfccaaususc-3' (SEQ ID NO: 50); t) the sense strand comprises 5'-asusuggucaUfCfCfcagaacuacaL02-3' (SEQ ID NO: 49), and the antisense strand comprises 5'-usGfsuagUfucugggaUfgAfccaaususc-3' (SEQ ID NO: 50); u) the sense strand comprises 5'-usasuuuaUfUfUfugagucugugL01-3' (SEQ ID NO: 51), and the antisense strand comprises 5'-csAfscagAfcucaaaaUfaAfauasgsg-3' (SEQ ID NO: 52); v) the sense strand comprises 5'-usasuuuaUfUfUfugagucugugL02-3' (SEQ ID NO: 51), and the antisense strand comprises 5'-csAfscagAfcucaaaaUfaAfauasgsg-3' (SEQ ID NO: 52); w) the sense strand comprises 5'-asusuuacugUfCfAfcgguucccaaL01-3' (SEQ ID NO: 55), and the antisense strand comprises 5'-usUfsgggAfaccgugaCfaGfuaaausgsc-3' (SEQ ID NO: 56); x) the sense strand comprises 5'-asusuuacugUfCfAfcgguucccaaL02-3' (SEQ ID NO: 55), and the antisense strand comprises 5'-usUfsgggAfaccgugaCfaGfuaaausgsc-3' (SEQ ID NO: 56).

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 hepatitis B virus infection.

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.