DsRNA molecule for inhibiting HBV gene expression and application thereof

Abstract

The invention provides a dsRNA molecule for inhibiting HBV gene expression and application of the dsRNA molecule. Specifically, the present disclosure provides a dsRNA molecule or ddsRNA molecule that inhibits hepatitis B virus (HBV) gene expression, comprising a sense strand and an antisense strand complementary to each other to form a double-stranded region, the sense strand and / or the antisense strand comprising or consisting of 15-25 nucleotides, the dsRNA molecule or ddsRNA molecule can be used for treating diseases caused by HBV infection, such as HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, HBV infection, in particular chronic hepatitis B (CHB) and / or chronic hepatitis D (CHD) diseases in humans.

Description

A dsRNA molecule for inhibiting HBV gene expression and its application

[0001] Cross-references

[0002] This disclosure claims priority to a Chinese patent application filed with the Patent Office of China on August 10, 2023, with application number 202311004622.7 and invention name “A dsRNA molecule for inhibiting HBV gene expression and its application,” the entire contents of which are incorporated by reference into this disclosure. Technical Field

[0003] The present disclosure belongs to the field of molecular biology, and specifically relates to a dsRNA molecule for inhibiting HBV gene expression and its application, as well as a method for reducing HBV gene expression levels using the dsRNA or its pharmaceutical composition. Background Art

[0004] Hepatitis B virus (HBV) is a strictly hepatotropic, double-stranded DNA virus. Globally, more than 400 million people are chronically infected with HBV and are therefore at increased risk of developing severe liver diseases such as chronic hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma (HCC), which results in an estimated 600,000 deaths annually. The natural evolution of chronic HBV infection consists of four stages: (1) an early "immune tolerance" stage with high levels of viral replication and minimal liver inflammation; (2) an immune response stage with marked liver inflammation and elevated serum transaminases; some patients also progress to the following stages: (3) a "non-replicating" stage with undetectable or low-level viremia (less than 2000 IU / ml by PCR-based assays) and resolution of liver inflammation; and (4) HBeAg-negative chronic hepatitis B, which is caused by the development of specific viral mutations that prevent HBeAg production but do not prevent viral replication. This form of chronic hepatitis B (CHB) is characterized by fluctuating serum HBV DNA levels, serum transaminase (ALT and AST) levels, and progressive liver disease. Longitudinal studies of patients with CHB have shown that the 5-year cumulative incidence of cirrhosis is 8%-20%, the 5-year cumulative incidence of hepatic decompensation is approximately 20%, and the annual incidence of HBV-related HCC is 2-5% at the time of cirrhosis.

[0005] Hepatitis D virus (HDV), also known as hepatitis delta virus, is a virus that requires the hepatitis B virus to replicate. HDV infection occurs simultaneously with HBV or as a dual infection. HBV-HDV co-infection is the most severe form of chronic viral hepatitis because it progresses more rapidly to liver-related death and hepatocellular carcinoma.

[0006] The HBV genome contains four overlapping open reading frames. Targeting the X gene can effectively inhibit the expression of all four HBV transcripts simultaneously, suppressing the secretion of HBsAg, HBeAg, and HBV DNA. Therefore, targeting the X gene is a potential HBV therapeutic target. HBeAg-negative chronic hepatitis B is at a relatively late stage in the natural history of chronic HBV. Studies have shown that in chimpanzees and humans, the majority of HBsAg in HBeAg-negative individuals is derived from integrated HBV DNA. These integrated DNA sites can disrupt the X gene's target site, resulting in off-target effects and reduced HBsAg inhibition. Based on these findings, simultaneously targeting both the X and S genes in the HBV genome is a promising approach for treating patients with chronic HBV.

[0007] The main goal of HBV treatment is to permanently suppress HBV replication and improve liver disease. The current standard of care for treating chronic HBV infection is daily oral nucleoside analogs or injections of pegylated interferon. However, the cure rate after treatment remains low (3-7% for pegylated interferon and 1-12% for nucleoside analogs) and drug resistance is easy to develop. Given the serious unmet clinical needs of existing anti-HBV treatments, the search for an effective anti-HBV drug or treatment is urgent.

[0008] Summary of the Invention

[0009] The present disclosure provides a dsRNA or ddsRNA molecule that inhibits hepatitis B virus gene expression, as well as methods and uses of the dsRNA or ddsRNA molecule for inhibiting or reducing hepatitis B virus gene expression and preventing or treating diseases or symptoms mediated by hepatitis B virus infection. The dsRNA or ddsRNA molecule promotes the specific degradation of hepatitis B virus transcripts through RNAi, thereby suppressing the expression and secretion levels of HBV antigens and HBV DNA.

[0010] The present disclosure provides a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting HBV gene expression, wherein the dsRNA comprises a sense strand and an antisense strand that complement each other to form a double-stranded region, wherein the sense strand and / or the antisense strand comprises at least 15, 16, 17, 18, 19 or 20 consecutive nucleotides of the nucleotide sequence of any sense sequence and / or antisense sequence listed in Table 1 or Table 2; optionally, the length of the double-stranded region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs.

[0011] In some embodiments, each strand of the dsRNA independently has 15-30 nucleotides, optionally independently has 19-23 nucleotides, further optionally independently has 21-23 nucleotides.

[0012] In some alternative embodiments, each strand of the dsRNA molecule has an overhang or blunt end. This includes 1-8 nucleotide overhangs, such as 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides, at the 5' and / or 3' ends of either or both strands, with the overhangs being arbitrarily selected from U, A, G, C, T, and dT. In some embodiments, the antisense strand of the dsRNA molecule has a 2-nucleotide overhang at its end; alternatively, the 3' end of the antisense strand of the dsRNA molecule has a 2-nucleotide overhang; further alternatively, when the sense strand of the dsRNA is 21 nucleotides, the 3' end of the antisense strand has a 2-nucleotide overhang, totaling 23 nucleotides.

[0013] In some embodiments, the present disclosure provides a dsRNA molecule comprising a sense strand and an antisense strand, wherein the nucleotide sequences of the sense strand and the antisense strand are shown in Tables 1 and 2. In Tables 1 and 2, each name number corresponds to a dsRNA molecule consisting of a sense strand and an antisense strand. For example, the dsRNA molecule numbered 101 in Table 2 consists of a sense strand and an antisense strand, the sequence of the sense strand is shown in SEQ ID NO. 271, and the sequence of the antisense strand is shown in SEQ ID NO. 272.

[0014] In some embodiments, the present disclosure provides a dsRNA molecule comprising a sense strand and an antisense strand, wherein the sense strand nucleotide sequence is shown in SEQ ID NO. 393 (5-3' direction), and / or the antisense strand nucleotide sequence is shown in SEQ ID NO. 394 (5-3' direction).

[0015] In some embodiments, the present disclosure provides a dsRNA molecule comprising a sense strand and an antisense strand, wherein the sense strand nucleotide sequence is shown in SEQ ID NO. 395 (5-3' direction), and / or the antisense strand nucleotide sequence is shown in SEQ ID NO. 396 (5-3' direction).

[0016] In some embodiments, the present disclosure provides a dsRNA molecule comprising a sense strand and an antisense strand, wherein the sense strand nucleotide sequence is shown in SEQ ID NO. 427 (5-3' direction), and / or the antisense strand nucleotide sequence is shown in SEQ ID NO. 428 (5-3' direction).

[0017] In some embodiments, at least one nucleotide in the dsRNA molecule is modified. The modification is selected from any one or more of the following: locked nucleic acid (LNA) modification, open ring or unlocked (UNA) modification, 2′-methoxyethyl modification, 2′-O-methyl modification, 2′-O-allyl modification, 2′-C-allyl modification, 2′-fluoro modification, 2′-deoxy modification, 2′-hydroxyl modification, phosphorothioate backbone modification, DNA modification, and fluorescent probe modification.

[0018] In some embodiments, all nucleotides in the dsRNA molecule are modified nucleotides.

[0019] In some embodiments, the modified nucleotides are each independently selected from a 2'-O-methyl modification and a 2'-fluoro modification.

[0020] In some embodiments, the dsRNA molecule comprises at least one phosphorothioate internucleotide linkage; alternatively, the dsRNA molecule comprises 6 phosphorothioate internucleotide linkages, and / or the phosphorothioate internucleotide linkages of the dsRNA molecule are located between the 3'-terminal three nucleotides of the antisense strand, between the 5'-terminal three nucleotides of the antisense strand, and / or between the 5'-terminal three nucleotides of the sense strand; further alternatively, the phosphorothioate internucleotide linkages of the dsRNA molecule are two phosphorothioate linkages between the 3'-terminal three nucleotides of the antisense strand, two phosphorothioate linkages between the 5'-terminal three nucleotides of the antisense strand, and two phosphorothioate linkages between the 5'-terminal three nucleotides of the sense strand.

[0021] In some embodiments, the modification methods of the dsRNA molecules provided by the present disclosure include: (1) sense strand: 17-21 nt in length, such as 21 nt; composed of alternating 2′-O-methyl modified regions and 2′-fluoro modified regions, each modified region is 1 to 3 nucleotides in length; the modification methods of the 5′ end and the first modified region from the 3′ end are the same; (2) antisense strand: 19-23 nt in length, such as 23 nt; composed of alternating 2′-O-methyl modified regions, 2′-fluoro modified regions, unmodified regions or DNA regions, each modified region is 1 to 5 nucleotides in length; and the continuous nucleotide region from the 2nd to the 5th position from the 5′ end and the continuous nucleotide region from the 1st to the 3rd position from the 3′ end are both connected by a thiophosphate backbone.

[0022] In another embodiment of the present disclosure, the dsRNA comprises: (1) the antisense strand has a 2-nucleotide overhang at the 3' end; (2) the antisense strand is modified with 2'-fluoro at least at positions 2, 14, and 16 from the 5' end, and positions not modified with 2'-fluoro are modified with 2'-O-methyl; (3) the antisense strand is modified with at least two phosphorothioate internucleotide linkages from the 3' end and the 5' end; (4) the sense strand is modified with continuous 2'-fluoro at position 7 and positions 9-11 from the 5' end, and positions not modified with 2'-fluoro are modified with 2'-O-methyl; (5) the sense strand is modified with at least two phosphorothioate internucleotide linkages from the 5' end; optionally, the 2-nucleotide overhang is a 5'(s)mN(s)mN3' structure overhang.

[0023] In some embodiments, the present disclosure provides a dsRNA molecule comprising a sense strand and an antisense strand, wherein:

[0024] (1) The sense strand sequence is shown in SEQ ID NO: 1, and the antisense strand sequence is shown in SEQ ID NO: 2;

[0025] (2) the sense strand sequence is shown in SEQ ID NO: 3, and the antisense strand sequence is shown in SEQ ID NO: 4;

[0026] (3) the sense strand sequence is shown in SEQ ID NO: 5, and the antisense strand sequence is shown in SEQ ID NO: 6;

[0027] (4) the sense strand sequence is shown in SEQ ID NO: 7, and the antisense strand sequence is shown in SEQ ID NO: 8;

[0028] (5) the sense strand sequence is shown in SEQ ID NO: 9, and the antisense strand sequence is shown in SEQ ID NO: 10;

[0029] (6) the sense strand sequence is shown in SEQ ID NO: 11, and the antisense strand sequence is shown in SEQ ID NO: 12;

[0030] (7) the sense strand sequence is shown in SEQ ID NO: 13, and the antisense strand sequence is shown in SEQ ID NO: 14;

[0031] (8) the sense strand sequence is shown in SEQ ID NO: 15, and the antisense strand sequence is shown in SEQ ID NO: 16;

[0032] (9) the sense strand sequence is shown in SEQ ID NO: 17, and the antisense strand sequence is shown in SEQ ID NO: 18;

[0033] (10) the sense strand sequence is shown in SEQ ID NO: 19, and the antisense strand sequence is shown in SEQ ID NO: 20;

[0034] (11) The sense strand sequence is shown in SEQ ID NO: 21, and the antisense strand sequence is shown in SEQ ID NO: 22;

[0035] (12) the sense strand sequence is shown in SEQ ID NO: 23, and the antisense strand sequence is shown in SEQ ID NO: 24;

[0036] (13) the sense strand sequence is shown in SEQ ID NO: 25, and the antisense strand sequence is shown in SEQ ID NO: 26;

[0037] (14) the sense strand sequence is shown in SEQ ID NO: 27, and the antisense strand sequence is shown in SEQ ID NO: 28;

[0038] (15) the sense strand sequence is shown in SEQ ID NO: 29, and the antisense strand sequence is shown in SEQ ID NO: 30;

[0039] (16) the sense strand sequence is shown in SEQ ID NO: 31, and the antisense strand sequence is shown in SEQ ID NO: 32;

[0040] (17) The sense strand sequence is shown in SEQ ID NO: 33, and the antisense strand sequence is shown in SEQ ID NO: 34;

[0041] (18) The sense strand sequence is shown in SEQ ID NO: 35, and the antisense strand sequence is shown in SEQ ID NO: 36;

[0042] (19) the sense strand sequence is shown in SEQ ID NO: 37, and the antisense strand sequence is shown in SEQ ID NO: 38;

[0043] (20) the sense strand sequence is shown in SEQ ID NO: 39, and the antisense strand sequence is shown in SEQ ID NO: 40;

[0044] (21) the sense strand sequence is shown in SEQ ID NO:41, and the antisense strand sequence is shown in SEQ ID NO:42;

[0045] (22) the sense strand sequence is shown in SEQ ID NO: 43, and the antisense strand sequence is shown in SEQ ID NO: 44;

[0046] (23) the sense strand sequence is shown in SEQ ID NO:45, and the antisense strand sequence is shown in SEQ ID NO:46;

[0047] (24) the sense strand sequence is as shown in SEQ ID NO: 47, and the antisense strand sequence is as shown in SEQ ID NO: 48; or

[0048] (25) the sense strand sequence is shown in SEQ ID NO: 49, and the antisense strand sequence is shown in SEQ ID NO: 50;

[0049] Among them, Am, Um / Tm, Cm, and Gm represent 2′-O-methyl-modified ribonucleotides A, U, C, and G, respectively; Af, Uf / Tf, Cf, and Gf represent 2′-fluoro-modified ribonucleotides A, U, C, and G, respectively; and (s) indicates that the two nucleotides are connected by a phosphorothioate backbone. When the nucleic acid sequence in the sequence listing represents an RNA sequence, "T" represents uridine. Furthermore, in the context of RNA, unless otherwise specified, "T" and "U" refer to uracil or uridine; similarly, in the context of RNA, Um and Tm, Uf and Tf refer to uracil or uridine with 2′-O-methyl modification, 2′-fluoro modification, respectively.

[0050] In some embodiments, the dsRNA is E13, E14, E31, E32, E46, E47, E83, E86, E90, E91, E92, ES6, E104, E105, E126, E159, E161, E162, E163, E164, E176, E178, E179, E180, or EX1;

[0051] The dsRNA molecule consists of a sense strand and an antisense strand, wherein:

[0052] (1) E13 molecule: the sense strand sequence is shown in SEQ ID NO: 1, and the antisense strand sequence is shown in SEQ ID NO: 2;

[0053] (2) E14 molecule: the sense strand sequence is shown in SEQ ID NO: 3, and the antisense strand sequence is shown in SEQ ID NO: 4;

[0054] (3) E31 molecule: the sense strand sequence is shown in SEQ ID NO: 5, and the antisense strand sequence is shown in SEQ ID NO: 6;

[0055] (4) E32 molecule: the sense strand sequence is shown in SEQ ID NO: 7, and the antisense strand sequence is shown in SEQ ID NO: 8;

[0056] (5) E46 molecule: the sense strand sequence is shown in SEQ ID NO: 9, and the antisense strand sequence is shown in SEQ ID NO: 10;

[0057] (6) E47 molecule: the sense strand sequence is shown in SEQ ID NO: 11, and the antisense strand sequence is shown in SEQ ID NO: 12;

[0058] (7) E83 molecule: the sense strand sequence is shown in SEQ ID NO: 13, and the antisense strand sequence is shown in SEQ ID NO: 14;

[0059] (8) E86 molecule: the sense strand sequence is shown in SEQ ID NO: 15, and the antisense strand sequence is shown in SEQ ID NO: 16;

[0060] (9) E90 molecule: the sense strand sequence is shown in SEQ ID NO: 17, and the antisense strand sequence is shown in SEQ ID NO: 18;

[0061] (10) E91 molecule: the sense strand sequence is shown in SEQ ID NO: 19, and the antisense strand sequence is shown in SEQ ID NO: 20;

[0062] (11) E92 molecule: the sense strand sequence is shown in SEQ ID NO: 21, and the antisense strand sequence is shown in SEQ ID NO: 22;

[0063] (12) ES6 molecule: the sense strand sequence is shown in SEQ ID NO: 23, and the antisense strand sequence is shown in SEQ ID NO: 24;

[0064] (13) E104 molecule: the sense strand sequence is shown in SEQ ID NO: 25, and the antisense strand sequence is shown in SEQ ID NO: 26;

[0065] (14) E105 molecule: the sense strand sequence is shown in SEQ ID NO: 27, and the antisense strand sequence is shown in SEQ ID NO: 28;

[0066] (15) E126 molecule: the sense strand sequence is shown in SEQ ID NO: 29, and the antisense strand sequence is shown in SEQ ID NO: 30;

[0067] (16) E159 molecule: the sense strand sequence is shown in SEQ ID NO: 31, and the antisense strand sequence is shown in SEQ ID NO: 32;

[0068] (17) E161 molecule: the sense strand sequence is shown in SEQ ID NO: 33, and the antisense strand sequence is shown in SEQ ID NO: 34;

[0069] (18) E162 molecule: the sense strand sequence is shown in SEQ ID NO: 35, and the antisense strand sequence is shown in SEQ ID NO: 36;

[0070] (19) E163 molecule: the sense strand sequence is shown in SEQ ID NO: 37, and the antisense strand sequence is shown in SEQ ID NO: 38;

[0071] (20) E164 molecule: the sense strand sequence is shown in SEQ ID NO: 39, and the antisense strand sequence is shown in SEQ ID NO: 40;

[0072] (21) E176 molecule: the sense strand sequence is shown in SEQ ID NO: 41, and the antisense strand sequence is shown in SEQ ID NO: 42;

[0073] (22) E178 molecule: the sense strand sequence is shown in SEQ ID NO: 43, and the antisense strand sequence is shown in SEQ ID NO: 44;

[0074] (23) E179 molecule: the sense strand sequence is shown in SEQ ID NO: 45, and the antisense strand sequence is shown in SEQ ID NO: 46;

[0075] (24) E180 molecule: the sense strand sequence is shown in SEQ ID NO: 47, and the antisense strand sequence is shown in SEQ ID NO: 48; or

[0076] (25) EX1 molecule: the sense strand sequence is shown in SEQ ID NO:49, and the antisense strand sequence is shown in SEQ ID NO:50.

[0077] In Tables 5 and 6, the dsRNA molecules corresponding to the designation E+numbers consist of a modified sense strand and a modified antisense strand. For example, the E13 dsRNA molecule consists of a modified sense strand and a modified antisense strand. The sequence of the sense strand is shown in SEQ ID NO. 1, and the sequence of the antisense strand is shown in SEQ ID NO. 2. The designations and numbers for other dsRNA molecules corresponding to the designations E+numbers are similarly deduced.

[0078] In some embodiments, the dsRNA molecule is E13, E14, E31, E32, E46, E47, E90, ES6, E162, E163, or E179.

[0079] In some embodiments, the above-described dsRNA molecules are conjugated to at least one ligand.

[0080] In some embodiments, the conjugated ligand can improve the cellular uptake, intracellular targeting, half-life, or drug metabolism or kinetics of the dsRNA molecule.

[0081] In some embodiments, the ligand-conjugated dsRNA has enhanced affinity or cellular uptake for a selected target (e.g., a specific tissue type, cell type, organelle, etc.), such as a hepatocyte, compared to a dsRNA molecule not conjugated to the ligand. Ligand conjugation does not interfere with the activity of the dsRNA.

[0082] In some embodiments, the ligand is conjugated to the 3' end, 5' end and / or the middle of the sense and / or antisense strands of the dsRNA molecule; alternatively, the ligand is conjugated to the 3' end of the sense strand of the dsRNA molecule.

[0083] In some optional embodiments, the ligand is selected from the following: cholesterol, biotin, vitamins, galactose derivatives or analogs, lactose derivatives or analogs, N-acetylgalactosamine derivatives or analogs, N-acetylglucosamine derivatives or analogs.

[0084] In some embodiments, the ligand targets a cell surface receptor and includes a galactose, galactosamine, lactose, or N-acetylgalactosamine / glucosamine moiety.

[0085] In some embodiments, the ligand can optionally be targeted to the liver, particularly to parenchymal cells of the liver.

[0086] In some alternative embodiments, the ligand targets the ASGPR receptor.

[0087] In some optional embodiments, the ligand may also be human serum albumin (HSA), hyaluronic acid, polypeptide, etc.

[0088] In some embodiments, the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

[0089] In some embodiments, the ligand is L96, which is linked to the dsRNA molecule via a phosphoester bond, and its structure is as shown in Formula I:

[0090] In some embodiments, the dsRNA has the structure of Formula II:

[0091] In some embodiments, the dsRNA is GE13, GE14, GE31, GE32, GE46, GE47, GE83, GE86, GE90, GE91, GE92, GES6, GE104, GE105, GE126, GE159, GE161, GE162, GE163, GE164, GE176, GE178, GE179, GE180, or GEX1. In Tables 5 and 6, the dsRNA molecules corresponding to the name number GE+number consist of a modified sense strand and a modified antisense strand, and the 3' end of the sense strand is linked to L96 according to Formula II. For example, the GE13 dsRNA molecule consists of a modified sense strand and a modified antisense strand. The 3' end of the sense strand is linked to L96 according to Formula II. The sequence of the sense strand is shown in SEQ ID NO. 1, and the sequence of the antisense strand is shown in SEQ ID NO. 2. The names and numbers of other GE+numbers are similar.

[0092] The present disclosure also provides a molecule comprising two or more dsRNA molecules, wherein the dsRNA molecules are bridged by a linker; the two or more linked dsRNA molecules can be the same or different.

[0093] In some embodiments, the linker can be a nucleotide sequence, such as RNA, DNA, or a hybrid molecule. In some embodiments, the linker can be entirely RNA, DNA, or a hybrid molecule. In particular, the nucleotide sequence can be uuu or (dT)n, where n=1-20, for example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; alternatively, the linker is (dT)3, i.e., dTdTdT.

[0094] In some embodiments, the linker can also be selected as a functional molecule, such as -(CH2) 12 -(C12 linker), -(CH2)6-SS-(CH2)6-(C6-SS-C6 linker), -CH2CH2O-(CH2CH2)n-CH2CH2O, n=1-20, for example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0095] In some embodiments, the linker may also be 1+1+1GalNAc (also referred to as GALNAC3 in this disclosure), and the structure of 1+1+1GalNAc is shown in Formula III:

[0096] In some embodiments, the linker bridges the sense strands of the different dsRNA molecules.

[0097] In some alternative embodiments, the linker bridges the sense strands of two different dsRNA molecules to form a dual-targeting dsRNA (ddsRNA) molecule. The antisense strands of different dsRNA molecules are not directly connected to each other, but become part of the ddsRNA molecule by forming a double-stranded region with the corresponding sense strand.

[0098] In some more optional embodiments, the linker bridges the sense strands of two different dsRNA molecules, and the linker bridges the 3' end of the sense strand of the first dsRNA molecule and the 5' end of the sense strand of the second dsRNA molecule, and the linker structure is shown in Formula IV:

[0099] In some embodiments, the different linked dsRNA molecules target different genes.

[0100] In some embodiments, the bridged sense strand is sequentially linked to a dsRNA molecule targeting the HBV S gene and a dsRNA molecule targeting the HBV X gene in a 5'-3' direction, or the bridged sense strand is sequentially linked to a dsRNA molecule targeting the HBV X gene and a dsRNA molecule targeting the S gene in a 5'-3' direction; further alternatively, the bridged sense strand is sequentially linked to a dsRNA molecule targeting the HBV S gene and a dsRNA molecule targeting the X gene in a 5'-3' direction; further alternatively, the nucleotide sequence of the dsRNA molecule targeting the HBV S gene is shown in Table 1, and the nucleotide sequence of the dsRNA molecule targeting the HBV X gene is shown in Table 2.

[0101] In some embodiments, at least one nucleotide in the ddsRNA molecule is modified. The modification is selected from any one or more of the following: locked nucleic acid (LNA) modification, open ring or unlocked (UNA) modification, 2′-methoxyethyl modification, 2′-O-methyl modification, 2′-O-allyl modification, 2′-C-allyl modification, 2′-fluoro modification, 2′-deoxy modification, 2′-hydroxyl modification, phosphorothioate backbone modification, DNA modification, fluorescent probe modification. In some embodiments, all nucleotides in the ddsRNA molecule are modified nucleotides.

[0102] In some embodiments, the modified nucleotides in the ddsRNA molecule are each independently selected from a 2'-O-methyl modification and a 2'-fluoro modification.

[0103] In some embodiments, the ddsRNA molecule comprises at least one phosphorothioate internucleotide linkage; optionally, the exposed 5' end and / or 3' end of the bridged sense strand and / or antisense strand in the ddsRNA molecule comprises at least one phosphorothioate internucleotide linkage; further optionally, the three nucleotides at the exposed 5' end and / or 3' end of the bridged sense strand and / or antisense strand in the ddsRNA molecule are linked by a phosphorothioate internucleotide linkage; further optionally, the three nucleotides at the exposed 5' end and 3' end of the bridged sense strand in the ddsRNA molecule are linked by a phosphorothioate internucleotide linkage, and the three nucleotides at the exposed 5' end and 3' end of the two antisense strands are linked by a phosphorothioate internucleotide linkage, or the three nucleotides at the exposed 5' end of the bridged sense strand in the ddsRNA molecule are linked by a phosphorothioate internucleotide linkage, and the three nucleotides at the exposed 5' end and 3' end of the two antisense strands are linked by a phosphorothioate internucleotide linkage. The definition herein is only for the presence of an exposed 3' end and / or 5' end, and does not necessarily mean that an exposed 3' end and / or 5' end must exist.

[0104] In some embodiments, the modification patterns of different dsRNA molecules in the ddsRNA molecules provided by the present disclosure include: (1) sense strand: 17-21 nt in length, such as 21 nt; composed of alternating 2′-O-methyl modified regions and 2′-fluoro modified regions, each modified region is 1 to 3 nucleotides in length; the modification pattern of the 5′ end and the first modified region from the 3′ end is the same; (2) antisense strand: 19-23 nt in length, such as 23 nt; composed of alternating 2′-O-methyl modified regions, 2′-fluoro modified regions, unmodified regions or DNA regions, each modified region is 1 to 5 nucleotides in length; and the continuous nucleotide region from the 2nd to the 5th position from the 5′ end and the continuous nucleotide region from the 1st to the 3rd position from the 3′ end are both connected by a thiophosphate backbone.

[0105] In another embodiment of the present disclosure, the ddsRNA molecule includes one, two, three, four, or five of the following structures: (1) the antisense chain has a 2-nucleotide overhang at the 3' end (optionally, an overhang of a 5'(s)mN(s)mN3' structure); (2) at least the 2nd, 14th, and 16th positions of the antisense chain from the 5' end are modified with 2'-fluoro, and the positions not modified with 2'-fluoro are modified with 2'-O-methyl; (3) at least the antisense chain from the 3' end and the 5' end are modified with 2'-O-methyl. There are two phosphorothioate internucleotide bonds; (4) after bridging, the sense strand for different targets is modified with continuous 2'-fluoro at position 7 and positions 9-11 from its own 5' end, and the positions not modified with 2'-fluoro are modified with 2'-O-methyl; (5) the sense strand after bridging has at least two phosphorothioate internucleotide bonds from the 5' end; optionally, when the linker is 1+1+1GALNAC, the sense strand after bridging has at least two phosphorothioate internucleotide bonds from the 3' end. In some embodiments, the nucleotide sequences of the sense strand and the antisense strand of the ddsRNA molecule are selected from the group consisting of:

[0106] (1) A sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO. 17 and the E162a sense sequence SEQ ID NO. 443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 18 and SEQ ID NO. 36;

[0107] (2) a sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO.17 and the E179a sense sequence SEQ ID NO.445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.18 and SEQ ID NO.46;

[0108] (3) a sense strand nucleotide sequence consisting of the E46 sense sequence SEQ ID NO.9 and the E162a sense sequence SEQ ID NO.443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.10 and SEQ ID NO.36;

[0109] (4) a sense strand nucleotide sequence consisting of the E46 sense sequence SEQ ID NO.9 and the E179a sense sequence SEQ ID NO.445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.10 and SEQ ID NO.46;

[0110] (5) a sense strand nucleotide sequence consisting of the E31 sense sequence SEQ ID NO. 5 and the E162a sense sequence SEQ ID NO. 443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 6 and SEQ ID NO. 36;

[0111] (6) a sense strand nucleotide sequence consisting of the E31 sense sequence SEQ ID NO. 5 and the E179a sense sequence SEQ ID NO. 445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 6 and SEQ ID NO. 46;

[0112] (7) a sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E162a sense sequence SEQ ID NO.443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.36;

[0113] (8) a sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E179a sense sequence SEQ ID NO.445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.46;

[0114] (9) A sense strand nucleotide sequence consisting of the ES6 sense sequence SEQ ID NO. 23 and the E162a sense sequence SEQ ID NO. 443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 24 and SEQ ID NO. 36;

[0115] (10) A sense strand nucleotide sequence consisting of the ES6 sense sequence SEQ ID NO. 23 and the E179a sense sequence SEQ ID NO. 445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 24 and SEQ ID NO. 46;

[0116] (11) a sense strand nucleotide sequence consisting of the E14 sense sequence SEQ ID NO. 3 and the E162a sense sequence SEQ ID NO. 443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 4 and SEQ ID NO. 36;

[0117] (12) a sense strand nucleotide sequence consisting of the E14 sense sequence SEQ ID NO. 3 and the E179a sense sequence SEQ ID NO. 445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 4 and SEQ ID NO. 46;

[0118] (13) The sense strand nucleotide sequence consisting of the E32 sense sequence SEQ ID NO.7 and the E163a sense sequence SEQ ID NO.444 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.38;

[0119] (14) a sense strand nucleotide sequence consisting of the E32 sense sequence SEQ ID NO.7 and the E179a sense sequence SEQ ID NO.445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.46;

[0120] (15) A sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO. 17 and the E163a sense sequence SEQ ID NO. 444 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 18 and SEQ ID NO. 38;

[0121] (16) A sense strand nucleotide sequence consisting of the E47 sense sequence SEQ ID NO.11 and the E163a sense sequence SEQ ID NO.444 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.12 and SEQ ID NO.38;

[0122] (17) A sense strand nucleotide sequence consisting of the E47 sense sequence SEQ ID NO.11 and the E179a sense sequence SEQ ID NO.445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.12 and SEQ ID NO.46;

[0123] (18) The sense strand nucleotide sequence consisting of the E46 sense sequence SEQ ID NO.9 and the E163a sense sequence SEQ ID NO.444 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.10 and SEQ ID NO.38;

[0124] (19) The sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E163a sense sequence SEQ ID NO.444 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.38;

[0125] (20) The sense chain nucleotide sequence consisting of the E47 sense sequence SEQ ID NO.11 and the E162a sense sequence SEQ ID NO.443 is connected by a linker in the direction from 5'>3', and the antisense chain nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.12 and SEQ ID NO.36;

[0126] (21) The sense strand nucleotide sequence consisting of the E32 sense sequence SEQ ID NO.7 and the E162a sense sequence SEQ ID NO.443 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.36;

[0127] (22) The sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO.17 and the E162b sense sequence SEQ ID NO.446 was connected via a linker in the direction from 5'>3', and the antisense strand consisting of the nucleotide sequences SEQ ID NO.18 and SEQ ID NO.36;

[0128] (23) The sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO.17 and the E179b sense sequence SEQ ID NO.448 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.18 and SEQ ID NO.46;

[0129] (24) The sense strand nucleotide sequence consisting of the E46 sense sequence SEQ ID NO.9 and the E162b sense sequence SEQ ID NO.446 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.10 and SEQ ID NO.36;

[0130] (25) The sense chain nucleotide sequence consisting of the E46 sense sequence SEQ ID NO.9 and the E179b sense sequence SEQ ID NO.448 is connected by a linker in the direction from 5'>3', and the antisense chain nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.10 and SEQ ID NO.46;

[0131] (26) The sense strand nucleotide sequence consisting of the E31 sense sequence SEQ ID NO. 5 and the E162b sense sequence SEQ ID NO. 446 was connected via a linker in the direction from 5' > 3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 6 and SEQ ID NO. 36;

[0132] (27) The sense strand nucleotide sequence consisting of the E31 sense sequence SEQ ID NO. 5 and the E179b sense sequence SEQ ID NO. 448 was connected via a linker in the direction from 5' > 3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 6 and SEQ ID NO. 46;

[0133] (28) The sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E162b sense sequence SEQ ID NO.446 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.36;

[0134] (29) The sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E179b sense sequence SEQ ID NO.448 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.46;

[0135] (30) The sense strand nucleotide sequence consisting of the ES6 sense sequence SEQ ID NO. 23 and the E162b sense sequence SEQ ID NO. 446 was connected by a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 24 and SEQ ID NO. 36;

[0136] (31) The sense strand nucleotide sequence consisting of the ES6 sense sequence SEQ ID NO. 23 and the E179b sense sequence SEQ ID NO. 448 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 24 and SEQ ID NO. 46;

[0137] (32) The sense strand nucleotide sequence consisting of the E14 sense sequence SEQ ID NO. 3 and the E162b sense sequence SEQ ID NO. 446 was connected via a linker in the direction from 5' > 3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 4 and SEQ ID NO. 36;

[0138] (33) The sense strand nucleotide sequence consisting of the E14 sense sequence SEQ ID NO. 3 and the E179b sense sequence SEQ ID NO. 448 was connected via a linker in the direction from 5' > 3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 4 and SEQ ID NO. 46;

[0139] (34) The sense strand nucleotide sequence consisting of the E32 sense sequence SEQ ID NO.7 and the E163b sense sequence SEQ ID NO.447 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.38;

[0140] (35) The sense strand nucleotide sequence consisting of the E32 sense sequence SEQ ID NO.7 and the E179b sense sequence SEQ ID NO.448 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.46;

[0141] (36) The sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO.17 and the E163b sense sequence SEQ ID NO.447 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.18 and SEQ ID NO.38;

[0142] (37) The sense chain nucleotide sequence consisting of the E47 sense sequence SEQ ID NO.11 and the E163b sense sequence SEQ ID NO.447 was connected by a linker in the direction from 5'>3', and the antisense chain nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.12 and SEQ ID NO.38;

[0143] (38) The sense chain nucleotide sequence consisting of the E47 sense sequence SEQ ID NO.11 and the E179b sense sequence SEQ ID NO.448 was connected by a linker in the direction from 5'>3', and the antisense chain nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.12 and SEQ ID NO.46;

[0144] (39) The sense strand nucleotide sequence consisting of the E46 sense sequence SEQ ID NO.9 and the E163b sense sequence SEQ ID NO.447 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.10 and SEQ ID NO.38;

[0145] (40) The sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E163b sense sequence SEQ ID NO.447 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.38;

[0146] (41) a sense strand nucleotide sequence consisting of the sense sequence of E47 SEQ ID NO.11 and the sense sequence of E162b SEQ ID NO.446 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.12 and SEQ ID NO.36; or

[0147] (42) The sense strand nucleotide sequence consisting of the E32 sense sequence SEQ ID NO.7 and the E162b sense sequence SEQ ID NO.446 was connected via a linker in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.36;

[0148] Wherein, Am, Um / Tm, Cm, and Gm represent 2'-O-methyl-modified ribonucleotides A, U, C, and G, respectively; Af, Uf / Tf, Cf, and Gf represent 2'-fluoro-modified ribonucleotides A, U, C, and G, respectively; and (s) indicates that the two nucleotides are connected by a phosphorothioate backbone. In the above-mentioned ddsRNA molecules, taking the nucleotide sequence of the first molecule as an example, the E90 sense sequence SEQ ID NO. 17 and the E90 antisense sequence SEQ ID NO. 18 complement each other to form a double-stranded region (as shown in Table 5); the E162a sense sequence SEQ ID NO. 443 differs from the E162 sense sequence SEQ ID NO. 35 only in the phosphate linkage; the nucleoside composition remains unchanged, thus not affecting the complementarity between the E162a sense sequence SEQ ID NO. 443 and the E162 antisense sequence SEQ ID NO. 36 to form a double-stranded region. Similarly, taking the nucleotide sequence of the 22nd molecule as an example, the E90 sense sequence SEQ ID NO. 17 and the E90 antisense sequence SEQ ID NO. 18 complement each other to form a double-stranded region (as shown in Table 5); the E162b sense sequence SEQ ID NO. 446 differs from the E162 sense sequence SEQ ID NO. 35 only in the phosphate linkage; the nucleoside composition remains unchanged, thus not affecting the complementarity between the E162b sense sequence SEQ ID NO. 446 and the E162 antisense sequence SEQ ID NO. 36 to form a double-stranded region.

[0149] In some embodiments, after the sense strands of two different dsRNAs are bridged via a linker to form a ddsRNA molecule, the ddsRNA molecule is conjugated to at least one ligand.

[0150] In some embodiments, the conjugated ligand can improve the cellular uptake, intracellular targeting, half-life, or drug metabolism or kinetics of the dsRNA molecule.

[0151] In some embodiments, the ligand-conjugated dsRNA has enhanced affinity or cellular uptake for a selected target (e.g., a specific tissue type, cell type, organelle, etc.), such as hepatocytes, compared to the dsRNA not conjugated to the ligand. Ligand conjugation does not interfere with the activity of the dsRNA.

[0152] In some embodiments, the ligand is conjugated to the 3' end, 5' end and / or the middle of the sense and / or antisense strand of the ddsRNA molecule; alternatively, the ligand is conjugated to the 3' end of the sense strand of the ddsRNA molecule.

[0153] In some optional embodiments, the ligand is selected from the following: cholesterol, biotin, vitamins, galactose derivatives or analogs, lactose derivatives or analogs, N-acetylgalactosamine derivatives or analogs, N-acetylglucosamine derivatives or analogs.

[0154] In some embodiments, the ligand targets a cell surface receptor and includes a galactose, galactosamine, lactose, or N-acetylgalactosamine / glucosamine moiety.

[0155] In some embodiments, the ligand can optionally be targeted to the liver, particularly to parenchymal cells of the liver.

[0156] In some alternative embodiments, the ligand targets the ASGPR receptor.

[0157] In some optional embodiments, the ligand may also be human serum albumin (HSA), hyaluronic acid, polypeptide, etc.

[0158] In some embodiments, the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

[0159] In some embodiments, the ligand is L96, which is linked to the ddsRNA molecule via a phosphoester bond, and its structure is as shown in Formula I:

[0160] In some embodiments, the ddsRNA has the structure of Formula V:

[0161] In some embodiments, two different dsRNA sense strands are bridged via a linker, such as 1+1+1 GalNAc. 1+1+1 GalNAc can serve as both a linker and a ligand, eliminating the need for further coupling of a ligand to the 3' end of the sense strand. The structure of the 1+1+1 GalNAc is shown in Formula III:

[0162] Further optionally, the ddsRNA has the structure of Formula VI:

[0163] In some further alternative embodiments, the ddsRNA molecule is selected from the group consisting of:

[0164] (1) The sense strand nucleotide sequence of E90 sense sequence SEQ ID NO.17 and the sense strand nucleotide sequence of E162a sense sequence SEQ ID NO.443 were connected in the direction from 5'>3' via the linker dTdTdT, and the 3' end of the sense strand of E162a was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO.18 and SEQ ID NO.36;

[0165] (2) A sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO. 17 and the E179a sense sequence SEQ ID NO. 445 connected in a 5'>3' direction via a linker dTdTdT, with L96 connected to the 3' end of the E179a sense sequence, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 18 and SEQ ID NO. 46;

[0166] (3) A sense strand nucleotide sequence consisting of the E46 sense sequence SEQ ID NO. 9 and the E162a sense sequence SEQ ID NO. 443 connected in a 5'>3' direction via a linker dTdTdT, with L96 connected to the 3' end of the E162a sense sequence, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 10 and SEQ ID NO. 36;

[0167] (4) A sense strand nucleotide sequence consisting of the E46 sense sequence SEQ ID NO. 9 and the E179a sense sequence SEQ ID NO. 445 connected in a 5'>3' direction via a linker dTdTdT, with L96 connected to the 3' end of the E179a sense sequence, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 10 and SEQ ID NO. 46;

[0168] (5) The sense strand nucleotide sequence of E31 sense sequence SEQ ID NO. 5 and the sense strand nucleotide sequence of E162a sense sequence SEQ ID NO. 443 were connected in the direction of 5'>3' via the linker dTdTdT, and the 3' end of the sense strand of E162a sense sequence was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO. 6 and SEQ ID NO. 36;

[0169] (6) A sense strand nucleotide sequence consisting of the E31 sense sequence SEQ ID NO. 5 and the E179a sense sequence SEQ ID NO. 445 connected in a 5'>3' direction via a linker dTdTdT, with L96 connected to the 3' end of the E179a sense sequence, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 6 and SEQ ID NO. 46;

[0170] (7) The sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO. 1 and the E162a sense sequence SEQ ID NO. 443 was connected via a linker dTdTdT in the direction from 5' > 3', and the 3' end of the E162a sense sequence was connected to L96, and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 2 and SEQ ID NO. 36;

[0171] (8) The sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E179a sense sequence SEQ ID NO.445 was connected via a linker dTdTdT in the direction from 5'>3', and the 3' end of the E179a sense sequence was connected to L96, and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.46;

[0172] (9) The sense strand nucleotide sequence of ES6 sense sequence SEQ ID NO. 23 and the sense strand nucleotide sequence of E162a sense sequence SEQ ID NO. 443 were connected in the direction from 5'>3' by the linker dTdTdT, and the 3' end of the sense strand of E162a was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO. 24 and SEQ ID NO. 36;

[0173] (10) The sense strand nucleotide sequence of ES6 sense sequence SEQ ID NO. 23 and the sense strand nucleotide sequence of E179a sense sequence SEQ ID NO. 445 were connected in the direction from 5'>3' by the linker dTdTdT, and the 3' end of the sense strand of E179a sense sequence was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO. 24 and SEQ ID NO. 46;

[0174] (11) The sense strand nucleotide sequence of E14 sense sequence SEQ ID NO. 3 and the sense strand nucleotide sequence of E162a sense sequence SEQ ID NO. 443 were connected in the direction from 5'>3' by the linker dTdTdT, and the 3' end of the sense strand of E162a sense sequence was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO. 4 and SEQ ID NO. 36;

[0175] (12) The sense sequence of E14 SEQ ID NO. 3 and the sense sequence of E179a SEQ ID NO. 445 were connected in the direction from 5'>3' by the linker dTdTdT to form a sense strand nucleotide sequence, and the 3' end of the sense sequence of E179a was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO. 4 and SEQ ID NO. 46;

[0176] (13) The sense sequence of E32 SEQ ID NO.7 and the sense sequence of E163a SEQ ID NO.444 were connected in the direction from 5'>3' by the linker dTdTdT to form a sense strand nucleotide sequence, and the 3' end of the sense sequence of E163a was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO.8 and SEQ ID NO.38;

[0177] (14) The sense sequence of E32 SEQ ID NO.7 and the sense sequence of E179a SEQ ID NO.445 were connected in the direction from 5'>3' by the linker dTdTdT to form a sense strand nucleotide sequence, and the 3' end of the sense sequence of E179a was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO.8 and SEQ ID NO.46;

[0178] (15) The sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO. 17 and the E163a sense sequence SEQ ID NO. 444 was connected via a linker dTdTdT in the direction from 5' > 3', and the 3' end of the E163a sense sequence was connected to L96, and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 18 and SEQ ID NO. 38;

[0179] (16) The sense sequence of E47 SEQ ID NO.11 and the sense sequence of E163a SEQ ID NO.444 were connected in the direction from 5'>3' by the linker dTdTdT to form a sense strand nucleotide sequence, and the 3' end of the sense sequence of E163a was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO.12 and SEQ ID NO.38;

[0180] (17) The sense sequence of E47 SEQ ID NO.11 and the sense sequence of E179a SEQ ID NO.445 were connected in the direction from 5'>3' by the linker dTdTdT to form a sense strand nucleotide sequence, and the 3' end of the sense sequence of E179a was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO.12 and SEQ ID NO.46;

[0181] (18) The sense sequence of E46 SEQ ID NO. 9 and the sense sequence of E163a SEQ ID NO. 444 were connected in the direction from 5'>3' by the linker dTdTdT to form a sense strand nucleotide sequence, and the 3' end of the sense sequence of E163a was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO. 10 and SEQ ID NO. 38;

[0182] (19) The sense strand nucleotide sequence of E13 sense sequence SEQ ID NO.1 and the sense strand nucleotide sequence of E163a sense sequence SEQ ID NO.444 were connected in the direction from 5'>3' by the linker dTdTdT, and the 3' end of the sense strand of E163a sense sequence was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO.2 and SEQ ID NO.38;

[0183] (20) The sense sequence of E47 SEQ ID NO.11 and the sense sequence of E162a SEQ ID NO.443 were connected in the direction from 5'>3' by the linker dTdTdT to form a sense strand nucleotide sequence, and the 3' end of the sense sequence of E162a was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO.12 and SEQ ID NO.36;

[0184] (21) The sense sequence of E32 SEQ ID NO.7 and the sense sequence of E162a SEQ ID NO.443 were connected in the direction from 5'>3' by the linker dTdTdT to form a sense strand nucleotide sequence, and the 3' end of the sense sequence of E162a was connected to L96, and the antisense strand nucleotide sequence was composed of the nucleotide sequences of SEQ ID NO.8 and SEQ ID NO.36;

[0185] In some further alternative embodiments, the ddsRNA molecule is selected from the group consisting of:

[0186] (1) The sense strand consisting of the E90 sense sequence SEQ ID NO. 17 and the E162b sense sequence SEQ ID NO. 446 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand consisting of the nucleotide sequences SEQ ID NO. 18 and SEQ ID NO. 36;

[0187] (2) a sense strand consisting of the E90 sense sequence SEQ ID NO.17 and the E179b sense sequence SEQ ID NO.448 connected in a 5'>3' direction via a linker GALNAC3, and an antisense strand consisting of the nucleotide sequences SEQ ID NO.18 and SEQ ID NO.46;

[0188] (3) A sense strand consisting of the E46 sense sequence SEQ ID NO.9 and the E162b sense sequence SEQ ID NO.446 connected in a 5'>3' direction via a linker GALNAC3, and an antisense strand consisting of the nucleotide sequences SEQ ID NO.10 and SEQ ID NO.36;

[0189] (4) a sense strand consisting of the E46 sense sequence SEQ ID NO.9 and the E179b sense sequence SEQ ID NO.448 connected in a 5'>3' direction via a linker GALNAC3, and an antisense strand consisting of the nucleotide sequences SEQ ID NO.10 and SEQ ID NO.46;

[0190] (5) A sense strand consisting of the E31 sense sequence SEQ ID NO. 5 and the E162b sense sequence SEQ ID NO. 446 connected in a 5'>3' direction via a linker GALNAC3, and an antisense strand consisting of the nucleotide sequences SEQ ID NO. 6 and SEQ ID NO. 36;

[0191] (6) A sense strand consisting of the E31 sense sequence SEQ ID NO. 5 and the E179b sense sequence SEQ ID NO. 448 connected in a 5'>3' direction via a linker GALNAC3, and an antisense strand consisting of the nucleotide sequences SEQ ID NO. 6 and SEQ ID NO. 46;

[0192] (7) The sense strand consisting of the E13 sense sequence SEQ ID NO.1 and the E162b sense sequence SEQ ID NO.446 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.36;

[0193] (8) The sense strand consisting of the E13 sense sequence SEQ ID NO.1 and the E179b sense sequence SEQ ID NO.448 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.46;

[0194] (9) The sense strand consisting of the ES6 sense sequence SEQ ID NO. 23 and the E162b sense sequence SEQ ID NO. 446 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 24 and SEQ ID NO. 36;

[0195] (10) The sense strand consisting of the ES6 sense sequence SEQ ID NO. 23 and the E179b sense sequence SEQ ID NO. 448 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 24 and SEQ ID NO. 46;

[0196] (11) The sense strand consisting of the E14 sense sequence SEQ ID NO. 3 and the E162b sense sequence SEQ ID NO. 446 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO. 4 and SEQ ID NO. 36;

[0197] (12) The sense strand consisting of the E14 sense sequence SEQ ID NO.3 and the E179b sense sequence SEQ ID NO.448 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.4 and SEQ ID NO.46;

[0198] (13) The sense strand consisting of the E32 sense sequence SEQ ID NO.7 and the E163b sense sequence SEQ ID NO.447 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.38;

[0199] (14) The sense strand consisting of the E32 sense sequence SEQ ID NO.7 and the E179b sense sequence SEQ ID NO.448 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.46;

[0200] (15) The sense strand consisting of the E90 sense sequence SEQ ID NO.17 and the E163b sense sequence SEQ ID NO.447 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.18 and SEQ ID NO.38;

[0201] (16) The sense strand consisting of the E47 sense sequence SEQ ID NO.11 and the E163b sense sequence SEQ ID NO.447 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.12 and SEQ ID NO.38;

[0202] (17) The sense strand consisting of the E47 sense sequence SEQ ID NO.11 and the E179b sense sequence SEQ ID NO.448 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.12 and SEQ ID NO.46;

[0203] (18) The sense strand consisting of the E46 sense sequence SEQ ID NO.9 and the E163b sense sequence SEQ ID NO.447 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.10 and SEQ ID NO.38;

[0204] (19) The sense strand consisting of the E13 sense sequence SEQ ID NO.1 and the E163b sense sequence SEQ ID NO.447 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.38;

[0205] (20) a sense strand composed of the sense sequence of E47 SEQ ID NO.11 and the sense sequence of E162b SEQ ID NO.446 connected in a 5'>3' direction via a linker GALNAC3, and an antisense strand composed of the nucleotide sequences of SEQ ID NO.12 and SEQ ID NO.36; or

[0206] (21) The sense strand consisting of the E32 sense sequence SEQ ID NO.7 and the E162b sense sequence SEQ ID NO.446 was connected via the linker GALNAC3 in the direction from 5'>3', and the antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.36;

[0207] Among them, Am, Um / Tm, Cm and Gm represent 2′-O-methyl modified ribonucleotides A, U, C and G, respectively; Af, Uf / Tm, Cf and Gf represent 2′-fluoro modified ribonucleotides A, U, C and G, respectively; (s) indicates that the two nucleotides are connected by a thiophosphate backbone.

[0208] In some embodiments, the ddsRNA molecule is G1, G2, G3, G4, G5, G6, G10, G11, G12, G13, G14, G15, G16, G17, G18, G19, G20, G21, G22, G23, G24, G25, G26, GN1, GN2, GN3, GN4, GN5, GN6, GN10, GN11, GN12, GN13, GN14, GN15, GN16, GN17, GN18, GN19, GN20, GN21, GN22, GN23, GN24, GN25, or GN26. In Table 9, the ddsRNA molecules corresponding to the name number G+number consist of one sense strand (the sense strand is connected to two modified sense sequences via a linker dTdTdT) and two modified antisense strands, and the 3' end of the sense strand is connected to L96 in the manner of Formula V. In Table 9, the ddsRNA molecules corresponding to the name number GN+number consist of one sense strand (the sense strand is connected to two modified sense sequences via a linker GALNAC3 in the structure of Formula VI) and two modified antisense strands, and no additional ligand is conjugated.

[0209] In some embodiments, the present disclosure also provides mixed dsRNA molecules comprising two or more dsRNA molecules described above.

[0210] In some embodiments, the present disclosure provides a mixed dsRNA molecule comprising the two dsRNA molecules described above.

[0211] In some embodiments, the mixed dsRNA molecule includes two dsRNA molecules targeting different HBV genes; alternatively, the two dsRNAs target the HBV S gene and the HBV X gene, respectively. The nucleotide sequence of the dsRNA molecule targeting the HBV S gene is shown in Table 1, and the nucleotide sequence of the dsRNA molecule targeting the HBV X gene is shown in Table 2.

[0212] In some embodiments, the mixed dsRNA molecule is selected from the group consisting of:

[0213] (1) E90 and E162;

[0214] (2) E90 and E179;

[0215] (3) E46 and E162;

[0216] (4) E46 and E179;

[0217] (5) E31 and E162;

[0218] (6) E31 and E179;

[0219] (7) E13 and E162;

[0220] (8) E13 and E179;

[0221] (9)ES6 and E162;

[0222] (10)ES6 and E179;

[0223] (11) E14 and 162;

[0224] (12) E14 and E179

[0225] (13) E32 and E163;

[0226] (14) E32 and E163;

[0227] (15) E90 and E163;

[0228] (16) E47 and E163;

[0229] (17) E47 and E179;

[0230] (18) E46 and E163;

[0231] (19) E13 and E163;

[0232] (20) E47 and E162;

[0233] (21)E32 and E162.

[0234] In some embodiments, the mixed dsRNA molecule is selected from the group consisting of:

[0235] (1) E90 and E162;

[0236] (2) E90 and E179;

[0237] (3) E46 and E162;

[0238] (4) E46 and E179;

[0239] (5) E31 and E162;

[0240] (6) E31 and E179;

[0241] (7) E13 and E162;

[0242] (8) E13 and E179;

[0243] (9)ES6 and E162;

[0244] (10)ES6 and E179;

[0245] (11) E14 and 162;

[0246] (12) E14 and E179

[0247] (13) E32 and E163;

[0248] (14) E32 and E163;

[0249] (15) E90 and E163;

[0250] (16) E47 and E163;

[0251] (17) E47 and E179;

[0252] (18) E46 and E163;

[0253] (19) E13 and E163;

[0254] (20) E47 and E162;

[0255] (21) E32 and E162;

[0256] Each dsRNA was conjugated with an L96 ligand at the 3' end of the sense strand.

[0257] In some alternative embodiments, the dsRNA molecule, ddsRNA molecule or mixed dsRNA molecule is capable of inhibiting the expression of all transcripts of the HBV virus.

[0258] In another aspect, the present disclosure also relates to biological materials associated with dsRNA molecules, ddsRNA molecules, or mixed dsRNA molecules;

[0259] In some embodiments, the dsRNA molecule, ddsRNA molecule, or mixed dsRNA molecule-associated biological material can be selected from any one of the following:

[0260] (A) a DNA molecule capable of producing the dsRNA molecule, ddsRNA molecule or mixed dsRNA molecule;

[0261] (B) a vector capable of expressing the dsRNA molecule, ddsRNA molecule or mixed dsRNA molecule;

[0262] (C) a cell comprising the dsRNA molecule, ddsRNA molecule or mixed dsRNA molecule;

[0263] (D) a reagent or a kit containing the dsRNA molecule, ddsRNA molecule or mixed dsRNA molecule or the DNA molecule or the vector;

[0264] (E) A pharmaceutical composition consisting of the dsRNA molecule, ddsRNA molecule or mixed dsRNA molecule, and other pharmaceutically acceptable components.

[0265] In some embodiments, the pharmaceutical composition comprises a pharmacologically effective amount of the dsRNA molecules, ddsRNA molecules or mixed dsRNA molecules of the present disclosure, and other pharmaceutically acceptable components. The "effective amount" refers to the amount of the dsRNA molecules that can effectively produce the expected pharmacological therapeutic effect.

[0266] In some embodiments, "other components" are pharmaceutically acceptable excipients, including but not limited to water, saline, glucose, buffer (such as PBS), excipients, diluents, disintegrants, binders, lubricants, sweeteners, flavorings, preservatives or combinations thereof.

[0267] In another aspect, the present disclosure relates to the use of the above-mentioned dsRNA molecules, ddsRNA molecules, mixed dsRNA molecules or related biological materials for preventing and / or treating diseases mediated by HBV virus infection.

[0268] In some embodiments, the present disclosure relates to the use of the above-mentioned dsRNA molecules, ddsRNA molecules, mixed dsRNA molecules or related biomaterials for alleviating symptoms of diseases mediated by HBV viral infection.

[0269] In some embodiments, the present disclosure provides a method for preventing and / or treating a disease mediated by HBV viral infection, comprising administering a therapeutically effective amount of the above-mentioned dsRNA molecules, ddsRNA molecules, mixed dsRNA molecules, or biological materials related to the above-mentioned dsRNA molecules, ddsRNA molecules, or mixed dsRNA to an individual in need of preventing and / or treating a disease mediated by HBV viral infection.

[0270] In some embodiments, the present disclosure provides the aforementioned dsRNA molecules, ddsRNA molecules, mixed dsRNA molecules, or biological materials related to the aforementioned dsRNA molecules, ddsRNA molecules, or mixed dsRNA molecules for use in preventing and / or treating diseases mediated by HBV viral infection.

[0271] In some embodiments, the present disclosure provides use of the aforementioned dsRNA molecules, ddsRNA molecules, mixed dsRNAs, or biological materials related to the aforementioned dsRNA molecules, ddsRNA molecules, or mixed dsRNAs in the preparation of medicaments for preventing and / or treating diseases mediated by HBV viral infection.

[0272] In some embodiments, the diseases mediated by HBV virus infection include but are not limited to: human chronic hepatitis B (CHB), chronic hepatitis D (CHD), hepatocellular carcinoma, fatty liver, hyperbilirubinemia, cirrhosis, liver cancer, etc.

[0273] In some embodiments, the present disclosure further provides any of the following uses: use of the dsRNA molecules, ddsRNA molecules, or mixed dsRNA molecules, or biomaterials related to the dsRNA molecules, ddsRNA molecules, or mixed dsRNA molecules, for inhibiting HBV virus expression of HBsAg, HBeAg, and HBV DNA, or for preparing products for inhibiting HBV virus expression of HBsAg, HBeAg, and HBV DNA. The inhibition of HBsAg, HBeAg, and HBV DNA expression refers to inhibiting or reducing gene expression levels in humans, monkeys, or mice infected with HBV virus in vivo or in vitro. The inhibition of HBV expression refers to inhibiting or reducing HBsAg, HBeAg, and HBV DNA expression levels of the HBV virus by at least 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%. Detection of target gene, target RNA, or target protein levels can be used to predict or assess activity, efficacy, or therapeutic outcomes.

[0274] In some embodiments, the cell is a mammalian cell carrying or transfected with HBV virus, such as a primate cell or a human cell;

[0275] Optionally, the HBV virus titer is expressed at a high level in the cells;

[0276] And / or, optionally, the cells are derived from the brain, salivary gland, heart, spleen, lung, liver, kidney, intestine, or tumor; further optionally, the cells are liver cancer cells.

[0277] In some embodiments, the cells are selected from HepG2.2.15, DE19, DES19 cells, primary cynomolgus monkey cells carrying HBV virus, and primary human hepatocytes carrying HBV virus.

[0278] In some embodiments, the dsRNA molecules, ddsRNA molecules, mixed dsRNA molecules or related biological materials can be administered by any suitable means, such as parenteral administration, including intramuscular, intravenous, intraarterial, peritoneal, or subcutaneous injection. Administration includes, but is not limited to, single administration or multiple administration.

[0279] In some embodiments, a single dose of the pharmaceutical composition can provide long-lasting, reduced expression of HBsAg, HBeAg, and HBV DNA for at least 3, 5, 7, 10, 14 days or longer.

[0280] In some embodiments, the dsRNA molecules, ddsRNA molecules, mixed dsRNA molecules, or biomaterials are used to reduce serum HBsAg, HBeAg, and HBV DNA, or to prepare a product for reducing serum HBsAg, HBeAg, and HBV DNA. The reduction in serum HBsAg, HBeAg, and HBV DNA concentrations involves reducing serum concentrations in a mouse model of HBV infection. For example, the serum HBsAg, HBeAg, and HBV DNA concentrations or levels are reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98%.

[0281] In some embodiments, the use of the dsRNA molecule, ddsRNA molecule, mixed dsRNA molecule or the biomaterial in treating a disease mediated by HBV virus infection or in preparing a product for treating a disease mediated by HBV virus infection;

[0282] In some embodiments, the dsRNA molecule, ddsRNA molecule, mixed dsRNA molecule or the biological material is used to alleviate the symptoms of a disease mediated by HBV virus infection or to prepare a product for alleviating the symptoms of a disease mediated by HBV virus infection.

[0283] In some embodiments, the HBV infection-mediated disease or symptom can be caused by HBV infection and can be regulated by downregulating HBV-expressed antigens and genomes. The treatment refers to the alleviation, reduction, or cure of the HBV infection-mediated disease or symptom, such as a reduction in serum HBsAg, HBeAg, and HBV DNA levels. For example, the serum HBsAg, HBeAg, and HBV DNA levels or concentrations are reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0284] In some embodiments, the present disclosure provides use of the ligand-conjugated dsRNA molecule, ddsRNA molecule, or mixed dsRNA in preparing a liver-targeted drug; the liver-targeted drug can be used to treat liver diseases mediated by HBV viral infection.

[0285] On the other hand, the present disclosure also provides methods and / or combination therapies for treating subjects suffering from conditions that would benefit from inhibition or reduction of expression of HBV virus-associated antigens and viral genomes, such as diseases associated with HBV viral infection, such as chronic hepatitis B and chronic hepatitis D, wherein these methods and / or combination therapies use RNAi compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of all transcripts of the HBV virus.

[0286] The innovations of the present disclosure are as follows: 1. The modified dsRNA, ddsRNA, or mixed dsRNA molecules possess high stability and inhibitory activity. 2. While maintaining high inhibitory activity and stability, the dsRNA, ddsRNA, or mixed dsRNA molecules conjugated with a ligand also exhibit good liver targeting and the ability to promote cellular endocytosis, thereby reducing the impact on other tissues or organs and the amount of dsRNA, ddsRNA, or mixed dsRNA molecules used, thereby alleviating toxicity and lowering costs. Furthermore, the dsRNA, ddsRNA, or mixed dsRNA molecules conjugated with a ligand can enter target cells and tissues without the need for transfection reagents, reducing the negative effects of transfection reagents, such as cell or tissue toxicity. This opens up the possibility of targeted therapy. Although many modifications can be attempted to improve the performance of dsRNA, ddsRNA molecules or mixed dsRNA, these attempts are generally difficult to demonstrate both mediating RNA interference and having improved stability in serum (e.g., having increased resistance to nucleases and / or extended duration). The modified dsRNA, ddsRNA molecules or mixed dsRNA disclosed herein have high stability while maintaining high inhibitory activity, achieving unexpected technical effects.

[0287] definition:

[0288] "G," "C," "A," "T," and "U" generally represent nucleotides based on guanine, cytosine, adenine, thymine, and uracil, respectively. GalNAc: N-acetylgalactosamine.

[0289] Modification: N = RNA; dN = DNA; Nm = 2'OMe modification; Nf = 2'F modification; (s) = PS backbone (i.e., 5'-thio-modified phosphate backbone) BRIEF DESCRIPTION OF THE DRAWINGS

[0290] Figure 1: High-throughput screening results of naked dsRNA targeting the S gene in four subtypes.

[0291] Figure 2: High-throughput screening results of naked dsRNA targeting gene X in four subtypes.

[0292] Figure 3: High-throughput screening results of modified dsRNA targeting the S gene in four subtypes. The results corresponding to each dsRNA molecule target subtype A, subtype B, subtype C, and subtype D from left to right.

[0293] Figure 4: High-throughput screening results of modified dsRNA targeting gene X in four subtypes. The results corresponding to each dsRNA molecule target subtype A, subtype B, subtype C, and subtype D from left to right.

[0294] Figure 5: HBsAg, HBeAg and HBV DNA inhibition rate of dual-target ddsRNA molecules in HepG2.2.15 cells.

[0295] Figure 6: Effects of dual-target ddsRNA molecules on HepG2.2.15 cell activity.

[0296] Figure 7: HBsAg, HBeAg and HBV DNA inhibition rate of dual-target ddsRNA molecules in HepG2.2.15 cells. DETAILED DESCRIPTION

[0297] For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which the technology belongs. All technical and patent disclosures cited in this application are incorporated herein by reference in their entirety.

[0298] As used in this disclosure, the term "nucleic acid molecule" may be used to refer to any molecule having a nucleotide sequence composed of two or more nucleotides connected, wherein the connection method includes: connection through a phosphate bond, through a modified phosphate bond (such as a phosphorothioate bond) or through other connection methods.

[0299] As used in this disclosure, the term "nucleotide sequence" refers to a polynucleotide chain composed of nucleotides arranged in a certain order. In this application, this polynucleotide chain can constitute a nucleic acid molecule or a segment of a chain of a nucleic acid molecule. Therefore, a "nucleotide sequence" can be represented by a specific polynucleotide sequence composed of various nucleotides (e.g., ATCG) (e.g., any one of SEQ ID NOs: 1 to 100), or it can be represented by nucleotides from a certain position to a certain position in a sequence. Without limitation, a "nucleotide sequence" can be RNA or DNA, or a hybrid molecule of RNA and DNA, and can also incorporate non-natural nucleotides or artificially modified nucleotides.

[0300] The term "nucleotide", in addition to referring to naturally occurring ribonucleotides or deoxyribonucleotide monomers, is also understood in this application to refer to related structural variants thereof, including derivatives and analogs, which are functionally equivalent with respect to the specific context in which the nucleotide is used, unless the context clearly indicates otherwise. For example, "nucleotide" refers to a deoxyribonucleotide or a ribonucleotide. A nucleotide can be a standard nucleotide (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), a nucleotide isomer, or a nucleotide analog. A nucleotide analog refers to a nucleotide with a modified purine or pyrimidine base or a modified ribose moiety. A nucleotide analog can be a naturally occurring nucleotide (e.g., inosine, etc.) or a non-naturally occurring nucleotide. Non-limiting examples of modifications on the sugar or base portion of a nucleotide include the addition (or removal) of an acetyl group, an amino group, a carboxyl group, a carboxymethyl group, a hydroxyl group, a methyl group, a phosphoryl group, and a thiol group, and the substitution of the carbon and nitrogen atoms of the base by other atoms (e.g., 7-deazapurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholino oligonucleotides. In some embodiments, the "nucleotides" of the present application do not include non-natural nucleotides with modified bases. In some embodiments, the "nucleotides" of the present application do not include nucleotides with modified bases. In the present application, "G", "C", "A", "T" and "U" generally represent nucleotides with guanine, cytosine, adenine, thymine and uracil as bases, respectively. However, in the context of RNA and in RNA sequences, unless otherwise specified, "T" refers to uridine or uracil. It should be understood that in the context of nucleotide sequences in this application, "nucleotides", "nucleotide residues" and "bases" can be used interchangeably. The number of nucleotide pairs and base pairs is in bp, and one bp is one nucleotide pair or one base pair. The number of nucleotides is in nt, and one nt is one nucleotide. In the present application, a polynucleotide is a DNA or RNA nucleotide, which generally contains an unlimited number of nucleotides. An oligonucleotide is a shorter polynucleotide, such as 10 to 100 nucleotides.

[0301] As used in this disclosure, the formation of hydrogen bonds between bases or nucleotides according to the Watson-Crick base pairing principle is referred to as complementarity, pairing, or matching, such as pairing of A with T or U, and C with G or I. Base pairing other than these is referred to as non-complementarity. When referring to "substantially complementary" between polynucleotide sequences or between nucleic acid chains or between a polynucleotide sequence and a nucleic acid chain, it means that the polynucleotide sequences or between nucleic acid chains or between a polynucleotide sequence and a nucleic acid chain are completely complementary or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary. When complementary, one or more mismatches may be allowed, and one or more bulges may be allowed in the polynucleotide sequence or nucleic acid chain, so that the complementary regions formed may be continuous or intermittent. If one of the two complementary polynucleotide sequences is shorter than the other, the number of nucleotides in the shorter one is used for calculation. For example, if the sense sequence is 21 nt and the antisense sequence is 23 nt, and the complementary region formed is 21 bp long, then the calculation is 21 / 21, which is 100% complementary, i.e., completely complementary.

[0302] As used in this disclosure, antisense sequence and sense sequence have the common meanings in the art, specifically with respect to the target sequence HBV X gene mRNA sequence and S gene mRNA of this application, that is, the antisense sequence refers to the sequence in the nucleic acid molecule that is substantially complementary to the target gene mRNA sequence, and the sense sequence refers to the sequence in the nucleic acid molecule that has at least 10 or more consecutive identical nucleotides with the target gene mRNA sequence. In this application, "sense strand" and "sense strand" have the same meaning, which corresponds to the sense strand, which corresponds to the antisense strand. Similarly, "sense sequence" and "sense sequence" have the same meaning,

[0303] As used in this disclosure, "dsRNA" is double-stranded RNA, which comprises two antiparallel and substantially complementary nucleic acid chains. Unless otherwise specified in this application, the length of the double-stranded RNA is not particularly limited. Since siRNA (small interfering RNA) is an RNA with a double-stranded structure, the term "dsRNA" covers siRNA, which can play an RNAi role and is the main tool for achieving RNAi. dsRNA also includes double-stranded RNA that is longer than siRNA, and the length greater than siRNA can refer to that its sense chain is longer than siRNA, or its antisense chain is longer than siRNA, or its sense chain and antisense chain are both longer than siRNA. After a double-stranded RNA that is usually longer than the siRNA sequence it contains enters the cell, it will be decomposed into siRNA by a type III endonuclease called Dicer. When "siRNA" is incorporated into the RNA-induced silencing complex (RISC), one or more helicases in RISC unwind the siRNA double helix. When bound to a targeted mRNA complementary to the antisense strand in the siRNA, one or more endonucleases within the RISC will cleave the target, inducing gene silencing. Typically, most or all of the nucleotides in each chain of the dsRNA molecule are ribonucleotides, but this does not exclude the inclusion of one or more non-ribonucleotides, such as deoxyribonucleotides and / or non-natural nucleotides, in any one or both chains. In some embodiments, the dsRNA molecule does not contain non-natural nucleotides. In some embodiments, each nucleotide in the dsRNA is a ribonucleotide. As used herein, the dsRNA may contain one or more chemically modified nucleotides or may not contain chemically modified nucleotides.

[0304] As used in this disclosure, "overhang" is relative to "blunt end" or "blunt end", which means that there is no single unpaired nucleotide at the 3' and / or 5' end of a double-stranded nucleotide; that is, "overhang" refers to a double-stranded nucleotide in which one chain has more nucleotides than the other chain, resulting in the presence of unpaired nucleotides at the 3' and / or 5' ends of the two chains. In this application, "overhang" and "overhang" have the same meaning. In this disclosure, the description of the overhang of the 5'(s)mN(s)mN3' structure indicates that the two protruding ribonucleotides at the 3' end are both 2'-O-methyl modified, and the three ribonucleotides at the 3' end are connected by phosphorothioate bonds.

[0305] As used in this disclosure, the 3' end and the 5' end have the common meanings in the art. If the direction is not explicitly specified (i.e., "the 3' end direction and the 5' end direction"), they include the meanings of "3' end" and "5' end". "3' end" and "5' end" emphasize the specific end position, such as: the three nucleotides at the 5' end are the three nucleotides counted from the first nucleotide at the 5' end (including it) in the 3' direction, and the three nucleotides at the 3' end are the three nucleotides counted from the first nucleotide at the 3' end (including it) in the 5' direction. In this application, if it does not cause ambiguity to those skilled in the art, no distinction is made between the 5' end and the 5' end, and no distinction is made between the 3' end and the 3' end.

[0306] As used herein, "exposed 5' end" refers to the first nucleotide from the 5' terminus that is no longer attached to other structures, such as linkers or ligands, in the 5' direction. "Exposed 3' end" refers to the first nucleotide from the 3' terminus that is no longer attached to other structures, such as linkers or ligands, in the 3' direction. The concept of "exposed" is in contrast to the concept of being "structure-internal" or "sequence-internal."

[0307] As used in this disclosure, "close," "near," or "far" when describing nucleotide positions on the same sequence or nucleotide chain refers to the number of nucleotides separating the two nucleotide positions.

[0308] As used in this disclosure, the chemical modification methods of nucleotides are divided into phosphate modification, base modification and ribose modification according to the position of the chemical modification, and the chemical modification method has accelerated the development of siRNA drugs. Based on the uniqueness of the drug, the specific application of the chemical modification method still needs to be explored in different specific scenarios. "Ribose modification" refers to chemical modification at various positions of the sugar ring of ribonucleic acid, including substituent modification, such as: 2'-modification (such as: 2'-O-methyl, 2'-fluoro, 2'-MOE), 5' modification (such as: 5'-Mo) and isomerization modification (such as: LNA, GNA modification, etc.). "Phosphate" modification mainly refers to the modification of the phosphodiester backbone between the ribose on siRNA, mainly including thiophosphate (PS), dithiophosphate (PS2), methyl phosphate (MP), methoxypropyl phosphate (MOP) and peptide nucleic acid (PNA). Base modification refers to chemical modification on the base of siRNA, which is mainly divided into three forms: purine modification (such as N6-methyladenosine), pyrimidine modification (such as 5-methylcytidine) and base substitution. When applying a specific modification method to a specific siRNA, it is necessary to consider the impact of different amounts of modification at different positions on the overall function of the siRNA. These different overall modification rules are referred to as "motifs" in this application. In this application, N appears in the context of the motif of the nucleotide sequence, representing ribonucleotides, dN represents deoxyribonucleotides (DNA), Nm represents 2'-O-Me ribose-modified ribonucleotides or 2'-O-methyl-modified ribonucleotides; Nf represents 2'-F ribose-modified ribonucleotides or 2'-fluororibose-modified ribonucleotides; s represents a thiophosphate backbone, i.e., a 5'-thio-modified phosphate backbone, or a thiophosphate bond.

[0309] As used herein, "GalNAc" or "N-acetylgalactosamine" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose. Unless otherwise specified, the term "GalNAc" or "N-acetylgalactosamine" includes both the β form and the α form. Optionally, the GalNAc compound of the present application is the β form, i.e., 2-(acetylamino)-2-deoxy-β-D-galactopyranose. Accordingly, a GalNAc derivative refers to a chemical derivative containing the above-mentioned GalNAc structure.

[0310] The term "solid support" or "solid phase carrier" refers to an inorganic particle, polymer or other solid material as a matrix, which can be linked to the target compound by surface modification with active groups (such as NH2) for the synthesis of oligonucleotides. Examples of solid phase carriers include but are not limited to controlled pore glass (CPG) and polystyrene (PS).

[0311] The term "conjugate" or "conjugate molecule" refers to a compound formed by covalent linkage of chemical moieties.

[0312] As used in this disclosure, the term "about" refers to the typical error range for each value that is readily known to those skilled in the art. Reference to an "about" value or parameter in this application includes (and describes) embodiments directed to that value or parameter itself. As used in this disclosure, when the term "about" precedes a numerical value, it means within a range of 10% above or below that numerical value. For example, "about 100" encompasses 90 and 110.

[0313] As used in this disclosure, the singular forms "a," "an," and "the" include plural forms unless otherwise indicated.

[0314] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0315] It should be understood that the present disclosure includes the various aspects, embodiments, and combinations of the aspects and / or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Within the scope of the technical concept of the present application, the technical solution of the present application may be subjected to a variety of simple modifications, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the contents disclosed herein and fall within the scope of protection of the present application.

[0316] Unless otherwise indicated, the practice of this application will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. All cells were obtained from the Committee for the Preservation of Type Cultures of the Chinese Academy of Sciences or from other sources available to the public, and all other reagents were commercially available.

[0317] It should be understood that the present application includes various aspects, embodiments and combinations of the aspects and / or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Other aspects, improvements and modifications within the scope of the present application will be apparent to those skilled in the art to which the present application belongs. Therefore, those of ordinary skill in the art will recognize that the scope of the present application also includes the improvements and modifications to the aspects and embodiments.

[0318] Example 1: Single-target dsRNA activity screening experiment

[0319] 1. dsRNA Design

[0320] Sequences were designed targeting different sites in the X gene (see Table 1) and S gene (see Table 2) for HBV subtypes A (GeneBank: AF305422.1), B (GeneBank: KP341008.1), C (GeneBank: KM999991), and D (GeneBank: KX470733). The sequence of AD-66810, disclosed in CN108064294A or CN112673103A, was used as a positive control sequence in this patent.

[0321] Table 1: High-throughput screening of dsRNA sequences targeting the S gene

[0322] Table 2: High-throughput screening of dsRNA sequences targeting gene X

[0323] 2. Synthesis and Purification of dsRNA / ddsRNA (Conjugate)

[0324] The dsRNA disclosed herein comprises ribonucleotides or 2'-methoxy or 2'-fluoro modified oligonucleotides, which are synthesized according to a theoretical yield of 1 μmol using a 1 μmol universal Frit vector. All oligonucleotides were prepared on an LK-192X synthesizer using a CPG carrier containing the GalNAc derivative L96 or a protected group. According to sequence requirements, all phosphoramidite monomers corresponding to the nucleoside were diluted in anhydrous acetonitrile at a ratio of 1:40 (g / mL) and coupled twice for 3 minutes. Deprotection was performed using 3% TCA, activation was performed using 0.3 M benzylthiotetrazolyl in acetonitrile, and capping and oxidation were performed using CAPA / CAPB and 50 mM I2 solutions, respectively. After trityl-off synthesis, the solid support was transferred to a 2 mL centrifuge tube, 1.2 mL of ammonia was added, and the tube was heated in a 65°C oven for 3 hours to remove the protecting groups. The tube was then cooled to room temperature and concentrated under vacuum for 30 minutes. The solution was then filtered through a 0.22 μm filter into a vial. Single-stranded fragments were purified using a semi-preparative reverse-phase instrument with an elution gradient of 7% to 30% (ACN:100 mM TEAA) over 10 minutes at a flow rate of 5 mL / min. After purification, the fragment was concentrated under vacuum and dried at room temperature. The sample was dissolved in water and purified on a GE Each solution was desalted on a Hi-Trap desalting column to elute the final oligonucleotide product. All identities and purity were confirmed using ESI-MS and IEX HPLC, respectively. Concentration was determined using a UV-visible microplate reader. Equimolar amounts of the sense and antisense strands were mixed and transferred to a new shipping tube. The mixture was heated at 95°C for 5 minutes, slowly annealed to room temperature, and finally dried using a vacuum concentrator at room temperature to yield the final product.

[0325] 3. High-throughput screening of unmodified dsRNA activity in vitro

[0326] In order to screen for dsRNA that can effectively inhibit multiple subtypes of HBV, we verified the effects in four subtypes: A, B, C, and D.

[0327] 1. Construction of detection plasmid

[0328] The recombinant plasmid was constructed using the psicheck-2 plasmid (GenScript Biotech, psicheck-2 is a dual-luciferase reporter gene plasmid that can express firefly luciferase and Renilla luciferase), and the HBV X or S gene of the four AD subtypes was inserted into the 5'XhoI and 3'NotI cloning sites of the psicheck-2 plasmid, respectively.

[0329] 2. Co-transfection of X or S gene dsRNA and recombinant plasmid into 293T cells

[0330] 293T human embryonic kidney cells were purchased from ATCC; other reagents were commercially available.

[0331] Cell line transfection: cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C constant temperature incubator. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were plated for transfection. The concentration of 293T human embryonic kidney cells was adjusted to 3×10 5 / ml, add 0.1ml of cell solution to each well of a 96-well plate and incubate overnight. Prepare the transfection complex: Mix 8μL Opti-MEM, 8ng of recombinant plasmid, and 2μL of 10nM dsRNA. Combine 9.5μL Opti-MEM and 0.5μL Lipofectamine 2000 transfection reagent, let stand for 5 minutes, then combine the two mixtures and let stand for 20 minutes. Add the above transfection complex to a 96-well plate and incubate in a 5% CO2, 37°C incubator for 6 hours. A control group treated with only culture medium was used. Aspirate the supernatant, add 0.1mL of complete culture medium to each well, continue incubation for 24 hours, and collect the samples.

[0332] 3. DLR detection and analysis

[0333] Detection was performed using the Dual-Luciferase Reporter Assay System kit (Promega). Cells were lysed, collected, and processed according to the kit instructions. The fluorescence intensities of firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferases were measured sequentially using an Infinite Eplex microplate reader (TECAN). The ratio of Renilla (Renilla reniformis) to firefly (Photinus pyralis) luciferase fluorescence intensity was calculated and normalized to the control group, where activity was assumed to be 100%. Tables 3 and 4 show the results of DLR detection targeting the S and X genes, respectively, and the average expression levels of the dual-luciferase reporter genes in the dsRNA treatment groups relative to the control group.

[0334] Table 3: Results of high-throughput 10 nM single-dose screening targeting the S gene in 293T cells

[0335] The results of Table 3 are shown in Figure 1. We selected dsRNA sequences that showed significant inhibition of S gene expression in the four subtypes A, B, C, and D as candidate sequences, and selected 12 of them for further sequence modification.

[0336] Table 4: Results of high-throughput 10 nM single-dose screening targeting gene X in 293T cells

[0337] The results in Table 4 are shown in Figure 2. We selected dsRNA sequences that significantly inhibited gene X expression in all four subtypes, A, B, C, and D, as candidate sequences. We selected 13 of these candidate sequences for further sequence modification. We optimized and validated potential candidate dsRNAs targeting both genes X and S.

[0338] Example 2: Single-target optimization of dsRNA in vitro activity high-throughput screening assay

[0339] To further confirm the ability of dsRNA molecules to target and inhibit either gene X or gene S, we performed sequence modification and optimization (Tables 5 and 6). 293T cells were transfected, and the transfection and detection evaluation procedures were the same as in Example 1. Tables 7 and 8 show the results of DLR detection targeting gene S and gene X, respectively, representing the average expression levels of the dual-luciferase reporter gene in the dsRNA test group relative to the control group.

[0340] Table 5: Optimized dsRNA sequences targeting the S gene

[0341] In the table above, each name number represents a dsRNA molecule containing the sense and antisense sequences in the row in which it is located.

[0342] Table 6: Optimized dsRNA sequences targeting gene X

[0343] In the table above, each name number represents a dsRNA molecule containing the sense and antisense sequences in the row in which it is located.

[0344] Table 7: Screening results of 10 nM single dose targeting S gene

[0345] Table 8: Screening results of 10 nM single dose targeting gene X

[0346] The results in Tables 7 and 8 are shown in Figures 3 and 4, respectively. The optimized dsRNAs demonstrated excellent potential for targeting gene X and gene S, respectively. We synthesized dual-target ddsRNA molecules using the same synthesis procedures as in Example 1, selecting the best performing dsRNA sequences for each gene.

[0347] Example 3: Dual-target optimization of ddsRNA in vitro activity screening and detection

[0348] 1. Dual-target ddsRNA optimization

[0349] Sequences E13, E14, E31, E32, E46, E47, E90, and ES6 were selected for the S gene, and sequences E162, E163, and E179 were selected for the X gene for the construction of ddsRNA molecules. The sense sequence in the ddsRNA sequence is sequentially linked, in 5'-3' order, to the sense sequence of an siRNA targeting the S gene, a linker, and the sense sequence of an siRNA targeting the X gene. If a ligand is present, the sense sequence in the ddsRNA sequence is sequentially linked, in 5'-3' order, to the sense sequence of an siRNA targeting the S gene, a linker, the sense sequence of an siRNA targeting the X gene, and the ligand L96 linked via a phosphate bond (the specific structure is shown in Formula V).

[0350] The optimized ddsRNA sequences for the S gene and the X gene are shown in Table 9:

[0351] The ddsRNA molecules numbered G1-G26 in Table 9 have the structure of Formula V, wherein E13, E14, E31, E32, E46, E47, E90, and ES6 siRNAs targeting the S gene serve as the second siRNA (i.e., the portion not connected to L96 in Formula V, the portion labeled SS-2 and AS-2 in Formula V), and E162, E163, and E179 siRNAs targeting the X gene serve as the first siRNA (i.e., the portion connected to L96 in Formula V, the portion labeled SS-1 and AS-1 in Formula V). The linker dTdTdT itself is also a short nucleotide sequence, which is connected to the first and second siRNAs respectively via conventional phosphate bonds to achieve the purpose of bridging. When constructing the V ddsRNA molecule, the siRNA targeting the S gene does not undergo any changes; the sense sequence of the siRNA targeting the X gene is connected to the linker at its 5' end and to L96 at its 3' end, so that the nucleotides of the sense sequence of the siRNA targeting the X gene are in the structure. Therefore, the 5' and 3' ends of the sense sequence of the siRNA targeting the X gene no longer use phosphorothioate bonds to connect the nucleotides, but only use conventional phosphate bonds to connect. The naked sequence and other modifications are exactly the same as the sense sequences of E162, E163, and E179, and the different sense sequences are represented by E162a, E163a, and E179a, respectively (sequences are shown in SEQ ID NOs. (See Figures 443-445.) The sense sequences of E162a, E163a, and E179a differ only in the phosphate linkage compared to the sense sequences of E162, E163, and E179; their nucleotide composition remains unchanged, which does not affect their ability to form complementary double-stranded regions with the antisense sequences of E162, E163, and E179, respectively. Therefore, in this construct, the antisense sequence targeting the portion of gene X is identical to the antisense sequences of E162, E163, and E179.

[0352] The ddsRNA molecules numbered GN1-GN26 in Table 9 have the structure of Formula VI, wherein E13, E14, E31, E32, E46, E47, E90, and ES6 siRNAs targeting the S gene serve as the first siRNA (i.e., the portions labeled SS-1 and AS-1 in Formula VI), and E162, E163, and E179 siRNAs targeting the X gene serve as the second siRNA (i.e., the portions labeled SS-2 and AS-2 in Formula VI). When constructing the ddsRNA molecule of Formula VI, the siRNA targeting the S gene does not undergo any changes; the sense sequence of the siRNA targeting the X gene is connected to the linker at its 5' end and exposed at its 3' end, so that the 5' terminal nucleotide of the sense sequence of the siRNA targeting the X gene is in the structure and no longer uses a phosphorothioate bond for nucleotide connection, but only uses a conventional phosphate bond for connection. The 3' end is exposed, so the terminal three nucleotides are connected by phosphorothioate bonds. The naked sequence and other modifications are exactly the same as the sense sequences of E162, E163, and E179, and the different sense sequences are represented by E162b, E163b, and E179b, respectively (sequences are shown in SEQ ID NOs. (See Figures 446-448.) The sense sequences of E162b, E163b, and E179b differ only in the phosphate linkage compared to the sense sequences of E162, E163, and E179; their nucleotide composition remains unchanged, which does not affect their ability to form complementary double-stranded regions with the antisense sequences of E162, E163, and E179, respectively. Therefore, in this construct, the antisense sequence targeting the portion of gene X is identical to the antisense sequences of E162, E163, and E179.

[0353] Table 9: Structure of dual-target coupled ddsRNA molecules

[0354] In the table above, each name number represents a ddsRNA molecule containing the sense and antisense sequences in its row.

[0355] 2. Dual-target optimized ddsRNA transfection into HepG2.2.15 cells

[0356] HepG2.2.15 hepatocytes can secrete HBsAg, HBeAg and HBV DNA and are currently a common cell line used for in vitro evaluation of HBV drug efficacy. HepG2.2.15 cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO2, 37°C constant temperature incubator. When the cells were in the logarithmic growth phase and in good condition (70% confluence), they were plated and transfected. The cell density was adjusted to 2×10 cells per well. 5 The cells were plated into 24-well plates. The working final concentrations of ddsRNA were 1nM and 0.02nM transfection systems, respectively. Prepare the transfection complex: mix 250μL Opti-MEM and 5μL ddsRNA, mix 250μL Opti-MEM and 1.5μL RNAiMax transfection reagent, let it stand for 5 minutes, then mix the above two mixtures and let it stand for 20 minutes. Add the above transfection complex to a 24-well plate and incubate in a 5% CO2, 37°C constant temperature incubator for 6 hours. The group treated with only culture medium was used as the control group. Aspirate the supernatant, add 1mL complete culture medium to each well, continue culturing for 6 days, and then collect the cell supernatant for HBsAg, HBeAg and HBV DNA detection. In addition, this embodiment also uses the detection method of the present application when detecting the efficacy of two single-target siRNAs simply mixed in a molar ratio of 1:1. The only difference is that when preparing the transfection complex, the ddsRNA is replaced with a mixture of two single-target siRNAs.

[0357] 3. Detection of HBsAg, HBeAg, and HBV DNA

[0358] Cell supernatants were collected from each treatment group and assayed for HBsAg and HBeAg secretion using ELISA, and HBV DNA secretion using QPCR. Cell viability was also assessed using CellTiter-Glo. Three replicates were performed for each treatment group, with the control group serving as a negative control. The inhibition rate for each treatment group was calculated as (control group content - treatment group content) / control group content x 100%. Inhibition rate data were statistically analyzed using GarphPad software. The results are shown in Tables 10 and 11.

[0359] Table 10 Results of 1 nM single dose screening in HepG2.2.15 cells

[0360] The results of Table 10 are shown in Figure 5. At a concentration of 1 nM, the dual-target ddsRNA molecules we designed and the positive control AD-66810 can significantly inhibit the expression and secretion of HBsAg, HBeAg and HBV DNA. Except for the G22 molecule, the inhibition rates of other candidate ddsRNA molecules on various indicators are all as high as 85% or more. At the same time, the E90 and E162 candidate sequences also showed efficient inhibition of various indicators, proving that targeting the S gene or the X gene alone can also effectively inhibit HBsAg, HBeAg and HBV DNA. In addition, we further tested the effects of each molecule in Table 10 on cell activity. The results are shown in Figure 6. The relative cell activity of the ddsRNA molecules of the present application was maintained above 95%, which was higher than the positive control, and no obvious cytotoxicity was observed.

[0361] To further characterize the in vitro inhibitory effect of each ddsRNA molecule, we performed downregulation at a drug concentration of 0.02 nM and compared it with more simple mixtures of single-target siRNAs. The results are shown in Table 11.

[0362] Table 11 Screening results of 0.02 nM single dose in HepG2.2.15 cells

[0363] The results of Table 11 are shown in Figure 7. Our multiple candidate ddsRNA molecules showed significantly better effects than AD-66810 in inhibiting HBsAg, HBeAg and HBV DNA, such as: G1, G3, G4, G5, G6, G10, G11, G12, G13, G14, G19, G20, G21, G23, G24, G25, G26, E46 / E162.

[0364] Our data further demonstrate that our dual-target coupling strategy may be a more effective HBV treatment strategy. In vitro data demonstrate that our candidate targets and drug design strategy are superior to currently available HBV drugs.

[0365] The above description is merely an optional embodiment, which is intended to be an example and not to limit the combination of features necessary for implementing the present application. The titles provided are not intended to limit the various embodiments of the present application. Terms such as "comprise", "contain" and "include" are not intended to be limiting. In addition, unless otherwise indicated, plural forms are included when there is no numeral modification, and "or" and "or" mean "and / or". Unless otherwise defined herein, the meaning of all technical and scientific terms used herein is the same as that generally understood by those skilled in the art. All publications and patents mentioned in this application are incorporated herein by reference. Without departing from the scope and spirit of the present application, various modifications and variations of the methods and compositions described herein are obvious to those skilled in the art. Although the present application has been described by specific optional embodiments, it should be understood that the present application claimed for protection should not be unduly limited to these specific embodiments. In fact, various variations of the described modes for implementing the present application that are obvious to those skilled in the relevant art are intended to be included within the scope of the appended claims.

Claims

1. A dsRNA molecule for inhibiting HBV gene expression, comprising a sense strand and an antisense strand that complement each other to form a double-stranded region, wherein the sense strand and / or the antisense strand comprises at least 15, 16, 17, 18, 19 or 20 consecutive nucleotides of the nucleotide sequence of any sense sequence and / or antisense sequence listed in Table 1 or Table 2; Optionally, each strand of the dsRNA independently has 15-30 nucleotides; further optionally, each strand independently has 19-23 nucleotides; further optionally, each strand independently has 21-23 nucleotides; And / or, optionally, the double-stranded region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs in length.

2. The dsRNA molecule according to claim 1, characterized in that: The nucleotide sequence of the sense strand and the nucleotide sequence of the antisense strand are shown in Table 1 or Table 2.

3. The dsRNA molecule according to claim 1 or 2, characterized in that: At least one nucleotide in the dsRNA molecule is modified, and the modification is selected from any one or more of the following: locked nucleic acid (LNA) modification, open ring or unlocked (UNA) modification, 2′-methoxyethyl modification, 2′-O-methyl modification, 2′-O-allyl modification, 2′-C-allyl modification, 2′-fluoro modification, 2′-deoxy modification, 2′-hydroxyl modification, thiophosphate backbone modification, DNA modification, fluorescent probe modification; optionally, all nucleotides in the dsRNA molecule are modified nucleotides.

4. The dsRNA molecule according to any one of claims 1 to 3, characterized in that: The dsRNA molecule comprises at least one phosphorothioate internucleotide linkage; optionally, the dsRNA molecule comprises 6 phosphorothioate internucleotide linkages, and / or, the phosphorothioate internucleotide linkages of the dsRNA molecule are located between the three nucleotides at the 3' end of the antisense strand, between the three nucleotides at the 5' end of the antisense strand, and / or between the three nucleotides at the 5' end of the sense strand; further optionally, the phosphorothioate internucleotide linkages of the dsRNA molecule are two phosphorothioate linkages between the three nucleotides at the 3' end of the antisense strand, two phosphorothioate linkages between the three nucleotides at the 5' end of the antisense strand, and two phosphorothioate linkages between the three nucleotides at the 5' end of the sense strand.

5. The dsRNA molecule according to claim 4, characterized in that: dsRNA includes: (1) The antisense strand has a 2-nucleotide overhang at the 3' end; (2) The antisense strand is 2'-fluorinated at least at the 2nd, 14th, and 16th positions from the 5' end, and the positions not modified by 2'-fluorinated are 2'-O-methyl modified; (3) The antisense strand is modified by at least two phosphorothioate internucleotide linkages from the 3' end and the 5' end; (4) The sense strand is continuously 2'-fluorinated at the 7th position and positions 9-11 from the 5' end, and the positions not modified by 2'-fluorinated are 2'-O-methyl modified; (5) The sense strand is modified by at least two phosphorothioate internucleotide linkages from the 5' end.

6. The dsRNA molecule according to claim 5, characterized in that: The dsRNA molecule comprises a sense strand and an antisense strand, wherein: (1) the sense strand sequence is as shown in SEQ ID NO: 1, and the antisense strand sequence is as shown in SEQ ID NO: 2; (2) the sense strand sequence is as shown in SEQ ID NO:3, and the antisense strand sequence is as shown in SEQ ID NO:4; (3) the sense strand sequence is as shown in SEQ ID NO:5, and the antisense strand sequence is as shown in SEQ ID NO:6; (4) the sense strand sequence is as shown in SEQ ID NO:7, and the antisense strand sequence is as shown in SEQ ID NO:8; (5) the sense strand sequence is as shown in SEQ ID NO:9, and the antisense strand sequence is as shown in SEQ ID NO:10; (6) the sense strand sequence is as shown in SEQ ID NO: 11, and the antisense strand sequence is as shown in SEQ ID NO: 12; (7) the sense strand sequence is as shown in SEQ ID NO: 13, and the antisense strand sequence is as shown in SEQ ID NO: 14; (8) the sense strand sequence is as shown in SEQ ID NO: 15, and the antisense strand sequence is as shown in SEQ ID NO: 16; (9) the sense strand sequence is as shown in SEQ ID NO: 17, and the antisense strand sequence is as shown in SEQ ID NO: 18; (10) the sense strand sequence is as shown in SEQ ID NO: 19, and the antisense strand sequence is as shown in SEQ ID NO: 20; (11) the sense strand sequence is shown in SEQ ID NO: 21, and the antisense strand sequence is shown in SEQ ID NO: 22; (12) the sense strand sequence is as shown in SEQ ID NO: 23, and the antisense strand sequence is as shown in SEQ ID NO: 24; (13) the sense strand sequence is shown in SEQ ID NO: 25, and the antisense strand sequence is shown in SEQ ID NO: 26; (14) the sense strand sequence is shown in SEQ ID NO: 27, and the antisense strand sequence is shown in SEQ ID NO: 28; (15) the sense strand sequence is shown in SEQ ID NO:29, and the antisense strand sequence is shown in SEQ ID NO:30; (16) the sense strand sequence is shown in SEQ ID NO:31, and the antisense strand sequence is shown in SEQ ID NO:32; (17) the sense strand sequence is shown in SEQ ID NO:33, and the antisense strand sequence is shown in SEQ ID NO:34; (18) the sense strand sequence is shown in SEQ ID NO:35, and the antisense strand sequence is shown in SEQ ID NO:36; (19) the sense strand sequence is shown in SEQ ID NO:37, and the antisense strand sequence is shown in SEQ ID NO:38; (20) the sense strand sequence is shown in SEQ ID NO:39, and the antisense strand sequence is shown in SEQ ID NO:40; (21) the sense strand sequence is shown in SEQ ID NO:41, and the antisense strand sequence is shown in SEQ ID NO:42; (22) the sense strand sequence is shown in SEQ ID NO:43, and the antisense strand sequence is shown in SEQ ID NO:44; (23) the sense strand sequence is shown in SEQ ID NO:45, and the antisense strand sequence is shown in SEQ ID NO:46; (24) the sense strand sequence is as shown in SEQ ID NO:47, and the antisense strand sequence is as shown in SEQ ID NO:48; or (25) the sense strand sequence is shown in SEQ ID NO:49, and the antisense strand sequence is shown in SEQ ID NO:50; Wherein, Am, Um / Tm, Cm and Gm represent ribonucleotides A, U, C and G modified by 2′-O-methyl, respectively; Af, Uf / Tf, Cf and Gf represent ribonucleotides A, U, C and G modified by 2′-fluoro, respectively; (s) represents that the two nucleotides are connected by a phosphorothioate bond; Or, the dsRNA molecule consists of a sense strand and an antisense strand, wherein: (1) E13 molecule: the sense strand sequence is shown in SEQ ID NO: 1, and the antisense strand sequence is shown in SEQ ID NO: 2; (2) E14 molecule: the sense strand sequence is shown in SEQ ID NO:3, and the antisense strand sequence is shown in SEQ ID NO:4; (3) E31 molecule: the sense strand sequence is shown in SEQ ID NO:5, and the antisense strand sequence is shown in SEQ ID NO:6; (4) E32 molecule: the sense strand sequence is shown in SEQ ID NO:7, and the antisense strand sequence is shown in SEQ ID NO:8; (5) E46 molecule: the sense strand sequence is shown in SEQ ID NO:9, and the antisense strand sequence is shown in SEQ ID NO:10; (6) E47 molecule: the sense strand sequence is shown in SEQ ID NO: 11, and the antisense strand sequence is shown in SEQ ID NO: 12; (7) E83 molecule: the sense strand sequence is shown in SEQ ID NO: 13, and the antisense strand sequence is shown in SEQ ID NO: 14; (8) E86 molecule: the sense strand sequence is shown in SEQ ID NO: 15, and the antisense strand sequence is shown in SEQ ID NO: 16; (9) E90 molecule: the sense strand sequence is shown in SEQ ID NO: 17, and the antisense strand sequence is shown in SEQ ID NO: 18; (10) E91 molecule: the sense strand sequence is shown in SEQ ID NO: 19, and the antisense strand sequence is shown in SEQ ID NO: 20; (11) E92 molecule: the sense strand sequence is shown in SEQ ID NO: 21, and the antisense strand sequence is shown in SEQ ID NO: 22; (12) ES6 molecule: the sense strand sequence is shown in SEQ ID NO: 23, and the antisense strand sequence is shown in SEQ ID NO: 24; (13) E104 molecule: the sense strand sequence is shown in SEQ ID NO: 25, and the antisense strand sequence is shown in SEQ ID NO: 26; (14) E105 molecule: the sense strand sequence is shown in SEQ ID NO: 27, and the antisense strand sequence is shown in SEQ ID NO: 28; (15) E126 molecule: the sense strand sequence is shown in SEQ ID NO: 29, and the antisense strand sequence is shown in SEQ ID NO: 30; (16) E159 molecule: the sense strand sequence is shown in SEQ ID NO:31, and the antisense strand sequence is shown in SEQ ID NO:32; (17) E161 molecule: the sense strand sequence is shown in SEQ ID NO: 33, and the antisense strand sequence is shown in SEQ ID NO: 34; (18) E162 molecule: the sense strand sequence is shown in SEQ ID NO: 35, and the antisense strand sequence is shown in SEQ ID NO: 36; (19) E163 molecule: the sense strand sequence is shown in SEQ ID NO: 37, and the antisense strand sequence is shown in SEQ ID NO: 38; (20) E164 molecule: the sense strand sequence is shown in SEQ ID NO:39, and the antisense strand sequence is shown in SEQ ID NO:40; (21) E176 molecule: the sense strand sequence is shown in SEQ ID NO:41, and the antisense strand sequence is shown in SEQ ID NO:42; (22) E178 molecule: the sense strand sequence is shown in SEQ ID NO:43, and the antisense strand sequence is shown in SEQ ID NO:44; (23) E179 molecule: the sense strand sequence is shown in SEQ ID NO:45, and the antisense strand sequence is shown in SEQ ID NO:46; (24) E180 molecule: the sense strand sequence is shown in SEQ ID NO:47, and the antisense strand sequence is shown in SEQ ID NO:48; or (25) EX1 molecule: The sense strand sequence is shown in SEQ ID NO:49, and the antisense strand sequence is shown in SEQ ID NO:

50.

7. The dsRNA molecule according to any one of claims 1 to 6, characterized in that: The dsRNA is conjugated to at least one ligand; optionally, the ligand is conjugated to the 3' end, 5' end and / or the middle of the sequence of the sense strand and / or antisense strand of the dsRNA molecule; further optionally, the ligand is conjugated to the 3' end of the sense strand of the dsRNA molecule.

8. The dsRNA molecule according to claim 7, characterized in that: The ligand is selected from the following: cholesterol, biotin, vitamins, galactose derivatives or analogs, lactose derivatives or analogs, N-acetylgalactosamine derivatives or analogs, N-acetylglucosamine derivatives or analogs; optionally, the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker; further optionally, the ligand is L96 connected to the dsDNA molecule via a phosphate bond; its structure is as shown in Formula I: Further optionally, the dsRNA has a structure as shown in Formula II:

9. A dual-targeting ddsRNA molecule targeting HBV X gene and S gene, characterized in that: The ddsRNA comprises two different dsRNA molecules bridged by a linker, wherein the linker is selected from the group consisting of: (1) a nucleotide sequence, optionally, the nucleotide sequence is RNA, DNA or a mixed molecule; (2) a functional molecule, optionally, the functional molecule is selected from one or more of the following: -(CH2) 12 -, -(CH2)6-SS-(CH2)6-, -CH2CH2O-(CH2CH2)n-CH2CH2O-, wherein n=1-20, further optionally, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; or (3) 1+1+1GalNAc, the structure of 1+1+1GalNAc is shown in Formula III: Optionally, the linker is 1+1+1GalNAc or uuu or (dT)n, n=1-20, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; further optionally, the linker is (dT)3, i.e. dTdTdT.

10. The ddsRNA molecule according to claim 9, characterized in that: The linker bridges the sense strands of two different dsRNA molecules, and the linker bridges the 3' end of the sense strand of the first dsRNA molecule and the 5' end of the sense strand of the second dsRNA molecule. The structure of the linker is shown in Formula IV: Optionally, the sense strand after bridging is sequentially connected with a dsRNA molecule targeting HBV S gene and a dsRNA molecule targeting X gene in the 5'-3' direction, or the sense strand after bridging is sequentially connected with a dsRNA molecule targeting HBV X gene and a dsRNA molecule targeting S gene in the 5'-3' direction; further optionally, the sense strand after bridging is sequentially connected with a dsRNA molecule targeting HBV S gene and a dsRNA molecule targeting X gene in the 5'-3' direction; further optionally, the nucleotide sequence of the dsRNA molecule targeting HBV S gene is shown in Table 1, and the nucleotide sequence of the dsRNA molecule targeting HBV X gene is shown in Table 2.

11. The ddsRNA molecule according to any one of claims 9 to 10, characterized in that: At least one nucleotide in the ddsRNA molecule is modified, and the modification is selected from any one or more of the following: locked nucleic acid (LNA) modification, open ring or unlocked (UNA) modification, 2′-methoxyethyl modification, 2′-O-methyl modification, 2′-O-allyl modification, 2′-C-allyl modification, 2′-fluoro modification, 2′-deoxy modification, 2′-hydroxyl modification, thiophosphate backbone modification, DNA modification, fluorescent probe modification; optionally, all nucleotides in the dsRNA molecule are modified nucleotides.

12. The ddsRNA molecule according to claim 11, characterized in that: The ddsRNA molecule contains at least one phosphorothioate internucleotide bond; optionally, the exposed 5' end and / or 3' end of the bridged sense strand and / or antisense strand in the ddsRNA molecule contains at least one phosphorothioate internucleotide bond; further optionally, the three nucleotides at the exposed 5' end and / or 3' end of the bridged sense strand and / or antisense strand in the ddsRNA molecule are connected by phosphorothioate internucleotide bonds.

13. The ddsRNA molecule according to claim 12, characterized in that: The ddsRNA molecule includes one, two, three, four, five or six of the following structures: (1) the antisense chain has a 2-nucleotide overhang at the 3' end; (2) at least the 2nd, 14th and 16th positions of the antisense chain from the 5' end are 2'-fluoro modified, and the positions not modified by 2'-fluoro are 2'-O-methyl modified; (3) the antisense chain has at least two thiophosphate internucleotide bonds from the 3' end and the 5' end; (4) after bridging, the sense chain for different targets has continuous 2'-fluoro modifications from the 7th position and 9-11 positions from its own 5' end, and the positions not modified by 2'-fluoro are 2'-O-methyl modified; (5) the sense chain after bridging has at least two thiophosphate internucleotide bonds from the 5' end; (6) the sense chain after bridging has at least two thiophosphate internucleotide bonds from the 3' end.

14. The ddsRNA molecule according to claim 13, characterized in that: The ddsRNA is selected from the group consisting of: (1) A sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO.17 and the E162a sense sequence SEQ ID NO.443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.18 and SEQ ID NO.36; (2) a sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO.17 and the E179a sense sequence SEQ ID NO.445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.18 and SEQ ID NO.46; (3) a sense strand nucleotide sequence composed of the sense sequence of E46 SEQ ID NO.9 and the sense sequence of E162a SEQ ID NO.443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence composed of the nucleotide sequences of SEQ ID NO.10 and SEQ ID NO.36; (4) a sense strand nucleotide sequence composed of the sense sequence of E46 SEQ ID NO.9 and the sense sequence of E179a SEQ ID NO.445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence composed of the nucleotide sequences of SEQ ID NO.10 and SEQ ID NO.46; (5) a sense strand nucleotide sequence consisting of the E31 sense sequence SEQ ID NO.5 and the E162a sense sequence SEQ ID NO.443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.6 and SEQ ID NO.36; (6) a sense strand nucleotide sequence consisting of the E31 sense sequence SEQ ID NO.5 and the E179a sense sequence SEQ ID NO.445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.6 and SEQ ID NO.46; (7) a sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E162a sense sequence SEQ ID NO.443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.36; (8) a sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E179a sense sequence SEQ ID NO.445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.46; (9) A sense strand nucleotide sequence consisting of the ES6 sense sequence SEQ ID NO.23 and the E162a sense sequence SEQ ID NO.443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.24 and SEQ ID NO.36; (10) A sense strand nucleotide sequence consisting of the ES6 sense sequence SEQ ID NO.23 and the E179a sense sequence SEQ ID NO.445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.24 and SEQ ID NO.46; (11) a sense strand nucleotide sequence consisting of the E14 sense sequence SEQ ID NO.3 and the E162a sense sequence SEQ ID NO.443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.4 and SEQ ID NO.36; (12) a sense strand nucleotide sequence consisting of the E14 sense sequence SEQ ID NO.3 and the E179a sense sequence SEQ ID NO.445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.4 and SEQ ID NO.46; (13) a sense strand nucleotide sequence consisting of the E32 sense sequence SEQ ID NO.7 and the E163a sense sequence SEQ ID NO.444 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.38; (14) a sense strand nucleotide sequence consisting of the E32 sense sequence SEQ ID NO.7 and the E179a sense sequence SEQ ID NO.445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.46; (15) a sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO.17 and the E163a sense sequence SEQ ID NO.444 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.18 and SEQ ID NO.38; (16) a sense strand nucleotide sequence consisting of the sense sequence of E47 SEQ ID NO.11 and the sense sequence of E163a SEQ ID NO.444 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.12 and SEQ ID NO.38; (17) a sense strand nucleotide sequence consisting of the E47 sense sequence SEQ ID NO.11 and the E179a sense sequence SEQ ID NO.445 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.12 and SEQ ID NO.46; (18) a sense strand nucleotide sequence consisting of the sense sequence of E46 SEQ ID NO.9 and the sense sequence of E163a SEQ ID NO.444 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.10 and SEQ ID NO.38; (19) a sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E163a sense sequence SEQ ID NO.444 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.38; (20) a sense strand nucleotide sequence consisting of the sense sequence of E47 SEQ ID NO.11 and the sense sequence of E162a SEQ ID NO.443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.12 and SEQ ID NO.36; (21) a sense strand nucleotide sequence consisting of the E32 sense sequence SEQ ID NO.7 and the E162a sense sequence SEQ ID NO.443 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.36; (22) connecting the sense sequence of E90 SEQ ID NO.17 and the sense sequence of E162b SEQ ID NO.446 via a linker in the direction from 5'>3' to form a sense strand nucleotide sequence, and an antisense strand consisting of nucleotide sequences SEQ ID NO.18 and SEQ ID NO.36; (23) a sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO.17 and the E179b sense sequence SEQ ID NO.448 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.18 and SEQ ID NO.46; (24) a sense strand nucleotide sequence consisting of the sense sequence of E46 SEQ ID NO.9 and the sense sequence of E162b SEQ ID NO.446 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.10 and SEQ ID NO.36; (25) a sense strand nucleotide sequence consisting of the sense sequence of E46 SEQ ID NO.9 and the sense sequence of E179b SEQ ID NO.448 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.10 and SEQ ID NO.46; (26) a sense strand nucleotide sequence consisting of the E31 sense sequence SEQ ID NO.5 and the E162b sense sequence SEQ ID NO.446 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.6 and SEQ ID NO.36; (27) a sense strand nucleotide sequence consisting of the E31 sense sequence SEQ ID NO.5 and the E179b sense sequence SEQ ID NO.448 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.6 and SEQ ID NO.46; (28) a sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E162b sense sequence SEQ ID NO.446 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.36; (29) a sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E179b sense sequence SEQ ID NO.448 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.46; (30) a sense strand nucleotide sequence consisting of the ES6 sense sequence SEQ ID NO.23 and the E162b sense sequence SEQ ID NO.446 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.24 and SEQ ID NO.36; (31) a sense strand nucleotide sequence consisting of the ES6 sense sequence SEQ ID NO.23 and the E179b sense sequence SEQ ID NO.448 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.24 and SEQ ID NO.46; (32) a sense strand nucleotide sequence consisting of the E14 sense sequence SEQ ID NO.3 and the E162b sense sequence SEQ ID NO.446 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.4 and SEQ ID NO.36; (33) a sense strand nucleotide sequence consisting of the E14 sense sequence SEQ ID NO.3 and the E179b sense sequence SEQ ID NO.448 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.4 and SEQ ID NO.46; (34) a sense strand nucleotide sequence consisting of the sense sequence of E32 SEQ ID NO.7 and the sense sequence of E163b SEQ ID NO.447 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.8 and SEQ ID NO.38; (35) a sense strand nucleotide sequence consisting of the E32 sense sequence SEQ ID NO.7 and the E179b sense sequence SEQ ID NO.448 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.46; (36) a sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO.17 and the E163b sense sequence SEQ ID NO.447 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.18 and SEQ ID NO.38; (37) A sense strand nucleotide sequence consisting of the E47 sense sequence SEQ ID NO.11 and the E163b sense sequence SEQ ID NO.447 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.12 and SEQ ID NO.38; (38) a sense strand nucleotide sequence consisting of the E47 sense sequence SEQ ID NO.11 and the E179b sense sequence SEQ ID NO.448 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.12 and SEQ ID NO.46; (39) a sense strand nucleotide sequence consisting of the E46 sense sequence SEQ ID NO.9 and the E163b sense sequence SEQ ID NO.447 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.10 and SEQ ID NO.38; (40) a sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E163b sense sequence SEQ ID NO.447 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.38; (41) a sense strand nucleotide sequence consisting of the E47 sense sequence SEQ ID NO.11 and the E162b sense sequence SEQ ID NO.446 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.12 and SEQ ID NO.36; or (42) a sense strand nucleotide sequence consisting of the E32 sense sequence SEQ ID NO.7 and the E162b sense sequence SEQ ID NO.446 connected by a linker in the direction from 5'>3', and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.36; Among them, Am, Um / Tm, Cm and Gm represent ribonucleotides A, U, C and G modified by 2′-O-methyl, respectively; Af, Uf / Tf, Cf and Gf represent ribonucleotides A, U, C and G modified by 2′-fluoro, respectively; (s) means that the two nucleotides are connected by a phosphorothioate bond.

15. The ddsRNA molecule according to any one of claims 9 to 14, characterized in that: The ddsRNA molecule is conjugated to at least one ligand, and optionally, the ligand is conjugated to the 3' end, 5' end and / or the middle of the sequence of the sense strand and / or antisense strand of the ddsRNA molecule; further optionally, the ligand is conjugated to the 3' end of the sense strand of the ddsRNA molecule.

16. The ddsRNA molecule according to claim 15, characterized in that: The ligand is selected from the group consisting of cholesterol, biotin, vitamins, galactose derivatives or analogs, lactose derivatives or analogs, N-acetylgalactosamine derivatives or analogs, N-acetylglucosamine derivatives or analogs; Optionally, the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker; further optionally, the ligand is L96, whose structure is as shown in Formula I: Further optionally, the ddsRNA has a structure of Formula V:

17. The ddsRNA molecule according to claims 9-14, characterized in that: When the linker is 1+1+1GalNAc, the ddsRNA has a structure of Formula VI:

18. The ddsRNA molecule according to claim 16, characterized in that: The ddsRNA molecule is selected from the group consisting of: (1) A sense strand nucleotide sequence consisting of the sense sequence of E90 SEQ ID NO.17 and the sense sequence of E162a SEQ ID NO.443 was connected in the direction of 5'>3' by a linker dTdTdT, and the 3' end of the sense sequence of E162a was connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.18 and SEQ ID NO.36; (2) a sense strand nucleotide sequence composed of the sense sequence of E90 SEQ ID NO.17 and the sense sequence of E179a SEQ ID NO.445 connected in the direction of 5'>3' by a linker dTdTdT, and the 3' end of the sense sequence of E179a is connected to L96, and an antisense strand nucleotide sequence composed of the nucleotide sequences of SEQ ID NO.18 and SEQ ID NO.46; (3) A sense strand nucleotide sequence composed of the sense sequence of E46 SEQ ID NO.9 and the sense sequence of E162a SEQ ID NO.443 connected in the direction of 5'>3' by a linker dTdTdT, and the 3' end of the sense sequence of E162a is connected to L96, and an antisense strand nucleotide sequence composed of the nucleotide sequences of SEQ ID NO.10 and SEQ ID NO.36; (4) The sense strand nucleotide sequence of E46 sense sequence SEQ ID NO.9 and E179a sense sequence SEQ ID NO.445 were connected in the direction of 5'>3' through the linker dTdTdT to form a sense strand nucleotide sequence and the sense sequence of E179a sense sequence The 3' end of the column is connected to L96 and an antisense strand nucleotide sequence consisting of nucleotide sequences SEQ ID NO.10 and SEQ ID NO.46; (5) A sense strand nucleotide sequence composed of the sense sequence of E31 SEQ ID NO.5 and the sense sequence of E162a SEQ ID NO.443 connected in the direction of 5'>3' by a linker dTdTdT, and the 3' end of the sense sequence of E162a is connected to L96, and an antisense strand nucleotide sequence composed of the nucleotide sequences of SEQ ID NO.6 and SEQ ID NO.36; (6) A sense strand nucleotide sequence consisting of the sense sequence of E31 SEQ ID NO.5 and the sense sequence of E179a SEQ ID NO.445 connected in the direction of 5'>3' by a linker dTdTdT, and the 3' end of the sense sequence of E179a is connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.6 and SEQ ID NO.46; (7) A sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E162a sense sequence SEQ ID NO.443 connected in the direction of 5'>3' by a linker dTdTdT, and the 3' end of the E162a sense sequence is connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.36; (8) A sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E179a sense sequence SEQ ID NO.445 connected in the direction from 5'>3' by a linker dTdTdT, and the 3' end of the E179a sense sequence is connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.46; (9) A sense strand nucleotide sequence consisting of the ES6 sense sequence SEQ ID NO.23 and the E162a sense sequence SEQ ID NO.443 connected in the direction of 5'>3' by a linker dTdTdT, and the 3' end of the E162a sense sequence is connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.24 and SEQ ID NO.36; (10) A sense strand nucleotide sequence consisting of the ES6 sense sequence SEQ ID NO.23 and the E179a sense sequence SEQ ID NO.445 connected in the direction from 5'>3' by a linker dTdTdT, and the 3' end of the E179a sense sequence is connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.24 and SEQ ID NO.46; (11) A sense strand nucleotide sequence consisting of the E14 sense sequence SEQ ID NO.3 and the E162a sense sequence SEQ ID NO.443 connected in the direction from 5'>3' by a linker dTdTdT, and the 3' end of the E162a sense sequence is connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.4 and SEQ ID NO.36; (12) A sense strand nucleotide sequence consisting of the E14 sense sequence SEQ ID NO.3 and the E179a sense sequence SEQ ID NO.445 connected in the direction from 5'>3' by a linker dTdTdT, and the 3' end of the E179a sense sequence is connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.4 and SEQ ID NO.46; (13) A sense strand nucleotide sequence consisting of the sense sequence of E32 SEQ ID NO.7 and the sense sequence of E163a SEQ ID NO.444 was connected in the direction of 5'>3' by a linker dTdTdT, and the 3' end of the sense sequence of E163a was connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.8 and SEQ ID NO.38; (14) A sense strand nucleotide sequence consisting of the sense sequence of E32 SEQ ID NO.7 and the sense sequence of E179a SEQ ID NO.445 was connected in the direction of 5'>3' by a linker dTdTdT, and the 3' end of the sense sequence of E179a was connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.8 and SEQ ID NO.46; (15) A sense strand nucleotide sequence consisting of the E90 sense sequence SEQ ID NO.17 and the E163a sense sequence SEQ ID NO.444 connected in the direction from 5'>3' by a linker dTdTdT, and the 3' end of the E163a sense sequence is connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.18 and SEQ ID NO.38; (16) A sense strand nucleotide sequence consisting of the sense sequence of E47 SEQ ID NO.11 and the sense sequence of E163a SEQ ID NO.444 was connected in the direction of 5'>3' via a linker dTdTdT, and the 3' end of the sense sequence of E163a was connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.12 and SEQ ID NO.38; (17) A sense strand nucleotide sequence consisting of the sense sequence of E47 SEQ ID NO.11 and the sense sequence of E179a SEQ ID NO.445 was connected in the direction of 5'>3' via a linker dTdTdT, and the 3' end of the sense sequence of E179a was connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.12 and SEQ ID NO.46; (18) A sense strand nucleotide sequence consisting of the sense sequence of E46 SEQ ID NO.9 and the sense sequence of E163a SEQ ID NO.444 connected in the direction of 5'>3' by a linker dTdTdT, and the 3' end of the sense sequence of E163a is connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.10 and SEQ ID NO.38; (19) A sense strand nucleotide sequence consisting of the E13 sense sequence SEQ ID NO.1 and the E163a sense sequence SEQ ID NO.444 connected in the direction from 5'>3' by a linker dTdTdT, and the 3' end of the E163a sense sequence is connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.38; (20) A sense strand nucleotide sequence consisting of the sense sequence of E47 SEQ ID NO.11 and the sense sequence of E162a SEQ ID NO.443 was connected in the direction of 5'>3' via a linker dTdTdT, and the 3' end of the sense sequence of E162a was connected to L96, and an antisense strand nucleotide sequence consisting of the nucleotide sequences of SEQ ID NO.12 and SEQ ID NO.36; (21) The sense strand nucleotide sequence of E32 sense sequence SEQ ID NO.7 and E162a sense sequence SEQ ID NO.443 were connected in the direction of 5'>3' through the linker dTdTdT to form the sense strand nucleotide sequence and the sense strand nucleotide sequence of E162a sense sequence SEQ ID NO.443 was connected in the direction of 5'>3' through the linker dTdTdT The 3' end of the sequence is connected to L96 and an antisense strand nucleotide sequence consisting of nucleotide sequences SEQ ID NO.8 and SEQ ID NO.36; Among them, Am, Um / Tm, Cm and Gm represent ribonucleotides A, U, C and G modified by 2′-O-methyl, respectively; Af, Uf / Tf, Cf and Gf represent ribonucleotides A, U, C and G modified by 2′-fluoro, respectively; (s) means that the two nucleotides are connected by a phosphorothioate bond; (dTdTdT) means that the two dsRNA molecules are bridged by three deoxythymidine nucleotide linkers.

19. The ddsRNA molecule according to claim 17, characterized in that: The ddsRNA molecule is selected from the group consisting of: (1) A sense strand consisting of the sense sequence of E90 SEQ ID NO.17 and the sense sequence of E162b SEQ ID NO.446 was connected via a linker 1+1+1GALNAC in the direction from 5'>3', and an antisense strand consisting of the nucleotide sequences of SEQ ID NO.18 and SEQ ID NO.36; (2) a sense strand composed of the sense sequence of E90 SEQ ID NO.17 and the sense sequence of E179b SEQ ID NO.448 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence composed of the nucleotide sequences of SEQ ID NO.18 and SEQ ID NO.46; (3) A sense strand consisting of the sense sequence of E46 SEQ ID NO.9 and the sense sequence of E162b SEQ ID NO.446 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.10 and SEQ ID NO.36; (4) A sense strand consisting of the sense sequence of E46 SEQ ID NO.9 and the sense sequence of E179b SEQ ID NO.448 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.10 and SEQ ID NO.46; (5) a sense strand composed of the sense sequence of E31 SEQ ID NO.5 and the sense sequence of E162b SEQ ID NO.446 connected in the direction of 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence composed of the nucleotide sequences of SEQ ID NO.6 and SEQ ID NO.36; (6) A sense strand consisting of the sense sequence of E31 SEQ ID NO.5 and the sense sequence of E179b SEQ ID NO.448 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.6 and SEQ ID NO.46; (7) A sense strand consisting of the E13 sense sequence SEQ ID NO.1 and the E162b sense sequence SEQ ID NO.446 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.36; (8) A sense strand consisting of the E13 sense sequence SEQ ID NO.1 and the E179b sense sequence SEQ ID NO.448 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.46; (9) A sense strand consisting of the ES6 sense sequence SEQ ID NO.23 and the E162b sense sequence SEQ ID NO.446 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.24 and SEQ ID NO.36; (10) A sense strand consisting of the ES6 sense sequence SEQ ID NO.23 and the E179b sense sequence SEQ ID NO.448 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.24 and SEQ ID NO.46; (11) A sense strand consisting of the E14 sense sequence SEQ ID NO.3 and the E162b sense sequence SEQ ID NO.446 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.4 and SEQ ID NO.36; (12) a sense strand composed of the sense sequence of E14 SEQ ID NO.3 and the sense sequence of E179b SEQ ID NO.448 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence composed of the nucleotide sequences of SEQ ID NO.4 and SEQ ID NO.46; (13) A sense strand consisting of the sense sequence of E32 SEQ ID NO.7 and the sense sequence of E163b SEQ ID NO.447 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.38; (14) A sense strand consisting of the sense sequence of E32 SEQ ID NO.7 and the sense sequence of E179b SEQ ID NO.448 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.8 and SEQ ID NO.46; (15) A sense strand consisting of the E90 sense sequence SEQ ID NO.17 and the E163b sense sequence SEQ ID NO.447 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.18 and SEQ ID NO.38; (16) A sense strand consisting of the sense sequence of E47 SEQ ID NO.11 and the sense sequence of E163b SEQ ID NO.447 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.12 and SEQ ID NO.38; (17 A sense strand consisting of the sense sequence of E47 SEQ ID NO.11 and the sense sequence of E179b SEQ ID NO.448 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.12 and SEQ ID NO.46; (18) A sense strand consisting of the sense sequence of E46 SEQ ID NO.9 and the sense sequence of E163b SEQ ID NO.447 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.10 and SEQ ID NO.38; (19) A sense strand consisting of the E13 sense sequence SEQ ID NO.1 and the E163b sense sequence SEQ ID NO.447 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence consisting of the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.38; (20) a sense strand composed of the sense sequence of E47 SEQ ID NO.11 and the sense sequence of E162b SEQ ID NO.446 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence composed of the nucleotide sequences of SEQ ID NO.12 and SEQ ID NO.36; or (21) a sense strand composed of the sense sequence of E32 SEQ ID NO.7 and the sense sequence of E162b SEQ ID NO.446 connected in the direction from 5'>3' by a linker 1+1+1GALNAC, and an antisense strand nucleotide sequence composed of the nucleotide sequences of SEQ ID NO.8 and SEQ ID NO.36; Among them, Am, Um / Tm, Cm and Gm represent ribonucleotides A, U, C and G modified by 2′-O-methyl, respectively; Af, Uf / Tf, Cf and Gf represent ribonucleotides A, U, C and G modified by 2′-fluoro, respectively; (s) means that the two nucleotides are connected by a phosphorothioate bond.

20. A mixed dsRNA molecule comprising two dsRNA molecules according to any one of claims 1 to 8; optionally, the two dsRNAs target HBV S gene and HBV X gene, respectively.

21. The mixed dsRNA molecule according to claim 20, characterized in that: The hybrid dsRNA molecule is selected from the group consisting of: (1) E90 and E162; (2) E90 and E179; (3) E46 and E162; (4) E46 and E179; (5) E31 and E162; (6) E31 and E179; (7) E13 and E162; (8) E13 and E179; (9)ES6 and E162; (10)ES6 and E179; (11) E14 and 162; (12) E14 and E179 (13) E32 and E163; (14) E32 and E163; (15) E90 and E163; (16) E47 and E163; (17) E47 and E179; (18) E46 and E163; (19) E13 and E163; (20) E47 and E162; (21) E32 and E162; Optionally, each dsRNA in the mixed dsRNA is conjugated with an L96 ligand at the 3' end of the sense strand.

22. A biological material associated with the dsRNA molecule of any one of claims 1 to 8, the ddsRNA molecule of any one of claims 9 to 19, or the mixed dsRNA molecule of any one of claims 20 to 21, selected from any one of the following: (A) a DNA molecule capable of producing the dsRNA molecule, ddsRNA molecule or mixed dsRNA molecule; (B) a vector comprising the dsRNA molecule, ddsRNA molecule or mixed dsRNA molecule; (C) a cell comprising the dsRNA molecule, ddsRNA molecule or mixed dsRNA molecule; (D) a reagent or a kit containing the dsRNA molecule, ddsRNA molecule or mixed dsRNA molecule or the DNA molecule or the vector; (E) A pharmaceutical composition, consisting of the dsRNA molecule, ddsRNA molecule or mixed dsRNA molecule and other pharmaceutically acceptable components.

23. Use of the dsRNA molecule of any one of claims 1 to 8, the ddsRNA molecule of any one of claims 9 to 19, the mixed dsRNA molecule of any one of claims 20 to 21 or the biomaterial of claim 22 in the preparation of a medicament for preventing and / or treating a disease mediated by HBV virus infection; optionally, the disease mediated by HBV virus infection comprises: Human chronic hepatitis B (CHB), chronic hepatitis D (CHD), hepatic diabetes, fatty liver, hyperbilirubinemia, cirrhosis, and liver cancer.

24. A method for preventing and / or treating a disease mediated by HBV virus infection, the method comprising administering to an individual in need thereof a therapeutically effective amount of a dsRNA molecule according to any one of claims 1 to 8, a ddsRNA molecule according to any one of claims 9 to 19, or a mixed dsRNA molecule according to any one of claims 20 to 21, or a biomaterial according to claim 22; Optionally, the disease mediated by HBV virus infection includes: Human chronic hepatitis B (CHB), chronic hepatitis D (CHD), hepatic diabetes, fatty liver, hyperbilirubinemia, cirrhosis, and liver cancer.

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