Oligonucleotide for inhibiting hepatitis b virus, conjugate, composition and use thereof

Abstract

An oligonucleotide, a conjugate, a composition and the use thereof. The oligonucleotide can inhibit the gene expression of a hepatitis B virus, and can efficiently inhibit the generation of HBsAg and total HBV DNAs, and therefore is expected to realize functional healing of hepatitis B.

Description

An oligonucleotide, conjugate, composition for inhibiting hepatitis B virus and its uses Technical Field

[0001] This disclosure pertains to the field of biomedicine, specifically relating to an oligonucleotide that inhibits the expression of the hepatitis B virus (HBV) gene, a conjugate containing the oligonucleotide, a composition thereof, and its use, as well as a method for inhibiting the expression of the hepatitis B virus gene in cells in vivo or in vitro, and a method for treating and / or preventing diseases, conditions, and / or symptoms at least partially mediated by the expression of the hepatitis B virus gene. Background Technology

[0002] Hepatitis B virus (HBV) is a hepatotropic virus belonging to the Hepatoviridae family. A complete viral particle is a spherical object with a diameter of 42 nm. The HBV genome is generally about 3.2 kb in length and contains four open reading frames (ORFs): S, C, P, and X, which have significant sequence overlap. ORF-C encodes HBe antigen (HBeAg) and HBc antigen (HBcAg), ORF-S encodes HBs antigen (HBsAg), ORF-P encodes viral DNA polymerase, and ORF-X encodes the X protein. HBcAg plays a crucial role in HBV infection, reflecting the presence of Dane particles in serum and HBV replication in the liver. It can also interact and complement other HBV serological markers. However, due to the strong affinity of HBcAg antibodies, they rapidly bind to HBcAg in serum, forming immune complexes, making it difficult to detect free HBcAg in serum. Mutations in the pre-C region gene can affect HBeAg production, resulting in negative antigen tests. This does not reflect a reduction or disappearance of HBV replication; rather, it may increase the risk of severe hepatitis. The X protein plays a significant role in regulating HBV DNA replication. The X gene region contains core promoter and enhancer elements related to transcription; mutations in this region can affect HBV transcriptional and translational levels. HBsAg is the antigen that HBV infects through the NTCP of liver parenchymal cells. This antigen appears in the blood circulation of patients in the early stages of HBV infection and can last for months, years or even a lifetime. It is the most commonly used indicator for diagnosing HBV infection.

[0003] The spontaneous error rate and lack of proofreading activity of HBV viral polymerase are the reasons for the numerous subtypes of the virus. Based on genotypic differences of >7.5%–8%, HBV is mainly divided into 10 genotypes, A, J, and D. The predominant subtypes and subtypes of the virus circulating in different regions vary greatly, as do natural infection histories, clinical manifestations, and responses to antiviral drugs. HBV infection is a global epidemic, with HBV types A, B, C, and D being the main infectious subtypes worldwide, accounting for approximately 80% of all infections.

[0004] HBV is highly adaptable to the external environment, surviving for at least 6 months at 30℃-32℃ and up to 15 years at ~20℃. Several key immunological characteristics of HBV allow it to persist for a long time and be difficult to eliminate. Its replication within hepatocytes goes undetected by the innate immune system, generating and secreting large amounts of viral antigens, gradually altering and depleting the function of HBV-specific T and B cells. Therefore, infection caused by this virus has become a global public health problem, with over 350 million people worldwide chronically infected. Hepatitis B patients can be classified as follows: a) HBeAg-positive chronic hepatitis B: positive serum HBsAg, HBV DNA, and HBeAg, negative anti-HBe, persistently or repeatedly elevated serum ALT, or hepatitis lesions on liver histology. b) HBeAg-negative chronic hepatitis B: positive serum HBsAg and HBV DNA, persistently negative HBeAg, positive or negative anti-HBe, persistently or repeatedly abnormal serum ALT, or hepatitis lesions on liver histology. Studies have found that 51.3% of patients with chronic hepatitis B (CHB) are HBeAg negative. As HBsAg is the main route of HBV infection, serum HBsAg positivity is one of the markers of HBV infection. Therefore, detecting serum HBsAg expression levels is particularly important for evaluating the effectiveness of HBV treatment.

[0005] Currently, there is no cure for chronic hepatitis B (CHB). Nucleotide analogues (NAs) and pegylated interferon (PEG-IFN) are the mainstream treatment options. NA treatment effectively inhibits HBV DNA replication, but has almost no effect on reducing HBsAg levels and requires lifelong medication; the disease rebounds once medication is discontinued. PEG-IFN treatment can provide long-term immune control, but the rate of achieving immune control is low. Some reports indicate that only 3% of individuals receiving PEG-IFN treatment achieved HBsAg clearance after 48 weeks. Furthermore, it has been found that the probability of HBsAg clearance is even lower in CHB patients with high HBsAg expression treated with PEG-IFN. Summary of the Invention

[0006] This disclosure provides an oligonucleotide that inhibits the expression of hepatitis B virus genes, which can effectively inhibit the production of HBsAg and total HBV DNA, enhance the body's immune function and stimulate the production of HBsAb, and is expected to achieve a functional cure for hepatitis B.

[0007] This disclosure also provides an RNAi reagent comprising at least one of the oligonucleotides of this disclosure, conjugates comprising the oligonucleotides, and compositions comprising the oligonucleotides.

[0008] This disclosure also provides a method for inhibiting hepatitis B virus gene expression in cells in vivo or in vitro using the RNAi reagent of this disclosure. The RNAi reagent of this disclosure can effectively reduce HBV DNA production, thereby reducing HBV DNA expression levels in subjects (e.g., human or animal subjects).

[0009] The RNAi reagent disclosed herein can be used to treat and / or prevent diseases, conditions, and / or symptoms at least partially mediated by hepatitis B virus gene expression, including but not limited to chronic hepatitis B (CHB), acute hepatitis B, hepatitis B-related liver diseases (e.g., hepatitis B-related cirrhosis, hepatitis B-related liver cancer), co-infection with hepatitis D virus (HDV), co-infection with hepatitis C virus (HCV), and human immunodeficiency virus (HIV). The methods disclosed herein include administering the RNAi reagent to a subject (e.g., a human or animal subject) using methods known in the art (e.g., subcutaneous injection or intravenous administration).

[0010] The oligonucleotides for inhibiting hepatitis B virus gene expression disclosed herein comprise a sense strand (SS) and / or an antisense strand (AS), wherein the sense strand and antisense strand may be partially, substantially, or completely complementary to each other. The length of each of the sense strand and antisense strand of the oligonucleotides disclosed herein may be 19-30 nucleotides, preferably 19-23 nucleotides.

[0011] In some implementations, the length of the sense strand of the oligonucleotide is 19 nucleotides and the length of the antisense strand is 21 nucleotides.

[0012] In some implementations, the sense and antisense strands of the oligonucleotide are at least 85% complementary in bases.

[0013] In some implementations, at least 16 nucleotides are anticomplementary between the sense and antisense strands of the oligonucleotide to form a double strand.

[0014] In some implementations, there are 0, 1, 2, or 3 base mismatches between the sense and antisense strands of the oligonucleotide.

[0015] In some implementations, the antisense strand of the oligonucleotide is at least 76% base-complementary to the HBV RNA transcript.

[0016] In some implementations, there are 0, 1, 2, 3, or 4 base mismatches between the nucleotides of the antisense strand of the oligonucleotide and the HBV mRNA.

[0017] In some embodiments, the sense and antisense strands of the oligonucleotide are at least partially anticomplementary to form a double strand, wherein the sense strand comprises, consists of, or is substantially composed of 19 nucleotides, defined sequentially from the 5' end to the 3' end of the sequence as positions 1 to 19; and the antisense strand comprises, consists of, or is substantially composed of 21 nucleotides, defined sequentially from the 5' end to the 3' end of the sequence as positions 1 to 21.

[0018] In some embodiments, the nucleotides in the sense strand and / or antisense strand of the oligonucleotide are each independently composed of at least one of the following: ribonucleotides containing guanine as a base, ribonucleotides containing adenine as a base, ribonucleotides containing cytosine as a base, ribonucleotides containing uracil as a base, and deoxyribonucleotides containing thymine as a base.

[0019] In some implementations, the sense strand, antisense strand, and duplex of the oligonucleotide are shown in Table 1.

[0020] In some implementations, the antisense strand comprises, is composed of, or is substantially composed of a sequence differing from any of the following sequences by 0, 1, 2, 3, or 4 nucleotides:

[0021] 5'-UUGUAAGUUGGCGAGAAAGGG-3'(SEQ ID NO:25)

[0022] 5'-AUACUUUCCAAUCAAUAGGGG-3'(SEQ ID NO:33)

[0023] 5'-UAUACAUGCAUAUAAAGGCUU-3' (SEQ ID NO: 27);

[0024] The differences are caused, for example, by substitution, insertion, deletion, or inversion of nucleotides in the listed nucleotide sequences.

[0025] In some implementations, the positive strand comprises, is composed of, or is substantially composed of a sequence that differs from any of the following sequences by 0, 1, 2, or 3 nucleotides:

[0026] 5'-CUUUCUCGCCAACUUACAA-3'(SEQ ID NO:74)

[0027] 5'-CCUAUUGAUUGGAAAGUAU-3'(SEQ ID NO:82)

[0028] 5'-GCCUUUAUAUGCAUGUAUA-3' (SEQ ID NO:76);

[0029] The differences are caused, for example, by substitution, insertion, deletion, or inversion of nucleotides in the listed nucleotide sequences.

[0030] The oligonucleotides disclosed herein exhibit highly efficient inhibitory effects on the production of total HBV RNA from different HBV subtypes in vivo or in vitro; optionally, the oligonucleotides are conjugated to a target ligand.

[0031] In some implementations, the positive chain contains oligonucleotides composed of, or substantially composed of, the sequences of SEQ ID NO:25 or SEQ ID NO:33, which exhibit good viral inhibitory activity against HBVA, B, C, and D models, as well as representative Chinese HBV.

[0032] In some embodiments, the oligonucleotide comprises or is any one of the duplexes BPR3012003, BPR3014004, and BPR3013024 shown in Table 1.

[0033] In some embodiments, the oligonucleotide is a short interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), or a dicer substrate.

[0034] In some embodiments, the oligonucleotide may be a single-stranded oligonucleotide, such as antisense oligonucleotides (ASO).

[0035] The oligonucleotides provided in this disclosure, each nucleotide in its sense strand and / or antisense strand, are independently modified or unmodified nucleotides.

[0036] In some embodiments, the modification includes at least one of the following: modification of the ribose of the nucleotide, modification of the phosphate backbone of the nucleotide, or modification of replacing the nucleotide with a nucleotide analog.

[0037] In some embodiments, the nucleotide modification is performed using a selection of nucleotides modified with 2'-methoxy (2'-O-methyl, 2-OMe), 2'-methoxyethyl (2'-O-MOE), 2'-deoxy-2'-fluoro (2'-F), 2'-arabino-fluoro (2'-Ara-F), 2'-deoxy (2'-deoxy)ribonucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, locked nucleic acid (LNA), 2',3'-unlocked nucleic acid (UNA), and glycerol nucleic acid. Nucleic Acid (GNA), L-2'-O-methyl modified nucleotide, L-2'-deoxy-2'-fluororibonucleotide, L-2'-deoxyribonucleotide, threonine nucleic acid (TNA), 4'-modified threonine nucleic acid, reverse nucleotide, reverse 2'-O-methyl modified nucleotide, reverse 2'-deoxyribonucleotide, reverse abase-free nucleotide, 5'-phosphorothiocate (PS) modified nucleotide, and 5'-(E)-vinylphosphonate modified ribonucleotide (VP).

[0038] In some embodiments, the -OH at the 2' position of the glycosyl group of some or all of the nucleotides in the sense and antisense strands of the oligonucleotide is replaced by fluorine, methoxy, or deoxygenated (modified), and the phosphate ester group between at least two terminal nucleotides is thiolated.

[0039] In some embodiments, the modified nucleotide is selected from at least one of 2'-methoxy modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, and 5'-thiophosphate modified nucleotides.

[0040] In some embodiments, the modified nucleotide is selected from at least one of 2'-methoxy modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 5'-thiophosphate modified nucleotides, and 2'-deoxyribonucleotides.

[0041] In some embodiments, the modified nucleotide is selected from at least one of 2'-methoxy modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 5'-thiophosphate modified nucleotides, 2'-deoxyribonucleotides, and L-2'-deoxy-2'-fluororibonucleotides.

[0042] In some embodiments, some or all of the nucleotides in the sense strand and the antisense strand are at least one of 2'-methoxy modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, and 2'-deoxyribonucleotides.

[0043] In some embodiments, at least two nucleotides at the 5' end and / or at least two nucleotides at the 3' end of the sense strand and / or the antisense strand are linked by phosphate thioester groups.

[0044] In some implementations, each nucleotide in the positive strand is an independently modified nucleotide.

[0045] In some implementations, each nucleotide in the antisense strand is an independently modified nucleotide.

[0046] In some implementations, the sense and antisense strands can be included in siRNA, miRNA, shRNA, or butyrate substrates to achieve gene silencing.

[0047] In some embodiments, in the sense strand, positions 5, 7, 8, and 9 are nucleotides modified with 2'-deoxy-2'-fluoro, and the remaining nucleotides are nucleotides modified with 2'-methoxy; in the antisense strand, positions 6, 14, and 16 are nucleotides modified with 2'-deoxy-2'-fluoro, position 2 is modified with 2'-deoxyribonucleotide, and the remaining nucleotides are nucleotides modified with 2'-methoxy; optionally, the 5' terminal nucleotide and / or the 3' terminal nucleotide of the sense strand and / or the antisense strand are nucleotides modified with 5'-thiophosphate; or, the two or three nucleotides between the 5' terminals and the two or three nucleotides at the 3' terminals of the sense strand and the antisense strand are all nucleotides modified with 5'-thiophosphate.

[0048] In some embodiments, in the sense strand, positions 5, 7, 8, and 9 are nucleotides modified with 2'-deoxy-2'-fluoro, and the remaining nucleotides are nucleotides modified with 2'-methoxy; in the antisense strand, positions 2, 6, 14, and 16 are nucleotides modified with 2'-deoxy-2'-fluoro, position 9 is modified with 2'-deoxyribonucleotide, and the remaining nucleotides are nucleotides modified with 2'-methoxy; optionally, the 5' terminal nucleotide and / or the 3' terminal nucleotide of the sense strand and / or the antisense strand are nucleotides modified with 5'-thiophosphate; or, the two or three nucleotides between the 5' terminals and the two or three nucleotides at the 3' terminals of the sense strand and the antisense strand are all nucleotides modified with 5'-thiophosphate.

[0049] In some embodiments, in the sense strand, positions 5, 7, 8, and 9 are nucleotides modified with 2'-deoxy-2'-fluoro, and the remaining nucleotides are nucleotides modified with 2'-methoxy; in the antisense strand, position 1 is a nucleotide modified with 5'-vinylphosphonate-2'-methoxy, positions 2, 6, 14, and 16 are nucleotides modified with 2'-deoxy-2'-fluoro, and the remaining nucleotides are nucleotides modified with 2'-methoxy; optionally, the 5' terminal nucleotide and / or the 3' terminal nucleotide of the sense strand and / or the antisense strand are nucleotides modified with 5'-thiophosphate; or, the two or three nucleotides between the 5' terminals of the sense strand and the antisense strand, as well as the two or three nucleotides at the 3' terminals, are all nucleotides modified with 5'-thiophosphate.

[0050] In some embodiments, in the sense strand, positions 5, 7, 8, and 9 are nucleotides modified with 2'-deoxy-2'-fluoro, and the remaining nucleotides are nucleotides modified with 2'-methoxy; in the antisense strand, positions 2, 6, 8, 9, 14, and 16 are nucleotides modified with 2'-deoxy-2'-fluoro, and the remaining nucleotides are nucleotides modified with 2'-methoxy; optionally, the 5' terminal nucleotide and / or the 3' terminal nucleotide of the sense strand and / or the antisense strand are nucleotides modified with 5'-thiophosphate; or, the two or three nucleotides between the 5' terminals and the two or three nucleotides at the 3' terminals of the sense strand and the antisense strand are all nucleotides modified with 5'-thiophosphate.

[0051] In some embodiments, in the sense strand, positions 5, 7, 8, and 9 are nucleotides modified with 2'-deoxy-2'-fluoro, and the remaining nucleotides are nucleotides modified with 2'-methoxy; in the antisense strand, positions 2, 14, and 16 are nucleotides modified with 2'-deoxy-2'-fluoro, positions 5 and 7 are nucleotides modified with 2'-deoxyribonucleotide, and the remaining nucleotides are nucleotides modified with 2'-methoxy; optionally, the 5' terminal nucleotide and / or the 3' terminal nucleotide of the sense strand and / or the antisense strand are nucleotides modified with 5'-thiophosphate; or, the nucleotides between two or three nucleotides at the 5' end and between two or three nucleotides at the 3' end of the sense strand and the antisense strand are both nucleotides modified with 5'-thiophosphate.

[0052] In some embodiments, in the sense strand, positions 5, 7, 8, and 9 are nucleotides modified with 2'-deoxy-2'-fluoro, and the remaining nucleotides are nucleotides modified with 2'-methoxy; in the antisense strand, positions 2, 6, 14, and 16 are nucleotides modified with 2'-deoxy-2'-fluoro, and the remaining nucleotides are nucleotides modified with 2'-methoxy; optionally, the 5' terminal nucleotide and / or the 3' terminal nucleotide of the sense strand and / or the antisense strand are nucleotides modified with 5'-thiophosphate; or, the nucleotides between two or three nucleotides at the 5' end and between two or three nucleotides at the 3' end of the sense strand and the antisense strand are both nucleotides modified with 5'-thiophosphate.

[0053] In some embodiments, the oligonucleotide is any one of BPR3012003, BPR3014004, and BPR3013024. In the sense strand, positions 5, 7, 8, and 9 are 2'-deoxy-2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides. At most three nucleotides at the 5' end of the sense strand are linked by thiophosphate groups. The targeting ligands include, but are not limited to, monomers or combinations thereof of L96, (BT-021), and (BT-057) described later. In the antisense strand, positions 2, 14, and 16 are 2'-deoxy-2'-fluoro-modified nucleotides, positions 5 and 7 are 2'-deoxyribonucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides. The three nucleotides at the 5' end and the three nucleotides at the 3' end of the antisense strand are linked by thiophosphate groups. The oligonucleotides in this embodiment, conjugated with different targeting ligands, all exhibit good in vivo HBsAg inhibition effects.

[0054] In some embodiments, the oligonucleotide is any one of BPR3012003, BPR3014004, and BPR3013024, and in the positive strand, positions 5, 7, 8, and 9 are nucleotides modified with 2'-deoxy-2'-fluoro, the remaining nucleotides are nucleotides modified with 2'-methoxy, the three nucleotides at the 3' end of the positive strand are linked by phosphate thioester groups, and the 5' end of the positive strand is connected to (BT-057)(BT-021)(BT... -021) or (BT-021)(BT-021)(BT-021) or s(BT-021)s(BT-021) conjugation; in the antisense strand, positions 2, 14, and 16 are nucleotides modified with 2'-deoxy-2'-fluoride, positions 5 and 7 are nucleotides modified with 2'-deoxyribonucleotides, and the remaining nucleotides are nucleotides modified with 2'-methoxy. The three nucleotides at the 5' end and the three nucleotides at the 3' end of the antisense strand are linked by thiophosphate groups.

[0055] In some embodiments, the oligonucleotide is any one of BPR3012003, BPR3014004, and BPR3013024, and in the sense strand, positions 5, 7, 8, and 9 are 2'-deoxy-2'-fluoro-modified nucleotides, the remaining nucleotides are 2'-methoxy-modified nucleotides, the two nucleotides at the 3' end of the sense strand are linked by a thiophosphate group, and the 5' end of the sense strand is conjugated with (BT-021)(BT-021) or (BT-057)(BT-021), and the 3' end is conjugated with s(BT-021); in the antisense strand, positions 2, 14, and 16 are 2'-deoxy-2'-fluoro-modified nucleotides, positions 5 and 7 are 2'-deoxyribonucleotides, the remaining nucleotides are 2'-methoxy-modified nucleotides, and the three nucleotides at the 5' end and the three nucleotides at the 3' end of the antisense strand are linked by a thiophosphate group.

[0056] In some embodiments, the oligonucleotide is any one of BPR3012003, BPR3014004, and BPR3013024. In the sense strand, positions 5, 7, 8, and 9 are 2'-deoxy-2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides. The two nucleotides at the 3' end of the sense strand are linked by a thiophosphate group, and the two nucleotides at the 5' end of the sense strand are linked by a thiophosphate group and conjugated to s(BT-021), with the 3' end conjugated to s(BT-021). In the antisense strand, positions 2, 14, and 16 are 2'-deoxy-2'-fluoro-modified nucleotides, positions 5 and 7 are 2'-deoxyribonucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides. The three nucleotides at the 5' end and the three nucleotides at the 3' end of the antisense strand are linked by a thiophosphate group.

[0057] The modification schemes provided in this disclosure are applicable to all sequences provided in Table 1, and are not limited to the sequences specifically verified in the examples. Other unverified RNAi reagents containing modified sequences have the same or similar technical effects as the RNAi reagent sequences verified in the examples.

[0058] In some implementations, oligonucleotides are obtained by independently modifying each nucleotide in the sense and antisense strands shown in Table 1.

[0059] In some embodiments, the antisense strand of the oligonucleotide comprises, is composed of, or is substantially composed of a sequence differing from any one of the following sequences by 0, 1, 2, 3, or 4 nucleotides, wherein the 5' and 3' ends of said sequences each contain two phosphate thioesters:

[0060] 5'-AmUfAmCmdTUmdTCmCmAmAmUmCmAfAmUfAmGmGmGmGm-3' (SEQ ID NO: 196)

[0061] 5'-AmUfAmCmUmUfUmCfCfAmAmUmCmAfAmUfAmGmGmGmGm-3' (SEQ ID NO: 194)

[0062] 5'-AmUfAmCmUmUfUmCmCmAmAmUmCmAfAmUfAmGmGmGmGm-3' (SEQ ID NO: 195)

[0063] 5'-UmUfGmUmAmAfGmUfUfGmGmCmGmAfGmAfAmAmGmGmGm-3' (SEQ ID NO: 202)

[0064] 5'-UmUfGmUmAmAfGmUmUmGmGmCmGmAfGmAfAmAmGmGmGm-3' (SEQ ID NO: 203)

[0065] 5'-UmUfGmUmdAAmdGUmUmGmGmCmGmAfGmAfAmAmGmGmGm-3' (SEQ ID NO: 204)

[0066] 5'-UmAfUmAmCmAfUmGfCfAmUmAmUmAfAmAfGmGmCmUmUm-3' (SEQ ID NO: 206)

[0067] 5'-UmAfUmAmCmAfUmGmCmAmUmAmUmAfAmAfGmGmCmUmUm-3' (SEQ ID NO: 207)

[0068] 5'-UmAfUmAmdCAmdTGmCmAmUmAmUmAfAmAfGmGmCmUmUm-3'(SEQ ID NO:208)

[0069] 5'-AmdTAmCmUmUfUmCmCmAmAmUmCmAfAmUfAmGmGmGmGm-3'(SEQ ID NO:247)

[0070] 5'-AmUfAmCmUmUfUmCmdCAmAmUmCmAfAmUfAmGmGmGmGm-3'(SEQ ID NO:248)

[0071] 5'-VPAmUfAmCmUmUfUmCmCmAmAmUmCmAfAmUfAmGmGmGmGm-3'(SEQ ID NO:249)

[0072] 5'-UmdTGmUmAmAfGmUmUmGmGmCmGmAfGmAfAmAmGmGmGm-3'(SEQ ID NO:250)

[0073] 5'-UmUfGmUmAmAfGmUmdTGmGmCmGmAfGmAfAmAmGmGmGm-3'(SEQ ID NO:251)

[0074] 5'-VPUmUfGmUmAmAfGmUmUmGmGmCmGmAfGmAfAmAmGmGmGm-3'(SEQ ID NO:252)

[0075] 5'-UmAfUmAmCmAfUmGmdCAmUmAmUmAfAmAfGmGmCmUmUm-3'(SEQ ID NO:253)

[0076] 5'-VPUmAfUmAmCmAfUmGmCmAmUmAmUmAfAmAfGmGmCmUmUm-3'(SEQ ID NO:254);

[0077] In this context, the uppercase letters “G”, “C”, “A”, and “U” represent ribonucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively; the lowercase letter “m” indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy modified nucleotide; the lowercase letter “f” indicates that the nucleotide adjacent to the left of the letter f is a 2'-deoxy-2'-fluoro modified nucleotide; VP represents a ribonucleotide to the right containing a 5'-vinylphosphonate modified nucleotide; and the lowercase letter “d” indicates that the nucleotide adjacent to the right of the letter d is a 2'-deoxyribonucleotide, where dT represents thymine deoxyribonucleotide.

[0078] In some embodiments, the positive strand of the oligonucleotide comprises, is composed of, or is substantially composed of a sequence differing from any one of the following sequences by 0, 1, 2, or 3 nucleotides, wherein the 5' end and / or 3' end of said sequence each contains 1 or 2 phosphate thioesters:

[0079] 5'-CmCmUmAmUfUmGfAfUfUmGmGmAmAmAmGmUmAmUm-3' (SEQ ID NO: 193)

[0080] 5'-CmUmUmUmCfUmCfGfCfCmAmAmCmUmUmAmCmAmAm-3' (SEQ ID NO: 201)

[0081] 5'-GmCmCmUmUfUmAfUfAfUmGmCmAmUmGmUmAmUmAm-3' (SEQ ID NO: 205);

[0082] In this context, the uppercase letters “G”, “C”, “A”, and “U” represent ribonucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively; the lowercase letter “m” indicates that the nucleotide adjacent to the left of the letter m is a nucleotide modified with 2'-methoxy; and the lowercase letter “f” indicates that the nucleotide adjacent to the left of the letter f is a nucleotide modified with 2'-deoxy-2'-fluoro.

[0083] The meanings of the symbols in the sequence disclosed herein can also be found in Table 3.

[0084] In some embodiments, the oligonucleotide comprises or is any one of the duplexes BPR301400401, BPR301400403, BPR301400404, BPR301401404, BPR301200301, BPR301200303, BPR301200304, BPR301302402, BPR301302403, BPR301302404, BRP301400421, BRP301400422, BRP301400423, BPR301200321, BPR301200322, BPR301200323, BPR301302421, BPR301302422, and BPR301302423 shown in Table 2.

[0085] In some embodiments, the oligonucleotide is delivered to hepatocytes and / or a subject via a targeting ligand, a lipid nanoparticle (LNP), or a lipid nanoparticle analogue, wherein the hepatocytes are in vivo or in vitro cells; preferably, the oligonucleotide is conjugated to a targeting ligand compound, wherein the ligand compound is a compound with affinity for the ASGPR protein; preferably, the oligonucleotide is contained in a lipid nanoparticle, wherein the lipid nanoparticle is selected from at least one of the group consisting of cationic lipid formulations, phospholipid formulations, cholesterol formulations, and polyethylene glycol-lipid formulations. Optionally, the oligonucleotide is contained in a viral vector, wherein the viral vector is selected from at least one of the group consisting of adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, SV40 vectors, polyomavirus vectors, papillomavirus vectors, microRNA viral vectors, anthrax virus vectors, helper-dependent adenoviruses, or gutless adenoviruses.

[0086] This disclosure also provides a conjugate comprising the oligonucleotide of this disclosure and a targeting ligand, wherein the positive strand of the oligonucleotide is conjugated to the targeting ligand.

[0087] In some embodiments, the targeting ligand is conjugated to the 5' end and / or the 3' end of the positive chain; preferably, the targeting ligand is conjugated to the 3' end of the positive chain; or, the targeting ligand is conjugated to the 5' end of the positive chain; or, the targeting ligand is conjugated to both the 5' end and the 3' end of the positive chain.

[0088] In some implementations, the targeting ligand comprises N-acetylgalactosamine (GalNAc), for example, N-acetylgalactosamine is covalently conjugated to the sense chain in a monovalent, divalent, or trivalent state.

[0089] In some embodiments, the targeting ligand is a galactosamide compound or analogue with ASGPR protein affinity, preferably having the structure shown in formula (I) or formula (II), wherein R is a C1-C8 straight-chain or branched alkyl group. Indicates the connection position of covalent chemical bonds:

[0090] In some embodiments, the conjugate comprises an oligonucleotide conjugate formed by the oligonucleotide and one or more targeting ligands, or a racemic, stereoisomer, isotopic label, or pharmaceutically acceptable salt thereof; wherein the targeting ligand is selected from structures such as L96, (BT-021), and (BT-057).

[0091] Preferably, one or more structures such as L96, (BT-021), and (BT-057) are included at the 3'-end, 5'-end, or mid-chain position of one or two oligonucleotide chains of the oligonucleotide conjugate; or a combination of structures such as L96, (BT-021), and (BT-057) are included.

[0092] Preferably, the number of the structures is 1 to 6, more preferably 1 to 4;

[0093] in The coordinates indicate the location of the covalent chemical bond, and the structure is connected to the oligonucleotide or the above structure via a phosphate ester (PO) or a phosphate thioester (PS).

[0094] When (BT-021) is attached to the 5'-end of the oligonucleotide chain, its structure is as follows:

[0095] When (BT-021) is attached to the 3'-end of an oligonucleotide chain, its structure is as follows:

[0096] In some embodiments, the conjugates are shown in Table 4.

[0097] In some embodiments, the conjugate comprises a double-stranded structure such as SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224.

[0098] In some embodiments, the conjugate is BRP400451, BPR301400454, BPR301400455, BPR301200354, BPR301200355, BPR301200356, BPR301400461, BPR301400463, BPR301400464, BPR301400466, BPR301400471, or BPR301400482.

[0099] The oligonucleotides or conjugates disclosed herein may be incorporated into compositions. Therefore, this disclosure also provides a composition comprising one or more oligonucleotides or conjugates disclosed herein, and pharmaceutically acceptable diluents, carriers, and / or excipients.

[0100] In some embodiments, the compositions provided in this disclosure are pharmaceutical compositions.

[0101] In the compositions disclosed herein, pharmaceutically acceptable diluents, carriers, and / or excipients may be any suitable diluents, carriers, and / or excipients conventionally used in the art.

[0102] In some embodiments, the diluent is selected from at least one of physiological saline, buffer solution, dextran solution, water, and glycerol. Preferably, the diluent is a biological isotonic solution, such as physiological saline or phosphate buffer.

[0103] In some embodiments, the composition may further comprise one or more additional therapeutic agents, such as those beneficial for treating and / or preventing diseases, symptoms, and / or signs at least partially mediated by HBV gene expression. Preferably, the therapeutic agent is selected from at least one of the following groups: hepatitis B virus vaccine, pegylated interferon α (PEG-IFNα), interferon α-2β, recombinant human interleukin-7, FXR agonist, Toll-like receptor 7 / 8 (TLR7 / 8) agonist, checkpoint inhibitor (e.g., PD-1 inhibitor, PD-L1 inhibitor, TIGIT inhibitor), nucleotide analogue (e.g., tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir, telbivudine, clavidine, etc.), polymerase inhibitor nucleotides, antisense oligonucleotides, hepatitis B virus neutralizing monoclonal antibodies, core protein inhibitors, capsid assembly regulators, core protein allosteric regulators, NTCP inhibitors, and siRNAs that inhibit hepatitis B virus gene expression.

[0104] This disclosure also provides a method for preparing the composition, comprising mixing one or more oligonucleotides or conjugates of this disclosure (and optionally additional therapeutic agents) with at least one pharmaceutically acceptable diluent, carrier, and / or excipient.

[0105] This disclosure also provides the RNAi reagents as pharmaceuticals (e.g., oligonucleotides, conjugates, or compositions provided in this disclosure).

[0106] This disclosure also provides said RNAi reagents (e.g., oligonucleotides, conjugates, or compositions provided in this disclosure) for the treatment and / or prevention of diseases, conditions, and / or symptoms at least partially mediated by hepatitis B virus gene expression.

[0107] This disclosure also provides the use of the RNAi reagent (e.g., the oligonucleotide, conjugate, or composition provided herein) in the preparation of a medicament or formulation for inhibiting hepatitis B virus gene expression in cells in vivo or in vitro.

[0108] In some implementations, the cells are in the subject's body.

[0109] In some embodiments, the cells are human cancer cells transfected with hepatitis B virus plasmids, such as HepG2. In some specific embodiments, the cells are HepG2.2.15.

[0110] In some implementations, the subject is a mammal, such as a human, mouse, or marmot.

[0111] In some embodiments, the expression of the hepatitis B virus gene is suppressed by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

[0112] This disclosure also provides the use of the RNAi reagent (e.g., the oligonucleotide, conjugate, or composition provided herein) in the preparation of medicaments for treating and / or preventing diseases, conditions, and / or symptoms at least partially mediated by hepatitis B virus gene expression.

[0113] This disclosure also provides a method for treating and / or preventing diseases, conditions, and / or symptoms at least partially mediated by hepatitis B virus gene expression, comprising administering a therapeutically effective amount of the RNAi reagent of this disclosure (e.g., oligonucleotides, conjugates, or compositions provided in this disclosure) to a subject in need.

[0114] In some embodiments, in any of the methods described above, the hepatitis B virus gene is type A HBV, type B HBV, type C HBV, type D HBV, type E HBV, type F HBV, type G HBV, type H HBV, type I HBV, type J HBV, or recombinant HBV; preferably, the hepatitis B virus gene is type A HBV, type B HBV, type C HBV, or type D HBV.

[0115] In some embodiments, in any of the methods described above, the disease includes at least one of chronic hepatitis B (CHB), acute hepatitis B, hepatitis B-related liver disease (e.g., hepatitis B-related cirrhosis, hepatitis B-related liver cancer), co-infection with hepatitis D virus (HDV), co-infection with hepatitis C virus (HCV), and co-infection with human immunodeficiency virus (HIV).

[0116] In some implementations, in any of the methods described above, the subject is a mammal, such as a human, mouse, or marmot.

[0117] In some embodiments, in any of the methods described above, the RNAi reagent is administered to the subject orally, via the intestine, via the mucosa, via the subcutaneous, via the parenteral route, or via other routes; the parenteral route includes subcutaneous injection or infusion, intravenous injection or infusion, and intramuscular injection or infusion; the other routes include nasal, vaginal, rectal, sublingual, or inhalation.

[0118] In some embodiments, in any of the methods described above, the concentration of the RNAi reagent is 0.01 nM to 100 nM, for example, 0.01 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM or any value within the range thereof.

[0119] In some embodiments, in any of the methods described above, the dosage of the RNAi reagent is about 1-300 mg / kg body weight, such as 1 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, 20 mg / kg body weight, 50 mg / kg body weight, 100 mg / kg body weight, 150 mg / kg body weight, 200 mg / kg body weight, 250 mg / kg body weight, 300 mg / kg body weight, or any value within the range thereof.

[0120] In some embodiments, in any of the methods described above, the RNAi reagent is administered once or more daily, weekly, every two weeks, every three weeks, every one month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or every twelve months, for example, daily, weekly, every two weeks, every three weeks, every one month, every two months, every three months, every four months, every five months, every six months, every seven months, or every... Apply 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 times every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months.

[0121] In some embodiments, in any of the methods described above, the total number of times the RNAi reagent is applied can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50. For example, the RNAi reagent can be applied approximately 1, 2, 3, or 4 times.

[0122] This disclosure also provides a kit comprising the RNAi reagents provided herein (e.g., oligonucleotides, conjugates, or compositions provided herein), and optionally instructions for use. Beneficial effects

[0123] (i) The oligonucleotides disclosed herein possess efficacy distinct from current clinically preferred hepatitis B treatments. They not only inhibit HBV DNA but also directly disrupt the template for HBV translation proteins, preventing the formation of HBV HBsAg and HBeAg at the source and continuously inhibiting their expression. In some individuals, this leads to the production of surface antibodies HBsAb and HBeAb, making it easier to trigger the immune shield and achieve functional cure. Specifically, the oligonucleotides disclosed herein exhibit significant inhibitory effects on the most common HBV types A, B, C, and D, and demonstrate good inhibitory effects on the representative Chinese HBV strain.

[0124] (ii) In the oligonucleotides disclosed herein, some or all of the nucleotides can be modified by various methods such as ribose modification, phosphate backbone modification, or replacement with nucleotide analogs to enhance the stability and activity of the oligonucleotides in vivo.

[0125] (iii) The oligonucleotide delivery method disclosed herein can effectively increase the delivery effect of oligonucleotides in vivo and prolong their effect, enabling them to inhibit HBV-related components in vivo for a long time and with high efficiency. Attached Figure Description

[0126] Figure 1 shows the serum HBsAg levels in Tg-HBV mice after a single subcutaneous administration of 3 mg / kg siRNAs compounds.

[0127] Figure 2 shows the HBV DNA levels in the serum of AAV-HBV mice after a single subcutaneous administration of 3 mg / kg siRNAs.

[0128] Figure 3 shows the serum HBsAg levels in AAV-HBV mice after a single subcutaneous administration of 3 mg / kg siRNAs compound.

[0129] Figure 4 shows the HBeAg levels in the serum of AAV-HBV mice after a single subcutaneous administration of 3 mg / kg siRNAs compounds. Detailed Implementation

[0130] The present disclosure will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Unless otherwise specified, the technical means used in the embodiments are all conventional operations in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0131] As used herein, the terms “deoxyribonucleic acid” and “DNA” refer to a nucleotide or polynucleotide that contains at least one sugar moiety having an H instead of an OH at its 2' position.

[0132] The oligonucleotide sequences used in this paper were designed based on the HBV reference genotype information used in the experimental models: HBV type A reference sequence from the National Center for Biotechnology Information (NCBI): AF305422 (SEQ ID NO:1); HBV type D NCBI reference sequence: U95551 (SEQ ID NO:4). The B2 and C2 HBV reference sequences (SEQ ID NO:2 and SEQ ID NO:3) are from SEQ ID NO:3 and SEQ ID NO:4 of patent application number 202310689271.1.

[0133] As used herein, the terms “siRNA,” “RNAi reagent,” and “oligonucleotide” refer to compositions comprising RNA or RNA-like (e.g., chemically modified RNA, or chemically modified RNA conjugated with a target ligand) oligonucleotide molecules capable of degrading or inhibiting the translation of target mRNA in a sequence-specific manner. While not wishing to be limited by any particular theory, the RNAi reagents disclosed herein can induce RNA interference through an RNA interference mechanism, namely through the formation of an RNA-induced silencing complex (RISC) interaction. Methods for RISC-induced gene expression inhibition or silencing in cells or animals are well known in the art. In this document, “oligonucleotide” and “siRNA” are sometimes used interchangeably.

[0134] Oligonucleotides typically contain a sense strand and an antisense strand. The functional strand in siRNA is the antisense strand, which forms a RISC with the corresponding intracellular protein. The AS portion of this RISC specifically recognizes and captures the target RNA through its inverse complementarity. Other proteins within the RISC then target and cleave the target RNA, thereby disabling its function. The antisense strand's inverse complementarity to the target RNA can be partially or completely complementary to the target RNA. Theoretically, siRNAs containing or partially containing this disclosed siRNA, or siRNAs integrated into other vectors, can also function.

[0135] As used herein, the term "overhang," also known as "nucleotide overhang," refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of siRNA. A nucleotide overhang exists, for example, when the 3' end of one strand of siRNA extends beyond the 5' end of the other strand, or vice versa. siRNA may include an overhang having at least one nucleotide; this overhang may contain at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang may include or consist of nucleotides / nucleotide analogs (including deoxynucleotides / nucleosides). One or more overhangs may be located on the sense strand, the antisense strand, or a combination thereof. Additionally, one or more nucleotides of the overhang may be present at the 5' end, the 3' end, or both ends of the antisense strand of the siRNA.

[0136] As used herein, the term "complementary" refers to the ability of polynucleotides to pair with each other to form base pairs. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide chains. Complementary polynucleotide chains can pair bases in a Watson-Crick manner (e.g., A with T, A with U, C with G), or in any other manner that allows for the formation of duplexes, such as the Hoogsteen base pair manner or the wobble base pair manner.

[0137] As used herein, the term "conjugate" or "coupler" refers to a new compound formed by the covalent linking (coupling) of two or more compound molecules through a bivalent or multivalent compound molecule with a linking function. The conjugate can be represented as GalNAc-siRNA, where GalNAc can be L96 (GalNAc3 ligand), serving as a liver-targeting delivery vector. L96 can form a conjugate by coupling with either the 3' end of the positive strand of the siRNA or the 5' end of the positive strand of the siRNA.

[0138] As used in this article, the phrase “inhibit hepatitis B virus gene expression” includes inhibiting the expression of any HBV gene (such as the HBV gene in mice, the HBV gene in rats, or the HBV gene in humans) as well as variants or mutants of the HBV gene that encodes the HBV protein.

[0139] Suppression of HBV gene expression includes any level of HBV gene suppression, such as at least partial suppression of HBV gene expression, like at least about 20% suppression. In some embodiments, the suppression is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0140] HBV gene expression can be assessed based on the levels of any variable associated with HBV gene expression, such as HBV mRNA levels, HBV RNA transcription protein levels (e.g., HBsAg, HBeAg, etc.), or HBV DNA levels. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. A control level can be any type of control level used in the art, such as baseline levels before administration or levels determined from similar untreated or controlled (e.g., buffer-only control or inert agent control) subjects, cells, or samples.

[0141] Subjects may include any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-human primates, sheep, dogs, cats, horses, cattle, etc. In some embodiments, the subject is a human. In some embodiments, the subject is a mouse or a groundhog.

[0142] As used herein, the terms "therapeutic effective amount," "therapeutic effective dose," and "effective amount" refer to the amount of the siRNA, conjugate, or composition of this disclosure, administered alone or in combination with other therapeutic agents to cells, tissues, or subjects, that effectively prevents or improves the symptoms of one or more diseases or conditions or the progression of such diseases or conditions. The effective amount for a specific subject can vary depending on various factors, such as the disease to be treated, the patient's overall health status, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity. When administered to an individual as a single active ingredient, the therapeutic effective amount refers to that single ingredient. When administered in combination, the therapeutic effective amount refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether they are administered in combination, continuously, or simultaneously. The therapeutic effective amount will typically reduce symptoms by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0143] The term "treatment" refers to a favorable or desired outcome, including, for example, a reduction in the expression level of HBV RNA in serum or plasma. The term "treatment" also includes, but is not limited to, the relief or improvement of symptoms in one or more of the following conditions: chronic hepatitis B (CHB), acute hepatitis B, hepatitis B-related liver diseases (e.g., hepatitis B-related cirrhosis, hepatitis B-related liver cancer), co-infection with hepatitis D virus (HDV), co-infection with hepatitis C virus (HCV), or co-infection with human immunodeficiency virus (HIV).

[0144] The terms “treatment” and “improvement” are used interchangeably in this document. These terms refer to methods of achieving beneficial or desired results, including but not limited to therapeutic benefits.

[0145] siRNA

[0146] This article provides siRNAs for inhibiting hepatitis B virus gene expression. Each siRNA contains a sense strand and an antisense strand.

[0147] The disclosed siRNA, or RNAi reagent containing the siRNA, comprises a core sequence consisting of 2-18 ribonucleotides starting at the 5' end of the antisense strand. Its main function is to participate in the formation of the RISC complex and to be anticomplementary to HBV mRNA. The nucleosides or nucleotide analogs in the antisense strand are allowed to have one, two, three, or four mismatches with the target RNA. The sense strand, as the anticomplementary sequence in the RNAi reagent, forms a duplex with the antisense strand, allowing the presence of nucleotide analogs complementary to those at corresponding positions on the antisense strand, and allowing zero, one, two, or three mismatches between the sense and antisense strands.

[0148] For an oligonucleotide to cleave target mRNA, the secondary structures formed during transcription of the target mRNA in vivo, or protein binding, must not affect the binding of the antisense strand. The majority of the nucleotides in each strand of an oligonucleotide are ribonucleotides, and one or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used herein, an oligonucleotide may contain chemically modified ribonucleotides; an oligonucleotide may contain substantial modifications at multiple nucleotide sites. As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide bond, and / or a modified nucleobase. Therefore, the term "modified nucleotide" encompasses substitutions, additions, or removals (e.g., functional groups or atoms) at internucleotide bonds, sugar moieties, or bases. Suitable modifications for nucleotides used in this disclosure include all types of modifications disclosed herein or known in the art.

[0149] Chemical modifications to the nucleotides and nucleotide analogs of the antisense and sense strands can, to some extent, protect the stability of oligonucleotides in vivo and within cells. Appropriate modifications can not only improve sequence stability in blood, interstitial fluid, and cells, but also maintain or enhance the formation and stability of RISC. Oligonucleotides themselves do not have specific cell-targeting functions. The delivery compounds disclosed herein, based on N-acetylgalactosamine (GalNAc), can specifically target and deliver siRNA to hepatocytes via the ASGPR receptor. Furthermore, since the delivery-related compounds are located at both ends of the siRNA structure, in addition to providing specific delivery functionality, they can also, to some extent, protect the stability of the oligonucleotide, thus achieving a long-lasting effect. The oligonucleotides disclosed herein include sense and antisense strands, and include, but are not limited to: short interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), or dicer substrates; the antisense strands disclosed herein include antisense oligonucleotides (ASO). The antisense strand of the siRNA described in this article is at least partially complementary to the targeted RNA.

[0150] In some implementations, the sense strand of siRNA has 19 nucleotides and the antisense strand has 21 nucleotides.

[0151] In some implementations, at least 15 nucleotides are reverse complementary between the sense and antisense strands to form a double strand.

[0152] In some implementations, at least 76% of the nucleotides in the antisense strand are reverse complementary to hepatitis B virus mRNA.

[0153] In some implementations, there are 0, 1, 2, 3, or 4 base mismatches between the nucleotides in the antisense strand and the hepatitis B virus mRNA.

[0154] In some embodiments, the 3' end of the antisense strand has a two-base overhang. Preferably, the two-base overhang can be any two modified or unmodified nucleotides.

[0155] Table 1 shows the duplexes composed of the sense and antisense strands of unmodified HBV siRNA. All sequences are shown in the 5' to 3' orientation. The duplex number refers to the number of the duplex assigned to the two strands in the same row of Table 1.

[0156] In some embodiments, an siRNA comprising or consisting of a double strand as shown in Table 1 of this disclosure, or an RNAi reagent comprising the siRNA, is administered to the subject.

[0157] Table 1. The duplex of the sense and antisense strands of unmodified HBV siRNA.

[0158] Modification scheme

[0159] To increase the specificity, stability, and effectiveness of oligonucleotides, this disclosure modifies at least one nucleotide in the sense and antisense strands of the siRNA. For example, siRNAs comprising or composed of the duplexes shown in Table 1 may have 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotide modifications.

[0160] This disclosure classifies nucleotide modifications in siRNA into three main categories: ribose group modification, backbone modification (such as phosphate group modification), and base modification (such as replacing nucleotides with nucleotide analogs). In this disclosure, by replacing nucleotides with nucleotide analogs, on the one hand, the introduction of exogenous substances can reduce the recognition of siRNA by enzymes in cells and body fluids; on the other hand, after siRNA metabolism, the nucleotide analogs themselves can inhibit the reverse transcription of HBV RNA, thereby further inhibiting the generation of HBV DNA.

[0161] The modified nucleotides are selected from: nucleotides modified with 2'-methoxy (2'-O-methyl, 2-OMe), nucleotides modified with 2'-methoxyethyl (2'-O-MOE), nucleotides modified with 2'-deoxy-2'-fluoro (2'-F), nucleotides modified with 2'-arabino-fluoro (2'-Ara-F), nucleotides modified with 2'-deoxy (2'-deoxy) ribonucleotides, nucleotides modified with 2'-amino, nucleotides modified with 2'-alkyl, locked nucleic acids (LNA), 2',3'-broken nucleotide mimics (UNA), and glycerol nucleic acids. Acid (GNA), L-2'-O-methyl modified nucleotides, L-2'-deoxy-2'-fluororibonucleotides, L-2'-deoxyribonucleotides, threonine nucleic acid (TNA), 4'-modified threonine nucleic acid, reverse nucleotides, reverse 2'-O-methyl modified nucleotides, reverse 2'-deoxyribonucleotides, reverse abase-free nucleotides, 5'-phosphate-thiocate (PS) modified nucleotides, and 5'-vinylphosphonate modified ribonucleotides (5'-(E)-vinylphosphonate (VP)).

[0162] Among them, 2'-methoxy modified nucleotides refer to nucleotides formed by replacing the 2' hydroxyl group of the ribosome with a methoxy group, and their structure is shown in A1.

[0163] 2'-Methoxyethyl modified nucleotides are nucleotides formed by replacing the hydrogen atom of the 2'-hydroxyl group of the ribosome with a methoxyethyl group, and their structure is shown in A2.

[0164] 2'-deoxy-2'-fluorinated nucleotides are nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with fluorine, and their structure is shown in A3.

[0165] 2-Deoxy-2-fluoro-arabinofuranose nucleotide is a nucleotide formed by replacing the 2'-hydroxyl group of arabinofuranose with a fluorine atom, and its structure is shown in A4.

[0166] 2'-Deoxyribonucleotide (DNA) is a nucleotide formed by replacing the 2' hydroxyl group of the ribose with hydrogen, and its structure is shown in A5.

[0167] A 2'-amino modified nucleotide is a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with an amino group, and its structure is shown in A6.

[0168] 2'-alkyl modified nucleotides are nucleotides formed by replacing the hydrogen atom of the 2'-hydroxyl group of the ribosome with an alkyl group, and their structure is shown in A7.

[0169] Locked nucleic acids are nucleotides in which an oxymethylene bridge is formed at the 2'-O and 4'-C positions of the ribosomes, and their structure is shown in A8.

[0170] 2',3'-broken nucleotide mimics are nucleotides formed by breaking the carbon-carbon bonds at the 2' and 3' positions of the ribosome, as shown in A9.

[0171] Glycerol-containing nucleic acids are nucleotides in which the pentose sugar is replaced by ethylene glycol, and their structure is shown in A10.

[0172] L-2'-deoxyribonucleotide refers to a nucleotide in which the chirality of the 1', 3', and 4' positions of the ribosome is completely opposite to that of the natural nucleotide, and the hydroxyl group at the 2' position is replaced by hydrogen. Its structure is shown in A11.

[0173] Threonine nucleic acid refers to nucleotides with a threon structure, as shown in A12.

[0174] The structure of 4'-modified threonine is shown in A13, where R represents an alkyl group from C10 to C30;

[0175] A reverse nucleotide is a nucleotide in which the 3' position of the ribosome is replaced by a phosphate group and the 5' position is a hydroxyl group, as shown in A14. Unless otherwise specified in this document, "reverse nucleotide" does not include any substitution of other site groups (e.g., the hydroxyl group at the 2' position).

[0176] A reverse 2'-O-methyl modified nucleotide is a nucleotide in which the 3' position of the ribosome is replaced by a phosphate group, the 5' position is a hydroxyl group, and the 2' position of the hydroxyl group is replaced by a methoxy group. Its structure is shown in A15.

[0177] Reverse 2'-deoxynucleotides are nucleotides in which the 3' position of the ribosome is replaced by a phosphate group, the 5' position is a hydroxyl group, and the 2' position of the hydroxyl group is replaced by hydrogen. Their structure is shown in A16.

[0178] Reverse abase-free nucleotides are pentose sugars whose 2' and 3' positions are replaced by phosphate, and their structure is shown in A17.

[0179] L-2'-deoxy-2'-substituted ribonucleotide, the structure of which is shown in A18, where X includes O, S, Se, CH2, CH-CH3, and R includes H, OH, OMe, F, OMOE, etc. Preferably, X represents O and R represents F, and the structure is shown in A19.

[0180] The structure of the L-2'-O-methyl modified nucleotide is shown in A20.

[0181] In some implementations, modification methods such as 2'-methoxy modification, 2'-deoxy-2'-fluorination modification, and 5'-thiophosphate modification are employed.

[0182] In some implementations, modification methods such as 2'-methoxy modification, 2'-deoxy-2'-fluoro modification, 5'-thiophosphate modification, and 2'-deoxyribonucleotide modification are employed.

[0183] In some implementations, modification methods such as 2'-methoxy, 2'-deoxy-2'-fluoro, 5'-thiophosphate, 2'-deoxyribonucleotide, and L-2'-deoxy-2'-fluororibonucleotide are employed.

[0184] In the siRNA disclosed herein, at least one of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of both the sense and antisense strands is a phosphate ester group with a modifying group. The phosphate ester group with the modifying group is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom. For example, replacing a non-bridging oxygen atom in the phosphodiester bond with a sulfur atom, or replacing the phosphodiester bond with a thiophosphate ester bond, i.e., the connection between two nucleotides is a thiophosphate ester group connection. This modification can stabilize the structure of the siRNA and maintain high base pairing specificity and high affinity.

[0185] The structural formula of the thiophosphate group mentioned in this article is shown in A21:

[0186] Table 2 shows some of the modified bichains of this disclosure and their corresponding justice and antisense chains.

[0187] In some implementations, an RNAi reagent containing or derived from the polynucleotide sequences shown in Table 2 is applied to cells and / or subjects.

[0188] In some embodiments, the sequences shown in Table 2 may be linked (also referred to herein as “conjugations”) to compounds capable of delivering RNAi reagents to cells and / or tissues in a subject, wherein the antisense sequence comprises one or two phosphate thioesters at its 5' and / or 3' ends, and the sense sequence comprises two phosphate thioesters at its 5' and 3' ends. In Examples 4, 5, and 6, the sequences comprise two phosphate thioesters at both the 5' and 3' ends of both the sense and reactive strands.

[0189] Those skilled in the art will recognize that the siRNA described herein can be obtained using conventional siRNA preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis). Solid-phase synthesis is already available as a commercially available custom service. Modified nucleotides can be introduced into the siRNA described herein using appropriately modified nucleotide monomers; methods for preparing appropriately modified nucleotide monomers and methods for introducing modified nucleotides into siRNA are also well known to those skilled in the art.

[0190] Table 2. Modified bichains and their corresponding positive and negative chains.

[0191] Table 3 provides an abbreviation for the nucleotides or modified nucleotides used in this disclosure.

[0192] Table 3. Explanation of Abbreviations for Nucleotides or Modified Nucleotides

[0193] siRNA delivery and conjugates

[0194] The siRNA disclosed herein primarily silences the transcription or reverse transcription of the target RNA by forming a RISC complex. Theoretically, various vectors can be used to deliver the HBV siRNA provided herein into cells. For example, it can be delivered to hepatocytes and / or subjects via targeting ligands, lipid nanoparticles (LNPs), or lipid nanoparticle analogs, wherein the hepatocytes are in vivo or in vitro cells. In some embodiments, the siRNA is transcribed in a DNA or RNA vector. The preparation and use of vectors for delivering the sequence to cells and / or subjects are well known in the art. Specifically, viral vector systems include, but are not limited to, (a) adenovirus vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) piconemavirus vectors; (i) anthrax virus vectors, such as smallpox, such as vaccinia virus vectors or avipox, such as canary pox or fowl pox; and (j) helper-dependent or gutless adenoviruses.

[0195] The HBV siRNA disclosed herein can be delivered to hepatocytes via compounds carrying GalNAc, including but not limited to: the L96 delivery system in some embodiments of this disclosure, Arrowhead's NAG37 delivery system, and other delivery systems carrying GalNAc.

[0196] The HBV siRNA disclosed herein can be delivered into cells by encapsulation or conjugation of lipid nanoparticles (LNPs) or LNP analogs and nanoparticles. For example, the HBV siRNA can be coupled with specific ligands to enter cells by specifically recognizing cell surface proteins or other physicochemical structures.

[0197] As used herein, the term "delivery" means to promote or influence cellular uptake or absorption. The uptake or absorption of HBV siRNA can occur through independent diffusion or active cellular processes, or through the use of delivery agents, targeting agents, etc., that can associate with the HBV siRNA disclosed herein.

[0198] In some implementations, the HBV siRNA disclosed herein is transfected into cells via liposome encapsulation.

[0199] This disclosure provides a conjugate comprising an oligonucleotide of the present disclosure and a targeting ligand, wherein the sense and / or antisense strands of the oligonucleotide are conjugated to the targeting ligand. It is understood that the conjugate may optionally also include a linker, which, together with the targeting ligand, serves as a targeting group, with the oligonucleotide, the linker, and the targeting ligand sequentially linked.

[0200] In some implementations, N-acetylgalactosamine (GalNAc) is used as a delivery carrier for siRNA drugs, specifically delivering the drug to hepatocytes to reduce or inhibit HBV gene expression, thereby inhibiting HBV DNA, HBsAg, and / or HBeAg levels in subjects (e.g., human or animal subjects), thereby achieving the purpose of preventing and / or treating diseases, conditions, and / or symptoms mediated by HBV gene expression, including but not limited to chronic hepatitis B, acute hepatitis B, hepatitis B-related liver diseases (e.g., hepatitis B-related cirrhosis, hepatitis B-related liver cancer), co-infection with hepatitis D virus, co-infection with hepatitis C virus, and co-infection with human immunodeficiency virus.

[0201] In some embodiments, the targeting ligand (conjugation group) may be attached to the end of the siRNA strand, such as the 5' and / or 3' end of the sense strand, and / or the 5' and / or 3' end of the antisense strand. When the conjugation group is attached to the end of the siRNA strand, it is typically attached to a phosphate group of a nucleotide. When the targeting ligand is a combination of multiple targeting ligands, the ligands are linked by phosphate groups. The targeting ligands may be L96, (BT-021), (BT-057), or combinations thereof.

[0202] Table 4 shows some of the conjugates disclosed herein, which comprise chemically modified sense and antisense strand sequences and one or more targeting ligands. The targeting ligands in Table 4 are represented by L96, (BT-021), and (BT-057), respectively, where L96 is the delivery agent disclosed in WO2015006740. s(BT-021) represents a thiophosphate ester as the delivery ligand, and L96, (BT-021), and (BT-057) represent phosphate esters as the delivery ligands.

[0203] In some implementations, the conjugate administered to the subject comprises the double strand shown in column 1 of Table 4, wherein the double strand is...

[0204] The above-mentioned conjugates can be synthesized using methods that have been described in detail in the prior art.

[0205] Table 4 Conjugates

[0206] Composition

[0207] This disclosure also provides compositions comprising the oligonucleotides or siRNAs or conjugates of this disclosure as active ingredients and pharmaceutically acceptable diluents, carriers, and / or excipients. The purpose of these compositions is to facilitate administration to organisms, thereby promoting the absorption of the active ingredient and enabling it to exert its biological activity.

[0208] Pharmaceutically acceptable diluents, carriers and / or excipients as used in this disclosure include any pharmaceutically acceptable diluents, carriers and / or excipients known in the art and suitable therefor, including but not limited to saline, buffered saline, dextran, water, glycerol and combinations thereof.

[0209] In some embodiments, the composition comprises the HBV siRNA disclosed herein and physiological saline.

[0210] In other embodiments, the composition comprises the HBV siRNA disclosed herein and a hydrochloride buffer.

[0211] In some embodiments, the composition further comprises at least one additional therapeutic agent. Preferably, the therapeutic agent is selected from hepatitis B virus vaccines, pegylated interferon α (PEG-IFNα), interferon α-2β, recombinant human interleukin-7, FXR agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, checkpoint inhibitors (e.g., PD-1 / PD-L1 inhibitors, TIGIT inhibitors), nucleotide analogs (e.g., tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir, telbivudine, clavidine), polymerase inhibitor nucleotides, antisense oligonucleotides (ASO), hepatitis B virus neutralizing monoclonal antibodies, core protein inhibitors, capsid assembly regulators, core protein allosteric regulators, NTCP inhibitors, and siRNAs that inhibit hepatitis B virus gene expression.

[0212] Purpose, target population, and administration method

[0213] This disclosure provides the use of oligonucleotides or siRNAs, conjugates, or compositions in the preparation of medicaments for the treatment and / or prevention of diseases, conditions, and / or symptoms mediated by hepatitis B virus gene expression.

[0214] This disclosure provides an oligonucleotide, conjugate, or pharmaceutical composition for inhibiting hepatitis B virus gene expression in cells, either in vivo or in vitro. In some embodiments, HBV gene expression is reduced or inhibited by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

[0215] This disclosure also provides a method for treating and / or preventing diseases, conditions and / or symptoms at least partially mediated by hepatitis B virus gene expression, comprising administering a therapeutically effective amount of the RNAi reagent of this disclosure (e.g., oligonucleotides, conjugates or compositions provided in this disclosure) to a subject in need.

[0216] In some embodiments of treatment and prevention provided in this disclosure, the treatment can be applied to cells, tissues, and organs of different species of mammals such as humans, mice, and marmots.

[0217] In some implementations, the HBV siRNA disclosed herein can be administered to human cancer cells such as HepG2 that have been transfected with HBV plasmids.

[0218] In other embodiments, the HBV siRNA disclosed herein can be applied to human cancer cells HepG 2.2.15.

[0219] Therefore, the HBV siRNA, conjugates, and pharmaceutical compositions disclosed herein can be used to treat HBV-related diseases or conditions in humans and non-human subjects, and can also be used for in vivo and in vitro HBV virus suppression. Common HBV infection-related diseases in humans include chronic hepatitis B (CHB), acute hepatitis B, hepatitis B-related cirrhosis and liver cancer, co-infection with other hepatitis viruses such as HCV and HDV, and the treatment of some other HBV-related liver diseases; the HBV siRNA, conjugates, and pharmaceutical compositions disclosed herein are applicable to all of these conditions.

[0220] The drug can be administered to patients via any suitable route known in the art. For example, in vitro cell administration routes include liposome-mediated transfection and specific-mediated endocytosis (such as ASGPR-mediated endocytosis). In some embodiments, HBV siRNA, conjugates, and pharmaceutical compositions are transfected via liposome encapsulation. Regarding in vivo administration in animals, generally, the methods of this disclosure can be implemented using any medically acceptable mode of administration, where acceptable means any mode that produces an effective level of treatment for HBV-related disease or condition without causing clinically unacceptable adverse effects. The HBV siRNA, conjugates, and pharmaceutical compositions of this disclosure can be administered orally, enterically, transmucosally, subcutaneously, and / or parenterally. Parenterally routes include subcutaneous, intravenous, intramuscular injection, or infusion techniques. Other routes include, but are not limited to, nasal (e.g., via a gastronasal tube), percutaneously, vaginally, rectally, sublingually, and inhalation.

[0221] In some embodiments, the HBV siRNA of this disclosure, conjugated with a specific liver-targeting delivery system, is administered subcutaneously in a biological isotonic solution such as saline or a buffer solution. Due to the presence of the specific delivery system of this disclosure, the HBV siRNA conjugate or pharmaceutical composition can also be administered via intravenous blood injection.

[0222] The dosage of the RNAi reagent disclosed herein can be determined based on the patient's weight, age, sex, disease severity, etc. Based on the amount of siRNA contained therein, the dosage of the RNAi reagent and composition disclosed herein is approximately 1-300 mg / kg body weight.

[0223] The dosing frequency can be daily, weekly, every two weeks, every three weeks, every one month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or annually, once or more.

[0224] The application cycle can be once or more daily, weekly, bi-weekly, bi-weekly, monthly, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or annually. The total number of applications of the RNAi reagent can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. For example, the RNAi reagent can be applied approximately 1, 2, 3, or 4 times.

[0225] In some embodiments, the RNAi reagents and compositions of this disclosure may be packaged in a kit. The RNAi reagents and pharmaceutically acceptable diluents, carriers, or excipients in the kit are provided in liquid or dry form. In some embodiments, the kit includes instructions on how to mix the RNAi reagents with pharmaceutically acceptable diluents, carriers, or excipients or other components.

[0226] Combination of treatment regimens

[0227] The oligonucleotides (or siRNAs) disclosed herein are designed to target multiple HBV RNA transcripts, thereby inhibiting the expression of HBV-related proteins and suppressing HBV RNA reverse transcription, thus inhibiting viral replication. Therefore, the oligonucleotides, conjugates, or compositions disclosed herein can be used in combination with at least one HBV infection-related treatment. The antiviral treatments for HBV-related patients include, but are not limited to: HBV vaccines, pegylated interferon α (PEG-IFNα), interferon α-2β, recombinant human interleukin-7, FXR agonists and Toll-like receptor 7 / 8 (TLR7 / 8) agonists, checkpoint inhibitors (e.g., PD-1 / PD-L1 inhibitors, TIGIT inhibitors), nucleoside analogs (e.g., tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), and telbivudine), polymerase inhibitor nucleotides, ASO, HBV neutralizing monoclonal antibodies, core protein inhibitors, capsid assembly regulators, core protein allosteric regulators, NTCP inhibitors, and other siRNAs, which can be used in combination with the RNAi agents disclosed herein to achieve multi-faceted inhibition of HBV activity. The drug combination therapy can be implemented as an integrated whole, administered individually and multiple times, or through physical mixing.

[0228] Methods for evaluating the efficacy of HBV RNAi treatment

[0229] As a multi-genome subtype virus, HBV exhibits genomic differences of >7.5%–8% across subtypes, thus single-model screening often fails to provide a comprehensive assessment. Given that the main HBV genotypes worldwide are A, B, C, and D, this publication systematically evaluates the inhibitory effects of HBV siRNA sequences on HBV models under different genotypes.

[0230] In some embodiments, the inhibitory effect of HBV siRNA sequences on HBV in HepG2 type B and type C HBV plasmids was evaluated.

[0231] In some embodiments, the inhibitory effect of HBV siRNA sequences on type D HBV in HepG2.2.15 was evaluated.

[0232] In some embodiments, the inhibitory effect of HBV siRNA sequences on HBV in A-type HBV transgenic mice was evaluated.

[0233] In other embodiments, the inhibitory effect of HBV siRNA sequences on HBV in type D AAV-HBV mice was evaluated.

[0234] In the above embodiments, this disclosure evaluated the inhibitory effect of HBV siRNA sequences on four major HBV genotypes in vivo and in vitro. To assess the potential differences in siRNA drug sensitivity due to certain HBV genomic mutations or sequencing errors, we evaluated the siRNA using the Chinese HBV reference in vitro screening system constructed in application number 202310689271.1. Referring to the methods described herein, the efficacy results of the siRNAs screened in this disclosure against representative Chinese infectious HBV in vitro screening systems are illustrated in Example 5 of this disclosure.

[0235] The HBV siRNA disclosed herein directly targets HBV RNA. In some embodiments, the activity of the RNAi reagent is assessed by the difference between the total HBV RNA amount and the negative control. The total HBV RNA amount is quantified by RT-qPCR, and the expression level of the target gene HBV RNA in each sample is calculated using the ΔΔCt relative quantification method. The relative expression level of the target gene is measured using a 2-1T / T ratio. -ΔΔCT The calculation formula is as follows: ΔCT = Average Ct value of target gene - Average Ct value of internal reference gene; ΔCT = ΔCT (drug-treated group) - ΔCT (PBS or saline control group); Relative expression level of HBV RNA = 2 -ΔΔCT Inhibition percentage (%) = (1 - (HBV RNA in sample / HBV RNA in control)) × 100%.

[0236] In some embodiments, the HBV RNA originates from within the cell.

[0237] In other embodiments, the HBV RNA is derived from serum or plasma.

[0238] In some embodiments, this disclosure evaluates the inhibitory effect of HBV siRNA on HBV RNA transcription proteins HBsAg and HBeAg. The protein sources for detection can be in vitro cell supernatant, serum, or plasma. HBsAg, as one of the proteins transcribed from HBV RNA, is a major pathway for HBV infection and an indicator of functional cure in clinical chronic hepatitis B patients. HBeAg, as another transcribed protein of HBV RNA, also reflects the activity of HBV siRNA based on the expression levels of these two proteins. The expression levels of HBsAg and HBeAg proteins are quantified using ELISA. The protein content in the sample is calculated using a standard curve according to the instructions. The inhibition rate is calculated as follows: Inhibition percentage (%) = (1 - (protein content in sample / protein content in control)) × 100%.

[0239] HBV DNA can be generated by HBV reverse transcriptase using HBV RNA as a template. The inhibitory effect of HBV siRNA on HBV RNA can inhibit the generation of HBV DNA to a certain extent.

[0240] In some embodiments, this disclosure measures the inhibitory activity of HBV siRNA agents against HBV DNA. HBV DNA is quantified using RT-qPCR. In some embodiments, the HBV DNA originates from intracellular sources; in other embodiments, the HBV DNA originates from serum or plasma.

[0241] In some in vitro embodiments, the main reagents used included DMEM / F12 culture medium (gibco catalog number 11330032), Cell Counting Kit-CCK8 (Li Ji, catalog number AC11L057), 96-well kit (QIAGEN, catalog number 74182), FastKing RT kit (Tiangen, catalog number KR116-02), Lipofectamine RNAiMax (catalog number: Invitrogen-13778-150), and 96-well plate (Costar 3599).

[0242] In some in vivo embodiments, the main reagents used include a hepatitis B virus nucleic acid assay kit (Sansure), a hepatitis B virus surface antigen assay kit (Mike), a hepatitis B virus e antigen assay kit (Mike), an anti-hepatitis B virus CoreAntigen antibody (Abcam catalog number ab115992), a biotinylated anti-hepatitis B virus Surface Antigen (Ad / Ay) antibody (Abcam catalog number ab68518), and an UltraSYBR Mixture (Low ROX) quantitative PCR kit (Kangwei Century catalog number CW2601M). One-Step gDNA Removal and cDNA Synthesis SuperMix (Tengen Biotech, product number AT311-02), Tissue Genomic DNA Extraction Kit (Tiangen Biotech, product number DP304-03), Mammalian Total Protein Extraction Kit (DE101-01), Easy II Protein Quantitative Kit (BCA) (DQ111-01), TransZol (ET101-01).

[0243] In some in vivo embodiments, the main reagents used include QIAamp 96DNABlood Kit (12) (Qiangen catalog number 51162), FasStart Universal Probe Mast (ROX) (Roche catalog number 04914058001), Quantitative Detection Kit for Hepatitis B Surface Antigen (Autobio catalog number CL 0310), and Human HBeAg ELISA Kit (Autobio catalog number CL 0312).

[0244] In some embodiments, the main instruments used include a fluorescence qPCR instrument (Applied Biosystems, QuantStudio™ 7Flex System) and a multi-functional plate reader (Molecular Devices, SpectraMaxiD3).

[0245] In some embodiments, the main instruments used include a biohazard level 2 safety cabinet (Thermo, 1300 SERIES A2), a refrigerated centrifuge (Beckman, 20R), analytical balance (Sartorius, BS110S), electronic balance (Changsha Xiangping Technology (Balance) Co., Ltd., ES-300S), ultra-micro spectrophotometer (Thermo, Nanodrop one).

[0246] The technical solution of the present invention will be further explained below with reference to specific embodiments.

[0247] Unless otherwise specified, all raw materials or reagents used in the embodiments and comparative examples of this invention are commercially available products.

[0248] Example 1: Preparation of siRNA

[0249] The oligonucleotide synthesis process in the RNAi reagent shown in Table 2 of this disclosure uses an insoluble polymer as a carrier and phosphoramidite nucleoside monomers as starting materials. Through solid-phase synthesis and cleavage / deprotection reactions, crude single-stranded oligonucleotides are obtained. After purification and ultrafiltration, two single-stranded intermediates are obtained. These two intermediates are annealed to form a double-stranded siRNA with complementary base pairing, which is then freeze-dried to obtain siRNA. The specific reaction process is as follows:

[0250] Deprotection reaction: Under room temperature conditions (20-25℃), the protecting group DMTr (dimethoxytriphenylmethyl) on the carrier / nucleotide is removed with dichloroacetic acid to obtain an active hydroxyl group that can undergo coupling reaction. The deprotecting agent is a dichloromethane solution of dichloroacetic acid (3% v / v).

[0251] Coupling reaction: The nucleotide phosphoramidite monomer and the activator are simultaneously fed into a solid-phase synthesis column. The phosphoramidite group is activated and undergoes a coupling condensation reaction with the active hydroxyl group to generate a phosphite triester. The activator is a 0.6 M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT).

[0252] Oxidation reaction: Under the action of iodine water as an oxidant, the triphosphite generated by the upper coupling condensation reaction is converted into a stable triphosphate. The oxidant is a 0.04M iodine / water / pyridine solution with a ratio of v(water):v(pyridine) = 1:9. Thioation reaction: Under the action of the thioreagent ADTT (hydrogenated yellow element), the triphosphite generated by the upper coupling condensation is converted into a stable trithiophosphate.

[0253] Capping reaction: The active hydroxyl groups that have not fully reacted during the coupling reaction are capped so that they do not participate in subsequent reactions. The capping reagents are CapA (acetic anhydride / acetonitrile) and Cap B (N-methylimidazolium:Py:acetonitrile = 2:3:5).

[0254] Repeat the above cyclic reaction until a complete oligonucleotide sequence is synthesized. After completing the four-step cyclic reaction for all nucleoside bases, remove the 5'-DMTr protecting group from the nucleotide ends, and then remove the cyanoethyl protecting group with 20% diethylamine. The solid-phase reaction is then complete. Wash the solid support with acetonitrile and dry it with an inert gas. Collect the solid support and use an ammonia deprotection reagent (concentrated ammonia) to cleave and deprotect the solid support, separating the oligonucleotide from the solid support and removing various protecting groups from the nucleoside bases. After filtration to remove the solid support, wash the solid support with 50% ethanol, combine the mother liquors, and concentrate the resulting solution to obtain a crude solution containing the desired oligonucleotide single strand.

[0255] Example 2: Synthesis of (BT-057)

[0256] Compound 79 (2.0 g, 7.7 mmol, 1.0 eq.) was weighed and dissolved in 20 mL of pyridine, then purged with nitrogen. The mixture was cooled to 0 °C in an ice-water bath, and 2.6 g (7.7 mmol, 1.0 eq.) of 4,4'-bismethoxytriphenylmethyl chloride was added in portions. After the addition was complete, the mixture was allowed to return to room temperature and reacted overnight. The reaction was quenched with water after TLC monitoring. The mixture was extracted twice with ethyl acetate, and the organic phases were combined, dried, concentrated, and purified by column chromatography to give 803.3 g of the compound, yield: 75.6%.

[0257] Compound 80 (2.0 g, 3.6 mmol, 1.0 eq.) and 4-dimethylaminopyridine (87 mg, 0.7 mmol, 0.2 eq.) were weighed into a 100 mL single-necked flask. After nitrogen purging protection, 20 mL of anhydrous dichloromethane and 0.93 g (7.2 mmol, 2.0 eq.) of N,N-diisopropylethylamine were added. Then, 1.0 g (4.3 mmol, 1.5 eq.) of 2-cyanoethyl N,N-diisopropylchlorophosphine was added dropwise using a syringe. After the addition was complete, the reaction was allowed to proceed at room temperature for 1 h. The reaction was monitored by TLC until it was complete. After removing dichloromethane by concentration under reduced pressure at room temperature, the mixture was purified by reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase: 0.01% ammonium bicarbonate) to obtain 1.6 g of compound (BT-057), yield: 59.3%. NMR(400MHz,DMSO-d6)δ7.41(d,J=7.8Hz,2H),7.36–7.15(m,7H),6.87(dd,J=8.8,6.7Hz, 4H),3.90(tt,J=10.7,5.4Hz,1H),3.73(d,J=2.7Hz,6H),3.68–3.47(m,3H),3.03(ddd,J=3 0.6,9.5,5.3Hz,2H),2.73(t,J=5.9Hz,1H),2.59(dt,J=10.0,4.5Hz,1H),1.7–1.43(m,2H ),1.35–1.17(m,24H),1.13(t,J=6.7Hz,9H),1.02(d,J=6.7Hz,3H),0.88–0.80(m,3H).31P NMR(162MHz,DMSO)δ147.57,147.25.MS(ESI):m / zcalcd for C46H69N2O5P[M+H]+:761.50,found:761.34.

[0258] Example 3: Synthesis and Solid Support Loading Example of (BT-021)

[0259] 1. Synthesis of Compound 57

[0260] Compound 103 (30 g, 77.09 mmol) was dissolved in 300 mL of dry 1,2-DCE. The system was placed at 0 °C, and trimethylsilyl trifluoromethanesulfonate (21 mL, 115.64 mmol, 1.5 eq.) was added. The reaction system was stirred at 0 °C for 10 min under nitrogen protection, and then placed in an oil bath at 50 °C for 12 h. The reaction of the starting material was monitored by TLC and LC-MS to ensure complete reaction. The system was then placed at room temperature, and... The mixture was stirred with a molecular sieve for 30 min, then anhydrous 3-pentanol (12.7 mL, 115.64 mmol, 1.5 eq.) was added. The system was stirred at room temperature under nitrogen protection for 12 h. The reaction was monitored by TLC and LC-MS. After the reaction was complete, triethylamine was added until the system was neutral. The mixture was then extracted successively with water, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1 to 1:2) to give a white solid compound 105 (19 g, two-step yield: 60%). MS (ESI): m / z calcd for C19H31NO9[M+H]+: 418.20, found: 418.21

[0261] Compound 105 (58 g, 139.568 mmol) was dissolved in 300 mL of dry NH3 / MeOH solution. The system was stirred at room temperature for 12 h under nitrogen protection, and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was directly concentrated under reduced pressure to obtain the crude product. The crude product was not separated and purified and was directly added to the subsequent reaction. MS (ESI): m / z calcd for C13H25NO6[M+H]+: 292.17, found: 292.19.

[0262] Compound 106 (40.6 g, 139.57 mmol) was dissolved in dry pyridine (800 mL), and TBDPSCl (47 mL, 181.44 mmol, 2 eq.) and 4-dimethylaminopyridine (5.12 g, 41.87 mmol, 0.3 eq.) were added. The system was stirred at room temperature for 12 h under nitrogen protection, and the reaction was monitored by TLC and LC-MS. After the reaction was complete, subsequent reactions were carried out directly without post-treatment. MS (ESI): m / z calcd for C29H43NO6Si[M+H]+: 530.29, found: 530.26.

[0263] The reaction system of compound 107 was placed at 0 °C, and benzoyl chloride (48 mL, 418.8 mmol, 2 eq.) was added dropwise, followed by the addition of 4-dimethylaminopyridine (6.8 g, 55.8 mmol, 0.4 eq.). The system was stirred at room temperature for 12 h under nitrogen protection, and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was diluted with ethyl acetate and extracted successively with water, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (petroleum ether:ethyl acetate = 4:1 to 2:1) to obtain a white solid compound 108, which was directly used for the next step. MS (ESI): m / z calcd for C43H51NO8Si[M+H]+: 738.34, found: 738.33.

[0264] Compound 108 (103 g, 139.57 mmol) was dissolved in anhydrous tetrahydrofuran (800 mL), and triethylamine trihydrofluoric acid (114 mL, 697.85 mmol, 5.0 eq.) was added. The reaction system was stirred at 50 °C under nitrogen protection, and the reaction was monitored by TLC and LC-MS. After about 12 h at room temperature, the reaction was complete. The reaction system was directly concentrated under reduced pressure to remove most of the solvent, diluted with ethyl acetate, and extracted successively with water, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1 to 1:1) to give a white solid compound 109 (147 g, yield from compound 105 onwards: 65%). MS (ESI): m / z calcd for C27H33NO8[M+H]+: 500.22, found: 500.23.

[0265] Compound 109 (10 g, 20.031 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL). The system was placed at 0 °C, and PPh3 (10.5 g, 40.062 mmol, 2 eq.) and DPPA (8.6 mL, 40.062 mmol, 2 eq.) were added. DIAD (7.9 mL, 40.062 mmol, 2 eq.) was added dropwise. The system was stirred at 0 °C under nitrogen protection for 4 h, and then the system was placed at room temperature for about 12 h. The reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was directly concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1 to 3:1) to give a white solid compound 57 (4.7 g, 44%). 1H NMR(400MHz,DMSO-d6)δ8.01–7.91(m,3H),7.75–7.68(m,3H),7.63–7.53(m,3H) ),7.40(t,J=7.8Hz,2H),5.62(d,J=3.4Hz,1H),5.34(dd,J=11.1,3.4Hz,1H),4 .77(d,J=8.5Hz,1H),4.26–4.15(m,2H),3.60–3.49(m,2H),3.32(d,J=3.7Hz,1 H),1.70(s,3H),1.61–1.42(m,4H),0.87(dt,J=17.7,7.4Hz,6H).MS(ESI):m / z calcd for C27H32N4O7[M+H]+:525.23,found:525.24.

[0266] 2. Synthesis of compound (BT-021)

[0267] Compound 101 (50 g, 0.55 mol, 1.0 eq.) was weighed and dissolved in 500 mL of acetonitrile. After purging with nitrogen, 167 g of triethylamine (1.65 mol, 3.0 eq.) and 160 g of ethyl trifluoroacetate (1.1 mol, 2.0 eq.) were added, and the mixture was reacted overnight at room temperature. After the reaction was completed by TLC monitoring, the solvent was removed by direct concentration under reduced pressure to obtain an oily crude product.

[0268] The crude oily product was dissolved in 400 mL of pyridine, purged with nitrogen, and cooled to 0 °C in an ice-water bath. 186 g (0.55 mol, 1.0 eq.) of 4,4'-bismethoxytriphenylmethyl chloride was added in portions. After the addition was complete, the mixture was allowed to return to room temperature and reacted overnight. The reaction was quenched with water after TLC monitoring. The mixture was extracted twice with ethyl acetate, and the organic phases were combined, dried, concentrated, and purified by column chromatography to give 102,233 g of the compound. The two-step yield was 86.9%.

[0269] Compound 102 (233 g, 0.47 mol, 1.0 eq.) was dissolved in 1.5 L of methanol, and 53.4 g (0.95 mol, 2.0 eq.) of potassium hydroxide (prepared as a 3.0 mol / L aqueous solution) was added. The reaction was carried out at room temperature for 2 h. After the reaction was completed by TLC monitoring, the solvent was removed by concentration under reduced pressure, followed by rapid column chromatography to obtain compound 103, 174 g, yield: 92.8%. 1H NMR (400MHz, DMSO-d6) δ7.40(d,J=7.5Hz,2H),7.33–7.18(m,7H),6.88(d,J=8.5Hz,4H),4.75(s,1H),3.73(s,6H),3.56(p,J=6.1Hz,1H),2.9 4(dd,J=9.1,5.4Hz,1H),2.83(dd,J=9.0,6.1Hz,1H),2.69(dd,J=12.7,4.0Hz,1H),2.49–2.42(m,1H),1.97(d,J=81.8Hz,1.7H).MS(ESI):m / z calcd for C24H27NO4[MH]-:392.19,found:392.16.

[0270] Compound 104 (5.0 g, 43.5 mmol, 1.0 eq.) was weighed and dissolved in 50 mL of acetonitrile. After purging with nitrogen, triethylamine (15.0 g, 130.5 mmol, 3.0 eq.) and ethyl trifluoroacetate (14.1 g, 87.0 mmol, 2.0 eq.) were added, and the mixture was reacted overnight at room temperature. After the reaction was completed, the product was concentrated under reduced pressure by TLC to obtain a crude oil product (100% yield was directly used for the next reaction).

[0271] The crude oily product and compound 103 (17.1 g, 43.5 mmol, 1.0 eq.) were dissolved in 100 mL of N,N-dimethylformamide. Under nitrogen protection, N,N-diisopropylethylamine (11.2 g, 87.0 mmol, 2.0 eq.) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) (19.8 g, 52.2 mmol, 1.2 eq.) were added, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by TLC, the reaction was quenched with water, extracted with ethyl acetate, and the organic phase was washed with saturated brine. The mixture was separated, dried, concentrated, and purified by column chromatography to give compound 110, 17.5 g. The two-step yield was 68.2%.

[0272] Compound 110 (17.5 g, 29.8 mmol, 1.0 eq.) was dissolved in 200 mL of methanol, and 3.4 g (59.7 mmol, 2.0 eq.) of potassium hydroxide (prepared as a 3.0 mol / L aqueous solution) was added. The reaction was carried out at room temperature for 2 h. After the reaction was completed by TLC monitoring, the solvent was removed by concentration under reduced pressure, followed by rapid column purification to obtain compound 111, 13.8 g, yield: 95.1%.

[0273] Compound 111 (1.7 g, 3.5 mmol, 1.0 eq.) and acetylacetic acid (0.4 g, 4.2 mmol, 1.2 eq.) were weighed and dissolved in 10 mL of N,N-dimethylformamide. Under nitrogen protection, 0.9 g (7.0 mmol, 2.0 eq.) of N,N-diisopropylethylamine and 1.6 g (4.2 mmol, 1.2 eq.) of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate were added, and the reaction was carried out at room temperature for 2 h. After the reaction was monitored by TLC, the reaction was quenched with water, extracted with ethyl acetate, and the organic phase was washed with saturated brine. The mixture was separated, dried, concentrated, and purified by reverse-phase preparative column chromatography (5%-95% acetonitrile / water, aqueous phase: 0.01% ammonium bicarbonate) to give compound 113, 1.2 g of white solid, yield: 60.9%.

[0274] Compound 113 (570 mg, 1.0 mmol, 1.0 eq.) and compound 57 (524 mg, 1.0 mmol, 1.0 eq.) were weighed and dissolved in 10 mL of methanol. Copper sulfate pentahydrate (500 mg, 2.0 mmol, 2.0 eq.) and sodium ascorbate (400 mg, 2.0 mmol, 2.0 eq.) were dissolved in 10 mL of water and added to the system. The reaction was monitored by TLC until completion. After concentration, the mixture was purified by reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase 0.01% ammonium bicarbonate) to obtain compound 114560 mg, yield: 51.2%.

[0275] Compound 114 (560 g, 0.51 mmol, 1.0 eq.) and 4-dimethylaminopyridine (12 mg, 0.1 mmol, 0.2 eq.) were weighed into a 100 mL single-necked flask. After nitrogen purging protection, 10 mL of anhydrous dichloromethane and 132 mg (1.02 mmol, 2.0 eq.) of N,N-diisopropylethylamine were added dropwise using a syringe. Then, 181 mg (0.77 mmol, 1.5 eq.) of 2-cyanoethyl N,N-diisopropylchlorophosphine (CEP-Cl) was added dropwise. After the addition was complete, the reaction was allowed to proceed at room temperature for 1 h. The reaction was monitored by TLC until it was complete. After removing dichloromethane by concentration under reduced pressure at room temperature, the compound (BT-021) was purified by reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase: 0.01% ammonium bicarbonate) to obtain 430 mg of white solid, yield: 65.2%. NMR(400MHz,DMSO-d6)δ7.98–7.91(m,3H),7.91–7.80(m,1H),7.78–7.63(m,4H),7 .59(t,J=7.5Hz,3H),7.46–7.36(m,4H),7.36–7.16(m,7H),6.93–6.82(m,4H),5.6 6(d,J=3.5Hz,1H),5.34(dt,J=11.3,3.7Hz,1H),4.59(dt,J=9.8,6.3Hz,2H),4.52 –4.41(m,2H),4.22(tq,J=16.2,7.4,6.9Hz,2H),4.05(dt,J=10.3,5.2Hz,1H),3.8 3(dq,J=12.9,6.3Hz,1H),3.73(d,J=2.4Hz,7H),3.57(ddt,J=23.6,10.2,6.3Hz,2 H),3.38(s,1H),3.27(dt,J=16.0,5.8Hz,3H),3.09–2.98(m,1H),2.92–2.68(m,4H ),2.68–2.51(m,2H),2.07(s,3H),1.90–1.77(m,1H),1.68(d,J=1.7Hz,5H),1.48– 1.22(m,5H),1.21–1.10(m,8H),1.03(dd,J=6.8,4.4Hz,3H),0.83–0.62(m,6H).31P NMR(162MHz, DMSO)δ148.36,148.32,148.22,148.16,148.05,147.87.MS(ESI):m / zcalcd for C70H86N8O14P[MH]-:1293.60,found:1293.63.

[0276] 3. Loading a solid-phase synthesis support

[0277] Compound 114 (420 mg, 0.35 mmol) and 4-dimethylaminopyridine (8.5 mg, 0.07 mmol) were added to a 100 mL single-necked flask. After nitrogen purging protection, 30 mL of anhydrous dichloromethane and succinic anhydride (70 mg, 0.7 mmol) were added, and the reaction was allowed to proceed overnight. The reaction was monitored by TLC until completion. After removing dichloromethane by concentration under reduced pressure at room temperature, the mixture was purified by reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase 0.01% ammonium bicarbonate) to obtain 350 mg of compound (BT-021)a.

[0278] Compound (BT-021)a was coupled with an amino support (polystyrene resin (PS) or CPG powder) through a coupling reaction and a capping reaction to prepare a pre-loaded (BT-021) solid-phase synthesis support. After the support loading was determined by analysis, it was used as a support for subsequent solid-phase synthesis to synthesize oligonucleotides with 3'-terminal conjugated ligands.

[0279] Example 4: Synthesis of GalNAc-RNAi reagent

[0280] 1. Following the process steps shown below, L96 (GalNAc3) and an amino carrier (polystyrene resin or CPG powder) were coupled and capped to prepare a solid-phase synthetic carrier preloaded with L96. The carrier loading was measured by analysis.

[0281] 2. Load a commercially available general-purpose solid-phase support (Tianjin Nankai Hecheng Technology Co., Ltd.) or the above-mentioned solid-phase synthesis support into a solid-phase reaction column. In a nucleic acid synthesizer, following the standard oligonucleotide solid-phase synthesis method, using modified nucleotide monomers, apply the dedimethoxytriphenylmethylation reaction, coupling reaction, oxidation reaction, and capping reaction as described in Example 1. By cyclically repeating these four steps, an oligonucleotide sequence (modified SS chain) of the L96, (BT-021), and / or (BT-057) monomer combination is synthesized. If the target product has a thiophosphate modification, a thiolation reaction is used instead of an oxidation reaction; that is, the oxidation reaction in Example 1 is replaced by the following thiolation reaction: under the action of the thiolation reagent PADS / ADTT, the triphosphite generated in the previous coupling condensation step is converted into a stable triphosphite. After synthesizing the desired sequence, the cyanoethyl protecting group is removed with 20% diethylamine, and the solid-phase support is dried by argon gas. The support is then deprotected by ammonolysis in concentrated ammonia at 55°C for 5 to 16 hours. After ammonia desorption and protection are completed, the carrier is removed by filtration; the ammonia hydrolysate is concentrated to remove ammonia water, and the remaining concentrated sample is sent for LC-MS analysis to confirm that the sample molecular weight is consistent with the theoretical molecular weight.

[0282] 3. Oligonucleotide conjugate samples coupled with L96 or (BT-021), (BT-057) monomer combinations were purified by ion exchange on an AKTAPure 150, followed by gel column desalting to obtain single-stranded samples that met the requirements.

[0283] 4. Using the general-purpose solid-phase synthesis support Nitto Phase HLUny Linker, antisense chains (modified AS chains) were synthesized according to the standard solid-phase synthesis method.

[0284] 5. Add the modified SS chain and the modified AS chain to the annealing container in an equimolar ratio, heat to 55°C, hold for about 30 minutes, and then allow to cool naturally to room temperature.

[0285] 6. The annealed double-stranded samples were dispensed into freeze-drying containers according to the required amount and then freeze-dried to obtain GalNAc-siRNA conjugates.

[0286] Example 5: In vitro screening of siRNA duplexes (HepG2.2.15)

[0287] This embodiment uses the HepG2.2.15 cell line containing the D-type HBV genome for in vitro evaluation of RNAi reagents. On day 0, 22,500 cells / well were seeded into 96-well plates, and cells were transfected with different concentrations of RNAi reagent using RNAiMax. On day 3, the culture medium was replaced with fresh medium free of the compound. On day 6, cells were collected, and intracellular HBV RNA was detected by RT-qPCR. GAPDH gene RNA was also detected as an internal control. The positive control (PC) was AD-66810 from CN 110913898A. Both the test siRNA and the control were measured at a single concentration of 0.1 nM in triplicate. A six-well phosphate buffer solution was used as a negative control. siRNAs showing superior HBV RNA inhibition rates compared to the positive control were selected.

[0288] Table 5 shows the inhibition rates of various HBV RNAi reagents on HBV RNA expression at single concentrations. The results indicate that the following reagents showed the highest inhibition rates: BPR301328101, BPR301100301, BPR301328001, BPR301320001, BPR301319601, BPR301400601, BPR301400701, BPR301304801, BPR301400501, BPR301200301, BPR301302401, BPR301299301, BPR301299201, BPR301299101, BPR301400401, and BPR301400301. 1. BPR301300201, BPR301200201, BPR301297301, BPR301296901, BPR301296501, BPR301296301, BPR301296201, BPR301293601, BPR301292001, BPR301100201, BPR301100101, BPR301400101, BPR301290201, BPR301200308, and BPR301400408 all showed better HBV RNA inhibition rates in HepG2.2.15 than the positive reference.

[0289] Table 5. Inhibition rates of HBV RNAi reagents on HBV RNA expression at single concentrations.

[0290] Example 6: Screening of siRNA duplexes in a representative Chinese HBV in vitro screening system

[0291] The specific experimental methods and reagents used in this embodiment are as follows: On day 0, plasmid DNA constructed based on B2 HBV (SEQ ID NO:2) and C2 HBV (SEQ ID NO:3) was transfected into HepG2 cells, and the transfected cells were seeded into 96-well plates at a density of 22,500 cells per well. On day 1, the test compound was transfected into the cells using RNAiMax. The control compound vir-2218 (a compound disclosed in US11492623B2) was initially concentrated at 10 nM, and then diluted to 1 nM, 0.5 nM, and 0.1 nM. PBS was used as the negative control, and each concentration was tested in triplicate. Cells were cultured at 5% CO2 and 37°C for 3 days, and then the medium was replaced with fresh medium without the compound and cultured for another 3 days. On day 6, the cell supernatant was collected to measure HBsAg, and RNA was extracted from the cells. RT-qPCR was used to detect the target gene mRNA in the samples. GAPDH, used as an internal reference gene, was also detected. Intracellular RNA was extracted using the RNeasy kit (QIAGEN-74182) according to the instructions, and then reverse transcribed into cDNA using the FastKing RT Kit (containing gDNase) (TIANGEN-KR116). HBV mRNA expression was then measured using real-time quantitative PCR. The relative expression level of the target gene was determined using a 2-1 -ΔΔCT The calculation formula is as follows: ΔCT = Average CT value of target gene - Average CT value of internal reference gene; ΔCT = ΔCT (drug-treated group) - ΔCT (control group); Relative mRNA expression level = 2 -ΔΔCT

[0292] Data analysis: HBV RNA inhibition rate (%) = (1 - HBV RNA level in the test sample / HBV RNA level in the control PBS) × 100% HBV.

[0293] Surface antigens were detected using an ELISA method, following the instructions of the Antu Bio kit (catalog number: Antu Bio-CL 0310). The method is briefly described below: Equilibrate the kit to room temperature for 1 hour, add 50 μl of sample to each well, then add 50 μl of enzyme conjugate and incubate at 37°C for 60 minutes. Wash the plate 6 times, add 50 μl of luminescent substrate, and incubate at room temperature in the dark for 10 minutes. Finally, measure the luminescence intensity. Unless otherwise specified in this article, these reagents can be obtained from any molecular biology reagent supplier under their trade names.

[0294] Table 6 shows the inhibition results of various HBV RNAi reagents on the expression of representative Chinese HBV-related HBsAg and HBV RNA at multiple concentrations. The results indicate that BPR301400401, BPR301400408, BPR301200301, and BPR301200308, at high, medium, and low concentrations, showed better or comparable inhibition rates of HBV RNA and HBsAg in the in vitro screening system for representative Chinese HBV compared to the positive reference.

[0295] Table 6. Inhibition results of various HBV RNAi reagents on the expression of representative HBV-related HBsAg and HBV RNA in Chinese individuals at multiple concentrations.

[0296] Example 7: In vivo evaluation of siRNA double strands (Tg-mice)

[0297] This embodiment uses a transgenic HBV mouse model to evaluate the in vivo efficacy of the HBV siRNA reagent. The transgenic mouse strain was C57B / 6N-Tg(1.28HBV) / Vst, purchased from Beijing Vitonda Biotechnology Co., Ltd. All mice were grouped according to their serum HBsAg levels and body weight before grouping. Mice with lower HBsAg levels and lighter body weight were removed from the experiment. The selected mice were evenly distributed into each group, ensuring that there were no statistically significant differences in serum HBsAg levels and body weight (P>0.5). Each group consisted of 4 males, and the dosage was 3 mg / kg, administered subcutaneously in physiological saline. A negative control group (NC) and a positive control group (PC) were set up. The positive control was AD-66810 from CN 110913898 A. The day of the first administration was designated as day 0, the day before administration was day -1, the day after administration was day 1, and so on. On day -7, serum was collected from all mice via the orbital venous plexus. The collected blood samples were incubated at room temperature for 30 minutes, centrifuged at 5000 rpm for 10 minutes, and the supernatant was used for HBV modeling detection. On day 0, animals selected based on the day -7 results were grouped. Blood samples were collected on days 7, 14, 21, 28, 35, and 42 after drug administration. HBsAg levels were measured by ELISA. The effect of siRNA duplexes on expression is summarized in Figure 1, where HBsAg detection was terminated on day 28 after administration of compounds BPR301400461, BPR301400463, BPR301400464, and BPR301400466. The difference in the logarithm of the relative expression values ​​before and after drug administration was used to describe the extent of downregulation of HBV expression components. The downregulation results of conjugates on HBsAg are shown in Table 7. Except for the BPR301200355 sample, whose HBsAg knockdown endpoint data was lower than that of the positive control, the maximum knockdown and detection endpoint data of HBsAg in other samples were better than those of the positive control.

[0298] Table 7 shows the downregulation results of the conjugates on HBsAg.

[0299] Example 8: In vivo evaluation of siRNA double strands (AAV mice)

[0300] This embodiment uses an AAV-HBV mouse model to evaluate the in vivo potency of the HBV siRNA reagent. Using 1x10 11The vgrAAV8-1.3HBV (type D) virus model was established by infecting 5-week-old male C57BL / 6 mice with the virus via tail vein injection. Each group consisted of 3-5 animals, with a positive control group (PC) and a negative control group (NC). The positive control was consistent with the previous example. The siRNA duplexes used for evaluation included: BPR301400463, BPR301400471, and BPR301400482. The day of the first administration was designated as day 0, the day before administration as day-1, the day after administration as day 1, and so on. On day-2 (27 days post-injection), serum was collected from all mice injected with the rAAV8-1.3HBV virus via the submandibular vein. Blood samples were collected and left at room temperature for 30 minutes, then centrifuged at 5000 rpm for 10 minutes. The supernatant was used to detect HBV DNA and HBsAg levels. Grouping and baseline determination were performed based on serum HBV DNA, HBsAg levels, and body weight. Mice with abnormal parameters were removed to ensure that there were no statistically significant differences in HBV DNA, HBsAg levels, and body weight among the groups (P>0.05). The dosage was 3 mg / kg, and the administration route was a single subcutaneous dose. The solvent and negative control were physiological saline. The blood collection time points and detection indicators after administration are shown in Figures 2, 3, and 4.

[0301] The expression values ​​of the BPR301400463, BPR301400471, and BPR301400482 siRNA duplex compounds used in the evaluation were log10-logarithmic changes and normalized relative to the untreated values. After a single dose, HBsAg expression was downregulated by >1.1 Log10 (IU / mL) for 42 days; HBV DNA expression was downregulated by >1.2 Log10 (IU / mL) for 28 days; and HBeAg expression was downregulated by >0.6 Log10 (IU / mL) for 28 days. The specific inhibitory effects on HBV DNA, HBsAg, and HBeAg are shown in Figures 2, 3, and 4.

[0302] The specific embodiments of this disclosure have been described in detail above. However, this disclosure is not limited to the specific details in the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0303] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0304] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

[0305] Sequence information:

Claims

1. An oligonucleotide for inhibiting hepatitis B virus gene expression, comprising a sense strand and / or an antisense strand, wherein the sense strand and the antisense strand are at least partially anticomplementary to form a duplex; the sense strand comprises 19 nucleotides, and the antisense strand comprises 19 nucleotides; wherein, At least 85% of the nucleotides in the sense strand are anticomplementary to the antisense strand to form a double strand, and at least 76% of the nucleotides in the antisense strand are anticomplementary to hepatitis B virus mRNA.

2. The oligonucleotide according to claim 1, wherein, The sense strand consists of 19-30 nucleotides, preferably 19-25 nucleotides, and more preferably 19-23 nucleotides; the antisense strand consists of 19-30 nucleotides, preferably 19-27 nucleotides, and more preferably 21-23 nucleotides. Preferably, the sense strand consists of 19 nucleotides and the antisense strand consists of 21 nucleotides.

3. The oligonucleotide according to claim 1 or 2, wherein, The nucleotides in the sense strand and / or the antisense strand are each independently selected from at least one group consisting of ribonucleotides containing guanine as a base, ribonucleotides containing adenine as a base, ribonucleotides containing cytosine as a base, ribonucleotides containing uracil as a base, and deoxyribonucleotides containing thymine as a base. Preferably, positions 1 to 19 of the justice chain and positions 1 to 19 of the antisense chain are oppositely complementary, forming a double chain; and / or The 20th and 21st positions of the antisense strand are each independently selected from at least one group consisting of ribonucleotides containing guanine as a base, ribonucleotides containing adenine as a base, ribonucleotides containing cytosine as a base, ribonucleotides containing uracil as a base, and deoxyribonucleotides containing thymine as a base.

4. The oligonucleotide according to any one of claims 1-3, wherein, The justice chain, the antisense chain, and the bichain are shown in Table 1.

5. The oligonucleotide according to any one of claims 1-4, wherein, The antisense strand comprises, is composed of, or is substantially composed of a sequence differing from any of the following sequences by 0, 1, 2, 3, or 4 nucleotides: 5'-UUGUAAGUUGGCGAGAAAGGG-3'(SEQ ID NO:25) 5'-AUACUUUCCAAUCAAUAGGGG-3'(SEQ ID NO:33) 5'-UAUACAUGCAUAUAAAGGCUU-3' (SEQ ID NO: 27).

6. The oligonucleotide according to any one of claims 1-5, wherein, The positive chain comprises, consists of, or is substantially composed of sequences differing from any of the following sequences by 0, 1, 2, or 3 nucleotides: composition: 5'-CUUUCUCGCCAACUUACAA-3'(SEQ ID NO:74) 5'-CCUAUUGAUUGGAAAGUAU-3'(SEQ ID NO:82) 5'-GCCUUUAUAUGCAUGUAUA-3' (SEQ ID NO:76).

7. The oligonucleotide according to any one of claims 1-6, wherein, The oligonucleotide comprises or is any one of the duplexes BPR3012003, BPR3014004, and BPR3013024: BPR3012003: Antonym: 5'-UUGUAAGUUGGCGAGAAAGGG-3' (SEQ ID NO:25) Chain of Justice: 5'-CUUUCUCGCCAACUUACAA-3'(SEQ ID NO:74) BPR3014004: Antonym: 5'-AUACUUUCCAAUCAAUAGGGG-3' (SEQ ID NO:33) Chain of Justice: 5'-CCUAUUGAUUGGAAAGUAU-3'(SEQ ID NO:82) BPR3013024: Antonym: 5'-UAUACAUGCAUAUAAAGGCUU-3' (SEQ ID NO:27) Justice Chain: 5'-GCCUUUAUAUGCAUGUAUA-3'(SEQ ID NO:76).

8. The oligonucleotide according to any one of claims 1-7, wherein, The oligonucleotide is a short interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), or a dicer substrate; the oligonucleotide can also be a single-stranded oligonucleotide, which is an antisense oligonucleotide (ASO).

9. The oligonucleotide according to any one of claims 1-8, wherein, Each nucleotide in the sense strand and / or the antisense strand is independently a modified nucleotide or an unmodified nucleotide; Preferably, in the modification of the nucleotide, the modified nucleotide is selected from nucleotides modified with 2'-methoxy (2'-O-methyl, 2-OMe), nucleotides modified with 2'-methoxyethyl (2'-O-MOE), nucleotides modified with 2'-deoxy-2'-fluoro (2'-F), nucleotides modified with 2'-arabino-fluoro (2'-Ara-F), nucleotides with 2'-deoxyribonucleotides, nucleotides modified with 2'-amino, nucleotides modified with 2'-alkyl, locked nucleic acids (LNA), 2',3'-unlocked nucleic acid (UNA), and glycerol nucleic acids. At least one of the following groups: acid (GNA), L-2'-O-methyl modified nucleotide, L-2'-deoxy-2'-fluororibonucleotide, L-2'-deoxyribonucleotide, threonine nucleic acid (TNA), 4'-modified threonine nucleic acid, reverse nucleotide, reverse 2'-O-methyl modified nucleotide, reverse 2'-deoxyribonucleotide, reverse abase-free nucleotide, 5'-phosphorothiocate (PS) modified nucleotide, and 5'-vinylphosphonate modified ribonucleotide (5'-(E)-vinylphosphonate (VP).

10. The oligonucleotide according to any one of claims 1-9, wherein, The modified nucleotide is selected from at least one of the following modification sequences: 2'-methoxy modified nucleotide, 2'-deoxy-2'-fluoro modified nucleotide, 5'-thiophosphate modified nucleotide, 2'-deoxyribonucleotide, and L-2'-deoxy-2'-fluororibonucleotide. Preferably, the modified nucleotide is selected from at least one of the modification sequences consisting of 2'-methoxy modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, and 5'-thiophosphate modified nucleotides.

11. The oligonucleotide according to any one of claims 1-10, wherein, The 5th, 7th, 8th, and 9th positions of the positive strand are nucleotides modified with 2'-deoxy-2'-fluoro, and the remaining nucleotides are nucleotides modified with 2'-methoxy.

12. The oligonucleotide according to any one of claims 1-11, wherein, The antisense strand has nucleotides modified with 2'-deoxy-2'-fluoro at positions 2, 6, 8, 9, 14, and 16, and the remaining nucleotides are nucleotides modified with 2'-methoxy. Preferably, the 2nd, 6th, 14th, and 16th positions of the antisense strand are nucleotides modified with 2'-deoxy-2'-fluoro, and the remaining nucleotides are nucleotides modified with 2'-methoxy. Preferably, the 6th, 14th, and 16th positions of the antisense strand are nucleotides modified with 2'-deoxy-2'-fluoro, the 2nd position is modified with 2'-deoxyribonucleotide, and the remaining nucleotides are nucleotides modified with 2'-methoxy. Preferably, the 2nd, 6th, 14th, and 16th positions of the antisense strand are nucleotides modified with 2'-deoxy-2'-fluoro, the 9th position is modified with 2'-deoxyribonucleotide, and the remaining nucleotides are nucleotides modified with 2'-methoxy. Preferably, the first position of the antisense strand is a ribonucleotide modified with 5'-vinylphosphonate-2'-methoxy, the second, sixth, 14th, and 16th positions are nucleotides modified with 2'-deoxy-2'-fluoro, and the remaining nucleotides are nucleotides modified with 2'-methoxy. Preferably, the 2nd, 14th, and 16th positions of the antisense strand are nucleotides modified with 2'-deoxy-2'-fluoro, the 5th and 7th positions are modified with 2'-deoxyribonucleotides, and the remaining nucleotides are nucleotides modified with 2'-methoxy.

13. The oligonucleotide according to any one of claims 1-12, wherein, The sense strand and / or the antisense strand are connected by at least two nucleotides at their 5' ends and / or at least two nucleotides at their 3' ends via phosphate thioester groups; In the sense strand and the antisense strand, each nucleotide is independently modified; the oligonucleotide is obtained by independently modifying each nucleotide in the sense strand and the antisense strand shown in Table 1.

14. The oligonucleotide according to any one of claims 1-13, wherein, The justice chain, the antisense chain, and the bichain are shown in Table 2.

15. The oligonucleotide according to any one of claims 1-14, wherein, The antisense strand comprises, is composed of, or is substantially composed of a sequence differing from any of the following sequences by 0, 1, 2, 3, or 4 nucleotides, wherein the 5' and 3' ends of the sequence each contain two phosphate thioesters: 5'-AmUfAmCmdTUmdTCmCmAmAmUmCmAfAmUfAmGmGmGmGm-3' (SEQ ID NO: 196) 5'-AmUfAmCmUmUfUmCfCfAmAmUmCmAfAmUfAmGmGmGmGm-3'(SEQ ID NO:194) 5'-AmUfAmCmUmUfUmCmCmAmAmUmCmAfAmUfAmGmGmGmGm-3'(SEQ ID NO:195) 5'-UmUfGmUmAmAfGmUfUfGmGmCmGmAfGmAfAmAmGmGmGm-3'(SEQ ID NO:202) 5'-UmUfGmUmAmAfGmUmUmGmGmCmGmAfGmAfAmAmGmGmGm-3'(SEQ ID NO:203) 5'-UmUfGmUmdAAmdGUmUmGmGmCmGmAfGmAfAmAmGmGmGm-3'(SEQ ID NO:204) 5'-UmAfUmAmCmAfUmGfCfAmUmAmUmAfAmAfGmGmCmUmUm-3'(SEQ ID NO:206) 5'-UmAfUmAmCmAfUmGmCmAmUmAmUmAfAmAfGmGmCmUmUm-3'(SEQ ID NO:207) 5'-UmAfUmAmdCAmdTGmCmAmUmAmUmAfAmAfGmGmCmUmUm-3'(SEQ ID NO:208) 5'-AmdTAmCmUmUfUmCmCmAmAmUmCmAfAmUfAmGmGmGmGm-3'(SEQ ID NO:247) 5'-AmUfAmCmUmUfUmCmdCAmAmUmCmAfAmUfAmGmGmGmGm-3'(SEQ ID NO:248) 5'-VPAmUfAmCmUmUfUmCmCmAmAmUmCmAfAmUfAmGmGmGmGm-3'(SEQ ID NO:249) 5'-UmdTGmUmAmAfGmUmUmGmGmCmGmAfGmAfAmAmGmGmGm-3'(SEQ ID NO:250) 5'-UmUfGmUmAmAfGmUmdTGmGmCmGmAfGmAfAmAmGmGmGm-3'(SEQ ID NO:251) 5'-VPUmUfGmUmAmAfGmUmUmGmGmCmGmAfGmAfAmAmGmGmGm-3'(SEQ ID NO:252) 5'-UmAfUmAmCmAfUmGmdCAmUmAmUmAfAmAfGmGmCmUmUm-3'(SEQ ID NO:253) 5'-VPUmAfUmAmCmAfUmGmCmAmUmAmUmAfAmAfGmGmCmUmUm-3' (SEQ ID NO: 254); In this context, the uppercase letters "G", "C", "A", and "U" represent ribonucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively; the lowercase letter "m" indicates that the nucleotide adjacent to the left of the letter m is 2'-methoxy modified; the lowercase letter "f" indicates that the nucleotide adjacent to the left of the letter f is 2'-deoxy-2'-fluoro modified; VP represents a ribonucleotide with 5'-vinylphosphonate modified on the right; and the lowercase letter "d" indicates that the nucleotide adjacent to the right of the letter d is 2'-deoxyribonucleotide modified, where dT represents thymine deoxyribonucleotide.

16. The oligonucleotide according to any one of claims 1-15, wherein, The positive strand comprises, is composed of, or is substantially composed of a sequence differing from any of the following sequences by 0, 1, 2, or 3 nucleotides, wherein the 5' end and / or 3' end of the sequence each contain 1 or 2 phosphate thioesters: 5'-CmCmUmAmUfUmGfAfUfUmGmGmAmAmAmGmUmAmUm-3' (SEQ ID NO: 193) 5'-CmUmUmUmCfUmCfGfCfCmAmAmCmUmUmAmCmAmAm-3' (SEQ ID NO: 201) 5'-GmCmCmUmUfUmAfUfAfUmGmCmAmUmGmUmAmUmAm-3' (SEQ ID NO: 205); In this context, the uppercase letters "G", "C", "A" and "U" represent ribonucleotides containing guanine, cytosine, adenine and uracil as bases, respectively; the lowercase letter "m" indicates that the nucleotide adjacent to the left of the letter m is a nucleotide modified with 2'-methoxy; and the lowercase letter "f" indicates that the nucleotide adjacent to the left of the letter f is a nucleotide modified with 2'-deoxy-2'-fluoro.

17. The oligonucleotide according to any one of claims 1-16, wherein, The oligonucleotides comprise or are duplexes BPR301400401, BPR301400403, BPR301400404, BPR301401404, BPR301200301, BPR301200303, BPR301200304, BPR301302402, BPR301302403, BPR301302404, BRP301400421, BRP301 Any one of 400422, BRP301400423, BPR301200321, BPR301200322, BPR301200323, BPR301302421, BPR301302422, and BPR301302423, wherein the antisense sequence comprises one or two thiophosphates at its 5' end and / or 3' end, and the sense sequence comprises two thiophosphates at its 5' end and 3' end.

18. The oligonucleotide according to any one of claims 1-17, wherein, The oligonucleotides are delivered to hepatocytes and / or the subject via a targeting ligand, lipid nanoparticles (LNPs), or lipid nanoparticle analogs, wherein the hepatocytes are in vivo or in vitro cells. Preferably, the oligonucleotide is conjugated to a targeting ligand compound, wherein the ligand compound is a compound with affinity for the ASGPR protein; Preferably, the oligonucleotide is contained in nanolipid particles, and the nanolipids are selected from at least one group consisting of cationic lipid formulations, phospholipid formulations, cholesterol formulations, and polyethylene glycol-lipid formulations.

19. A conjugate comprising an oligonucleotide as described in any one of claims 1-18, and a targeting ligand conjugated to the positive strand of the oligonucleotide.

20. The conjugate according to claim 19, wherein, The targeting ligand is conjugated to the 5' end and / or 3' end of the positive chain; Preferably, the targeting ligand is conjugated to the 3' end of the positive chain; or, the targeting ligand is conjugated to the 5' end of the positive chain; or, the targeting ligand is conjugated to both the 5' and 3' ends of the positive chain.

21. The conjugate according to claim 19 or 20, wherein, The targeting ligand is a galactosamide compound or its analogue with ASGPR protein affinity, preferably having the structure shown in formula (I) or formula (II), wherein R is a C1-C8 straight-chain or branched alkyl group. Indicates the connection position of covalent chemical bonds:

22. The conjugate according to any one of claims 19-21, comprising an oligonucleotide conjugate formed from the oligonucleotide and one or more targeting ligands, or a racemic, stereoisomer, isotopic label, or pharmaceutically acceptable salt thereof, wherein, The targeting ligand is selected from structures such as L96, (BT-021), and (BT-057); Preferably, one or more structures such as L96, (BT-021), and (BT-057) are included at the 3'-end, 5'-end, or mid-chain position of one or two oligonucleotide chains of the oligonucleotide conjugate; or a combination of structures such as L96, (BT-021), and (BT-057) are included. Preferably, the number of the structures is 1 to 6, more preferably 1 to 4; in The term indicates the location of the covalent chemical bond, and the structure is linked to an oligonucleotide or the above structure via a phosphate ester (PO) or a thiophosphate ester (PS).

23. The conjugate according to any one of claims 19-22, wherein, The conjugates are shown in Table 4; preferably, the conjugates comprise, for example, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:2 ... The double-stranded structure of NO:224; more preferably, the conjugate is BRP301400451, BPR301400454, BPR301400455, BPR301200354, BPR301200355, BPR301200356, BPR301400461, BPR301400463, BPR301400464, BPR301400466, BPR301400471 or BPR301400482.

24. A composition comprising an oligonucleotide as described in any one of claims 1-18 or a conjugate as described in any one of claims 19-23, and a pharmaceutically acceptable diluent, carrier, and / or excipient.

25. The composition according to claim 24, wherein, The diluent is selected from at least one of the following groups: physiological saline, buffer solution, dextran solution, water, and glycerol. Preferably, the diluent is a biological isotonic solution, such as physiological saline or phosphate buffer.

26. The composition according to claim 24 or 25, wherein, The composition may contain at least one additional therapeutic agent; Preferably, the therapeutic agent is selected from at least one of the following groups: hepatitis B virus vaccine, pegylated interferon α (PEG-IFNα), interferon α-2β, recombinant human interleukin-7, FXR agonist, Toll-like receptor 7 / 8 (TLR7 / 8) agonist, checkpoint inhibitor (e.g., PD-1 inhibitor, PD-L1 inhibitor, TIGIT inhibitor), nucleotide analogue (e.g., tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir, telbivudine, clavidine, etc.), polymerase inhibitor nucleotide, antisense oligonucleotide (ASO), hepatitis B virus neutralizing monoclonal antibody, core protein inhibitor, capsid assembly regulator, core protein allosteric regulator, NTCP inhibitor, and siRNA that inhibits hepatitis B virus gene expression.

27. The oligonucleotide as described in any one of claims 1-18, the conjugate as described in any one of claims 19-23, or the composition as described in any one of claims 24-26 as a medicine.

28. An oligonucleotide of any one of claims 1-18, a conjugate of any one of claims 19-23, or a composition of any one of claims 24-26 for the treatment and / or prevention of diseases, conditions, and / or symptoms mediated at least in part by hepatitis B virus gene expression.

29. Use of the oligonucleotide of any one of claims 1-18, the conjugate of any one of claims 19-23, or the composition of any one of claims 24-26 in the preparation of a medicament for the treatment and / or prevention of diseases, symptoms, and / or conditions mediated at least in part by hepatitis B virus gene expression.

30. The oligonucleotide, conjugate, or composition according to claim 27 or 28, or the use according to claim 29, wherein, The diseases mentioned include at least one of the following: chronic hepatitis B (CHB), acute hepatitis B, hepatitis B-related liver diseases (e.g., hepatitis B-related cirrhosis, hepatitis B-related liver cancer), co-infection with hepatitis D virus (HDV), co-infection with hepatitis C virus (HCV), and co-infection with human immunodeficiency virus (HIV).

31. The oligonucleotide, conjugate, or composition according to claim 27 or 28, or the use according to claim 29, wherein, The hepatitis B virus gene is type A HBV, type B HBV, type C HBV, type D HBV, type E HBV, type F HBV, type G HBV, type H HBV, type I HBV, type J HBV, or recombinant HBV; Preferably, the hepatitis B virus gene is type A HBV, type B HBV, type C HBV, or type D HBV.

32. Use of the oligonucleotide of any one of claims 1-18, the conjugate of any one of claims 19-23, or the composition of any one of claims 24-26 in the preparation of a medicament or formulation for inhibiting the expression of hepatitis B virus gene in cells in vivo or in vitro; Preferably, the cells are in the body of the subject; more preferably, the subject is a mammal, such as a human, mouse, or marmot. Preferably, the cells are human cancer cells transfected with hepatitis B virus plasmids, such as HepG2; Preferably, the cells are in vitro cells, and the oligonucleotide, the conjugate, or the composition is contacted with the in vitro cells via liposome-mediated transfection and / or specifically mediated endocytosis (e.g., anti-desialylglycoprotein receptor (ASGPR)-mediated endocytosis). Preferably, the expression of the hepatitis B virus gene is suppressed by at least 50%; more preferably, the expression of the hepatitis B virus gene is suppressed by at least 90%.

33. A method for treating and / or preventing diseases, symptoms and / or conditions mediated at least in part by hepatitis B virus gene expression, comprising administering to a subject in need a therapeutically effective amount of the oligonucleotide of any one of claims 1-18, the conjugate of any one of claims 19-23 or the composition of any one of claims 24-26; Preferably, the subject is a mammal, such as a human, mouse, or marmot; Preferably, the hepatitis B virus gene is type A HBV, type B HBV, type C HBV, type D HBV, type E HBV, type F HBV, type G HBV, type H HBV, type I HBV, or type J HBV, or recombinant HBV; more preferably, the hepatitis B virus gene is type A HBV, type B HBV, type C HBV, or type D HBV. Preferably, the disease includes at least one of the following: chronic hepatitis B (CHB), acute hepatitis B, hepatitis B-related liver disease (e.g., hepatitis B-related cirrhosis, hepatitis B-related liver cancer), co-infection with hepatitis D virus (HDV), co-infection with hepatitis C virus (HCV), and co-infection with human immunodeficiency virus (HIV). Preferably, the oligonucleotide, the conjugate, or the composition is administered to the subject via oral, enteric, mucosal, subcutaneous, parenteral, or other routes; the parenteral route includes subcutaneous injection or infusion, intravenous injection or infusion, and intramuscular injection or infusion; the other routes include nasal, vaginal, rectal, sublingual, or inhalation. Preferably, the concentration of the oligonucleotide, the conjugate, or the composition is 0.01 nM to 100 nM; Preferably, the dosage of the oligonucleotide, the conjugate, or the composition is about 1-300 mg / kg body weight; Preferably, the oligonucleotide, the conjugate, or the composition is applied once or more daily, weekly, bi-weekly, 3-weekly, monthly, 2-monthly, 3-monthly, 4-monthly, 5-monthly, 6-monthly, 7-monthly, 8-monthly, 9-monthly, 10-monthly, 11-monthly, or 12-monthly.

34. A kit comprising the oligonucleotide of any one of claims 1-18, the conjugate of any one of claims 19-23, or the composition of any one of claims 24-26; Optionally, the kit also includes instructions for use.

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