siRNA for inhibition of hepatitis B virus and its application

Abstract

Provided are an siRNA for inhibiting hepatitis B virus and the use thereof. Specifically provided is an oligonucleotide for inhibiting HBV or a pharmaceutically acceptable salt thereof. The oligonucleotide comprises an antisense strand, and the antisense strand comprises at least one modified nucleotide, wherein the nucleotide sequence of the antisense strand comprises a sequence or a fragment as set forth in any one of SEQ ID NOs: 3-35. The siRNA has a significant inhibitory effect on HBsAg, HBV DNA and HBeAg produced by cccDNA and HBV DNA integrated into the host genome, both in vivo and in vitro, and has lasting efficacy. Compared with an original sequence, the optimized sequence not only improves the inhibitory effect and the efficacy cycle, but also reduces the off-target toxicity.

Description

siRNA for inhibiting hepatitis B virus and its use

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 2023112745419 filed on September 28, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure belongs to the field of biomedicine and relates to a small interfering RNA (siRNA) for inhibiting hepatitis B virus and its use, specifically comprising double-stranded modified siRNA, and conjugates and salts thereof; the use is for preparing drugs for treating hepatitis B virus or chronic infection. Background Art

[0004] Hepatitis B is a viral disease caused by the hepatitis B virus (HBV). It is primarily transmitted through blood, sexual intercourse, and mother-to-child transmission. The World Health Organization estimates that approximately 250 million people worldwide have chronic HBV infection. After several years of infection, approximately 15-40% of patients with chronic HBV infection develop severe adverse outcomes, such as cirrhosis, liver failure, and hepatocellular carcinoma. Currently, nearly one million patients die annually from HBV-related complications (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 continuously 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). ​​Currently, 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.

[0005] 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 sustained disappearance of HBsAg after drug discontinuation, HBV DNA below the detection limit, normal liver enzymes, and improvement of liver tissue pathology. Among them, HBsAg disappearance 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.

[0006] In 1998, two American scientists, Andrew Fire and Craig Mello, discovered a novel biological mechanism: small RNA molecules can mediate the degradation of specific mRNAs (Fire, Andrew, et al. Nature 391, 6669 (1998): 806-811). This mechanism is activated when double-stranded RNA molecules are present in cells, resulting in RNA interference (RNA interference). This discovery heralded the beginning of a new research field, and earned the two scientists the 2006 Nobel Prize in Physiology or Medicine. When double-stranded RNA binds to the protein complex Dicer, Dicer cleaves the dsRNA into fragments. Another protein complex, RISC, then binds to these fragments. One strand of the siRNA double strand is removed, but the other strand remains bound to the RISC (RNA-induced silencing complex). Guided by the single-stranded RNA, RISC recognizes and degrades the target gene's mRNA, inhibiting the expression of a specific protein and thus specifically silencing the gene.

[0007] RNA interference has opened up a new area of ​​application for genetic technology. Double-stranded RNA molecules have been artificially designed to silence specific genes in humans, animals, or plants. These artificially designed double-stranded RNA molecules (siRNA) for gene silencing are introduced into cells and activate the RNA interference mechanism to degrade the corresponding mRNA. Currently, 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 siRNA therapies are in the research and development stage or have been approved for marketing, showing excellent therapeutic effects. Since the first siRNA drug was launched in 2018, at least four siRNAs have been approved for marketing in the European Union or the United States. Therefore, using RNA interference technology to inhibit the expression of specific target genes has become an effective way to treat diseases.

[0008] Currently, there are HBV siRNA drugs in the clinical stage, which have shown certain effects in inhibiting HBsAg, HBV DNA and HBeAg, but their toxicity and safety issues cannot be ignored. Therefore, it is urgent to develop HBV siRNA drugs with excellent effects and high safety.

[0009] Summary of the Invention

[0010] To address the problems existing in the prior art, the present invention provides an siRNA for inhibiting hepatitis B virus and its use. The siRNA exhibits significant in vivo and in vitro inhibitory effects on HBsAg, HBV DNA, and HBeAg produced by cccDNA and HBV DNA integrated into the host genome, with sustained efficacy. The optimized sequence, compared to the original sequence, not only enhances the inhibitory effect and duration of efficacy but also reduces off-target toxicity.

[0011] In one aspect, the present disclosure provides an oligonucleotide for inhibiting HBV or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises a sense chain and an antisense chain, wherein the nucleotide sequence of the sense chain comprises a sequence or fragment shown in SEQ ID NO.1; and the nucleotide sequence of the antisense chain comprises a sequence or fragment shown in SEQ ID NO.3.

[0012] 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.

[0013] In another aspect, the present disclosure provides use of the aforementioned oligonucleotide or a pharmaceutically acceptable salt and / or composition thereof in the preparation of a medicament for reducing at least one of HBsAg, HBeAg and HBV DNA in the serum of a subject.

[0014] In another aspect, the present disclosure provides use of the oligonucleotide or a pharmaceutically acceptable salt thereof and / or composition in the preparation of a medicament for treating HBV infection or hepatitis B, and hepatitis B virus combined with hepatitis D virus infection.

[0015] In another aspect, the present disclosure provides a method for reducing at least one of HBsAg, HBeAg, and HBV DNA in serum, comprising administering to a subject a therapeutically effective amount of the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof.

[0016] 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

[0017] Figure 1 shows the siRNA synthesis process.

[0018] FIG2 shows the inhibitory effects of a single subcutaneous administration of compounds at different concentrations on serum HBsAg, HBeAg and HBV DNA in HBV transgenic mice.

[0019] FIG3 shows the inhibitory effects of compounds AL0107045, AL0107048, AL0107049, AL0107050, AL0107051, AL0107052, AL0107053, and AL0107054 on serum HBV DNA, HBeAg, and HBsAg in HBV transgenic mice after a single subcutaneous administration.

[0020] FIG4 shows the inhibitory effects of compounds L0107045, AL0107057, and AL0107058 on serum HBV DNA, HBeAg, and HBsAg in HBV transgenic mice after a single subcutaneous administration.

[0021] FIG5 shows the effect curves of different compounds on body weight changes in male and female SD rats.

[0022] FIG6 shows the effects of different compounds on the expression of aspartate aminotransferase in male and female SD rats. DETAILED DESCRIPTION

[0023] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are common procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0024] As used herein, "about" or "approximately" refers to a value similar to the reference value. In certain embodiments, unless otherwise indicated or otherwise apparent from the context, the term "approximately" or "about" refers to a range of values ​​that fall 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 numeral would exceed 100% of the possible value).

[0025] 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 four open reading frames and seven proteins. The seven proteins encoded by HBV include small (S), medium (M), and large (L) surface antigens (HBsAg) or envelope (Env) proteins, pre-Core protein, core protein, viral polymerase (Pol), and HBx protein. 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. Pre-Core is a core protein with an N-terminal signal peptide and undergoes proteolytic processing at its N- and C-termini prior to secretion from infected cells, forming the so-called hepatitis B e-antigen (HBeAg). The HBx protein is required for efficient transcription of covalently closed circular DNA (cccDNA). HBx is not a viral structural protein. With the exception of the core and polymerase, which share mRNA, all viral proteins of HBV have their own mRNA. With the exception of the pre-Core protein, HBV viral proteins do not undergo post-translational proteolytic processing.

[0026] 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 can be a polypeptide, a fragment or epitope thereof, or a combination of multiple HBV polypeptides, portions or derivatives thereof. The HBV antigen is capable of eliciting 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 that protects the subject from the viral disease or infection. For example, the HBV antigen can comprise a polypeptide or 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).

[0027] 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, against 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 icosahedral capsids. 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.

[0028] As used herein, the term "hepatitis B virus-related disease" or "HBV-related disease" refers to 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.

[0029] As used herein, term " complementary " refers to the structural relationship that allows nucleotide to form base pairs with each other between nucleotide (for example, on relative nucleic acid or on two nucleotides on the relative region of single nucleic acid chain).For example, the purine nucleotides complementary to the pyrimidine nucleotides of 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 Watson-Crick (Watson-Crick) mode or in any other manner that allows to form a stable duplex.In some embodiments, two nucleic acids can have and be complementary to each other to form the nucleotide sequence of complementary region, as described herein.

[0030] As used herein, the term "(siRNA) off-target effects" (OTEs) refers to the non-specific effects of siRNA during its action, which may affect genes other than the target gene, leading to non-target gene silencing and resulting in siRNA off-target effects. Both the inherent properties of siRNA and its delivery method can lead to reduced specificity and the generation of OTEs. siRNA-related OTEs are divided into three major types: microRNA-like off-target effects, immune stimulation, and RNAi element saturation.

[0031] As used herein, the term "strand" refers to a single continuous sequence of nucleotides linked together by internucleotide bonds (e.g., phosphodiester bonds, phosphorothioate bonds). In some embodiments, the strand has two free ends, e.g., a 5'-end and a 3'-end.

[0032] As used herein, the term "deoxyribonucleotide" refers to a nucleotide that has a hydrogen at the 2' position of its pentose sugar compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide that has a modification or substitution of one or more atoms other than the 2' position, including a modification or substitution in or of a sugar, a phosphate group, or a base.

[0033] As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide that is substantially in duplex form. In some embodiments, the complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed between the antiparallel sequence of the nucleotides of the covalently separated nucleic acid chains. In some embodiments, the complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed between the antiparallel sequence of the nucleotides of the covalently attached nucleic acid chains. In some embodiments, the 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 (for example, via a hairpin) to provide the complementary antiparallel sequence of the nucleotides of 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 an overhang at one or both ends. In some embodiments, a double-stranded oligonucleotide comprises the antiparallel sequence of nucleotides, which are partially complementary, and therefore, can have one or more mispairings, and the mispairings can include internal mispairings or terminal mispairings.

[0034] As used herein, the term "oligonucleotide" refers to a short nucleic acid, for example, a short nucleic acid less than 100 nucleotides in length. The oligonucleotide can comprise ribonucleotides, deoxyribonucleotides and / or modified nucleotides, including, for example, modified ribonucleotides. The oligonucleotide can be single-stranded or double-stranded. The oligonucleotide may or may not have a duplex region. As one set of non-limiting examples, the oligonucleotide can 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.

[0035] As used herein, the term "nucleotide analogue" refers to non-standard nucleotides, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs can be modified at any position to change certain chemical properties of the nucleotide, but still retain the ability of the nucleotide analog to perform its intended function. The nucleotide analogs herein can be used as nucleotide monomers to replace the nucleotides on the main chain. Specifically, the nucleotide analogs include but are not limited to: 2'-methoxyethyl modified (moe) nucleotides, 2'-methoxy (m) modified nucleotides, 2'-deoxy-2'-fluoro (f) modified nucleotides, thiophosphate (s) modified nucleotides, adenosine-2' phosphate (A-2'-5'), uridine-2' phosphate (U-2'-5'), guanosine-2' phosphate (G-2'-5'), guanosine-diol nucleic acid (Ggn), adenosine-diol nucleic acid (Agn), N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide (GNF-BX) modified nucleotides or 5'-phosphate mimetic modified nucleotides.

[0036] 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: N-terminal, PAZ, MID, and PIWI, and two connecting domains: L1 and L2. It is a member of the Argonaute protein (AGO) family. AGO2 protein is widely expressed in organisms 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, thereby playing a key role in various pathophysiological processes. It is known that the 5'-terminal phosphate group can enhance the interaction between certain nucleic acid inhibitor molecules and AGO2 protein.

[0037] As used herein, (Agn) refers to adenosine-diol nucleic acid (GNA), (Cgn) refers to cytidine-diol nucleic acid (GNA), (Ggn) refers to guanosine-diol nucleic acid (GNA), and (Tgn) refers to thymidine-diol nucleic acid (GNA).

[0038] 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 the functional delivery of nucleic acids to target cells or reduce possible toxic side effects of nucleic acids, such as increasing their stability to nucleases and improving their affinity for target binding. The modified oligonucleotides herein may comprise one or more chemically modified ribonucleotides of either or both of the antisense and sense strands. The modification may be a modification on the nucleotide, a modification between nucleotides, or a substitution of a nucleotide analog. The modification on the nucleotide may be a chemical modification on the base, ribose and / or phosphate. The chemical modifications described herein include modifications on the nucleotide, modifications between nucleotides, or a substitution of a nucleotide analog. The modification described herein is selected from at least one of the following: 2'-methoxyethyl (moe) modification, 2'-methoxy (m) modification, 2'-deoxy-2'-fluoro (f) modification, phosphorothioate (s), adenosine-2' phosphate (A-2'-5'), uridine-2' phosphate (U-2'-5'), guanosine-2' phosphate (G-2'-5'), guanosine-diol nucleic acid (Ggn), adenosine-diol nucleic acid (Agn), uridine-diol nucleic acid (Ugn), N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide (GNF-BX), 5'-vinylphosphonic acid (Vp) modification or 5' vinylphosphonate-2'-N-acetyl modification. Specifically, the ribose modification includes any one or more of 2'-methoxyethyl modification (moe), 2'-methoxy (m) modification, N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide (GNF-BX) modification, 5'-vinylphosphonic acid (Vp) modification, 2'-deoxy-2'-fluoro (f) modification, and 5' vinylphosphonate-2'-N-acetyl modification. The modification between the nucleotides includes modification of the phosphodiester bond in the phosphate group, such as thiophosphate (s) modification. The thiophosphate modification is formed by replacing at least one oxygen atom in the phosphodiester bond with a sulfur atom. Herein, the replacement of the nucleoside analogs, such as adenosine-2' phosphate (A-2'-5') can replace the adenosine analog at the corresponding position, uridine-2' phosphate (U-2'-5') can replace the uridine analog at the corresponding position, guanosine-2' phosphate (G-2'-5') can replace the guanosine analog at the corresponding position, guanosine-diol nucleic acid (Ggn) can replace the guanosine analog at the corresponding position, adenosine-diol nucleic acid (Agn) can replace the adenosine analog at the corresponding position, uridine-diol nucleic acid (Ugn) can replace the uridine analog at the corresponding position, 5' vinylphosphonate-2'-N-acetyl-uridine (APU001) replaces the uridine analog at the corresponding position. The corresponding replacement of the specific positions of the nucleoside analogs in the present invention can be found in Tables 1 and 2 of the present invention.

[0039] As used herein, the term "inhibit" means that gene expression is reduced when the cell, cell population, or tissue is treated with the siRNA, siRNA conjugates, and pharmaceutical compositions of the present disclosure, compared to a cell, cell population, or tissue that has not been so treated. The term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "inhibit," and other similar terms, and includes any level of inhibition. Preferably, inhibition includes statistically significant inhibition or clinically significant inhibition.

[0040] As used herein, the term "pharmaceutical composition" refers to a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with a pharmaceutically acceptable excipient for administration to a mammal (eg, a human) in need thereof.

[0041] As used herein, "subject" refers to 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.

[0042] The term "therapeutically effective amount" refers to an amount of a compound or molecule of the present 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.

[0043] In one aspect, the present disclosure provides an oligonucleotide for inhibiting HBV or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises a sense chain and an antisense chain, wherein the nucleotide sequence of the sense chain comprises a sequence or fragment shown in SEQ ID NO.1; and the nucleotide sequence of the antisense chain comprises a sequence or fragment shown in SEQ ID NO.3.

[0044] In some embodiments, the antisense strand is further modified, and the modification is selected from: one or more of: 2'-methoxyethyl modification, 2'-methoxy (m) modification, 2'-deoxy-2'-fluoro (f) modification, thiophosphate (s) modification, adenosine-2' phosphate (A-2'-5') modification, uridine-2' phosphate (U-2'-5') modification, guanosine-2' phosphate (G-2'-5') modification, guanosine-diol nucleic acid (Ggn) modification, adenosine-diol nucleic acid (Agn) modification, N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide (GNF-BX) modification or 5'-phosphate mimetic modification.

[0045] In some embodiments, the modification is selected from one or more of a 2'-methoxyethyl modification, an adenosine-2' phosphate (A-2'-5') modification, a uridine-2' phosphate (U-2'-5') modification, a guanosine-2' phosphate (G-2'-5') modification, a guanosine-diol nucleic acid (Ggn) modification, an adenosine-diol nucleic acid (Agn) modification, an N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide (GNF-BX) modification, or a 5'-phosphate mimetic modification.

[0046] In some embodiments, any one of the nucleic acid positions 1 to 8 from the 5' end of the antisense strand is modified accordingly.

[0047] In some embodiments, the modified antisense strand comprises a sequence or fragment represented by any one of SEQ ID NOs. 4-35.

[0048] In some embodiments, the modification refers to a modification on a nucleotide, a modification between nucleotides, or a modification by substitution of a nucleotide analog.

[0049] In some embodiments, the modification is selected from one or more of: 2'-methoxyethyl modification, 2'-methoxy (m) modification, 2'-deoxy-2'-fluoro (f) modification, thiophosphate (s) group modification, adenosine-2' phosphate (A-2'-5') modification, uridine-2' phosphate (U-2'-5') modification, guanosine-2' phosphate (G-2'-5') modification, guanosine-diol nucleic acid (Ggn) modification, adenosine-diol nucleic acid (Agn) modification, uridine-diol nucleic acid (Ugn) modification, N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide (GNF-BX) modification or 5'-phosphate mimetic modification.

[0050] In some embodiments, the ribose modification includes any one or more of a 2'-methoxyethyl modification (moe), a 2'-methoxy (m) modification, an N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide (GNF-BX) modification, a 5'-vinylphosphonic acid (Vp) modification, a 2'-deoxy-2'-fluoro (f) modification, and a 5' vinylphosphonate-2'-N-acetyl modification.

[0051] In some embodiments, the base modification includes but is not limited to a 5' vinylphosphonate modification or a 5' vinylphosphonate-2'-N-acetyl modification. In the case of these two modifications, the base modification is located on the 5' terminal nucleic acid of the antisense strand.

[0052] In some embodiments, the modification between the nucleotides includes modification of the phosphodiester bond in the phosphate group, such as phosphorothioate(s) modification. The phosphorothioate modification is formed by replacing at least one oxygen atom in the phosphodiester bond with a sulfur atom.

[0053] In some embodiments, the 5'-phosphate mimetic is 5'-oxymethylphosphonate, 5'-vinylphosphonic acid (Vp), 5'vinylphosphonate-2'-N-acetyl, or 5'-malonylphosphonate; preferably 5'-vinylphosphonic acid (Vp) or 5'vinylphosphonate-2'-N-acetyl.

[0054] In some embodiments, the 5'-phosphate mimetic is modified at the 5'-end of the antisense strand, preferably the 5'-phosphate mimetic is 5'-vinylphosphonic acid (Vp).

[0055] In an exemplary embodiment, the 5' terminal nucleotide of the antisense strand of the oligonucleotide is modified with Vp to be a compound having the following structural formula:

[0056] Wherein Base is a pyrimidine or purine base, Y is other nucleoside on the oligonucleotide, and A is selected from any one of the substituents conventionally selected in the art, such as halogen (such as F), OH, OCH3, OCF3, OCH2CH3, 2'-methoxyethyl, 2'-N-acetyl, etc.

[0057] In some embodiments, the 5'-phosphate mimetic modified nucleotide is 5' vinylphosphonate-2'-methoxy-uridine.

[0058] In some embodiments, the 5'-phosphate mimetic modified nucleotide is 5' vinylphosphonate-2'-N-acetyl-uridine (APU001).

[0059] In some embodiments, all nucleotides of the sense and antisense strands are modified nucleotides.

[0060] In some embodiments, the oligonucleotide comprises at least one modified internucleotide linkage.

[0061] In some embodiments, the at least one modified internucleotide linkage is a phosphorothioate linkage.

[0062] 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 a 5'-phosphate group are often 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 degradation by phosphatases or other enzymes to increase the cellular uptake of the oligonucleotide or improve the pharmacokinetic properties of the oligonucleotide.

[0063] In some embodiments, the 4'-carbon of the pentose sugar of the 5'-terminal nucleotide of the antisense strand comprises a phosphate analog modification that replaces the -CH2OH attached to the 4'-carbon of the pentose sugar.

[0064] In some embodiments, the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

[0065] In the present invention, the terms "phosphate mimetic", "phosphate analogue" and "phosphonate" are used interchangeably, and all refer to a phosphate 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.

[0066] 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 can be:

[0067] Where U is uracil base.

[0068] 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 represented by the formula -O-CH2-PO(OH)2 or -O-CH2-PO(OR)2, wherein R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, CHOCH2CH2Si(CH3)3, or a protecting group. In some embodiments, the alkyl group is CH2CH3. Typically, R is independently selected from H, CH3, or CH2CH3.

[0069] In some embodiments, the modification refers to a substitution, such as a substitution of the nucleotide sequence of SEQ ID NO: 3 in the antisense strand. In some embodiments, the single nucleotide "APU001" in the antisense strand of the oligonucleotide replaces the 2'-methoxy-modified uridine "Um" at the 5' end of the antisense strand; in some embodiments, the single nucleotide "Ggn" in the antisense strand of the oligonucleotide replaces the nucleotide "Gm" with a 2'-methoxy modification; in some embodiments, the single nucleotide "Ggn" in the antisense strand of the oligonucleotide replaces the nucleotide "Gf" or Gfs with a 2'-deoxy-2'-fluoro modification; in some embodiments, the single nucleotide "Ugn" in the antisense strand of the oligonucleotide replaces the nucleotide "Um" with a 2'-methoxy modification; in some embodiments, In some embodiments, the single nucleotide "Ugn" in the antisense strand replaces the nucleotide "Uf" with a 2'-deoxy-2'-fluoro modification; in some embodiments, the single nucleotide "Agn" in the antisense strand of the oligonucleotide replaces the nucleotide "Am" with a 2'-methoxy modification; in some embodiments, the single nucleotide "Agn" in the antisense strand of the oligonucleotide replaces the nucleotide "Af" with a 2'-deoxy-2'-fluoro modification; in some embodiments, the single nucleotide "U-2'-5'" in the antisense strand of the oligonucleotide replaces the nucleotide "Um" with a 2'-methoxy modification; in some embodiments, the single nucleotide "U-2'-5'" in the antisense strand of the oligonucleotide replaces In some embodiments, the antisense strand of the oligonucleotide is substituted for the nucleotide "Uf" with a 2'-deoxy-2'-fluoro modification; in some embodiments, the antisense strand of the oligonucleotide is substituted for the nucleotide "Am" with a 2'-methoxy modification; in some embodiments, the antisense strand of the oligonucleotide is substituted for the nucleotide "Af" with a 2'-deoxy-2'-fluoro modification; in some embodiments, the antisense strand of the oligonucleotide is substituted for the nucleotide "Gm" with a 2'-methoxy modification; in some embodiments, the antisense strand of the oligonucleotide is substituted for the nucleotide "G-2'-5'" with a 2'-deoxy -2'-fluoro modified nucleotide "Gf"; in some embodiments, the antisense strand of the oligonucleotide has a single nucleotide "GNF-BX" substituted for a nucleotide "Am" with a 2'-methoxy modification; in some embodiments, the antisense strand of the oligonucleotide has a single nucleotide "GNF-BX" substituted for a nucleotide "Af" with a 2'-deoxy-2'-fluoro modification; in some embodiments, the antisense strand of the oligonucleotide has a single nucleotide "GNF-BX" substituted for a nucleotide "Um" with a 2'-methoxy modification; in some embodiments, the antisense strand of the oligonucleotide has a single nucleotide "GNF-BX" substituted for a nucleotide "Gm" with a 2'-methoxy modification;In some embodiments, the single nucleotide "G(moe)" in the antisense strand of the oligonucleotide replaces the nucleotide "Gfs" with a 3'-phosphorothioate group and a 2'-deoxy-2'-fluoro modification; in some embodiments, the single nucleotide "G(moe)" in the antisense strand of the oligonucleotide replaces the nucleotide "Gm" with a 2'-methoxy modification; in some embodiments, the single nucleotide "G(moe)" in the antisense strand of the oligonucleotide replaces the nucleotide "Gf" with a 2'-deoxy-2'-fluoro modification; in some embodiments, the single nucleotide "U(moe)" in the antisense strand of the oligonucleotide replaces the nucleotide "Um" with a 2'-methoxy modification; in some embodiments, the single nucleotide "A(moe)" in the antisense strand of the oligonucleotide replaces the nucleotide "Am" with a 2'-methoxy modification; in some embodiments, the single nucleotide "A(moe)" in the antisense strand of the oligonucleotide replaces the nucleotide "Af" with a 2'-deoxy-2'-fluoro modification.

[0070] In some embodiments, the position where the substitution occurs is preferably at any nucleotide from the 1st to the 8th position from the 5' end of the antisense chain. In a more preferred embodiment, APU001 is a uridine analog that replaces the 5' terminal nucleotide of the antisense chain, and A-2'-5', U-2'-5', G-2'-5', Ggn, Agn, Ugn, and GNF-BX optionally replace the corresponding nucleotides from the 2nd to the 8th position from the 5' end of the antisense chain.

[0071] In the present invention, the specific substitution methods and positions of nucleotides can be found in the detailed information in Table 1 and Table 2.

[0072] 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.

[0073] In some embodiments, the sense strand is 19 bases in length. In some embodiments, the antisense strand is 21 bases in length.

[0074] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand selected from the group consisting of:

[0075] (1) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 3;

[0076] (2) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 4;

[0077] (3) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 5;

[0078] (4) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 6;

[0079] (5) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 7;

[0080] (6) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 8;

[0081] (7) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 9;

[0082] (8) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 10;

[0083] (9) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 11;

[0084] (10) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 12;

[0085] (11) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 13;

[0086] (12) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 14;

[0087] (13) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 15;

[0088] (14) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 16;

[0089] (15) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 17;

[0090] (16) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 18;

[0091] (17) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 19;

[0092] (18) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 20;

[0093] (19) the sense strand represented by SEQ ID NO: 1, and the antisense strand represented by SEQ ID NO: 21;

[0094] (20) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 22;

[0095] (21) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 23;

[0096] (22) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 24;

[0097] (23) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 25;

[0098] (24) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 26;

[0099] (25) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 27;

[0100] (26) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 28;

[0101] (27) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 29;

[0102] (28) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 30;

[0103] (29) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 31;

[0104] (30) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 32;

[0105] (31) the sense strand shown in SEQ ID NO: 2, and the antisense strand shown in SEQ ID NO: 33;

[0106] (32) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 34;

[0107] (33) The sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 35.

[0108] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand selected from the group consisting of:

[0109] (1) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 5;

[0110] (2) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 6;

[0111] (3) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 7;

[0112] (4) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 8;

[0113] (5) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 9;

[0114] (6) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 10;

[0115] (7) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 11;

[0116] (8) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 12;

[0117] (9) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 13;

[0118] (10) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 14;

[0119] (11) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 15;

[0120] (12) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 16;

[0121] (13) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 17;

[0122] (14) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 18;

[0123] (15) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 19;

[0124] (16) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 20;

[0125] (17) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 21;

[0126] (18) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 22;

[0127] (19) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 23;

[0128] (20) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 24;

[0129] (21) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 25;

[0130] (22) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 26;

[0131] (23) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 27;

[0132] (24) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 28;

[0133] (25) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 29;

[0134] (26) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 30;

[0135] (27) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 31;

[0136] (28) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 32;

[0137] (29) the sense strand shown in SEQ ID NO: 2, and the antisense strand shown in SEQ ID NO: 33;

[0138] (30) the sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 34;

[0139] (31) The sense strand shown in SEQ ID NO: 1, and the antisense strand shown in SEQ ID NO: 35.

[0140] In some embodiments, at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands.

[0141] Targeting ligands 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.

[0142] In some embodiments, the targeting ligand is conjugated to the sense strand of the oligonucleotide at the 3 '-end.

[0143] In some embodiments, the targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a lipid.

[0144] In some embodiments, the targeting ligand comprises an amino sugar.

[0145] In some embodiments, the 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 chain of the oligonucleotide in a trivalent state to form an oligonucleotide sequence with a GalNAc carrier.

[0146] In some embodiments, the GalNAc moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

[0147] In some embodiments, the targeting ligand has the following structure:

[0148] In some embodiments, the targeting ligand is conjugated to the sense strand of the oligonucleotide at the 3'-end via a linker to form the following conjugate:

[0149] X may be selected from S or O.

[0150] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand selected from the group consisting of:

[0151] (1) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 3;

[0152] (2) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 4;

[0153] (3) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 27;

[0154] (4) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 28;

[0155] (5) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 29;

[0156] (6) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 30;

[0157] (7) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 31;

[0158] (8) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 32;

[0159] (9) the sense strand shown in SEQ ID NO: 37, and the antisense strand shown in SEQ ID NO: 39;

[0160] (10) the sense strand shown in SEQ ID NO: 38, and the antisense strand shown in SEQ ID NO: 33;

[0161] (11) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 34;

[0162] (12) The sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 35.

[0163] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand selected from the group consisting of:

[0164] (1) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 27;

[0165] (2) the sense strand shown in SEQ ID NO:36, and the antisense strand shown in SEQ ID NO:28;

[0166] (3) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 29;

[0167] (4) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 30;

[0168] (5) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 31;

[0169] (6) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 32;

[0170] (7) the sense strand shown in SEQ ID NO: 37, and the antisense strand shown in SEQ ID NO: 39;

[0171] (8) the sense strand shown in SEQ ID NO: 38, and the antisense strand shown in SEQ ID NO: 33;

[0172] (9) the sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 34;

[0173] (10) The sense strand shown in SEQ ID NO: 36, and the antisense strand shown in SEQ ID NO: 35.

[0174] 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.

[0175] In some embodiments, the composition is in the form of oral administration, intravenous injection, subcutaneous injection or intramuscular injection, preferably subcutaneous injection.

[0176] 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.

[0177] In another aspect, the present disclosure provides a use of the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof or the aforementioned composition in the preparation of a medicament for reducing at least one of HBsAg, HBeAg and HBV DNA in the serum of a subject.

[0178] In another aspect, the present disclosure provides a use of the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof or the aforementioned composition in the preparation of a medicament for treating HBV infection or hepatitis B, and / or hepatitis B virus combined with hepatitis D virus infection.

[0179] In some embodiments, the HBV infection is a chronic HBV infection.

[0180] In some embodiments, the hepatitis B is chronic hepatitis B.

[0181] In another aspect, the present disclosure provides a method for treating HBV infection or hepatitis B, and / or hepatitis B virus combined with hepatitis D virus infection, comprising administering a therapeutically effective amount of the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof or the aforementioned composition to a subject in need.

[0182] In some embodiments, the HBV infection is a chronic HBV infection.

[0183] In some embodiments, the hepatitis B is chronic hepatitis B.

[0184] In another aspect, the present disclosure provides the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof or the aforementioned composition for use in treating HBV infection or hepatitis B, and / or hepatitis B virus combined with hepatitis D virus infection.

[0185] In some embodiments, the HBV infection is a chronic HBV infection.

[0186] In some embodiments, the hepatitis B virus is chronic hepatitis B virus. Various preparations have been developed to promote the use of oligonucleotides. For example, oligonucleotides can be delivered to a subject or a cellular environment using a preparation that minimizes degradation, promotes delivery and / or intake, or provides another beneficial property for the oligonucleotides in the preparation. In some embodiments, provided herein are siRNAs or compositions thereof comprising oligonucleotides (for example, 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 administered to a subject (in the direct environment of a target cell 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 for delivering oligonucleotides for reducing HBsAg, HBeAg, and HBV DNA, as disclosed herein. In some embodiments, oligonucleotides are formulated in a buffer solution, such as a phosphate-buffered saline solution, a liposome, a micellar structure, and a shell. In some embodiments, the naked oligonucleotide or its conjugate is formulated in water or an aqueous solution (e.g., pH adjusted water). In some embodiments, the naked oligonucleotide or its conjugate is formulated in an alkaline buffered aqueous solution (e.g., PBS).

[0187] 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.

[0188] 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 can be otherwise formulated for administration to a cell, tissue, organ, or body of a subject in need thereof.

[0189] In some embodiments, preparations as disclosed herein include excipients. In some embodiments, excipients give stability of the improvement of active ingredient, absorption of improvement, solubility and / or therapeutic enhancement to the composition. In some embodiments, excipients are buffers (for example, sodium citrate, sodium phosphate, tris alkali or sodium hydroxide) or vehicles (for example, buffered solution, petrolatum, dimethyl sulfoxide or mineral oil). In some embodiments, oligonucleotides are freeze-dried for extending their shelf life, and then solution is made before use (for example, being applied to a subject). Therefore, the excipient in the composition comprising any one of the oligonucleotides described herein can be a lyoprotectant (for example, mannitol, lactose, polyethylene glycol or polyvinyl pyrrolidone) or a collapse temperature modifier (for example, dextran, ficoll or gelatin).

[0190] In some embodiments, the pharmaceutical composition is formulated to be compatible with the 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.

[0191] The pharmaceutical composition suitable for injectable use includes sterile aqueous solution (when water-soluble) or dispersion and the sterile powder for the temporary preparation of sterile injectable solution or dispersion.For intravenous or subcutaneous administration, suitable carrier includes 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 a suitable mixture thereof. In many cases, it is preferred to include isotonic agents in the composition, for example sugar, polyols such as mannitol, sorbitol and sodium chloride. Sterile injectable solution can be prepared by: a desired amount of oligonucleotide and the desired above-enumerated composition or combination are incorporated into the solvent of selection, followed by filtration sterilization.

[0192] In some embodiments, the composition may contain at least about 0.1% or more of a therapeutic agent (e.g., an oligonucleotide 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. One 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.

[0193] Even though many of the embodiments relate to liver-targeted delivery of any of the oligonucleotides disclosed herein, targeting other tissues is also contemplated.

[0194] In some embodiments, the administration of oligonucleotides as described herein results in a reduction in the level of HBsAg, HBeAg and HBV DNA. In some embodiments, the reduction in 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. Appropriate control levels can be the level of HBsAg, HBeAg and HBV DNA in the serum of a subject not contacted with an oligonucleotide 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.

[0195] 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. Transgenics can be delivered using viral vectors (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or non-viral vectors (e.g., plasmid or synthetic mRNA). In some embodiments, the transgenic can be injected directly into the subject.

[0196] 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 any one 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, arms and legs numbness, 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 related to hypercholesterolemia, atherosclerosis and / or one or more symptoms or complications thereof.

[0197] 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., brain cell death due to inadequate blood and oxygen flow to the brain), feelings of weakness, confusion, difficulty speaking, dizziness, difficulty walking or standing upright, blurred vision, numbness in the face, arms, and legs, severe headache, loss of consciousness, peripheral arterial disease, and / or kidney problems (e.g., chronic kidney disease).

[0198] In certain aspects, the present disclosure provides methods 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 encoding the same or a transgene) to the subject. In some embodiments, the subject to be treated is a subject who would benefit therapeutically from a reduction in the amount of, for example, HBsAg, HBeAg and HBV DNA in the serum.

[0199] The methods described herein generally involve administering to a subject an effective amount (i.e., an amount capable of producing the desired therapeutic result) of an oligonucleotide. A therapeutically acceptable amount can be an amount capable of treating a disease or condition. The appropriate dosage for any one 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 concurrently.

[0200] 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, intraarterially, intramuscularly), topically (e.g., epidermally, by inhalation, via eye drops, or through a mucous membrane), or by direct injection into a target organ (e.g., the subject's liver). Typically, the oligonucleotides disclosed herein are administered intravenously or subcutaneously.

[0201] In some embodiments, the oligonucleotide is administered at a dose 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 dose 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.

[0202] As a set of non-limiting examples, the oligonucleotides of the disclosure would typically be administered yearly, twice yearly, quarterly (once every three months), bimonthly (once every two months), monthly, semi-monthly, or weekly.

[0203] 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, cows, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.

[0204] For purposes of clarity and conciseness, 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.

[0205] Hereinafter, the present disclosure will be described in more detail with reference to specific examples. However, the examples are for illustrative purposes only and have no limiting effect on the present disclosure.

[0206] Example

[0207] Example 1: Design of siRNA

[0208] First, a computer-based algorithm was used to generate candidate oligonucleotide sequences complementary to hepatitis B virus genotype D (subtype ayw, V01460J02203). Some of these sequences were also complementary to genotypes A, B, and C or had no more than two mismatches. The siRNAs were designed as double-stranded sequences with a 19 / 21 pairing pattern for the sense and antisense strands, with the antisense strand possessing two overhanging ends complementary to the mRNA sequence. In some complementary sequences, the first and / or 19th bases on the 5' end of the sense strand were substituted with bases that were different from those in the HBV mRNA, and the corresponding bases on the antisense strand were altered accordingly, forming a pair with the sense strand. In some sequences, bases were substituted at specific positions within the sense strand, forming mismatches with corresponding positions on the antisense strand. In some sequences, deoxyribonucleotides were substituted for ribonucleotides at specific positions. In some sequences, hypoxanthine (I) was substituted for U, A, or G at specific positions on the antisense strand, forming wobble pairs with the sense strand. Table 1 lists the designed siRNA-modified oligonucleotide sequences. Table 2 lists the modified oligonucleotide sequences after optimization based on AL0105138.

[0209] Table 1. Modified oligonucleotides

[0210] Note: "G", "C", "A", "U", "T" and "I" generally represent nucleotides based on guanine, cytosine, adenine, uracil, thymine and hypoxanthine, respectively; modifications: d represents deoxynucleotide; m represents 2'-methoxy; f represents 2'-deoxy-2'-fluoro; s represents phosphorothioate; Vp represents vinylphosphonic acid (modified at the 5' end of the antisense strand). In the sequence listing of this application, according to the rules of the ST.26 sequence listing, "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.

[0211] Table 2. Modified oligonucleotides

[0212] Note: "G", "C", "A", "U", "T" and "I" usually represent nucleotides with guanine, cytosine, adenine, uracil, thymine and hypoxanthine as bases, respectively; modifications: d represents deoxynucleotide; m represents 2'-methoxy; f represents 2'-deoxy-2'-fluoro; Moe represents 2'-O-methoxyethyl; s represents 3'-thiophosphate; Vp represents 5'-vinylphosphonic acid (modified at the 5' end of the antisense strand).

[0213] A-2'-5' stands for adenosine-2' phosphate; U-2'-5' stands for uridine-2' phosphate; G-2'-5' stands for guanosine-2' phosphate; Ggn stands for guanosine-diol nucleic acid; Agn stands for adenosine-diol nucleic acid; Ugn stands for uridine-diol nucleic acid; GNF-BX stands for N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide; APU001 stands for 5' vinylphosphonate-2'-N-acetyl-uridine. Sequences 4-35 were used as a reference, with nucleic acid analogs replacing the corresponding positions.

[0214] Tables 3 and 4 list the designed vectored siRNA sequences.

[0215] Table 3. siRNA sequences with vectors

[0216] Table 4. siRNA sequences with vectors

[0217] Note: "G", "C", "A", "U", "T" and "I" usually represent nucleotides with guanine, cytosine, adenine, uracil, thymine and hypoxanthine as bases, respectively; modifications: d represents deoxynucleotide; m represents 2'-methoxy; f represents 2'-deoxy-2'-fluoro; Moe represents 2'-O-methoxyethyl; s represents 3'-thiophosphate; Vp represents 5'-vinylphosphonic acid (modified at the 5' end of the antisense strand).

[0218] A-2'-5' stands for adenosine-2' phosphate; U-2'-5' stands for uridine-2' phosphate; G-2'-5' stands for guanosine-2' phosphate; Ggn stands for guanosine-diol nucleic acid; Agn stands for adenosine-diol nucleic acid; Ugn stands for uridine-diol nucleic acid; GNF-BX stands for N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide; APU001 stands for 5' vinylphosphonate-2'-N-acetyl-uridine. Sequences 4-35 were used as a reference, with nucleic acid analogs replacing the corresponding positions.

[0219] Wherein, L96 represents a carrier. Alternatively, the structural formula of the ligand conjugated to the oligonucleotide is as follows:

[0220] Furthermore, the ligand is conjugated to the sense strand of the oligonucleotide at the 3'-end via a linker to form the following conjugate:

[0221] X may be selected from S or O.

[0222] Example 2: Preparation of siRNA

[0223] Abbreviations for nucleotide monomers used in nucleic acid sequence representations. It will be understood that these monomers, when present in an oligonucleotide, are interconnected by 5'-3' phosphodiester bonds unless otherwise indicated. L96 was prepared using the method described in patent CN104717982B.

[0224] The specific process of preparing oligonucleotides is as follows.

[0225] (1) Preparation of siRNA

[0226] 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.

[0227] Solid-phase synthesis (Figure 1): Sense and antisense strands are synthesized separately on a solid support using phosphoramidite technology using an automated oligonucleotide synthesizer. Examples of such synthesizers include the AKTA Oligopilot (Cytiva) and the Dr. Oligo 192XLc (Kunshan Berleke Precision Instrument Co., Ltd.). Solid-phase synthesis begins at the 3' end of the sequence and sequentially couples monomers into the sequence. Each coupling of a phosphoramidite monomer involves four chemical steps: 1) unblocking or deprotection (removal of the hydroxyl protecting group); 2) coupling; 3) oxidation; and 4) capping. All phosphoramidite monomers, reagents, and purification consumables used were commercially available, including various phosphoramidite monomers (e.g., 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (e.g., 40 wt% aqueous methylamine solution, 28 wt% aqueous ammonium hydroxide solution) purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used herein are described in US20130178612A1 and US2015100197A1, among others; the synthesis methods for sequences containing VPUm and APU structures are described in J. Med. Chem. 2018, 61, 734-744.

[0228] (2) Preparation of double-stranded RNA reagent

[0229] The synthesis process of the sense chain is as follows:

[0230] Oligonucleotides are synthesized using the solid-phase phosphoramidite method. Reactions are performed on a computer-controlled synthesizer in a stainless steel synthesis column. Sense strand synthesis begins with a solid-phase support loaded with a targeting ligand (e.g., L96), or directly from the solid-phase support. The solid-phase synthesizer controls different pipelines to inject different raw materials, reagents, and solvents in the order of 3' to 5' of the sequence, connecting the phosphoramidite nucleoside monomers one by one. The reaction process involves four cycles: DMT protection group removal, condensation, oxidation or thiolation, and end-capping. Each cycle connects one nucleotide unit to produce an oligonucleotide sequence of 19 or 21 nucleotide units. After synthesis, the protecting group (2-cyanoethyl) is removed on the solid-phase synthesis column, and the synthesized sequence is cleaved from the solid-phase support by aminolysis. The resulting product is filtered, the filter cake is washed with ethanol, and the filtrate and washings are collected and concentrated to obtain the crude sense strand. The crude product is purified by chromatography (SOURCE 15Q) and lyophilized to obtain the desired sense strand.

[0231] The synthesis process of the antisense strand is as follows:

[0232] The synthesis of the antisense strand is similar to that of the sense strand. Different raw materials, reagents, and solvents are injected into the different pipelines in the order of 3' to 5' of the sequence 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 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. The filter cake is filtered and 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), ultrafiltration, and lyophilization to obtain the target product antisense strand.

[0233] The process of preparing double-stranded siRNA is as follows:

[0234] Dissolve the AS and SS strands separately in injection water, mix at a defined ratio (1.01:1.0-1.2:1.0), incubate at 30-50°C for 30-90 minutes, cool to room temperature, and freeze-dry to obtain double-stranded siRNA.

[0235] According to the same method, the double-stranded siRNA reagent conjugates in Tables 1 and 2 above were prepared.

[0236] Example 3: Evaluation of in vitro anti-HBV activity of modified sequences using HepG2.2.15 cells

[0237] (1) Culture of HepG2.2.15 cells:

[0238] HepG2.2.15 cells are HepG2 cells transfected with a recombinant HBV-DNA plasmid. They can stably produce HBV virus and are a commonly used in vitro model for HBV drug screening. Cells were provided by Shanghai WuXi AppTec Pharmaceutical Development Co., Ltd. and cultured in a 37°C, 5% CO2 incubator using Dulbecco's Membrane Medium (Gibco-11995-065) supplemented with 10% FBS (ExCell Bio-FSP500), InvitroGRO CP Medium (Bioreclamation IVT-S03316), and 1% penicillin-streptomycin (Hyclone-SV30010).

[0239] (2) Plating and transfection

[0240] On day 0, the test compound was diluted with DPBS (KCl, 0.2 g / L; KH2PO4, 0.2 g / L; NaCl, 8 g / L; Na2HPO4·7H2O, 2.16 g / L; adjusted to pH 7.0) to a final concentration of 1 nM and 0.1 nM. HepG2.2.15 cells were taken from the culture flask, washed with DPBS, and digested with trypsin.

[0241] After collecting the cells, count them and adjust the cells to the appropriate density. 4 Cells were seeded into 96-well plates at a density of 100 cells / mL. Simultaneously, the test compounds were transfected into the cells using Lipofectamine RNAiMax (Invitrogene, 13378-150). Three replicate wells were set up for each concentration point of each test compound.

[0242] (3) Sampling and testing

[0243] 72 hours after transfection with the test compound, cell supernatants were collected and assayed for HBsAg expression using an ELISA. Cell culture supernatants were diluted with DPBS and assayed for HBsAg using an HBsAg ELISA kit (Cat. No.: Antu Biotech-CL0310). The method is briefly described as follows: 50 μL of each standard, sample, and control was added to the assay plate. Then, 50 μL of enzyme conjugate was added to each well. The plate was incubated at 37°C for 60 minutes. The plate was washed with wash buffer and aspirated dry. Then, 50 μL of premixed luminescent substrate was added. The plate was incubated at room temperature in the dark for 10 minutes. Luminescence was measured using a microplate reader.

[0244] (4) Data Analysis

[0245] HBsAg inhibition rate (%) = (1-HBsAg value of sample / HBsAg value of DPBS control group) × 100%.

[0246] The calculation results of the in vitro inhibitory activity of HepG2.2.15 cells are shown in Table 5.

[0247] Table 5. In vitro inhibitory activity of HepG2.2.15 cells (n=3)

[0248] The results showed that, with the exception of AL0105125, AL0105128, and AL0105134, which had relatively low inhibition efficiencies against HBsAg, all other siRNA drugs achieved an inhibition efficiency of over 70% at a concentration of 0.1 nM and over 40% at a concentration of 0.01 nM. AL0105133 and AL0105138 had particularly prominent inhibitory effects. AL0105137 and AL0105139, obtained by Vp modification at the 5' end of the antisense strands of AL0105133 and AL0105138, further enhanced their inhibitory effects.

[0249] Example 4: Evaluation of the in vitro anti-HBV activity of modified sequences using HepG2.2.15 cells

[0250] (1) Culture of HepG2.2.15 cells:

[0251] HepG2.2.15 cells are HepG2 cells transfected with a recombinant HBV-DNA plasmid. They can stably produce HBV virus and are a commonly used in vitro model for HBV drug screening. Cells were provided by Shanghai WuXi AppTec Pharmaceutical Development Co., Ltd. and cultured in a 37°C, 5% CO2 incubator using Dulbecco's Membrane Medium (Gibco-11995-065) supplemented with 10% FBS (ExCell Bio-FSP500), InvitroGRO CP Medium (Bioreclamation IVT-S03316), and 1% penicillin-streptomycin (Hyclone-SV30010).

[0252] (2) Plating and transfection

[0253] On day 0, the test compound was diluted with DPBS (KCl, 0.2 g / L; KH2PO4, 0.2 g / L; NaCl, 8 g / L; Na2HPO4·7H2O, 2.16 g / L; adjusted to pH 7.0) to a final concentration of 1 nM and 0.1 nM. HepG2.2.15 cells were taken from the culture flask, washed with DPBS, and digested with trypsin.

[0254] After collecting the cells, count them and adjust the cells to the appropriate density. 4Cells were seeded into 96-well plates at a density of 100 cells / mL. Simultaneously, the test compounds were transfected into the cells using Lipofectamine RNAiMax (Invitrogene, 13378-150). Three replicate wells were set up for each concentration point of each test compound.

[0255] (3) Sampling and testing

[0256] 72 hours after transfection with the test compound, cell supernatants were collected and assayed for HBsAg expression using an ELISA. Cell culture supernatants were diluted with DPBS and assayed for HBsAg using an HBsAg ELISA kit (Cat. No.: Antu Biotech-CL0310). The method is briefly described as follows: 50 μL of each standard, sample, and control was added to the assay plate. Then, 50 μL of enzyme conjugate was added to each well. The plate was incubated at 37°C for 60 minutes. The plate was washed with wash buffer and aspirated dry. Then, 50 μL of premixed luminescent substrate was added. The plate was incubated at room temperature in the dark for 10 minutes. Luminescence was measured using a microplate reader.

[0257] (4) Data Analysis

[0258] HBsAg inhibition rate (%) = (1-HBsAg value of sample / HBsAg value of DPBS control group) × 100%.

[0259] The calculation results of the in vitro inhibitory activity of HepG2.2.15 cells are shown in Table 6.

[0260] Table 6. In vitro inhibitory activity of HepG2.2.15 cells (n=3)

[0261] The results showed that except for a few compounds AL0105140, AL0105147, AL0105148, AL0105149 and AL0105151, other compounds all showed a high level of HBsAg inhibition, with an average inhibition rate of more than 75%. The inhibition rates of individual compounds AL0105141 and AL0105143 were above 90%.

[0262] Example 5: Evaluation of in vitro off-target activity of modified sequences using HepG2.2.15 cells

[0263] (1) Experimental methods

[0264] This experiment used the psi-CHECK2 (Promega™) plasmid system to assess the inherent ability of guide and follower strands to influence on-target and off-target effects by monitoring changes in target sequence expression levels. Two psi-CHECK2 plasmids were prepared for each siRNA, one containing the complete target sequence (a sequence that is completely complementary to the guide or follower strand of the corresponding siRNA) and one containing a sequence that does not completely match the target sequence. These sequences were cloned downstream of the coding region of the Renilla luciferase reporter gene. RNAi activity or seed-mediated activity of siRNAs targeting any of the above sequences can lead to cleavage of the fusion mRNA, subsequent degradation, or translational inhibition. Any of these conditions will result in reduced protein expression.

[0265] (2) Data Analysis

[0266] Inhibition efficiency (%) = 1-(Ratio reporter + siRNA / Ratio reporter only) * 100%

[0267] IC 50 It was calculated using the "[Inhibitor] vs. Normalized Response - Variable Slope" function in Graphpad Prism 7 using the following formula:

[0268] (Y=100 / (1+(X^HillSlope) / (IC 50 ^HillSlope)))

[0269] The experimental results are shown in Table 7.

[0270] Table 7. In vitro inhibitory activity of HepG2.2.15 cells (n=3)

[0271] The results showed that in terms of on-target ability, except for AL0105161, whose inhibition efficiency was significantly reduced, the inhibition efficiency of other siRNA drugs was equal to or better than that of AL0105138, especially AL0105157, AL0105158, AL0105159, AL0105160, and AL0105164, which could significantly enhance the inhibition efficiency of AL0105138; in terms of off-target ability, after different optimization of AL0105138, all groups of compounds could significantly improve the off-target effect of AL0105138.

[0272] Example 6: Evaluation of the Anti-HBV Activity of Compounds at Different Concentrations in Vivo Using HBV Transgenic Mice

[0273] The experiment used SPF-grade male C57B / 6N-Tg(1.28HBV) / Vst mice, 6-8 weeks of age (Beijing Weitongda Biotechnology Co., Ltd., Animal Production Certificate SCXK(Beijing)2019-0002). Each mouse was housed individually in individual 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."

[0274] The animals were acclimated for one week before the experiment. Based on the quantitative results of serum HBsAg (primary) and HBV DNA (secondary), the animals were randomly divided into nine groups of five. The day of drug administration was designated as D1, and blood was collected before drug administration on the day of administration for the determination of HBsAg, HBV DNA, and HBeAg.

[0275] All animals were dosed subcutaneously on day 1 with a single injection of 1 mg / kg, 3 mg / kg, and 9 mg / kg for AL0107039 and AL0107044, and 3 mg / kg for AL0107043 and AL0107045, in a 5 mL / kg volume. A control group received an equivalent dose of saline. Animals in each group were observed 15 minutes to 1 hour after the last animal in each group was dosed.

[0276] On days 8, 15, 22, 29, 35, and 42 after administration, 100 μL of blood was collected from the orbital cavity and centrifuged to separate the serum. 10 μL of serum was diluted 50-fold with PBS and then sent for testing. Serum HBsAg, HBeAg, and HBV DNA after dilution were measured by Beijing Dian Medical Laboratory Co., Ltd.

[0277] 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 measurements before treatment: residual inhibition rate = value measured at different times / pre-treatment base value * 100%.

[0278] The experimental results are shown in Table 8 and Figure 2.

[0279] Table 8. Residual rates of HBsAg, HBeAg and HBV DNA in serum of HBV transgenic mice after single subcutaneous administration of different concentrations

[0280] The test results in Figure 2 and Table 8 indicate that both compounds AL0107039 and AL0107044 exhibited significant inhibitory effects on HBsAg, HBV DNA, and HBeAg in HBV transgenic mice in a dose-dependent manner, with significant inhibitory efficiency demonstrated even at the lowest dose of 1 mg / kg. The inhibitory effect of AL0107044 was superior to that of AL0107039, and that of AL0107045 was superior to that of AL0107043.

[0281] Example 7: Evaluation of the Anti-HBV Activity of Compounds in Vivo Using HBV Transgenic Mice

[0282] The experiment used SPF-grade male C57B / 6N-Tg(1.28HBV) / Vst mice, 6-8 weeks of age (Beijing Weitongda Biotechnology Co., Ltd., Animal Production Certificate SCXK(Beijing)2019-0002). Each mouse was housed individually in individual 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."

[0283] The animals were acclimated for one week before the experiment. Based on the quantitative results of serum HBsAg (primary) and HBV DNA (secondary), the animals were randomly divided into nine groups of five. The day of drug administration was designated as D1, and blood was collected before drug administration on the day of administration for the determination of HBsAg, HBV DNA, and HBeAg.

[0284] All animals were administered a single subcutaneous injection of 3 mg / kg in a 5 mL / kg volume on Day 1. A control group received an equivalent dose of saline. Animals in each group were observed 15 minutes to 1 hour after the last animal in each group was administered.

[0285] On days 8, 15, 22, and 29 after administration, 100 μL of blood was collected from the orbital cavity and centrifuged to separate the serum. 10 μL of serum was diluted 50-fold with PBS and then sent for testing. Serum HBsAg, HBeAg, and HBV DNA were measured by Beijing Dian Medical Laboratory Co., Ltd.

[0286] 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 measurements before treatment: residual inhibition rate = value measured at different times / pre-treatment base value * 100%.

[0287] The experimental results are shown in Tables 9-11 and Figure 3.

[0288] Table 9. Inhibitory effect of single subcutaneous administration on serum HBV DNA in HBV transgenic mice [Log10 HBV DNA (IU / mL)] (mean ± SD)

[0289] Table 10. Inhibitory effect of single subcutaneous administration on serum HBeAg in HBV transgenic mice [Log 10 HBeAg (IU / mL)] (mean ± standard deviation)

[0290] Table 11. Inhibitory effect of single subcutaneous administration on serum HBsAg in HBV transgenic mice [Log 10 HBsAg (IU / mL)] (mean ± SD)

[0291] From the test results in Tables 9-11 and Figure 3, it can be seen that all groups of compounds have significant inhibitory effects on HBsAg, HBV DNA and HBeAg in HBV transgenic mice, among which AL0107045 and AL0107048 have the best inhibitory effects; the inhibitory efficiency of the optimized compounds (AL0107049-AL0107054) is slightly lower than that of the original compound AL0107048; among the optimized compounds (AL0107049-AL0107054), the inhibitory efficiency of AL0107049 and AL0107053 is relatively good.

[0292] Example 8: Evaluation of the Anti-HBV Activity of Different Compounds in Vivo Using HBV Transgenic Mice

[0293] The experiment used SPF-grade male C57B / 6N-Tg(1.28HBV) / Vst mice, 6-8 weeks of age (Beijing Weitongda Biotechnology Co., Ltd., Animal Production Certificate SCXK(Beijing)2019-0002). Each mouse was housed individually in individual 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."

[0294] The animals were acclimated for one week before the experiment. Based on the quantitative results of serum HBsAg (primary) and HBV DNA (secondary), the animals were randomly divided into 10 groups of 5 animals each. The day of drug administration was designated as D1, and blood was collected before drug administration on the day of administration for the determination of HBsAg, HBV DNA, and HBeAg.

[0295] All animals received a single subcutaneous injection of 3 mg / kg (5 mL / kg) on ​​day 1. A saline control group received the same dose of saline. On days 7, 14, 21, 28, and 35 after dosing, 100 μL of blood was collected from the orbital cavity. Serum was separated by centrifugation, and 10 μL of serum was diluted 50-fold with PBS and submitted for testing. Serum HBsAg, HBeAg, and HBV DNA were measured by Beijing Dian Medical Laboratory Co., Ltd.

[0296] The experimental data were statistically analyzed using Graphpad Prism statistical analysis software.

[0297] The experimental results are shown in Tables 12-14 and Figure 4.

[0298] Table 12. Inhibitory effect of single subcutaneous administration on serum HBV DNA in HBV transgenic mice [Log 10 HBV DNA (IU / mL)] (mean ± SD)

[0299] Table 13. Inhibitory effect of single subcutaneous administration on serum HBeAg in HBV transgenic mice [Log 10 HBeAg (IU / mL)] (mean ± standard deviation)

[0300] Table 14. Inhibitory effect of single subcutaneous administration on serum HBsAg in HBV transgenic mice [Log 10 HBsAg (IU / mL)] (mean ± SD)

[0301] As shown in Figures 12-14 and Figure 4, the compounds AL0107045, AL0107057, and AL0107058 all demonstrated significant inhibitory effects on HBsAg, HBV DNA, and HBeAg in HBV transgenic mice, with the AL0107058 compound being particularly significant. Compared to the original sequence AL0107045, AL0107058 demonstrated unexpectedly enhanced inhibitory effects on HBsAg, HBV DNA, and HBeAg, which persisted throughout the entire experimental period.

[0302] Example 9: In vivo toxicity evaluation of compounds at different concentrations using SD rats

[0303] Eighty-six quarantined SPF-grade Sprague-Dawley rats (half male and half female) were randomly assigned by weight to a vehicle control group (N group, 0.9% sodium chloride injection), AL0107045S group, AL7045M group, AL7045H group, AL0107057S group, AL0107057M group, AL0107057H group, AL0107058S group, AL0107058M group, and AL0107058H group, with eight animals per group (half male and half female). After grouping, the test article / vehicle was administered subcutaneously three times (on days 1, 15, and 29 for the AL0107045, AL0107057, and AL0107058 studies). The day of the first administration of the test article was designated as day 1. The doses of each test article in Groups S, M, and H were 50.0, 100.0, and 300.0 mg / kg / dose, respectively. Group N was administered 0.9% sodium chloride injection. The administration volume for each group was 5.0 mL / kg / dose. Body weights were measured on the 1st, 5th, 7th, 12th, 14th, 19th, 21st, 26th, and 28th sections of the study. On the day of the final administration, all animals were fasted overnight. After intraperitoneal anesthesia, blood was collected from the peritoneal vein for serum biochemical analysis. The experimental results are shown in Figures 5-6.

[0304] The results of weight changes (Figure 5) clearly show that animals in all AL0107045 dose groups showed slower weight gain compared to the saline group from days 7 to 14 of the experiment, and even experienced negative weight gain from days 12 to 14. This was consistent across both sexes, with weight gain returning to normal from days 14 to 28. There was no significant difference in weight gain between animals in all AL0107057 and AL0107058 dose groups compared to the saline group.

[0305] The results of aspartate aminotransferase ( FIG6 ) showed that, obviously, the levels of animals in all AL0107045 dose groups were significantly higher than those in the saline group; all AL0107057 and AL0107058 dose groups had no significant effect on aspartate aminotransferase.

[0306] Based on the weight changes and aspartate aminotransferase results, it can be inferred that the AL0107045 compound has a significant toxic effect on rats, causing weight loss during the experiment. In addition, blood biochemical analysis after the experiment showed a significant increase in aspartate aminotransferase levels, indicating that the AL0107045 compound has a certain toxic reaction on the liver. The AL0107057 and AL0107058 compounds were optimized based on AL0107045. The results also showed that there was no significant difference in weight gain and aspartate aminotransferase levels compared with the control group. It is obvious that the AL0107057 and AL0107058 groups of compounds optimized the toxic reaction of AL0107045.

[0307] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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.

[0308] The above-described embodiments merely represent several implementation methods of the present disclosure. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person of ordinary skill in the art could make various modifications and improvements without departing from the spirit of the present disclosure, all of which fall within the scope of protection of the present disclosure. Therefore, the scope of protection of the present patent shall be determined by the appended claims.

Claims

1. An oligonucleotide for inhibiting hepatitis B virus (HBV) or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises a sense strand (SS) and an antisense strand (AS), wherein the nucleotide sequence SS comprises the sequence presented in SEQ ID NO: 1, or a fragment thereof; the nucleotide sequence AS comprises the sequence presented in SEQ ID NO: 3 or a fragment thereof.

2. An oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, where preferably AS is further modified, and the modification is selected from one or more of 2'-methoxyethyl modification, 2'-methoxy (m) modification, 2'-deoxy-2'-fluoro (f) modification, modification with phosphorothioate group(s), adenosine 2'-phosphate (A-2'-5') modification, uridine 2'-phosphate (U-2'-5') modification, guanosine 2'-phosphate (G-2'-5') modification, guanosine-glycolic nucleic acid (Ggn) modification, adenosine-glycolic nucleic acid (Agn) modification, N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide (GNF-BX) modification, or 5'-phosphate mimetic modification; preferably, the modification is selected from one or more of a 2'-methoxyethyl modification, an adenosine 2'-phosphate (A-2'-5') modification, a uridine 2'-phosphate (U-2'-5') modification, a guanosine 2'-phosphate (G-2'-5') modification, a guanosine-glycolic nucleic acid (Ggn) modification, adenosine-glycolic nucleic acid (Agn) modification, a N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide (GNF-BX) modification, or a 5'-phosphate mimetic modification; preferably, the modification is a combination of either a guanosine glycollate nucleic acid (Ggn) modification or an adenosine 2'-phosphate (A-2'-5') modification with a 5'-phosphate mimetic modification; preferably, the modification is located at any of the nucleotides in positions 1 to 8, starting from the 5' end of AS; preferably, the modified AS comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 4-35, or a fragment thereof.

3. An oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1 or claim 2, wherein the 5'-phosphate mimetic is selected from one or more of 5'-hydroxymethylphosphonate, 5'-vinylphosphonic acid (Vp), 5'-vinylphosphonate-2'-N-acetyl or 5'-malonylphosphonate, and more preferably is one or more of 5'-vinylphosphonic acid (Vp) or 5'-vinylphosphonate-2'-N-acetyl, and more preferably is 5'-vinylphosphonic acid (Vp); preferably the 5'-phosphate mimetic is modified at the 5'-end of AS.

4. An oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the oligonucleotide comprises at least one modified internucleotide linkage; and preferably, at least one modified internucleotide linkage is a phosphorothioate linkage.

5. An oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein the oligonucleotide comprises a combination of SS and AS selected from the group consisting of: (1) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 3; (2) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 4; (3) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 5; (4) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 6; (5) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 7; (6) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 8; (7) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 9; (8) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 10; (9) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 11; (10) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 12; (11) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 13; (12) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 14; (13) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 15; (14) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 16; (15) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 17; (16) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 18; (17) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 19; (18) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 20; (19) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 21; (20) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 22; (21) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 23; (22) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 24; (23) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 25; (24) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 26; (25) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 27; (26) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 28; (27) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 29; (28) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 30; (29) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 31; (30) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 32; (31) SS as shown in SEQ ID NO: 2 and AS as shown in SEQ ID NO: 33; (32) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO: 34; (33) SS as shown in SEQ ID NO: 1 and AS as shown in SEQ ID NO:

35.

6. An oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands; preferably, the targeting ligand is conjugated to the 3' end of the SS oligonucleotide; preferably, the targeting ligand comprises a carbohydrate, amino sugar, cholesterol, polypeptide or lipid; preferably, the targeting ligand comprises an amino sugar; preferably, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety; preferably, 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 3' end of the SS oligonucleotide via a linker to form the following conjugate: , where X can be chosen from S or O.

7. An oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-6, wherein the oligonucleotide comprises a combination of SS and AS selected from the group consisting of: (1) SS as shown in SEQ ID NO: 36 and AS as shown in SEQ ID NO: 3; (2) SS as shown in SEQ ID NO: 36 and AS as shown in SEQ ID NO: 4; (3) SS as shown in SEQ ID NO: 36 and AS as shown in SEQ ID NO: 27; (4) SS as shown in SEQ ID NO: 36 and AS as shown in SEQ ID NO: 28; (5) SS as shown in SEQ ID NO: 36 and AS as shown in SEQ ID NO: 29; (6) SS as shown in SEQ ID NO: 36 and AS as shown in SEQ ID NO: 30; (7) SS as shown in SEQ ID NO: 36 and AS as shown in SEQ ID NO: 31; (8) SS as shown in SEQ ID NO: 36 and AS as shown in SEQ ID NO: 32; (9) SS as shown in SEQ ID NO: 37 and AS as shown in SEQ ID NO: 39; (10) SS as shown in SEQ ID NO: 38 and AS as shown in SEQ ID NO: 33; (11) SS as shown in SEQ ID NO: 36 and AS as shown in SEQ ID NO: 34; and (12) SS as shown in SEQ ID NO: 36 and AS as shown in SEQ ID NO:

35.

8. A composition comprising an oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 and, optionally, a pharmaceutically acceptable carrier.

9. A method for reducing at least one of hepatitis B surface antigen (HBsAg), hepatitis B e-antigen (HBeAg) and HBV DNA in the serum of a subject, comprising administering a therapeutically effective amount of an oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-7 or a composition according to claim 8 to a subject in need thereof.

10. A method for treating HBV infection or hepatitis B and / or HBV-HDV co-infection, comprising administering a therapeutically effective amount of an oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-7 or a composition according to claim 8 to a subject in need thereof; preferably, HBV infection is chronic HBV infection; Preferably hepatitis B is CHB.

11. An oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 or a composition according to claim 8 for use in the treatment of HBV infection or hepatitis B and / or HBV-HDV co-infection; preferably, HBV infection is chronic HBV infection; Preferably hepatitis B is CHB.