Double-stranded rnai agent for targeting and regulating HBV gene expression and use thereof

Abstract

To provide a double-stranded RNAi agent for targeting, regulating and controlling HBV gene expression, and use thereof.SOLUTION: The double-stranded RNAi agent comprises an antisense strand and a sense strand complementary to the antisense strand forming a double-stranded region, wherein the nucleotide sequence of the antisense strand is represented by a specific sequence, or the nucleotide sequence of the antisense strand is a modified sequence of the specific sequence. It can significantly reduce the expression of one or more HBV genes in cells and animals, block the viral life cycle, and can be used to develop drugs for treating diseases related to HBV gene expression.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The present disclosure relates to the technical field of nucleic acid modification, and in particular to double-stranded RNAi agents and their uses for targeting, regulating and controlling HBV gene expression. [Background technology]

[0002] Nucleic acid drugs, especially oligonucleotide drugs, are widely used due to their easy synthesis and relatively high activity. Oligonucleotide drugs generally include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), and nucleic acid aptamers.

[0003] Oligonucleotides are short DNA or RNA molecules, or oligomers, that tend to bind to complementary oligonucleotides (DNA or RNA) in a sequence-specific manner, thereby forming duplexes or, less commonly, hybrids. This fundamental property has led to their widespread use in genetic testing, research, and medicine. In nature, oligonucleotides are typically small RNA molecules that play a role in regulating gene expression, or intermediates obtained by degradation of larger nucleic acid molecules.

[0004] RNA interference (RNAi) is a natural defense mechanism against foreign genes. siRNAs can downregulate target genes by recognizing specific sequences and degrading the target mRNA.

[0005] Classical RNAi molecules consist of a typical 19+2 nucleotide polymeric structure (a double helix structure consisting of a 21-nucleotide RNA molecule and a 19-nucleobase nucleotide molecule with a 2-nucleotide 3' overhang). One strand of the siRNA (the guide strand or antisense strand) is complementary to the target gene's mRNA transcript, while the other strand is classified as the passenger strand (or sense strand). The siRNA (antisense strand) guides the alpha globulin (AGO2) complex to complement the target transcript and become part of the RNA-induced silencing complex (RISC). Perfect complementarity between the siRNA (antisense strand) and the target results in cleavage of the target transcript at positions 10–11 of the guide strand (antisense strand) through catalytic action of the AGO2 protein.

[0006] siRNA functions through Watson-Crick base pairing with mRNA. However, it has advantages over small molecule drugs and monoclonal antibodies, which require recognition of the complex spatial structure of specific proteins. Therefore, many diseases cannot be treated with small molecules or monoclonal antibodies because they cannot recognize molecular structures with high activity, affinity, or binding specificity for the target molecule. The mechanism of action of siRNA drugs allows for the regulation and control of target protein expression at the genetic level, providing greater target specificity than small molecule drugs or antibody drugs. This mechanism, based on the principle of base-complementary pairing, broadens the therapeutic scope of siRNA, simplifies its design, and shortens the research and development cycle.

[0007] The nucleotides in natural oligonucleotides are linked by phosphodiester bonds. Under physiological conditions, they are particularly sensitive to nucleases. Therefore, natural, unstructured, and unmodified oligonucleotide drugs are easily and rapidly degraded by nucleases in the body, resulting in relatively low activity and poor druggability. Chemical modification of the oligonucleotide structure is an effective way to improve its activity, increasing its stability against nucleases and its affinity for RNA, further facilitating cellular endocytosis and tissue targeting, thereby effectively regulating and controlling target gene expression.

[0008] Based on the basic structure of an oligonucleotide: the base, sugar ring, phosphate backbone and termini, chemical modifications can be performed in four parts:

[0009] 1) Base modifications: These are mainly divided into three types: purine modifications, pyrimidine modifications, and base substitutions. Purine modifications include N6-methyladenosine, N1-methyladenosine, and 7-methylguanylate modifications. Pyrimidine modifications include 3-methyluridine, 5-methyluridine, 5-methylcytosine, N4-acetylcytidine, pseudouridine, thiouridine, propyneuridine, and dihydrouridine.

[0010] 2) Sugar ring modification: This can be divided into modifications and substitutions of the sugar ring. Modifications of the sugar ring include 2'-modification, 4'-modification, 5'-modification, isomer modification, and combinations of these modifications. The most common 2'-modifications in siRNA are 2'-OMe (2'-methoxy) and 2'-F (2'-fluoro). Compared with native siRNA, siRNAs modified with both 2'-OMe and 2'-F have higher Tm values, better serum stability, and better activity.

[0011] 3) Modification of the phosphate backbone: mainly by phosphorothioate modification, methyl phosphate, selenophosphate, boranophosphate, dithiophosphate modification, and modification by replacing the bridging oxygen atoms of the phosphodiester bond linkage region with sulfur atoms; the phosphate groups between nucleosides are completely replaced with groups that do not contain phosphorus atoms, for example, P atoms are replaced with C, S, and N atoms to form guanidino groups, S-methylthiourea, etc.

[0012] 4) Terminal modification: covalent attachment of special groups at the 5' and / or 3' ends of the sense strand and phosphorylation modification of the 5' end of the antisense strand.

[0013] Hepatitis B virus (HBV) is a double-stranded, hepatotropic virus that infects only humans and non-human primates, replicating primarily in the liver and can be transmitted from mother to child via blood (including minor trauma to the skin or mucous membranes) or sexual contact. HBV infection remains a major health problem worldwide, and chronic HBV infection is associated with a high rate of progression to liver fibrosis, cirrhosis, and even liver cancer.

[0014] The current standard of care for chronic HBV infection is oral nucleoside (nucleotide) analogs (NAs) and injectable interferon α. ​​NAs inhibit HBV replication by inhibiting HBV DNA synthesis. Most patients require long-term treatment, and the rate of virological relapse after discontinuation of the drug is high. Interferon α plays a dual role in immunomodulation and antiviral function through enhancing immune cell function, promoting cytokine expression, and inducing interferon signaling to encode multiple antiviral proteins. Interferon alone is only effective in a subset of patients and is relatively poorly tolerated. The 2022 "Guidelines for the Prevention and Treatment of Chronic Hepatitis B" recommends nucleoside (nucleotide) analogs such as entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, and tenofovir alafenamide, and recommended α interferons include pegylated α interferon.

[0015] Currently, the goal of chronic HBV infection treatment is to achieve functional cure—that is, the persistent undetectable HBV DNA and HBV surface antigen (HBsAg) levels after drug discontinuation, regardless of whether HBsAg serological conversion occurs. A sustained reduction in HBsAg levels and serological conversion are crucial for achieving functional cure, potentially alleviating liver inflammatory responses, improving liver tissue pathology, reducing the incidence of end-stage liver disease, and extending patient survival. Currently, clinical drug therapy has difficulty achieving functional cure or has extremely low functional cure rates. Therefore, further development of drugs that can downregulate HBsAg expression is necessary to achieve functional cure. Summary of the Invention [Problem to be solved by the invention]

[0016] To solve the technical problem of the lack of drugs capable of more effectively inhibiting HBV gene expression in the prior art, the present disclosure provides a double-stranded RNAi agent that targets, regulates, and controls HBV gene expression, and uses thereof. The present disclosure designs a series of unique siRNA sequences against the HBV genome sequence, and performs specific template modifications on the sequences.

[0017] Typically, siRNA monomers are modified with 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F). However, even if we consider only the combination of these two monomer modifications, the sense and antisense strands of siRNA have a total of 44 bases, i.e., 2 44 Furthermore, different configurations of the terminal thio modification further increase the number of possible modification schemes.

[0018] Even with the same siRNA sequence, different modification methods can result in significant differences in activity, and even with different siRNAs, the same modification method can result in significant differences in activity. Although there are several principles for siRNA modification design, previous studies have shown that it is impossible to accurately predict activity based on the modification method, meaning that there is no clear relationship between the modification method and activity. Therefore, screening for a highly active modification scheme from the countless possible modification combinations is extremely difficult.

[0019] The present disclosure chemically modifies designed siRNA sequences to screen for some special modified sequences that have significant inhibitory effects on HBV gene expression. [Means for solving the problem]

[0020] In one aspect, the disclosure provides a double-stranded RNAi agent comprising an antisense strand and a sense strand complementary to the antisense strand, the sense strand forming a double-stranded region, wherein the nucleotide sequence of the antisense strand is set forth in SEQ ID NO:8, 11, or 13, or the nucleotide sequence of the antisense strand is a modified sequence of the sequence set forth in SEQ ID NO:8, 11, or 13.

[0021] In another aspect, the disclosure also provides a conjugate comprising the double-stranded RNAi agent and a ligand conjugated to the double-stranded RNAi agent.

[0022] In another aspect, the present disclosure also provides a pharmaceutical composition comprising the double-stranded RNAi agent or conjugate and a pharmaceutically acceptable carrier.

[0023] In another aspect, the disclosure also provides a kit, including Kit A, wherein Kit A includes one or more of the double-stranded RNAi agent, the conjugate, or the pharmaceutical composition.

[0024] In another aspect, the present disclosure provides the use of the aforementioned double-stranded RNAi agent, conjugate or pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease associated with HBV gene expression.

[0025] In another aspect, the present disclosure also provides a method for reducing HBV gene expression or inhibiting HBV replication for non-prophylactic and / or therapeutic purposes, the method comprising administering to a subject one or more of the double-stranded RNAi agent, the conjugate, the pharmaceutical composition, and the kit.

[0026] The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present disclosure, provided that they are consistent with common knowledge in the art.

[0027] All reagents and materials used in this disclosure are commercially available. [Effects of the Invention]

[0028] The positive and progressive effects of the present disclosure are as follows:

[0029] (1) Either the present sequence or the modified sequence has a significant inhibitory effect on HBsAg, which is significantly superior to the positive sequence.

[0030] (2) When conjugated to GalNAc compounds, the modified sequence can be efficiently delivered to the liver of animals, significantly inhibiting HBV gene expression and significantly reducing the levels of HBsAg, HBeAg, and HBV DNA in serum.

[0031] (3) Each sequence modified with the modified template of the present application all has significantly improved HBV inhibitory activity compared to relatively similar sequences disclosed in the prior art.

[0032] (4) In the present disclosure, we have discovered that siRNAs with similar sequences have very different activities.

[0033] (5) This disclosure also reveals that different sequences have different sensitivities to various modification templates, and it is unclear which modification template can be used to modify an siRNA sequence to have high activity. [Brief explanation of the drawings]

[0034] [Figure 1A] Serum HBsAg levels in AAV-HBV model mice at a level of 3 mg / kg of candidate siRNA sequences. [Figure 1B] Serum HBV DNA levels in AAV-HBV model mice at a level of 3 mg / kg of candidate siRNA sequences. [Figure 1C] Serum HBeAg levels in AAV-HBV model mice at a level of 3 mg / kg of candidate siRNA sequences. [Figure 1D] Serum HBsAb levels in AAV-HBV model mice at a level of 3 mg / kg of candidate siRNA sequences. [Figure 1E] Serum ALT levels in AAV-HBV model mice at a level of 3 mg / kg of candidate siRNA sequences. [Figure 1F] This shows the change in body weight of AAV-HBV model mice at the level of 3 mg / kg of candidate siRNA sequences. [Figure 2A] Serum HBsAg levels in mice after two challenges. [Figure 2B] Serum HBV DNA levels in mice after two challenges. [Figure 2C] Serum HBeAg levels in mice after two challenges. [Figure 2D] Serum HBbAg levels in mice after two challenges. [Figure 2E] Serum ALT levels in mice after two challenges. [Figure 2F] Changes in mouse body weight after two challenges. [Figure 3A]HBsAg levels in AAV-HBV model mice at a level of 0.5 mg / kg of candidate siRNA sequences. [Figure 3B] HBV DNA levels in AAV-HBV model mice at a level of 0.5 mg / kg of candidate siRNA sequences. [Figure 3C] This shows the HBeAg levels in AAV-HBV model mice at a level of 0.5 mg / kg of candidate siRNA sequences. [Figure 3D] HBsAb levels in AAV-HBV model mice at a level of 0.5 mg / kg of candidate siRNA sequences. [Figure 3E] ALT levels in AAV-HBV model mice at a level of 0.5 mg / kg of candidate siRNA sequences. [Figure 3F] This shows the change in body weight of AAV-HBV model mice at the level of 0.5 mg / kg of candidate siRNA sequences. [Figure 4A] HBsAg levels in AAV-HBV model mice with different GalNAc-coupled candidate siRNA sequences. [Figure 4B] HBV DNA levels in AAV-HBV model mice with different GalNAc-coupled candidate siRNA sequences. [Figure 4C] 1 shows HBeAg levels in AAV-HBV model mice with different GalNAc-coupled candidate siRNA sequences. [Figure 4D] Figure 1 shows the ALT levels of AAV-HBV model mice with different GalNAc-coupled candidate siRNA sequences. [Figure 4E] 1 shows changes in body weight of AAV-HBV model mice with different GalNAc-coupled candidate siRNA sequences. DETAILED DESCRIPTION OF THE INVENTION

[0035] In order that this disclosure may be more readily understood, certain terms are first defined. Furthermore, it should be noted that when a value or range of values ​​for a parameter is listed, it is intended that intermediate values ​​and ranges among these listed values ​​are also part of this disclosure.

[0036] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or a plurality of elements, such as more than one element.

[0037] As used herein, the term "including" means "including but not limited to," and these terms are used interchangeably.

[0038] As used herein, the term "or" is used to mean, and they are used interchangeably with, the term "and / or," unless the context clearly indicates otherwise.

[0039] As used herein, the term "approximately" or "about" as applied to one or more target values ​​refers to a value similar to the stated reference value. In certain embodiments, unless otherwise specified or clear from the context, the term "approximately" or "about" refers to a range of values ​​that is within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in either direction (higher or lower) of the stated reference value (unless such number exceeds 100% of a possible value).

[0040] As used herein, "HBV" refers to hepatitis B virus, including hepatitis B virus genotypes A, B, C, D, E, F, G, H, I, J and subtypes thereof, and is not limited to a particular genotype.

[0041] "G," "C," "A," and "U" typically represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. "T" and "dT" may be used interchangeably herein and refer to deoxyribonucleotides whose nucleobase is thymine, such as deoxyribothymine, 2'-deoxythymidine, or thymidine. However, it should be understood that the terms "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" may also refer to modified nucleotides (described in more detail below) or alternative replacement moieties. Those skilled in the art will appreciate that guanine, cytosine, adenine, and uracil can be substituted with other moieties without substantially altering the base-pairing properties of an oligonucleotide (including nucleotides having such replacement moieties). For example, but not limited to, a nucleotide containing inosine as a base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, in the nucleotide sequences of the present disclosure, nucleotides containing uracil, guanine, or adenine may be substituted with nucleotides containing, for example, inosine, and sequences containing such substitutions are examples of the present disclosure.

[0042] The terms "RNAi agent" and "RNA interference agent" are used interchangeably herein and are encompassed by the terms defined herein, and refer to agents that mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. RNAi agents induce sequence-specific degradation of mRNA through a process called RNA interference (RNAi). RNAi agents regulate (e.g., inhibit) the expression of HBV in cells, e.g., cells within the body of a subject (e.g., a mammalian subject). RNAi molecules include single-stranded RNAi molecules, double-stranded siRNAs, and short hairpin RNAs (shRNAs).

[0043] The term "small interfering ribonucleic acid" or "siRNA" refers to a small interfering ribonucleic acid (RNAi) molecule. This is a double-stranded RNA molecule, also known in the art as short interfering RNA or silencing RNA. siRNA typically consists of a sense strand (also known as a passenger strand) and an antisense strand (also known as a leading strand), each of which is 17-30 nucleotides in length, typically 19-25 nucleosides. The antisense strand is complementary (at least 95% complementary, fully complementary, etc.) to the target nucleic acid (suitably the mature mRNA sequence), and the sense strand is complementary to the antisense strand, whereby the sense and antisense strands form a duplex or duplex region. The siRNA strands may form a blunt-ended duplex, or preferably, the 3' ends of the sense and antisense strands may form a 3' overhang, e.g., 1, 2, or 3 nucleotides, which may form a RISC substrate in vivo, similar to the product generated by Dicer. Effective extended forms of Dicer substrates are described in US8349809 and US8513207, which are incorporated herein by reference. In some embodiments, both the sense and antisense strands have a 2-nucleotide 3' overhang. Thus, the double-stranded region may be, for example, 17-25 nucleotides in length, e.g., 21-23 nucleotides in length.

[0044] The term "antisense strand" refers to the strand of RNAi (e.g., dsRNA) that comprises a region that is substantially complementary to a target sequence. As used herein, the term "complementary region" refers to the region of the antisense strand that is substantially complementary to a sequence (e.g., a target sequence) defined herein. If the complementary region is not completely complementary to the target sequence, mismatches may occur in the internal or terminal regions of the molecule. Usually, the most tolerable mismatches are in the terminal regions, for example, within 5, 4, 3, or 2 nucleotides of the 5'-end and / or 3'-end.

[0045] As used herein, the term "sense strand" refers to the strand of an RNAi that includes a region that is substantially complementary to a region of the antisense strand, as that term is defined herein.

[0046] As used herein, the term "inhibition" can be used interchangeably with "reduction," "silencing," "downregulation," "suppression," and other similar terms, and includes any level of inhibition.

[0047] As used herein, the phrase "inhibiting HBV gene expression" includes inhibiting the expression of HBV DNA, HBV mRNA, HBsAg, HBeAg, hepatitis B core antigen (HBcAg), and the like.

[0048] "Inhibiting the expression of HBV antigens" includes inhibiting the expression of HBsAg, HBeAg, and HBcAg proteins.

[0049] "Inhibiting HBV gene expression" includes inhibiting HBV DNA, HBV mRNA, HBsAg, HBeAg, HBcAg at any level, e.g., inhibiting HBV DNA, HBV mRNA, HBsAg, HBeAg, HBcAg expression at least partially, e.g., by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0050] HBV gene expression can be assessed based on the level of any variable associated with HBV gene expression, such as, for example, HBV DNA level, HBV mRNA level, HBV antigen protein level, or HBV viral particle level. Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level utilized in the art, such as a baseline level before administration or a level determined from a similar untreated or control-treated subject, cell, or sample (e.g., a buffer-only control or an inactive agent control).

[0051] As used herein, a "patient" or "subject" is intended to include a human or non-human animal, preferably a mammal such as a mouse. Most preferably, the subject or patient is a human.

[0052] As used herein, "HBV gene expression-associated disease" includes any disease associated with an HBV gene or protein. The disease may be caused, for example, by overproduction of an HBV antigen protein, mutation of an HBV gene, abnormal degradation of an HBV antigen protein, or abnormal interaction of an HBV antigen protein with other proteins or other endogenous or exogenous substances. Exemplary HBV-associated diseases include hepatitis associated with HBV infection, such as chronic hepatitis B and acute hepatitis B, as well as HBV / hepatitis D virus (HDV) coinfection and HBV / HIV coinfection-associated diseases.

[0053] As used herein, a "therapeutically effective amount" is intended to include the amount of an RNAi agent that, when administered to a patient to treat an HBV-related disease, is sufficient to treat the disease (e.g., reduce, ameliorate, or maintain an existing disease or one or more disease symptoms). The "therapeutically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity, medical history, age, weight, family history, genetic makeup, the stage of the pathological process mediated by HBV expression, type of previous or concurrent treatment (if any), and other personal characteristics of the patient being treated.

[0054] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent sufficient to prevent or ameliorate the disease or one or more symptoms of the disease when administered to a subject who has not yet experienced or exhibited symptoms of HBV-related disease but is susceptible to the disease. Ameliorating the disease includes slowing the progression of the disease or reducing the severity of subsequent disease. The "prophylactically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the degree of risk for the disease, medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment (if any), and other personal characteristics of the patient being treated.

[0055] A "therapeutically effective amount" or a "prophylactically effective amount" also includes that amount of an RNAi agent that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agents used in the methods of the present disclosure can be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0056] As used herein, the term "sample" includes similar fluids, cells, or tissues isolated from a subject, as well as collections of fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, etc. Tissue samples can include samples from tissues, organs, or localized regions. For example, samples may be derived from specific organs, parts of organs, or fluids or cells within these organs. In some examples, samples can be taken from the liver (e.g., the entire liver or specific parts of the liver, or specific types of cells within the liver, e.g., hepatocytes). In preferred embodiments, a "sample from a subject" refers to blood or plasma taken from the subject. In other embodiments, a "sample from a subject" refers to liver tissue (or subcomponents thereof) from the subject.

[0057] In one aspect, the disclosure provides a double-stranded RNAi agent comprising an antisense strand and a sense strand complementary to the antisense strand, the sense strand forming a double-stranded region, wherein the nucleotide sequence of the antisense strand is set forth in SEQ ID NO:8, 11, or 13, or the nucleotide sequence of the antisense strand is a modified sequence of the sequence set forth in SEQ ID NO:8, 11, or 13.

[0058] In some embodiments, the double-stranded RNAi agent comprises an oligonucleotide duplex comprising a paired sense strand and an antisense strand, wherein the sense strand has the sequence set forth in SEQ ID NO: 1, 4, or 6, or a fragment thereof, or a modified version of said sequence or fragment.

[0059] In some embodiments, the sense strand and the antisense strand comprise at least one modified nucleotide.

[0060] In some embodiments, the double-stranded RNAi agent functions to inhibit HBV gene expression.

[0061] In some embodiments, the at least one modified nucleotide is one or more selected from the group consisting of a deoxy-nucleotide, a 3'-terminal deoxy-thymine nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxy-modified nucleotide, a 2'-O-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising an unnatural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexynyl-modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methyl phosphate group, a nucleotide comprising a 5'-phosphate, and a nucleotide comprising a 5'-phosphate mimic.

[0062] In some embodiments, at least one strand of the double-stranded RNAi agent comprises a 3' overhang of at least two nucleotides.

[0063] In some embodiments, the double-stranded region of the double-stranded RNAi agent is 20 pairs of nucleotides.

[0064] In some embodiments, the sense strand of the double-stranded RNAi agent has 20 nucleotides and the antisense strand has 22 nucleotides.

[0065] In some embodiments, all modifications contained in the nucleotides in the sense and antisense strands are chemical modifications of the 2' position of the nucleotide ribose.

[0066] In some embodiments, the chemical modification at the 2'-position of the nucleotide ribose is one or more selected from the group consisting of 2'-methoxy, 2'-O-methoxyethyl, 2'-fluoro, 2'-benzyloxy, 2'-methylcarbonylamino, and 2'-pyridylmethoxy.

[0067] In some embodiments, the chemical modification at the 2' position of the nucleotide ribose is 2'-methoxy or 2'-fluoro.

[0068] In some embodiments, the nucleotides are linked by 3',5'-phosphodiester bonds.

[0069] In some embodiments, the 3',5'-phosphodiester bond comprises a thio modification.

[0070] In some embodiments, the 5' carbon atom of the 5'-terminal nucleotide glycoside of the antisense strand is phosphorylated.

[0071] The phosphorylated group at the 5'-position of the phosphorylation includes one or more selected from a 5'-vinylphosphonate group, a 5'-methylphosphonate group, a 5'-C-methylphosphate group, a 5'-phosphorothioate group, and a 5'-phosphate group, and has the following structure: TIFF2026009862000002.tif107151 (R is hydrogen, hydroxyl, amine group, C 1~4 Alkyl, aromatic groups, C 1~4 Alkoxy, C 1~4 alkylcarbonylamino, or halogen; The base is any one selected from adenine, guanine, cytosine, thymine, and uracil.

[0072] In some embodiments, the terminal nucleotides of the sequence are linked by a 3',5'-phosphodiester bond that includes a thio modification to form a chirally pure 3',5'-phosphorothioate diester bond.

[0073] In some embodiments, the sense strand and antisense strand comprise 1-3 thio linkages at their 5' ends, and the antisense strand comprises 1-3 thio linkages at their 3' ends.

[0074] In some embodiments, the antisense strand employs any of the modification schemes described in the table below. TIFF2026009862000003.tif239156 TIFF2026009862000004.tif227156 or TIFF2026009862000005.tif239158 TIFF2026009862000006.tif227158 and / or the sense strand employs any of the modification methods described in the table below. TIFF2026009862000007.tif243158 where 2'-OMe is 2'-methoxy, 2'-F is 2'-fluoro, PS is a phosphorothioate backbone, and EVP is 5'-(E)-vinylphosphonate.

[0075] In some embodiments, the double-stranded RNAi agent is modified as follows: The siRNA-modified template in which modification A was adopted in the antisense strand and modification scheme a in the sense strand was named DV27P. The siRNA-modified template in which modification A was adopted in the antisense strand and modification method b in the sense strand was named DV29P. The siRNA-modified template in which modification B was adopted for the antisense strand and modification method a for the sense strand was named DV26P. The siRNA-modified template in which modification B was adopted for the antisense strand and modification method b for the sense strand was named DV28P. The siRNA-modified template in which modification C was adopted for the antisense strand and modification method b for the sense strand was named DV32P. The siRNA-modified template in which modification D was adopted for the antisense strand and modification method b for the sense strand was named DV34P. The siRNA-modified template in which modification E was adopted for the antisense strand and modification method a for the sense strand was named DV25P. The siRNA-modified template in which modification F was adopted in the antisense strand and modification method b was adopted in the sense strand was named DV33P.

[0076] In some embodiments, the antisense strand uses modified groups at positions 2 to 8 from the 5' end, and the modified groups are one or more types selected from UNA, GNA, and DNA, and the structures of the UNA and GNA are as follows: TIFF2026009862000008.tif52101

[0077] The base is any one selected from adenine, guanine, cytosine, thymine, and uracil.

[0078] In some embodiments, the double-stranded RNAi agent comprises any one selected from the following oligonucleotide duplexes consisting of a pair of a sense strand and an antisense strand: (1) the sense strand has a sequence shown in SEQ ID NO: 81, and the antisense strand has a sequence shown in SEQ ID NO: 179, 180, 181, 182, or 184; (2) the sense strand has the sequence shown in SEQ ID NO: 82, and the antisense strand has the sequence shown in SEQ ID NO: 179, 180, or 183; (3) the sense strand has a sequence shown in SEQ ID NO: 83, and the antisense strand has a sequence shown in SEQ ID NO: 185 or 186; (4) the sense strand has the sequence shown in SEQ ID NO: 84, and the antisense strand has the sequence shown in SEQ ID NO: 186; (5) the sense strand has the sequence shown in SEQ ID NO: 85, and the antisense strand has the sequence shown in SEQ ID NO: 186; (6) the sense strand has a sequence shown in SEQ ID NO: 96, and the antisense strand has a sequence shown in SEQ ID NO: 201, 202, 203, or 204; (7) the sense strand has a sequence shown in SEQ ID NO: 97, and the antisense strand has a sequence shown in SEQ ID NO: 201 or 202; (8) the sense strand has a sequence shown in SEQ ID NO: 98, and the antisense strand has a sequence shown in SEQ ID NO: 205 or 206; (9) the sense strand has the sequence shown in SEQ ID NO: 99, and the antisense strand has the sequence shown in SEQ ID NO: 206; (10) The sense strand has a sequence shown in SEQ ID NO: 100, and the antisense strand has a sequence shown in SEQ ID NO: 206; (11) The sense strand has a sequence shown in SEQ ID NO: 106, and the antisense strand has a sequence shown in SEQ ID NO: 213, 214, 215, or 216; (12) The sense strand has a sequence shown in SEQ ID NO: 107, and the antisense strand has a sequence shown in SEQ ID NO: 214 or 213; (13) The sense strand has a sequence shown in SEQ ID NO: 108, and the antisense strand has a sequence shown in SEQ ID NO: 217 or 218; (14) The sense strand has a sequence shown in SEQ ID NO: 109, and the antisense strand has a sequence shown in SEQ ID NO: 218; (15) The sense strand has a sequence shown in SEQ ID NO: 110, and the antisense strand has a sequence shown in SEQ ID NO: 218; (16) The sense strand has the sequence shown in SEQ ID NO: 306, and the antisense strand has the sequence shown in SEQ ID NO: 321.

[0079] The double-stranded RNA (dsRNA) reagent (double-stranded RNAi agent) of the present disclosure may optionally be conjugated to one or more ligands. The ligand can be attached to the 3'-end, 5'-end, or both ends of the sense strand, the antisense strand, or both strands. For example, the ligand may be conjugated to the sense strand. In a preferred embodiment, the ligand is attached to the 3'-end of the sense strand. In one preferred embodiment, the ligand is a GalNAc ligand.

[0080] In another aspect, the disclosure provides a conjugate comprising a double-stranded RNAi agent as described above and a ligand conjugated to the double-stranded RNAi agent.

[0081] In some embodiments, the ligand is conjugated to the 3'-end or 5'-end of the oligonucleotide sense strand.

[0082] In some embodiments, the ligand is one or more GalNAc derivatives attached by a bivalent or trivalent branched conjugate, or a GalNAc derivative attached by a monovalent conjugate.

[0083] In some embodiments, the ligand is: TIFF2026009862000009.tif47100 (wherein X is hydrogen or a hydroxy protecting group or H, wherein the hydroxy protecting group comprises acetyl, benzoyl, or isobutyryl; Y is an amine protecting group or H, wherein the amine protecting group is formyl, acetyl, propionyl, n-butyryl, or isobutyryl; n is an integer between 0 and 20; and q, r, and s are independently integers between 1 and 7.)

[0084] In some embodiments, the ligand is: TIFF2026009862000010.tif43120

[0085] In some embodiments, the ligand is: TIFF2026009862000011.tif21120 (wherein X is oxygen, nitrogen, or sulfur, Y is an alkyl or aromatic group; R1 is oxygen or sulfur; R2 is hydrogen, an amine group, or C 1~4 Alkyl, aromatic groups, C 1~4 alkoxy or halogen; A is -(CH2) a -, -(CH2CH2O) b -, -((CH2) c NHCO) d -or-((CH2) c CONH) d where a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5; B is -(CH2) e where e is an integer from 0 to 7; L is -CONH- or -NHCO-; X1 is -(CH2) f -or-(CH2CH2O) f CH2-, and f is an integer of 1 to 5; X2 is -(CH2) gand g is an integer from 1 to 6; Y1 is 0 or 1, Y2 is 0, 1 or 2; Y3 is 1, 2 or 3; m is an integer from 0 to 4, n is an integer from 0 to 4.

[0086] In some embodiments, the ligand is G4, G5, G6, or G7. TIFF2026009862000012.tif176137

[0087] In some embodiments, the conjugate has the structure shown below: TIFF2026009862000013.tif182137

[0088] In some embodiments, the ligand is: TIFF2026009862000014.tif37128 (wherein X is oxygen, nitrogen, or sulfur, Y is an alkyl or aromatic group; R1 is oxygen or sulfur; R2 is hydrogen, an amine group, or C 1~4 Alkyl, aromatic groups, C 1~4 alkoxy or halogen; A is -(CH2) a -, -(CH2CH2O) b -, -((CH2) c NHCO) d -or-((CH2) c CONH) d where a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5; B is -(CH2) e where e is an integer from 0 to 7; L is -CONH- or -NHCO-; X1 is -(CH2) f-or-(CH2CH2O) f CH2-, and f is an integer of 1 to 5; X2 is -(CH2) g and g is an integer from 1 to 6; Y1 is 0 or 1, Y2 is 0, 1 or 2; Y3 is 1, 2 or 3; m is an integer from 0 to 4, n is an integer from 0 to 4.

[0089] In some embodiments, the ligand is G101, G102, G103, G105, or G106. TIFF2026009862000015.tif13082 TIFF2026009862000016.tif19296

[0090] In some embodiments, the conjugate has the structure shown below: TIFF2026009862000017.tif202115 TIFF2026009862000018.tif135133

[0091] In some embodiments, the conjugate comprises any one selected from the following oligonucleotide duplexes consisting of a pair of a sense strand and an antisense strand: (1) the sense strand has a sequence shown in SEQ ID NO: 326, 327, or 328, and the antisense strand has a sequence shown in SEQ ID NO: 179; (2) the sense strand has a sequence shown in SEQ ID NO: 329, 330, or 331, and the antisense strand has a sequence shown in SEQ ID NO: 181; (3) the sense strand has a sequence shown in SEQ ID NO: 338 or 341, and the antisense strand has a sequence shown in SEQ ID NO: 201; (4) The sense strand has the sequence shown in SEQ ID NO: 350, 351, or 352, and the antisense strand has the sequence shown in SEQ ID NO: 321.

[0092] In some embodiments, the conjugate functions to inhibit HBV gene expression.

[0093] In another aspect, the disclosure provides a pharmaceutical composition comprising the aforementioned double-stranded RNAi agent or the aforementioned conjugate, and a pharmaceutically acceptable carrier.

[0094] In one embodiment, a pharmaceutical composition is provided that includes a double-stranded RNAi agent described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the double-stranded RNAi agent can be used to treat diseases or conditions associated with HBV gene expression or activity, such as chronic hepatitis B. Such pharmaceutical compositions are formulated based on the mode of delivery. One example includes compositions formulated for systemic delivery via parenteral delivery, such as intravenous (IV) delivery. Another example includes compositions formulated for direct delivery to the brain parenchyma, such as via intracerebral infusion, such as via continuous pump infusion.

[0095] The pharmaceutical composition comprising the double-stranded RNAi agent of the present disclosure can be, for example, a solution with or without buffer, or a composition comprising pharmaceutically acceptable carrier.Such compositions include, for example, aqueous or crystalline compositions, liposome preparations, micelle preparations, emulsions, and gene therapy vectors.

[0096] In the method of the present disclosure, the double-stranded RNAi agent can be administered in a solution. The free double-stranded RNAi agent can be administered in a non-buffered solution such as saline or water. Alternatively, the free siRNA can be administered in a suitable buffer solution. The buffer solution can include acetate, citrate, prolamin, carbonate, phosphate, or any combination thereof. In one preferred embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolality of the buffer containing the double-stranded RNAi agent can be adjusted to be suitable for administration to a subject.

[0097] In some embodiments, the buffer solution further comprises a reagent for controlling the osmolality of the solution so that the osmolality is maintained at a desired value, e.g., a physiological value in human plasma. Solutes that can be added to the buffer solution to control the osmolality include, but are not limited to, proteins, peptides, amino acids, non-metabolizable polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the reagent for controlling the osmolality of the solution is a salt. In some embodiments, the reagent for controlling the osmolality of the solution is sodium chloride or potassium chloride.

[0098] The pharmaceutical compositions of the present disclosure can be administered at a dose sufficient to inhibit HBV gene expression. Typically, a suitable dose of the double-stranded RNAi agent of the present disclosure ranges from about 0.001 to about 200.0 mg / kg / day, typically from about 0.1 to 50 mg / kg / day. For example, the double-stranded RNAi agent (e.g., dsRNA) can be administered at a dose of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4 ,3.5,3.6,3.7,3.8,3.9,4,4.1,4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5,5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9,6,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7 .8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg.

[0099] The pharmaceutical composition can be administered once a day, or the double-stranded RNAi agent can be administered as two, three or more divided doses at appropriate intervals throughout the day, or can be administered by continuous infusion or delivery through controlled-release preparations.In this case, the amount of double-stranded RNAi agent contained in each sub-dose needs to be reduced accordingly to achieve the total daily dose.The dosage unit can also be formulated to be delivered over several days, for example, by using a conventional sustained-release preparation that continuously releases the double-stranded RNAi agent over several days.Sustained-release preparations are well known in the art and are particularly useful for delivering reagents to specific sites, and therefore can be used with the reagents of the present disclosure.In this embodiment, the dosage unit contains multiples of the corresponding daily dose.

[0100] In other embodiments, a single dose of the pharmaceutical composition can be administered in extended intervals, with subsequent doses administered at intervals of no more than 3, 4, or 5 days, or no more than 1, 2, 3, or 4 weeks. In some embodiments of the present disclosure, a single dose of the pharmaceutical composition of the present disclosure is administered weekly. In other embodiments of the present disclosure, a single dose of the pharmaceutical composition of the present disclosure is administered monthly.

[0101] Those skilled in the art will understand that certain factors, including but not limited to the severity of the disease or condition, previous treatment, the subject's general health and / or age, and other current diseases, can affect the dose and schedule required to effectively treat a subject. Furthermore, treating a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. As described elsewhere herein, the effective dose and in vivo half-life of each double-stranded RNAi agent included in the present disclosure can be estimated using conventional methods or based on in vivo tests using appropriate animal models.

[0102] The pharmaceutical compositions of the present disclosure can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (e.g., via a skin patch); pulmonary, e.g., by inhalation or insufflation of a powder or aerosol, including via a nebulizer; intratracheal; intranasal; epidermal, transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, e.g., via an implant device; or intracranial administration, e.g., intraparenchymal, intrathecal, or intraventricular administration.

[0103] The double-stranded RNAi agents used in the compositions and methods of the present disclosure can be prepared for delivery in membranous molecular assemblies such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer (e.g., one bilayer or multiple bilayers). Liposomes include unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the double-stranded RNAi agent composition. The lipophilic material typically separates the aqueous exterior from the aqueous interior, which does not contain the double-stranded RNAi agent composition (although in some instances, does contain the double-stranded RNAi agent composition). Liposomes are useful for transporting and delivering active ingredients to the site of action. Because the liposome membrane structurally resembles a biological membrane, upon administration of liposomes to a tissue, the liposome bilayer fuses with the cell membrane bilayer. When liposome and cell fuse, the aqueous contents inside, including double-stranded RNAi agent, are delivered into the cell, where this double-stranded RNAi agent can specifically bind to target RNA and mediate RNAi.In some cases, these liposomes are specifically targeted, for example, directing this double-stranded RNAi agent to specific cell type.

[0104] Liposomes containing double-stranded RNAi agents can be prepared in various ways. In one example, the lipid components of liposomes are dissolved in detergent, and the lipid components form micelles. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and be non-ionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholic acid, and lauroyl sarcosine. The double-stranded RNAi agent is then added to the micelles containing the lipid components. The cationic group of the lipid interacts with the double-stranded RNAi agent and condenses around the double-stranded RNAi agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation of double-stranded RNAi agents.

[0105] Double-stranded RNAi agents, such as dsRNAs of this disclosure, can be fully encapsulated within a lipid preparation (eg, an LNP or other nucleic acid-lipid particle).

[0106] The term "lipid nanoparticle (LNP)" used herein refers to stable nucleic acid-lipid particles.LNPs contain cationic lipids, non-cationic lipids, and lipids that prevent the particles from aggregating (for example, PEG-lipid conjugates).LNPs have a long circulation life after intravenous (iv) injection and accumulate at distant sites (for example, sites physically distant from the administration site), making them very useful for synthetic applications.

[0107] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) ranges from about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0108] In some embodiments, the double-stranded RNAi agent or the conjugate is administered in an unbuffered solution.

[0109] In some embodiments, the non-buffered solution is saline or water.

[0110] In some embodiments, the double-stranded RNAi agent or the conjugate is administered in a buffered solution.

[0111] In some embodiments, the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

[0112] In some embodiments, the buffer solution is a phosphate buffer solution.

[0113] In some embodiments, the double-stranded RNAi agent or the conjugate is formulated in a lipid preparation for delivery in a membranous molecular assembly.

[0114] In some embodiments, the lipid preparation is a nucleic acid-lipid particle.

[0115] In some embodiments, the lipid preparation is a lipid nanoparticle.

[0116] In some embodiments, the mass / mass ratio of lipid to said double-stranded RNAi agent or said conjugate is 1:1 to 50:1, 1:1 to 25:1, 3:1 to 15:1, 4:1 to 10:1, 5:1 to 9:1, or 6:1 to 9:1.

[0117] In some embodiments, the lipid nanoparticles comprise cationic lipids, neutral lipids, structured lipids, and polymer-conjugated lipids.

[0118] In some embodiments, the cationic lipid is a compound of formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof: TIFF2026009862000019.tif3563(G1 is C 1~6 alkylene, and G2 is C 2~8 alkylene, and G3 is C 1~3 alkylene, and L1 is C 6~15 is a straight chain alkyl, and L2 is C 12~25It is a branched alkyl.

[0119] In some embodiments, the cationic lipid is YK-009 having the structure of formula (II) (see Patent CN114044741B). TIFF2026009862000020.tif3494

[0120] In some embodiments, the cationic lipid is a compound of formula (II), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof: TIFF2026009862000021.tif3362(G1 is C 2~8 alkylene, and G2 is C 2~8 alkylene, L1 is -C(O)O- or -OC(O)-, L2 is -C(O)O- or -OC(O)-, and R1 is C 6~25 is a straight or branched alkyl; R2 is C 6~25 is a straight-chain or branched alkyl, G3 is HO(CH2)2- or HO(CH2)3-, G4 is HO(CH2)2- or HO(CH2)3-, and L is (CH2)2- or -(CH2)3- or -(CH2)4-.

[0121] In some embodiments, the cationic lipid is YK-401 having the structure of formula (II-I) or YK-402 having the structure of formula (II-II) (see Patent CN115784921B). TIFF2026009862000022.tif85118

[0122] In some embodiments, the cationic lipid is a compound of formula (III), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof: TIFF2026009862000023.tif3280(G1 is C 1~6 alkylene, and G2 is C 2~8 alkylene, and R1 is C 6~20 is a straight or branched alkyl; R2 is C 12~25is a branched alkyl, and G3 is HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CHO(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2-, or CH3CH2NH(CH2)2-.

[0123] In some embodiments, the cationic lipid is YK-201 having the structure of formula (III-I) or YK-202 having the structure of formula (III-II) (see Patent CN115677518B). TIFF2026009862000024.tif88144

[0124] In some embodiments, the cationic lipid is a compound of formula (IV), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof: TIFF2026009862000025.tif3276 (G1 is C 1~8 alkylene, and G2 is C 2~8 alkylene, and R1 is C 6~25 is a straight or branched alkyl; R2 is C 12~25 is a straight-chain or branched alkyl, and G is HO(CH)N(R)CHCH(OH)CH—, where R is —CH, —CHCH, or —CHCHOH.

[0125] In some embodiments, the cationic lipid is YK-305 having the structure of formula (IV-I) or YK-310 having the structure of formula (IV-II) (see Patent CN115745820B). TIFF2026009862000026.tif94130

[0126] In some embodiments, the cationic lipid is a compound of formula (V), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof: TIFF2026009862000027.tif3178(G 1 and G 2 are each independently an unsubstituted C6 to C 10 alkylene, and G 3 is unsubstituted C1 to C 12 alkylene, and R 1 and R 2 are independently C6 to C 24 Alkyl or C6-C 24 alkenyl, and R 3 is OR 5 , N, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and R 4 is C1~C 12 is a hydrocarbon group, and R 5 is H or a C1-C6 hydrocarbon group.

[0127] In some embodiments, the cationic lipid is ALC0315 (see patent CN108368028B) having the structure of formula (VI): TIFF2026009862000028.tif51127

[0128] In some embodiments, the cationic lipid is a compound of formula (VI), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof: TIFF2026009862000029.tif41122(R4 is -(CH2) n Q and -(CH2) n CHQR, where Q is -OR, -OH, -O(CH2) nand n is selected from the group consisting of N(R), -OC(O)R, -CX, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)R, -N(H)S(O)R, -N(R)C(O)N(R), -N(H)C(O)N(R), -N(H)C(O)N(H)(R), -N(R)C(S)N(R), -N(H)C(S)N(R), -N(R)S(O)R, and heterocycle.

[0129] In some embodiments, the cationic lipid is SM102 (see Patent CN110520409A) having the structure of formula (VI-I): TIFF2026009862000030.tif39140

[0130] In some embodiments, the cationic lipid is the compound DLIN-MC3-DMA (see CN102625696B) of formula (VII), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof. TIFF2026009862000031.tif24144

[0131] In some embodiments, the cationic lipid comprises one or more selected from YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA.

[0132] In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is between 1:1 and 10:1.

[0133] In some embodiments, the molar ratio of the cationic lipid to the structural lipid is between 1:1 and 5:1.

[0134] In some embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-70):(0.5-5).

[0135] In some embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-45):(0.5-5).

[0136] In some embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is 50:10:38.5:1.5 or 49:10:39.5:1.5.

[0137] In some embodiments, the neutral lipid comprises one or more selected from phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and derivatives thereof.

[0138] In some embodiments, the neutral lipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16).0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine The phosphatidylcholine may be one or more selected from the group consisting of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0139] In some embodiments, the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1,2-distearoyl-sn-glycero-3-phosphocholine.

[0140] In some embodiments, the structured lipid is one or more selected from cholesterol, a non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and a corticosteroid.

[0141] In some embodiments, the structural lipid is cholesterol.

[0142] In some embodiments, the polymer-conjugated lipid is one or more selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0143] In some embodiments, the polymer-conjugated lipid is one or more selected from distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

[0144] The pharmaceutical compositions of the present disclosure include, but are not limited to, solution, emulsion, and liposome-containing preparation.These compositions can be made with various components, including, but not limited to, preformed liquid, self-emulsifying solid, and self-emulsifying semisolid.When treating liver disease (such as liver cancer), liver-targeting preparations are particularly preferred.

[0145] The pharmaceutical compositions of the present disclosure (which can be conveniently provided in unit dosage form) can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include combining the active ingredients with the pharmaceutical carrier(s) or excipient(s). Generally, these pharmaceutical compositions are prepared by uniformly and intimately mixing the active ingredients with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0146] The pharmaceutical compositions of the present disclosure can be prepared into any of a number of possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gelatin capsules, suppositories, and enemas. The pharmaceutical compositions of the present disclosure can also be prepared as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may also contain substances that increase the viscosity of the suspension. Such substances include, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0147] Certain pharmaceutical compositions of the present disclosure also incorporate carrier compounds into their formulations. As used herein, "carrier compound" or "carrier" may refer to a nucleic acid or its analog that is inert (i.e., has no biological activity itself) but is recognized as a nucleic acid during biological processes, such as by degrading biologically active nucleic acids or promoting their removal from the circulation, thereby reducing their bioavailability. Coadministration of a nucleic acid and a carrier compound (usually in excess) can significantly reduce the amount of nucleic acid recovered in the liver, kidney, or other extracirculatory reservoirs. This is thought to be the result of competition between the carrier compound and the nucleic acid for a common receptor. For example, coadministration with polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4'isothiocyanatostilbene-2,2'-disulfonic acid can reduce the recovery of partially phosphorothioated dsRNA in liver tissue.

[0148] Compared to carrier compounds, "pharmaceutical carriers" or "excipients" are pharmaceutically acceptable solvents, suspending agents, or other pharmaceutically inert vehicles for delivering one or more nucleic acids to animals.The excipients may be liquid or solid, and are selected to provide the desired volume, consistency, etc. when combined with the nucleic acids and other components of a particular pharmaceutical composition, taking into account the intended mode of administration. Common pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized cornstarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, cornstarch, polyethylene glycol, sodium benzoate, sodium acetate, and the like); disintegrants (e.g., starch, sodium starch glycolate, and the like); and wetting agents (e.g., sodium lauryl sulfate, and the like).

[0149] Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not toxically react with nucleic acids can also be used to prepare the pharmaceutical compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0150] Preparations for topical administration of nucleic acids include solutions of nucleic acids in sterile or non-sterile aqueous, non-aqueous, or liquid or solid oil bases in common solvents such as alcohol. These solutions may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not toxically react with nucleic acids can be used.

[0151] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0152] The dosage forms, carrier compounds, pharmaceutical carriers, excipients, etc. of the above pharmaceutical compositions are described in US Pat. No. 10,125,369 B2, which is incorporated herein by reference.

[0153] In another aspect, the disclosure includes Kit A, wherein said Kit A comprises a kit including one or more of the double-stranded RNAi agents, conjugates, or pharmaceutical compositions described above.

[0154] In some embodiments, the kit further comprises Kit B, wherein Kit B comprises: (1) other drugs that reduce HBV gene expression, or compositions containing the drugs that reduce HBV gene expression; (2) One or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0155] As used herein, "other drugs that reduce HBV gene expression, or compositions comprising drugs that reduce HBV gene expression" refers to drugs that do not include the double-stranded RNAi agents, conjugates, or pharmaceutical compositions disclosed herein.

[0156] In another aspect, the present disclosure provides the use of the aforementioned double-stranded RNAi agent, conjugate or pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease associated with HBV gene expression.

[0157] In some embodiments, the HBV gene expression-associated disease is a disease type selected from chronic hepatitis B, liver fibrosis, cirrhosis, liver cancer, acute hepatitis B, and hepatitis D virus-associated disease associated with HBV infection.

[0158] In another aspect, the present disclosure provides methods for reducing HBV gene expression or inhibiting HBV replication for non-prophylactic and / or therapeutic purposes, the method comprising administering to a subject one or more of the aforementioned double-stranded RNAi agents, conjugates, pharmaceutical compositions, and kits.

[0159] As used herein, "for non-prophylactic and / or therapeutic purposes" refers to reducing HBV gene expression or inhibiting HBV replication for purposes of scientific research, such as in a laboratory.

[0160] Nucleotide abbreviations used herein are as follows: A = adenosine-3'-phosphate Am = 2'-methoxyadenosine-3'-phosphate Ams = 2'-methoxyadenosine-3'-phosphorothioate Af = 2'-fluoroadenosine-3'-phosphate Afs = 2'-fluoroadenosine-3'-phosphorothioate G = guanosine-3'-phosphate Gm = 2'-methoxyguanosine-3'-phosphate Gms = 2'-methoxyguanosine-3'-phosphorothioate Gf = 2'-fluoroguanosine-3'-phosphate Gfs = 2'-fluoroguanosine-3'-phosphorothioate C = cytidine-3'-phosphate Cm = 2'-methoxycytidine-3'-phosphate Cms = 2'-methoxycytidine-3'-phosphorothioate Cf = 2'-fluorocytidine-3'-phosphate Cfs = 2'-fluorocytidine-3'-phosphorothioate U = uridine-3'-phosphate Um = 2'-methoxyuridine-3'-phosphate Ums = 2'-methoxyuridine-3'-phosphorothioate Uf = 2'-fluorouridine-3'-phosphate Ufs = 2'-fluorouridine-3'-phosphorothioate AmsEVP = 5'-(E)-vinylphosphonate-2'-methoxyadenosine-3'-phosphorothioate UmsEVP = 5'-(E)-vinylphosphonate-2'-methoxyuridine-3'-phosphorothioate A(gna) = adenosine-diol nucleic acid C(gna) = cytidine-diol nucleic acid G(gna) = guanosine-diol nucleic acid T(gna) = thymidine-diol nucleic acid U(gna) = uridine-diol nucleic acid dA = deoxyadenosine-3'-phosphate dG = deoxyguanosine-3'-phosphate dC = deoxycytidine-3'-phosphate dT = deoxythymidine-3'-phosphate

[0161] The following examples are used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and all the raw materials used are commercially available.

[0162] Example 1: Synthesis of DV29P template-modified small interfering oligonucleotides A total of 36 siRNA base sequences were designed, with Alnylam's VIR-2218 serving as the positive reference, the sequence number of which was APC-VIR in this example, and ANC-DV29P serving as the negative control. The base sequences were modified using the DV29P template. Positions 7, 9, 11, and 17 of the sense strand were 2'-F modified, with the remaining positions 2'-OMe modified. Positions 2, 4, 5, 6, 14, and 16 of the antisense strand were 2'-F modified, with the remaining positions 2'-OMe modified. Additionally, there were two thio modifications at the 5' end of the sense strand. There were two thio modifications at the 5' and 3' ends of the antisense strand. There was one EVP modification at the 5' end of the antisense strand. The sequences of the siRNAs modified using the DV29P template are listed in Table 1.

[0163] 1. Synthesis of DV29P template modified sequence A194-DV29P The basic sequence of the small interfering ribonucleic acid with SEQ ID NO: A194-DV29P as set forth in Table 1: Sense strand: 5'-UCGUGUUACAGGCGGGGUUUU-3' (SEQ ID NO: 15) Antisense strand: 5'-AAAACCCCGCCUGUAACACGAGA-3' (SEQ ID NO: 47) Positions 7, 9, 11, and 17 of the sense strand are 2'-F modified, and the remaining positions are 2'-OMe modified. Positions 2, 4, 5, 6, 14, and 16 of the antisense strand are 2'-F modified, and the remaining positions are 2'-OMe modified. In addition, the sense strand has two thio modifications at its 5' end. The antisense strand has two thio modifications at its 5' and 3' ends. The antisense strand has one EVP modification at its 5' end. Equipment and reagents: Keika 192P model DNA / RNA automatic synthesizer. The solid support is a general-purpose support of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (manufacturer: Cytiva).

[0164] Preparation method Nucleotide monomer solutions such as DMT-A-OMe phosphoramidite monomer (Formula 1), DMT-C-OMe phosphoramidite monomer (Formula 2), DMT-G-OMe phosphoramidite monomer (Formula 3), DMT-U-OMe phosphoramidite monomer (Formula 4), DMT-AF phosphoramidite monomer (Formula 5), ​​DMT-CF phosphoramidite monomer (Formula 6), DMT-GF phosphoramidite monomer (Formula 7), DMT-UF phosphoramidite monomer (Formula 8), vinyl-(E)-phosphonate-A-OMe phosphoramidite monomer (Formula 9), and vinyl-(E)-phosphonate-U-OMe phosphoramidite monomer (Formula 10) were prepared in acetonitrile according to the monomer concentration of 0.15 M. TIFF2026009862000032.tif89116 TIFF2026009862000033.tif135118 The seventh base from the 5' end of the antisense strand of the positive reference sequence APC-VIR contains a diol nucleic acid (GNA) modification, and the GNA monomer structure is as follows: TIFF2026009862000034.tif91156

[0165] Prepare in the following steps: (1) Deprotection The DMT protecting group was removed using 3% dichloroacetic acid in toluene as the deprotecting reagent, followed by rinsing with acetonitrile. (2) Coupling Using 0.25 M 5-ethylthiotetrazolyl in acetonitrile as the activating agent, an acetonitrile solution of each nucleotide monomer was coupled, followed by rinsing with acetonitrile. (3) Oxidation / sulfurization Oxidation: Oxidation was carried out using 0.05 M iodine in pyridine / water (90 / 10) as the oxidizing agent, followed by rinsing with acetonitrile. Sulfurization: Sulfurization was carried out using a 3% solution of xanthan hydride in pyridine as the sulfurizing agent, followed by rinsing with acetonitrile. (4) Hydroxyl group protection Hydroxyl group protection was achieved using 10% acetic anhydride in tetrahydrofuran (CAPA) and tetrahydrofuran / pyridine / nitromethylimidazole 74 / 10 / 16 (v / v / v) (CAPB) as hydroxyl group protecting reagents, followed by rinsing with acetonitrile. The above procedure was repeated, and cycles were run according to the set sequence to obtain the fully protected product. (5) The DMT protecting group of the last nucleotide was removed using a toluene solution of 3% dichloroacetic acid as a deprotecting reagent, followed by rinsing with acetonitrile. (6) Aminolysis and purification The solid support was transferred to a reactor, concentrated aqueous ammonia (25-28%) was added, and aminolysis was maintained at 60°C for 12 hours. After that, the system was cooled to room temperature, the mixture was filtered, and rinsed with a mixed solution of purified water and ethanol. The filtrates were combined, passed through a chromatography column, concentrated, and freeze-dried to obtain the product. (7) Annealing The purified sense and antisense strands were mixed in a 1:1 ratio, heated to 95°C for 3 minutes, and then slowly cooled to room temperature to form a duplex. A194-DV29P purity: 90.52%; observed molecular weight: 14727.09.

[0166] 2. Synthesis of other sequences The other sequences listed in Table 1 were also synthesized according to the above method.

[0167] [Table 1] TIFF2026009862000036.tif219164 TIFF2026009862000037.tif219164 TIFF2026009862000038.tif219164 TIFF2026009862000039.tif70164

[0168] Example 2: Inhibitory effect of DV29P template-modified sequence on HBsAg and HBeAg The DV29P template-modified siRNA sequences synthesized in Example 1 were transfected into HepG2.2.15 cells via lipid nanoparticles (LNPs), and then the inhibitory effects of each sequence on HBsAg and HBeAg were detected using ELISA technology.

[0169] 1. Experimental Materials Test sample: DV29P template modified siRNA sequence listed in Table 1 (synthesized in Example 1). Cell type: HepG2.2.15 cells Drug solvent: sterile enzyme-free water, Gibco Opti-MEM (Thermo Fisher Scientific).

[0170] 2. Experimental Method ELISA was used to detect the inhibition of the samples against HBsAg and HBeAg in HepG2.2.15 cell line.

[0171] 2.1 Cell culture Subculture: HepG2.2.15 cells were subcultured in DMEM / F12 medium containing 10% fetal bovine serum, 370 μg / ml geneticin, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin in a cell culture incubator at 37°C and 5% CO2, with passage once every 3 days. The cells were digested with 0.25% trypsin, subcultured, and centrifuged at 800 rpm for 3 minutes. The supernatant was discarded, and fresh medium was added for subculture. Plating culture: HepG2.2.15 cells were plated and cultured in DMEM / F12 medium containing 10% fetal bovine serum, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin.

[0172] 2.2 Cell transfection Preparation of transfection mixture: Lipofectamine TMRNAiMAX (Thermo Fisher Scientific) and Opti-MEM were mixed at a ratio of 2:98 and vortexed to mix evenly. Preparation of transfection complexes: 30 μL of siRNA solution diluted with Opti-MEM was added to 30 μL of transfection mixture at a 1:1 (v / v) ratio, vortexed to mix evenly, and then allowed to stand at room temperature for 15 minutes to obtain transfection complexes. Preparation of transfection reagent for the transfection control group: 15 μL of the prepared transfection mixture was added to 15 μL of Opti-MEM, vortexed to mix evenly, and left to stand at room temperature for 15 minutes. The prepared transfection complexes were added to a 96-well cell culture plate (15 µL per well, three replicate wells per array) so that the final siRNA concentration in each well was 0.3 nM. The cell suspension (2.25 x 10 4 After uniformly mixing using the cross method, the cells were placed in a cell culture incubator at 37°C with 5% CO2 and cultured.

[0173] 2.3 Detection of HBsAg and HBeAg in cell supernatants 1) Collection of cell supernatant a. On the third day after cell transfection, the liquid was replaced, the cell supernatant was discarded, 150 μL / well of fresh medium was added, and the culture was continued. b. Six days after transfection, cell supernatants were collected for detection of HBsAg and HBeAg contents. 2) Quantitative detection of HBs and HBe The concentrations of HBsAg and HBeAg were detected using a hepatitis B virus e antigen detection kit (CL0310 manufactured by Autobio Diagnostics) and a hepatitis B virus surface antigen detection kit (CL0312 manufactured by Autobio Diagnostics). The specific steps are as follows: a. The kit and test samples were allowed to come to room temperature. b. 50 μL each of test samples, standards, negative controls, and positive references was added to a well plate. c. 50 μL of enzyme conjugate was added to each well, mixed thoroughly, and incubated at 37°C for 60 minutes. d. The liquid was removed from the well plate, washed five times with washing solution, and finally the well plate was tapped dry on absorbent paper. e. Luminescent substrates A and B were mixed in equal proportions, added at 50 μL / well, and allowed to react in the dark at room temperature for 3 minutes. f. Luminescence values ​​were measured using a microplate reader.

[0174] 2.4 Data Processing The formula for calculating the HBsAg and HBeAg inhibition rates is as follows: HBsAg inhibition rate (%) = (1 - HBsAg expression level in sample / HBsAg expression level in transfection control group on the same plate) x 100% HBeAg inhibition rate (%) = (1 - HBeAg expression level in sample / HBeAg expression level in transfection control group in the same plate) x 100%. HBsAg inhibition rate relative to the positive reference (assuming the inhibition rate of the positive reference is 1) = HBsAg inhibition rate (%) in the sample / Inhibition rate (%) of the positive reference on the same plate It should be understood that a value greater than 1 indicates that the sequence has a better inhibitory effect on HBsAg than the positive reference, and a value less than 1 indicates that the sequence has a lesser inhibitory effect on HBsAg than the positive reference. Percent inhibition of HBeAg relative to the positive reference (assuming the positive reference inhibition rate is 1) = Percent inhibition of HBeAg in the sample / Percent inhibition of the positive reference in the same plate It should be understood that a value greater than 1 indicates that the sequence has a better inhibitory effect on HBeAg than the positive reference, and a value less than 1 indicates that the sequence has a lesser inhibitory effect on HBeAg than the positive reference.

[0175] 2.5 IC50 experiment In this experiment, the concentration of each sequence was diluted four-fold starting from 10 nM, resulting in eight concentrations (10 nM, 2.5 nM, 0.625 nM, 0.15625 nM, 39.06 pM, 9.77 pM, 2.44 pM, and 0.61 pM). The inhibition rate of each sequence at each concentration was measured and plotted to calculate the IC50 concentration of each sequence and the positive control.

[0176] 2.6 Cytotoxicity experiments For IC50 experiments, cell supernatants were collected and cell viability was measured using the CellTiter-Glo® (Promega) kit. Briefly, CellTiter-Glo reagent and culture medium were mixed at a 1:1 ratio, and 100 μL was added to each well. After incubation at room temperature for 10 minutes, luminescence signals were detected using a microplate reader. The formula for calculating cell viability is as follows: Cell viability (%) = (sample signal value - blank control mean value) / (transfection control mean value - blank control mean value) x 100%.

[0177] 3. Experimental Results The experimental results showed that the sequences B207S-DV29P, B1572-DV29P, B1575-DV29P, B418S-DV29P, A206-DV29P, A416-DV29P, A1548-DV29P, A1550-DV29P, and A1573-DV29P had significantly better inhibitory effects on HBsAg than other sequences, and also had significantly better inhibitory effects on HBeAg than other sequences. The expression inhibition rates for HBsAg and HBeAg at a single concentration of each sequence and the inhibition rates for the positive reference sequence APC-VIR are shown in Tables 3, 4, 5, and 6. As a result of the single-concentration screening experiment, several sequences modified with template DV29P, including B207S-DV29P, B1572-DV29P, B1575-DV29P, B418S-DV29P, A206-DV29P, A416-DV29P, A1548-DV29P, A1550-DV29P, and A1573-DV29P (specific sequences are shown in Table 2), ranked higher than the positive reference APC-VIR in terms of inhibition rate against HBsAg and HBeAg. The IC50 and cytotoxicity results for each sequence are shown in Table 7. The IC50 experiment results showed that the 11 candidate sequences modified with the modified template DV29P designed in this disclosure had IC50 values ​​against HBsAg ranging from 0.01 to 0.08 nM, all superior to the positive reference (0.1777 nM). For example, the IC50 values ​​for A261-DV29P, A1573-DV29P, and A206-DV29P were 0.0169 nM, 0.0226 nM, and 0.0226 nM, respectively. These sequences can effectively inhibit HBsAg expression even at low concentrations. The IC50 values ​​for these 11 candidate sequences against HBeAg ranged from 0.06 to 0.4 nM, all superior to the positive reference (0.7685 nM). For example, the IC50 values ​​of A1573-DV29P, A206-DV29P, and A1550-DV29P were 0.0684 nM, 0.1105 nM, and 0.1202 nM, respectively. The cytotoxicity results showed that, except for A416-DV29P, which had a cell viability of 81.82% at the highest test concentration of 10 nM, the cell viability of all the remaining sequences exceeded 90% and no obvious cytotoxicity was observed, demonstrating the wide concentration range of the sequences. Based on the above results, nine sequences were selected from these 11 sequences for further optimization, and the sequences B207S-DV29P, B1572-DV29P, B1575-DV29P, B418S-DV29P, A206-DV29P, A416-DV29P, A1548-DV29P, A1550-DV29P, and A1573-DV29P were used as candidate sequences.

[0178] [Table 2]

[0179] (i) Inhibitory effect of each sequence at a single concentration on HBV gene expression after modification with the DV29P template. (1) Sequences with better inhibitory effects against HBsAg than the positive reference are shown in Table 3 and include B207S-DV29P, B1575-DV29P, B1572-DV29P, B418S-DV29P, A261-DV29P, A206-DV29P, A1550-DV29P, A1573-DV29P, A1548-DV29P, and A416-DV29P. Among these, the four sequences B207S-DV29P, B1575-DV29P, B1572-DV29P, and B418S-DV29P all had inhibitory rates against HBsAg of over 80% at a concentration of 0.3 nM. For example, the inhibition rate of B207S-DV29P reached 93.37%, which was 23.59% higher than that of the positive reference.

[0180] [Table 3]

[0181] (2) Sequences with a lower inhibitory effect on HBsAg than the positive reference are shown in Table 4. For example, the inhibitory rate of sequence B416S-DV29P on HBsAg is only 6.98%.

[0182] [Table 4]

[0183] (3) The sequences with better inhibitory effects on HBeAg than the positive reference are shown in Table 5, among which B1575-DV29P, B207S-DV29P, B1572-DV29P, and B418S-DV29P ranked first, 38.62%, 22.07%, 17.10%, and 13.24% better than the positive reference, respectively.

[0184] [Table 5]

[0185] (4) Sequences with a lower inhibitory effect on HBeAg than the positive reference are shown in Table 6. For example, the inhibitory rate of the sequence A1520-DV29P was only 0.28% at a concentration of 0.3 nM.

[0186] [Table 6]

[0187] (ii) IC50 experiments and cytotoxicity results Nine excellent sequences were selected for single-concentration experiments: B207S-DV29P, B1572-DV29P, B1575-DV29P, B418S-DV29P, A206-DV29P, A416-DV29P, A1548-DV29P, A1550-DV29P, and A1573-DV29P. The IC50 values ​​of these candidate sequences against HBsAg and HBeAg were lower than those of the positive control, demonstrating that their inhibitory effects were superior to those of the positive control. For example, the IC50 values ​​for A261-DV29P, A1573-DV29P, and A206-DV29P against HBsAg were 0.019 nM, 0.022 nM, and 0.024 nM, respectively, and the IC50 values ​​for A1573-DV29P, B207S-DV29P, A206-DV29P, and A1550-DV29P against HBeAg were 0.079 nM, 0.093 nM, 0.115 nM, and 0.138 nM, respectively. Specific results are shown in Table 7. The cytotoxicity results showed that, except for sequence A416-DV29P, which had a cell viability of 81.82% at the highest test concentration of 10 nM, the cell viability of all the remaining sequences exceeded 90% and no obvious cytotoxicity was observed, demonstrating the wide concentration range of the sequences. Specific results are shown in Table 7.

[0188] [Table 7]

[0189] Example 3: Inhibitory effect of unmodified sequences on HBV genes In this example, several unmodified sequences corresponding to the modified sequences in Example 1 were synthesized and transfected into HepG2.2.15 cells via lipid nanoparticles (LNPs). ELISA technology was used to detect the inhibitory effect of each sequence on HBsAg and HBeAg, and unmodified siRNA sequences with relatively superior inhibitory effects were screened.

[0190] 1. Experimental Materials Test sample The unmodified small interfering RNA sequences are shown in Table 8. All sequences were synthesized according to the method in Example 1.

[0191] [Table 8]

[0192] 2. Experimental Method See Example 2, the screening concentration was set at 0.3 nM.

[0193] 3. Experimental Results The experimental results showed that the sequences B207S, B1572, B1575, B418S, A206, A416, A1548, A1550, and A1573 had significantly better inhibitory effects on HBsAg and HBeAg than the other sequences. As shown in Table 9, the expression inhibition rates of each sequence, including B207S, B1575, B418S, A1573, B1572, A261, A206, A1550, A416, and A1548, at a single concentration were superior to those of the positive reference sequences. Among them, the inhibition rates of B207S against HBsAg and HBeAg were 77.02% and 40.74%, respectively, significantly superior to the 61.45% and 20.85% of the positive reference sequence APC.

[0194] [Table 9]

[0195] Example 4: Inhibitory effect of different template-modified sequences on HBsAg and HBeAg In this example, eight modified templates designed in this disclosure (DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, and DV34P) and existing disclosed templates (DV30P, DV31P, DV21P, and DV22P) were used to modify a total of 15 unmodified sequences: A205, B208, B207S, A261, B262, B264, A1550, B1556, B1560, B1575, A1573, B1572, B418S, A416, and B416S. After delivery to HepG2.2.15 cells via lipid nanoparticles (LNPs), the inhibitory effects of each sequence on HBsAg and HBeAg were detected using ELISA technology.

[0196] 1. Experimental Materials Test samples: 15 siRNA basic sequences modified using different templates (DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, DV34P, DV30P, DV31P, DV21P, DV22P) as shown in Table 11 (see Example 1 for synthesis method). Cell type: HepG2.2.15 cells Drug Solvent: Sterile enzyme-free water, Gibco Opti-MEM The principles of modification using the modification template of the present disclosure are as follows. When the antisense strand is 23 nucleotides in length, the antisense strand employs one or a combination of two or more of the modification schemes shown in Table 10-1 below.

[0197] [Table 10-1] TIFF2026009862000049.tif200164When the antisense strand is 22 nucleotides in length, the antisense strand employs one or a combination of two or more of the modification schemes shown in Table 10-2 below.

[0198] [Table 10-2] TIFF2026009862000051.tif199164When the sense strand is 21 nucleotides in length, the sense strand employs one or a combination of two of the modification schemes shown in Table 10-3 below.

[0199] [Table 10-3] When the sense strand is 20 nucleotides in length, the sense strand employs one or a combination of two of the modification schemes shown in Table 10-4 below.

[0200] [Table 10-4] In the above Tables 10-1 to 10-4, 2'-OMe is 2'-methoxy, 2'-F is 2'-fluoro, PS is a phosphorothioate backbone, and EVP is 5'-(E)-vinylphosphonate. The siRNA-modified template in which modification A was adopted in the antisense strand and modification scheme a in the sense strand was named DV27P. The siRNA-modified template in which modification A was adopted in the antisense strand and modification method b in the sense strand was named DV29P. The siRNA-modified template in which modification B was adopted for the antisense strand and modification method a for the sense strand was named DV26P. The siRNA-modified template in which modification B was adopted for the antisense strand and modification method b for the sense strand was named DV28P. The siRNA-modified template in which modification C was adopted for the antisense strand and modification method b for the sense strand was named DV32P. The siRNA-modified template in which modification D was adopted for the antisense strand and modification method b for the sense strand was named DV34P. The siRNA-modified template in which modification E was adopted for the antisense strand and modification method a for the sense strand was named DV25P. The siRNA-modified template in which modification F was adopted in the antisense strand and modification method b was adopted in the sense strand was named DV33P. The synthesis method for each sequence was the same as in Example 1.

[0201] [Table 11] TIFF2026009862000055.tif224164 TIFF2026009862000056.tif224164 TIFF2026009862000057.tif224164 TIFF2026009862000058.tif225164 TIFF2026009862000059.tif224164 TIFF2026009862000060.tif223164 TIFF2026009862000061.tif224164 TIFF2026009862000062.tif224164 TIFF2026009862000063.tif224164 TIFF2026009862000064.tif223164 TIFF2026009862000065.tif224164 TIFF2026009862000066.tif224164 TIFF2026009862000067.tif224164 TIFF2026009862000068.tif224164 TIFF2026009862000069.tif113164

[0202] 2. Experimental Method Referring to Example 2, the concentration for single concentration screening experiments was set at 0.3 nM.

[0203] 3. Experimental Results The experimental results showed that the sequences B207S-DV32P, A1550-DV27P, B1572-DV27P, B1575-DV27P, B207S-DV29P, A1573-DV29P, B1575-DV29P, B418S-DV34P, and A416-DV27P had significantly better inhibitory effects on HBsAg than the positive reference sequence, and significantly better inhibitory effects on HBeAg than the positive reference sequence.

[0204] The expression inhibition rates for HBsAg and HBeAg at a single concentration for each sequence and the inhibition rate for the positive reference sequence APC-VIR are shown in Table 12-41. The overall ranking is expressed as the inhibition rate ranking of the sequence relative to the positive reference.

[0205] The results of single-concentration screening experiments showed that some base sequences, when modified with the modified templates DV25P-29P and DV32P-34P designed in the present disclosure, had better inhibitory effects on HBsAg and HBeAg than the positive reference sequence. Among them, the modified sequences designed based on the base sequences B207S and B1575 ranked highest in inhibitory rate against HBsAg, demonstrating the sensitivity of the target. Among them, the sequences B207S-DV29P, B207S-DV32P, B1575-DV29P, B1575-DV26P, and B1575-DV27P all had inhibitory rates against HBsAg of over 90%. For example, the inhibitory rates of the sequences B207S-DV29P and B1575-DV29P against HBsAg were 92.11% and 91.78%, respectively. Among them, the modified sequences designed based on the basic sequences B1575 and A1573 ranked highest in inhibitory rate against HBeAg, indicating the sensitivity of the target. Among them, the sequences B1575-DV29P, B1575-DV26P, B1575-DV27P, and B1575-DV32P all had inhibitory rates against HBeAg of over 60%.

[0206] Even if the siRNA sequence is the same, its activity may be significantly different when modified with different modification templates.For example, when the basic sequence B1556 is modified with the modified sequence DV27P of the present disclosure, the inhibition rate of HBsAg can be significantly improved by 48.63%, compared with when the sequence is modified with the modified sequence DV28P of the present disclosure.

[0207] Although the basic sequences are similar, the sensitivity to each modified template is different.For example, when the basic sequence B1572 is modified with the modified template DV27P designed in the present disclosure, it has the highest inhibitory rate against HBsAg and HBeAg, while when the basic sequences A1573 and B1575 are modified with the template DV29P, it has the highest inhibitory rate against HBsAg and HBeAg.Therefore, it is unclear which modified template can be used to modify siRNA sequence to have high activity.

[0208] The IC50 test and cytotoxicity results for each sequence are shown in Table 42. The experimental results showed that the IC50 values ​​for the 11 sequences tested against HBsAg ranged from 0.05 nM to 0.15 nM, all lower than the 0.3362 nM of the positive reference sequence, demonstrating that their inhibitory effects were superior to those of the positive reference. For example, the IC50 values ​​for B1572-DV27P, B1550-DV27P, and B207S-DV32P against HBsAg were 0.0538 nM, 0.0570 nM, and 0.0572 nM, respectively. With the exception of sequence B261-DV26P, the IC50 values ​​for the remaining 10 sequences against HBeAg ranged from 0.2 nM to 0.85 nM, all lower than the 1.89 nM of the positive reference sequence, demonstrating that their inhibitory effects were superior to those of the positive reference. For example, the IC50 values ​​of the sequences B1550-DV27P, B1572-DV27P, and B1575-DV26P against HBeAg were 0.2073 nM, 0.2143 nM, and 0.2923 nM, respectively. The cytotoxicity results showed that the cell viability of these 11 sequences exceeded 90% at the highest tested concentration of 10 nM, without any obvious cytotoxicity, indicating that the sequences have a wide concentration range.

[0209] Based on the above results, 10 sequences, B207S-DV29P, B207S-DV32P, B1575-DV29P, B1575-DV26P, B1575-DV27P, B1572-DV27P, B1573-DV29P, B1550-DV27P, B416-DV27P, and B418S-DV34P, can be used as candidate sequences.

[0210] (i) Inhibitory effect of each basic sequence after template modification on HBsAg and HBeAg (1) Basic arrangement A1550

[0211] [Table 12]

[0212] [Table 13]

[0213] When the basic sequence A1550 was modified with the modified templates DV25P-29P and DV32P-34P designed in the present disclosure, the inhibitory rates against HBsAg and HBeAg were all improved compared to when modified with the modified templates disclosed in the prior art. For example, when modified with templates DV26P and DV27P, the inhibitory rates against HBsAg of B1550-DV26P and B1550-DV27P were improved by 25.36% and 25.51%, respectively, and the inhibitory rates against HBeAg were improved by 24.77% and 26.55%, respectively. Specific results are shown in Tables 12 and 13.

[0214] (2) Basic sequence B1556

[0215] [Table 14]

[0216] [Table 15]

[0217] When the base sequence B1556 was modified with the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure, the HBsAg inhibition rates of B1556-DV26P and B1556-DV27P were improved by 37.46% and 47.61%, respectively, compared to when modified with the modified templates disclosed in the prior art. The HBeAg inhibition rates of B1556-DV26P and B1556-DV27P were improved by 25.29% and 31.73%, respectively. The specific results are shown in Tables 14 and 15.

[0218] (3) Basic sequence B1560

[0219] [Table 16]

[0220] [Table 17]

[0221] When the base sequence B1560 was modified with the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure, the inhibitory rates of B1560-DV26P and B1560-DV27P against HBsAg were improved by 35.82% and 43.60%, respectively, compared to the modified templates disclosed in the prior art. The inhibitory rates against HBeAg were improved by 20.84% ​​and 28.54%, respectively, when modified with templates DV26P and DV27P. Specific results are shown in Tables 16 and 17. From this, the following was learned:

[0222] 1) For the basic sequences A1550, B1556, and B1560, when modified with the modified templates DV25P-29P and DV32P-34P designed in the present disclosure, the inhibitory activity against HBsAg and HBeAg was significantly superior to that of the modified templates disclosed in the prior art. When modified with DV26P and DV27P, the inhibitory rate against HBsAg and HBeAg was the highest and significantly superior to that of the modified templates disclosed in the prior art.

[0223] 2) Even if the siRNA sequence is the same, its activity may differ significantly when modified with different modification templates. For example, when the basic sequence B1556 is modified with the modified sequence DV27P of the present disclosure, the inhibition rate against HBsAg can be significantly improved by 48.63% compared to when the sequence is modified with the modified sequence DV28P of the present disclosure.

[0224] 3) Even siRNAs with similar sequences have significantly different activities. For example, the DV29P modified sequence A1550-DV29P showed a 40.86% higher inhibitory rate against HBsAg than A1556-DV29P.

[0225] (4) Basic sequence B1572

[0226] [Table 18]

[0227] [Table 19]

[0228] When the base sequence B1572 was modified with the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure, the inhibitory rates of B1572-DV26P and B1572-DV27P against HBsAg were improved by 26.84% and 27.59%, respectively, compared to the modified templates disclosed in the prior art. The inhibitory rates against HBeAg were improved by 32.02% and 36.74%, respectively, when modified with templates DV26P and DV27P. Specific results are shown in Tables 18 and 19.

[0229] (5) Basic sequence A1573

[0230] [Table 20]

[0231] [Table 21]

[0232] When the base sequence A1573 was modified with the modified templates DV26P-29P, DV32P, and DV34P designed in the present disclosure, the inhibitory rates against HBsAg and HBeAg were all significantly improved compared to the modified templates disclosed in the prior art. For example, when modified with template DV29P, the inhibitory rates of A1573-DV29P against HBsAg and HBeAg were improved by 16.08% and 32.68%, respectively. Specific results are shown in Tables 20 and 21.

[0233] (6) Basic sequence B1575

[0234] [Table 22]

[0235] [Table 23]

[0236] When the base sequence B1575 was modified with the modified templates DV26P-29P, DV32P, and DV34P designed in the present disclosure, the inhibitory rates against HBsAg and HBeAg were all significantly improved compared to the modified templates disclosed in the prior art. For example, when modified with template DV29P, the inhibitory rates of B1575-DV29P against HBsAg and HBeAg were improved by 31.19% and 46.17%, respectively. Specific results are shown in Tables 22 and 23. From this, the following was learned:

[0237] 1) When the base sequences B1572, A1573, and B1575 were modified with the modified templates DV26P-29P, DV32P, and DV34P designed in the present disclosure, the inhibitory effects against HBsAg and HBeAg were all significantly superior to those of the modified templates disclosed in the prior art. When the base sequence B1572 was modified with the modified templates DV26P and DV27P of the present disclosure, the inhibitory rates against HBsAg and HBeAg were the highest and significantly superior to those of the modified templates disclosed in the prior art. When the base sequences A1573 and B1575 were modified with the modified template DV29P of the present disclosure, the inhibitory rates against HBsAg and HBeAg were the highest and significantly superior to those of the modified templates disclosed in the prior art.

[0238] 2) Even if the siRNA sequence is the same, modification with different modification templates can significantly alter its activity. For example, when the base sequence B1575 was modified with the modified sequence DV29P of the present disclosure, the inhibition rate against HBsAg was significantly improved by 29.99% compared to when the sequence was modified with the modified sequence DV28P of the present disclosure.

[0239] 3) Even siRNAs with similar sequences have different sensitivities to each modified template. For example, when the base sequence B1572 was modified with the modified template DV27P designed in the present disclosure, the inhibitory rate against HBsAg and HBeAg was highest, while when the base sequences A1573 and B1575 were modified with the template DV29P, the inhibitory rate against HBsAg and HBeAg was highest.

[0240] (7) Basic Array B205

[0241] [Table 24]

[0242] [Table 25]

[0243] When the base sequence B205 was modified with the modified templates DV26P-29P, DV32P, and DV34P designed in the present disclosure, the inhibition rates against HBsAg and HBeAg were all significantly improved compared to the modified templates disclosed in the prior art. For example, when modified with template DV28P, the inhibition rates of B205-DV28P against HBsAg and HBeAg were improved by 36.55% and 25.36%, respectively. Specific results are shown in Tables 24 and 25.

[0244] (8) Basic arrangement B207S

[0245] [Table 26]

[0246] [Table 27]

[0247] When the basic sequence B207S was modified with the modified templates DV25P-29P and DV32P-34P designed in the present disclosure, the inhibitory rates against HBsAg and HBeAg were all significantly improved compared to the modified templates disclosed in the prior art. For example, when modified with templates DV29P and DV32P, the inhibitory rates of B207S-DV29P against HBsAg and HBeAg were improved by 35.37% and 26.71%, respectively, and the inhibitory rates of B207S-DV32P against HBsAg and HBeAg were improved by 34.98% and 18.95%, respectively. Specific results are shown in Tables 26 and 27.

[0248] (9) Basic sequence B208

[0249] [Table 28]

[0250] [Table 29]

[0251] For the base sequence B208, when modified with the modified templates DV26P-29P, DV32P, and DV34P designed in the present disclosure, the inhibitory rates of B208-DV26P against HBsAg and HBeAg were most significantly improved when modified with template DV26P, compared with the modified templates disclosed in the prior art, with increases of 37.63% and 19.38%, respectively. The specific results are shown in Tables 28 and 29. From this, the following was learned:

[0252] 1) When the basic sequences B205, B207S, and B208 were modified with the modified templates DV25P-29P and DV32P-34P designed in the present disclosure, their inhibitory effects against HBsAg and HBeAg were significantly superior to those of the modified templates disclosed in the prior art. When the basic sequence B205 was modified with the modified template DV28P designed in the present disclosure, the inhibitory rate of this sequence against HBsAg and HBeAg was the highest and significantly superior to those of the modified templates disclosed in the prior art. When the basic sequence B207S was modified with the modified template DV29P designed in the present disclosure, the inhibitory rate of this sequence against HBsAg and HBeAg was the highest and significantly superior to those of the modified templates disclosed in the prior art. For the basic sequence B208, when modified with the modified template DV26P designed in the present disclosure, the inhibitory rate of this sequence against HBsAg and HBeAg was the highest and was significantly superior to that of the modified templates disclosed in the prior art.

[0253] 2) Even if the siRNA sequence is the same, its activity may differ significantly when modified with different modification templates. For example, when the basic sequence B208 was modified with the modified sequence DV26P of the present disclosure, the inhibition rate against HBsAg was significantly improved by 21.96% compared to when the sequence was modified with the modified sequence DV29P of the present disclosure.

[0254] 3) Even siRNAs with similar sequences have significantly different activities. For example, the DV29P modified sequence B207S-DV29P showed a 45.84% higher inhibitory rate against HBsAg compared with B208-DV29P and a 23.77% higher inhibitory rate compared with B205-DV29P.

[0255] 4) Although the base sequences are similar, the sensitivity to each modified template was found to be different. For example, when the base sequence B205 was modified with the modified template DV28P designed in the present disclosure, the inhibitory rates against HBsAg and HBeAg were the highest, at 78.48% and 35.95%, respectively. On the other hand, when the base sequence B207S was modified with the template DV29P, the inhibitory rates against HBsAg and HBeAg were the highest, at 92.11% and 55.18%, respectively. When the base sequence B208 was modified with the template DV26P, the inhibitory rates against HBsAg and HBeAg were the highest, at 68.23% and 41.45%, respectively.

[0256] (10) Basic Array A261

[0257] [Table 30]

[0258] [Table 31]

[0259] For the base sequence A261, when modified with the modified templates DV26P-29P, DV32P, and DV34P designed in the present disclosure, the inhibitory rates against HBsAg and HBeAg were all significantly improved compared to the modified templates disclosed in the prior art. For example, when modified with template DV26P, the inhibitory rates of B261-DV26P against HBsAg and HBeAg were improved by 16.82% and 11.89%, respectively. Specific results are shown in Tables 30 and 31.

[0260] (11) Basic sequence B262

[0261] [Table 32]

[0262] [Table 33]

[0263] When the base sequence B262 was modified with the modified templates DV26P-29P, DV32P, and DV34P designed in the present disclosure, the inhibition rates against HBsAg and HBeAg were all significantly improved compared to the modified templates disclosed in the prior art. For example, when modified with template DV26P, the inhibition rates of B261-DV26P against HBsAg and HBeAg were improved by up to 28.99% and 17.93%, respectively. Specific results are shown in Tables 32 and 33.

[0264] (12) Basic sequence B264

[0265] [Table 34]

[0266] [Table 35]

[0267] When the base sequence B264 was modified with the modified templates DV26P-29P, DV32P, and DV34P designed in the present disclosure, the inhibition rates against HBsAg and HBeAg were all significantly improved compared to the modified templates disclosed in the prior art. For example, when modified with template DV29P, the inhibition rates of B264-DV29P against HBsAg and HBeAg were improved by 35.22% and 13.12%, respectively. Specific results are shown in Tables 34 and 35. From this, the following was learned:

[0268] 1) When basic sequences A261, B262, and B264 were modified with the modified templates DV26P-29P, DV32P, and DV34P designed in the present disclosure, their inhibitory effects against HBsAg and HBeAg were all significantly superior to those of the modified templates disclosed in the prior art. When basic sequence A261 was modified with the modified template DV26P designed in the present disclosure, the inhibitory rate of this sequence against HBsAg was the highest, and when modified with the DV27P template, the inhibitory rate of this sequence against HBeAg was the highest, significantly superior to those of the modified templates disclosed in the prior art. When basic sequence B262 was modified with the modified template DV28P designed in the present disclosure, the inhibitory rate of this sequence against HBsAg and HBeAg was the highest, significantly superior to those of the modified templates disclosed in the prior art. For the basic sequence B264, when modified with the modified template DV29P designed in the present disclosure, the inhibitory rate of the sequence against HBsAg and HBeAg was the highest and was significantly superior to that of the modified templates disclosed in the prior art.

[0269] 2) Even if the siRNA sequence is the same, its activity may differ significantly when modified with different modification templates. For example, when the basic sequence B264 was modified with the modification template DV27P of the present disclosure, the inhibition rate against HBsAg was significantly reduced by 20.16% compared to when it was modified with the DV29P template.

[0270] 3) Even siRNAs with similar sequences exhibited significantly different activities. For example, the DV29P modified sequence A261-DV29P exhibited a 40.21% higher inhibitory rate against HBsAg compared with B262-DV29P and a 30.46% higher inhibitory rate compared with B264-DV29P.

[0271] 4) Although the base sequences are similar, the sensitivity to each modified template was found to be different. For example, the base sequence B262 showed the highest inhibitory rate against HBsAg and HBeAg when modified with the modified template DV28P designed in this disclosure, while the base sequence B264 showed the highest inhibitory rate against HBsAg and HBeAg when modified with the template DV29P.

[0272] (13) Basic Array A416

[0273] [Table 36]

[0274] [Table 37]

[0275] When the basic sequence A416 was modified with the modified templates DV26P-29P, DV32P, and DV34P designed in the present disclosure, the inhibition rates against HBsAg and HBeAg were all significantly improved compared to the modified templates disclosed in the prior art. For example, when modified with DV27P, the inhibition rates against HBsAg and HBeAg were improved by 29.19% and 33.83%, respectively. Specific results are shown in Tables 36 and 37.

[0276] (14) Basic arrangement B416S

[0277] [Table 38]

[0278] [Table 39]

[0279] When the base sequence B416Sn was modified with the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure, the inhibition rates against HBsAg and HBeAg were all lower, less than 25%, compared to the modified templates disclosed in the prior art. Specific results are shown in Tables 38 and 39.

[0280] (15) Basic arrangement B418S

[0281] [Table 40]

[0282] [Table 41]

[0283] When the basic sequence B418S was modified with the modified templates DV26P-29P, DV32P, and DV34P designed in the present disclosure, the inhibition rates against HBsAg and HBeAg were all significantly improved compared to the modified templates disclosed in the prior art. For example, when modified with the DV34P template, the inhibition rates against HBsAg and HBeAg were improved by 25.62% and 26.91%, respectively. Specific results are shown in Tables 40 and 41. From this, the following was learned:

[0284] 1) When the basic sequences A416 and B418S were modified with the modified templates DV26P-29P, DV32P, and DV34P designed in the present disclosure, all of their inhibitory effects against HBsAg and HBeAg were significantly superior to those of the modified templates disclosed in the prior art. When the basic sequence A416 was modified with the modified template DV27P designed in the present disclosure, the inhibitory rate of this sequence against HBsAg and HBeAg was the highest. When the basic sequence B418S was modified with DV34P, the inhibitory rate of this sequence against HBsAg and HBeAg was the highest. On the other hand, when the basic sequence B416S was used with the modified templates DV26P-28P, DV32P, and DV34P designed in the present disclosure and the modified templates disclosed in the prior art, all of the sequences had low inhibitory rates against HBsAg and HBeAg, less than 25%.

[0285] 2) Even if the siRNA sequence is the same, its activity may differ significantly when modified with different modification templates. For example, when the basic sequence A416 was modified with the modification template DV27P of the present disclosure, the inhibitory rate against HBsAg was improved by 16.79% compared to when it was modified with the DV32P template.

[0286] 3) Even siRNAs with similar sequences have significantly different activities. For example, the DV29P modified sequence A416-DV29P showed a 72.41% higher inhibitory rate against HBsAg than B416S-DV29P.

[0287] 4) Although the base sequences are similar, the sensitivity to each modified template was found to be different. For example, the base sequence A416 exhibited the highest inhibitory rate against HBsAg and HBeAg when modified with the modified template DV27P designed in this disclosure, while the base sequence B418S exhibited the highest inhibitory rate against HBsAg and HBeAg when modified with the template DV34P.

[0288] (ii) IC50 experiments and cytotoxicity results A total of 12 sequences, including B207S-DV29P, B207S-DV32P, B1575-DV29P, B1575-DV26P, B1575-DV27P, B1572-DV27P, B1573-DV29P, B1550-DV27P, B261-DV26P, B416-DV27P, and B418S-DV34P, as well as one positive reference, APC-VIR, were selected as dominant sequences in single-concentration screening experiments, and IC50 experiments were performed to measure the IC50 concentration of each sequence.

[0289] [Table 42]

[0290] The IC50 experiments showed that the IC50 values ​​of these 11 sequences against HBsAg ranged from 0.05 nM to 0.15 nM, all lower than the 0.3362 nM of the positive reference sequence, demonstrating their superior inhibitory effects. For example, the IC50 values ​​of sequences B1572-DV27P, B1550-DV27P, and B207S-DV32P against HBsAg were 0.0538 nM, 0.0570 nM, and 0.0572 nM, respectively, indicating that these sequences could effectively inhibit HBsAg expression at low concentrations. With the exception of sequence B261-DV26P, the remaining 10 sequences had IC50 values ​​between 0.2 nM and 0.85 nM against HBeAg, all lower than the 1.89 nM of the positive reference sequence, demonstrating their superior inhibitory effects. For example, the IC50 values ​​of the sequences B1550-DV27P, B1572-DV27P, and B1575-DV26P against HBeAg were 0.2073 nM, 0.2143 nM, and 0.2923 nM, respectively, indicating that these sequences can effectively inhibit HBeAg expression at low concentrations. The experimental results are shown in Table 42.

[0291] The cytotoxicity results showed that the cell viability of all 11 sequences exceeded 90% at the highest test concentration of 10 nM, with no obvious cytotoxicity observed, indicating that the sequences had a wide concentration range. Specific results are shown in Table 42. Example 5: Comparison of the inhibitory effect of sequences disclosed in the prior art on HBV genes

[0292] In this example, the HBV gene inhibition efficiency was compared between unmodified sequences disclosed in the prior art that are identical to or similar to the basic sequences B207S, B1575, B1550, B1572, A416, B1573, and B418S disclosed herein, sequences modified with the disclosed existing modified templates DV30P, DV31P, DV21P, and DV22P, and sequences modified with the modified templates DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, and DV34P disclosed herein.

[0293] 1. Experimental Materials Test sample (1) Sequences disclosed in the prior art are shown in Table 43.

[0294] [Table 43] (2) In Example 4, siRNA sequences modified using modified templates DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, DV34P, DV30P, DV31P, DV21P, and DV22P and the corresponding unmodified base sequences were employed. Cell type: HepG2.2.15 cells Drug Solvent: Sterile enzyme-free water, Gibco Opti-MEM

[0295] 2. Experimental Method Referring to Example 2, the concentration for single concentration screening experiments was set at 0.3 nM.

[0296] 3. Experimental Results The experimental results showed that the inhibitory rates of the unmodified basic sequences B207S, A1550, B1572, B1575, A1573, B418S, and A416 of the present disclosure against HBsAg and HBeAg were significantly better than those of the similar sequences P206, P413, P1551, and APC, and the inhibitory rates were further significantly improved after template modification.

[0297] Tables 44 and 45 show a comparison of the inhibition rates against HBsAg and HBeAg, respectively, between sequences disclosed in the prior art and sequences relatively close to the sequences of the present disclosure.

[0298] As can be seen from Table 43, compared with sequence P206 disclosed in the prior art, sequence B207S of the present disclosure is shifted one base backward and its length is reduced by one base. Compared with P206, the inhibitory rates of B207S against HBsAg and HBeAg were improved by 15.57% and 11.69%, respectively. When B207S was modified with the template of the present disclosure, the inhibitory efficiency was further improved. For example, when modified with DV32P, the inhibitory rates of B207S-DV32P against HBsAg and HBeAg were improved by 28.92% and 20.55%, respectively. Furthermore, it was significantly superior to the modified templates DV30P, DV31P, DV21P, and DV22P disclosed in the prior art.

[0299] Compared with the sequence P413 disclosed in the prior art, the sequence A416 of the present disclosure is shifted three bases backward. Compared with P413, the inhibitory rates of A416 against HBsAg and HBeAg were improved by 5.95% and 7.42%, respectively. When A416 was modified with the template of the present disclosure, the inhibitory efficiency was further improved. For example, after modification with DV27P, the inhibitory rates of B416-DV27P against HBsAg and HBeAg were improved by 24.16% and 21.26%, respectively. It is also significantly superior to the modified templates DV30P, DV31P, DV21P, and DV22P disclosed in the prior art.

[0300] Compared with the sequence P1551 disclosed in the prior art, the sequence A1550 of the present disclosure is shifted forward by one base. Compared with P1551, the inhibitory rates of A1550 against HBsAg and HBeAg are improved by 7.98% and 9.03%, respectively. When A1550 is modified with the template of the present disclosure, the inhibitory efficiency is further improved. For example, after modification with DV27P, the inhibitory rates of B1550-DV27P against HBsAg and HBeAg are improved by 23.96% and 26.06%, respectively, and are significantly superior to the modified templates DV30P, DV31P, DV21P, and DV22P disclosed in the prior art.

[0301] Compared with the sequence APC disclosed in the prior art, the sequences B1575, B1573, and B1572 of the present disclosure are shifted forward by 4, 6, and 7 bases, respectively, and extended by 2 bases. Compared with APC, the inhibitory rates of B1575, B1573, and B1572 against HBsAg were improved by 13.46%, 10.80%, and 9.94%, respectively, and when modified with the template of the present disclosure, the inhibitory efficiency was further improved. For example, the sequence B1575-DV29P showed the most significant improvement, with an increase of 29.28%, and the inhibitory rate against HBeAg was improved by 24.25%. The sequence B1575-DV26P showed an improved inhibition rate of 27.18% against HBsAg and 16.55% against HBeAg, which was significantly superior to the modified templates DV30P, DV31P, DV21P, and DV22P disclosed in the prior art.

[0302] [Table 44] TIFF2026009862000103.tif174164

[0303] [Table 45] TIFF2026009862000105.tif175164

[0304] Example 6: Inhibitory effects of sequences employing specific off-target prevention designs on HBV and off-target genes In practical applications of siRNA, there are many cases where the expression of non-target mRNAs that are only partially complementary to the guide strand (antisense strand) is inhibited. Research by Alnylam Pharmaceuticals has shown that the hepatotoxicity of N-acetylgalactosamine (GalNAc)-coupled siRNA is mainly due to off-target effects caused by gene inhibition of incorrect targets via a recognition mechanism such as microRNA (miRNA).

[0305] To solve this problem, heat-labile nucleotide modifications such as glycol nucleotide (GNA), unlocked nucleotide (UNA), or DNA can be used at positions 6 and 7 of the antisense strand seed region to disrupt the antisense strand seed region, thereby affecting siRNA binding to non-target mRNA through seed region recognition, significantly reducing off-target effects and hepatotoxicity. Alnylam Pharmaceuticals has adopted a GNA modification at position 7 of the siRNA antisense strand in its latest fifth-generation template design to reduce siRNA off-target effects.

[0306] In this example, the base sequences B207S, B1575, A1550, B1572, A1573, A261, A416, and B418S were used to study off-target and off-target prevention. To evaluate the off-target status of each sequence, the inhibitory efficiency of each sequence against target and off-target genes was compared by qPCR in cell experiments.

[0307] In this example, to reduce the off-target effects of the sequences, the sequences B418S-DV34P and B416-DV27P, which have potential off-target effects, were modified with DNA or GNA at positions 6 and 7 of the antisense strand, and their activity and off-target effects were compared with sequences without the anti-off-target modifications.

[0308] 1. Experimental Materials Test samples: Template-modified sequences listed in Table 46 and corresponding sequences employing off-target effect prevention designs (see Example 1 for synthesis methods). In the sequence numbers, d67B indicates that the sequence employs the d67B off-target prevention modification, d7B indicates that the sequence employs the d7B off-target prevention modification, and + indicates that the sequence employs the GNA off-target prevention modification. Cell type: HepG2.2.15 cells Drug solvent: Sterile enzyme-free water, Gibco Opti-MEM.

[0309] [Table 46]

[0310] 2. Experimental Method The qRT-PCR method was used to detect the inhibition of samples against HBV and off-target genes in HepG2.2.15 cells.

[0311] 2.1 Cell culture Subculture: HepG2.2.15 cells were subcultured in DMEM / F12 medium containing 10% fetal bovine serum, 370 μg / ml geneticin, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin in a cell culture incubator at 37°C and 5% CO2, with passage once every 3 days. The cells were digested with 0.25% trypsin, subcultured, and centrifuged at 800 r / min for 3 minutes. The supernatant was discarded, and fresh medium was added for subculture. Plating culture: HepG2.2.15 cells were plated and cultured in DMEM / F12 medium containing 10% fetal bovine serum, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin.

[0312] 2.2 Cell transfection Preparation of transfection reagent: Lipofectamine RNAiMAX and Opti-MEM were mixed at a ratio of 2:98 and vortexed to mix evenly. Preparation of transfection complexes: 30 μL of siRNA solution diluted with Opti-MEM was added to 30 μL of transfection mixture at a 1:1 (v / v) ratio, vortexed to mix evenly, and then allowed to stand at room temperature for 15 minutes to obtain transfection complexes. Preparation of transfection reagent for the transfection control group: 15 μL of the prepared transfection mixture was added to 15 μL of Opti-MEM, vortexed to mix evenly, and left to stand at room temperature for 15 minutes. The prepared transfection complexes were added to a 96-well cell culture plate (15 μL per well, three replicate wells per array) so that the final siRNA concentrations in each well were 0.065, 0.14, and 0.28 nM, respectively. Three concentrations of each siRNA were prepared, and each concentration was set up in three replicate wells. The cell suspension (2.25 × 10 4 After uniformly mixing using the cross method, the cells were placed in a cell culture incubator at 37°C with 5% CO2 and cultured.

[0313] 2.3 RNA extraction and reverse transcription Forty-eight hours after transfection, the culture medium was removed and the cells were harvested for RNA extraction. Total RNA was extracted using the RNeasy® 96 Kit (QIAGEN-74182) according to the kit's instructions. The RNA was then reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (Vazyme) according to the kit's instructions.

[0314] 2.4 RT-qPCR HBV and off-target gene cDNAs were detected by qPCR, with GAPDH cDNA detected in parallel as an internal control. Eight μL of the prepared qPCR reaction mixture and 2 μL of sample cDNA were added to a 384-well plate. The SYBR qPCR reaction program was as follows: 50°C for 2 minutes, 95°C for 2 minutes, then cycling to 95°C for 5 seconds, followed by 60°C for 30 seconds, for a total of 40 cycles. The final melting curve was 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds.

[0315] 2.5 Data Analysis Based on the CT value of each sample, the ΔΔCT relative quantification method was used to calculate the target gene RNA expression level in the sample. The relative expression level of the target gene was calculated by 2 -ΔΔCT was expressed as:

[0316] The calculation formula is as follows: ΔCT = average CT value of target gene - average CT value of GAPDH ΔΔCT = ΔCT (experimental group) - ΔCT (transfection control group) Relative expression level of target gene mRNA = 2 -ΔΔCT Gene expression inhibition rate (%) = (1 - mRNA expression level of sample / mRNA expression level of transfection control group) x 100%

[0317] 3. Experimental Results Experimental results showed that the sequences B207S, B1575, A1550, B1572, A1573, and A261 had no off-target effects. The sequences B418S-DV34P and A416-DV27P, when designed to prevent off-targeting, had significantly reduced inhibitory effects on off-target genes without affecting the inhibitory effect on the target gene HBV. This indicates that the off-targeting design does not affect the inhibitory effect of the sequences modified with the template of the present disclosure on the HBV gene, but can significantly inhibit off-target effects.

[0318] (i) qPCR analysis of potential off-target effects and off-target prevention effects of each sequence (1) The inhibitory efficiency of each sequence against target and off-target genes is shown in Table 47. The results show that sequence A416 exhibited a significant concentration-dependent inhibitory effect against the SLCO2B1 gene, with a maximum inhibition rate of 40.05%. B418S exhibited a consistent concentration-dependent inhibition against the SLC41A2 gene, with a maximum inhibition rate of 24.13%. The remaining sequences, B207S, A1550, and A416, all exhibited inhibition rates of less than 20% against off-target genes, indicating no off-target effects. The two sequences, A416 and BS18S, also have potential off-target effects due to the large number of potential off-target genes overall.

[0319] [Table 47]

[0320] (2) The off-target prevention d7B and d67B designs were applied to sequences A416 and BS18S. The inhibitory effects of the off-target prevention designed sequences and sequences without the off-target prevention design on off-target genes and target genes are shown in Table 48. The results showed that when modified with the off-target prevention d7B modification, the IC50 of sequence C416-DV27Pd7B against the off-target gene SLCO2B1 was improved by 5.69-fold, and the maximum inhibition rate was reduced by 9.54%. The maximum inhibition rate of sequence C418S-DV34Pd7B against the off-target gene SLC41A2 was reduced by 16.91%, which was significantly better than the off-target prevention modifications d67B and GNA. The IC50 of sequence C418S-DV34Pd7B against the target gene HBV was 0.22 nM, which is close to the value of sequence B418S-DV34P without the off-target prevention modification. The maximum inhibition rates for the target genes were approximately 81% in all cases, which indicated that the inhibitory effect of the sequence C418S-DV34Pd7B on the target genes was not affected by the d7B modification.

[0321] [Table 48]

[0322] Example 7: Inhibitory effect of sequences modified with the modified template (high dose) of the present disclosure against HBV in mice In this example, we selected several sequences, including the base sequences B418S, A416, B1550, B207S, B1575, B1572, and A1573, and modified them. For example, we coupled all of them to GalNAc ligands using template modifications alone or both template modifications and off-target prevention design. Using an AAV-HBV mouse model, we analyzed the in vivo efficacy of these RNAi agents after repeated administration at high doses, such as the 3 mg / kg level. After infection with a recombinant adeno-associated virus (AAV) carrying a replication-competent HBV genome, this mouse model continuously produced HBV virions and HBV antigens for over a year without seroconversion, recapitulating some of the immunological characteristics of clinical chronic hepatitis B patients. Therefore, this model is also being used to evaluate novel immune-based and antiviral therapies. In this example, an AAV-HBV mouse model was used to detect the inhibitory effects of the above sequences on HBsAg, HBeAg, and HBV DNA in serum at different time points, and the effect of each sequence on the reconstitution of adaptive immune function in mice at specific time points.

[0323] 1. Experimental Materials experimental drug The 3' end of the sense strand of each sequence in Table 49 (including sequences using template modification only and sequences using template modification and off-target prevention design) is coupled to GalNAc ligand G5, and the structural formula is as follows: TIFF2026009862000109.tif41123 For the coupling method between oligonucleotide and ligand G5, refer to the preparation method in Example 3 of patent application CN116854754A. The oligonucleotide and ligand G5 form a conjugate as shown below. TIFF2026009862000110.tif36139 The specific sequence of each sequence is as shown in Table 49. In the SEQ ID NO:, G5 indicates coupling of the sequence to GalNAc ligand G5, and GL indicates coupling of the sequence to GalNAc ligand L96. The structural formula of L96 is as follows: TIFF2026009862000111.tif47126

[0324] [Table 49] TIFF2026009862000113.tif114164

[0325] Preparation of test drug: Drug solvent: PBS buffer Preparation conditions: sterile environment Labeling method: The prepared dosage formulation should be labeled, and the topic number, name, concentration, quantity, preparation date, preparer, storage conditions, etc. should be written on the outer box. Storage conditions: Prepare immediately before use, and store any remaining sample at -80°C.

[0326] Experimental Animal Information: Species / strain: AAV-HBV mouse Grade: SPF Gender: Male Quantity: 100 animals Age: 7 weeks Weight: 19-24g Origin: Guangdong Zhiyuan Biomedical Science and Technology Co., Ltd. Production license number: SCXK(Guangdong)2021-0057

[0327] Animal Ethics Committee (IACUC): After receiving the experimental animals, they were housed at Guangzhou JINNIO Biotechnology Co., Ltd. under license number JENNIO (JINBIN) 2019-0002. This project was reviewed by the Experimental Animal Ethics Committee of Guangzhou JINNIO Biotechnology Co., Ltd., and the IACUC number is JENNIO-IACUC-2024-A005. The study process was carried out in strict accordance with IACUC requirements to ensure animal welfare.

[0328] Breeding and care Rearing conditions: After being received, the experimental animals were reared at Guangzhou Jijou Biotechnology Co., Ltd. They were reared in a rearing cage with a length × width × height of 29.0 cm × 18.5 cm × 13.0 cm. The set temperature range was 20 - 26°C, the set humidity range was 40% - 70%, and the lights automatically turned on, with 12 hours of light and darkness alternating repeatedly.

[0329] The criteria for the rearing environmental conditions refer to the national standard GB14925 - 2010 of the People's Republic of China.

[0330] The animals were able to freely ingest feed and drinking water. This feed was irradiated sterilized experimental mouse maintenance feed provided by Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., and the production license number was Su Feed Certificate (2019) No. 01008. The detection of the nutritional components of the feed refers to the national standard GB14924.3 - 2010 of the People's Republic of China, and the detection of the content of contaminants refers to the national standard GB14924.2 - 2001 of the People's Republic of China. The feed supplier provides a test report for each batch. The drinking water was reverse osmosis water and stored in a drinking water bottle. The detection of the drinking water refers to the national standard GB5750 - 2006 of the People's Republic of China and was sent to a third - party inspection agency for inspection once a year.

[0331] The animal dressing was a corn - core dressing provided by Guangzhou Saibenuo Biotechnology Co., Ltd. The production license number of the animal dressing was SCXK (Beijing) 2019 - 0004. The detection of the content of contaminants in the dressing refers to the national standard GB14924.2 - 2001 of the People's Republic of China, and the dressing supplier provides a test report for each batch.

[0332] The animal rearing cages and dressings were replaced at least once a week. Before being used in a barrier environment, all animal rearing cages and dressings were autoclaved with a pulsating vacuum sterilizer. The animal rearing cages were cleaned, disinfected, and wiped at least once a week.

[0333] The animal rearing and observation rooms, including shelves, floors, tables, etc., were cleaned and disinfected daily.

[0334] Disinfectants used in barrier environments include 6.67% bromogenitic acid solution, 0.5% 84 disinfectant solution, 75% disinfectant solution, and 0.08% decylmethylammonium bromide solution. These four disinfectants must be used in sequence and cannot be mixed.

[0335] 2. Experimental Method 2.1 AAV-HBV mouse modeling Definition of test day: The day on which the animals were administered the vehicle or test drug was defined as day 0.

[0336] One hundred SPF-grade C57BL / 6 male mice were acclimated in a barrier facility for 7 days and observed daily. Modeling was performed after confirming that the mice were healthy and normal. rAAV8-1.3HBV (Guangzhou Paijin Biotechnology Co., Ltd., product name: AAV8 [HBV-D, ayw] (D#2012), batch number: HBV101-6) was injected into the tail vein of the mice at a dose of 1 × 10 per mouse. 11 GC / 100 μL was injected into the mice. At the 5th and 6th week after modeling (D-14 and D-7), blood was collected from the animals and centrifuged to obtain plasma, which was used to detect the contents of HBV DNA, HBsAg, and HBeAg.

[0337] 2.2 Animal Grouping and Dosing Definition of test day: The day on which the animals received vehicle or test drug was defined as day 0 (D0).

[0338] The animals were divided into groups according to the indicators at week 6 (D-7) of modeling. 72 animals were selected from the animals that successfully modeled and randomly divided into 12 groups of 6 animals each based on HBsAg. After confirming that the mean HBsAg levels between groups were similar and that there were no statistical differences in HBV DNA and HBeAg between groups, the drugs were administered at week 7 (D0) of modeling after grouping. Details of grouping and administration are shown in Table 50.

[0339] [Table 50]

[0340] 2.3 Secondary challenge experiment On Day 60 of administration, three mice in the C207S-DV32PG5 group were challenged with 8 μg of pAAV-HBV1.2 plasmid (Fenghui Biotechnology) hydrodynamically injected into the tail vein at an injection volume of 100 μL / g, while the remaining three mice received no treatment. Three mice in the blank group were similarly challenged and served as controls.

[0341] 2.4 Observations and Monitoring Indicators (1) General Observations During the modeling and testing periods, animals were observed daily and recorded, including whether they were dead or moribund, food and water intake, injuries, excrement, appearance and coat, mental state, and activity.

[0342] (2) Weight Acclimation Period: Animals were weighed and recorded upon receipt and at the end of the acclimation period. During the study period, animals were weighed weekly and recorded. If drug administration or blood sampling was required on the same day, weights were measured before surgery and before sacrifice.

[0343] (3) Measurement of serum HBs antigen, HBe antigen, HBV DNA, HBsAb, and ALT levels At week 5 (D-14), week 6 (D-7), before drug administration (D0), and one week after drug administration, 200 μL of blood was collected from the orbital canthus of each animal. Anticoagulation was performed using EDTA-k2 anticoagulation tubes. Plasma was collected after centrifugation at 1000 g for 10 minutes. Mice were challenged on D60. 200 μL of blood was collected from the orbital canthus on D63, D67, D74, and D81. Anticoagulation was performed using EDTA-k2 anticoagulation tubes. Plasma was collected after centrifugation at 1000 g for 10 minutes. 20 μL of plasma was added to 80 μL of PBS9 and vortexed to homogenize. HBsAg, HBV DNA, HBeAg, and ALT index detection were performed. 15 μL of plasma was added to 210 μL of PBS and vortexed to homogenize. HBsAb index detection was performed. All treated samples were detected by Guangzhou Huayin Medical Testing Center Co., Ltd., and the remaining plasma was stored at -80°C.

[0344] 3. Experimental Results The experimental results showed that, compared with the vehicle control group and the negative control group, the sequences B207S-DV32P, A1550-DV27P, B1572-DV27P, B1575-DV27P, B207S-DV29P, A1573-DV29P, B1575-DV29P, B418S-DV34Pd7B, and A416-DV27Pd7B could be targeted to the liver after coupling with conjugate G5 and could continuously and significantly inhibit the levels of HBsAg, HBeAg, and HBV DNA in plasma.

[0345] Among these, the sequences C207S-DV32PG5, C207S-DV29PG5, and C418S-DV34Pd7BG5 demonstrated superior efficacy in reducing HBsAg, HBV DNA, and HBeAg, significantly superior to the positive reference sequence VIR-2218-GL. Furthermore, sequences C207S-DV32PG5 and C207S-DV29PG5 ultimately achieved negative conversion of HBsAg and HBV DNA in all mice in the group, and all mice produced high levels of antibodies. Finally, three of six mice in the C418S-DV34Pd7BG5 sequence group also achieved negative conversion of HBsAg and produced high levels of antibodies.

[0346] Results from the secondary challenge experiment showed that HBsAg, HBeAg, and HBV DNA appeared in the blank group (a group not infected with HBV virus before D56) after challenge, indicating the effectiveness of the challenge method. After challenge, HBV DNA in mice in the C207S-DV32PG5 group initially increased but then decreased to the detection limit. Serum HBsA antibody levels significantly increased, while HBsAg remained below the detection limit, demonstrating that the challenge was effective and that hepatitis B mice treated with this small molecule nucleic acid sequence were protected from re-challenge with HBV virus. This demonstrates that hepatitis B mice treated with this small molecule nucleic acid sequence are protected from re-challenge with HBV virus. (i) Time course of HBsAg, HBeAg, HBV DNA, HBsAb, and ALT levels in mice of each sequence group

[0347] The time-dependent changes in HBsAg, HBeAg, HBV DNA, HBsAb, ALT, and body weight levels in mice in each group are shown in Tables 51-59 and Figures 1A-1F. As can be seen from the tables, the sequences C207S-DV32PG5, C207S-DV29PG5, and C418S-DV34Pd7BG5 demonstrated excellent efficacy in reducing HBsAg, HBV DNA, and HBeAg, significantly superior to the positive reference sequence VIR-2218-GL. Furthermore, the sequences C207S-DV32PG5 and C207S-DV29PG5 ultimately achieved negative conversion of HBsAg and HBV DNA in all mice in the group, all of which produced high levels of antibodies. In the C207S-DV32PG5 group, HBsAg and HBV DNA conversion was achieved in all mice in the group on days 14 and 21 after administration, and this state continued until the end of the experiment. All mice were considered to have achieved functional cure. In the C418S-DV34Pd7BG5 group, three of six mice ultimately achieved HBsAg conversion and also produced relatively high levels of antibodies. The above data indicated that the C207S-DV32PG5, C207S-DV29PG5, and C418S-DV34Pd7BG5 sequences were superior in terms of efficacy.

[0348] [Table 51]

[0349] [Table 52]

[0350] [Table 53]

[0351] [Table 54]

[0352] [Table 55]

[0353] [Table 56]

[0354] [Table 57]

[0355] [Table 58]

[0356] [Table 59]

[0357] (ii) Protective effect of sequence CS7S-DV32PG5 against secondary challenge in mice Mice in the CS7S-DV32PG5 group were injected with HBV plasmid via the tail vein on day 60 via high-pressure hydrodynamic injection to simulate secondary HBV infection. The experimental results, shown in Figures 2A-2F, demonstrate that HBsAg, HBeAg, and HBV DNA appeared in the blank group (a group not infected with HBV virus before day 56) after high-pressure hydrodynamic injection of HBV plasmid, demonstrating the effectiveness of the challenge method. In mice in the challenge group of C207S-DV32PG5, HBV DNA initially increased after challenge but then declined to the limit of detection. Serum HBsAg antibodies significantly increased, while HBsAg remained below the limit of detection, demonstrating the effectiveness of the challenge and that hepatitis B mice treated with this small molecule nucleic acid sequence are protected from re-challenge with HBV virus.

[0358] Example 8: Inhibitory effect of sequences modified with the modified template (low dose) of the present disclosure on HBV in mice In this example, several sequences, such as sequences C207S-DV32PG5, C207S-DV29PG5, C418S-DV34Pd7BG5, and C1575-DV27PG5 from Example 7 (see Table 49), were selected as examples, and the in vivo efficacy of these RNAi agents after multiple administrations at low doses, such as at the 0.5 mg / kg level, was analyzed in an AAV-HBV mouse model.

[0359] 1. Experimental Materials The sequences C207S-DV32PG5, C207S-DV29PG5, C418S-DV34Pd7BG5, C1575-DV27PG5, and VIR-2218-GL of Example 7 are shown in Table 49. There is also entecavir (ETV).

[0360] 2. Experimental Method As in Example 7, grouping and dosing details are shown in Table 60.

[0361] [Table 60]

[0362] 3. Experimental Results The time course of HBsAg, HBeAg, HBV DNA, HBsAb, and ALT levels in mice in each group are shown in Tables 61-68 and Figures 3A-3F. The experimental results showed that the sequences C207S-DV32PG5 and C207S-DV29PG5 were significantly superior to the positive reference sequence VIR-2218-GL in inhibiting HBsAg, HBV DNA, and HBeAg. Among them, C207S-DV32PG5 was the most effective, reducing HBsAg by approximately 2.9 log10 on day 56, which was approximately 1.4 log10 higher than C207S-DV29PG5. On day 56, three mice were HBsAg negative, and four mice produced antibodies. None of the mice in the C207S-DV29PG5 group were HBsAg negative. The above data again demonstrate the superiority of sequence C207S-DV32PG5 in terms of efficacy.

[0363] [Table 61]

[0364] [Table 62]

[0365] [Table 63]

[0366] [Table 64]

[0367] [Table 65]

[0368] [Table 66]

[0369] [Table 67]

[0370] [Table 68]

[0371] Example 9: Inhibitory effect of delivery ligand-modified sequences of the present disclosure against HBV in mice In this example, several sequences containing the base sequences B418S and B207S were selected as examples, and these sequences were modified, for example, by using template modification alone or by using both template modification and off-target prevention design to couple different delivery ligands to these sequences. The in vivo efficacy of these RNAi agents was analyzed using an AAV-HBV mouse model.

[0372] 1. Experimental Materials experimental drug The 3' end of the sense strand of each sequence in Table 69 (including sequences using only template modifications and sequences using both template modifications and off-target prevention designs) is coupled to a delivery ligand, G5, G101, or G103, and has the following structural formula: TIFF2026009862000133.tif45134 TIFF2026009862000134.tif12482 For the coupling method between oligonucleotide and ligand G5, G101 or G103, refer to the preparation method in Example 3 of patent application CN116854754A. The oligonucleotide and the ligand G5, G101, or G103 form the conjugates shown below. TIFF2026009862000135.tif107145 TIFF2026009862000136.tif66112

[0373] The specific sequence of each sequence is shown in Table 69. In the SEQ ID NO: G5 indicates coupling of the sequence to delivery ligand G5, G101 indicates coupling of the sequence to delivery ligand G101, G103 indicates coupling of the sequence to delivery ligand G103, and GL indicates coupling of the sequence to delivery ligand L96. The structural formula of L96 is as follows: TIFF2026009862000137.tif47126

[0374] [Table 69] TIFF2026009862000139.tif108164

[0375] Preparation of test drugs Same as in Example 7.

[0376] Experimental Animal Information Species / Strain: AAV-HBV mice Grade: SPF Gender: Male Quantity: 82 animals Age: 5 weeks Body weight: 19 - 24 g Source: Beijing Charles River Laboratory Animal Technology Co., Ltd. Production license number: SCXK(Beijing)2021 - 0006

[0377] Institutional Animal Care and Use Committee (IACUC) After receiving the experimental animals, they were housed at Beijing Charles River Laboratory Animal Technology Co., Ltd. with the use permission number: SCXK(Beijing)2022 - 0013. This project was reviewed by the Experimental Animal Ethics Committee of Beijing Charles River Laboratory Animal Technology Co., Ltd., and the IACUC number is VST - SY - 24062701. The test process was carried out strictly in accordance with the requirements of IACUC to ensure animal welfare.

[0378] Husbandry and Management The animals were housed in an IVC negative pressure barrier environment using plastic (polycarbonate) cages (volume 370×157×180 mm). Since the experimental animals were male and aggressive, they were housed individually in separate cages. The animals were housed at Beijing Charles River Laboratory Animal Technology Co., Ltd. The use and testing of animal feed, bedding, and drinking water were all performed in accordance with GB14925-2010, "Laboratory Animal Environment and Facilities" specifications. Control of the animal housing environment, including temperature, humidity, pressure difference, noise, light intensity, ventilation rate, and ammonia concentration, was provisionally implemented in accordance with GB50447-2008, "Technical Specifications for the Construction of Laboratory Animal Facilities." The temperature in the animal room was controlled at 20–26°C (with a diurnal temperature difference of ≤4°C), and the relative humidity was controlled at 40–70%. Artificial lighting was used, with alternating 12-hour periods of light and darkness. Compressed wood chip bedding was purchased from Beijing Keyao Cooperative Feed Co., Ltd. (Batch No.: 23109613), and growth and breeding diets for mice and rats were purchased from Beijing Keyao Cooperative Feed Co., Ltd. (Batch No.: 23103313). Records of animal housing management and environmental control during the experiment were kept by Beijing Charles River Laboratory Animal Technology Co., Ltd.

[0379] 2. Experimental Method 2.1 AAV-HBV mouse modeling Definition of test day: The day on which the animals were administered the vehicle or test drug was defined as day 0. One hundred SPF-grade C57BL / 6 male mice were acclimated in a barrier facility for 7 days and observed daily. Modeling was performed after confirming that the mice were healthy and normal. A mouse animal model of persistent HBV infection was established by tail vein injection of rAAV8-1.3HBV. The injected dose of AAV virus was 1.00 × 10 vg / mouse. AAV virus was diluted in sterile PBS at 5.00 × 10 10 The virus was diluted to 1000 μg / mL and 200 μL was injected into each mouse. Serum HBV DNA, HBeAg, and HBsAg contents were measured starting 4 weeks after virus injection.

[0380] 2.2 Animal herding and administration Definition of test day: The day on which the animals received vehicle or test drug was defined as day 0 (D0). On day -2, blood was collected from the submandibular space of all mice, and plasma was collected. The collected blood samples were anticoagulated with EDTA and centrifuged at 5000 rpm for 10 minutes, and the supernatant was used for HBV modeling detection. On day 0, 72 animals were selected based on the detection results on day -2 and divided into groups of 6 animals each, for a total of 12 groups. The grouping and administration details are shown in Table 70.

[0381] [Table 70]

[0382] 2.3 Observations and Monitoring Indicators (1) General Observations During the modeling and testing periods, animals were observed daily and recorded, including whether they were dead or moribund, food and water intake, injuries, excrement, appearance and coat, mental state, and activity.

[0383] (2) Weight Acclimation Period: Animals were weighed and recorded upon receipt and at the end of the acclimation period. During the study period, animals were weighed weekly and recorded. If drug administration or blood sampling was required on the same day, weights were measured before surgery and before sacrifice.

[0384] (3) Measurement of serum HBs antigen, HBe antigen, HBV DNA, and ALT levels Submandibular blood samples were collected from all animals on days -2, 6, 13, 20, 27, and 34, and plasma was collected. The collected blood samples were anticoagulated with EDTA and centrifuged at 5000 rpm for 10 minutes to separate the serum. After blood collection, the serum was separated and diluted with PBS solution before testing. 10 μL of serum was collected from each sample and diluted to 500 μL with PBS (50-fold dilution) and sent to Beijing Di'an Medical Testing Laboratory Co., Ltd. for serum HBV DNA, HBeAg, and HBsAg detection. 30 μL of serum was collected from each sample and diluted to 120 μL with PBS (4-fold dilution) and sent to Beijing Di'an Medical Testing Laboratory Co., Ltd. for serum ALT detection. The remaining plasma was stored at -80°C.

[0385] 3. Experimental Results The time course of HBsAg, HBeAg, HBV DNA, ALT levels, and body weight for each group of mice are shown in Tables 71-75 and Figures 4A-4E. Experimental results showed that the efficacy of all sequences was superior to that of the positive reference sequence VIR-2218-GL. Among them, C207S-DV29PG101, C207S-DV29PG103, C207S-DV32PG101, and C207S-DV32PG103 demonstrated superior efficacy in reducing HBsAg, HBV DNA, and HBeAg, significantly superior to that of the positive reference sequence VIR-2218-GL. Even at 28 days after administration, HBsAg and HBV DNA levels were reduced by more than 3 log10, with no rebound observed, demonstrating excellent anti-HBV efficacy.

[0386] [Table 71]

[0387] [Table 72]

[0388] [Table 73]

[0389] Table 74

[0390] Table 75

Claims

1. It is a double-stranded RNAi agent, The double-stranded RNAi agent comprises an antisense strand and a sense strand complementary to the antisense strand that forms a double-stranded region, wherein the nucleotide sequence of the antisense strand is represented by SEQ ID NO: 8, or the nucleotide sequence of the antisense strand is a modified sequence of the sequence represented by SEQ ID NO: 8, and the nucleotide sequence of the sense strand is represented by SEQ ID NO: 1, or the nucleotide sequence of the sense strand is a modified sequence of the sequence represented by SEQ ID NO:

1.

2. The double-stranded RNAi agent according to claim 1, characterized in that the sense strand and antisense strand each contain at least one modified nucleotide.

3. The double-stranded RNAi agent according to claim 1, characterized in that the double-stranded RNAi agent has the function of inhibiting HBV gene expression.

4. The double-stranded RNAi agent according to claim 2, characterized in that at least one of the modified nucleotides is one or more selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxythymine nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, debasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramides, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexyl-modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methyl phosphate group, and nucleotides containing a 5'-phosphate.

5. The double-stranded RNAi agent according to claim 1, characterized in that the antisense strand of the double-stranded RNAi agent includes 3' overhangs of two nucleotides.

6. The double-stranded RNAi agent according to claim 1, characterized in that the double-stranded region of the double-stranded RNAi agent is 20 pairs of nucleotides.

7. The double-stranded RNAi agent according to claim 1, characterized in that the sense strand of the double-stranded RNAi agent has 20 nucleotides and the antisense strand has 22 nucleotides.

8. The double-stranded RNAi agent according to claim 1, characterized in that all modifications included in the nucleotides in the sense strand and antisense strand are chemical modifications at the 2' position of the nucleotide ribose.

9. The double-stranded RNAi agent according to claim 8, characterized in that the chemical modification at the 2' position of the nucleotide ribose is one or more selected from the group consisting of 2'-methoxy, 2'-O-methoxyethyl, 2'-fluoro, 2'-benzyloxy, 2'-methylcarbonylamino, and 2'-pyridylmethoxy.

10. The double-stranded RNAi agent according to claim 9, characterized in that the chemical modification at the 2' position of the nucleotide ribose is 2'-methoxy or 2'-fluoro.

11. The double-stranded RNAi agent according to claim 1, characterized in that the nucleotides are linked together by 3',5'-phosphodiester bonds.

12. The double-stranded RNAi agent according to claim 11, characterized in that the 3',5'-phosphodiester bond includes a thio modification.

13. The double-stranded RNAi agent according to claim 1, characterized in that the carbon atom at the 5' position of the nucleotide glycoside at the 5' end of the antisense strand is phosphorylated.

14. The double-stranded RNAi agent according to claim 13, characterized in that the phosphorylation group at the 5' position of the phosphorylation comprises one or more selected from a 5'-vinylphosphonate group, a 5'-methylphosphonate group, a 5'-C-methylphosphate group, a 5'-phosphorothioate group, and a 5'-phosphate group, and its structure is as follows. (R represents hydrogen, hydroxyl, amine group, C) 1～4 Alkyl, aromatic group, C 1～4 Alkoxy, C 1～4 Alkylcarbonylamino or halogen, The base is one of the following selected from adenine, guanine, cytosine, thymine, and uracil.

15. The double-stranded RNAi agent according to claim 1, characterized in that the terminal nucleotides of the sequence are linked by 3',5'-phosphodiester bonds containing thio modifications, forming chiral, pure 3',5'-phosphorothioate diester bonds.

16. The double-stranded RNAi agent according to claim 15, characterized in that the sense strand and antisense strand each contain 1 to 3 thio links at their 5' ends, and the antisense strand each contains 1 to 3 thio links at its 3' end.

17. The aforementioned antisense chain is modified using one of the modification methods listed in the table below. and / or, the sense chain may be modified using one of the modification methods listed in the table below. The double-stranded RNAi agent according to claim 1, characterized in that, here, 2'-OMe is 2'-methoxy, 2'-F is 2'-fluoro, PS is a phosphorothioate skeleton, and EVP is 5'-(E)-vinylphosphonate.

18. The modification method for the double-stranded RNAi agent is as follows: Modification method A is used for the antisense chain and modification method a is used for the sense chain. Modification method A is used for the antisense chain, and modification method b is used for the sense chain. Modification method B is used for the antisense chain and modification method a is used for the sense chain. Modification method B is used for the antisense chain and modification method b is used for the sense chain. Modification method C is used for the antisense chain, and modification method b is used for the sense chain. Modification method D is used for the antisense chain, and modification method b is used for the sense chain. Modification method E is used on the antisense chain and modification method a is used on the sense chain, or The double-stranded RNAi agent according to claim 17, characterized in that modification method F is used on the antisense strand and modification method b is used on the sense strand.

19. The double-stranded RNAi agent according to claim 1, characterized in that the antisense strand has a modifying group at positions 2 to 8 from the 5' end, the modifying group is one or more selected from UNA, GNA, and DNA, and the structures of UNA and GNA are as follows. (The base is one selected from adenine, guanine, cytosine, thymine, and uracil.)

20. The double-stranded RNAi agent according to any one of claims 1 to 19, characterized in that it comprises one selected from the following oligonucleotide double-stranded bodies in which a sense strand and an antisense strand are paired. (1) The sense strand has the sequence indicated by SEQ ID NO: 81, and the antisense strand has the sequence indicated by SEQ ID NO: 179, 180, 181, 182, or 184. (2) The sense strand has the sequence indicated by SEQ ID NO: 82, and the antisense strand has the sequence indicated by SEQ ID NO: 179, 180, or 183. (3) The sense strand has the sequence indicated by SEQ ID NO: 83, and the antisense strand has the sequence indicated by SEQ ID NO: 185 or 186. (4) The sense strand has the sequence indicated by SEQ ID NO: 84, and the antisense strand has the sequence indicated by SEQ ID NO:

186. (5) The sense strand has the sequence indicated by SEQ ID NO: 85, and the antisense strand has the sequence indicated by SEQ ID NO:

186.

21. It is a conjugate, The conjugate is characterized by comprising a double-stranded RNAi agent according to claim 1 and a ligand to be conjugated to the double-stranded RNAi agent.

22. The conjugate according to claim 21, characterized in that the ligand is conjugated to the 3'-terminus or 5'-terminus of the oligonucleotide sense chain.

23. The conjugate according to claim 21, characterized in that the ligand is one or more GalNAc derivatives bound by a divalent or trivalent branched conjugate, or a GalNAc derivative bound by a monovalent conjugate.

24. The conjugate according to claim 21, characterized in that the ligand is as follows. (Here, X is hydrogen or one hydroxy protecting group, the hydroxy protecting group includes acetyl, benzoyl, or isobutyryl; Y is an amine protecting group or H, the amine protecting group is formyl, acetyl, propionyl, n-butyryl, or isobutyryl; n is an integer between 0 and 20; and q, r, and s are independently integers between 1 and 7.)

25. The conjugate according to claim 24, characterized in that the ligand is as follows.

26. The conjugate according to claim 21, characterized in that the ligand is as follows. (Here, X is oxygen, nitrogen, or sulfur, Y is an alkyl or aromatic group, R 1 It is oxygen or sulfur, R 2 It consists of hydrogen, an amine group, and C 1～4 Alkyl, aromatic group, C 1～4 It is an alkoxy or halogen, A is -(CH 2 ) a -, -(CH 2 CH 2 O) b -, -((CH 2 ) c NHCO) d - or -((CH 2 ) c CONH) d -, where a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5. B is - (CH 2 ) e - and here e is an integer from 0 to 7, L is -CONH- or -NHCO-, X 1 is, -(CH 2 ) f - or - (CH 2 CH 2 O) f CH 2 - and f is an integer from 1 to 5, X 2 is, -(CH 2 ) g - and g is an integer from 1 to 6. Y 1 is 0 or 1, Y 2 is 0, 1, or 2, Y 3 is 1, 2, or 3, m is an integer between 0 and 4. n is an integer between 0 and 4.

27. The conjugate according to claim 26, characterized in that the ligand is G4, G5, G6, or G7.

28. The conjugate according to claim 27, characterized in that the conjugate has the structure shown below.

29. The conjugate according to claim 21, characterized in that the ligand is as follows. (Here, X is oxygen, nitrogen, or sulfur, Y is an alkyl or aromatic group, R 1 It is oxygen or sulfur, R 2 It consists of hydrogen, an amine group, and C 1～4 Alkyl, aromatic group, C 1～4 It is an alkoxy or halogen, A is - (CH 2 ) a -, - (CH 2 CH 2 O) b -, -((CH 2 ) c NHKCO) d - or - ((CH 2 ) c CONH) d - where a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5. B is - (CH 2 ) e - and here e is an integer from 0 to 7, L is -CONH- or -NHCO-, X 1 is, -(CH 2 ) f - or - (CH 2 CH 2 O) f CH 2 - and f is an integer from 1 to 5, X 2 is, -(CH 2 ) g - and g is an integer from 1 to 6. Y 1 is 0 or 1, Y 2 is 0, 1, or 2, Y 3 is 1, 2, or 3, m is an integer between 0 and 4. n is an integer between 0 and 4.

30. The conjugate according to claim 29, characterized in that the ligand is G101, G102, G103, G105, or G106.

31. The conjugate according to claim 30, characterized in that the conjugate has the structure shown below.

32. The conjugate according to claim 21, characterized in that the conjugate includes one selected from the following oligonucleotide double-stranded bodies in which a sense strand and an antisense strand are paired. (1) The sense strand has the sequence indicated by SEQ ID NO: 326, 327, or 328, and the antisense strand has the sequence indicated by SEQ ID NO:

179. (2) The sense strand has the sequence indicated by SEQ ID NO: 329, 330, or 331, and the antisense strand has the sequence indicated by SEQ ID NO:

181.

33. The conjugate according to any one of claims 21 to 32, characterized in that the conjugate has the function of inhibiting HBV gene expression.

34. A pharmaceutical composition, The pharmaceutical composition is characterized by comprising a double-stranded RNAi agent according to claim 1 or a conjugate according to claim 21, and a pharmaceutically acceptable carrier.

35. The pharmaceutical composition according to claim 34, characterized in that the double-stranded RNAi agent or the conjugate is administered in a non-buffered solution.

36. The pharmaceutical composition according to claim 35, characterized in that the non-buffered solution is saline solution or water.

37. The pharmaceutical composition according to claim 34, characterized in that the double-stranded RNAi agent or the conjugate is administered in a buffer solution.

38. The pharmaceutical composition according to claim 37, characterized in that the buffer solution comprises an acetate, a citrate, a prolamin, a carbonate, or a phosphate, or any combination thereof.

39. The pharmaceutical composition according to claim 38, characterized in that the buffer solution is a phosphate buffer solution.

40. The pharmaceutical composition according to claim 37, characterized in that the double-stranded RNAi agent or the conjugate is prepared into a lipid preparation for delivery within a membrane molecular assembly.

41. The pharmaceutical composition according to claim 40, characterized in that the lipid preparation is nucleic acid-lipid particles.

42. The pharmaceutical composition according to claim 41, characterized in that the lipid preparation is lipid nanoparticles.

43. The pharmaceutical composition according to claim 40, characterized in that the mass / mass ratio of lipid to the double-stranded RNAi agent or the conjugate is 1:1 to 50:1, 1:1 to 25:1, 3:1 to 15:1, 4:1 to 10:1, 5:1 to 9:1, or 6:1 to 9:

1.

44. The pharmaceutical composition according to claim 42, characterized in that the lipid nanoparticles include cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids.

45. The pharmaceutical composition according to claim 44, characterized in that the cationic lipid is a compound having the structure of formula (I), its N-oxide, a pharmaceutically acceptable salt, or a stereoisomer. (G 1 C 1～6 It is alkylene, G 2 C 2～8 It is alkylene, G 3 C 1～3 It is alkylene, L 1 C 6～15 It is a linear alkyl group, L 2 C 12～25 It is a branched alkyl group.

46. The pharmaceutical composition according to claim 45, characterized in that the cationic lipid is YK-009 having a (I-I) structure.

47. The pharmaceutical composition according to claim 44, characterized in that the cationic lipid is a compound having the structure of formula (II), or its N-oxide, a pharmaceutically acceptable salt, or a stereoisomer. (G 1 is C 2～8 alkylene, G 2 is C 2～8 alkylene, L 1 is -C(O)O- or -OC(O)-, L 2 is -C(O)O- or -OC(O)-, R 1 is C 6～25 straight-chain or branched alkyl, R 2 is C 6～25 straight-chain or branched alkyl, G 3 is HO(CH 2 ) 2 - or HO(CH 2 ) 3 - and G 4 is HO(CH 2 ) <​​​​​​​​​​​​​​​​​

48. The pharmaceutical composition according to claim 47, characterized in that the cationic lipid is YK-401 having a (II-I) structure or YK-402 having a (II-II) structure.

49. The pharmaceutical composition according to claim 44, characterized in that the cationic lipid is a compound having the structure of formula (III), or its N-oxide, a pharmaceutically acceptable salt, or a stereoisomer. (G 1 C 1～6 It is alkylene, G 2 C 2～8 It is alkylene, R 1 C 6～20 It is a linear or branched alkyl group, R 2 C 12～25 It is a branched alkyl group, G 3 , HO(CH 2 ) 2 N(CH 3 ) (CH 2 ) 2 -, HO(CH 2 ) 2 N(CH 2 CH 3 ) (CH 2 ) 2 -, (HO(CH 2 ) 2 ) 2 N(CH 2 ) 2 - , CH 3 O(CH 2 ) 2 N(CH 3 ) (CH 2 ) 2 -, (CH 3 ) 2 N(CH 2 ) 3 SC(O)O(CH 2 ) 2 -, (CH 3 ) 2 N(CH 2 ) 3 SC(O)-, CH 3 NH(CH 2 ) 2 N(CH 3 ) (CH 2 ) 2 - or CH 3 CH 2 NH(CH 2 ) 2 - That is the case.

50. The pharmaceutical composition according to claim 49, characterized in that the cationic lipid is YK-201 having a (III-I) structure or YK-202 having a (III-II) structure.

51. The pharmaceutical composition according to claim 44, characterized in that the cationic lipid is a compound having the structure of formula (IV), its N-oxide, a pharmaceutically acceptable salt, or a stereoisomer. (G 1 C 1～8 It is alkylene, G 2 C 2～8 It is alkylene, R 1 C 6～25 It is a linear or branched alkyl group, R 2 C 12～25 It is a linear or branched alkyl group, G 3 , HO(CH 2 ) 2 N(R) 3 )CH 2 CH(OH)CH 2 - and here, R 3 is, -CH 3 ien-CH 2 CH 3 or -CH 2 CH 2 It is OH.

52. The pharmaceutical composition according to claim 51, characterized in that the cationic lipid is YK-305 having a structure of formula (IV-I) or YK-310 having a structure of formula (IV-II).

53. The pharmaceutical composition according to claim 44, characterized in that the cationic lipid is a compound having the structure of formula (V), or its N-oxide, a pharmaceutically acceptable salt, or a stereoisomer. (G 1 and G 2 These are, independently, unsubstituted C 6 ~C 10 It is alkylene, G 3 This is an unsubstituted C 1 ~C 12 It is alkylene, R 1 and R 2 Each of them is independent of C 6 ~C 24 Alkyl or C 6 ~C 24 It is an alkenyl, R 3 OR 5 , N, -C(=O)OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 And R 4 C 1 ~C 12 It is a hydrocarbon group, and R 5 is H or C 1 ~C 6 It is a hydrocarbon group.

54. The pharmaceutical composition according to claim 53, characterized in that the cationic lipid is ALC0315 having a (V-I) structure.

55. The pharmaceutical composition according to claim 44, characterized in that the cationic lipid is a compound having the structure of formula (VI), or its N-oxide, a pharmaceutically acceptable salt, or a stereoisomer. (R 4 is, -(CH 2 ) n Q and - (CH 2 ) n Selected from CHQR, where Q is -OR, -OH, -O(CH 2 ) n N(R) 2 , -OC(O)R, -CX 3 , -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O) 2 R, -N(H)S(O) 2 R, -N(R)C(O)N(R) 2 , -N(H)C(O)N(R) 2 , -N(H)C(O)N(H)(R), -N(R)C(S)N(R) 2 , -N(H)C(S)N(R) 2 , -N(H)C(S)N(H)(R), -N(R)S(O) 2 R 8 (Selected from the group consisting of , and complex algebras, where n is 1, 2, or 3.)

56. The pharmaceutical composition according to claim 55, characterized in that the cationic lipid is SM102 having a structure of formula (VI-I).

57. The pharmaceutical composition according to claim 44, characterized in that the cationic lipid is DLIN-MC3-DMA, a compound having the structure of formula (VII), or its N-oxide, a pharmaceutically acceptable salt, or a stereoisomer.

58. The pharmaceutical composition according to claim 44, characterized in that the cationic lipid comprises one or more selected from YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA.

59. The pharmaceutical composition according to claim 44, characterized in that the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 10:

1.

60. The pharmaceutical composition according to claim 44, characterized in that the molar ratio of the cationic lipid to the structural lipid is 1:1 to 5:

1.

61. The pharmaceutical composition according to claim 44, characterized in that the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-70):(0.5-5).

62. The pharmaceutical composition according to claim 61, characterized in that the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-45):(0.5-5).

63. The pharmaceutical composition according to claim 62, characterized in that the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 50:10:38.5:1.5 or 49:10:39.5:1.

5.

64. The pharmaceutical composition according to claim 44, characterized in that the neutral lipid comprises one or more selected from phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, and sterol.

65. The aforementioned neutral lipids are 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diundecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, and 1,2-dilinoleoyl -sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,The pharmaceutical composition according to claim 64, characterized in that it is one or more selected from 2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, and mixtures thereof.

66. The pharmaceutical composition according to claim 65, characterized in that the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1,2-distearoyl-sn-glycero-3-phosphocholine.

67. The pharmaceutical composition according to claim 44, characterized in that the structural lipid is one or more selected from cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and corticosteroids.

68. The pharmaceutical composition according to claim 67, characterized in that the structural lipid is cholesterol.

69. The pharmaceutical composition according to claim 44, characterized in that the polymer-conjugated lipid is one or more selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

70. The pharmaceutical composition according to claim 69, characterized in that the polymer-conjugated lipid is one or more selected from distearoylphosphatidylethanolamine polyethylene glycol 2000, dimyristoylglycerol-3-methoxypolyethylene glycol 2000, and methoxypolyethylene glycol ditetradecylacetamide.

71. It's a kit, The kit comprises Kit A, wherein Kit A comprises one or more of the double-stranded RNAi agents described in Claim 1 and the conjugates described in Claim 21.

72. The aforementioned kit further comprises kit B, and kit B is (1) Other drugs that reduce HBV gene expression, or compositions containing the drugs that reduce HBV gene expression. (2) One or more of the following: diagnostic agents, oncolytic agents, inhibitors of inhibitory molecules, and vaccines. (3) One or two of the following: chemotherapeutic agents and cytotoxic agents, The kit according to claim 71, characterized in that it includes one or more of the following.

73. The kit further comprises kit B, the kit B is One or more of the following: contrast agents, hormone preparations, targeted small molecule preparations, proteasome inhibitors, cytokines, and activators of costimulatory molecules. The kit according to claim 71, characterized in that it includes one or two of the following.

74. Use of the double-stranded RNAi agent according to claim 1 or the conjugate according to claim 21 in the preparation of a drug for the prevention and / or treatment of chronic hepatitis B, hepatic fibrosis, cirrhosis, liver cancer, acute hepatitis B, and hepatitis D virus-related diseases with HBV / infection.

75. A method for reducing HBV gene expression or inhibiting HBV replication in vitro for nonpreventive and / or therapeutic purposes, The method comprises administering to a subject one or more of the double-stranded RNAi agent described in claim 1 and the conjugate described in claim 21.

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