SiRNA for interfering hepatitis B virus and application thereof

Abstract

The present disclosure relates to siRNA for interfering with hepatitis B virus and uses thereof. Specifically, the invention relates to an oligonucleotide for inhibiting HBV or a pharmaceutically acceptable salt thereof, the oligonucleotide comprises an antisense strand, and the antisense strand has a sequence as shown in any one of SEQ ID NO: 251-345 or a fragment thereof, or a modification sequence of the sequence or the fragment thereof. The siRNA has a remarkable inhibition effect on HBsAg, HBV DNA and HBeAg generated by HBV DNA integrated by cccDNA and a host genome in vivo and in vitro, and the drug effect is lasting.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a small interfering RNA (siRNA) for inhibiting the expression of hepatitis B virus gene and its use, specifically comprising double-stranded modified or unmodified siRNA, and conjugates and salts thereof; the use is for preparing drugs for treating hepatitis B virus or chronic infection. Background Art

[0002] Although the use of hepatitis B virus (HBV) vaccines has effectively prevented the prevalence of HBV infection, HBV infection remains the primary cause of viral hepatitis and its complications, and is also a major public health challenge that must be faced. About 250 million people worldwide have chronic HBV infection. After several years of infection, about 15-40% of patients with chronic HBV infection have serious adverse outcomes, such as cirrhosis, liver failure, and hepatocellular carcinoma. Currently, nearly 1 million patients die from HBV-related complications each year (Nicolini LA, et al. Int. J. Environ. Res. Public Health 2019, 16, 3307). The basis of chronic hepatitis B infection is the persistent presence of covalently closed circular DNA (cccDNA) of hepatitis B virus in infected hepatocytes as a reserve and template for viral replication. At the same time, as a byproduct of viral replication, HBV DNA can also be randomly integrated into the host genome. Although the integrated HBV sequence cannot maintain viral replication, it can continue to produce viral proteins, such as hepatitis B surface antigen (HBsAg) and transcriptional regulatory HBV x protein (Suarez AAR, et al. Liver International. 2021; 41, Suppl. 1: 15-23). ​​At present, the treatment of hepatitis B is relatively limited, mainly including nucleoside analogs (NUCs) entecavir, tenofovir, etc., and polyethylene glycol-modified interferon α (Peg-IFN-α), but these treatments are usually difficult to eliminate the virus, and HBsAg disappearance is still rare. Therefore, the above treatment methods require long-term medication to maintain viral suppression and prevent viral recurrence after drug withdrawal.

[0003] The cost of lifelong treatment or intervention is a heavy economic burden for patients, so there is an urgent need to develop new treatment strategies to achieve "functional cure" for chronic hepatitis B infection (CHB), that is, the continued disappearance of HBsAg after drug withdrawal, HBV DNA below the detection limit, normal liver enzymes, and improvement of liver tissue pathology. Among them, the disappearance of HBsAg is an indication of deep suppression of HBV replication and the only indication for safe cessation of treatment. Therefore, CHB is an unmet clinical need that requires continuous efforts to develop new molecules, combination therapies, and completely innovative treatment strategies to achieve the goal of HBV clearance.

[0004] In 1998, two American scientists, Andrew Fire and Craig Mello, discovered a new biological mechanism, that is, small RNA molecules can mediate the degradation of specific mRNA (Fire, Andrew, et al. Nature 391.6669 (1998): 806-811.). When RNA molecules appear in cells in double-stranded form, this mechanism will be induced and activated, that is, RNA interference occurs. This discovery heralds the beginning of a new research field, and the two scientists also won the Nobel Prize in Physiology and Medicine in 2006. When double-stranded RNA binds to the protein complex Dicer, Dicer cuts dsRNA into fragments, and then another protein complex RISC binds to these fragments. One strand of the siRNA double strand is removed, but the other strand is still bound to the RISC (RNA-induced silencing complex) complex. RISC recognizes and degrades the mRNA of the target gene through the guidance of single-stranded RNA, inhibits the expression of specific proteins, and then specifically leads to gene silencing.

[0005] RNA interference has opened up a new field for the application of genetic technology. Double-stranded RNA molecules have been artificially designed to silence specific genes in humans, animals or plants. This artificially designed double-stranded RNA molecule (siRNA) for gene silencing is introduced into cells and activates the RNA interference mechanism to degrade the corresponding mRNA. At present, this method is an important research tool in biology and biomedicine. In addition, a large number of siRNA drugs have been developed to treat viral infections, cardiovascular diseases, cancer, endocrine disorders and many other diseases. Most of the siRNA therapies that are in the research and development stage or have been approved for marketing have shown good therapeutic effects. Since the first siRNA drug was launched in 2018, at least 4 siRNAs have been approved for marketing in the European Union or the United States. Therefore, the use of RNA interference technology to inhibit the expression of specific target genes has become an effective way to treat diseases.

[0006] Currently, there are HBV siRNA drugs in the clinical stage, which have shown certain effects in inhibiting HBsAg, HBV DNA and HBeAg, but the proportion of patients achieving functional cure is still low. It is urgent to develop more excellent HBV siRNA drugs in order to achieve the goal of functional cure. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the purpose of the present disclosure is to provide a siRNA for interfering with hepatitis B virus and its use. The siRNA has a significant inhibitory effect on HBsAg, HBVDNA and HBeAg produced by cccDNA and HBV DNA integrated into the host genome, and some siRNAs have an inhibitory efficiency of more than 99% on HBsAg after a single administration, and some siRNAs are still effective after a single administration for 42 days, with a long-lasting efficacy.

[0008] In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting HBV, wherein the oligonucleotide comprises an antisense strand having a sequence as shown in any one of SEQ ID NOs: 251-345 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

[0009] In some embodiments of the present disclosure, the oligonucleotide further comprises a sense strand having a sequence as shown in any one of SEQ ID NOs: 1-95 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof.

[0010] In another aspect, the present disclosure provides a composition comprising the oligonucleotide or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0011] In another aspect, the present disclosure provides use of the oligonucleotide or a pharmaceutically acceptable salt thereof and / or a composition thereof in the preparation of a medicament for reducing at least one of HBsAg, HBeAg and HBV DNA in serum.

[0012] In another aspect, the present disclosure provides use of the oligonucleotide or a pharmaceutically acceptable salt thereof and / or a composition thereof in the preparation of a medicament for treating HBV infection or hepatitis B virus, and hepatitis B virus combined with hepatitis D virus infection.

[0013] In another aspect, the present disclosure provides a method for reducing at least one of HBsAg, HBeAg and HBV DNA in serum, comprising administering a therapeutically effective amount of the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof to a subject.

[0014] In another aspect, the present disclosure provides a method for treating HBV infection or hepatitis B, comprising administering to a subject a therapeutically effective amount of the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The siRNA synthesis process is shown.

[0016] Figure 2 The results show the inhibitory effect on serum HBsAg, HBeAg and HBV DNA in HBV transgenic mice after a single subcutaneous administration of 3 mg / kg.

[0017] Figure 3 The results show the inhibitory effect on serum HBsAg, HBeAg and HBV DNA in HBV transgenic mice after a single subcutaneous administration of 3 mg / kg.

[0018] Figure 4 The results show the inhibitory effect on serum HBsAg, HBeAg and HBV DNA in HBV transgenic mice after a single subcutaneous administration of 3 mg / kg.

[0019] Figure 5 The results show the inhibitory effect of a single subcutaneous administration of different concentrations on serum HBsAg, HBeAg and HBV DNA in HBV transgenic mice. DETAILED DESCRIPTION

[0020] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0021] About: As used herein, the term "about" or "approximately" as applied to one or more target values ​​refers to a value similar to the reference value. In certain embodiments, unless otherwise specified or in addition apparent from the context, the term "approximately" or "approximately" refers to a range of values ​​falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in either direction (greater than or less than) (unless such numerals will exceed 100% of possible values).

[0022] As used herein, "hepatitis B virus" or "HBV" refers to a virus of the Hepadnaviridae family. HBV is a small (e.g., 3.2 kb) hepadnavirus that encodes 4 open reading frames and 7 proteins. The 7 proteins encoded by HBV include small (S), medium (M) and large (L) surface antigens (HBsAg) or envelope (Env) proteins, pre-Core proteins, core proteins, viral polymerase (Pol) and HBx proteins. HBV expresses three surface antigens or envelope proteins, L, M and S, of which S is the smallest and L is the largest. The extra domains in the M and L proteins are named Pre-S2 and Pre-S1, respectively. The core protein is a subunit of the viral nucleocapsid. Pol is required for the synthesis of viral DNA (reverse transcriptase, RNaseH and primers), which occurs in the nucleocapsid located in the cytoplasm of infected hepatocytes. PreCore is a core protein with an N-terminal signal peptide and is proteolytically processed at its N and C termini before secretion from infected cells, the so-called hepatitis B e-antigen (HBeAg). The HBx protein is required for the efficient transcription of covalently closed circular DNA (cccDNA). HBx is not a viral structural protein. In addition to the core and polymerase that share mRNA, all viral proteins of HBV have their own mRNA. Except for the protein pre-Core, HBV viral proteins do not undergo post-translational proteolytic processing.

[0023] As used herein, the terms "HBV antigen", "antigenic polypeptide of HBV", "HBV antigenic polypeptide", "HBV antigenic protein", "HBV immunogenic polypeptide" and "HBV immunogen" all refer to polypeptides that are capable of inducing an immune response, such as a humoral and / or cell-mediated response, against HBV in a subject. The HBV antigen may be a polypeptide, a fragment or an epitope thereof, or a combination, a portion or a derivative thereof of a plurality of HBV polypeptides. The HBV antigen is capable of inducing a protective immune response in a host, such as inducing an immune response against a viral disease or infection, and / or generating immunity (i.e., vaccination) against a viral disease or infection in a subject, which protects the subject from a viral disease or infection. For example, the HBV antigen may comprise a polypeptide or an immunogenic fragment thereof from any HBV protein, such as HBeAg, pre-core protein, HBsAg (S, M or L protein), core protein, viral polymerase or HBx protein derived from any HBV genotype (such as genotype A, B, C, D, E, F, G and / or H, or a combination thereof).

[0024] As used herein, each of the terms "HBV core antigen", "HBcAg" and "core antigen" refers to an HBV antigen that is capable of inducing an immune response, such as a humoral and / or cell-mediated response, to the HBV core protein in a subject. Each of the terms "core", "core polypeptide" and "core protein" refers to the HBV viral core protein. The full-length core antigen is typically 183 amino acids in length and includes an assembly domain (amino acids 1-149) and a nucleic acid binding domain (amino acids 150-183). The 34-residue nucleic acid binding domain is essential for pregenomic RNA encapsidation. This domain also acts as a nuclear import signal. It contains 17 arginine residues and is highly alkaline, consistent with its function. The HBV core protein is dimerized in solution, and the dimers self-assemble into an icosahedral capsid. Each dimer of the core protein has four α-helical bundles flanked by α-helical domains at both ends. Truncated HBV core proteins lacking nucleic acid binding domains can also form capsids.

[0025] As used herein, the term "hepatitis B virus-related disease" or "HBV-related disease" is a disease or condition caused by or associated with HBV infection or replication. The term "HBV-related disease" includes diseases, disorders or conditions that benefit from reduced HBV gene expression or replication. Non-limiting examples of HBV-related diseases include, for example, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0026] As used herein, the term "complementary" refers to a structural relationship between nucleotides (e.g., on two nucleotides on a relative nucleic acid or on a relative region of a single nucleic acid chain) that allows nucleotides to form base pairs with each other. For example, a purine nucleotide complementary to a pyrimidine nucleotide of a nucleic acid relative to a nucleic acid can be base-paired together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides can be base-paired in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. In some embodiments, two nucleic acids can have a nucleotide sequence that is complementary to each other so as to form a complementary region, as described herein.

[0027] As used herein, the term "strand" refers to a single continuous sequence of nucleotides linked together by internucleotide bonds (eg, phosphodiester bonds, phosphorothioate bonds). In some embodiments, the strand has two free ends, eg, a 5'-end and a 3'-end.

[0028] As used herein, the term "deoxyribonucleotide" refers to a nucleotide having a hydrogen at the 2' position of its pentose compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having a modification or substitution of one or more atoms other than at the 2' position (including a modification or substitution in or of a sugar, a phosphate group, or a base).

[0029] As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide that is substantially in the form of a duplex. In some embodiments, complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed between the antiparallel sequences of the nucleotides of the covalently separated nucleic acid chains. In some embodiments, complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed between the antiparallel sequences of the nucleotides of the covalently attached nucleic acid chains. In some embodiments, complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed from a single nucleic acid chain, and the single nucleic acid chain is folded (e.g., via a hairpin) to provide a complementary antiparallel sequence of the nucleotides of the base pairing together. In some embodiments, a double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are completely duplexed from each other. However, in some embodiments, a double-stranded oligonucleotide comprises partially duplexed, for example, two covalently separated nucleic acid chains with overhangs at one or both ends. In some embodiments, a double-stranded oligonucleotide comprises an antiparallel sequence of nucleotides, which is partially complementary, and therefore, one or more mispairings may be present, and the mispairings may include internal mispairings or terminal mispairings.

[0030] As used herein, the term "oligonucleotide" refers to a short nucleic acid, such as a short nucleic acid less than 100 nucleotides in length. The oligonucleotide may comprise ribonucleotides, deoxyribonucleotides and / or modified nucleotides, including, for example, modified ribonucleotides. The oligonucleotide may be single-stranded or double-stranded. The oligonucleotide may or may not have a duplex region. As a group of non-limiting examples, the oligonucleotide may be, but is not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide, short siRNA or single-stranded siRNA. In some embodiments, the double-stranded oligonucleotide is an RNAi oligonucleotide.

[0031] As used herein, the term "AGO2 protein" refers to Argonaute 2 protein, i.e., eukaryotic translation initiation factor 2C subunit 2 (EIF2C2), which contains four globular domains, namely, N-terminal (amino-terminal), PAZ, MID and PIWI, and two connecting domains, namely, L1 and L2, and belongs to the Argonaute protein (AGO) family. AGO2 protein is widely expressed in vivo and is a core component of the RNA-induced silencing complex. It has endoribonuclease activity and can inhibit the expression of target genes by promoting the maturation of miRNA and regulating its biosynthesis and function, thus playing a key role in a variety of pathological and physiological processes. It is known that the 5'-terminal phosphate group can enhance the interaction between certain nucleic acid inhibitor molecules and AGO2 protein.

[0032] As used herein, "modified sequence of nucleotides", "modified sequence of antisense strand and fragments thereof", "modified sequence of sense strand or fragments thereof" or "modified sequence" refers to chemical modification of nucleotide sequences to enhance functional delivery of nucleic acids to target cells, such as increasing their stability to nucleases and improving their affinity for target binding. The modified nucleic acids herein may comprise one or more chemically modified ribonucleotides of either or both of the antisense strand and the sense strand. The modified nucleotides may be chemical modifications on the base, ribose and / or backbone. The chemical modifications described herein include ribose modifications and / or backbone modifications. The modifications of the nucleotides described herein are selected from at least one of the following modifications: 2'-methoxyethyl modified (moe) nucleotides, 2'-methoxy (m) modified nucleotides, 2'-deoxy-2'-fluoro (f) modified nucleotides, phosphorothioate (s) nucleotides and 5'-vinylphosphonic acid (Vp) modified nucleotides. Specifically, the ribose modification includes any one or more of 2'-methoxyethyl modification (moe), 2'-methoxy (m) modification, 5'-vinylphosphonic acid (Vp) modification and 2'-deoxy-2'-fluoro (f) modification, and the backbone modification includes modification of the phosphate portion of the nucleoside, such as thiophosphate (s) modification.

[0033] As used herein, the term "pharmaceutical composition" refers to a mixture or solution comprising a therapeutically effective amount of an active drug ingredient and a pharmaceutically acceptable excipient for administration to a mammal (eg, a human) in need thereof.

[0034] As used herein, "subject" means any animal, preferably a mammal, more preferably a human, who is to undergo or has undergone treatment according to the methods of the embodiments of the present application. The term "mammal" as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more preferably humans.

[0035] The term "therapeutically effective amount" refers to an amount of a compound or molecule of the invention that, when administered to a subject, (i) treats or prevents a particular disease, condition or disorder, (ii) reduces, ameliorates or eliminates one or more symptoms of a particular disease, condition or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition or disorder as described herein. The therapeutically effective amount depends on the compound, the disease state being treated, the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending medical or veterinary doctor and other factors.

[0036] In one aspect, the present disclosure provides an oligonucleotide for inhibiting HBV or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises an antisense strand having a sequence as shown in any one of SEQ ID NOs: 251-345 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

[0037] In some embodiments, the oligonucleotide further comprises a sense strand having a sequence as shown in any one of SEQ ID NOs: 1-95 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof.

[0038] In some embodiments, the antisense strand consists of a sequence as shown in any one of SEQ ID NOs: 251-345 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

[0039] In some embodiments, the sense strand consists of a sequence as shown in any one of SEQ ID NOs: 1-95 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

[0040] In some embodiments, the modified sequence of the antisense strand comprises a sequence as shown in any one of SEQ ID NOs: 346-459 or a fragment thereof.

[0041] In some embodiments, the modified sequence of the sense strand comprises a sequence as shown in any one of SEQ ID NOs: 96-209 or a fragment thereof.

[0042] In some embodiments, the antisense strand consists of a sequence as shown in any one of SEQ ID NOs: 346-459 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

[0043] In some embodiments, the sense strand consists of a sequence as shown in any one of SEQ ID NOs: 96-209 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

[0044] In some embodiments, the aforementioned oligonucleotides comprise at least one modified nucleotide.

[0045] In some embodiments, the modified nucleotide comprises at least one modification as described below:

[0046] 2'-methoxyethyl modified (moe) nucleotides, 2'-methoxy (m) modified nucleotides, 2'-deoxy-2'-fluoro (f) modified nucleotides, phosphorothioate (s) based nucleotides and / or 5'-vinylphosphonic acid (Vp) modified nucleotides; a preferred embodiment is that the 5'-vinylphosphonic acid (Vp) modification is a modification on the 5' terminal nucleoside of the antisense strand of the oligonucleotide.

[0047] In an exemplary embodiment, the 5' terminal nucleotide of the antisense strand of the oligonucleotide is modified by Vp to be a compound having the following structural formula:

[0048]

[0049] Wherein Base is a pyrimidine or purine base, Y is another nucleoside on the oligonucleotide, and A is selected from halogen (such as F), OH, OCH 3 、OCF 3 、OCH 2 CH 3 , 2'-methoxyethyl, etc., any of the substituents conventionally selected in the art.

[0050] In some embodiments, all nucleotides of the oligonucleotide are modified.

[0051] In some embodiments, the aforementioned oligonucleotides comprise at least one modified internucleotide linkage.

[0052] In some embodiments, the at least one modified internucleotide bond is a phosphorothioate bond.

[0053] In some embodiments, the 5'-terminal phosphate group of the oligonucleotide can enhance the interaction between the oligonucleotide and the Argonaut 2 protein. However, oligonucleotides with 5'-phosphate groups are generally easily degraded by phosphatases or other enzymes, limiting their bioavailability in vivo. In some embodiments, the oligonucleotide includes a 5' phosphate analog that is effective against phosphatases or other enzyme degradation to increase the cellular uptake of the oligonucleotide, or improve the pharmacokinetic properties of the oligonucleotide.

[0054] In some embodiments, the 4'-carbon of the pentose of the 5'-terminal nucleotide of the antisense strand comprises a phosphate analog modification that replaces the -CH 2 OH.

[0055] In some embodiments, the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

[0056] In the present invention, the terms "phosphate mimetic", "phosphate analogue" and "phosphonate" are used interchangeably, all of which refer to a phosphate ester in which at least one "O" is replaced by a "C". An exemplary structure is as follows: wherein R' is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and the like.

[0057] In some embodiments, the phosphate analog is a vinylphosphonate, wherein the vinyl group is bound to the 4'-carbon of the oligonucleotide pentose, replacing the -CH2OH on the original pentose. An exemplary structure may be:

[0058]

[0059] Where U is uracil base.

[0060] In some embodiments, the phosphate analog is an oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bound to the 4'-carbon of the sugar of the oligonucleotide. In some embodiments, the oxymethylphosphonate is of the formula -O-CH 2 -PO(OH) 2 or -O-CH 2 -PO(OR) 2 represents, wherein R is independently selected from H, CH 3 , alkyl group, CH 2 CH 2 CN, CH 2 OCOC(CH 3 ) 3 , CH 2 OCH 2 CH 2 Si(CH3 ) 3 In some embodiments, the alkyl group is CH 2 CH 3 Typically, R is independently selected from H, CH 3 or CH 2 CH 3 .

[0061] In some embodiments, the oligonucleotide is an oligonucleotide with a chain length of 19 to 21 base pairs, for example, a chain length of 19, 20, or 21 base pairs. The oligonucleotide includes a sense strand and an antisense strand with the same or different numbers of base pairs.

[0062] In some embodiments, the sense strand is 19 bases in length. In some embodiments, the antisense strand is 21 bases in length.

[0063] In some embodiments, the antisense strand has a sequence shown in any one of SEQ ID Nos. 251 to 345 or a fragment thereof, or a sequence or fragment with chemical modification.

[0064] In some embodiments, the sense strand has a sequence shown in any one of SEQ ID No. 1-95 or a fragment thereof, or a sequence or fragment with chemical modification.

[0065] In some embodiments, the chemical modification includes 2'-methoxyethyl modification (moe), 2'-methoxy (m) modification, 2'-deoxy-2'-fluoro (f) modification, thiophosphate (s) modification and 5'-vinylphosphonic acid (Vp) modification. In some embodiments, the chemical modification includes ribose modification and / or backbone modification, wherein the ribose modification includes any one or more of 2'-methoxyethyl modification (moe), 2'-methoxy (m) modification, 5'-vinylphosphonic acid (Vp) modification and 2'-deoxy-2'-fluoro (f) modification, and the backbone modification includes modification of the phosphate moiety, such as thiophosphate (s).

[0066] In some embodiments, the modified sequence or fragment of the sense strand includes 3 or 4 2'-deoxy-2'-fluoro (f) modifications. In some embodiments, the modified sequence or fragment of the sense strand includes 2 phosphorothioate (s) modifications. In some embodiments, the modified sequence or fragment of the sense strand includes 15 or 16 2'-methoxy (m) modifications. In some embodiments, the sense strand includes 3 2'-deoxy-2'-fluoro (f) modifications and 16 2'-methoxy (m) modifications.

[0067] In some embodiments, the modified sequence or fragment of the antisense strand includes 6 2'-deoxy-2'-fluoro (f) modifications. In some embodiments, the modified sequence or fragment of the antisense strand includes 15 2'-methoxy (m) modifications. In some embodiments, the modified sequence or fragment of the antisense strand includes 3 or 4 thiophosphate (s) modifications. In some embodiments, the modified sequence or fragment of the antisense strand includes 0 or 1 2'-methoxyethyl (moe) modifications. For example, the antisense strand modified sequence or fragment has a sequence shown in SEQ ID NO: 265 or 360.

[0068] In some embodiments, the first nucleotide at the 5' end of the antisense strand is a 5'-vinylphosphonic acid (Vp) modified nucleotide.

[0069] In some embodiments, the first nucleotide at the 5' end of the antisense strand is a nucleotide modified with 5'-vinylphosphonic acid (Vp) and 2'-methoxy (m), for example, the modified sequence of the antisense strand is any one of SEQ ID NOs: 373, 374, 456, 457 and 459. In some embodiments, the aforementioned oligonucleotide comprises a combination of the following sense strands and antisense strands:

[0070] (1) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 251;

[0071] (2) the sense strand set forth in SEQ ID NO:2, and the antisense strand set forth in SEQ ID NO:252;

[0072] (3) the sense strand shown in SEQ ID NO:4, and the antisense strand shown in SEQ ID NO:254;

[0073] (4) the sense strand set forth in SEQ ID NO:6, and the antisense strand set forth in SEQ ID NO:256;

[0074] (5) the sense strand set forth in SEQ ID NO:7, and the antisense strand set forth in SEQ ID NO:257;

[0075] (6) the sense strand set forth in SEQ ID NO:8, and the antisense strand set forth in SEQ ID NO:258;

[0076] (7) the sense strand shown in SEQ ID NO:9, and the antisense strand shown in SEQ ID NO:259;

[0077] (8) the sense strand shown in SEQ ID NO:10, and the antisense strand shown in SEQ ID NO:260;

[0078] (9) the sense strand shown in SEQ ID NO:11, and the antisense strand shown in SEQ ID NO:261;

[0079] (10) the sense strand represented by SEQ ID NO:12, and the antisense strand represented by SEQ ID NO:262;

[0080] (11) the sense strand shown in SEQ ID NO:13, and the antisense strand shown in SEQ ID NO:263;

[0081] (12) the sense strand represented by SEQ ID NO:14, and the antisense strand represented by SEQ ID NO:264;

[0082] (13) the sense strand represented by SEQ ID NO:15, and the antisense strand represented by SEQ ID NO:265;

[0083] (14) the sense strand represented by SEQ ID NO:16, and the antisense strand represented by SEQ ID NO:266;

[0084] (15) the sense strand represented by SEQ ID NO:17, and the antisense strand represented by SEQ ID NO:267;

[0085] (16) the sense strand represented by SEQ ID NO:18, and the antisense strand represented by SEQ ID NO:268;

[0086] (17) the sense strand represented by SEQ ID NO:19, and the antisense strand represented by SEQ ID NO:269;

[0087] (18) the sense strand shown in SEQ ID NO:20, and the antisense strand shown in SEQ ID NO:270;

[0088] (19) the sense strand represented by SEQ ID NO:21, and the antisense strand represented by SEQ ID NO:271;

[0089] (20) the sense strand represented by SEQ ID NO:22, and the antisense strand represented by SEQ ID NO:272;

[0090] (21) the sense strand shown in SEQ ID NO:23, and the antisense strand shown in SEQ ID NO:273;

[0091] (22) the sense strand shown in SEQ ID NO:24, and the antisense strand shown in SEQ ID NO:274;

[0092] (23) the sense strand represented by SEQ ID NO:25, and the antisense strand represented by SEQ ID NO:275;

[0093] (24) the sense strand represented by SEQ ID NO:26, and the antisense strand represented by SEQ ID NO:276;

[0094] (25) the sense strand represented by SEQ ID NO:39, and the antisense strand represented by SEQ ID NO:289;

[0095] (26) the sense strand represented by SEQ ID NO:40, and the antisense strand represented by SEQ ID NO:290;

[0096] (27) the sense strand represented by SEQ ID NO:41, and the antisense strand represented by SEQ ID NO:291;

[0097] (28) the sense strand represented by SEQ ID NO:42, and the antisense strand represented by SEQ ID NO:292;

[0098] (29) the sense strand represented by SEQ ID NO:46, and the antisense strand represented by SEQ ID NO:296;

[0099] (30) the sense strand represented by SEQ ID NO:48, and the antisense strand represented by SEQ ID NO:298;

[0100] (31) the sense strand represented by SEQ ID NO:54, and the antisense strand represented by SEQ ID NO:304;

[0101] (32) the sense strand represented by SEQ ID NO:58, and the antisense strand represented by SEQ ID NO:308;

[0102] (33) the sense strand represented by SEQ ID NO:59, and the antisense strand represented by SEQ ID NO:309;

[0103] (34) the sense strand represented by SEQ ID NO:60, and the antisense strand represented by SEQ ID NO:310;

[0104] (35) the sense strand represented by SEQ ID NO:61, and the antisense strand represented by SEQ ID NO:311;

[0105] (36) the sense strand represented by SEQ ID NO:62, and the antisense strand represented by SEQ ID NO:312;

[0106] (37) the sense strand represented by SEQ ID NO:63, and the antisense strand represented by SEQ ID NO:313;

[0107] (38) the sense strand represented by SEQ ID NO:64, and the antisense strand represented by SEQ ID NO:314;

[0108] (39) the sense strand represented by SEQ ID NO:67, and the antisense strand represented by SEQ ID NO:317;

[0109] (40) the sense strand represented by SEQ ID NO:69, and the antisense strand represented by SEQ ID NO:319;

[0110] (41) the sense strand represented by SEQ ID NO:70, and the antisense strand represented by SEQ ID NO:320;

[0111] (42) the sense strand represented by SEQ ID NO:71, and the antisense strand represented by SEQ ID NO:321;

[0112] (43) the sense strand represented by SEQ ID NO:72, and the antisense strand represented by SEQ ID NO:322;

[0113] (44) the sense strand represented by SEQ ID NO:73, and the antisense strand represented by SEQ ID NO:323;

[0114] (45) the sense strand represented by SEQ ID NO:74, and the antisense strand represented by SEQ ID NO:324;

[0115] (46) the sense strand represented by SEQ ID NO:75, and the antisense strand represented by SEQ ID NO:325;

[0116] (47) the sense strand represented by SEQ ID NO:76, and the antisense strand represented by SEQ ID NO:326;

[0117] (48) the sense strand represented by SEQ ID NO:77, and the antisense strand represented by SEQ ID NO:327;

[0118] (49) the sense strand represented by SEQ ID NO:78, and the antisense strand represented by SEQ ID NO:328;

[0119] (50) the sense strand represented by SEQ ID NO:79, and the antisense strand represented by SEQ ID NO:329;

[0120] (51) the sense strand represented by SEQ ID NO:80, and the antisense strand represented by SEQ ID NO:330;

[0121] (52) the sense strand represented by SEQ ID NO:81, and the antisense strand represented by SEQ ID NO:331;

[0122] (53) the sense strand represented by SEQ ID NO:82, and the antisense strand represented by SEQ ID NO:332;

[0123] (54) the sense strand represented by SEQ ID NO:83, and the antisense strand represented by SEQ ID NO:333;

[0124] (55) the sense strand represented by SEQ ID NO:84, and the antisense strand represented by SEQ ID NO:334;

[0125] (56) the sense strand represented by SEQ ID NO:85, and the antisense strand represented by SEQ ID NO:335;

[0126] (57) the sense strand represented by SEQ ID NO:86, and the antisense strand represented by SEQ ID NO:336;

[0127] (58) the sense strand represented by SEQ ID NO:91, and the antisense strand represented by SEQ ID NO:341;

[0128] (59) the sense strand represented by SEQ ID NO:92, and the antisense strand represented by SEQ ID NO:342;

[0129] (60) the sense strand represented by SEQ ID NO:94, and the antisense strand represented by SEQ ID NO:344;

[0130] (61) The sense strand represented by SEQ ID NO:95, and the antisense strand represented by SEQ ID NO:345.

[0131] In some embodiments, the aforementioned oligonucleotide comprises a combination of sense strands and antisense strands selected from the following:

[0132] (1) the sense strand shown in SEQ ID NO:96, and the antisense strand shown in SEQ ID NO:346;

[0133] (2) the sense strand set forth in SEQ ID NO:97, and the antisense strand set forth in SEQ ID NO:347;

[0134] (3) the sense strand shown in SEQ ID NO:99, and the antisense strand shown in SEQ ID NO:349;

[0135] (4) the sense strand shown in SEQ ID NO: 101, and the antisense strand shown in SEQ ID NO: 351;

[0136] (5) the sense strand set forth in SEQ ID NO:102, and the antisense strand set forth in SEQ ID NO:352;

[0137] (6) the sense strand shown in SEQ ID NO:103, and the antisense strand shown in SEQ ID NO:353;

[0138] (7) the sense strand shown in SEQ ID NO:104, and the antisense strand shown in SEQ ID NO:354;

[0139] (8) the sense strand represented by SEQ ID NO:105, and the antisense strand represented by SEQ ID NO:355;

[0140] (9) the sense strand represented by SEQ ID NO:106, and the antisense strand represented by SEQ ID NO:356;

[0141] (10) the sense strand represented by SEQ ID NO:107, and the antisense strand represented by SEQ ID NO:357;

[0142] (11) the sense strand shown in SEQ ID NO:108, and the antisense strand shown in SEQ ID NO:358;

[0143] (12) the sense strand shown in SEQ ID NO:109, and the antisense strand shown in SEQ ID NO:359;

[0144] (13) the sense strand shown in SEQ ID NO: 110, and the antisense strand shown in SEQ ID NO: 360;

[0145] (14) the sense strand represented by SEQ ID NO:111, and the antisense strand represented by SEQ ID NO:361;

[0146] (15) the sense strand represented by SEQ ID NO:112, and the antisense strand represented by SEQ ID NO:362;

[0147] (16) the sense strand represented by SEQ ID NO:113, and the antisense strand represented by SEQ ID NO:363;

[0148] (17) the sense strand represented by SEQ ID NO:114, and the antisense strand represented by SEQ ID NO:364;

[0149] (18) the sense strand represented by SEQ ID NO:115, and the antisense strand represented by SEQ ID NO:365;

[0150] (19) the sense strand represented by SEQ ID NO:116, and the antisense strand represented by SEQ ID NO:366;

[0151] (20) the sense strand represented by SEQ ID NO:117, and the antisense strand represented by SEQ ID NO:367;

[0152] (21) the sense strand represented by SEQ ID NO:118, and the antisense strand represented by SEQ ID NO:368;

[0153] (22) the sense strand represented by SEQ ID NO:119, and the antisense strand represented by SEQ ID NO:369;

[0154] (23) the sense strand represented by SEQ ID NO:120, and the antisense strand represented by SEQ ID NO:370;

[0155] (24) the sense strand represented by SEQ ID NO:121, and the antisense strand represented by SEQ ID NO:371;

[0156] (25) the sense strand represented by SEQ ID NO:138, and the antisense strand represented by SEQ ID NO:388;

[0157] (26) the sense strand represented by SEQ ID NO:139, and the antisense strand represented by SEQ ID NO:389;

[0158] (27) the sense strand represented by SEQ ID NO:140, and the antisense strand represented by SEQ ID NO:390;

[0159] (28) the sense strand represented by SEQ ID NO:141, and the antisense strand represented by SEQ ID NO:391;

[0160] (29) the sense strand represented by SEQ ID NO:145, and the antisense strand represented by SEQ ID NO:395;

[0161] (30) the sense strand represented by SEQ ID NO:147, and the antisense strand represented by SEQ ID NO:397;

[0162] (31) the sense strand represented by SEQ ID NO:153, and the antisense strand represented by SEQ ID NO:403;

[0163] (32) the sense strand represented by SEQ ID NO:154, and the antisense strand represented by SEQ ID NO:404;

[0164] (33) the sense strand represented by SEQ ID NO:155, and the antisense strand represented by SEQ ID NO:405;

[0165] (34) the sense strand represented by SEQ ID NO:156, and the antisense strand represented by SEQ ID NO:406;

[0166] (35) the sense strand represented by SEQ ID NO:157, and the antisense strand represented by SEQ ID NO:407;

[0167] (36) the sense strand represented by SEQ ID NO:158, and the antisense strand represented by SEQ ID NO:408;

[0168] (37) the sense strand represented by SEQ ID NO:159, and the antisense strand represented by SEQ ID NO:409;

[0169] (38) the sense strand represented by SEQ ID NO:160, and the antisense strand represented by SEQ ID NO:410;

[0170] (39) the sense strand represented by SEQ ID NO:163, and the antisense strand represented by SEQ ID NO:413;

[0171] (40) the sense strand represented by SEQ ID NO:165, and the antisense strand represented by SEQ ID NO:415;

[0172] (41) the sense strand represented by SEQ ID NO:166, and the antisense strand represented by SEQ ID NO:416;

[0173] (42) the sense strand represented by SEQ ID NO:167, and the antisense strand represented by SEQ ID NO:417;

[0174] (43) the sense strand represented by SEQ ID NO:168, and the antisense strand represented by SEQ ID NO:418;

[0175] (44) the sense strand represented by SEQ ID NO:169, and the antisense strand represented by SEQ ID NO:419;

[0176] (45) the sense strand represented by SEQ ID NO: 170, and the antisense strand represented by SEQ ID NO: 420;

[0177] (46) the sense strand represented by SEQ ID NO: 171, and the antisense strand represented by SEQ ID NO: 421;

[0178] (47) the sense strand represented by SEQ ID NO:172, and the antisense strand represented by SEQ ID NO:422;

[0179] (48) the sense strand represented by SEQ ID NO:173, and the antisense strand represented by SEQ ID NO:423;

[0180] (49) the sense strand represented by SEQ ID NO: 174, and the antisense strand represented by SEQ ID NO: 424;

[0181] (50) the sense strand represented by SEQ ID NO:175, and the antisense strand represented by SEQ ID NO:425;

[0182] (51) the sense strand represented by SEQ ID NO: 176, and the antisense strand represented by SEQ ID NO: 426;

[0183] (52) the sense strand represented by SEQ ID NO: 177, and the antisense strand represented by SEQ ID NO: 427;

[0184] (53) the sense strand represented by SEQ ID NO:178, and the antisense strand represented by SEQ ID NO:428;

[0185] (54) the sense strand represented by SEQ ID NO: 179, and the antisense strand represented by SEQ ID NO: 429;

[0186] (55) the sense strand represented by SEQ ID NO: 180, and the antisense strand represented by SEQ ID NO: 430;

[0187] (56) the sense strand represented by SEQ ID NO: 181, and the antisense strand represented by SEQ ID NO: 431;

[0188] (57) the sense strand represented by SEQ ID NO:182, and the antisense strand represented by SEQ ID NO:432;

[0189] (58) the sense strand represented by SEQ ID NO:183, and the antisense strand represented by SEQ ID NO:433;

[0190] (59) the sense strand represented by SEQ ID NO:184, and the antisense strand represented by SEQ ID NO:434;

[0191] (60) the sense strand represented by SEQ ID NO:185, and the antisense strand represented by SEQ ID NO:435;

[0192] (61) the sense strand represented by SEQ ID NO: 190, and the antisense strand represented by SEQ ID NO: 440;

[0193] (62) the sense strand represented by SEQ ID NO: 191, and the antisense strand represented by SEQ ID NO: 441;

[0194] (63) the sense strand represented by SEQ ID NO:193, and the antisense strand represented by SEQ ID NO:443;

[0195] (64) the sense strand represented by SEQ ID NO: 194, and the antisense strand represented by SEQ ID NO: 444;

[0196] (65) the sense strand represented by SEQ ID NO: 197, and the antisense strand represented by SEQ ID NO: 447;

[0197] (66) the sense strand represented by SEQ ID NO: 199, and the antisense strand represented by SEQ ID NO: 449;

[0198] (67) the sense strand represented by SEQ ID NO:200, and the antisense strand represented by SEQ ID NO:450;

[0199] (68) the sense strand represented by SEQ ID NO:201, and the antisense strand represented by SEQ ID NO:451;

[0200] (69) the sense strand represented by SEQ ID NO:202, and the antisense strand represented by SEQ ID NO:452;

[0201] (70) the sense strand represented by SEQ ID NO:203, and the antisense strand represented by SEQ ID NO:453;

[0202] (71) the sense strand represented by SEQ ID NO:205, and the antisense strand represented by SEQ ID NO:435;

[0203] (72) the sense strand represented by SEQ ID NO:206, and the antisense strand represented by SEQ ID NO:456;

[0204] (73) the sense strand represented by SEQ ID NO:207, and the antisense strand represented by SEQ ID NO:457;

[0205] (74) the sense strand represented by SEQ ID NO:208, and the antisense strand represented by SEQ ID NO:458;

[0206] (75) The sense strand represented by SEQ ID NO:209, and the antisense strand represented by SEQ ID NO:459.

[0207] In some embodiments, the aforementioned oligonucleotide comprises a combination of sense strands and antisense strands selected from the following:

[0208] (1) the sense strand shown in SEQ ID NO:97, and the antisense strand shown in SEQ ID NO:347;

[0209] (2) the sense strand shown in SEQ ID NO: 101, and the antisense strand shown in SEQ ID NO: 351;

[0210] (3) the sense strand shown in SEQ ID NO: 102, and the antisense strand shown in SEQ ID NO: 352;

[0211] (4) the sense strand set forth in SEQ ID NO:103, and the antisense strand set forth in SEQ ID NO:353;

[0212] (5) the sense strand set forth in SEQ ID NO:104, and the antisense strand set forth in SEQ ID NO:354;

[0213] (6) the sense strand set forth in SEQ ID NO:105, and the antisense strand set forth in SEQ ID NO:355;

[0214] (7) the sense strand represented by SEQ ID NO:106, and the antisense strand represented by SEQ ID NO:356;

[0215] (8) the sense strand shown in SEQ ID NO:108, and the antisense strand shown in SEQ ID NO:358;

[0216] (9) the sense strand shown in SEQ ID NO:109, and the antisense strand shown in SEQ ID NO:359;

[0217] (10) the sense strand shown in SEQ ID NO:110, and the antisense strand shown in SEQ ID NO:360;

[0218] (11) the sense strand shown in SEQ ID NO:111, and the antisense strand shown in SEQ ID NO:361;

[0219] (12) the sense strand shown in SEQ ID NO:112, and the antisense strand shown in SEQ ID NO:362;

[0220] (13) the sense strand shown in SEQ ID NO:113, and the antisense strand shown in SEQ ID NO:363;

[0221] (14) the sense strand represented by SEQ ID NO:114, and the antisense strand represented by SEQ ID NO:364;

[0222] (15) the sense strand represented by SEQ ID NO:115, and the antisense strand represented by SEQ ID NO:365;

[0223] (16) the sense strand represented by SEQ ID NO:116, and the antisense strand represented by SEQ ID NO:366;

[0224] (17) the sense strand represented by SEQ ID NO:117, and the antisense strand represented by SEQ ID NO:367;

[0225] (18) the sense strand represented by SEQ ID NO:118, and the antisense strand represented by SEQ ID NO:368;

[0226] (19) the sense strand represented by SEQ ID NO:119, and the antisense strand represented by SEQ ID NO:369;

[0227] (20) the sense strand represented by SEQ ID NO:166, and the antisense strand represented by SEQ ID NO:416;

[0228] (21) the sense strand represented by SEQ ID NO:167, and the antisense strand represented by SEQ ID NO:417;

[0229] (22) the sense strand represented by SEQ ID NO:168, and the antisense strand represented by SEQ ID NO:418;

[0230] (23) the sense strand represented by SEQ ID NO:169, and the antisense strand represented by SEQ ID NO:419;

[0231] (24) the sense strand represented by SEQ ID NO: 170, and the antisense strand represented by SEQ ID NO: 420;

[0232] (25) the sense strand represented by SEQ ID NO: 171, and the antisense strand represented by SEQ ID NO: 421;

[0233] (26) the sense strand represented by SEQ ID NO:172, and the antisense strand represented by SEQ ID NO:422;

[0234] (27) the sense strand represented by SEQ ID NO:173, and the antisense strand represented by SEQ ID NO:423;

[0235] (28) the sense strand represented by SEQ ID NO:174, and the antisense strand represented by SEQ ID NO:424;

[0236] (29) the sense strand represented by SEQ ID NO:175, and the antisense strand represented by SEQ ID NO:425;

[0237] (30) the sense strand represented by SEQ ID NO:176, and the antisense strand represented by SEQ ID NO:426;

[0238] (31) the sense strand represented by SEQ ID NO:177, and the antisense strand represented by SEQ ID NO:427;

[0239] (32) the sense strand represented by SEQ ID NO:178, and the antisense strand represented by SEQ ID NO:428;

[0240] (33) the sense strand represented by SEQ ID NO:179, and the antisense strand represented by SEQ ID NO:429;

[0241] (34) the sense strand represented by SEQ ID NO: 180, and the antisense strand represented by SEQ ID NO: 430;

[0242] (35) the sense strand represented by SEQ ID NO:181, and the antisense strand represented by SEQ ID NO:431;

[0243] (36) the sense strand represented by SEQ ID NO:182, and the antisense strand represented by SEQ ID NO:432;

[0244] (37) the sense strand represented by SEQ ID NO:183, and the antisense strand represented by SEQ ID NO:433;

[0245] (38) the sense strand represented by SEQ ID NO:184, and the antisense strand represented by SEQ ID NO:434;

[0246] (39) the sense strand represented by SEQ ID NO:185, and the antisense strand represented by SEQ ID NO:435;

[0247] (40) the sense strand represented by SEQ ID NO: 190, and the antisense strand represented by SEQ ID NO: 440;

[0248] (41) The sense strand represented by SEQ ID NO:191, and the antisense strand represented by SEQ ID NO:441.

[0249] In some embodiments, the aforementioned oligonucleotide comprises a combination of sense strands and antisense strands selected from the following:

[0250] (1) the sense strand shown in SEQ ID NO:210, and the antisense strand shown in SEQ ID NO:351;

[0251] (2) the sense strand set forth in SEQ ID NO:211, and the antisense strand set forth in SEQ ID NO:355;

[0252] (3) the sense strand set forth in SEQ ID NO:212, and the antisense strand set forth in SEQ ID NO:359;

[0253] (4) the sense strand set forth in SEQ ID NO:213, and the antisense strand set forth in SEQ ID NO:361;

[0254] (5) the sense strand set forth in SEQ ID NO:214, and the antisense strand set forth in SEQ ID NO:362;

[0255] (6) the sense strand set forth in SEQ ID NO:215, and the antisense strand set forth in SEQ ID NO:363;

[0256] (7) the sense strand set forth in SEQ ID NO:216, and the antisense strand set forth in SEQ ID NO:365;

[0257] (8) the sense strand represented by SEQ ID NO:217, and the antisense strand represented by SEQ ID NO:366;

[0258] (9) the sense strand shown in SEQ ID NO:218, and the antisense strand shown in SEQ ID NO:367;

[0259] (10) the sense strand represented by SEQ ID NO:219, and the antisense strand represented by SEQ ID NO:361;

[0260] (11) the sense strand shown in SEQ ID NO:220, and the antisense strand shown in SEQ ID NO:374;

[0261] (12) the sense strand represented by SEQ ID NO:221, and the antisense strand represented by SEQ ID NO:376;

[0262] (13) the sense strand represented by SEQ ID NO:222, and the antisense strand represented by SEQ ID NO:377;

[0263] (14) the sense strand represented by SEQ ID NO:223, and the antisense strand represented by SEQ ID NO:375;

[0264] (15) the sense strand represented by SEQ ID NO:224, and the antisense strand represented by SEQ ID NO:388;

[0265] (16) the sense strand represented by SEQ ID NO:225, and the antisense strand represented by SEQ ID NO:391;

[0266] (17) the sense strand represented by SEQ ID NO:226, and the antisense strand represented by SEQ ID NO:395;

[0267] (18) the sense strand represented by SEQ ID NO:227, and the antisense strand represented by SEQ ID NO:443;

[0268] (19) the sense strand represented by SEQ ID NO:228, and the antisense strand represented by SEQ ID NO:407;

[0269] (20) the sense strand represented by SEQ ID NO:229, and the antisense strand represented by SEQ ID NO:410;

[0270] (21) the sense strand represented by SEQ ID NO:230, and the antisense strand represented by SEQ ID NO:416;

[0271] (22) the sense strand represented by SEQ ID NO:231, and the antisense strand represented by SEQ ID NO:444;

[0272] (23) the sense strand represented by SEQ ID NO:232, and the antisense strand represented by SEQ ID NO:422;

[0273] (24) the sense strand represented by SEQ ID NO:233, and the antisense strand represented by SEQ ID NO:429;

[0274] (25) the sense strand represented by SEQ ID NO:234, and the antisense strand represented by SEQ ID NO:432;

[0275] (26) the sense strand represented by SEQ ID NO:235, and the antisense strand represented by SEQ ID NO:441;

[0276] (27) the sense strand represented by SEQ ID NO:236, and the antisense strand represented by SEQ ID NO:445;

[0277] (28) the sense strand represented by SEQ ID NO:237, and the antisense strand represented by SEQ ID NO:446;

[0278] (29) the sense strand represented by SEQ ID NO:238, and the antisense strand represented by SEQ ID NO:447;

[0279] (30) the sense strand represented by SEQ ID NO:239, and the antisense strand represented by SEQ ID NO:448;

[0280] (31) the sense strand represented by SEQ ID NO:240, and the antisense strand represented by SEQ ID NO:449;

[0281] (32) the sense strand represented by SEQ ID NO:241, and the antisense strand represented by SEQ ID NO:450;

[0282] (33) the sense strand represented by SEQ ID NO:242, and the antisense strand represented by SEQ ID NO:451;

[0283] (34) the sense strand represented by SEQ ID NO:243, and the antisense strand represented by SEQ ID NO:452;

[0284] (35) the sense strand represented by SEQ ID NO:244, and the antisense strand represented by SEQ ID NO:453;

[0285] (36) the sense strand represented by SEQ ID NO:245, and the antisense strand represented by SEQ ID NO:454;

[0286] (37) the sense strand represented by SEQ ID NO:246, and the antisense strand represented by SEQ ID NO:455;

[0287] (38) the sense strand represented by SEQ ID NO:247, and the antisense strand represented by SEQ ID NO:456;

[0288] (39) the sense strand represented by SEQ ID NO:248, and the antisense strand represented by SEQ ID NO:457;

[0289] (40) the sense strand represented by SEQ ID NO:249, and the antisense strand represented by SEQ ID NO:458;

[0290] (41) The sense strand represented by SEQ ID NO:250, and the antisense strand represented by SEQ ID NO:459.

[0291] In some embodiments, at least one nucleotide of the aforementioned oligonucleotides is conjugated to one or more targeting ligands.

[0292] The targeting ligand can be any ligand capable of targeting a specific receptor. Examples are: folate, GalNAc, galactose, mannose, mannose-6P, sugar clusters (such as GalNAc clusters, mannose clusters, galactose clusters) or an aptamer. A cluster is a combination of two or more sugar units. These targeting ligands also include integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. These ligands can also be based on nucleic acids, such as an aptamer. The aptamer can be unmodified or have any combination of modifications disclosed herein.

[0293] In some embodiments, each targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a lipid.

[0294] In some embodiments, the targeting ligand comprises an aminosugar.

[0295] In some embodiments, each targeting ligand comprises an N-acetylgalactosamine (GalNAc) portion, and the GalNAc is linked to the oligonucleotide in the following manner: N-acetylated galactosamine is covalently conjugated to the phosphate of the 3' terminal nucleotide of the sense strand of the oligonucleotide in a trivalent state to form an oligonucleotide sequence with a GalNAc carrier.

[0296] In some embodiments, wherein the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

[0297] In a specific embodiment, the GalNaAc has the structure as described below:

[0298]

[0299] In a preferred embodiment, the targeting ligand is conjugated to the sense strand of the oligonucleotide at the 3'-end via a linker to form the following conjugate:

[0300]

[0301] Wherein X can be S or O.

[0302] In another aspect, the present disclosure provides a composition comprising the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0303] In some embodiments, the composition is in the form of an oral agent, an intravenous injection, a subcutaneous injection, or an intramuscular injection, preferably a subcutaneous injection.

[0304] In some embodiments, the combination further comprises other drugs for treating HBV infection; preferably, nucleoside analogs, interferon-type antiviral drugs, as well as hepatitis B virus entry inhibitors, capsid assembly inhibitors, HBsAg secretion inhibitors, HBsAg antibodies, hepatitis B therapeutic vaccines, host immune modulators, apoptosis protein antagonists, etc.

[0305] In another aspect, the present disclosure provides use of the aforementioned oligonucleotide or a pharmaceutically acceptable salt or composition thereof in the preparation of a medicament for reducing at least one of HBsAg, HBeAg and HBV DNA.

[0306] In another aspect, the present disclosure provides use of the aforementioned oligonucleotide or a pharmaceutically acceptable salt or composition thereof in the preparation of a medicament for treating HBV infection or hepatitis B virus, and hepatitis B virus combined with hepatitis D virus infection.

[0307] In some embodiments, the HBV infection is a chronic HBV infection.

[0308] In some embodiments, the hepatitis B virus is chronic hepatitis B virus.

[0309] Various preparations have been developed to promote the use of oligonucleotides. For example, oligonucleotides can be delivered to subjects or cell environments using preparations that minimize degradation, promote delivery and / or intake, or provide another beneficial property for the oligonucleotides in the preparation. In some embodiments, provided herein are siRNAs or compositions thereof comprising oligonucleotides (e.g., single-stranded or double-stranded oligonucleotides) to reduce HBsAg, HBeAg and HBV DNA. Such siRNAs or compositions thereof can be suitably formulated so that when applied to subjects (in the direct environment of target cells or systemically), enough oligonucleotides of a portion enter cells to reduce HBsAg, HBeAg and HBV DNA. Any of various suitable oligonucleotide preparations can be used to deliver oligonucleotides for reducing HBsAg, HBeAg and HBV DNA, as disclosed herein. In some embodiments, oligonucleotides are formulated in buffer solutions, such as phosphate buffered saline solutions, liposomes, micellar structures and shells. In some embodiments, naked oligonucleotides or their conjugates are formulated in water or aqueous solutions (e.g., water adjusted by pH). In some embodiments, the naked oligonucleotide or its conjugate is formulated in an alkaline buffered aqueous solution (eg, PBS).

[0310] The preparation of oligonucleotides with cationic lipids can be used to facilitate transfection of oligonucleotides into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives and polycationic molecules (e.g., polylysine) can be used.

[0311] Thus, in some embodiments, the formulation comprises lipid nanoparticles. In some embodiments, the excipient comprises a liposome, lipid, lipid complex, microsphere, microparticle, nanosphere or nanoparticle, or may be otherwise formulated for administration to a cell, tissue, organ or body of a subject in need thereof.

[0312] In some embodiments, the preparation as disclosed herein comprises excipient.In some embodiments, excipient imparts the stability of the improvement of active ingredient, the absorption of improvement, the solubility of improvement and / or therapeutic enhancement to the composition.In some embodiments, excipient is a buffer (e.g., sodium citrate, sodium phosphate, tris alkali or sodium hydroxide) or a vehicle (e.g., a buffer solution, petrolatum, dimethyl sulfoxide or mineral oil).In some embodiments, the oligonucleotide is freeze-dried for extending its shelf life, and then a solution is made before use (e.g., being applied to a subject).Therefore, the excipient in the composition comprising any one of the oligonucleotides described herein can be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol or polyvinyl pyrrolidone) or a collapse temperature modifier (e.g., dextran, ficoll or gelatin).

[0313] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Typically, the route of administration is intravenous or subcutaneous.

[0314] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when water-soluble) or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL.TM. (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium, which contains, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. In many cases, it is preferred to include isotonic agents, for example sugars, polyols such as mannitol, sorbitol and sodium chloride in the composition. Sterile injectable solutions can be prepared by: the desired amount of oligonucleotides and the desired above-listed components are incorporated into the solvent of choice, followed by filtration sterilization.

[0315] In some embodiments, the composition may contain at least about 0.1% or more of a therapeutic agent (e.g., oligonucleotides for reducing HBsAg, HBeAg, and HBV DNA), although the percentage of one or more active ingredients may be between about 1% and about 80% or more of the weight or volume of the total composition. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore various dosages and treatment regimens may be desired.

[0316] Even though many of the embodiments relate to liver-targeted delivery of any of the oligonucleotides disclosed herein, targeting other tissues is also contemplated.

[0317] In some embodiments, the administration of oligonucleotides as described herein causes the reduction of the level of HBsAg, HBeAg and HBV DNA.In some embodiments, the reduction of the level of HBsAg, HBeAg and HBV DNA can be reduced to 1% or lower, 5% or lower, 10% or lower, 15% or lower, 20% or lower, 25% or lower, 30% or lower, 35% or lower, 40% or lower, 45% or lower, 50% or lower, 55% or lower, 60% or lower, 70% or lower, 80% or lower or 90% or lower compared to appropriate control levels.Suitable control levels can be the level of HBsAg, HBeAg and HBV DNA in the subject's serum not contacted with oligonucleotides as described herein.In some embodiments, the effect of oligonucleotide delivery to cells is evaluated after a limited period of time according to methods disclosed herein. For example, the levels of HBsAg, HBeAg and HBV DNA in serum can be analyzed at least 8 hours, 12 hours, 18 hours, 24 hours; or at least one, two, three, four, five, six, seven or fourteen days after the oligonucleotides are introduced into the cells.

[0318] In some embodiments, the oligonucleotide is delivered in the form of a transgenic engineered to express an oligonucleotide disclosed herein in a cell (e.g., in the form of an shRNA). In some embodiments, the oligonucleotide is delivered using a transgenic engineered to express any oligonucleotide disclosed herein. A viral vector (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or a non-viral vector (e.g., plasmid or synthetic mRNA) can be used to deliver the transgene. In some embodiments, the transgene can be injected directly into the subject.

[0319] Aspects of the present disclosure relate to methods for reducing HBsAg, HBeAg and HBV DNA for treating HBV infection in a subject, hepatitis B and / or one or more symptoms or complications thereof. In some embodiments, the method may include administering an effective amount of any of the oligonucleotides disclosed herein to a subject in need thereof. In some embodiments, such treatments may be used, for example, to reduce or prevent hypercholesterolemia (high levels of low-density lipoprotein (LDL)-cholesterol), atherosclerosis, coronary heart disease (e.g., coronary artery disease), angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmia, palpitations, stroke (i.e., due to insufficient blood and oxygen flow to the brain causing brain cell death), weakness, confusion, difficulty speaking, dizziness, difficulty walking or standing straight, blurred vision, face, numbness of arms and legs, severe headache, loss of consciousness, peripheral arterial disease and / or kidney problems (e.g., chronic kidney disease). In some embodiments, such treatments may be used, for example, to treat or prevent one or more symptoms associated with hypercholesterolemia, atherosclerosis and / or one or more symptoms or complications thereof.

[0320] Thus, in some embodiments, the present disclosure provides methods of treating a subject at risk for (or susceptible to) hypercholesterolemia, atherosclerosis, and / or one or more symptoms or complications thereof, including coronary heart disease (e.g., coronary artery disease), angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, irregular heartbeat, palpitations, stroke (i.e., death of brain cells due to insufficient blood and oxygen flow to the brain), feelings of weakness, confusion, difficulty speaking, dizziness, difficulty walking or standing upright, blurred vision, numbness of the face, arms and legs, severe headaches, loss of consciousness, peripheral arterial disease, and / or kidney problems (e.g., chronic kidney disease).

[0321] In certain aspects, the present disclosure provides a method for preventing a disease, disorder, symptom or condition as described herein in a subject by administering a therapeutic agent (e.g., an oligonucleotide or a vector or transgene encoding it) to the subject. In some embodiments, the subject to be treated is a subject who will benefit from the reduction in the amount of, for example, HBsAg, HBeAg and HBV DNA in serum.

[0322] The methods described herein generally involve administering to a subject an effective amount (i.e., an amount capable of producing a desired therapeutic outcome) of an oligonucleotide. A therapeutically acceptable amount can be an amount capable of treating a disease or condition. The appropriate dosage for any subject will depend on certain factors, including the subject's size, body surface area, age, the specific composition to be administered, one or more active ingredients in the composition, the time and route of administration, overall health, and other drugs administered simultaneously.

[0323] In some embodiments, any of the compositions disclosed herein is administered to a subject enterally (e.g., orally, through a gastric feeding tube, through a duodenal feeding tube, via gastrostomy or rectally), parenterally (e.g., subcutaneously, intravenously or intravenously, intraarterially or intramuscularly), topically (e.g., epidermally, by inhalation, via eye drops or through a mucosa), or by direct injection into a target organ (e.g., the subject's liver). Typically, the oligonucleotides disclosed herein are administered intravenously or subcutaneously.

[0324] In some embodiments, the oligonucleotide is administered at a dosage in the range of 0.1 mg / kg to 25 mg / kg (e.g., 1 mg / kg to 9 mg / kg). In some embodiments, the oligonucleotide is administered at a dosage in the range of 0.1 mg / kg to 9 mg / kg or in the range of 0.5 mg / kg to 9 mg / kg.

[0325] As a set of non-limiting examples, the oligonucleotides of the disclosure will typically be administered yearly, twice yearly, quarterly (once every three months), bimonthly (once every two months), monthly, semi-monthly, or weekly.

[0326] In some embodiments, the subject to be treated is a human (e.g., a human patient) or a non-human primate or other mammalian subject. Other exemplary subjects include domestic animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.

[0327] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

[0328] Hereinafter, the present disclosure will be described in more detail with reference to specific examples; however, the examples are only for illustrative purposes and have no limiting effect on the present disclosure.

[0329] Preparation Example

[0330] Preparation of siRNA

[0331] Abbreviations for nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are linked to each other via 5'-3' phosphodiester bonds unless otherwise specified. The preparation of L96 was carried out according to the method described in patent CN104717982B.

[0332] The preparation process of oligonucleotides is as follows:

[0333] (1) Preparation of siRNA

[0334] The siRNA sequence is synthesized separately on a solid support carrier via a sense strand (SS) and an antisense strand (AS), and is obtained after deprotection, cleavage, purification, annealing, purification and lyophilization.

[0335] Solid phase synthesis ( Figure 1 ): Use phosphoramidite technology to synthesize the sense strand and antisense strand separately on a solid support carrier using an oligonucleotide automatic synthesizer. The synthesizer is such as AKTA Oligopilot (Cytiva Company), Dr.Oligo192XLc (Kunshan Berrick Precision Instrument Co., Ltd.). Solid phase synthesis starts from the 3' end of the sequence, and the monomers are coupled into the sequence in sequence. Each coupling of a phosphoramidite monomer includes four chemical steps: 1) unblocking or deprotection (removal of hydroxyl protecting group); 2) coupling; 3) oxidation; 4) end-capping. The phosphoramidite monomers, reagents, and purification consumables used are all commercial circulating reagents and consumables, such as various phosphoramidite monomers (such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (such as 40wt% methylamine aqueous solution, 28wt% ammonium hydroxide aqueous solution, etc.) purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used in this article are described in US20130178612A1, US2015100197A1, etc.; the synthesis method containing VpUm and APU structure sequences is described in J.Med.Chem.2018,61,734-744.

[0336] (2) Preparation of double-stranded RNA reagent

[0337] The synthesis process of the positive chain is as follows:

[0338] The oligonucleotide is synthesized by solid phase phosphoramidite method. A computer-controlled synthesizer is used to react in a stainless steel synthesis column. In the synthesis of the sense chain, a solid phase support loaded with a targeting ligand (such as L96) is used as the starting point, or the solid phase support carrier is used as the starting point directly. Different raw materials, reagents and solvents are injected in the order of sequence 3' to 5' through different pipelines controlled by the solid phase synthesizer, and phosphoramidite nucleoside monomers are connected one by one. The reaction process includes four cycles of removal of DMT protecting group reaction, condensation reaction, oxidation or thiolation reaction, and end-capping reaction. Each cycle connects a nucleotide unit to obtain an oligonucleotide sequence of 19 or 21 nucleotide units. After the synthesis is completed, the protecting group (2-cyanoethyl) is removed on the solid phase synthesis column, and the synthesized sequence is cut from the solid phase support carrier by aminolysis reaction, filtered, the filter cake is washed with ethanol, the filtrate and washing liquid are collected, and concentrated to obtain a crude sense chain. The crude product is purified by chromatography (SOURCE 15Q) and freeze-dried to obtain the target product sense chain.

[0339] The synthesis process of the antisense strand is as follows:

[0340] The synthesis of the antisense strand is similar to that of the sense strand. Different raw materials, reagents and solvents are injected into different pipelines in the order of sequence 3' to 5' through the solid phase synthesizer, and the phosphoramidite nucleoside monomers are connected one by one. The reaction process includes four cycles of DMT protection group removal reaction, condensation reaction, oxidation or thiolation reaction, and end-capping reaction. Each cycle connects a nucleotide unit to obtain an oligonucleotide sequence of 21 or 23 nucleotide units. After the synthesis is completed, the protective group (2-cyanoethyl) is removed on the solid phase synthesis column, and then the synthesized sequence is cut from the solid phase support carrier by aminolysis reaction, filtered, and the filter cake is washed with ethanol. The filtrate and washing liquid are collected and concentrated to obtain the crude antisense strand. The crude product is purified by chromatography (SOURCE 15Q), ultrafiltered, and freeze-dried to obtain the target product antisense strand.

[0341] Preparation of siRNA double strands:

[0342] The AS chain and the SS chain were dissolved in injection water respectively, mixed in a determined ratio (1.01:1.0-1.2:1.0), incubated at 30-50°C for 30-90 minutes, cooled to room temperature, and freeze-dried to obtain a double-stranded siRNA product.

[0343] According to the same method, the double-stranded siRNA reagent conjugates shown in Tables 1, 2 and 3 below were prepared.

[0344] Example 1 Design of HBV siRNA

[0345] First, a computer-based algorithm was used to generate candidate oligonucleotide sequences complementary to the D genotype (subtype ayw, V01460 J02203) of the hepatitis B virus, some of which were also complementary to genotypes A, B, C, etc. or had no more than 2 mismatches. siRNA was designed as a double strand with a 19 / 21 pairing of the sense strand and the antisense strand, respectively. The antisense strand had two overhanging ends complementary to the mRNA sequence, and in some cases, the overhanging ends of the antisense strand were non-complementary UU; in some complementary paired sequences, the 1st and / or 19th bases at the 5' end of the sense strand were replaced with bases different from HBV mRNA, and the bases at the corresponding positions of the antisense strand were changed accordingly to form a pairing with the sense strand; in some sequences, the bases at individual positions within the sense strand were replaced to form a mismatch with the corresponding positions of the antisense strand; in some sequences, deoxyribonucleotides were used to replace ribonucleotides; in some sequences, hypoxanthine (I) was used to replace U, A or G at individual positions of the antisense strand to form a wobble pairing with the sense strand. Table 1 lists the designed siRNA oligonucleotide naked sequences.

[0346] Table 1: Naked oligonucleotide sequences

[0347]

[0348]

[0349]

[0350] Note: "G", "C", "A", "U", "T" and "I" usually represent nucleotides with guanine, cytosine, adenine, uracil, thymine and hypoxanthine as bases respectively; moe represents that the 2-position of the ribose of the nucleotide is modified with methoxyethyl.

[0351] Table 2 lists the designed modified oligonucleotide sequences.

[0352] Table 2: Modified oligonucleotides

[0353]

[0354]

[0355]

[0356]

[0357]

[0358] Note: "G", "C", "A", "U", "T" and "I" usually represent nucleotides with guanine, cytosine, adenine, uracil, thymine and hypoxanthine as bases, respectively; modification: d represents deoxynucleotide; m represents 2'-methoxy; f represents 2'-deoxy-2'-fluoro; s represents thiophosphate; Vp represents vinylphosphonic acid. In the sequence table of this application, according to the rules of ST.26 sequence table, "U" is replaced by "T" in all sequence "residue" items, "dT" is replaced by "n", and "I" is replaced by "n". In the modified sequence, except for Vp, other modifications represent modifications to the previous nucleotide.

[0359] Table 3 lists the designed vector-carrying siRNA sequences.

[0360] Table 3: siRNA sequences with vector

[0361]

[0362]

[0363]

[0364] Note: "G", "C", "A", "U", "T" and "I" usually represent nucleotides with guanine, cytosine, adenine, uracil, thymine and hypoxanthine as bases, respectively; modification: d represents deoxynucleotide; m represents 2'-methoxy; f represents 2'-deoxy-2'-fluoro; s represents 3'-phosphorothioate; Vp represents 5'-vinylphosphonic acid. In the sequence table of this application, according to the rules of ST.26 sequence table, "U" is replaced by "T" in all sequence "residue" items, "dT" is replaced by "n", and "I" is replaced by "n". In the modified sequence, except for Vp, other modifications represent modifications to the previous nucleotide.

[0365] Wherein, L96 represents a carrier. Alternatively, the structural formula of the ligand conjugated with the oligonucleotide is as follows:

[0366]

[0367] Further, the ligand is conjugated to the sense strand of the oligonucleotide at the 3'-end via a linker to form the following conjugate:

[0368] X may be selected from S or O.

[0369] Example 2 Evaluation of the in vitro anti-HBV activity of modified sequences using HepG2.2.15 cells

[0370] (1) Culture of HepG2.2.15 cells:

[0371] HepG2.2.15 cells are HepG2 cells transfected with HBV-DNA recombinant plasmids. They can stably produce HBV virus and are a commonly used in vitro model for HBV drug screening. The cells were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd. and placed at 37°C and 5% CO 2 The cells were cultured in a culture incubator using DMEM basal medium (Gibco-11995-065) supplemented with 10% FBS (ExCell Bio-FSP500), InvitroGRO CP Medium (BioreclamationIVT-S03316) and 1% penicillin-streptomycin (Hyclone-SV30010).

[0372] (2) Plating and transfection

[0373] On day 0, the test compound was firstly added to DPBS (KCl, 0.2 g / L; KH 2 PO 4 , 0.2g / L; NaCl, 8g / L; Na 2 HPO 4 7H 2 O, 2.16g / L; adjust pH = 7.0) to 2 concentrations, with a final concentration of 1nM and 0.1nM. Take the HepG2.2.15 cells from the culture flask, wash them with DPBS, and add trypsin for digestion.

[0374] After collecting the cells, count them and adjust the cells to an appropriate density. 4 The cells were inoculated into a 96-well plate at a density of 10 cells; while the cells were inoculated, the test compound was transfected into the cells using Lipofectamine RNAiMax (Invitrogene, 13378-150). Three replicate wells were set for each concentration point of each test compound.

[0375] (3) Sampling and testing

[0376] 72h after transfection of the test compound, the cell supernatant was collected and the expression level of HBsAg in the supernatant was detected by ELISA. The cell culture supernatant was diluted with DPBS and the HBsAg ELISA kit (Cat. No.: Antu Bio-CL0310) was used to detect the content of HBsAg. The method is briefly described as follows: 50μL of standard, sample and control were added to the test plate, and then 50μL of enzyme conjugate was added to each well, incubated at 37℃ for 60 minutes, washed with washing solution and dried, then 50μL of premixed luminescent substrate was added, incubated at room temperature in the dark for 10 minutes, and finally the luminescence value was determined by microplate reader.

[0377] (4) Data analysis

[0378] HBsAg inhibition rate (%) = (1-HBsAg value of sample / HBsAg value of DPBS control group) × 100%

[0379] Table 4. In vitro inhibitory activity of HepG2.2.15 cells (n=3)

[0380]

[0381] Except for a few compounds including AL0105003, AL0105005, AL0105025, AL0105026 and AL0105027, other compounds showed very high HBsAg inhibition levels, with average inhibition rates above 85%, and some compounds with inhibition rates above 99%.

[0382] Example 3 Evaluation of the in vitro anti-HBV activity of modified sequences using HepG2.2.15 cells

[0383] (1) Culture of HepG2.2.15 cells:

[0384] HepG2.2.15 cells are HepG2 cells transfected with HBV-DNA recombinant plasmids. They can stably produce HBV virus and are a commonly used in vitro model for HBV drug screening. The cells were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd. and placed at 37°C and 5% CO 2 The cells were cultured in a culture incubator using DMEM basal medium (Gibco-11995-065) supplemented with 10% FBS (ExCell Bio-FSP500), InvitroGRO CP Medium (BioreclamationIVT-S03316) and 1% penicillin-streptomycin (Hyclone-SV30010).

[0385] (2) Plating and transfection

[0386] On day 0, the test compound was first diluted with DPBS to two concentrations, with a final concentration of 0.1 nM and 0.01 nM. HepG2.2.15 cells were taken from the culture flask, washed with DPBS, and digested with trypsin. After collecting the cells, they were counted and adjusted to an appropriate cell density. 1.5×10 4 The cells were inoculated into a 96-well plate at a density of 10 cells; while the cells were inoculated, the test compound was transfected into the cells using Lipofectamine RNAiMax (Invitrogene, 13378-150). Three replicate wells were set for each concentration point of each test compound.

[0387] (3) Sampling and testing

[0388] 72h after transfection of the test compound, the cell supernatant was collected and the expression level of HBsAg in the supernatant was detected by ELISA. The cell culture supernatant was diluted with DPBS and the HBsAg ELISA kit (Cat. No.: Antu Bio-CL0310) was used to detect the content of HBsAg. The method is briefly described as follows: 50μL of standard, sample and control were added to the test plate, and then 50μL of enzyme conjugate was added to each well, incubated at 37℃ for 60 minutes, washed with washing solution and dried, then 50μL of premixed luminescent substrate was added, incubated at room temperature in the dark for 10 minutes, and finally the luminescence value was determined by microplate reader.

[0389] (4) Data analysis

[0390] HBsAg inhibition rate (%) = (1-HBsAg value of sample / HBsAg value of DPBS control group) × 100%

[0391] Table 5. In vitro inhibitory activity of HepG2.2.15 cells (n=3)

[0392]

[0393]

[0394] The bases of AL0105010 and AL0105016 were substituted at some positions of the sense strand or / and antisense strand to obtain AL0105056-5058, AL0105084-5099 and AL0105100-5122, respectively. The in vivo test results showed that the substitution of certain bases could significantly improve the inhibition efficiency of siRNA against HBsAg. For example, the inhibition rate of AL0105016 0.1nM against HBsAg was 74.50%, while the AL0105084, AL0105087, AL0105095-99 The inhibition rate of 0.1nM increased to more than 80%, and that of AL0105096-99 was more than 90%. In addition, the inhibition efficiency of AL0105085, AL0105086, AL0105088, AL0105089, and AL0105090 also increased to varying degrees; the inhibition rate of AL0105010 0.1nM on HbsAg was 83.65%, and the inhibition rates of AL0105100-0115, AL0105120, and AL0105121 obtained by base substitution were all increased to more than 90%. In summary, the new siRNAs obtained by base substitution at individual positions have improved activity, except for a few siRNAs that have reduced activity.

[0395] Example 4 Evaluation of the in vitro anti-HBV activity of modified sequences using HepG2.2.15 cells

[0396] (1) Experimental method is the same as in Example 3

[0397] (2) Data Analysis

[0398] HBsAg inhibition rate (%) = (1-HBsAg value of sample / HBsAg value of DPBS control group) × 100%

[0399] Table 6. In vitro inhibitory activity of HepG2.2.15 cells (n=3)

[0400]

[0401] The results showed that among the drugs with different modified combinations, except for AL0105125, AL0105128, and AL0105134, which had relatively low inhibition efficiency on HBsAg, all other siRNA drugs had an inhibition efficiency of more than 70% on HBsAg at a concentration of 0.1nM, and an inhibition efficiency of more than 45% on HBsAg at a concentration of 0.01nM.

[0402] Example 5 Evaluation of the Anti-HBV Activity of Compounds in Vivo Using HBV Transgenic Mice

[0403] The experiment used SPF male C57B / 6N-Tg(1.28HBV) / Vst mice aged 6-8 weeks (Beijing Weitongda Biotechnology Co., Ltd., Animal Production Certificate SCXK(Beijing)2019-0002). The mice were kept in single cages, and the use and testing of animal feed, bedding and drinking water were carried out in accordance with GB14925-2010 "Experimental Animal Environment and Facilities".

[0404] The animals were tested after one week of acclimatization. According to the quantitative test results of serum HBsAg (primary) and HBV DNA (secondary), the animals were randomly divided into 9 groups, with 5 animals in each group. The day of administration was recorded as D1, and blood was collected before administration on the day of administration for the determination of HBsAg, HBV DNA and HBeAg.

[0405] All animals were given subcutaneous injections on D1, all as a single injection, with a dose of 3 mg / kg and a volume of 5 mL / kg. A control group was given a saline solution, which was injected with the same dose of saline. The animals in each group were observed 15 minutes to 1 hour after the last animal in each group was given the drug.

[0406] On the 8th, 15th, 22nd and 29th days after administration, 100 μL of blood was collected from the eye sockets and centrifuged to separate the serum. 10 μL of serum was diluted 50 times with PBS and sent for testing. The diluted serum HBsAg, HBeAg and HBV DNA were tested by Beijing Dian Medical Testing Laboratory Co., Ltd.

[0407] The experimental data were statistically analyzed using Graphpad prism statistical analysis software. The calculation of the residual inhibition rate of HBV DNA, HBsAg and HBeAg was based on the average of the two measured values ​​before the drug: residual inhibition rate = different time determination values ​​ / pre-drug basic value * 100%.

[0408] The experimental results are shown in Table 7 and Figure 2 .

[0409] Table 7: Residual rates of serum HBsAg, HBeAg and HBV DNA in HBV transgenic mice after a single subcutaneous administration of 3 mg / kg

[0410]

[0411] from Figure 2 As shown in the test results of Table 7, the tested compounds have certain inhibitory effects on HBsAg, HBeAg and HBV DNA in the serum of HBV transgenic mice, among which the maximum inhibition rates of AL0107001, AL0107002, AL0107004 and AL0107008 on HBsAg are all greater than 80%, and the maximum inhibition rate of AL0107004 is about 98%. At the same time, these four compounds also show obvious reduction effects on HBeAg and HBV DNA.

[0412] Example 6 Evaluation of the Anti-HBV Activity of Compounds in Vivo Using HBV Transgenic Mice

[0413] The experiment used SPF male C57B / 6N-Tg(1.28HBV) / Vst mice aged 6-8 weeks (Beijing Weitongda Biotechnology Co., Ltd., Animal Production Certificate SCXK(Beijing)2019-0002). The mice were kept in single cages, and the use and testing of animal feed, bedding and drinking water were carried out in accordance with GB14925-2010 "Experimental Animal Environment and Facilities".

[0414] The animals were tested after one week of acclimatization. According to the quantitative test results of serum HBsAg (primary) and HBV DNA (secondary), the animals were randomly divided into 14 groups, with 5 animals in each group. The day of administration was recorded as D1, and blood was collected before administration on the day of administration for the determination of HBsAg, HBV DNA and HBeAg.

[0415] All animals were given subcutaneous injections on D1, all as a single injection, with a dose of 3 mg / kg and a volume of 5 mL / kg. A control group was given a saline solution, which was injected with the same dose of saline. The animals in each group were observed 15 minutes to 1 hour after the last animal in each group was given the drug.

[0416] On the 8th, 15th, 22nd and 29th days after administration, 100 μL of blood was collected from the eye sockets and centrifuged to separate the serum. 10 μL of serum was diluted 50 times with PBS and sent for testing. The diluted serum HBsAg, HBeAg and HBV DNA were tested by Beijing Dian Medical Testing Laboratory Co., Ltd.

[0417] The experimental data were statistically analyzed using Graphpad prism statistical analysis software. The calculation of the residual inhibition rate of HBV DNA, HBsAg and HBeAg was based on the average of the two measured values ​​before the drug: residual inhibition rate = different time determination values ​​ / pre-drug basic value * 100%.

[0418] The experimental results are shown in Table 8 and Figure 3 .

[0419] Table 8: Residual rates of serum HBsAg, HBeAg and HBV DNA in HBV transgenic mice after a single subcutaneous administration of 3 mg / kg

[0420]

[0421]

[0422] from Figure 3 As shown in the test results of Table 8, the tested compounds have certain inhibitory effects on HBsAg, HBeAg and HBV DNA in the serum of HBV transgenic mice, among which AL0107020, AL0107023, AL0107024, AL0107025, AL0107026, AL0107029 and AL01070230, a total of 8 compounds, have a maximum inhibition rate of HBsAg greater than 95%, and the maximum inhibition rate of AL0107030 is greater than 99%. At the same time, these compounds also show a significant reduction effect on HBeAg and HBV DNA.

[0423] Example 7 Evaluation of the Anti-HBV Activity of Compounds in Vivo Using HBV Transgenic Mice

[0424] The experiment used SPF male C57B / 6N-Tg(1.28HBV) / Vst mice aged 6-8 weeks (Beijing Weitongda Biotechnology Co., Ltd., Animal Production Certificate SCXK(Beijing)2019-0002). The mice were kept in single cages, and the use and testing of animal feed, bedding and drinking water were carried out in accordance with GB14925-2010 "Experimental Animal Environment and Facilities".

[0425] The animals were tested after one week of acclimatization. According to the quantitative test results of mouse serum HBsAg (primary) and HBV DNA (secondary), the animals were randomly divided into 18 groups, with 5 animals in each group. The day of drug administration was recorded as D1, and blood was collected before drug administration on the day of drug administration for the determination of HBsAg, HBV DNA and HBeAg.

[0426] All animals were given subcutaneous injections on D1, all as a single injection, with a dose of 3 mg / kg and a volume of 5 mL / kg. A control group was given a saline solution, which was injected with the same dose of saline. The animals in each group were observed 15 minutes to 1 hour after the last animal in each group was given the drug.

[0427] On the 8th, 15th, 22nd and 29th days after administration, 100 μL of blood was collected from the eye sockets and centrifuged to separate the serum. 10 μL of serum was diluted 50 times with PBS and sent for testing. The diluted serum HBsAg, HBeAg and HBV DNA were tested by Beijing Dian Medical Testing Laboratory Co., Ltd.

[0428] The experimental data were statistically analyzed using Graphpad prism statistical analysis software. The calculation of the residual inhibition rate of HBV DNA, HBsAg and HBeAg was based on the average of the two measured values ​​before the drug: residual inhibition rate = different time determination values ​​ / pre-drug basic value * 100%.

[0429] The experimental results are shown in Table 9 and Figure 4 .

[0430] Table 9: Residual rates of serum HBsAg, HBeAg and HBV DNA in HBV transgenic mice after a single subcutaneous administration of 3 mg / kg

[0431]

[0432]

[0433] from Figure 4From the test results in Table 9, it can be seen that the tested compounds have a certain inhibitory efficiency on HBsAg, HBeAg and HBV DNA in the serum of HBV transgenic mice, among which 14 compounds including AL0107002, AL0107004, AL0107025, AL0107029, AL0107030, AL0107032, AL0107033, AL0107035, AL0107036, AL0107037, AL0107038, AL0107039, AL0107041 and AL0107042 have a certain inhibitory efficiency on HBs The maximum inhibition rates of Ag were all greater than 90%, among which 11 compounds, including AL0107004, AL0107025, AL0107029, AL0107030, AL0107035, AL0107036, AL0107037, AL0107038, AL0107039, AL0107041 and AL0107042, had a maximum inhibition rate of greater than 95% on HBsAg, and a maximum inhibition efficiency of nearly 99%. At the same time, these compounds also showed a significant reduction effect on HBeAg and HBV DNA.

[0434] Example 8 Evaluation of the Anti-HBV Activity of Compounds at Different Concentrations Using HBV Transgenic Mice

[0435] The experiment used SPF male C57B / 6N-Tg(1.28HBV) / Vst mice aged 6-8 weeks (Beijing Weitongda Biotechnology Co., Ltd., Animal Production Certificate SCXK(Beijing)2019-0002). The mice were kept in single cages, and the use and testing of animal feed, bedding and drinking water were carried out in accordance with GB14925-2010 "Experimental Animal Environment and Facilities".

[0436] The animals were tested after one week of acclimatization. According to the quantitative test results of serum HBsAg (primary) and HBV DNA (secondary), the animals were randomly divided into 9 groups, with 5 animals in each group. The day of administration was recorded as D1, and blood was collected before administration on the day of administration for the determination of HBsAg, HBV DNA and HBeAg.

[0437] All animals were given subcutaneous injections on D1, all in a single injection, with doses of 1 mg / kg, 3 mg / kg and 9 mg / kg for AL0107039 and AL0107044, and 3 mg / kg for AL0107043 and AL0107045, with a volume of 5 mL / kg, and a control group of animals injected with the same dose of saline. The animals in each group were observed 15 minutes to 1 hour after the last animal in each group was given the drug.

[0438] On days 8, 15, 22, 29, 35, and 42 after administration, 100 μL of blood was collected from the eye sockets and centrifuged to separate serum. 10 μL of serum was diluted 50 times with PBS and sent for testing. The diluted serum HBsAg, HBeAg, and HBV DNA were tested by Beijing Dian Medical Testing Laboratory Co., Ltd.

[0439] The experimental data were statistically analyzed using Graphpad prism statistical analysis software. The calculation of the residual inhibition rate of HBV DNA, HBsAg and HBeAg was based on the average of the two measured values ​​before the drug: residual inhibition rate = different time determination values ​​ / pre-drug basic value * 100%.

[0440] The experimental results are shown in Table 10 and Figure 5 .

[0441] Table 10: Residual rates of serum HBsAg, HBeAg and HBV DNA in HBV transgenic mice after single subcutaneous administration of different concentrations

[0442]

[0443]

[0444] from Figure 5 As shown in the test results of Table 10, the two compounds AL0107039 and AL0107044 have significant inhibitory effects on HBsAg, HBV DNA and HBeAg in HBV transgenic mice, and have obvious dose dependence. The lowest dose of 1 mg / kg can show significant inhibitory efficiency. The inhibitory efficiency of the two compounds AL0107043 and AL0107045 at 3 mg / kg is equivalent to the inhibitory efficiency of the two compounds AL0107039 and AL0107044 at 9 mg / kg, showing a stronger inhibitory effect than the latter two compounds. Among them, after Day 15, the inhibitory effect of 3 mg / kg of AL0107045 was significantly better than the inhibitory efficiency of 9 mg / kg of AL0107039 and AL0107044.

[0445] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0446] The above-mentioned embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be subject to the attached claims.

Claims

1. An oligonucleotide for inhibiting HBV or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises an antisense strand having a sequence as shown in any one of SEQ ID NOs: 251-345 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof; Preferably, the oligonucleotide further comprises a sense strand having a sequence as shown in any one of SEQ ID NOs: 1-95 or a fragment thereof, or a modified sequence of the sequence or the fragment thereof; Preferably, the modified sequence of the antisense strand comprises a sequence as shown in any one of SEQ ID NOs: 346-459 or a fragment thereof; Preferably, the modified sequence of the sense strand comprises a sequence as shown in any one of SEQ ID NOs: 96-209 or a fragment thereof.

2. The oligonucleotide of claim 1 or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises at least one modified nucleotide; Preferably, the modified nucleotide comprises at least one modification as described below; 2'-methoxyethyl modified (moe) nucleotides, 2'-methoxy (m) modified nucleotides, 2'-deoxy-2'-fluoro (f) modified nucleotides, phosphorothioate (s) group modified nucleotides and 5'-phosphate mimetic modified nucleotides; Preferably, the 5'-phosphate mimetic is 5'-oxymethylphosphonate, 5'-vinylphosphonic acid (Vp) or 5'-malonylphosphonate; more preferably 5'-vinylphosphonic acid (Vp); Preferably, the 5'-phosphate mimetic is modified to the 5'-end of the antisense strand; Preferably, all nucleotides of the oligonucleotide are modified.

3. The oligonucleotide of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises at least one modified internucleotide bond; Preferably, said at least one modified internucleotide bond is a phosphorothioate bond.

4. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the oligonucleotide comprises a combination of a sense strand and an antisense strand selected from the group consisting of: (1) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 251; (2) the sense strand set forth in SEQ ID NO:2, and the antisense strand set forth in SEQ ID NO:252; (3) the sense strand set forth in SEQ ID NO:4, and the antisense strand set forth in SEQ ID NO:254; (4) the sense strand set forth in SEQ ID NO:6, and the antisense strand set forth in SEQ ID NO:256; (5) the sense strand set forth in SEQ ID NO:7, and the antisense strand set forth in SEQ ID NO:257; (6) the sense strand set forth in SEQ ID NO:8, and the antisense strand set forth in SEQ ID NO:258; (7) the sense strand shown in SEQ ID NO:9, and the antisense strand shown in SEQ ID NO:259; (8) the sense strand shown in SEQ ID NO:10, and the antisense strand shown in SEQ ID NO:260; (9) the sense strand shown in SEQ ID NO:11, and the antisense strand shown in SEQ ID NO:261; (10) the sense strand represented by SEQ ID NO:12, and the antisense strand represented by SEQ ID NO:262; (11) the sense strand shown in SEQ ID NO:13, and the antisense strand shown in SEQ ID NO:263; (12) the sense strand represented by SEQ ID NO:14, and the antisense strand represented by SEQ ID NO:264; (13) the sense strand represented by SEQ ID NO:15, and the antisense strand represented by SEQ ID NO:265; (14) the sense strand represented by SEQ ID NO:16, and the antisense strand represented by SEQ ID NO:266; (15) the sense strand represented by SEQ ID NO:17, and the antisense strand represented by SEQ ID NO:267; (16) the sense strand represented by SEQ ID NO:18, and the antisense strand represented by SEQ ID NO:268; (17) the sense strand represented by SEQ ID NO:19, and the antisense strand represented by SEQ ID NO:269; (18) the sense strand shown in SEQ ID NO:20, and the antisense strand shown in SEQ ID NO:270; (19) the sense strand represented by SEQ ID NO:21, and the antisense strand represented by SEQ ID NO:271; (20) the sense strand represented by SEQ ID NO:22, and the antisense strand represented by SEQ ID NO:272; (21) the sense strand shown in SEQ ID NO:23, and the antisense strand shown in SEQ ID NO:273; (22) the sense strand shown in SEQ ID NO:24, and the antisense strand shown in SEQ ID NO:274; (23) the sense strand represented by SEQ ID NO:25, and the antisense strand represented by SEQ ID NO:275; (24) the sense strand represented by SEQ ID NO:26, and the antisense strand represented by SEQ ID NO:276; (25) the sense strand represented by SEQ ID NO:39, and the antisense strand represented by SEQ ID NO:289; (26) the sense strand represented by SEQ ID NO:40, and the antisense strand represented by SEQ ID NO:290; (27) the sense strand represented by SEQ ID NO:41, and the antisense strand represented by SEQ ID NO:291; (28) the sense strand represented by SEQ ID NO:42, and the antisense strand represented by SEQ ID NO:292; (29) the sense strand represented by SEQ ID NO:46, and the antisense strand represented by SEQ ID NO:296; (30) the sense strand represented by SEQ ID NO:48, and the antisense strand represented by SEQ ID NO:298; (31) the sense strand represented by SEQ ID NO:54, and the antisense strand represented by SEQ ID NO:304; (32) the sense strand represented by SEQ ID NO:58, and the antisense strand represented by SEQ ID NO:308; (33) the sense strand represented by SEQ ID NO:59, and the antisense strand represented by SEQ ID NO:309; (34) the sense strand represented by SEQ ID NO:60, and the antisense strand represented by SEQ ID NO:310; (35) the sense strand represented by SEQ ID NO:61, and the antisense strand represented by SEQ ID NO:311; (36) the sense strand represented by SEQ ID NO:62, and the antisense strand represented by SEQ ID NO:312; (37) the sense strand represented by SEQ ID NO:63, and the antisense strand represented by SEQ ID NO:313; (38) the sense strand represented by SEQ ID NO:64, and the antisense strand represented by SEQ ID NO:314; (39) the sense strand represented by SEQ ID NO:67, and the antisense strand represented by SEQ ID NO:317; (40) the sense strand represented by SEQ ID NO:69, and the antisense strand represented by SEQ ID NO:319; (41) the sense strand represented by SEQ ID NO:70, and the antisense strand represented by SEQ ID NO:320; (42) the sense strand represented by SEQ ID NO:71, and the antisense strand represented by SEQ ID NO:321; (43) the sense strand represented by SEQ ID NO:72, and the antisense strand represented by SEQ ID NO:322; (44) the sense strand represented by SEQ ID NO:73, and the antisense strand represented by SEQ ID NO:323; (45) the sense strand represented by SEQ ID NO:74, and the antisense strand represented by SEQ ID NO:324; (46) the sense strand represented by SEQ ID NO:75, and the antisense strand represented by SEQ ID NO:325; (47) the sense strand represented by SEQ ID NO:76, and the antisense strand represented by SEQ ID NO:326; (48) the sense strand represented by SEQ ID NO:77, and the antisense strand represented by SEQ ID NO:327; (49) the sense strand represented by SEQ ID NO:78, and the antisense strand represented by SEQ ID NO:328; (50) the sense strand represented by SEQ ID NO:79, and the antisense strand represented by SEQ ID NO:329; (51) the sense strand represented by SEQ ID NO:80, and the antisense strand represented by SEQ ID NO:330; (52) the sense strand represented by SEQ ID NO:81, and the antisense strand represented by SEQ ID NO:331; (53) the sense strand represented by SEQ ID NO:82, and the antisense strand represented by SEQ ID NO:332; (54) the sense strand represented by SEQ ID NO:83, and the antisense strand represented by SEQ ID NO:333; (55) the sense strand represented by SEQ ID NO:84, and the antisense strand represented by SEQ ID NO:334; (56) the sense strand represented by SEQ ID NO:85, and the antisense strand represented by SEQ ID NO:335; (57) the sense strand represented by SEQ ID NO:86, and the antisense strand represented by SEQ ID NO:336; (58) the sense strand represented by SEQ ID NO:91, and the antisense strand represented by SEQ ID NO:341; (59) the sense strand represented by SEQ ID NO:92, and the antisense strand represented by SEQ ID NO:342; (60) the sense strand represented by SEQ ID NO:94, and the antisense strand represented by SEQ ID NO:344; (61) The sense strand represented by SEQ ID NO:95, and the antisense strand represented by SEQ ID NO:

345.

5. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the oligonucleotide comprises a combination of a sense strand and an antisense strand selected from the group consisting of: (1) the sense strand shown in SEQ ID NO:96, and the antisense strand shown in SEQ ID NO:346; (2) the sense strand set forth in SEQ ID NO:97, and the antisense strand set forth in SEQ ID NO:347; (3) the sense strand shown in SEQ ID NO:99, and the antisense strand shown in SEQ ID NO:349; (4) the sense strand shown in SEQ ID NO: 101, and the antisense strand shown in SEQ ID NO: 351; (5) the sense strand set forth in SEQ ID NO:102, and the antisense strand set forth in SEQ ID NO:352; (6) the sense strand shown in SEQ ID NO:103, and the antisense strand shown in SEQ ID NO:353; (7) the sense strand shown in SEQ ID NO:104, and the antisense strand shown in SEQ ID NO:354; (8) the sense strand represented by SEQ ID NO:105, and the antisense strand represented by SEQ ID NO:355; (9) the sense strand represented by SEQ ID NO:106, and the antisense strand represented by SEQ ID NO:356; (10) the sense strand represented by SEQ ID NO:107, and the antisense strand represented by SEQ ID NO:357; (11) the sense strand shown in SEQ ID NO:108, and the antisense strand shown in SEQ ID NO:358; (12) the sense strand shown in SEQ ID NO:109, and the antisense strand shown in SEQ ID NO:359; (13) the sense strand shown in SEQ ID NO: 110, and the antisense strand shown in SEQ ID NO: 360; (14) the sense strand represented by SEQ ID NO:111, and the antisense strand represented by SEQ ID NO:361; (15) the sense strand represented by SEQ ID NO:112, and the antisense strand represented by SEQ ID NO:362; (16) the sense strand represented by SEQ ID NO:113, and the antisense strand represented by SEQ ID NO:363; (17) the sense strand represented by SEQ ID NO:114, and the antisense strand represented by SEQ ID NO:364; (18) the sense strand represented by SEQ ID NO:115, and the antisense strand represented by SEQ ID NO:365; (19) the sense strand represented by SEQ ID NO:116, and the antisense strand represented by SEQ ID NO:366; (20) the sense strand represented by SEQ ID NO:117, and the antisense strand represented by SEQ ID NO:367; (21) the sense strand represented by SEQ ID NO:118, and the antisense strand represented by SEQ ID NO:368; (22) the sense strand represented by SEQ ID NO:119, and the antisense strand represented by SEQ ID NO:369; (23) the sense strand represented by SEQ ID NO:120, and the antisense strand represented by SEQ ID NO:370; (24) the sense strand represented by SEQ ID NO:121, and the antisense strand represented by SEQ ID NO:371; (25) the sense strand represented by SEQ ID NO:138, and the antisense strand represented by SEQ ID NO:388; (26) the sense strand represented by SEQ ID NO:139, and the antisense strand represented by SEQ ID NO:389; (27) the sense strand represented by SEQ ID NO:140, and the antisense strand represented by SEQ ID NO:390; (28) the sense strand represented by SEQ ID NO:141, and the antisense strand represented by SEQ ID NO:391; (29) the sense strand represented by SEQ ID NO:145, and the antisense strand represented by SEQ ID NO:395; (30) the sense strand represented by SEQ ID NO:147, and the antisense strand represented by SEQ ID NO:397; (31) the sense strand represented by SEQ ID NO:153, and the antisense strand represented by SEQ ID NO:403; (32) the sense strand represented by SEQ ID NO:154, and the antisense strand represented by SEQ ID NO:404; (33) the sense strand represented by SEQ ID NO:155, and the antisense strand represented by SEQ ID NO:405; (34) the sense strand represented by SEQ ID NO:156, and the antisense strand represented by SEQ ID NO:406; (35) the sense strand represented by SEQ ID NO:157, and the antisense strand represented by SEQ ID NO:407; (36) the sense strand represented by SEQ ID NO:158, and the antisense strand represented by SEQ ID NO:408; (37) the sense strand represented by SEQ ID NO:159, and the antisense strand represented by SEQ ID NO:409; (38) the sense strand represented by SEQ ID NO:160, and the antisense strand represented by SEQ ID NO:410; (39) the sense strand represented by SEQ ID NO:163, and the antisense strand represented by SEQ ID NO:413; (40) the sense strand represented by SEQ ID NO:165, and the antisense strand represented by SEQ ID NO:415; (41) the sense strand represented by SEQ ID NO:166, and the antisense strand represented by SEQ ID NO:416; (42) the sense strand represented by SEQ ID NO:167, and the antisense strand represented by SEQ ID NO:417; (43) the sense strand represented by SEQ ID NO:168, and the antisense strand represented by SEQ ID NO:418; (44) the sense strand represented by SEQ ID NO:169, and the antisense strand represented by SEQ ID NO:419; (45) the sense strand represented by SEQ ID NO: 170, and the antisense strand represented by SEQ ID NO: 420; (46) the sense strand represented by SEQ ID NO: 171, and the antisense strand represented by SEQ ID NO: 421; (47) the sense strand represented by SEQ ID NO:172, and the antisense strand represented by SEQ ID NO:422; (48) the sense strand represented by SEQ ID NO:173, and the antisense strand represented by SEQ ID NO:423; (49) the sense strand represented by SEQ ID NO:174, and the antisense strand represented by SEQ ID NO:424; (50) the sense strand represented by SEQ ID NO:175, and the antisense strand represented by SEQ ID NO:425; (51) the sense strand represented by SEQ ID NO: 176, and the antisense strand represented by SEQ ID NO: 426; (52) the sense strand represented by SEQ ID NO: 177, and the antisense strand represented by SEQ ID NO: 427; (53) the sense strand represented by SEQ ID NO:178, and the antisense strand represented by SEQ ID NO:428; (54) the sense strand represented by SEQ ID NO: 179, and the antisense strand represented by SEQ ID NO: 429; (55) the sense strand represented by SEQ ID NO: 180, and the antisense strand represented by SEQ ID NO: 430; (56) the sense strand represented by SEQ ID NO: 181, and the antisense strand represented by SEQ ID NO: 431; (57) the sense strand represented by SEQ ID NO:182, and the antisense strand represented by SEQ ID NO:432; (58) the sense strand represented by SEQ ID NO:183, and the antisense strand represented by SEQ ID NO:433; (59) the sense strand represented by SEQ ID NO:184, and the antisense strand represented by SEQ ID NO:434; (60) the sense strand represented by SEQ ID NO:185, and the antisense strand represented by SEQ ID NO:435; (61) the sense strand represented by SEQ ID NO: 190, and the antisense strand represented by SEQ ID NO: 440; (62) the sense strand represented by SEQ ID NO: 191, and the antisense strand represented by SEQ ID NO: 441; (63) the sense strand represented by SEQ ID NO:193, and the antisense strand represented by SEQ ID NO:443; (64) the sense strand represented by SEQ ID NO: 194, and the antisense strand represented by SEQ ID NO: 444; (65) the sense strand represented by SEQ ID NO: 197, and the antisense strand represented by SEQ ID NO: 447; (66) the sense strand represented by SEQ ID NO: 199, and the antisense strand represented by SEQ ID NO: 449; (67) the sense strand represented by SEQ ID NO:200, and the antisense strand represented by SEQ ID NO:450; (68) the sense strand represented by SEQ ID NO:201, and the antisense strand represented by SEQ ID NO:451; (69) the sense strand represented by SEQ ID NO:202, and the antisense strand represented by SEQ ID NO:452; (70) the sense strand represented by SEQ ID NO:203, and the antisense strand represented by SEQ ID NO:453; (71) the sense strand represented by SEQ ID NO:205, and the antisense strand represented by SEQ ID NO:435; (72) the sense strand represented by SEQ ID NO:206, and the antisense strand represented by SEQ ID NO:456; (73) the sense strand represented by SEQ ID NO:207, and the antisense strand represented by SEQ ID NO:457; (74) the sense strand represented by SEQ ID NO:208, and the antisense strand represented by SEQ ID NO:458; (75) The sense strand represented by SEQ ID NO:209, and the antisense strand represented by SEQ ID NO:

459.

6. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the oligonucleotide comprises a combination of a sense strand and an antisense strand selected from the group consisting of: (1) the sense strand shown in SEQ ID NO:97, and the antisense strand shown in SEQ ID NO:347; (2) the sense strand shown in SEQ ID NO: 101, and the antisense strand shown in SEQ ID NO: 351; (3) the sense strand shown in SEQ ID NO: 102, and the antisense strand shown in SEQ ID NO: 352; (4) the sense strand set forth in SEQ ID NO:103, and the antisense strand set forth in SEQ ID NO:353; (5) the sense strand set forth in SEQ ID NO:104, and the antisense strand set forth in SEQ ID NO:354; (6) the sense strand set forth in SEQ ID NO:105, and the antisense strand set forth in SEQ ID NO:355; (7) the sense strand represented by SEQ ID NO:106, and the antisense strand represented by SEQ ID NO:356; (8) the sense strand shown in SEQ ID NO:108, and the antisense strand shown in SEQ ID NO:358; (9) the sense strand shown in SEQ ID NO:109, and the antisense strand shown in SEQ ID NO:359; (10) the sense strand shown in SEQ ID NO:110, and the antisense strand shown in SEQ ID NO:360; (11) the sense strand shown in SEQ ID NO:111, and the antisense strand shown in SEQ ID NO:361; (12) the sense strand shown in SEQ ID NO:112, and the antisense strand shown in SEQ ID NO:362; (13) the sense strand shown in SEQ ID NO:113, and the antisense strand shown in SEQ ID NO:363; (14) the sense strand represented by SEQ ID NO:114, and the antisense strand represented by SEQ ID NO:364; (15) the sense strand represented by SEQ ID NO:115, and the antisense strand represented by SEQ ID NO:365; (16) the sense strand represented by SEQ ID NO:116, and the antisense strand represented by SEQ ID NO:366; (17) the sense strand represented by SEQ ID NO:117, and the antisense strand represented by SEQ ID NO:367; (18) the sense strand represented by SEQ ID NO:118, and the antisense strand represented by SEQ ID NO:368; (19) the sense strand represented by SEQ ID NO:119, and the antisense strand represented by SEQ ID NO:369; (20) the sense strand represented by SEQ ID NO:166, and the antisense strand represented by SEQ ID NO:416; (21) the sense strand represented by SEQ ID NO:167, and the antisense strand represented by SEQ ID NO:417; (22) the sense strand represented by SEQ ID NO:168, and the antisense strand represented by SEQ ID NO:418; (23) the sense strand represented by SEQ ID NO:169, and the antisense strand represented by SEQ ID NO:419; (24) the sense strand represented by SEQ ID NO: 170, and the antisense strand represented by SEQ ID NO: 420; (25) the sense strand represented by SEQ ID NO: 171, and the antisense strand represented by SEQ ID NO: 421; (26) the sense strand represented by SEQ ID NO:172, and the antisense strand represented by SEQ ID NO:422; (27) the sense strand represented by SEQ ID NO:173, and the antisense strand represented by SEQ ID NO:423; (28) the sense strand represented by SEQ ID NO:174, and the antisense strand represented by SEQ ID NO:424; (29) the sense strand represented by SEQ ID NO:175, and the antisense strand represented by SEQ ID NO:425; (30) the sense strand represented by SEQ ID NO:176, and the antisense strand represented by SEQ ID NO:426; (31) the sense strand represented by SEQ ID NO:177, and the antisense strand represented by SEQ ID NO:427; (32) the sense strand represented by SEQ ID NO:178, and the antisense strand represented by SEQ ID NO:428; (33) the sense strand represented by SEQ ID NO:179, and the antisense strand represented by SEQ ID NO:429; (34) the sense strand represented by SEQ ID NO: 180, and the antisense strand represented by SEQ ID NO: 430; (35) the sense strand represented by SEQ ID NO:181, and the antisense strand represented by SEQ ID NO:431; (36) the sense strand represented by SEQ ID NO:182, and the antisense strand represented by SEQ ID NO:432; (37) the sense strand represented by SEQ ID NO:183, and the antisense strand represented by SEQ ID NO:433; (38) the sense strand represented by SEQ ID NO:184, and the antisense strand represented by SEQ ID NO:434; (39) the sense strand represented by SEQ ID NO:185, and the antisense strand represented by SEQ ID NO:435; (40) the sense strand represented by SEQ ID NO: 190, and the antisense strand represented by SEQ ID NO: 440; (41) The sense strand represented by SEQ ID NO:191, and the antisense strand represented by SEQ ID NO:

441.

7. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein the oligonucleotide comprises a combination of a sense strand and an antisense strand selected from the group consisting of: (1) the sense strand shown in SEQ ID NO:210, and the antisense strand shown in SEQ ID NO:351; (2) the sense strand set forth in SEQ ID NO:211, and the antisense strand set forth in SEQ ID NO:355; (3) the sense strand set forth in SEQ ID NO:212, and the antisense strand set forth in SEQ ID NO:359; (4) the sense strand set forth in SEQ ID NO:213, and the antisense strand set forth in SEQ ID NO:361; (5) the sense strand set forth in SEQ ID NO:214, and the antisense strand set forth in SEQ ID NO:362; (6) the sense strand set forth in SEQ ID NO:215, and the antisense strand set forth in SEQ ID NO:363; (7) the sense strand set forth in SEQ ID NO:216, and the antisense strand set forth in SEQ ID NO:365; (8) the sense strand represented by SEQ ID NO:217, and the antisense strand represented by SEQ ID NO:366; (9) the sense strand shown in SEQ ID NO:218, and the antisense strand shown in SEQ ID NO:367; (10) the sense strand represented by SEQ ID NO:219, and the antisense strand represented by SEQ ID NO:361; (11) the sense strand shown in SEQ ID NO:220, and the antisense strand shown in SEQ ID NO:374; (12) the sense strand represented by SEQ ID NO:221, and the antisense strand represented by SEQ ID NO:376; (13) the sense strand represented by SEQ ID NO:222, and the antisense strand represented by SEQ ID NO:377; (14) the sense strand represented by SEQ ID NO:223, and the antisense strand represented by SEQ ID NO:375; (15) the sense strand represented by SEQ ID NO:224, and the antisense strand represented by SEQ ID NO:388; (16) the sense strand represented by SEQ ID NO:225, and the antisense strand represented by SEQ ID NO:391; (17) the sense strand represented by SEQ ID NO:226, and the antisense strand represented by SEQ ID NO:395; (18) the sense strand represented by SEQ ID NO:227, and the antisense strand represented by SEQ ID NO:443; (19) the sense strand represented by SEQ ID NO:228, and the antisense strand represented by SEQ ID NO:407; (20) the sense strand represented by SEQ ID NO:229, and the antisense strand represented by SEQ ID NO:410; (21) the sense strand represented by SEQ ID NO:230, and the antisense strand represented by SEQ ID NO:416; (22) the sense strand represented by SEQ ID NO:231, and the antisense strand represented by SEQ ID NO:444; (23) the sense strand represented by SEQ ID NO:232, and the antisense strand represented by SEQ ID NO:422; (24) the sense strand represented by SEQ ID NO:233, and the antisense strand represented by SEQ ID NO:429; (25) the sense strand represented by SEQ ID NO:234, and the antisense strand represented by SEQ ID NO:432; (26) the sense strand represented by SEQ ID NO:235, and the antisense strand represented by SEQ ID NO:441; (27) the sense strand represented by SEQ ID NO:236, and the antisense strand represented by SEQ ID NO:445; (28) the sense strand represented by SEQ ID NO:237, and the antisense strand represented by SEQ ID NO:446; (29) the sense strand represented by SEQ ID NO:238, and the antisense strand represented by SEQ ID NO:447; (30) the sense strand represented by SEQ ID NO:239, and the antisense strand represented by SEQ ID NO:448; (31) the sense strand represented by SEQ ID NO:240, and the antisense strand represented by SEQ ID NO:449; (32) the sense strand represented by SEQ ID NO:241, and the antisense strand represented by SEQ ID NO:450; (33) the sense strand represented by SEQ ID NO:242, and the antisense strand represented by SEQ ID NO:451; (34) the sense strand represented by SEQ ID NO:243, and the antisense strand represented by SEQ ID NO:452; (35) the sense strand represented by SEQ ID NO:244, and the antisense strand represented by SEQ ID NO:453; (36) the sense strand represented by SEQ ID NO:245, and the antisense strand represented by SEQ ID NO:454; (37) the sense strand represented by SEQ ID NO:246, and the antisense strand represented by SEQ ID NO:455; (38) the sense strand represented by SEQ ID NO:247, and the antisense strand represented by SEQ ID NO:456; (39) the sense strand represented by SEQ ID NO:248, and the antisense strand represented by SEQ ID NO:457; (40) the sense strand represented by SEQ ID NO:249, and the antisense strand represented by SEQ ID NO:458; (41) The sense strand represented by SEQ ID NO:250, and the antisense strand represented by SEQ ID NO:

459.

8. The oligonucleotide of any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands; Preferably, the targeting ligand is conjugated to the sense strand of the oligonucleotide at the 3'-end; Preferably, the targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide or a lipid; Preferably, the targeting ligand comprises an amino sugar; Preferably, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety; Preferably, wherein the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety or a tetravalent GalNAc moiety; Preferably, the targeting ligand has the following structure: Preferably, the targeting ligand is conjugated to the sense strand of the oligonucleotide at the 3'-end via a linker to form the following conjugate: X may be selected from S or O.

9. A composition comprising the oligonucleotide according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

10. Use of the oligonucleotide according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof or the composition according to claim 9 in the preparation of a medicament for reducing at least one of HBsAg, HBeAg and HBV DNA in serum.

11. Use of the oligonucleotide according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof or the composition according to claim 98 in the preparation of a medicament for treating HBV infection or hepatitis B virus, and / or hepatitis B virus combined with hepatitis D virus infection; Preferably, the HBV infection is a chronic HBV infection; Preferably, the hepatitis B virus is chronic hepatitis B virus.

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