Double-stranded ribonucleic acid for inhibiting hepatitis B virus

Abstract

Provided are a double-stranded ribonucleic acid for inhibiting hepatitis B virus, a pharmaceutically acceptable salt thereof or a ligand conjugate thereof, and a pharmaceutical composition comprising the double-stranded ribonucleic acid, the pharmaceutically acceptable salt thereof or the ligand conjugate thereof, and therapeutic uses of the double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof or a ligand conjugate thereof.

Description

Double-stranded RNA for inhibiting hepatitis B virus

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority and benefits of Chinese Patent Application No. 202210708546.7 filed with the State Intellectual Property Office of China on June 21, 2022, and the contents disclosed in said application are hereby incorporated by reference in their entirety. Technical Field

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

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

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

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

[0007] SUMMARY OF THE INVENTION

[0008] On the one hand, the present application provides a double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the double-stranded ribonucleic acid comprises a sense strand and an antisense strand, wherein the sense strand comprises 5'-CUGGCUCAGUUUACUAGUA-3' (SEQ ID NO: 1) and the length of the sense strand does not exceed 21 nucleotides, wherein the antisense strand comprises 5'-UACUAGUAAACUGAGCCAGGA-3' (SEQ ID NO: 2) and the length of the antisense strand does not exceed 23 nucleotides, and the sense strand or the antisense strand is optionally modified.

[0009] On the other hand, the present application provides a pharmaceutical composition comprising the double-stranded ribonucleic acid of the present application, a pharmaceutically acceptable salt thereof or a ligand conjugate thereof and a pharmaceutically acceptable carrier or excipient.

[0010] On the other hand, the present application provides a kit for treating and / or preventing hepatitis B virus infection, which includes the double-stranded ribonucleic acid, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present application; and optional instructions for use.

[0011] On the other hand, the present application provides a method for treating and / or preventing hepatitis B virus infection, which comprises administering the double-stranded ribonucleic acid, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present application to a subject for treatment and / or prevention (hereinafter also referred to as a subject).

[0012] On the other hand, the present application provides the use of the double-stranded ribonucleic acid, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present application in the preparation of a drug for treating and / or preventing hepatitis B virus infection.

[0013] On the other hand, the present application provides use of the double-stranded ribonucleic acid, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present application in treating and / or preventing hepatitis B virus infection.

[0014] In another aspect, the present application provides the double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, a ligand conjugate thereof, or a pharmaceutical composition of the present application for treating and / or preventing hepatitis B virus infection.

[0015] In some embodiments, the hepatitis B virus infection can be at any stage of the disease, such as acute hepatitis B or chronic hepatitis B, or liver diseases caused by hepatitis B virus infection include hepatitis, liver fibrosis, cirrhosis, liver failure or liver cancer. In some embodiments, the hepatitis B virus infection is chronic hepatitis B.

[0016] In some embodiments, the double-stranded RNA, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present application is used as a single therapeutic agent for treating and / or preventing hepatitis B virus infection.

[0017] In some embodiments, the double-stranded RNA, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present application is used together with other therapeutic agents for treating and / or preventing hepatitis B virus infection.

[0018] Detailed Description of the Invention

[0019] The exemplary embodiments of the present application will be described below, but those skilled in the art will understand that the scope of protection of the present application is not limited thereto. Instead, various modifications, changes or alterations may be made based on the spirit and concept of the present application, and the contents after such modifications, changes or alterations still fall within the scope of the present application.

[0020] The present application provides a double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the double-stranded ribonucleic acid comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides of 5'-CUGGCUCAGUUUACUAGUA-3' (SEQ ID NO: 1) and the length of the sense strand does not exceed 21 nucleotides, wherein the antisense strand comprises at least 15 consecutive nucleotides of 5'-UACUAGUAAACUGAGCCAGGA-3' (SEQ ID NO: 2) and the length of the antisense strand does not exceed 23 nucleotides, and the sense strand or the antisense strand is optionally modified.

[0021] The present application provides a double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the double-stranded ribonucleic acid comprises a sense strand and an antisense strand, wherein the sense strand comprises 5'-CUGGCUCAGUUUACUAGUA-3' (SEQ ID NO: 1) and the length of the sense strand does not exceed 21 nucleotides, wherein the antisense strand comprises 5'-UACUAGUAAACUGAGCCAGGA-3' (SEQ ID NO: 2) and the length of the antisense strand does not exceed 23 nucleotides, and the sense strand or the antisense strand is optionally modified.

[0022] The present application also provides a double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the double-stranded ribonucleic acid comprises a sense chain and an antisense chain, and the sense chain and the antisense chain have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the full length of the nucleotide sequence of the sense chain and the antisense chain, respectively.

[0023] In some embodiments, the double-stranded RNA, pharmaceutically acceptable salts thereof, or ligand conjugates thereof of the present application can be used to inhibit hepatitis B virus, particularly to inhibit hepatitis B virus gene expression.

[0024] In some embodiments, the double-stranded RNA, its pharmaceutically acceptable salt, or its ligand conjugate of the present application is an RNAi drug.

[0025] Double-stranded region length

[0026] In some embodiments, the double-stranded region of the double-stranded RNA has a length of 19-21 nucleotide pairs, for example, the double-stranded region of the double-stranded RNA has a length of 19, 20 or 21 nucleotide pairs.

[0027] protrusion

[0028] In some embodiments, the sense strand or antisense strand optionally includes an overhang at the 5' end and / or the 3' end.

[0029] In some embodiments, the overhang contains 1, 2, 3, 4, or 5 nucleotides. In some embodiments, the overhang contains 1 or 2 nucleotides.

[0030] In some embodiments, the sense strand optionally includes an overhang at the 5' end and / or the 3' end. In some embodiments, the sense strand optionally includes an overhang of 1, 2, 3, 4, or 5 nucleotides at the 5' end and / or the 3' end. In some embodiments, the sense strand optionally includes an overhang of 1 or 2 nucleotides at the 5' end and / or the 3' end.

[0031] In some embodiments, the antisense strand optionally includes an overhang at the 5' end and / or the 3' end. In some embodiments, the antisense strand optionally includes an overhang of 1, 2, 3, 4, or 5 nucleotides at the 5' end and / or the 3' end. In some embodiments, the antisense strand optionally includes an overhang of 1 or 2 nucleotides at the 5' end and / or the 3' end.

[0032] In some embodiments, the overhang is selected from unmodified or modified A, G, C, U, or T.

[0033] In some embodiments, the antisense strand optionally includes an overhang at the 5' end and / or the 3' end, wherein the overhang is selected from unmodified or modified GA. In some embodiments, the antisense strand optionally includes an overhang at the 3' end, wherein the overhang is selected from unmodified or modified GA.

[0034] In some embodiments, the overhang is selected from unmodified or modified U or T.

[0035] In some embodiments, when the overhang is one nucleotide, the overhang is selected from unmodified or modified U or T. In some embodiments, when the overhang is one nucleotide, the overhang is selected from 2'-O-methyl-modified U (hereinafter also referred to as u) or deoxythymidine nucleotide (hereinafter also referred to as dT).

[0036] In some embodiments, when the overhang is 2 nucleotides, the overhang is selected from unmodified or modified UU or TT. In some embodiments, when the overhang is 2 nucleotides, the overhang is selected from uu or dTdT.

[0037] In some embodiments, the sense strand optionally includes an overhang at the 5' end and / or the 3' end, wherein the overhang is selected from uu or dTdT.

[0038] In some embodiments, the antisense strand optionally includes an overhang at the 5' end and / or the 3' end, wherein the overhang is selected from uu or dTdT.

[0039] In some embodiments, the overhang is linked to the adjacent nucleotide via a phosphate group or a phosphorothioate group.

[0040] In some embodiments, one or more nucleotides in the overhang are linked by phosphate or phosphorothioate groups.

[0041] Modification

[0042] In some embodiments, the sense strand or the antisense strand is optionally modified.

[0043] In some embodiments, one or more nucleotides of the sense strand or antisense strand are modified.

[0044] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides of the sense strand are modified; or a range of nucleotides within any of the foregoing values ​​is modified, e.g., 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, or 1-10 nucleotides are modified.

[0045] In some embodiments, more than one, more than two, more than three, more than four, more than five, more than six, more than seven, more than eight, more than nine, more than ten, more than eleven, more than twelve, more than thirteen, more than fourteen, more than fifteen, more than sixteen, more than seventeen, more than eighteen, more than nineteen, more than twenty, or more than twenty-one nucleotides of the sense strand are modified; typically, more than ten, more than eleven, more than twelve, more than thirteen, more than fourteen, more than fifteen, more than sixteen, more than seventeen, more than eighteen, or more than nineteen nucleotides of the sense strand are modified.

[0046] In some embodiments, all nucleotides in the sense strand are modified.

[0047] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides of the antisense strand are modified; or a range of nucleotides within any of the foregoing values ​​is modified, e.g., 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, or 1-10 nucleotides are modified.

[0048] In some embodiments, more than one, more than two, more than three, more than four, more than five, more than six, more than seven, more than eight, more than nine, more than ten, more than eleven, more than twelve, more than thirteen, more than fourteen, more than fifteen, more than sixteen, more than seventeen, more than eighteen, more than nineteen, more than twenty-one, more than twenty-two, or more than twenty-three nucleotides of the antisense strand are modified; typically, more than ten, more than eleven, more than twelve, more than thirteen, more than fourteen, more than fifteen, more than sixteen, more than seventeen, more than eighteen, more than nineteen, more than twenty-one, or more than twenty-one nucleotides of the antisense strand are modified.

[0049] In some embodiments, all nucleotides in the antisense strand are modified.

[0050] In some embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified.

[0051] In some embodiments, the modification is selected from the group consisting of a sugar modification of a nucleotide, a modification of a linkage between nucleotides, or a terminal modification.

[0052] In some embodiments, the sugar modification of the nucleotide is selected from dehydroxylation, fluorination, amination, alkylation, hydroxyalkylation or hydroxyalkenylation.

[0053] In some embodiments, the sugar modification of the nucleotide occurs at the 2' position of the sugar group.

[0054] In some embodiments, the glycosyl modification is selected from 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-alkyl, 2'-O-alkyl, 2'-O-ether, 2'-O-alkenyl. In some embodiments, the glycosyl modification is selected from 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-methyl, 2'-ethyl, 2'-methyl-O-methyl, 2'-ethyl-O-methyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-ethyl-O-methyl, or 2'-O-allyl.

[0055] In some embodiments, the internucleotide linkage modification is selected from phosphorothioate (PS), phosphorodithioate (PS2), methylphosphonate (MP), methoxypropylphosphonate (MOP) or aminophosphonate. In some embodiments, the internucleotide linkage modification is selected from phosphorothioate (PS).

[0056] In some embodiments, the terminal modification is selected from a 5'-terminal modification or a 3'-terminal modification. In some embodiments, the terminal modification is selected from a 5'-phosphate, a 5'-methylphosphonate (5'-MP), a 5'-thiophosphate (5'-PS), or a 5'-(E)-vinylphosphonate (5'-(E)-VP).

[0057] In some embodiments, the modification is selected from 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-methyl, 2'-ethyl, 2'-methyl-O-methyl, 2'-ethyl-O-methyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-allyl, phosphorothioate, methylphosphonate, aminophosphonate, 5'-phosphate, 3'-phosphate, 5'-(E)-vinyl phosphate, or 3'-(E)-vinyl phosphate.

[0058] In some embodiments, the modification is selected from 2'-dehydroxylation, 2'-fluoro, 2'-O-methyl, 2'-O-ethyl, 2'-O-ethyl-O-methyl, 2'-O-allyl, phosphorothioate, or 5'-(E)-vinyl phosphate.

[0059] In some embodiments, the modification is selected from 2'-fluoro, 2'-O-methyl, phosphorothioate, or 5'-(E)-vinyl phosphate.

[0060] As will be described in detail below, when referring to the nth nucleotide of both the sense and antisense strands, the numbering starts from the 5' end.

[0061] In some embodiments, the space between the first and second nucleotides of the sense strand and / or the space between the second and third nucleotides of the sense strand is modified with a phosphorothioate group.

[0062] In some embodiments, the antisense strand is modified between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 19 and 20, and / or between nucleotides 20 and 21 with a phosphorothioate group.

[0063] In some embodiments, the 5'-end of the antisense strand is modified with 5'-(E)-vinyl phosphate.

[0064] In some embodiments, the sense strand has 2, 3, 4, or 5 nucleotides modified with 2'-fluoro. In some embodiments, the sense strand has 14, 15, 16, or 17 nucleotides modified with 2'-O-methyl.

[0065] In some embodiments, 0, 1, or 2 nucleotides from position 1 to 6 of the sense strand are modified with 2'-fluoro.

[0066] In some embodiments, 4, 5, or 6 nucleotides from positions 1 to 6 of the sense strand are modified with 2'-O-methyl.

[0067] In some embodiments, two or three nucleotides from position 7 to position 9 of the sense strand are modified with 2'-fluoro.

[0068] In some embodiments, 0 or 1 nucleotides from position 7 to 9 of the sense strand are modified with 2'-O-methyl.

[0069] In some embodiments, 0 or 1 nucleotides from position 10 to 19 of the sense strand are 2'-fluoro-modified.

[0070] In some embodiments, 8, 9, or 10 nucleotides from positions 10 to 19 of the sense strand are modified with 2'-O-methyl.

[0071] In some embodiments, the 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro-modified, and 0, 1 or 2 nucleotides among nucleotides 1 to 6 and / or 10 to 19 of the sense strand are also 2'-fluoro-modified. In some embodiments, the 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro-modified, and 0, 1 or 2 nucleotides among nucleotides 1 to 6 and 10 to 19 of the sense strand are also 2'-fluoro-modified. In some embodiments, the 7th, 8th and 9th nucleotides of the sense strand are 2'-fluoro-modified, and 1 nucleotide among nucleotides 1 to 6 and 10 to 19 of the sense strand is also 2'-fluoro-modified.

[0072] In some embodiments, the antisense strand has 2, 3, 4, 5, 6, or 7 nucleotides modified with 2'-fluoro.

[0073] In some embodiments, the antisense strand has 14, 15, 16, 17, 18, or 19 nucleotides modified with 2'-O-methyl.

[0074] In some embodiments, three or four nucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-fluorinated. In some embodiments, four nucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-fluorinated.

[0075] In some embodiments, three, four, five, or six nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-fluoro-modified. In some embodiments, four or six nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-fluoro-modified.

[0076] In some embodiments, nucleotides 2, 6, 14, and 16 of the antisense strand are 2'-fluoro-modified, and any one or two of nucleotides 1, 3 to 5, 7 to 13, 15, and / or 17 to 21 of the antisense strand are also 2'-fluoro-modified. In some embodiments, nucleotides 2, 6, 14, and 16 of the antisense strand are 2'-fluoro-modified, and any one or two of nucleotides 1, 3 to 5, 7 to 13, 15, and 17 to 21 of the antisense strand are also 2'-fluoro-modified.

[0077] In some embodiments, 14, 15, 16 or 17 nucleotides among nucleotides 1, 3 to 5, 7 to 13, 15, 17 to 21 of the antisense strand are modified with 2'-O-methyl.

[0078] In a specific embodiment, the 5th, 7th, 8th and 9th nucleotides of the sense strand are modified with 2'-fluoro, and the other nucleotides of the sense strand are modified with 2'-O-methyl, and the 2nd, 6th, 8th, 9th, 14th and 16th nucleotides of the antisense strand are modified with 2'-fluoro, and the other nucleotides of the antisense strand are modified with 2'-O-methyl.

[0079] In a specific embodiment, the 5th, 7th, 8th and 9th nucleotides of the sense strand are modified with 2'-fluoro, and the other nucleotides of the sense strand are modified with 2'-O-methyl, and the 2nd, 6th, 14th and 16th nucleotides of the antisense strand are modified with 2'-fluoro, and the other nucleotides of the antisense strand are modified with 2'-O-methyl.

[0080] In a specific embodiment, the 7th, 8th and 9th nucleotides of the sense strand are modified with 2'-fluoro, and the other nucleotides of the sense strand are modified with 2'-O-methyl, and the 2nd, 6th, 14th and 16th nucleotides of the antisense strand are modified with 2'-fluoro, and the other nucleotides of the antisense strand are modified with 2'-O-methyl.

[0081] In a specific embodiment, the 5th, 7th, 8th and 9th nucleotides of the sense strand are modified with 2'-fluoro, the other nucleotides of the sense strand are modified with 2'-O-methyl, and the 2nd, 6th, 8th, 9th, 14th and 16th nucleotides of the antisense strand are modified with 2'-fluoro, the other nucleotides of the antisense strand are modified with 2'-O-methyl, and between the 1st and 2nd nucleotides of the sense strand, between the 2nd and 3rd nucleotides of the sense strand, between the 1st and 2nd nucleotides of the antisense strand, between the 2nd and 3rd nucleotides of the antisense strand, between the 19th and 20th nucleotides of the antisense strand and / or between the 20th and 21st nucleotides of the antisense strand are modified with phosphorothioate groups. Optionally, the 5'-end of the antisense strand is modified with 5'-(E)-vinyl phosphate.

[0082] In a specific embodiment, the 5th, 7th, 8th and 9th nucleotides of the sense strand are modified with 2'-fluoro, the other nucleotides of the sense strand are modified with 2'-O-methyl, and the 2nd, 6th, 14th and 16th nucleotides of the antisense strand are modified with 2'-fluoro, the other nucleotides of the antisense strand are modified with 2'-O-methyl, and between the 1st and 2nd nucleotides of the sense strand, between the 2nd and 3rd nucleotides of the sense strand, between the 1st and 2nd nucleotides of the antisense strand, between the 2nd and 3rd nucleotides of the antisense strand, between the 19th and 20th nucleotides of the antisense strand and / or between the 20th and 21st nucleotides of the antisense strand are modified with phosphorothioate groups. Optionally, the 5'-end of the antisense strand is modified with 5'-(E)-vinyl phosphate.

[0083] In a specific embodiment, the 7th, 8th and 9th nucleotides of the sense strand are modified with 2'-fluoro, the other nucleotides of the sense strand are modified with 2'-O-methyl, the 2nd, 6th, 14th and 16th nucleotides of the antisense strand are modified with 2'-fluoro, the other nucleotides of the antisense strand are modified with 2'-O-methyl, and between the 1st and 2nd nucleotides of the sense strand, between the 2nd and 3rd nucleotides of the sense strand, between the 1st and 2nd nucleotides of the antisense strand, between the 2nd and 3rd nucleotides of the antisense strand, between the 19th and 20th nucleotides of the antisense strand and / or between the 20th and 21st nucleotides of the antisense strand are modified with phosphorothioate groups. Optionally, the 5'-end of the antisense strand is modified with 5'-(E)-vinyl phosphate.

[0084] ligand

[0085] In some embodiments, the ligand includes a branching group and a linking group.

[0086] In some embodiments, the ligand is linked to the double-stranded RNA through a linker. Herein, the double-stranded RNA linked to the ligand is referred to as a double-stranded RNA ligand conjugate.

[0087] In some embodiments, the ligand is connected to one or more targeting groups via a branching group. In some embodiments, the branching group comprises a targeting group.

[0088] In some embodiments, the ligand contains at least one targeting group. In some embodiments, the ligand contains one, two, three, four, or five targeting groups. In some embodiments, the ligand contains two, three, or four targeting groups. In some embodiments, the ligand contains three targeting groups.

[0089] In some embodiments, the targeting group is selected from a GalNAc group.

[0090] In some embodiments, the ligand contains one, two, three, four, or five GalNAc groups. In some embodiments, the ligand contains two, three, or four GalNAc groups. In some embodiments, the ligand contains three GalNAc groups.

[0091] In some embodiments, the branching group is selected from:

[0092] In some embodiments, the linking group is selected from:

[0093] In some embodiments, the ligand is selected from:

[0094] In some embodiments, the ligand is attached to the sense strand or the antisense strand. In some embodiments, the ligand is attached to the 5' or 3' end of the sense strand or the antisense strand. In some embodiments, the ligand is attached to the 5' or 3' end of the sense strand. In some embodiments, the ligand is attached to the 3' end of the sense strand.

[0095] In some embodiments, the part is connected to the sense strand or antisense strand of double-stranded RNA through a phosphate group or a thiophosphate group. In some embodiments, the part is connected to the sense strand through a phosphate group or a thiophosphate group. In some embodiments, the part is connected to the 3 ' end of the sense strand through a phosphate group or a thiophosphate group.

[0096] In some embodiments, the double-stranded ribonucleic acid ligand conjugate is as follows:

[0097] In some embodiments, the double-stranded ribonucleic acid ligand conjugate is selected from:

[0098] In some embodiments, the double-stranded ribonucleic acid ligand conjugate is selected from:

[0099] In some embodiments, the unmodified sense strand of the double-stranded RNA is selected from:

[0100] In some embodiments, the unmodified antisense strand of the double-stranded RNA is selected from:

[0101] In some embodiments, the modified sense strand of the double-stranded RNA is selected from:

[0102] In some embodiments, the modified antisense strand of the double-stranded ribonucleic acid is selected from:

[0103] The double-stranded RNA of the present application can be formed by including any one of the above-mentioned sense strands and any one of the above-mentioned antisense strands.

[0104] For example, the double-stranded ribonucleic acid comprises any one of the following sense strands: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42; and

[0105] The double-stranded ribonucleic acid comprises the antisense strand of any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:24, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64 or SEQ ID NO:65.

[0106] In some embodiments, the double-stranded RNA (unmodified) is selected from:

[0107] In some embodiments, the double-stranded ribonucleic acid (modified) is selected from:

[0108] In some embodiments, the double-stranded ribonucleic acid ligand conjugate is selected from:

[0109] Wherein, the L represents a ligand, and the ligand is as described above; in some embodiments, the ligand is selected from L01 ligand or L02 ligand. Wherein, VP- indicates that the nucleotide to the right of the hyphen is a nucleotide modified with (E)-vinyl phosphate. For example, the u at the 5'-end of SEQ ID NO:64 and SEQ ID NO:65 above is modified with 5'-(E)-vinyl phosphate. Wherein, the sequence ID number of the sense strand refers to the sequence ID number of the nucleic acid sequence in the sense strand. The dsRNA ligand conjugate serial number and the L at the 3' end of the sense strand sequence indicate that in the dsRNA ligand conjugate, a ligand is connected to the 3' end of the sense strand sequence.

[0110] Pharmaceutically acceptable salts

[0111] In some embodiments, the salt described above is selected from base addition salts, acid addition salts, and combinations thereof.

[0112] In some embodiments, the base addition salt is selected from sodium, potassium, calcium, ammonium, organic amine, magnesium salts and combinations thereof, and the acid addition salt is selected from inorganic acid salts, organic acid salts and combinations thereof.

[0113] In some embodiments, the inorganic acid is selected from hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and combinations thereof, and the organic acid is selected from acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and combinations thereof. Beneficial effects

[0114] The double-stranded RNA, its pharmaceutically acceptable salt or its ligand conjugate of the present application has a high coverage rate in different HBV genotypes and is suitable for the treatment and / or prevention of patients with multiple genotypes. The double-stranded RNA, its pharmaceutically acceptable salt or its ligand conjugate of the present application also has good HBV inhibitory activity and stability. In particular, the double-stranded RNA, its pharmaceutically acceptable salt or its ligand conjugate of the present application shows excellent HBV mRNA inhibition efficiency at both the cellular level and the animal level, and can effectively reduce the expression of hepatitis B virus surface antigen and e antigen, showing a good therapeutic effect on hepatitis B virus infection, especially chronic hepatitis B.

[0115] Definition and Description

[0116] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. The absence of a specific definition for a particular term or phrase should not be construed as undefined or ambiguous, but rather should be understood as meaning understood by one of ordinary skill in the art. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0117] In this application, unless otherwise stated, the terms "comprise, include and contain" or equivalents are open-ended expressions, which mean that in addition to the listed elements, components or steps, other unspecified elements, components or steps may also be included.

[0118] When any variable occurs more than once in a compound, nucleotide, or single-stranded or double-stranded structure, its definition in each instance is independent. For example, modifications described herein occur independently, meaning that, unless otherwise indicated, modifications to the sense strand do not affect modifications to the antisense strand, modifications to one nucleotide do not affect modifications to another nucleotide, and modifications to the sugar group of one nucleotide do not affect modifications to another sugar group of the same nucleotide. Such effects include whether a modification occurs and what type of modification is used.

[0119] Examples of HBV genome sequences or mRNA sequences of the present application can be obtained from published databases, such as Genbank, for example, HBV genome sequence (GenBank accession #EU554538.1). The term "HBV" used in the present application also refers to any naturally occurring DNA sequence variation of the HBV genome.

[0120] As known in the art, the term "interfering RNA" or "RNAi" or "interfering RNA sequence" refers to a single-stranded RNA (e.g., a mature miRNA) or double-stranded RNA (e.g., a duplex RNA such as siRNA, aiRNA, or pre-miRNA) that can reduce or inhibit the expression of a target gene or sequence when the interfering RNA and the target gene or sequence are in the same cell (e.g., by mediating the degradation of an mRNA complementary to the interfering RNA sequence or inhibiting the translation or transcription of an mRNA complementary to the interfering RNA sequence). The interfering RNA can have substantial or complete identity to the target gene or sequence, or can include mismatch regions (i.e., mismatch sequences).

[0121] The double-stranded RNA of the present application functions as an interfering RNA. Hereinafter, the double-stranded RNA of the present application may also be referred to as siRNA.

[0122] As known in the art, the term "mismatch region" or "mismatch sequence" refers to a portion of an interfering RNA (e.g., siRNA, aiRNA, miRNA) sequence that does not have 100% complementarity with its target sequence. An interfering RNA (e.g., siRNA, aiRNA, miRNA) can have at least 1, 2, 3, 4, 5, 6 or more mismatch regions. Mismatch regions can be continuous or can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides. Mismatch regions can include a single nucleotide or can include 2, 3, 4, 5, 6 or more nucleotides.

[0123] The term "identity" refers to the similarity between two nucleotide sequences or between two amino acid sequences. The identity of a sequence preferably relates to the percentage of nucleotides or amino acids in the same position in two or more sequences of the same length. Specifically, the "% identity" of two amino acid sequences or two nucleotide sequences can be determined as follows: compare sequences for the purpose of optimal comparison (for example, a room can be introduced in either sequence to optimally compare with another sequence) and compare the amino acids or nucleotides in the corresponding positions. Rooms are generally considered to be non-identical positions, regardless of their actual position in the comparison. "Optimal comparison" is generally the comparison of the two sequences that results in the highest percentage identity. Percent identity is determined by the number of identical nucleotides in the compared sequences (that is, % identity = number of identical positions / total number of positions × 100). The application's sequence identity is at least 80%, 85%, 90% or 95%, preferably at least 90%, and non-limiting examples include: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. Mathematical algorithm well known to those skilled in the art can be utilized to complete the determination of identity percentage between two sequences.

[0124] As known in the art, interfering RNA includes "small interfering RNA (siRNA)", which has a length of, for example, about 15-60, 15-50, 15-40, 15-30, 15-25, 19-25, 19-23, or 19-21 nucleotides. The double-stranded RNA of the present application can function as such siRNA and can have a corresponding length.

[0125] As used herein, the nucleotide positions of the sense strand or antisense strand are counted from the 5' end. For a dsRNA with a sense strand of 19 nucleotides and an antisense strand of 21 nucleotides, for example, position 1 of 5'-CUGGCUCAGUUUACUAGUA-3' (SEQ ID NO: 1) is C and position 19 is A; and position 1 of 5'-UACUAGUAAACUGAGCCAGGA-3' (SEQ ID NO: 2) is U and position 21 is A. For a dsRNA with a sense strand of more than 19 nucleotides and an antisense strand of more than 21 nucleotides, for example, if the sense strand has several nucleotides added to the end based on SEQ ID NO: 1 and the antisense strand has several nucleotides added to the end based on SEQ ID NO: 2, the counting method of the nucleotide positions of the sense strand or antisense strand will not change accordingly, that is, position 1 of the sense strand will still be position 1 of SEQ ID NO: 1, and position 1 of the antisense strand will still be position 1 of SEQ ID NO: 2.

[0126] As is known in the art, and unless otherwise indicated, the term "complementary" when used to describe the relationship between a first nucleic acid sequence and a second nucleic acid sequence refers to the ability of an oligonucleotide or polynucleotide containing the first nucleic acid sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleic acid sequence and form a double-stranded structure under specific conditions. As used herein, "complementary" sequences may also include a double-stranded structure formed by base pairing formed by non-Watson-Crick base pairing and / or non-natural or modified nucleotides, or may be a double-stranded structure formed entirely by base pairing formed by non-Watson-Crick base pairing and / or non-natural or modified nucleotides, as long as the above requirements with respect to their ability to hybridize are met.

[0127] As known in the art, a "fully complementary" sequence includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleic acid sequence with an oligonucleotide or polynucleotide comprising a second nucleic acid sequence over the entire length of the first nucleic acid sequence and the second nucleic acid sequence.

[0128] As known in the art, "substantially complementary" means that two nucleic acid sequences are completely complementary or at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the overlapping nucleotides are complementary.

[0129] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in terms of base pairing between the sense and antisense strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as can be understood from the context in which they are used.

[0130] As known in the art, "double-stranded ribonucleic acid," "double-stranded RNA," or "dsRNA" are used interchangeably. The term "dsRNA" comprises two antiparallel, complementary nucleic acid strands that have a "sense" or "antisense" orientation relative to a target RNA (e.g., an HBV gene). In embodiments of the present application, dsRNA can degrade target RNA (e.g., mRNA) through the RNA interference (RNAi) mechanism.

[0131] The double-stranded RNA of the present application contains a sense strand and an antisense strand. The term "sense strand" or "sense strand" refers to a single strand in the dsRNA double strand that is substantially complementary to a region of the antisense strand. The term "antisense strand," "guide strand," or "guide strand" refers to a single strand in the dsRNA double strand that is substantially complementary to a region of the target sequence. If the sense strand is not fully complementary to the antisense strand, mismatches may occur within the molecule or in the terminal regions. Generally, the most tolerable mismatches are within the terminal regions. If the antisense strand is not fully complementary to the target sequence, mismatches may occur within the molecule or in the terminal regions. Generally, the most tolerable mismatches are within the terminal regions.

[0132] As is known in the art, the double strands of dsRNA can have the same or different numbers of nucleotides. The length of the double stranded region formed by complementation can be any length that allows degradation of the target RNA, and possible lengths are in the range of about 9 to 36 pairs of nucleotides, such as 15 to 30 pairs, 16 to 28 pairs, 19 to 21 pairs, etc.

[0133] As is known in the art, outside the double-stranded region, dsRNA may include one or more nucleotide overhangs, which refer to at least one unpaired nucleotide / nucleoside analog. For example, when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand (or vice versa), an overhang is present. The nucleotides at the "overhang" may include 0-5 nucleotides, where "0" indicates no "overhang," and "5" indicates that there are 5 additional (i.e., not paired with the other single strand) nucleotides on the single strand of the dsRNA double strand. These optional "overhangs" may be located at the 5' and / or 3' end of any single strand in the two strands of the dsRNA. In some embodiments, the "overhang" includes 0-5 nucleotides. In some embodiments, the "overhang" includes 0-2 nucleotides. In some embodiments, the "overhang" at the 3' and / or 5' end of the sense strand of the dsRNA has 0-2 nucleotides. In some embodiments, the "overhang" at the 3' and / or 5' end of the antisense strand of the dsRNA has 0-2 nucleotides. The nucleotides forming the "overhang" can be A, G, C, U or T or modified structures thereof. The nucleotides forming the "overhang" can be U, T or dT or modified structures thereof. In some embodiments, the "overhang" includes but is not limited to "TT", "dTdT", "UU" or their corresponding modified structures, such as UU modified with a 2' methoxy group, i.e., uu. In some embodiments, the "overhang" at the 3' and / or 5' end of the antisense strand of the dsRNA is substantially complementary to the target RNA. In some embodiments, the "overhang" at the 3' and / or 5' end of the antisense strand of the dsRNA is completely complementary to the target RNA. In some embodiments, the "overhang" at the 3' end of the antisense strand of the dsRNA is completely complementary to the target RNA. In some embodiments, the "overhang" at the 3' end of the antisense strand of the dsRNA is selected from unmodified or modified GA. The term "blunt" or "blunt end" means that there are no unpaired nucleotides at the end of the dsRNA, i.e., there are no nucleotide overhangs. A dsRNA with "blunt ends" at both ends is a dsRNA that is double-stranded throughout its entire length, ie, has no nucleotide overhangs at either end of the molecule.

[0134] In the present application, the dsRNA or any single strand thereof is optionally modified, and both unmodified and modified ribonucleic acids are within the scope of protection of this application. The modification does not significantly weaken or abolish the dsRNA's ability to inhibit HBV gene expression. The modification of the dsRNA or any single strand thereof may be at the 5' and / or 3' termini, nucleotides, or internucleotide linkages. Synthesis or modification can be performed using methods known in the art.

[0135] In the present application, the modification of the nucleotide includes but is not limited to occurring on the sugar group of the nucleotide, including one or more substituted or removed sugar moieties, such as removing the hydroxyl group on the carbonyl, or fluorination, amination, alkylation, hydroxyalkylation or hydroxyalkenylation. The modification on the sugar group can occur at various positions on the sugar ring. Exemplarily, the modification on the sugar group of the nucleotide includes but is not limited to 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-methyl, 2'-ethyl, 2'-methyl-O-methyl, 2'-ethyl-O-methyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-ethyl-O-methyl or 2'-O-allyl, and the structure can be as follows:

[0136] Wherein Base represents base.

[0137] In the present application, the modification of the internucleotide linkage includes substitution or replacement of the atom or functional group of the phosphate group, such as phosphorothioate (PS), phosphorodithioate (PS2), methylphosphonate (MP), methoxypropylphosphonate (MOP) or aminophosphonate.

[0138] In this application, the capital letters G, C, A, U or T respectively represent nucleotides containing guanine, cytosine, adenine, uracil, and thymine as bases. The lowercase letters g, c, a, and u represent that the nucleotides represented by their corresponding capital letters are modified by 2'-methoxy groups, i.e., g, c, a, and u represent 2'-O-methyl G, 2'-O-methyl C, 2'-O-methyl A, and 2'-O-methyl U, respectively. The capital letter plus the lowercase letter f on the right side represents that the nucleotides represented by their corresponding capital letters are modified by 2'-fluorine groups, i.e., Gf, Cf, Af, and Uf represent 2'-fluorine G, 2'-fluorine C, 2'-fluorine A, and 2'-fluorine U, respectively. The lowercase letter s represents that the two nucleotide residues adjacent to the left and right of s are connected by a phosphorothioate group. For example, "csu" represents that the c and u residues are connected by a phosphorothioate group. VP- indicates that the nucleotide to the right of the hyphen is a (E)-vinyl phosphate-modified nucleotide, for example, "VP-u" indicates a (E)-vinyl phosphate-modified 2'-O-methyl U.

[0139] In this application, the 5'-end and / or 3'-end modifications refer to modifications occurring at the 5'-end and / or 3'-end of dsRNA or any single strand thereof, such as phosphorylation, conjugation, or reverse linkage. Taking the 5'-end as an example, it includes but is not limited to 5'-phosphate, 5'-methylphosphonate (5'-MP), 5'-thiophosphate (5'-PS), or 5'-(E)-vinylphosphonate (5'-(E)-VP), and the structure can be shown as follows:

[0140] wherein Base represents a base, and X is selected from a hydroxyl group or a 2' modification on a sugar group.

[0141] In the present application, the ligand is a group connected to the dsRNA, and the ligand includes a branched group and a linker, and the dsRNA, the linker and the branched group are connected in sequence (for example, as shown in Formula 104). The branched group contains at least one (for example, one, two, three, four or five) pharmaceutically acceptable targeting group, which targets the dsRNA to a specific tissue or enhances cellular absorption. The targeting group is, for example, but not limited to, a GalNAc (N-acetylgalactosamine, N-Acetylgalactosamine, for example, as shown in Formula 105) group. A plurality of the targeting groups are connected in series or in parallel through a branched group. Wherein, the GalNAc group can be monovalent, divalent, trivalent or tetravalent. The monovalent, divalent, trivalent, and tetravalent terms used herein refer to the molar ratio of dsRNA to GalNAc molecules in the dsRNA ligand conjugate formed by the dsRNA molecule and the ligand containing GalNAc as a targeting group being 1:1, 1:2, 1:3, and 1:4, respectively. In some embodiments, when the dsRNA of the present application is conjugated to a ligand containing GalNAc, the GalNAc molecule is trivalent or tetravalent. In some embodiments, when the dsRNA of the present application is conjugated to a ligand containing GalNAc, the GalNAc molecule is trivalent.

[0142] In the present application, the ligand can be attached to the phosphate group, 2'-hydroxyl group, 3'-hydroxyl group, or base of a nucleotide. The ligand can be attached to any nucleotide of the dsRNA, including but not limited to the 5' or 3' terminal nucleotide of the sense or antisense strand or a non-terminal intermediate nucleotide. When the ligand is attached to the end of the dsRNA strand, the ligand can be attached to the phosphate group of the nucleotide; when the ligand is attached to the intermediate nucleotide of the dsRNA, the ligand can be attached to the sugar ring or base of the nucleotide.

[0143] In this application, the types of ligands or preparation methods described herein can refer to methods known in the art, including but not limited to the ligands and preparation methods described in WO2009082607, WO2014025805, WO2015006740, and WO2021249484, the entire disclosures of which are incorporated herein by reference. Exemplary ligands include but are not limited to L01 or L02 described above.

[0144] In this application, unless otherwise specified, "conjugation" refers to the non-covalent or covalent attachment of two or more chemical moieties, each with a specific function, to one another; accordingly, "conjugate" refers to a compound formed by non-covalent or covalent attachment of these chemical moieties. In this application, conjugates attached to one another by covalent attachment are preferred.

[0145] In the present application, the ligand is linked to the 5' or 3' end of the sense strand or antisense strand. Preferably, the ligand is linked to the 5' or 3' end of the sense strand. More preferably, the ligand is linked to the 3' end of the sense strand. Exemplarily, the dsRNA ligand conjugate formed by linking the dsRNA to the ligand is shown in Formula 102 or Formula 103 below:

[0146] The compounds of the present application (e.g., the double-stranded RNA, pharmaceutically acceptable salts thereof, and ligand conjugates thereof) may exist in specific geometric or stereoisomeric forms, all of which are within the scope of the present application. The present application contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and racemic mixtures and other mixtures thereof, such as enantiomer- or diastereomer-enriched mixtures, all of which are within the scope of the present application. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present application.

[0147] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0148] Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

[0149] Unless otherwise specified, use a solid wedge key. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond and straight dashed key Indicate the relative configuration of stereocenters with a wavy line Indicates a wedge-shaped solid key or dotted wedge key Or use a wavy line Indicates a straight solid bond and / or straight dashed key

[0150] Unless otherwise indicated, the terms "enriched in one isomer", "isomerically enriched", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100%, and the content of that isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

[0151] Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

[0152] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary groups are cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art and then recovered to obtain the pure enantiomer. In addition, the separation of enantiomers and diastereoisomers is typically accomplished by using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate formation from an amine). The compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that constitute the compound. For example, compounds can be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I) or C-14( 14 C). For example, deuterated drugs can be formed by replacing hydrogen with heavy hydrogen. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and extended drug biological half-life. All isotopic variations of the compounds of this application, whether or not radioactive, are included within the scope of this application.

[0153] The term "treatment" means administering the compound or preparation described herein (e.g., the double-stranded RNA, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present invention) to improve or eliminate a disease or one or more symptoms associated with the disease, and includes: (i) inhibiting the disease or disease state, i.e., curbing its development; (ii) alleviating the disease or disease state, i.e., causing the disease or disease state to regress.

[0154] The term "prevention" means administering the compound or preparation described herein (e.g., the double-stranded RNA, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present invention) to prevent a disease or one or more symptoms associated with the disease, and includes preventing the occurrence of a disease or disease state in a subject, particularly when such subject is susceptible to the disease state but has not yet been diagnosed as having the disease state.

[0155] The terms "subject," "patient," or "subject" are used interchangeably herein to refer to an animal that has been the subject of treatment, observation, or experiment. In some embodiments, the subject is a mammal, preferably a primate, and more preferably a human.

[0156] The term "therapeutically effective amount" means an amount of a compound of the present application (e.g., a double-stranded RNA, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof) that (i) treats or prevents a specific disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. The amount of a compound of the present application that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the route of administration, and the age of the subject to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and the disclosure of this application.

[0157] The therapeutic dose of the compound of the present application may be determined based on, for example, the specific use of the treatment, the mode of administering the compound, the health and condition of the patient, and the judgment of the prescribing physician. The ratio or concentration of the compound of the present application in the pharmaceutical composition may not be fixed and depends on a variety of factors, including dosage, chemical properties (e.g., hydrophobicity), and route of administration. For example, the compound of the present application may be provided in a physiologically buffered saline solution containing about 0.1 to 10% w / v of the compound for parenteral administration. Some typical dosage ranges are about 1 μg / kg to about 1 g / kg body weight / day. In certain embodiments, the dosage range is about 0.01 mg / kg to about 100 mg / kg body weight / day. The dosage is likely to depend on such variables as the type and extent of the disease or condition, the general health status of the specific patient, the relative biological efficacy of the selected compound, the excipient formulation, and its route of administration. The effective dose can be obtained by extrapolation of a dose-response curve derived from an in vitro or animal model test system.

[0158] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and other animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0159] As the pharmaceutically acceptable salt, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids and the like can be mentioned.

[0160] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application (e.g., double-stranded RNA, pharmaceutically acceptable salts thereof, or ligand conjugates thereof) and pharmaceutically acceptable carriers, excipients, or vehicles. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism. In this article, the terms "pharmaceutical composition" and "preparation" have the same meaning and are used interchangeably. The carriers, excipients, or vehicles used herein include any and all solvents, diluents, or other liquid excipients, dispersants, or suspending agents, surfactants, isotonic agents, thickeners, or emulsifiers, preservatives, solid binders, lubricants, and the like that are suitable for the desired specific dosage form. Various carriers, excipients, or vehicles used to formulate pharmaceutically acceptable compositions and methods for their preparation can employ known techniques. Except for any conventional carrier medium that is incompatible with the compounds of the present application (e.g., produces any adverse biological effects or otherwise interacts in a harmful manner with any other component of the pharmaceutically acceptable composition), its use is encompassed within the scope of this application. In some specific embodiments, the carriers, adjuvants or excipients used herein are conventionally used in the field of dsRNA administration.

[0161] The pharmaceutical compositions of the present application can be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable carriers, excipients or vehicles, and can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.

[0162] Typical routes of administration of the compounds of the present application or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0163] The pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing methods, dissolution methods, granulation methods, sugar-coated pill making methods, grinding methods, emulsification methods, freeze-drying methods, etc.

[0164] The kit of the present application includes the double-stranded ribonucleic acid, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present application; and optionally, instructions for using the double-stranded ribonucleic acid, its pharmaceutically acceptable salt, its ligand conjugate, or pharmaceutical composition of the present application to treat and / or prevent hepatitis B virus infection.

[0165] The solvents used in this application are commercially available.

[0166] Unless otherwise specified, the solvent ratios used in column chromatography and preparative thin-layer silica gel chromatography in this application are all volume ratios.

[0167] It is known in the art that the modified nucleotide groups can be introduced into the dsRNA described herein by using nucleoside monomers with corresponding modifications. Those skilled in the art can obtain methods for preparing corresponding modified nucleoside monomers and methods for introducing modified nucleotide groups into dsRNA from the prior art. All modified nucleoside monomers can be obtained from commercial sources or prepared using known methods.

[0168] It is known in the art that the desired ribonucleic acid can be obtained by conventional ribonucleic acid preparation methods (such as solid phase synthesis and liquid phase synthesis) in the art, for example, the desired ribonucleic acid can be synthesized by phosphoramidite solid phase synthesis technology. The preparation method of the double-stranded ribonucleic acid of the present application, its pharmaceutically acceptable salt or its ligand conjugate comprises the following steps: according to the nucleotide type or order of the sense strand or antisense strand of the double-stranded ribonucleic acid, nucleotide monomers are sequentially connected in a 3' to 5' direction to synthesize the sense strand and antisense strand. The connection of each nucleotide monomer includes a four-step reaction of deprotection, coupling, capping, oxidation or sulfurization. Those skilled in the art can adopt conventional reaction conditions, the type and dosage of reagents, or adjust according to the test situation to achieve the deprotection, coupling, capping, oxidation or sulfurization reaction.

[0169] In some embodiments, for the synthesis of ribonucleic acid containing a ligand, the ligand can be connected to the ribonucleic acid through a coupling reaction during or after the synthesis of the oligoribonucleotide, or the ligand can be first connected to a solid phase support, and then the nucleoside monomer and the ligand-solid phase support can be connected in sequence in the 3' to 5' direction.

[0170] Methods for purification and desalting are well known to those skilled in the art. For example, RNA purification can be accomplished by preparative ion chromatography. For another example, RNA desalting can be accomplished by reverse phase chromatography purification or ultrafiltration centrifugation.

[0171] Annealing methods are well known to those skilled in the art. For example, the sense strand and the antisense strand can be mixed at a molar ratio of 1:1, heated to 70-95° C., and then cooled to room temperature to form a double-stranded structure.

[0172] During the synthesis process, the RNA concentration can be detected by, for example, ion exchange chromatography, or the molecular weight can be determined by liquid chromatography-mass spectrometry, or the concentration can be determined by microspectrophotometry to control the synthesis quality. Such detection methods are well known to those skilled in the art.

[0173] Unless otherwise specifically stated, singular terms encompass plural terms and plural terms encompass the singular. Unless otherwise specifically stated, the words "a" or "an" mean "at least one" or "at least one." Unless otherwise specified, the use of "or" means "and / or."

[0174] For the purposes of description and disclosure, all patents, patent applications, and other identified publications are expressly incorporated herein by reference. These publications are provided solely for their disclosure prior to the filing date of the present application. All statements regarding the dates of these documents or the representations of their contents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any citation of these publications herein does not constitute an admission that such publications become part of the common general knowledge in the art in any country. BRIEF DESCRIPTION OF THE DRAWINGS

[0175] FIG1 shows the serum HBV DNA levels in AAV-HBV mouse experiments.

[0176] FIG2 shows the serum HBsAg levels in AAV-HBV mouse experiments.

[0177] FIG3 shows the serum HBeAg level in AAV-HBV mouse experiments. DETAILED DESCRIPTION

[0178] The present invention is described in detail below by way of examples, but is not intended to constitute any adverse restriction to the present invention. The compound of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by the combination thereof with other chemical synthesis methods and equivalent replacement modes well known to those skilled in the art, and preferred embodiments include but are not limited to the embodiments of the present invention. It will be apparent to those skilled in the art that various changes and improvements will be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0179] Reagents and materials

[0180] Commercially available protected monomers used in the synthesis

[0181] Reagents used in synthesis

[0182] Preparation Example 1: Connecting a GalNAc ligand (a ligand containing a GalNAc group as a targeting group) to a solid support

[0183] Weigh 1.0 g of amino-CPG (amino loading: 30-40 μmol / g), add 5 mL of decapping agent, react for 1 minute, drain the reagent, and repeat this cycle four times. Based on a loading of 35 μmol, add 2 eq of GalNAc ligand / acetonitrile solution, followed by 2.5 eq of DIC and 2.5 eq of DMAP condensation reagent. Incubate at 25-30°C for 24 hours. After completion, filter out the solvent, rinse with anhydrous acetonitrile, then add the GalNAc ligand and condensation reagent in the aforementioned amounts, and react for an additional 24 hours. After the reaction, rinse the solid support with anhydrous acetonitrile, add 4 mL of a 1:1 mixture of Capping A and Capping B, react for 2 minutes, drain, then add 4 mL of the mixture, react for 2 minutes, drain, rinse with anhydrous acetonitrile, and vacuum dry at 30°C for 1 hour before use. This example uses the L01 ligand.

[0184] Preparation Example 2 Preparation of double-stranded ribonucleic acid

[0185] 2.1.1 Synthesis of GalNAc ligand-ssRNA (sense strand)

[0186] On a K&A nucleic acid synthesizer, the GalNAc ligand-solid phase support was placed into a synthesis column and ssRNA was synthesized using standard phosphoramidite technology. The specific steps included:

[0187] 1) DMT removal: Wash the resin twice with acetonitrile, then remove DMT from the resin with 3% trichloroacetic acid / DCM, and wash with acetonitrile 4-5 times.

[0188] 2) Condensation: Add monomers and condensation reagents to the synthesis column and condense at room temperature for 10 minutes. Wash with acetonitrile 4-5 times.

[0189] 3) Blocking: Block unreacted hydroxyl groups with Capping A first, then with Capping B. Wash with acetonitrile 4-5 times.

[0190] 4) Oxidation: Add an oxidizing agent for 2 minutes, then wash with acetonitrile 4 to 5 times.

[0191] Repeat steps 1) to 4) until all sequences are synthesized. Finally, remove DMT with a decapping agent and wash with acetonitrile 4 to 5 times.

[0192] Formation of phosphorothioate bonds: Replace the oxidizing reagent with a thio reagent and set the thio time to 10 minutes to complete the formation of phosphorothioate bonds.

[0193] 2.1.2 Synthesis of ssRNA (ligand-free sense strand)

[0194] On a K&A nucleic acid synthesizer, a universal CPG solid phase carrier was placed into a synthesis column and ssRNA was synthesized using standard phosphoramidite technology. The specific steps include:

[0195] 1) DMT removal: The resin was washed twice with acetonitrile, and then the DMT on the resin was removed with 3% trichloroacetic acid / DCM, and then washed with acetonitrile 4 to 5 times.

[0196] 2) Condensation: Add monomers and condensation reagents to the synthesis column and condense at room temperature for 10 minutes. Wash with acetonitrile 4-5 times.

[0197] 3) Blocking: Block unreacted hydroxyl groups with Capping A first, then with Capping B. Wash with acetonitrile 4-5 times.

[0198] 4) Oxidation: Add an oxidizing agent for 2 minutes, then wash with acetonitrile 4 to 5 times.

[0199] Repeat steps 1) to 4) until all sequences are synthesized. Finally, remove DMT with a decapping agent and wash with acetonitrile 4 to 5 times.

[0200] Formation of phosphorothioate bonds: Replace the oxidizing reagent with a thio reagent and set the thio time to 10 minutes to complete the formation of phosphorothioate bonds.

[0201] 2.2 Synthesis of asRNA (ligand-free antisense strand)

[0202] On a K&A nucleic acid synthesizer, place the CPG solid phase carrier into a synthesis column and perform synthesis according to the method described in 2.1.1 or 2.1.2.

[0203] 2.3 Separation of ssRNA from solid phase support

[0204] The lysis reagent was a 3:1 ratio of ammonia to ethanol. 5 ml of lysis reagent was added for every 100 mg of solid support and stirred at 65-70°C for 3 hours. After the reaction, the mixture was cooled to room temperature and precipitated with icy n-butanol. The mixture was then placed in a -20°C refrigerator for 30 minutes. The precipitate was centrifuged and washed with n-butanol twice. Finally, the precipitate was washed with acetone, centrifuged, and dried under vacuum to obtain the crude product for mass spectrometry analysis.

[0205] LC-MS (negative ion mode) conditions are as follows:

[0206] Chromatographic column: HILIC column

[0207] Mobile phase A: 20 mM ammonium formate, pH 6.2

[0208] Mobile phase B: 20 ​​mm ammonium formate + 95% ACN

[0209] Column temperature: 45°C

[0210] Flow rate: 1 mL / min.

[0211] 2.4 Annealing to form siRNA (dsRNA)

[0212] Dissolve the crude single-stranded RNA in water and remove the carrier by membrane filtration. Measure the concentrations of the sense and antisense strands using a microspectrophotometer. Add the sense and antisense strands at a molar ratio of 1:1 and mix. Denature at 94°C for 4 minutes. Anneal naturally and cool to room temperature.

[0213] 2.5 Purification of siRNA (dsRNA)

[0214] Purification was performed using a DNAPac RP 10x150mm 4μm column. Mobile phase A consisted of 0.1 mol / L triethylamine, pH 8.0, and mobile phase B consisted of 0.1 mol / L triethylamine, pH 8.0 + 50% ACN. Detection wavelengths were 215 nm and 260 nm. The main peak was collected and the solvent was removed by rotary evaporation to obtain the desired product.

[0215] 2.6 Desalination and content calibration

[0216] Use 3K ultrafiltration centrifuge tubes, 12000×g, centrifugation time 12min, ultrafiltration concentration and desalination; after the rotary evaporation liquid is concentrated, add water for replacement, and repeat more than 5 times.

[0217] Concentration determination: After concentration, the sample was measured using a micro-spectrophotometer.

[0218] Double-stranded RNA looks like this:

[0219] The pharmaceutically acceptable salt of the double-stranded RNA or its ligand conjugate (the ligand is selected from L01 ligand or L02 ligand) can be prepared according to the method described in this application or methods known in the art.

[0220] Experimental Example 1 In vitro screening of double-stranded RNA

[0221] Cell culture and transfection

[0222] Cos7 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in DMEM complete medium (Hyclone, catalog number SH30243.01) containing 10% fetal bovine serum (Gibco, catalog number 10099-141) at 37°C in an incubator containing 5% CO2. When the cells grew to near confluence, they were treated with trypsin to release the cells from the culture flask. Cos7 cells were plated at 2×10 4Cells were seeded into 96-well plates and cultured overnight. When the cell density reached 70-80%, Cos7 cells were transfected with pmirGLO-HBV plasmid containing 2.2 kb HBV genome sequence (GenBank accession #EU554538.1). 50 ng of plasmid was transfected into the cells using 0.3 μL Lipofectamine 5-10 at a concentration of 50 ng / well as described by the manufacturer. TM 3000 transfection reagent (Invitrogen, Cat. No. L3000015) was used for transfection. 6-8 hours after plasmid transfection, the culture medium was removed. siRNA was then transfected into Cos7 cells that had been transfected with the pmirGLO-HBV plasmid. siRNA transfection was performed by adding 0.5 μL of siRNA to 5 μL of OPTI-MEM medium and 0.3 μL of Lipofectamine TM RNAiMAX transfection reagent (Invitrogen, Cat. No. 13778150) was added to 5 μL of OPTI-MEM medium; the mixture was pipetted and allowed to stand at room temperature for 5 minutes. The complex was then added to a 96-well plate, and 90 μL of complete DMEM medium was added to each well. The cells were cultured for 48 hours. Two concentrations of each siRNA were used, with three replicates per well. Single-dose experiments were performed at final dsRNA concentrations of 10 nM and 1 nM. A dsRNA that did not bind to the target sequence was used as a control.

[0223] Detection

[0224] Remove the culture medium from the wells and add 150 μL of A mixture of Luciferase reagent (Promega) and DMEM complete medium (volume ratio = 1:1) was mixed thoroughly and incubated at room temperature for 10 minutes. The chemiluminescence value (Fir) was read using a Spark multifunctional microplate reader (Tecan). 75 μL of Reagent (Promega), mix thoroughly, incubate at room temperature for 10 min, and read the chemiluminescence value (Ren) using a Spark multifunctional microplate reader.

[0225] Calculate the luminescence ratio for each well as Ratio = Fir / Ren. The luminescence ratio (Ratio(test)) or Ratio(control) for each test or control group is the average of the Ratios for all wells. Normalize the luminescence ratio of each test group to the control group's luminescence ratio to obtain the ratio R (Ratio(test)) / Ratio(control), which represents the expression level of the firefly luciferase reporter gene. siRNA inhibition rate (%) = (1-R) ​​x 100%. The results are shown below:

[0226] The results showed that the dsRNA of the present application showed excellent activity at the cellular level, and the inhibitory activity was dose-dependent.

[0227] Experimental Example 2 Dual luciferase assay

[0228] Cell culture and transfection

[0229] Cos7 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in DMEM complete medium (Hyclone, Catalog No. SH30243.01) containing 10% fetal bovine serum (Gibco, Catalog No. 10099-141) at 37°C in an incubator containing 5% CO2. When the cells grew to near confluence, they were treated with trypsin to release the cells from the culture flask. Cos7 cells were plated at 2×10 4 Cells were seeded into 96-well plates and cultured overnight. When the cell density reached 70-80%, Cos7 cells were transfected with pmirGLO-HBV plasmid containing 2.2 kb HBV genome sequence (GenBank accession #EU554538.1). 50 ng of plasmid was transfected into the cells using 0.3 μL Lipofectamine 5-10 at a concentration of 50 ng / well as described by the manufacturer. TM 3000 transfection reagent (Invitrogen, cat. no. L3000015) was used for transfection. 6-8 hours after plasmid transfection, the culture medium was removed. siRNA was then transfected into Cos7 cells that had been transfected with pmirGLO-HBV plasmid. The siRNA transfection method was to dilute the siRNA 5-fold in RNase-free water to a concentration range of 2-0.0000256 μM; 0.5 μL of siRNA was added to 5 μL of OPTI-MEM medium, and 0.3 μL of Lipofectamine was added. TM RNAiMAX transfection reagent (Invitrogen, Cat. No. 13778150) was added to 5 μL of OPTI-MEM medium; the two were pipetted to mix thoroughly and allowed to stand at room temperature for 5 minutes. The complex was then added to a 96-well plate, and 90 μL of complete DMEM medium was added to each well. The cells were cultured for 48 hours. Eight concentrations of each siRNA were set, with three replicates for each concentration. A dsRNA that did not match the target sequence was used as a control.

[0230] Detection

[0231] Remove the culture medium from the wells and add 150 μL of A mixture of Luciferase reagent (Promega) and DMEM complete medium (volume ratio = 1:1) was mixed thoroughly and incubated at room temperature for 10 minutes. The chemiluminescence value (Fir) was read using a Spark multifunctional microplate reader (Tecan). 75 μL of Reagent (Promega), mix thoroughly, incubate at room temperature for 10 min, and read the chemiluminescence value (Ren) using a Spark multifunctional microplate reader.

[0232] Calculate the luminescence ratio for each well (Ratio = Fir / Ren). The luminescence ratio (Ratio(test)) or (Ratio(control)) for each test or control group is the average of all wells' Ratios. Normalize the luminescence ratio of each test group to the control group's luminescence ratio to obtain the ratio (Ratio(test)) / Ratio(control), which represents the expression level of the firefly luciferase reporter gene. siRNA inhibition rate (%) = (1-R) ​​× 100%.

[0233] The concentration-inhibition rate (%) data were processed using EXCEL software, and the EC50 of siRNA was calculated using a four-parameter nonlinear regression model. The results are shown below:

[0234] The results indicate that the dsRNA of the present application exhibits excellent activity at the cellular level and has a low EC50 value.

[0235] Experimental Example 3 Inhibitory activity in HepG2.2.15 cells

[0236] Cell culture and transfection

[0237] HepG2.2.15 cells were cultured in MEM complete medium (Gibco, Cat. No. 11095-080) containing 10% fetal bovine serum (Gibco, Cat. No. 10099-141) and 380 μg / mL G418 (Gibco, Cat. No. 10131-027) at 37°C in a 5% CO2 incubator. When the cells grew to near confluence, they were treated with trypsin to release the cells from the culture flask and plated at 2×10 4 Cells / well were seeded in a 96-well plate and cultured overnight. When the cell density reached 70-80%, siRNA was transfected. The siRNA transfection method was to dilute the siRNA 5-fold in RNase-free water to a concentration range of 2-0.0000256μM; 0.5μL siRNA was added to a 0.3μL Lipofectamine TMRNAiMAX transfection reagent (Invitrogen, cat. no. 13778150) was added to 10 μL of OPTI-MEM medium and pipetted to mix thoroughly. The culture medium was allowed to stand at room temperature for 5 minutes. The MEM medium in the 96-well plate was discarded and 90 μL of complete MEM medium was added to each well. The mixture was added to the 96-well plate at a volume of 10 μL / well. The 96-well plate was placed in a 37°C, 5% CO2 incubator and cultured for 6 days. The medium was changed every 3 days. The culture supernatant was collected on the last day for analysis. Eight concentrations were set for each siRNA. Three replicates were set for each concentration. A dsRNA that did not match the target sequence was selected as a control.

[0238] ELISA to detect the inhibitory effect of siRNA on HBsAg

[0239] Cell supernatants were collected and assayed for HBsAg levels using a Hepatitis B virus surface antigen (HBsAg) assay kit (Beijing Kemei Diagnostics Co., Ltd., Cat. No. 1010803042). The assay was performed according to the manufacturer's instructions, and chemiluminescence readings were obtained using a Spark multi-function microplate reader (Tecan). A standard curve was constructed using the standard sample, and the HBsAg concentration (ng / mL) in the sample was calculated. The concentration-inhibition rate (%) data were processed using Excel software, and the EC50 for siRNA inhibition of HBsAg was calculated using a four-parameter nonlinear regression model.

[0240] Inhibition rate (%) = (1-siRNA group concentration (ng / mL) / control group concentration (ng / mL)) × 100

[0241] ELISA to detect the inhibitory effect of siRNA on HBeAg

[0242] Cell supernatants were collected and assayed for HBeAg levels using a Hepatitis B virus e antigen (HBeAg) assay kit (Beijing Kemei Diagnostics Co., Ltd., Cat. No. 1010801042). The assay was performed according to the manufacturer's instructions, and chemiluminescence readings were obtained using a Spark multi-function microplate reader (Tecan). A standard curve was constructed using the standard sample, and HBeAg concentrations (NCU / mL) in the samples were calculated. Data comparing concentration and inhibition (%) were processed using Excel software, and the EC50 for siRNA inhibition of HBeAg was calculated using a four-parameter nonlinear regression model.

[0243] Inhibition rate (%) = (1-siRNA group concentration (NCU / mL) / control group concentration (NCU / mL)) × 100.

[0244] The result is as follows:

[0245] The results indicate that the dsRNA of the present application exhibits excellent activity at the cellular level, with low EC50 values ​​in both HBsAg inhibition and HBeAg inhibition.

[0246] On the other hand, the results also show that the dsRNA of the present application has a significant inhibitory effect on HBsAg, indicating the possibility that the dsRNA of the present application can functionally cure viral hepatitis B.

[0247] Experimental Example 4 AAV-HBV Mouse Experiment

[0248] The AAV-HBV mouse model was prepared according to the literature (Dong Xiaoyan et al., Chin J Biotech 2010, May 25; 26(5): 679-686). The rAAV8-1.3HBV, type D (ayw) was purchased from Beijing Wujiahe Gene Technology Co., Ltd., with a concentration of 1.78×10 12 viral genome (vg) / mL, batch number A2020081701. rAAV8-1.3HBV was pre-constituted with sterile PBS to a concentration of 5×10 11 vg / mL solution. 6-7 week old male C57BL / 6 mice (purchased from Shanghai Slake Laboratory Animal Co., Ltd.) were injected with 200 μL rAAV8-1.3HBV via the tail vein of each mouse, i.e., 1×10 11 On day 28 after virus injection, all mice were bled via orbital bleeding to collect serum, and the serum HBV DNA, HBsAg, and HBeAg levels were measured.

[0249] After successful animal model establishment, mice were randomly divided into seven groups (n=3 per group) based on serum HBV DNA, HBsAg, and HBeAg levels: PBS control, M01L01, M01EVPL01, M21L01, M21EVPL01, M22L01, and M22EVPL01. The day of the first administration was designated as day 0. Mice in the treatment groups received a single subcutaneous injection of the corresponding dsRNA ligand conjugate solution based on body weight at a dose of 9 mg / kg in a volume of 5 mL / kg. Mice in the PBS control group received a single subcutaneous injection of PBS in a volume of 5 mL / kg. Orbital blood was collected 2 days before administration and on days 7, 14, 21, 28, 35, 42, 56, 70, and 84 after administration. Serum was isolated and assayed for HBV DNA, HBsAg, and HBeAg levels. Blood (100 μL) was collected from the orbital vein. After the whole blood was allowed to stand at room temperature for 30 minutes, it was centrifuged at 3000 rpm at 4°C for 10 minutes to collect serum. Serum DNA was lysed and released according to the instructions of the Hepatitis B Virus Nucleic Acid Assay Kit (Shengxiang Biotechnology, Catalog No. 101604042). Quantitative PCR was performed to measure serum HBV DNA levels. Serum HBsAg and HBeAg levels were measured using the Cobas e immunoassay analyzer (Roche, Catalog No. Cobas8000) using the Hepatitis B Virus Surface Antigen Quantitative Detection Kit (Roche, 08814899190) and the Hepatitis B e Antigen Detection Kit (Roche, Catalog No. 11820583122).

[0250] The HBV DNA inhibition rate was calculated according to the following equation:

[0251] HBV DNA inhibition rate = (1-HBV DNA content after administration / HBV DNA content before administration) × 100%.

[0252] The HBV DNA content is expressed as the number of equivalents (IU) of HBV DNA per milliliter (mL) of serum.

[0253] The HBsAg inhibition rate was calculated according to the following equation:

[0254] HBsAg inhibition rate = (1 - HBsAg content after administration / HBsAg content before administration) × 100%. HBsAg content is expressed as IU HBsAg per mL of serum.

[0255] The HBeAg inhibition rate was calculated according to the following equation:

[0256] HBeAg inhibition rate = (1 - HBeAg content after administration / HBeAg content before administration) × 100%. The HBeAg content is expressed as the number of equivalents (U) of HBeAg per milliliter (mL) of serum.

[0257] The dsRNA ligand conjugates are shown in the following table:

[0258] Wherein, VP- indicates that the nucleotide to the right of the hyphen is a (E)-vinyl phosphate-modified nucleotide. For example, the 5'-terminal u in SEQ ID NO:64 and SEQ ID NO:65 above is modified with 5'-(E)-vinyl phosphate. Wherein, the sequence ID number of the sense strand refers to the sequence ID number of the nucleic acid sequence in the sense strand. The dsRNA ligand conjugate serial number and L01 at the 3' end of the sense strand sequence indicate that in the dsRNA ligand conjugate, the L01 ligand is attached to the 3' end of the sense strand sequence.

[0259] The results are shown in the following table and Figures 1-3:

[0260] Mouse serum HBV DNA inhibition rate

[0261] Mouse serum HBsAg inhibition rate

[0262] Mouse serum HBeAg suppression rate

[0263] In addition, the PBS control group showed no suppression of serum HBV DNA, HBsAg, and HBeAg throughout the 84-day monitoring period.

[0264] The results indicate that the dsRNA ligand conjugate of the present application also exhibits excellent activity at the animal level, with high inhibition rates in serum HBV DNA inhibition, serum HBsAg inhibition, and serum HBeAg inhibition, demonstrating potential for clinical application.

[0265] On the other hand, the results also show that the dsRNA ligand conjugate of the present application has a significant inhibitory effect on HBsAg, indicating the possibility that the dsRNA of the present application can functionally cure viral hepatitis B.

Claims

1. A double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof, wherein the double-stranded ribonucleic acid comprises a sense strand and an antisense strand, wherein the sense strand comprises 5'-CUGGCUCAGUUUACUAGUA-3' (SEQ ID NO: 1) and the length of the sense strand does not exceed 21 nucleotides, wherein the antisense strand comprises 5'-UACUAGUAAACUGAGCCAGGA-3' (SEQ ID NO: 2) and the length of the antisense strand does not exceed 23 nucleotides, and the sense strand or the antisense strand is optionally modified. 2 . The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to claim 1 , wherein the sense strand or the antisense strand optionally comprises an overhang at the 5′ end and / or the 3′ end.

3. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to claim 2, wherein the overhang contains 1, 2, 3, 4, or 5 nucleotides; or, the overhang contains 1 or 2 nucleotides.

4. The double-stranded ribonucleic acid, pharmaceutically acceptable salt thereof, or ligand conjugate thereof according to claim 2 or 3, wherein the overhang is selected from unmodified or modified A, G, C, U, or T; or selected from unmodified or modified U or T; or when the overhang is 1 nucleotide, the overhang is selected from u or dT; or when the overhang is 2 nucleotides, the overhang is selected from uu or dTdT; or when the overhang is 2 nucleotides, the overhang is selected from unmodified or modified GA.

5. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 2 to 4, wherein the overhang is linked to the adjacent nucleotide via a phosphate group or a phosphorothioate group; or, one or more nucleotides in the overhang are linked to each other via a phosphate group or a phosphorothioate group.

6. The double-stranded ribonucleic acid, pharmaceutically acceptable salt thereof, or ligand conjugate thereof according to any one of claims 1 to 5, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides of the sense strand are modified, or nucleotides within the range formed by any of the foregoing values ​​are modified, for example, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1 -15, 1-14, 1-13, 1-12, 1-11, or 1-10 nucleotides are modified; or more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 16, more than 17, more than 18, more than 19, or more than 20 nucleotides of the sense strand are modified; or all nucleotides of the sense strand are modified; and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides of the antisense strand are modified, or nucleotides within a range of any of the foregoing values ​​are modified, e.g., 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13 In some embodiments, the antisense strand comprises at least one, 1-12, 1-11, or 1-10 nucleotides that are modified; or more than one, more than two, more than three, more than four, more than five, more than six, more than seven, more than eight, more than nine, more than ten, more than 11, more than 12, more than 13, more than 14, more than 15, more than 16, more than 17, more than 18, more than 19, more than 20, more than 21, or more than 22 nucleotides of the antisense strand are modified; or all nucleotides of the antisense strand are modified.

7. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 6, wherein the modification is selected from the group consisting of sugar modification of nucleotides, modification of the linkage between nucleotides, and terminal modification.

8. The double-stranded ribonucleic acid, pharmaceutically acceptable salt thereof, or ligand conjugate thereof according to any one of claims 1 to 7, wherein the sugar modification of the nucleotide is selected from 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-alkyl, 2'-O-alkyl, 2'-O-ether, and 2'-O-alkenyl; or, the sugar modification of the nucleotide is selected from 2'-dehydroxylation, 2'-fluoro, 2'-amino, 2'-methyl, 2'-ethyl, 2'-methyl-O-methyl, 2'-ethyl-O-methyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-ethyl-O-methyl, or 2'-O-allyl; The internucleotide linkage modification is selected from phosphorothioate (PS), phosphorodithioate (PS2), methylphosphonate (MP), methoxypropylphosphonate (MOP) or aminophosphonate; or, the internucleotide linkage modification is selected from phosphorothioate (PS); and / or the terminal modification is selected from 5'-terminal modification or 3'-terminal modification; or, the terminal modification is selected from 5'-phosphate, 5'-methylphosphonate (5'-MP), 5'-phosphorothioate (5'-PS) or 5'-(E)-vinylphosphonate (5'-(E)-VP).

9. The double-stranded ribonucleic acid, pharmaceutically acceptable salt thereof, or ligand conjugate thereof according to any one of claims 1 to 8, wherein the 1st and 2nd nucleotides of the sense strand and / or the 2nd and 3rd nucleotides of the sense strand are modified with a phosphorothioate group, and the 1st and 2nd nucleotides of the antisense strand, the 2nd and 3rd nucleotides of the antisense strand, the 19th and 20th nucleotides of the antisense strand, and / or the 20th and 21st nucleotides of the antisense strand are modified with a phosphorothioate group.

10. The double-stranded ribonucleic acid, pharmaceutically acceptable salt thereof, or ligand conjugate thereof according to any one of claims 1 to 9, wherein 2, 3, 4, or 5 nucleotides of the sense strand are modified with 2'-fluoro, and 14, 15, 16, or 17 nucleotides of the sense strand are modified with 2'-O-methyl; and / or; The antisense strand has 2, 3, 4, 5, 6 or 7 nucleotides modified with 2'-fluoro, and the antisense strand has 14, 15, 16, 17, 18 or 19 nucleotides modified with 2'-O-methyl.

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

12. The double-stranded ribonucleic acid, pharmaceutically acceptable salt thereof, or ligand conjugate thereof according to any one of claims 1 to 11, wherein 3 or 4 nucleotides among nucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-fluoro-modified; or 3, 4, 5, or 6 nucleotides among nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-fluoro-modified; or nucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-fluoro-modified, and any one or two of nucleotides at positions 1, 3 to 5, 7 to 13, 15, and 17 to 21 of the antisense strand are also 2'-fluoro-modified; or 14, 15, 16, or 17 nucleotides among nucleotides at positions 1, 3 to 5, 7 to 13, 15, and 17 to 21 of the antisense strand are 2'-O-methyl-modified.

13. The double-stranded ribonucleic acid, pharmaceutically acceptable salt thereof, or ligand conjugate thereof according to any one of claims 1 to 12, wherein the ligand contains at least one targeting group; or the ligand contains one, two, three, four or five targeting groups. The double-stranded ribonucleic acid, pharmaceutically acceptable salt thereof, or ligand conjugate thereof according to claim 13 , wherein the targeting group is a GalNAc group.

15. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 14, wherein the ligand comprises the following branching group:

16. The double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 15, wherein the ligand is selected from:

17. The double-stranded ribonucleic acid, pharmaceutically acceptable salt thereof, or ligand conjugate thereof according to any one of claims 1 to 16, wherein the ligand is linked to the 5' or 3' end of the sense strand or antisense strand; or, the ligand is linked to the 5' or 3' end of the sense strand; or, the ligand is linked to the 3' end of the sense strand.

18. The double-stranded ribonucleic acid, or a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 17, wherein the double-stranded ribonucleic acid comprises any one of the following sense strands: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and The double-stranded ribonucleic acid comprises any one of the following antisense strands: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 6 NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64 or SEQ ID NO:

65.

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

65.

20. A pharmaceutical composition comprising the double-stranded ribonucleic acid, a pharmaceutically acceptable salt thereof, or a ligand conjugate thereof according to any one of claims 1 to 19, optionally comprising a pharmaceutically acceptable carrier or excipient.

21. Use of the double-stranded RNA according to any one of claims 1 to 19, its pharmaceutically acceptable salt or ligand conjugate thereof, or the pharmaceutical composition according to claim 20 in the preparation of a medicament for treating and / or preventing hepatitis B virus infection.

22. The use according to claim 21, wherein the double-stranded RNA, its pharmaceutically acceptable salt or ligand conjugate, or pharmaceutical composition is used together with other therapeutic agents.

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