siRNAs that target and regulate HBV gene expression and their applications

Abstract

This disclosure provides siRNAs that target and regulate HBV gene expression and their applications. The double-stranded RNAi agent comprises an antisense strand and a sense strand complementary to the antisense strand forming the double-stranded region. The nucleotide sequence of the antisense strand is shown in SEQ ID NO:13, or the nucleotide sequence of the antisense strand is a modified sequence of the sequence shown in SEQ ID NO:13. Cellular and animal experimental results show that the double-stranded RNAi agent provided in this disclosure can significantly reduce the expression of one or more HBV genes, block the viral life cycle, and can be used to develop drugs for treating HBV gene expression-related diseases.

Description

Technical Field

[0001] This disclosure relates to the field of nucleic acid modification technology, and more specifically, to siRNAs that target and regulate HBV gene expression and their applications. Background Technology

[0002] Nucleic acid drugs, especially oligonucleotide drugs, are widely used due to their simple synthesis and high activity. Oligonucleotide drugs typically include antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), and nucleic acid aptamers.

[0003] Oligonucleotides are a class of short DNA or RNA molecules, oligomers, that readily bind in a sequence-specific manner to their complementary oligonucleotides, DNA, or RNA, forming double strands, or less commonly, hybrids. This fundamental characteristic makes oligonucleotides widely applicable in gene detection, research, and medicine. In nature, oligonucleotides are typically small RNA molecules that play a role in gene expression regulation, or intermediates derived from the 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 breaking down target mRNA.

[0005] The effective molecule of classic RNAi consists of a signature 19+2 nucleotide polymer (a double helix composed of a 21-nucleotide RNA molecule and a 19-nucleotide nucleotide molecule with corresponding nucleobases, including a 2-nucleotide 3' overhang). One strand of the siRNA (guide strand or antisense strand) is complementary to the target gene's transcript mRNA, while the other strand is designated as the passenger strand (or sense strand). The siRNA (antisense strand) guides the alginate protein (AGO2) to complement the target transcript and becomes part of the RNA guided silencing complex (RISC). Perfect complementarity between the siRNA (antisense strand) and the target leads to a break in the target transcript at position 10-11 of the guide strand (antisense strand) catalyzed by the AGO2 protein.

[0006] siRNA has inherent advantages over small molecule and antibody drugs because it performs its function by completing Watson-Crick base pairing with mRNA, while small molecule and monoclonal antibody drugs need to recognize the complex spatial structures of specific proteins. Therefore, many diseases cannot be treated with small molecule and monoclonal antibody drugs because their target molecules are highly active and cannot recognize molecular structures with affinity and binding specificity. The mechanism of action of siRNA drugs allows for the regulation of target protein expression at the gene level, exhibiting target specificity compared to small molecule or antibody drugs. Its mechanism based on the principle of complementary base pairing also makes siRNA's therapeutic scope broader, its design simpler, and its development cycle shorter.

[0007] In natural oligonucleotides, nucleotides are linked by phosphodiester bonds. Under physiological conditions, they are particularly sensitive to nucleases. Therefore, natural, unmodified, and structurally unmodified oligonucleotide drugs are easily and rapidly degraded by nucleases in vivo, resulting in low activity and poor drug-likeness. Chemically modifying the structure of oligonucleotides is an effective way to improve their activity. This can enhance their stability to nucleases, their affinity for RNA, and better promote endocytosis and tissue targeting, thereby effectively regulating the expression of target genes.

[0008] Based on the basic structure of oligonucleotides—bases, sugar rings, phosphate backbone, and ends—chemical modification can be performed on four parts:

[0009] 1) Base modification: mainly divided into three forms: purine modification, pyrimidine modification, and base substitution. Purine modification includes N6-methyladenosine, N1-methyladenosine, and 7-methylguanylic acid modification; pyrimidine modification includes 3-methyluridine, 5-methyluridine, 5-methylcytosine, N4-acetylcytidine, pseudouridine, thiouridine, propynouridine, and dihydrouridine, etc.

[0010] 2) Glycocycle modification: This mainly includes glycocycle modification and substitution. Glycocycle modifications include 2'-modification, 4'-modification, 5'-modification, isomerization, and combinations of these modifications. The most common 2'-modifications of siRNA are 2'-OMe (2'-methoxy) and 2'-F (2'-fluoro) modifications. Compared to native siRNA, siRNA modified with both 2'-OMe and 2'-F exhibits higher Tm values, stronger serum stability, and better activity.

[0011] 3) Modification of the phosphate backbone: mainly the modification of thiophosphates; through methyl phosphate, selenophosphate, methylboryl phosphate, dithiophosphate, and the replacement of bridging oxygen atoms in the phosphate diester bond linkage region with sulfur atoms; the phosphate ester groups between nucleosides are completely replaced with groups that do not contain phosphorus atoms, such as replacing P atoms with C, S and N atoms to form guanidino, S-methylthiourea, etc.

[0012] 4) Terminal modification: Covalently conjugating special groups at the 5' end and / or 3' end of the sense chain and phosphorylating the 5' end of the antisense chain.

[0013] Hepatitis B virus (HBV) is a double-stranded hepatotropic virus that infects only humans and non-human primates. It replicates primarily in the liver and can be transmitted through mother-to-child transmission, blood (including minor skin and mucous membrane injuries), and sexual contact. HBV infection remains a major health problem worldwide, and chronic HBV infection has a high probability of developing into liver fibrosis, cirrhosis, and liver cancer.

[0014] The current standard of care for chronic HBV infection is treatment with oral nucleoside (acid) analogs (NAs) and injectable interferon-alpha. NAs inhibit HBV replication by suppressing HBV DNA synthesis; most patients require long-term treatment, and the virological relapse rate after discontinuation is high. Interferon-alpha exerts a dual role in immunomodulation and antiviral action by enhancing immune cell function, promoting cytokine expression, and inducing interferon signaling to encode multiple antiviral proteins. Interferon alone is only effective in some patients and is relatively poorly tolerated. The 2022 edition of the "Guidelines for the Prevention and Treatment of Chronic Hepatitis B" recommends entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, and tenofovir alafenamide, and pegylated interferon-alpha.

[0015] Currently, the treatment goal for chronic HBV infection is to achieve functional cure, which means that HBV DNA and HBV surface antigen (HBsAg) remain undetectable after drug discontinuation, with or without HBsAg seroconversion. Sustained reduction of HBsAg and seroconversion are crucial for achieving functional cure, as this is expected to alleviate liver inflammation, improve liver histopathology, reduce the incidence of end-stage liver disease, and prolong patient survival. Currently, achieving functional cure through clinical medication is difficult or the cure rate is extremely low; therefore, it is necessary to further develop drugs that downregulate HBsAg expression to achieve functional cure. Summary of the Invention

[0016] To address the technical problem of the lack of a drug that can more effectively inhibit HBV gene expression in the existing technology, this disclosure provides siRNAs that target and regulate HBV gene expression and their applications. This disclosure involves designing a series of unique siRNA sequences targeting the HBV genome sequence and modifying them with specific templates.

[0017] Typically, siRNA is modified with monomers containing 2'-methoxy (2'-OMe) and 2'-fluorinated (2'-F). However, even considering only the combination of these two monomer modifications, the siRNA has a total of 44 bases across the sense and antisense strands, meaning there are 2... 44 There are numerous possible combinations. Furthermore, the different terminal thiolation configurations further expand the number of possible modification schemes.

[0018] Different modifications to the same siRNA sequence can result in significant differences in activity, and even different siRNAs modified with the same method can exhibit vastly different activities. While there are some principles governing siRNA modification design, existing research has shown that activity cannot be accurately predicted based on the modification method; that is, there is no definite relationship between modification method and activity. Therefore, screening for highly active modification schemes from countless possible combinations is extremely difficult.

[0019] This disclosure identifies specific modified sequences that significantly inhibit HBV gene expression by chemically modifying designed siRNA sequences.

[0020] On one hand, this disclosure provides a double-stranded RNAi agent comprising an antisense strand and a sense strand complementary to the antisense strand forming the double-stranded region, wherein the nucleotide sequence of the antisense strand is as shown in SEQ ID NO: 13, or the nucleotide sequence of the antisense strand is a modified sequence of the sequence shown in SEQ ID NO: 13.

[0021] In another aspect, this 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, this disclosure also provides a pharmaceutical composition comprising the aforementioned double-stranded RNAi agent or conjugate, and a pharmaceutically acceptable carrier.

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

[0024] In another aspect, this disclosure also provides the use of the aforementioned double-stranded RNAi agents, conjugates, or pharmaceutical compositions in the preparation of medicaments for the prevention and / or treatment of HBV gene expression-related diseases.

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

[0026] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this disclosure.

[0027] All reagents and raw materials used in this disclosure are commercially available.

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

[0029] (1) Modified sequences, including the basic sequence B418S, have a significant inhibitory effect on HBsAg, which is significantly better than the Yangshen sequence.

[0030] (2) The modified sequence conjugates with GalNAc compound, which can be efficiently delivered to animal liver and significantly inhibit HBV gene expression, and significantly reduce the serum levels of HBsAg, HBeAg and HBV DNA.

[0031] (3) The sequences modified with the template of this disclosure are significantly enhanced in terms of HBV inhibition activity compared with the sequences disclosed in the prior art.

[0032] (4) This disclosure found that siRNAs with similar sequences have very different activities.

[0033] (5) This disclosure also found that different sequences are sensitive to different modification templates, and it is uncertain which modification template the siRNA sequence can use to achieve high activity. Attached Figure Description

[0034] Figure 1A The serum HBsAg level in AAV-HBV model mice at a level of 3 mg / kg for candidate siRNA sequences.

[0035] Figure 1B The serum HBV DNA level in AAV-HBV model mice at a level of 3 mg / kg for candidate siRNA sequences.

[0036] Figure 1C The serum HBeAg level in AAV-HBV model mice at a level of 3 mg / kg for candidate siRNA sequences.

[0037] Figure 1D The serum HBsAb level in AAV-HBV model mice at a level of 3 mg / kg for candidate siRNA sequences.

[0038] Figure 1E Serum ALT levels in AAV-HBV model mice at a level of 3 mg / kg for candidate siRNA sequences.

[0039] Figure 1F The change in body weight in AAV-HBV model mice at a level of 3 mg / kg for candidate siRNA sequences.

[0040] Figure 2A The value represents the serum HBsAg level in mice after a second challenge.

[0041] Figure 2B The value represents the serum HBV DNA level in mice after a second challenge.

[0042] Figure 2C The value represents the serum HBeAg level in mice after a second challenge.

[0043] Figure 2D The value represents the serum HBbAg level in mice after a second challenge.

[0044] Figure 2E Serum ALT levels in mice after a second challenge.

[0045] Figure 2F The change in mouse body weight after a second challenge with the virus.

[0046] Figure 3A The level of HBsAg in AAV-HBV model mice at 0.5 mg / kg of candidate siRNA sequence.

[0047] Figure 3B The level of HBV DNA in AAV-HBV model mice at a candidate siRNA sequence level of 0.5 mg / kg.

[0048] Figure 3C The HBeAg level in AAV-HBV model mice at a level of 0.5 mg / kg for candidate siRNA sequences.

[0049] Figure 3D The level of HBsAb in AAV-HBV model mice at a candidate siRNA sequence of 0.5 mg / kg.

[0050] Figure 3E The ALT level in AAV-HBV model mice at a level of 0.5 mg / kg for the candidate siRNA sequence.

[0051] Figure 3FThe change in body weight in AAV-HBV model mice at a level of 0.5 mg / kg for candidate siRNA sequences.

[0052] Figure 4A To determine the HBsAg level in AAV-HBV model mice by selecting candidate siRNA sequences that conjugate different GalNAc.

[0053] Figure 4B To determine the HBV DNA level in AAV-HBV model mice by using candidate siRNA sequences conjugated with different GalNAc.

[0054] Figure 4C To determine the HBeAg levels in AAV-HBV model mice by selecting candidate siRNA sequences that conjugate different GalNAc.

[0055] Figure 4D To determine the ALT levels in AAV-HBV model mice by using candidate siRNA sequences conjugated with different GalNAc.

[0056] Figure 4E Changes in body weight in AAV-HBV model mice conjugated with candidate siRNA sequences of different GalNAc. Detailed Implementation

[0057] To make this disclosure easier to understand, certain terms are first defined. Furthermore, it should be noted that whenever a range of values ​​or parameters is enumerated, the purpose is to indicate that intermediate values ​​and ranges of these referenced values ​​also become part of this disclosure.

[0058] The articles “a” and “an” as used in this article refer to one or more (i.e., at least one) grammatical objects of the article. By way of example, “an element” refers to one element or more elements, such as multiple elements.

[0059] The term “including” is used here to refer to the phrase “including but not limited to” and is used interchangeably with it.

[0060] The term “or” is used here to mean and / or the term “and / or” and is used interchangeably with it, unless the context clearly indicates otherwise.

[0061] As used herein, the term “about” or “approximately” when applied to one or more target values ​​means a value similar to the reference value. In some embodiments, unless otherwise stated or otherwise apparent from the context, the term “approximately” or “about” means a range of values ​​falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in any direction (unless such a number would exceed 100% of the possible value).

[0062] As used in this article, "HBV" refers to hepatitis B virus, including hepatitis B virus with genotypes A, B, C, D, E, F, G, H, I, J and their subtypes, and is not limited to any one genotype.

[0063] The letters “G,” “C,” “A,” and “U” typically represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. “T” and “dT” are used interchangeably herein and refer to deoxyribonucleotides in which the nucleobase is thymine, such as deoxyribothymine, 2'-deoxythymidine, or thymidine. However, it should be understood that the terms “ribonucleotide,” “nucleotide,” or “deoxyribonucleotide” can also refer to a modified nucleotide (as further detailed below) or an alternative substitution. Those skilled in the art will readily recognize that guanine, cytosine, adenine, and uracil can be substituted with other parts without substantially altering the base-pairing properties of an oligonucleotide (including a nucleotide with such a substitution). For example, and not limited to, nucleotides containing inosine as a base can base-pair with nucleotides containing adenine, cytosine, or uracil. Therefore, nucleotides containing uracil, guanine, or adenine can be substituted in the nucleotide sequence of this disclosure with a nucleotide containing, for example, inosine. Sequences containing such substitutions are embodiments of this disclosure.

[0064] The terms “RNAi agent” and “RNA interference agent” are used interchangeably herein and refer to the term as defined herein, which includes RNA agents and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. RNAi agents direct sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). RNAi agents regulate, for example, inhibit, the expression of HBV in cells such as those of a subject (e.g., a mammalian subject). RNAi molecules include single-stranded RNAi molecules and double-stranded siRNAs, as well as short hairpin RNAs (shRNAs).

[0065] The term "small interfering RNA" or "siRNA" refers to small interfering RNA (RNAi) molecules. It is a class of double-stranded RNA molecules, also known in the art as short interfering RNA or silent RNA. siRNAs typically contain a sense strand (also called a guest strand) and an antisense strand (also called a leader strand), each strand being 17 to 30 nucleotides in length, typically 19 to 25 nucleotides in length. The antisense strand is complementary to the target nucleic acid (e.g., at least 95% complementary, e.g., fully complementary) (suitably a mature mRNA sequence), and the sense strand is complementary to the antisense strand, such that the sense and antisense strands form a double-stranded structure or a double-stranded region. The siRNA strands may form blunt-ended double-stranded structures, or preferably, the 3′ ends of the sense and antisense strands may form 3′ overhangs, e.g., one, two, or three nucleotides, similar to the products produced by Dicer, which can form RISC substrates in vivo. Efficient extended forms of Dicer substrates have been described in US 8349809 and US8513207, which are incorporated herein by reference. In some embodiments, both the sense and antisense strands have a 3′ overhang of 2 nucleotides. Therefore, the length of the double-stranded region can be, for example, 17 to 25 nucleotides, such as 21 to 23 nucleotides.

[0066] The term “antisense strand” refers to the strand of RNAi (e.g., dsRNA) that includes a region substantially complementary to the target sequence. As used herein, the term “complementary region” refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., the target sequence). When the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the molecule or in terminal regions. Typically, the most permissible mismatches are in terminal regions, for example, within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

[0067] When used herein, the term “sense chain” refers to a chain of RNAi that includes regions substantially complementary to regions of the antisense chain (as defined herein).

[0068] The term “inhibition” as used in this article may be used interchangeably with “reduction,” “silence,” “downregulation,” “suppression,” and other similar terms, and includes any level of inhibition.

[0069] As used in this article, the phrase “inhibit HBV gene expression” includes inhibiting the expression of HBV DNA, HBV mRNA, HBsAg, HBeAg, hepatitis B core antigen (HBcAg), etc.

[0070] "Inhibiting the expression of HBV antigens" includes inhibiting the expression of HBsAg, HBeAg, and HBcAg proteins.

[0071] "Suppression of HBV gene expression" includes suppression of HBV DNA, HBV mRNA, HBsAg, HBeAg, and HBcAg at any level, such as at least partial suppression of the expression of HBV DNA, HBV mRNA, HBsAg, HBeAg, and HBcAg, such as suppression of 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%.

[0072] HBV gene expression can be assessed based on the levels of any variable associated with HBV gene expression, such as HBV DNA levels, HBV mRNA levels, HBV antigen protein levels, and HBV viral particle levels. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. A control level can be any type of control level utilized in the art, such as baseline levels before administration or levels determined from similar untreated or controlled (e.g., buffer-only or inert control) subjects, cells, or samples.

[0073] As used herein, "patient" or "subject" is intended to include human or non-human animals, preferably mammals such as mice. Most preferably, the subject or patient is a human.

[0074] As used herein, “HBV gene expression-related diseases” are intended to include any disease associated with the HBV gene or protein. Such diseases can be caused, for example, by overproduction of HBV antigen proteins, by HBV gene mutations, by abnormal cleavage of HBV antigen proteins, or by abnormal interactions between HBV antigen proteins and other proteins or other endogenous or exogenous substances. Exemplary HBV-related diseases include HBV infection-associated hepatitis, such as chronic hepatitis B, acute hepatitis B, and HBV / hepatitis D virus (HDV) co-infection and HBV / HIV co-infection-related diseases.

[0075] As used herein, “therapeutic effective dose” is intended to include the amount of RNAi agent that is sufficient to achieve treatment of an HBV-related disease (e.g., by weakening, improving, or maintaining the existing disease or symptoms of one or more diseases) when administered to a patient. This “therapeutic effective dose” can vary depending on the RNAi agent, how it is administered, the disease and its severity, and medical history, age, weight, family history, genetic makeup, stage of the HBV-mediated pathological process, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0076] As used herein, “preventive effective dose” refers to the amount of an RNAi agent sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to a subject who has not yet experienced or exhibited symptoms of an HBV-related disease but may be susceptible to it. Improving the disease includes slowing its progression or reducing the severity of subsequent disease development. This “preventive effective dose” can vary depending on the RNAi agent, how it is administered, the level of risk for the disease, and medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0077] "Therapeutic effective amount" or "prophylactic effective amount" also includes the amount of RNAi agent that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the methods of this disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0078] As used herein, the term "sample" includes similar fluids, cells, or tissues isolated from a subject, as well as a collection of fluids, cells, or tissues present in the subject. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs, or localized regions. For example, a sample may originate from a specific organ, a portion of an organ, or fluids or cells within those organs. In some embodiments, a sample may originate from the liver (e.g., the entire liver or segments of the liver, or certain types of cells in the liver, such as hepatocytes). In a preferred embodiment, "sample derived from a subject" means blood or plasma drawn from that subject. In other embodiments, "sample derived from a subject" means liver tissue (or a subcomponent thereof) derived from that subject.

[0079] In one aspect, this disclosure provides a double-stranded RNAi agent comprising an antisense strand and a sense strand complementary to the antisense strand forming the double-stranded region, wherein the nucleotide sequence of the antisense strand is as shown in SEQ ID NO: 13, or the nucleotide sequence of the antisense strand is a modified sequence of the sequence shown in SEQ ID NO: 13.

[0080] In some embodiments, the double-stranded RNAi agent comprises an oligonucleotide duplex consisting of a sense strand and an antisense strand: the sense strand has a sequence or a fragment thereof as shown in SEQ ID NO: 6, or a modified sequence thereof.

[0081] In some implementations, the sense and antisense strands contain at least one modified nucleotide.

[0082] In some implementations, the double-stranded RNAi agent has the function of inhibiting HBV gene expression.

[0083] In some embodiments, at least one of the modified nucleotides is selected from one or more of the group consisting of: deoxy-nucleotides, 3'-terminal deoxy-thymidine nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free 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, morpholinyl nucleotides, aminophosphates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimics.

[0084] In some embodiments, at least one strand of the double-stranded RNAi agent contains a 3' overhang of at least two nucleotides.

[0085] In some embodiments, the double-stranded region of the double-stranded RNAi agent consists of 20 pairs of nucleotides.

[0086] In some embodiments, the sense strand of the double-stranded RNAi agent has 20 nucleotides and the antisense strand has 22 nucleotides.

[0087] In some implementations, all modifications to the nucleotides on the sense and antisense strands are chemical modifications at the 2' position of the nucleotide ribose.

[0088] In some embodiments, the chemical modification at the 2' position of the nucleotide ribose is selected from one or more of the group consisting of: 2'-methoxy, 2'-O-methoxyethyl, 2'-fluoro, 2'-benzyloxy, 2'-methylcarbonylamino, and 2'-pyridinemethoxy.

[0089] In some embodiments, the 2' position of the nucleotide ribose is chemically modified with 2'-methoxy or 2'-fluoro.

[0090] In some embodiments, the nucleotides are linked by 3',5'-phosphodiester bonds.

[0091] In some embodiments, the 3',5'-phosphodiester bond comprises a thiolated modification.

[0092] In some embodiments, the 5' carbon atom of the antisense strand 5'-terminal nucleotide glycoside is phosphorylated.

[0093] In some embodiments, the phosphorylated 5'-phosphorylation group comprises one or more selected from: 5'-vinylphosphonate group, 5'-methylphosphonate group, 5'-C-methylphosphate group, 5'-thiophosphate group, and 5'-phosphate group, with the following structure:

[0094]

[0095] ;

[0096] R represents hydrogen, hydroxyl group, amino group, C 1-4 Alkyl, aromatic, C 1-4 Alkoxy, C 1-4 Alkyl carbonyl amino or halogen;

[0097] The bases are selected from any one of adenine, guanine, cytosine, thymine, and uracil.

[0098] In some embodiments, the terminal nucleotides are linked by thiolated 3',5'-phosphodiester bonds, forming chiral pure 3',5'-thiophosphodiester bonds.

[0099] In some implementations, the ends of the sense and antisense chains 5' contain 1-3 thioligases, and the ends of the antisense chains 3' contain 1-3 thioligases.

[0100] In some implementations, the antisense chain is modified in one of the following table formats:

[0101]

[0102]

[0103] ;

[0104] And / or, the chain of justice is modified in one of the following table formats:

[0105] ;

[0106] Wherein 2'-OMe is 2'-methoxy; 2'-F is 2'-fluoro; PS is the thiophosphate skeleton; and EVP is 5'-vinyl-(E)-phosphonate.

[0107] In some implementations, the double-stranded RNAi agent is modified in the following manner:

[0108] The siRNA template modified with modification A on the antisense strand and modification a on the sense strand was named DV27P.

[0109] The siRNA template modified with modification A on the antisense strand and modification b on the sense strand was named DV29P.

[0110] The antisense strand uses modification B, and the sense strand uses modification a. The siRNA modification template is named DV26P.

[0111] The siRNA template modified by B on the antisense strand and by b on the sense strand was named DV28P.

[0112] The siRNA template modified with C on the antisense strand and b on the sense strand was named DV32P.

[0113] The siRNA template modified with D on the antisense strand and b on the sense strand was named DV34P.

[0114] The siRNA template modified with modification E on the antisense strand and modification a on the sense strand is named DV25P; or,

[0115] The siRNA template modified with F on the antisense strand and b on the sense strand was named DV33P.

[0116] In some embodiments, the second to eighth positions of the antisense strand from the 5' end are modified with a group selected from one or more of UNA, GNA, and DNA, wherein the structures of UNA and GNA are as follows:

[0117] ;

[0118] The bases are selected from any one of adenine, guanine, cytosine, thymine, and uracil.

[0119] In some embodiments, the double-stranded RNAi agent comprises any oligonucleotide duplex selected from the following sense and antisense strand pairings:

[0120] (1) The positive chain has a sequence as shown in SEQ ID NO: 106; and the negative chain has a sequence as shown in SEQ ID NO: 213, 214, 215 or 216;

[0121] (2) The sense chain has a sequence as shown in SEQ ID NO: 107; and the antisense chain has a sequence as shown in SEQ ID NO: 214 or 213;

[0122] (3) The sense chain has a sequence as shown in SEQ ID NO: 108; and the antisense chain has a sequence as shown in SEQ ID NO: 217 or 218;

[0123] (4) The sense chain has the sequence shown in SEQ ID NO: 109; and the antisense chain has the sequence shown in SEQ ID NO: 218; and,

[0124] (5) The positive chain has a sequence as shown in SEQ ID NO: 110; and the negative chain has a sequence as shown in SEQ ID NO: 218.

[0125] (6) The positive chain has a sequence as shown in SEQ ID NO: 306; and the negative chain has a sequence as shown in SEQ ID NO: 321.

[0126] The double-stranded RNA (dsRNA) reagent (double-stranded RNAi agent) disclosed herein can optionally be conjugated to one or more ligands. The ligand can be attached to the sense strand, antisense strand, or both strands at the 3' end, 5' end, or both ends. For example, the ligand can be conjugated to the sense strand. In a preferred embodiment, the ligand binds to the 3' end of the sense strand. In a preferred embodiment, the ligand is a GalNAc ligand.

[0127] In another aspect, this disclosure provides a conjugate comprising the aforementioned double-stranded RNAi agent and a ligand conjugated to the double-stranded RNAi agent.

[0128] In some embodiments, the ligand is conjugated to the 3'-end or 5'-end of the sense strand of the oligonucleotide.

[0129] In some embodiments, the ligand is one or more GalNAc derivatives attached using divalent or trivalent branched conjugates, or GalNAc derivatives attached using monovalent conjugates.

[0130] In some implementations, the ligand is:

[0131] ,

[0132] Wherein, X is hydrogen or a hydroxyl protecting group or H, the hydroxyl protecting group including acetyl, benzoyl or isobutyryl; Y is an amine protecting group or H, the amine protecting group being formyl, acetyl, propionyl, n-butyryl or isobutyryl; n is an integer between 0 and 20; q, r and s are independently integers between 1 and 7.

[0133] In some implementations, the ligand is:

[0134] .

[0135] In some implementations, the ligand is:

[0136] ,

[0137] Where X is oxygen, nitrogen, or sulfur;

[0138] Y is an alkyl or aromatic group;

[0139] R1 is oxygen or sulfur;

[0140] R2 is hydrogen, amino group, or C. 1-4 Alkyl, aromatic, C 1-4 Alkoxy or halogen;

[0141] 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;

[0142] B is -(CH2) e -, where e is an integer between 0 and 7;

[0143] L is either -CONH- or -NHCO-;

[0144] X1 is -(CH2) f -or-(CH2CH2O) fCH2-, f is an integer from 1 to 5;

[0145] X2 is -(CH2) g -, g is an integer from 1 to 6;

[0146] Y1 is 0 or 1;

[0147] Y2 is 0, 1, or 2;

[0148] Y3 is 1, 2, or 3;

[0149] m is an integer between 0 and 4;

[0150] n is an integer between 0 and 4.

[0151] In some implementations, the ligand is G4, G5, G6, or G7:

[0152] ,

[0153] ,

[0154] ,or

[0155] .

[0156] In some embodiments, the conjugate has the following structure:

[0157] ,

[0158] ,

[0159] ,

[0160] ,or

[0161] .

[0162] In some implementations, the ligand is:

[0163]

[0164] Where X is oxygen, nitrogen, or sulfur;

[0165] Y is an alkyl or aromatic group;

[0166] R1 is oxygen or sulfur;

[0167] R2 is hydrogen, amino group, or C. 1-4 Alkyl, aromatic, C 1-4 Alkoxy or halogen;

[0168] 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;

[0169] B is -(CH2) e -, where e is an integer between 0 and 7;

[0170] L is either -CONH- or -NHCO-;

[0171] X1 is -(CH2) f -or-(CH2CH2O) f CH2-, f is an integer from 1 to 5;

[0172] X2 is -(CH2) g - g is an integer from 1 to 6;

[0173] Y1 is 0 or 1;

[0174] Y2 is 0, 1, or 2;

[0175] Y3 is 1, 2, or 3;

[0176] m is an integer between 0 and 4;

[0177] n is an integer between 0 and 4.

[0178] In some implementations, the ligand is G101, G102, G103, G105, or G106:

[0179] , , , ,or .

[0180] In some embodiments, the conjugate has the following structure:

[0181] ,

[0182] ,

[0183] ,

[0184] ,or

[0185] .

[0186] In some embodiments, the conjugate comprises any oligonucleotide duplex selected from the following sense and antisense strand pairings:

[0187] The positive chain has a sequence as shown in SEQ ID NO: 350, 351 or 352; and the negative chain has a sequence as shown in SEQ ID NO: 321.

[0188] In some embodiments, the conjugate has the function of inhibiting HBV gene expression.

[0189] In another aspect, this disclosure provides a pharmaceutical composition comprising the aforementioned double-stranded RNAi agent or the aforementioned conjugate, and a pharmaceutically acceptable carrier.

[0190] In one embodiment, a pharmaceutical composition comprising a double-stranded RNAi agent as described herein and a pharmaceutically acceptable carrier is provided herein. The pharmaceutical composition comprising the double-stranded RNAi agent can be used to treat diseases or conditions associated with the expression or activity of the HBV gene, such as chronic hepatitis B. Such pharmaceutical compositions are formulated based on delivery models. One example is a composition formulated for systemic administration via parenteral delivery, such as intravenous (IV) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma, such as by infusion into the brain, for example, via a continuous pump infusion.

[0191] Pharmaceutical compositions comprising the double-stranded RNAi agents of this disclosure may be, for example, solutions with or without a buffer solution or compositions containing a pharmaceutically acceptable carrier. Such compositions include, for example, aqueous or crystalline compositions, liposome formulations, micellar formulations, emulsions, and gene therapy carriers.

[0192] In the method disclosed herein, the double-stranded RNAi agent can be administered in a solution. A free double-stranded RNAi agent can be administered in a non-buffered solution, such as physiological saline or water. Alternatively, the free siRNA can also be administered in a suitable buffered solution. The buffered solution may include acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffered solution is phosphate-buffered saline (PBS). The pH and volumetric molar osmotic concentration of the buffer containing the double-stranded RNAi agent can be adjusted to suit its administration to the subject.

[0193] In some embodiments, the buffer solution further comprises a reagent for controlling the molar osmotic pressure concentration of the solution, such that the molar osmotic pressure concentration is maintained at a desired value, such as the physiological value in human plasma. Solutes that may be added to the buffer solution to control the molar osmotic pressure concentration include (but are not limited to) proteins, peptides, amino acids, non-metabolitic polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the reagent for controlling the molar osmotic pressure concentration of the solution is a salt. In some embodiments, the reagent for controlling the molar osmotic pressure concentration of the solution is sodium chloride or potassium chloride.

[0194] The pharmaceutical compositions disclosed herein can be administered at a dose sufficient to inhibit HBV gene expression. Typically, a suitable dose of the double-stranded RNAi agent of this disclosure is in the range of about 0.001 to about 200.0 mg per kilogram of body weight per day, and generally in the range of about 0.1 to 50 mg per kilogram of body weight per day. For example, double-stranded RNAi agents (e.g., dsRNA) can be administered at doses 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, 1 8, 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 approximately 50 mg / kg.

[0195] The pharmaceutical composition can be administered once daily, or the double-stranded RNAi agent can be administered two, three, or more sub-dose at appropriate intervals throughout the day, or even administered via continuous infusion or delivery using a controlled-release formulation. In this case, the amount of double-stranded RNAi agent contained in each sub-dose must be correspondingly less to achieve the total daily dose. Dosage units can also be compounded for delivery over several days, for example using conventional sustained-release formulations that provide sustained release of the double-stranded RNAi agent over a timeframe of several days. Sustained-release formulations are well known in the art and are particularly useful for delivering reagents at specific sites, thus allowing their use with the reagents disclosed herein. In this embodiment, the dosage unit comprises a corresponding plurality of daily doses.

[0196] In other embodiments, a single dose of the pharmaceutical composition can be administered continuously, such that subsequent doses are given at intervals of no more than 3, 4, or 5 days or at intervals of no more than 1, 2, 3, or 4 weeks. In some embodiments of this disclosure, a single dose of the pharmaceutical composition of this disclosure is given weekly. In other embodiments of this disclosure, a single dose of the pharmaceutical composition of this disclosure is given monthly.

[0197] Those skilled in the art will understand that certain factors can influence the dosage and timing required to effectively treat a subject, including (but not limited to) the severity of the disease or condition, prior treatment, the subject's overall health and / or age, and other pre-existing conditions. Furthermore, treating a subject with a therapeutically effective dose of the composition may comprise a single treatment or a series of treatments. As described elsewhere herein, the effective dose and in vivo half-life of the various double-stranded RNAi agents covered by this disclosure can be estimated using conventional methods or based on in vivo testing using suitable animal models.

[0198] Depending on whether local or systemic treatment is desired and depending on the area to be treated, the pharmaceutical compositions of this disclosure can be administered in a variety of ways. Administration can be local (e.g., via a skin patch); pulmonary; such as by inhalation or blowing in a powder or aerosol, including via a nebulizer; intratracheal; intranasal; epidermal; and percutaneous, oral, or parenteral administration. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, for example, via an implanted device; or intracranial, such as administration within the brain parenchyma, intrasheath, or ventricle.

[0199] Double-stranded RNAi agents used in the compositions and methods of this disclosure can be formulated for delivery in membrane-bound molecular aggregates, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids disposed in at least one bilayer (e.g., one or more bilayers). Liposomes comprise monolayered or multilayered vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the double-stranded RNAi agent composition. The lipophilic material separates the aqueous interior from an aqueous exterior that typically does not include the double-stranded RNAi agent composition (although in some instances it may include it). Liposomes are useful for the transfer and delivery of active ingredients to sites of action. Because the liposome membrane is structurally similar to a biological membrane, when a liposome is applied to a tissue, the liposome bilayer fuses with the cell membrane bilayer. As the liposome fuses with the cell, the aqueous contents, including the double-stranded RNAi agent, are delivered into the cell, wherein the double-stranded RNAi agent can specifically bind to a target RNA and can mediate RNAi. In some cases, these liposomes are also specifically targeted, for example, to guide the double-stranded RNAi agent to a specific cell type.

[0200] Liposomes containing a double-stranded RNAi agent can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent to form micelles. For example, the lipid component can be an amphiphilic cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include bile salts, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The double-stranded RNAi agent formulation is then added to micelles containing the lipid component. The cationic groups on the lipid interact with the double-stranded RNAi agent and condense around it to form liposomes. After condensation, the detergent is removed, for example by dialysis, to obtain the liposome formulation of the double-stranded RNAi agent.

[0201] Double-stranded RNAi agents, such as the dsRNA disclosed herein, can be completely encapsulated in lipid formulations (e.g., LNPs or other nucleic acid-lipid particles).

[0202] As used herein, the term "lipid nanoparticle (LNP)" refers to a stable nucleic acid-lipid particle. An LNP contains a cationic lipid, a non-cationic lipid, and a lipid that prevents the particle from aggregating (e.g., a PEG-lipid conjugate). LNPs are extremely useful for synthetic applications because they exhibit prolonged cycle life after intravenous (iv) injection and accumulate at distal sites (e.g., at sites physically separate from the administration site).

[0203] In one embodiment, the ratio of lipids to the drug (mass / mass ratio) (e.g., the ratio of lipids to dsRNA) will be in the range of 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.

[0204] In some embodiments, the double-stranded RNAi agent or the conjugate is administered in a non-buffered solution.

[0205] In some implementations, the non-buffered solution is saline or water.

[0206] In some embodiments, the double-stranded RNAi agent or the conjugate is administered with a buffer solution.

[0207] In some embodiments, the buffer solution comprises acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate, or any combination thereof.

[0208] In some embodiments, the buffer solution is a phosphate buffer salt.

[0209] In some embodiments, the double-stranded RNAi agent or the conjugate is formulated as a lipid formulation for delivery in a membrane-bound molecular assembly.

[0210] In some embodiments, the lipid formulation is a nucleic acid-lipid particle.

[0211] In some embodiments, the lipid formulation is lipid nanoparticles.

[0212] In some embodiments, the mass / mass ratio of lipid to the double-stranded RNAi agent or the conjugate is 1:1-50:1, 1:1-25:1, 3:1-15:1, 4:1-10:1, 5:1-9:1, or 6:1-9:1.

[0213] In some embodiments, the lipid nanoparticles include cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids.

[0214] In some embodiments, the cationic lipid is a compound of formula (I), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer.

[0215] (I),

[0216] G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; G3 is C 1～3 Alkylene; L1 is C6～15 Straight-chain alkyl; L2 is C 12～25 Branched alkyl groups.

[0217] In some embodiments, the cationic lipid is YK-009 of formula (II) (see patent CN114044741B):

[0218] (II).

[0219] In some embodiments, the cationic lipid is a compound of formula (II), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer.

[0220] (II),

[0221] G1 is C 2~8 Alkylene; G2 is C 2~8 Alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6~25 Straight-chain or branched alkyl; R2 is C 6~25 Straight-chain or branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2- or -(CH2)3- or -(CH2)4-.

[0222] In some embodiments, the cationic lipid is YK-401 with formula (II-I) or YK-402 with formula (II-II) (see patent CN115784921B):

[0223] (II-I),

[0224] (II-II).

[0225] In some embodiments, the cationic lipid is a compound of formula (III), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer.

[0226] (III),

[0227] G1 is C 1~6 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~20 Straight-chain or branched alkyl; R2 is C 12~25Branched alkyl; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(C H2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-.

[0228] In some embodiments, the cationic lipid is YK-201 with formula (III-I) or YK-202 with formula (III-II) (see patent CN115677518B):

[0229] (III-I),

[0230] (III-II).

[0231] In some embodiments, the cationic lipid is a compound of formula (IV), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer.

[0232] (IV),

[0233] G1 is C 1~8 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~25 Straight-chain or branched alkyl; R2 is C 12~25 Straight-chain or branched alkyl group; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3, -CH2CH3, or -CH2CH2OH. .

[0234] In some embodiments, the cationic lipid is YK-305 of formula (IV-I) or YK-310 of formula (IV-II) (see patent CN115745820B):

[0235] (IV-I),

[0236] (IV-II).

[0237] In some embodiments, the cationic lipid is a compound of formula (V), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer.

[0238] (V),

[0239] G 1 and G 2 Each is independently unsubstituted C6-C 10 Alkylene; G 3 For unsubstituted C1-C 12 Alkylene; R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl; R 3 OR 5 N, -C (=O) OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 ;R 4 For C1-C 12 hydrocarbon group; and R 5 It is an H or C1-C6 hydrocarbon group.

[0240] In some embodiments, the cationic lipid is ALC0315 of formula (VI) (see patent CN108368028B):

[0241] (VI).

[0242] In some embodiments, the cationic lipid is a compound of formula (VI), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer.

[0243] (VI),

[0244] R4 is selected from -(CH2). n Q and -(CH2) n CHQR; Q is selected from the following groups: -OR, -OH, -O(CH2). n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -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)2R8 and heterocycles; n is 1, 2 or 3.

[0245] In some embodiments, the cationic lipid is SM102 with the (VI-I) structure (see patent CN110520409A):

[0246] (VI-I).

[0247] In some embodiments, the cationic lipid is a compound of formula (VII) DLIN-MC3-DMA (see CN102625696B), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer.

[0248] (VII)

[0249] In some embodiments, the cationic lipids include one or more selected from YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA.

[0250] In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 10:1.

[0251] In some embodiments, the molar ratio of the cationic lipid to the structural lipid is 1:1 to 5:1.

[0252] In some embodiments, 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).

[0253] In some embodiments, 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).

[0254] In some embodiments, 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.

[0255] In some embodiments, the neutral lipids include one or more selected from: phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

[0256] In some embodiments, the neutral lipid is selected from one or more of the following: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether 1,2-Dilinoleoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidanoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME) 16.0PE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyl oleoyl 1-Steayl-2-oleoyl-stearoyl-ethanolamine (POPE), 1-stearoyl-2-oleoyl-stearoyl-ethanolamine (DSPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl-phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0257] In some embodiments, the neutral lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine and / or 1,2-distearate-sn-glycerol-3-phosphate choline.

[0258] In some embodiments, the structural lipid is selected from one or more of the following: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

[0259] In some implementations, the structural lipid is cholesterol.

[0260] In some embodiments, the polymeric conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0261] In some embodiments, the polymeric conjugated lipid is selected from one or more of the following: distearate phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecyl acetamide) (ALC-0159).

[0262] The pharmaceutical compositions disclosed herein include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be derived from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids. Particularly preferred are formulations targeting the liver when treating liver conditions such as liver cancer.

[0263] The pharmaceutical compositions disclosed herein (which can be conveniently present in unit dosage forms) can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include steps such as combining the active ingredients with the drug carrier or excipient. Generally, these pharmaceutical compositions are prepared by the following steps: uniformly and finely combining the active ingredients with a liquid carrier or a finely dispersed solid carrier, or both, and, if necessary, shaping the product.

[0264] The pharmaceutical compositions disclosed herein can be formulated into any of a number of possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft capsules, suppositories, and enemas. The pharmaceutical compositions disclosed herein can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, such substances including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0265] Some pharmaceutical compositions disclosed herein also incorporate a carrier compound into the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid or its analogue that is inert (i.e., not biologically active in itself) but is considered a nucleic acid in vivo, for example by reducing the bioavailability of a biologically active nucleic acid by degrading it or promoting its removal from circulation. Co-administration of a nucleic acid and a carrier compound (generally in excess of the latter) can result in a significant reduction in the amount of nucleic acid recovered from the liver, kidneys, or other external circulation reservoirs, presumably due to competition for a common receptor between the carrier compound and the nucleic acid. For example, co-administration with polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4'-isothiocyanate 2,2'-disulfonic acid can reduce the recovery of partially thiophosphated dsRNA from liver tissue.

[0266] Compared to carrier compounds, a "drug carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or other pharmaceutically inert medium used to deliver one or more nucleic acids to animals. The excipient can be liquid or solid, and when combined with nucleic acids and other components of a particular pharmaceutical composition, the excipient is selected to provide desired volume, consistency, etc., with reference to the intended manner of administration. Typical drug carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, or dicalcium phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silica, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0267] Pharmaceutically acceptable organic or inorganic excipients that are suitable for non-parenteral administration, do not react toxically with nucleic acids, and are suitable for formulating the pharmaceutical compositions disclosed herein may also be used. Suitable pharmaceutically acceptable carriers include, but are not limited to: water, saline solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silica, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0268] Formulations for topical administration of nucleic acids may include sterile or non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or nucleic acid solutions in liquid or solid oil matrices. These solutions may also include buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration and not toxic to nucleic acids may be used.

[0269] Suitable pharmaceutically acceptable excipients include, but are not limited to: water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0270] The dosage form, carrier compound, drug carrier, excipients, etc. of the above-mentioned pharmaceutical composition are described in US Patent 10125369B2, which is incorporated herein by reference.

[0271] In another aspect, this disclosure provides a kit comprising a box A, which includes one or more of the aforementioned double-stranded RNAi agents, conjugates, or pharmaceutical compositions.

[0272] In some embodiments, the pillbox further includes a pillbox B, which contains one or two of the following:

[0273] (1) Other drugs that reduce HBV gene expression or compositions containing said drugs that reduce HBV gene expression;

[0274] (2) One or more of the following groups: hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0275] The “other drugs that reduce HBV gene expression or compositions containing the drugs that reduce HBV gene expression” mentioned herein refer to drugs that do not contain the double-stranded RNAi agents, conjugates or pharmaceutical compositions provided in this disclosure.

[0276] On the other hand, this disclosure provides the use of the aforementioned double-stranded RNAi agents, conjugates, or pharmaceutical compositions in the preparation of medicaments for the prevention and / or treatment of HBV gene expression-related diseases.

[0277] In some implementations, the HBV gene expression-related diseases are selected from the following disease types: chronic hepatitis B, liver fibrosis, cirrhosis, liver cancer, acute hepatitis B, and diseases related to co-infection with HBV / hepatitis D virus.

[0278] In another aspect, this disclosure provides a method for reducing HBV gene expression or inhibiting HBV replication for non-preventive and / or therapeutic purposes, the method comprising applying one or more of the aforementioned double-stranded RNAi agents, conjugates, pharmaceutical compositions, and kits to a sample.

[0279] The term "non-preventive and / or therapeutic" as used in this article refers to reducing HBV gene expression or inhibiting HBV replication for research purposes, such as in a laboratory setting.

[0280] The nucleotide abbreviations for this article are as follows:

[0281] A = Adenosine-3'-phosphate

[0282] Am = 2'-methoxyadenosine-3'-phosphate

[0283] Ams = 2'-methoxyadenosine-3'-thiophosphate

[0284] Af = 2'-Fluoroadenosine-3'-phosphate

[0285] Afs = 2'-Fluoroadenosine-3'-Thiophosphate

[0286] G = Guanosine-3'-phosphate

[0287] Gm = 2'-methoxyguanosine-3'-phosphate

[0288] Gms = 2'-methoxyguanosine-3'-thiophosphate

[0289] Gf=2'-Fluoroguanosine-3'-phosphate

[0290] Gfs = 2'-Fluoroguanosine-3'-Thiophosphate

[0291] C=cytidine-3'-phosphate

[0292] Cm = 2'-methoxycytidine-3'-phosphate

[0293] Cms = 2'-methoxycytidine-3'-thiophosphate

[0294] Cf = 2'-Fluorocytidine-3'-phosphate

[0295] Cfs = 2'-Fluorocytidine-3'-Thiophosphate

[0296] U = uridine-3'-phosphate

[0297] Um = 2'-methoxyuridine-3'-phosphate

[0298] Ums = 2'-methoxyuridine-3'-thiophosphate

[0299] Uf = 2'-fluorouridine-3'-phosphate

[0300] Ufs = 2'-fluorouridine-3'-thiophosphate

[0301] AmsEVP = 5'-vinyl-(E)-phosphonate-2'-methoxyadenosine-3'-thiophosphate

[0302] UmsEVP = 5'-vinyl-(E)-phosphonate-2'-methoxyuridine-3'-thiophosphate

[0303] A(gna) = adenosine diol nucleic acid

[0304] C(gna) = cytidine-diol nucleic acid

[0305] G(gna) = guanosine-diol nucleic acid

[0306] T(gna) = thymidine-diol nucleic acid

[0307] U(gna) = uridine-diol nucleic acid

[0308] dA = deoxyadenosine-3'-phosphate

[0309] dG = deoxyguanosine-3'-phosphate

[0310] dC = deoxycytidine-3'-phosphate

[0311] dT = deoxythymidine-3'-phosphate

[0312] The following examples are used to illustrate this disclosure, but are not intended to limit the scope of this disclosure. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0313] Example 1: Synthesis of small interfering oligonucleotides modified with DV29P template

[0314] A total of 36 basic siRNA sequences were designed. Alnylam's VIR-2218 was used as a positive control (sequence number APC-VIR in this embodiment); ANC-DV29P was used as a negative control. The basic sequences were modified using the DV29P template. Positions 7, 9, 11, and 17 of the sense strand were modified with 2'-F, and the remaining positions were modified with 2'-OMe. Positions 2, 4, 5, 6, 14, and 16 of the antisense strand were modified with 2'-F, and the remaining positions were modified with 2'-OMe. In addition, there were two thiolation modifications at the 5' end of the sense strand; two thiolation modifications each at the 5' and 3' ends of the antisense strand; and one EVP modification at the 5' end of the antisense strand. The DV29P template-modified siRNA sequences are shown in Table 1.

[0315] 1. Synthesis of the DV29P template modification sequence A194-DV29P

[0316] The basic sequence of the small interfering RNA with sequence number A194-DV29P in Table 1 is as follows:

[0317] Justice Chain: 5'-UCGUGUUACAGGCGGGGUUUU-3' (SEQ ID NO: 15)

[0318] Antisense chain: 5'-AAAACCCCGCCUGUAACACGAGA-3' (SEQ ID NO: 47)

[0319] The positive chain has 2'-F modifications at positions 7, 9, 11, and 17, and 2'-OMe modifications at the remaining positions; the negative chain has 2'-F modifications at positions 2, 4, 5, 6, 14, and 16, and 2'-OMe modifications at the remaining positions. Additionally, the positive chain has two thio modifications at the 5' end; the negative chain has two thio modifications at both the 5' and 3' ends; and the negative chain has one EVP modification at the 5' end.

[0320] Instruments and reagents: CYTiva 192 P model DNA / RNA automated synthesizer, with cross-linked polystyrene beads as the solid carrier, model Primer support 5G Unylinker 350 (Cytiva manufacturer).

[0321] Preparation method:

[0322] The following nucleotide monomer solutions were prepared with acetonitrile at a monomer concentration of 0.15 M: DMT-A-OMe phosphorus amide monomer (Formula 1), DMT-C-OMe phosphorus amide monomer (Formula 2), DMT-G-OMe phosphorus amide monomer (Formula 3), DMT-U-OMe phosphorus amide monomer (Formula 4), DMT-AF phosphorus amide monomer (Formula 5), ​​DMT-CF phosphorus amide monomer (Formula 6), DMT-GF phosphorus amide monomer (Formula 7), DMT-UF phosphorus amide monomer (Formula 8), vinyl-(E)-phosphonate-A-OMe phosphorus amide monomer (Formula 9), and vinyl-(E)-phosphonate-U-OMe phosphorus amide monomer (Formula 10).

[0323] Formula 1 Formula 2

[0324] Formula 3 Formula 4

[0325] Formula 5 Formula 6

[0326] Formula 7 Formula 8

[0327] Formula 9 Formula 10

[0328] The APC-VIR antisense strand of the Yangshen sequence is modified with a GNA (glucan-containing nucleotide) at the 7th base starting from the 5' end. The GNA monomer structure is as follows:

[0329] (S)-GNA-U (S)-GNA-C

[0330] (S)-GNA-A (S)-GNA-G

[0331] Prepared using the following steps:

[0332] (1) Deprotection

[0333] The DMT protecting group was removed using a 3% dichloroacetic acid toluene solution as a deprotecting agent, followed by washing with acetonitrile.

[0334] (2) Coupling

[0335] The acetonitrile solution of each nucleotide monomer was coupled using a 0.25 M 5-ethylthiotetrazole solution as an activator, followed by rinsing with acetonitrile.

[0336] (3) Oxidation / sulfidation

[0337] Oxidation: Oxidation was performed using a 0.05 M iodine pyridine / water (90 / 10) solution as the oxidant, followed by rinsing with acetonitrile.

[0338] Vulcanization: Vulcanization is carried out using a pyridine solution of 3% hydroxanthin as a vulcanizing agent, followed by rinsing with acetonitrile.

[0339] (4) Hydroxyl protection

[0340] Hydroxyl protection was performed using a 10% tetrahydrofuran solution of acetic anhydride (CAP A) and tetrahydrofuran / pyridine / aziridine-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as hydroxyl protecting agents, followed by rinsing with acetonitrile.

[0341] Repeat the above operations in a cyclical manner according to the set sequence to obtain a fully protected product.

[0342] (5) Use a 3% dichloroacetic acid toluene solution as a deprotection agent to remove the DMT protecting group of the last nucleotide, and then wash with acetonitrile.

[0343] (6) Ammonolysis and purification

[0344] The solid support was transferred to the reactor, concentrated ammonia (25-28%) was added, and the mixture was kept at 60°C for 12 h for ammonolysis. The system was then cooled to room temperature, and the mixture was filtered, washed with a mixture of purified water and ethanol, and the filtrates were combined, passed through a chromatography column, concentrated, and lyophilized to obtain the product.

[0345] (7) Annealing

[0346] The purified sense and antisense strands were mixed in a 1:1 ratio, heated to 95°C and held for 3 minutes, and then slowly cooled to room temperature to form a double strand.

[0347] A194-DV29P purity: 90.52%; measured molecular weight: 14727.09.

[0348] 2. Synthesis of other sequences

[0349] Synthesize the other sequences listed in Table 1 using the method described above.

[0350] Table 1. DV29P template-modified siRNA sequences

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359] Example 2: Inhibitory effect of DV29P template modification sequence on HBsAg and HBeAg

[0360] The DV29P template-modified siRNA sequence synthesized in Example 1 was transfected into HepG2.2.15 cells via lipid nanoparticles (LNPs), and the inhibitory effects of each sequence on HBsAg and HBeAg were detected using ELISA technology.

[0361] 1. Experimental Materials

[0362] Test sample: DV29P template modified siRNA sequence listed in Table 1 (synthesized in Example 1).

[0363] Cell type: HepG2.2.15 cells

[0364] Drug solvent: sterile enzyme-free water, gibco Opti-MEM (Thermo Fisher Scientific).

[0365] 2. Experimental Methods

[0366] The inhibitory effect of the samples on HBsAg and HBeAg in the HepG2.2.15 cell line was detected by ELISA.

[0367] 2.1 Cell Culture

[0368] Subculture: HepG2.2.15 cells were subcultured in DMEM / F12 medium containing 10% fetal bovine serum, 370 μg / ml GENETICN, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin. The cells were incubated at 37°C in a cell culture incubator containing 5% CO2, and subcultured every three days. After digestion with 0.25% trypsin, the cells were centrifuged at 800 r / min for 3 min, the supernatant was discarded, and fresh medium was added for further subculturing.

[0369] Plate 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.

[0370] 2.2 Cell transfection

[0371] Preparation of transfection mixture: Lipofectamine TM RNAiMAX (Thermo Fisher Scientific) and Opti-MEM were mixed in a 2:98 ratio and then vortexed to mix.

[0372] Preparation of transfection complex: Take 30 μL of Opti-MEM diluted siRNA solution at a ratio of 1:1 (v / v) and add it to 30 μL of transfection mixture. Vortex mix and let stand at room temperature for 15 min to obtain transfection complex.

[0373] Transfection control group transfection reagent preparation: Add 15 μL of the prepared transfection mixture to 15 μL of Opti-MEM. Vortex mix and let stand at room temperature for 15 min.

[0374] Add the prepared transfection complex to a 96-well cell culture plate (15 μL per well, 3 replicates per sequence) to achieve a final siRNA concentration of 0.3 nM per well. Add 135 μL of cell suspension (containing 2.25 × 10⁻⁶ cells / well). 4 (1 cell). After mixing using the cross-hatching method, incubate in a 37°C, 5% CO2 cell culture incubator.

[0375] 2.3 Detection of HBsAg and HBeAg in cell supernatant

[0376] 1) Collection of cell supernatant

[0377] a. Change the medium on day 3 after cell transfection, discard the cell supernatant, and add 150 μL / well of fresh medium to continue culturing.

[0378] b. Collect cell supernatant on day 6 post-transfection for the detection of HBsAg and HBeAg levels.

[0379] 2) Quantitative detection of HBsAg and HBeAg

[0380] The concentrations of HBsAg and HBeAg were detected using the Hepatitis B e Antigen Detection Kit (Antu Bio CL0310) and the Hepatitis B Surface Antigen Detection Kit (Antu Bio CL0312). The specific operating steps are as follows:

[0381] a. Allow the kit and test samples to return to room temperature.

[0382] b. Add 50 μL each of the test sample, standard, negative control, and positive control to a well plate.

[0383] c. Add 50 μL of enzyme conjugate to each well. Mix thoroughly and incubate at 37°C for 60 min.

[0384] d. Remove the liquid from the orifice plate and wash it 5 times with the cleaning solution. Finally, pat the orifice plate dry on absorbent paper.

[0385] e. Mix luminescent substrates A and B in equal proportions and add 50 μL / well. React at room temperature in the dark for 3 min.

[0386] f. Measure the luminescence value using an ELISA reader.

[0387] 2.4 Data Processing

[0388] The formulas for calculating the inhibition rates of HBsAg and HBeAg are as follows:

[0389] HBsAg inhibition rate (%) = (1 - HBsAg expression level in sample / HBsAg expression level in control group in the same plate) × 100%;

[0390] HBeAg inhibition rate (%) = (1 - HBeAg expression level in sample / HBeAg expression level in control group in the same plate) × 100%.

[0391] The inhibition rate of HBsAg relative to Yangshen (Yangshen inhibition rate is set to 1) = HBsAg inhibition rate of sample (%) / Yangshen inhibition rate in the same plate (%). It should be understood that a value greater than 1 indicates that the inhibition effect of this sequence on HBsAg is better than that of Yangshen, and a value less than 1 indicates that the inhibition efficiency of this sequence on HBsAg is lower than that of Yangshen.

[0392] The inhibition rate of HBeAg relative to Yangshen (Yangshen inhibition rate is set to 1) = HBeAg inhibition rate of sample (%) / Yangshen inhibition rate in the same plate (%). It should be understood that a value greater than 1 indicates that the inhibitory effect of this sequence on HBeAg is better than that of Yangshen, and a value less than 1 indicates that the inhibition efficiency of this sequence on HBeAg is lower than that of Yangshen.

[0393] 2.5 IC50 Experiment

[0394] In this experiment, the concentration of each sequence was set starting from 10 nM, with 4-fold dilutions, for a total of 8 concentration points (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, plotted, and the IC50 concentration of each sequence and the ginseng was calculated.

[0395] 2.6 Cytotoxicity assay

[0396] When performing the IC50 experiment, after collecting the cell supernatant, CellTiter-Glo ® The (Promega) kit is used to determine cell viability. The method is briefly described as follows: Mix CellTiter-Glo reagent with culture medium at a 1:1 ratio, add 100 μL to each well, incubate at room temperature for 10 min, and then detect the luminescence signal value using a microplate reader.

[0397] The formula for calculating cell viability is: Cell viability (%) = (Sample signal value – Average value of blank control) / (Average value of transfection control – Average value of blank control) × 100%.

[0398] 3. Experimental Results

[0399] Experimental results showed that sequences B207S-DV29P, B1572-DV29P, B1575-DV29P, B418S-DV29P, A206-DV29P, A416-DV29P, A1548-DV29P, A1550-DV29P, and A1573-DV29P exhibited significantly better inhibitory effects on HBsAg and HBeAg than other sequences.

[0400] The inhibition rates of each sequence on the expression of HBsAg and HBeAg at single concentrations and their inhibition rates relative to the positive control sequence APC-VIR are shown in Tables 3, 4, 5, and 6.

[0401] The results of single-concentration screening experiments showed that some 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 (see Table 2 for specific sequences), had higher inhibition rates against HBsAg and HBeAg than Yangshen APC-VIR.

[0402] The IC50 and cytotoxicity results for each sequence are shown in Table 7. The IC50 results indicate that the 11 candidate sequences modified with the DV29P template designed in this publication exhibited IC50 values ​​against HBsAg ranging from 0.01 to 0.08 nM, all superior to the 0.1777 nM of Yangshen; for example, the IC50 values ​​of A261-DV29P, A1573-DV29P, and A206-DV29P were 0.0169 nM, 0.0226 nM, and 0.0226 nM, respectively. These sequences effectively inhibited HBsAg expression even at low concentrations. The IC50 values ​​of these 11 candidate sequences against HBeAg ranged from 0.06 to 0.4 nM, all superior to the 0.7685 nM of *Codonopsis pilosula*. 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. Cytotoxicity results showed that, among the 11 sequences, except for sequence A416-DV29P which exhibited a cell viability of 81.82% at the highest tested concentration of 10 nM, the cell viability of the remaining sequences was greater than 90%, showing no significant cytotoxicity, indicating that the sequences have a wide concentration selectivity range.

[0403] The design prioritizes HBsAg inhibition while also considering HBeAg inhibition. Based on the above results, 9 sequences were selected from these 11 sequences for further optimization. Sequences B207S-DV29P, B1572-DV29P, B1575-DV29P, B418S-DV29P, A206-DV29P, A416-DV29P, A1548-DV29P, A1550-DV29P, and A1573-DV29P can be considered as candidate sequences.

[0404] Table 2. siRNA sequences with superior inhibition rates against HBsAg and HBeAg compared to Yangshen.

[0405]

[0406]

[0407] (i) The inhibitory effect of each sequence on HBV gene expression at a single concentration after modification with DV29P template.

[0408] (1) The sequences that have a better inhibitory effect on HBsAg than Yangshen are shown in Table 3, including B207S-DV29P, B1575-DV29P, B1572-DV29P, B418S-DV29P, A261-DV29P, A206-DV29P, A1550-DV29P, A1573-DV29P, A1548-DV29P, and A416-DV29P. Among them, the four sequences B207S-DV29P, B1575-DV29P, B1572-DV29P, and B418S-DV29P all have an inhibition rate of more than 80% on HBsAg at a concentration of 0.3 nM. For example, the inhibition rate of B207S-DV29P reaches 93.37%, which is 23.59% higher than that of Yangshen.

[0409] Table 3 shows that the inhibition rate of HBsAg is higher than that of the DV29P modified sequence of *Syngonium oxyphylla*.

[0410]

[0411] (2) The sequences that are inferior to Yangshen in inhibiting HBsAg are shown in Table 4. For example, the inhibition rate of sequence B416S-DV29P against HBsAg is only 6.98%.

[0412] Table 4 shows that the inhibition rate of HBsAg is lower than that of the DV29P modified sequence of *Syngonium oxyphylla*.

[0413]

[0414] (3) The sequences that have better inhibitory effects on HBeAg than Yangshen are shown in Table 5. Among them, B1575-DV29P, B207S-DV29P, B1572-DV29P and B418S-DV29P are ranked first, and are 38.62%, 22.07%, 17.10% and 13.24% better than Yangshen, respectively.

[0415] Table 5 shows that the inhibition rate of HBeAg is higher than that of the DV29P modified sequence of Yangshen.

[0416]

[0417] (4) The sequences that are inferior to Yangshen in inhibiting HBeAg are shown in Table 6. For example, the inhibition rate of sequence A1520-DV29P is only 0.28% at a concentration of 0.3 nM.

[0418] Table 6 shows that the inhibition rate of HBeAg is lower than that of the DV29P modified sequence of Yangshen.

[0419]

[0420] (ii) IC50 assay and cytotoxicity results

[0421] Nine sequences, including B207S-DV29P, B1572-DV29P, B1575-DV29P, B418S-DV29P, A206-DV29P, A416-DV29P, A1548-DV29P, A1550-DV29P, and A1573-DV29P, were selected from the best sequences in the single-concentration experiments and IC50 experiments were conducted. The results showed that the IC50 values ​​of these candidate sequences against HBsAg and HBeAg were all lower than those against the positive control, demonstrating that their inhibitory effects were superior to those against the positive control. For example, the IC50 values ​​of 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 ​​of 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. Detailed results are shown in Table 7.

[0422] Cytotoxicity results showed that, except for sequence A416-DV29P, which had a cell viability of 81.82% at the highest tested concentration of 10 nM, the cell viability of the other sequences was greater than 90%, and they did not exhibit significant cytotoxicity, indicating that the sequences have a wide concentration selectivity range. Specific results are shown in Table 7.

[0423] Table 7 IC50 test results for HBsAg and HBeAg

[0424]

[0425] Example 3: Inhibitory effect of unmodified sequence on HBV gene

[0426] In this embodiment, some unmodified sequences corresponding to the modified sequences in Example 1 were synthesized and transfected into HepG2.2.15 cells via lipid nanoparticles (LNP). The inhibitory effects of each sequence on HBsAg and HBeAg were detected using ELISA technology, and unmodified siRNA sequences with better inhibitory effects were screened out.

[0427] 1. Experimental Materials

[0428] Test sample:

[0429] The unmodified small interfering RNA sequences listed in Table 8 were synthesized according to the method described in Example 1.

[0430] Table 8 Unmodified siRNA sequences

[0431]

[0432] Cell type: HepG2.2.15 cells

[0433] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM.

[0434] 2. Experimental Methods

[0435] Referring to Example 2, the screening concentration was set to 0.3 nM.

[0436] 3. Experimental Results

[0437] Experimental results showed that sequences B207S, B1572, B1575, B418S, A206, A416, A1548, A1550, and A1573 exhibited significantly better inhibitory effects on HBsAg and HBeAg than other sequences.

[0438] Table 9 lists the sequences that showed superior inhibition rates against HBsAg and HBeAg at single concentrations compared to those of Yangshen, including B207S, B1575, B418S, A1573, B1572, A261, A206, A1550, A416, and A1548. Among them, B207S achieved inhibition rates of 77.02% and 40.74% against HBsAg and HBeAg, respectively, which were significantly better than the 61.45% and 20.85% of the Yangshen APC sequence.

[0439] Table 9 shows that the inhibition rates of HBsAg and HBeAg are higher than those of the basic sequence of Yangshen.

[0440]

[0441] Example 4: Inhibitory effects of sequences modified with different templates on HBsAg and HBeAg

[0442] This embodiment uses 15 unmodified sequences (A205, B208, B207S, A261, B262, B264, A1550, B1556, B1560, B1575, A1573, B1572, B418S, A416, B416S) and 8 modified templates (DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, and DV34P) designed in this disclosure, as well as existing publicly available templates (DV30P, DV31P, DV21P, and DV22P), resulting in a total of 154 sequences. After transfection into HepG2.2.15 cells using lipid nanoparticles (LNPs), the inhibitory effects of each sequence on HBsAg and HBeAg were detected using ELISA.

[0443] 1. Experimental Materials

[0444] Test samples: The sequences listed in Table 11 are modified from 15 siRNA base sequences using different templates (DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, DV34P, DV30P, DV31P, DV21P, DV22P) (synthesis method is as described in Example 1).

[0445] Cell type: HepG2.2.15 cells

[0446] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM

[0447] The principles for modifying this template are as follows:

[0448] When the antisense strand is 23 nucleotides long, the antisense strand adopts one or more of the modifications shown in Table 10-1 below:

[0449] Table 10-1 Antisense Chain Modification Methods

[0450]

[0451]

[0452]

[0453] When the antisense strand is 22 nucleotides long, the antisense strand adopts one or more of the modifications shown in Table 10-2 below:

[0454] Table 10-2 Antisense Chain Modification Methods

[0455]

[0456]

[0457] When the length of the sense strand is 21 nucleotides, the sense strand adopts one or a combination of two of the modifications shown in Table 10-3 below:

[0458] Table 10-3 Methods of Modifying the Chain of Justice

[0459]

[0460] When the length of the sense strand is 20 nucleotides, the sense strand adopts one or a combination of two of the modifications shown in Table 10-4 below:

[0461] Table 10-4 Methods of Modifying the Chain of Justice

[0462]

[0463] In Tables 10-1 to 10-4 above, 2'-OMe is 2'-methoxy; 2'-F is 2'-fluoro; PS is the thiophosphate skeleton; and EVP is 5'-vinyl-(E)-phosphonate.

[0464] The antisense strand uses modification A, and the sense strand uses modification a. The siRNA modification template is named DV27P.

[0465] The siRNA template modified by A for the antisense strand and b for the sense strand was named DV29P.

[0466] The antisense strand uses modification B, and the sense strand uses modification a. The siRNA modification template is named DV26P.

[0467] The antisense strand uses modification B, and the sense strand uses modification b. The siRNA modification template is named DV28P.

[0468] The antisense strand uses modification C, and the sense strand uses modification b. The siRNA modification template is named DV32P.

[0469] The antisense strand uses modification D, and the sense strand uses modification b. The siRNA modification template is named DV34P.

[0470] The antisense strand uses modification E, and the sense strand uses modification a. The siRNA modification template is named DV25P.

[0471] The antisense strand uses modification F, and the sense strand uses modification b. The siRNA modification template is named DV33P.

[0472] The method for synthesizing each sequence is the same as in Example 1.

[0473] Table 11 Sequences modified with different modification templates

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505] 2. Experimental Methods

[0506] Referring to Example 2, the concentration for the single-concentration screening experiment was set to 0.3 nM.

[0507] 3. Experimental Results

[0508] Experimental results showed that sequences B207S-DV32P, A1550-DV27P, B1572-DV27P, B1575-DV27P, B207S-DV29P, A1573-DV29P, B1575-DV29P, B418S-DV34P, and A416-DV27P exhibited significantly better inhibitory effects on HBsAg and HBeAg than the Yangshen sequence.

[0509] The inhibition rates of each sequence on HBsAg and HBeAg expression at single concentrations and their inhibition rates relative to the Yangshen sequence APC-VIR are shown in Table 12-41. The overall ranking is represented by the inhibition rate ranking of the sequences relative to the Yangshen sequence.

[0510] Single-concentration screening experiments showed that some basic sequences modified with the designed templates DV25P-29P and DV32P-34P exhibited superior inhibitory effects against HBsAg and HBeAg compared to the positron-dependent sequences. Among these, the modified sequences designed using the basic sequences B207S and B1575 showed the highest inhibition rates against HBsAg, demonstrating the sensitivity of this target site. Sequences B207S-DV29P, B207S-DV32P, B1575-DV29P, B1575-DV26P, and B1575-DV27P all achieved inhibition rates exceeding 90% against HBsAg. For example, sequences B207S-DV29P and B1575-DV29P achieved inhibition rates of 92.11% and 91.78% against HBsAg, respectively. Among them, the modified sequences designed with the basic sequences B1575 and A1573 ranked highest in HBeAg inhibition rate, demonstrating the sensitivity of this target site. The sequences B1575-DV29P, B1575-DV26P, B1575-DV27P, and B1575-DV32P all showed HBeAg inhibition rates exceeding 60%.

[0511] The same siRNA sequence exhibits significant differences in activity when modified with different templates. For example, the basal sequence B1556, when modified with the DV27P template disclosed in this publication, shows a 48.63% increase in HBsAg inhibition rate compared to when modified with the DV28P template disclosed in this publication, which is a significant improvement.

[0512] Although the basic sequences are similar, their sensitivities to different modification templates vary. For example, the basic sequence B1572, modified with the DV27P template designed in this disclosure, exhibits the highest inhibition rate against HBsAg and HBeAg, while the basic sequences A1573 and B1575, modified with template DV29P, show the highest inhibition rates against HBsAg and HBeAg. Therefore, it is uncertain which modification template will result in high activity for the siRNA sequence.

[0513] The IC50 and cytotoxicity results for each sequence are shown in Table 42. The results indicate that the IC50 values ​​of the 11 sequences tested against HBsAg ranged from 0.05 nM to 0.15 nM, all lower than the 0.3362 nM of the Yangshen sequence, demonstrating superior inhibitory effects. For example, the IC50 values ​​of B1572-DV27P, B1550-DV27P, and B207S-DV32P against HBsAg were 0.0538 nM, 0.0570 nM, and 0.0572 nM, respectively. Except for sequence B261-DV26P, the IC50 values ​​of the other 10 sequences against HBeAg ranged from 0.2 nM to 0.85 nM, all lower than the 1.89 nM of the Yangshen sequence, demonstrating superior inhibitory effects. For example, the IC50 values ​​of sequences B1550-DV27P, B1572-DV27P, and B1575-DV26P against HBeAg were 0.2073 nM, 0.2143 nM, and 0.2923 nM, respectively. Cytotoxicity results showed that all 11 sequences exhibited cell viability greater than 90% at the highest tested concentration of 10 nM, without significant cytotoxicity, indicating a wide concentration selectivity range.

[0514] Based on the above results, the 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 considered as candidate sequences.

[0515] (i) The inhibitory effect of each basic sequence on HBsAg and HBeAg after template modification

[0516] (1) Basic sequence A1550

[0517] Table 12 Inhibition rate of HBsAg by template modification of the basic sequence A1550

[0518]

[0519] Table 13. HBeAg inhibition rate of the basic sequence A1550 after template modification.

[0520]

[0521] The basic sequence A1550, modified with the modified templates DV25P-29P and DV32P-34P designed in this disclosure, showed improved inhibition rates against HBsAg and HBeAg compared to modifications using existing modified templates. For example, after modification with templates DV26P and DV27P, the inhibition rates of B1550-DV26P and B1550-DV27P against HBsAg increased by 25.36% and 25.51%, respectively, and the inhibition rates against HBeAg increased by 24.77% and 26.55%, respectively. Specific results are shown in Tables 12 and 13.

[0522] (2) Basic sequence B1556

[0523] Table 14 Inhibition rate of HBsAg by template modification of the basic sequence B1556

[0524]

[0525] Table 15. HBeAg inhibition rate of the basic sequence B1556 after template modification.

[0526]

[0527] After the basic sequence B1556 was modified with the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure, compared with the modified templates disclosed in the prior art, the inhibition rates of B1556-DV26P and B1556-DV27P after modification with templates DV26P and DV27P were increased by 37.46% and 47.61% for HBsAg, and by 25.29% and 31.73% for HBeAg, respectively. Specific results are shown in Tables 14 and 15.

[0528] (3) Basic sequence B1560

[0529] Table 16 Inhibition rate of HBsAg by template modification of the basic sequence B1560

[0530]

[0531] Table 17. HBeAg inhibition rate of the basic sequence B1560 after template modification.

[0532]

[0533] The basic sequence B1560, after being modified with the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure, showed improved inhibition rates of HBsAg (by 35.82% and 43.60%, respectively) and HBeAg (by 20.84% ​​and 28.54%, respectively) after modification with templates DV26P and DV27P, compared with the modified templates disclosed in the prior art. Specific results are shown in Tables 16 and 17.

[0534] Therefore, we can conclude that:

[0535] 1) For the basic sequences A1550, B1556, and B1560, modification with the modified templates DV25P-29P and DV32P-34P designed in this disclosure significantly improved the inhibitory effects on HBsAg and HBeAg compared to the modified templates disclosed in the prior art. Modification with DV26P and DV27P resulted in the highest inhibition rates for HBsAg and HBeAg, which were also significantly better than the modified templates disclosed in the prior art.

[0536] 2) The same siRNA sequence exhibits significant differences in activity when modified with different templates. For example, the basic sequence B1556, when modified with the DV27P template disclosed in this publication, shows a 48.63% increase in HBsAg inhibition rate compared to when modified with the DV28P template disclosed in this publication, which is a significant improvement.

[0537] 3) siRNAs with similar sequences can have vastly different activities. For example, the DV29P modified sequence A1550-DV29P showed a 40.86% higher inhibition rate against HBsAg compared to A1556-DV29P.

[0538] (4) Basic sequence B1572

[0539] Table 18 Inhibition rate of HBsAg by template modification of the basic sequence B1572

[0540]

[0541] Table 19. HBeAg inhibition rate of the basic sequence B1572 after template modification.

[0542]

[0543] The basic sequence B1572, after being modified with the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure, showed improved inhibition rates of HBsAg (by 26.84% and 27.59%, respectively) and HBeAg (by 32.02% and 36.74%, respectively) after modification with templates DV26P and DV27P, compared with the modified templates disclosed in the prior art. Specific results are shown in Tables 18 and 19.

[0544] (5) Basic sequence A1573

[0545] Table 20 Inhibition rate of HBsAg by template modification of the basic sequence A1573

[0546]

[0547] Table 21. Inhibition rate of HBeAg by template modification of the basic sequence A1573

[0548]

[0549] The basic sequence A1573, after being modified with the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure, significantly improved the inhibition rates of HBsAg and HBeAg compared with the modified templates disclosed in the prior art. For example, after modification with template DV29P, the inhibition rates of A1573-DV29P against HBsAg and HBeAg increased by 16.08% and 32.68%, respectively. Specific results are shown in Tables 20 and 21.

[0550] (6) Basic sequence B1575

[0551] Table 22 Inhibition rate of HBsAg by template modification of the basic sequence B1575

[0552]

[0553] Table 23. HBeAg inhibition rate of the basic sequence B1575 after template modification.

[0554]

[0555] The basic sequence B1575 was modified using the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure. Compared with the modified templates disclosed in the prior art, all of these modifications significantly improved the inhibition rates of HBsAg and HBeAg. For example, after modification with template DV29P, the inhibition rates of B1575-DV29P against HBsAg and HBeAg increased by 31.19% and 46.17%, respectively. Specific results are shown in Tables 22 and 23.

[0556] Therefore, we can conclude that:

[0557] 1) For the basic sequences B1572, A1573, and B1575, modification with the disclosed templates DV26P-29P, DV32P, and DV34P significantly enhanced the inhibitory effects on HBsAg and HBeAg compared to existing modified templates. For the basic sequence B1572, modification with the disclosed templates DV26P and DV27P resulted in the highest inhibition rates for HBsAg and HBeAg, significantly superior to existing modified templates. For the basic sequences A1573 and B1575, modification with the disclosed template DV29P resulted in the highest inhibition rates for HBsAg and HBeAg, significantly superior to existing modified templates.

[0558] 2) The same siRNA sequence exhibits significant differences in activity when modified with different templates. For example, the basic sequence B1575, when modified with the DV29P template disclosed in this publication, showed a 29.99% increase in HBsAg inhibition rate compared to when modified with the DV28P template disclosed in this publication, which is a significant improvement.

[0559] 3) siRNAs with similar sequences have different sensitivities to different modified templates. For example, the basic sequence B1572, when modified with the modified template DV27P designed in this publication, has the highest inhibition rate against HBsAg and HBeAg, while the basic sequences A1573 and B1575, when modified with template DV29P, have the highest inhibition rates against HBsAg and HBeAg.

[0560] (7) Basic sequence B205

[0561] Table 24 Inhibition rate of HBsAg by template modification of the basic sequence B205

[0562]

[0563] Table 25. HBeAg inhibition rate of the basic sequence B205 after template modification.

[0564]

[0565] The basic sequence B205 was modified using the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure. Compared with the modified templates disclosed in the prior art, all of these modifications significantly improved the inhibition rates of HBsAg and HBeAg. For example, after modification with template DV28P, the inhibition rates of B205-DV28P against HBsAg and HBeAg increased by 36.55% and 25.36%, respectively. Specific results are shown in Tables 24 and 25.

[0566] (8) Basic sequence B207S

[0567] Table 26 Inhibition rate of HBsAg by template modification of the basic sequence B207S

[0568]

[0569] Table 27. HBeAg inhibition rate of the basic sequence B207S after template modification.

[0570]

[0571] The basic sequence B207S was modified using the modified templates DV25P-29P and DV32P-34P designed in this disclosure. Compared with the modified templates disclosed in the prior art, both modifications significantly improved the inhibition rates of HBsAg and HBeAg. For example, after modification with templates DV29P and DV32P, the inhibition rates of B207S-DV29P against HBsAg and HBeAg increased by 35.37% and 26.71%, respectively, and the inhibition rates of B207S-DV32P against HBsAg and HBeAg increased by 34.98% and 18.95%, respectively. Specific results are shown in Tables 26 and 27.

[0572] (9) Basic sequence B208

[0573] Table 28 Inhibition rate of HBsAg by template modification of the basic sequence B208

[0574]

[0575] Table 29. HBeAg inhibition rate of the basic sequence B208 after template modification

[0576]

[0577] The basic sequence B208 was modified using the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure. Compared with the modified templates disclosed in the prior art, the modification with template DV26P significantly improved the inhibition rate of B208-DV26P against HBsAg and HBeAg, increasing it by 37.63% and 19.38%, respectively. Specific results are shown in Tables 28 and 29.

[0578] Therefore, we can conclude that:

[0579] 1) For the basic sequences B205, B207S, and B208, modification with the modified templates DV25P-29P and DV32P-34P designed in this disclosure significantly enhances the inhibitory effects on HBsAg and HBeAg compared to existing modified templates. For the basic sequence B205, modification with the modified template DV28P designed in this disclosure results in the highest inhibition rate against HBsAg and HBeAg, significantly superior to existing modified templates. For the basic sequence B207S, modification with the modified template DV29P designed in this disclosure results in the highest inhibition rate against HBsAg and HBeAg, significantly superior to existing modified templates. For the basic sequence B208, modification with the modified template DV26P designed in this disclosure results in the highest inhibition rate against HBsAg and HBeAg, significantly superior to existing modified templates.

[0580] 2) The same siRNA sequence exhibits significant differences in activity when modified with different templates. For example, the basic sequence B208, when modified with the DV26P template disclosed in this publication, showed a 21.96% increase in HBsAg inhibition rate compared to when modified with the DV29P template disclosed in this publication, which is a significant improvement.

[0581] 3) siRNAs with similar sequences can have vastly different activities. For example, the DV29P modified sequence B207S-DV29P showed a 45.84% higher inhibition rate against HBsAg than B208-DV29P, and a 23.77% higher inhibition rate than B205-DV29P.

[0582] 4) It can be seen that although the basic sequences are similar, the sensitivity to each modified template is different. For example, the basic sequence B205, modified with the modified template DV28P designed in this disclosure, has the highest inhibition rate of HBsAg and HBeAg, which is 78.48% and 35.95%, respectively; while the basic sequence B207S, after being modified with template DV29P, has the highest inhibition rate of HBsAg and HBeAg, which is 92.11% and 55.18%, respectively; and the basic sequence B208, after being modified with template DV26P, has the highest inhibition rate of HBsAg and HBeAg, which is 68.23% and 41.45%, respectively.

[0583] (10) Basic sequence A261

[0584] Table 30 Inhibition rate of HBsAg by template modification of the basic sequence A261

[0585]

[0586] Table 31. Inhibition rate of HBeAg by template modification of the basic sequence A261

[0587]

[0588] The basic sequence A261 was modified using the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure. Compared with the modified templates disclosed in the prior art, all of these modifications significantly improved the inhibition rates of HBsAg and HBeAg. For example, after modification with template DV26P, the inhibition rates of B261-DV26P against HBsAg and HBeAg increased by 16.82% and 11.89%, respectively. Specific results are shown in Tables 30 and 31.

[0589] (11) Basic sequence B262

[0590] Table 32 Inhibition rate of HBsAg by template modification of the basic sequence B262

[0591]

[0592] Table 33. HBeAg inhibition rate of the basic sequence B262 after template modification.

[0593]

[0594] The basic sequence B262 was modified using the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure. Compared with the modified templates disclosed in the prior art, all of these modifications significantly improved the inhibition rates of HBsAg and HBeAg. For example, after modification with template DV26P, the inhibition rates of B261-DV26P against HBsAg and HBeAg were increased by up to 28.99% and 17.93%, respectively. Specific results are shown in Tables 32 and 33.

[0595] (12) Basic sequence B264

[0596] Table 34. Inhibition rate of HBsAg by template modification of the basic sequence B264

[0597]

[0598] Table 35. HBeAg inhibition rate of the basic sequence B264 after template modification.

[0599]

[0600] The base sequence B264 was modified using the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure. Compared with the modified templates disclosed in the prior art, all of these modifications significantly improved the inhibition rates of HBsAg and HBeAg. For example, after modification with template DV29P, the inhibition rates of B264-DV29P against HBsAg and HBeAg increased by 35.22% and 13.12%, respectively. Specific results are shown in Tables 34 and 35.

[0601] Therefore, we can conclude that:

[0602] 1) For the basic sequences A261, B262, and B264, the inhibitory effects on HBsAg and HBeAg after modification with the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure are significantly better than those of the modified templates disclosed in the prior art. For the basic sequence A261, the sequence with the modified template DV26P designed in this disclosure has the highest inhibition rate on HBsAg, and the sequence with the modified template DV27P has the highest inhibition rate on HBeAg, which is significantly better than that of the modified templates disclosed in the prior art. For the basic sequence B262, the sequence with the modified template DV28P designed in this disclosure has the highest inhibition rate on both HBsAg and HBeAg, which is significantly better than that of the modified templates disclosed in the prior art. For the basic sequence B264, the sequence with the modified template DV29P designed in this disclosure has the highest inhibition rate on both HBsAg and HBeAg, which is significantly better than that of the modified templates disclosed in the prior art.

[0603] 2) The same siRNA sequence exhibits significant differences in activity when modified with different templates. For example, the basic sequence B264, when modified with the DV27P template disclosed in this publication, showed a 20.16% reduction in HBsAg inhibition rate compared to modification with the DV29P template, which is a significant decrease.

[0604] 3) siRNAs with similar sequences can have vastly different activities. For example, the DV29P modified sequence A261-DV29P showed a 40.21% higher inhibition rate against HBsAg than B262-DV29P, and a 30.46% higher inhibition rate than B264-DV29P.

[0605] 4) It can be seen that although the basic sequences are similar, the sensitivity to each modified template is different. For example, the basic sequence B262, modified with the modified template DV28P designed in this disclosure, has the highest inhibition rate of HBsAg and HBeAg, while the basic sequence B264, after being modified with template DV29P, has the highest inhibition rate of HBsAg and HBeAg.

[0606] (13) Basic sequence A416

[0607] Table 36. Inhibition rate of HBsAg by template modification of the basic sequence A416

[0608]

[0609] Table 37. HBeAg inhibition rate after template modification of the basic sequence A416.

[0610]

[0611] The basic sequence A416 was modified using the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure. Compared with the modified templates disclosed in the prior art, all of these modifications significantly improved the inhibition rates of HBsAg and HBeAg. For example, the inhibition rates of HBsAg and HBeAg were increased by 29.19% and 33.83%, respectively, by using DV27P modification. Specific results are shown in Tables 36 and 37.

[0612] (14) Basic sequence B416S

[0613] Table 38 Inhibition rate of HBsAg by template modification of the basic sequence B416S

[0614]

[0615] Table 39. HBeAg inhibition rate of the basic sequence B416S after template modification.

[0616]

[0617] The basic sequence B416S, after being modified with the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure, showed lower inhibition rates of HBsAg and HBeAg, below 25%, compared with the modified templates disclosed in the prior art. Specific results are shown in Tables 38 and 39.

[0618] (15) Basic sequence B418S

[0619] Table 40 Inhibition rate of HBsAg by template modification of the basic sequence B418S

[0620]

[0621] Table 41. Inhibition rate of HBeAg by the basic sequence B418S after template modification.

[0622]

[0623] The basic sequence B418S was modified using the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure. Compared with the modified templates disclosed in the prior art, all of these modifications significantly improved the inhibition rates of HBsAg and HBeAg. For example, after modification with the DV34P template, the inhibition rates of HBsAg and HBeAg were increased by 25.62% and 26.91%, respectively. Specific results are shown in Tables 40 and 41.

[0624] Therefore, we can conclude that:

[0625] 1) For the basic sequences A416 and B418S, the inhibitory effects on HBsAg and HBeAg after modification with the modified templates DV26P-29P, DV32P, and DV34P designed in this disclosure are significantly better than those of the modified templates disclosed in the prior art. For the basic sequence A416, the inhibition rate of HBsAg and HBeAg is the highest after modification with the modified template DV27P designed in this disclosure. For the basic sequence B418S, the inhibition rate of HBsAg and HBeAg is the highest after modification with DV34P. However, for the basic sequence B416S, the inhibition rates of HBsAg and HBeAg are all low, below 25%, using both the modified templates DV26P-28P, DV32P, DV34P designed in this disclosure and the modified templates disclosed in the prior art.

[0626] 2) The same siRNA sequence exhibits significant differences in activity when modified with different templates. For example, the base sequence A416, when modified with the DV27P template disclosed in this publication, showed a 16.79% increase in HBsAg inhibition rate compared to modification with the DV32P template.

[0627] 3) siRNAs with similar sequences can have vastly different activities. For example, the DV29P modified sequence A416-DV29P showed a 72.41% higher inhibition rate against HBsAg compared to B416S-DV29P.

[0628] 4) It can be seen that although the basic sequences are similar, the sensitivity to each modified template is different. For example, the basic sequence A416 is modified with the modified template DV27P designed in this disclosure, and the inhibition rate of HBsAg and HBeAg is the highest. The basic sequence B418S is modified with the template DV34P and the inhibition rate of HBsAg and HBeAg is the highest.

[0629] (ii) IC50 assay and cytotoxicity results

[0630] Dominant sequences from the single-concentration screening experiment were selected, 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 APC-VIR sequence from *Codonopsis pilosula*, for a total of 12 sequences. IC50 experiments were conducted to determine the IC50 concentration of each sequence.

[0631] Table 42. Experimental results of IC50 for each modified sequence.

[0632]

[0633] The IC50 results 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 control sequence, demonstrating that their inhibitory effects were superior to those of the positive control sequence. 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 even at low concentrations. Except for sequence B261-DV26P, the IC50 values ​​of the other 10 sequences against HBeAg ranged from 0.2 nM to 0.85 nM, all lower than the 1.89 nM of the positive control sequence, demonstrating that their inhibitory effects were superior to those of the positive control sequence. For example, the IC50 values ​​of 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 could effectively inhibit HBeAg expression even at low concentrations. The experimental results are shown in Table 42.

[0634] Cytotoxicity results showed that all 11 sequences exhibited cell viability greater than 90% at the highest tested concentration of 10 nM, without significant cytotoxicity, indicating that the sequences have a wide concentration selectivity range. Specific results are shown in Table 42.

[0635] Example 5: Comparison of HBV gene inhibition effects with publicly available sequences in the prior art

[0636] This embodiment compares the HBV gene inhibition efficiency of unmodified sequences that are identical or similar to the basic sequences B207S, B1575, B1550, B1572, A416, B1573, and B418S disclosed in the prior art, with sequences modified by existing modified templates DV30P, DV31P, DV21P, and DV22P, and sequences modified by modified templates DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, and DV34P disclosed in the prior art.

[0637] 1. Experimental Materials

[0638] Test sample:

[0639] (1) Prior art disclosure sequence, see Table 43.

[0640] Table 43 Prior Art Disclosure Sequence

[0641]

[0642] Note: The sequences disclosed in the above patent applications are all unmodified RNA sequences.

[0643] (2) The siRNA sequences modified by the modified templates DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, DV34P, DV30P, DV31P, DV21P, and DV22P in Example 4, and the corresponding unmodified basic sequences.

[0644] Cell type: HepG2.2.15 cells

[0645] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM

[0646] 2. Experimental Methods

[0647] Referring to Example 2, the concentration for the single-concentration screening experiment was set to 0.3 nM.

[0648] 3. Experimental Results

[0649] Experimental results show that the unmodified base sequences B207S, A1550, B1572, B1575, A1573, B418S, and A416 disclosed in this paper have significantly better inhibition rates against HBsAg and HBeAg than those of sequence-similar sequences P206, P413, P1551, and APC. After template modification, the inhibition rate is further significantly improved.

[0650] Tables 44 and 45 show a comparison of the inhibition rates of HBsAg and HBeAg for sequences that are similar to those disclosed in the prior art and those disclosed herein.

[0651] As shown in Table 43, compared with the prior art disclosed sequence P206, the disclosed sequence B207S is shifted backward by one base and shortened by one base. Compared with P206, the inhibition rates of B207S against HBsAg and HBeAg are increased by 15.57% and 11.69%, respectively. After modification with the template disclosed in this paper, the inhibition efficiency of B207S is further improved. For example, after modification with DV32P, the inhibition rates of B207S-DV32P against HBsAg and HBeAg are increased by 28.92% and 20.55%, respectively, which is significantly better than the prior art disclosed modified templates DV30P, DV31P, DV21P, and DV22P.

[0652] Compared with the prior art disclosed sequence P413, the disclosed sequence A416 is shifted backward by 3 bases. Compared with P413, A416 increases the inhibition rate of HBsAg and HBeAg by 5.95% and 7.42%, respectively. After A416 is modified with the template disclosed in this invention, the inhibition efficiency is further improved. For example, after modification with DV27P, the inhibition rate of B416-DV27P against HBsAg and HBeAg is increased by 24.16% and 21.26%, respectively, which is significantly better than the prior art disclosed modified templates DV30P, DV31P, DV21P, and DV22P.

[0653] Compared with the prior art disclosed sequence P1551, the disclosed sequence A1550 is shifted forward by one base. Compared with P1551, the inhibition rates of A1550 against HBsAg and HBeAg are increased by 7.98% and 9.03%, respectively. After modification with the template disclosed in this invention, the inhibition efficiency of A1550 is further improved. For example, after modification with DV27P, the inhibition rates of B1550-DV27P against HBsAg and HBeAg are increased by 23.96% and 26.06%, respectively, which is significantly better than the prior art disclosed modified templates DV30P, DV31P, DV21P, and DV22P.

[0654] Compared with the existing APC sequence, the sequences B1575, B1573, and B1572 in this disclosure are shifted forward by 4, 6, and 7 bases, respectively, and their lengths are all extended by 2 bases. Compared with APC, the inhibition rates of HBsAg by B1575, B1573, and B1572 are increased by 13.46%, 10.80%, and 9.94%, respectively. After modification with the template of this disclosure, the inhibition efficiency is further improved. For example, the B1575-DV29P sequence shows the most significant improvement, increasing the inhibition rate by 29.28%, while also increasing the inhibition rate of HBeAg by 24.25%. The B1575-DV26P sequence increases the inhibition rate of HBsAg by 27.18% and the inhibition rate of HBeAg by 16.55%, and is significantly superior to the existing modified templates DV30P, DV31P, DV21P, and DV22P.

[0655] Table 44 Comparison of HBsAg inhibition rates between prior art and the sequences disclosed herein

[0656]

[0657]

[0658] Table 45 Comparison of HBeAg inhibition rates between prior art and the present disclosure sequences.

[0659]

[0660]

[0661] Example 6: Inhibitory effect of a sequence designed with specific anti-off-target effects on HBV and off-target genes.

[0662] In practical applications of siRNA, there are numerous cases where the expression of non-target mRNAs with only partial complementarity to the guide strand (antisense strand) is suppressed. Research by Alniram indicates that the hepatotoxicity of n-acetylgalactosamine (GalNAc)-conjugated siRNA is primarily attributed to off-target effects caused by gene suppression of incorrect targets through a microRNA-like recognition mechanism.

[0663] To address this issue, thermally unstable nucleotide modifications, such as glycol-treated nucleic acids (GNAs), unlocking nucleotides (UNAs), or DNA, can be made at sites 6 and 7 of the antisense strand seed region. These modifications disrupt the seed region, affecting the binding of siRNA to non-target mRNAs via seed region recognition, thereby significantly reducing off-target effects and mitigating hepatotoxicity. Alniram, in its latest fifth-generation template design, employs a GNA modification at site 7 of the siRNA antisense strand to reduce off-target effects.

[0664] In this embodiment, the basic sequences B207S, B1575, A1550, B1572, A1573, A261, A416, and B418S were used to study off-target effects and prevention of off-target effects. In order to evaluate the off-target effects of each sequence, this embodiment used qPCR to compare the inhibition efficiency of each sequence on the target gene and off-target genes in cell experiments.

[0665] To reduce off-target effects of the sequences, this embodiment modifies the sequences B418S-DV34P and B416-DV27P, which have potential off-target effects, at positions 6 and 7 of the antisense strand with DNA or GNA to prevent off-target effects, and compares their activity and off-target effects with those of sequences without such modifications.

[0666] 1. Experimental Materials

[0667] Test samples: Template-modified sequences listed in Table 46 and corresponding sequences designed to prevent off-target interference (synthesis method as described in Example 1). In the sequence number, d67B indicates that the sequence is modified with d67B to prevent off-target interference, d7B indicates that the sequence is modified with d7B to prevent off-target interference, and + indicates that the sequence is modified with GNA to prevent off-target interference.

[0668] Cell type: HepG2.2.15 cells

[0669] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM.

[0670] Table 46 Template modification and anti-miss design sequence

[0671]

[0672] 2. Experimental Methods

[0673] The qRT-PCR method was used to detect the inhibitory effect of the samples on HBV and off-target genes in HepG2.2.15 cells.

[0674] 2.1 Cell Culture

[0675] Subculture: HepG2.2.15 cells were subcultured in DMEM / F12 medium containing 10% fetal bovine serum, 370 μg / ml GENETICN, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin. The cells were incubated at 37°C in a cell culture incubator containing 5% CO2, and subcultured every three days. After digestion with 0.25% trypsin, the cells were centrifuged at 800 r / min for 3 min, the supernatant was discarded, and fresh medium was added for further subculturing.

[0676] Plate 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.

[0677] 2.2 Cell transfection

[0678] Transfection reagent preparation: Mix Lipofectamine RNAiMAX and Opti-MEM at a ratio of 2:98 and vortex thoroughly.

[0679] Preparation of transfection complex: Take 30 μL of Opti-MEM diluted siRNA solution at a ratio of 1:1 (v / v) and add it to 30 μL of transfection mixture. Vortex mix and let stand at room temperature for 15 min to obtain transfection complex.

[0680] Transfection control group transfection reagent preparation: Add 15 μL of the prepared transfection mixture to 15 μL of Opti-MEM. Vortex mix and let stand at room temperature for 15 min.

[0681] Add the prepared transfection complex to a 96-well cell culture plate (15 μL per well, 3 replicates per sequence) to achieve final siRNA concentrations of 0.065, 0.14, and 0.28 nM per well, with 3 concentrations for each siRNA and 3 replicates for each concentration. Add 135 μL of cell suspension (containing 2.25 × 10⁻⁶ cells / well). 4 (1 cell). After mixing using the cross-hatching method, incubate in a 37°C, 5% CO2 cell culture incubator.

[0682] 2.3 RNA extraction and reverse transcription

[0683] Forty-eight hours after transfection, the culture medium was removed and cells were collected for RNA extraction. Total RNA was extracted using the RNeasy® 96 Kit (QIAGEN-74182) according to the kit instructions. Subsequently, the RNA was reverse transcribed into cDNA using HiScript Ⅲ RT SuperMix for qPCR (Vazyme) according to the kit instructions.

[0684] 2.4 RT-qPCR

[0685] HBV and off-target gene cDNA were detected by qPCR, with GAPDH cDNA used as an internal control for parallel detection. 8 μL of prepared qPCR reaction solution and 2 μL of sample cDNA were added to each of 384 wells. The SYBR qPCR program was as follows: 50 °C for 2 min, 95 °C for 2 min, then cycling at 95 °C for 5 s, followed by 60 °C for 30 s, for a total of 40 cycles. The final melting curve was prepared by heating at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s.

[0686] 2.5 Data Analysis

[0687] The expression level of the target gene RNA in each sample was calculated based on the CT value, using the ΔΔCT relative quantification method. The relative expression level of the target gene was calculated using a 22T / T ratio. -ΔΔCT express.

[0688] The calculation formula is as follows:

[0689] ΔCT = Average CT value of the target gene - Average CT value of GAPDH;

[0690] ΔΔCT = ΔCT (experimental group) - ΔCT (transfection control group);

[0691] Relative expression level of target gene mRNA = 2 -ΔΔCT

[0692] Gene expression inhibition rate (%) = (1 – mRNA expression level of sample / mRNA expression level of transfected control group) × 100%

[0693] 3. Experimental Results

[0694] Experimental results showed that sequences B207S, B1575, A1550, B1572, A1573, and A261 had no off-target effects. Sequences B418S-DV34P and A416-DV27P, after adopting an off-target prevention design, significantly reduced the inhibitory effect on off-target genes without affecting the inhibitory effect on the target gene HBV. This indicates that adopting an off-target prevention design does not affect the inhibitory effect of the template modification sequences disclosed in this paper on the HBV gene, but can significantly inhibit off-target effects.

[0695] (i) qPCR analysis of the potential off-target effects and off-target prevention effects of each sequence

[0696] (1) The inhibition efficiency of each sequence on the target gene and off-target genes is shown in Table 47. The results show that the inhibition of gene SLCO2B1 by sequence A416 is significantly concentration-dependent, with a maximum inhibition rate of 40.05%. The inhibition of gene SLC41A2 by sequence B418S is somewhat concentration-dependent, with a maximum inhibition rate of 24.13%. The inhibition rates of off-target genes by the remaining sequences B207S, A1550, and A416 are all below 20%, indicating no off-target effect. The A416 and BS18S sequences also have a relatively large number of potential off-target genes, therefore sequences A416 and BS18S have potential off-target effects.

[0697] Table 47. Repressive effects of each sequence on off-target and target genes.

[0698]

[0699] (2) Off-target prevention d7B and d67B were designed for sequences A416 and BS18S. The inhibitory effects of the anti-off-target design sequence and the undesigned sequence on off-target genes and target genes are shown in Table 48. The results showed that after anti-off-target modification d7B, the IC50 of sequence C416-DV27Pd7B on off-target gene SLCO2B1 was increased by 5.69 times and the maximum inhibition rate was reduced by 9.54%. The maximum inhibition rate of sequence C418S-DV34Pd7B on off-target gene SLC41A2 was reduced by 16.91%, which was significantly better than anti-off-target modification d67B and GNA modification. Meanwhile, the IC50 of the C418S-DV34Pd7B sequence against the target gene HBV is 0.22 nM, which is quite similar to that of the unmodified B418S-DV34P sequence. The maximum inhibition rate of the target gene is about 81% for both sequences, indicating that the d7B modification does not affect the inhibitory effect of the C418S-DV34Pd7B sequence on the target gene.

[0700] Table 48. Inhibitory effects of off-target modified sequences on off-target and target genes.

[0701]

[0702] Example 7: Inhibitory effect of the sequence modified with the template of this disclosure (high dose) on HBV in mice.

[0703] This embodiment exemplarily selects several sequences, including the basic sequences B418S, A416, B1550, B207S, B1575, B1572, and A1573. These sequences are modified, for example, by template modification only, or by simultaneous template modification and off-target design, and all are conjugated to GalNAc ligands. The in vivo efficacy of these RNAi agents after repeated administration at high doses, such as 3 mg / kg, is analyzed using an AAV-HBV mouse model. This mouse model, after infection with recombinant adeno-associated virus (AAV) carrying a replicating HBV genome, can continuously produce HBV viral particles and HBV antigens without seroconversion for more than one year, reproducing some immunological characteristics of clinical chronic hepatitis B patients. Therefore, this model is also used to evaluate novel immuno-based therapies and antiviral treatments. In this embodiment, using the AAV-HBV mouse model, the inhibitory effects of the above sequences on serum HBsAg, HBeAg, and HBV DNA at different time points, and the effects of each sequence on the reconstitution of adaptive immune function in mice at specific time points are examined.

[0704] 1. Experimental Materials

[0705] Test drug:

[0706] The sequences in Table 49, including those using only template modification, those using template modification and those designed to prevent off-target effects, are coupled to the GalNAc ligand G5 at the 3' end of the positive strand, with the following structural formulas:

[0707]

[0708] The method for conjugating oligonucleotides with ligand G5 is described in Example 3 of patent application CN116854754A.

[0709] The oligonucleotide forms a conjugate with ligand G5 as shown below:

[0710]

[0711] The specific sequences are shown in Table 49. In the sequence number, G5 indicates that the sequence is coupled with GalNAc ligand G5, and GL indicates that the sequence is coupled with GalNAc ligand L96.

[0712] The structural formula of L96 is as follows:

[0713]

[0714] Table 49 siRNA sequences in animal experiments

[0715]

[0716]

[0717] Preparation of the test drug:

[0718] Drug solvent: PBS buffer

[0719] Preparation conditions: Sterile environment

[0720] Labeling method: The prepared drug formulations are labeled with tags, and the outer packaging indicates the project number, name, concentration, quantity, preparation date, preparer, and storage conditions;

[0721] Storage conditions: Prepared for immediate use, and the remaining samples are stored at -80°C.

[0722] Experimental animal information:

[0723] Species / strain: AAV-HBV mice

[0724] Grade: SPF

[0725] Gender: Male

[0726] Quantity:​​​​​​​​​​​​​​​​​​​​​​​​​​The standard of feeding environmental conditions refers to the national standard of the People's Republic of China GB14925-2010.

[0736] Animals are fed freely and provided with drinking water freely. The feed is irradiated and sterilized maintenance feed for experimental mice, provided by Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., with the production license number of Su Feed License (2019) 01008. The detection of feed nutritional components refers to the national standard of the People's Republic of China GB14924.3-2010, and the detection of pollutant content refers to the national standard of the People's Republic of China GB14924.2-2001. The feed supplier provides the test report for each batch. The drinking water is reverse osmosis water, contained in drinking water bottles. The detection of drinking water refers to the national standard of the People's Republic of China GB5750-2006, and it is sent to a third-party testing agency for testing once a year.

[0737] The animal bedding is corncob bedding, provided by Guangzhou Saibenuo Biotechnology Co., Ltd., with the production license number of animal bedding SCXK (Beijing) 2019-0004. The detection of pollutant content in bedding refers to the national standard of the People's Republic of China GB14924.2-2001, and the bedding supplier provides the test report for each batch.

[0738] The animal feeding cages and bedding are replaced at least once a week. All animal feeding cages and bedding enter the barrier environment for use after being sterilized by high pressure in a pulsating vacuum sterilizer; the animal feeding cage racks are cleaned and disinfected by wiping at least once a week.

[0739] The animal feeding and observation room is cleaned and disinfected every day, including the plate rack, floor, desktop, etc.

[0740] The disinfectants used in the barrier environment include: 6.67% bromogeramine solution, 0.5% 84 disinfectant, 75% disinfectant, 0.08% baidusha. The four disinfectants are used in rotation and cannot be mixed.

[0741] 2. Experimental methods

[0742] 2.1 AAV-HBV mouse modeling

[0743] Definition of test date: The day when animals are administered the vehicle or the test drug is defined as day 0.

[0744] 100 SPF-grade C57BL / 6 male mice are adaptively fed in the barrier facility for 7 days, and observed daily. After confirming that the mice are healthy and normal, modeling is carried out. The mice are injected with rAAV8-1.3HBV (Guangzhou Pizhen Biotechnology Co., Ltd., name: AAV8[HBV-D,ayw](D#2012), batch number: HBV101-6) via the tail vein, and each mouse is injected with 1×10 11GC / 100μL. Blood was collected from the animals at 5 and 6 weeks after modeling (D-14, D-7), centrifuged, and plasma was collected to detect HBV DNA, HBsAg, and HBeAg levels.

[0745] 2.2 Animal grouping and administration

[0746] Trial date definition: The day on which the animal is given the solvent or test drug is defined as day 0 (D 0).

[0747] Animals were grouped according to indicators at week 6 of modeling (D-7). Seventy-two animals with successful modeling were randomly divided into 12 groups of 6 animals each, based on HBsAg indicators. The average HBsAg levels were ensured to be the same across groups, and there were no statistically significant differences in HBV DNA and HBeAg indicators among groups. Drug administration was initiated at week 7 of modeling (D0). Details of grouping and drug administration are shown in Table 50.

[0748] Table 50 Group Setup and Dosing Details

[0749]

[0750] Note: sc: subcutaneous injection.

[0751] 2.3 Secondary challenge experiment

[0752] At drug administration D 60, three mice in the C207S-DV32PG5 group were challenged by hydrodynamic injection of 8 μg pAAV-HBV1.2 plasmid (Fenghui Biotechnology) via the tail vein at an injection volume of 100 μL / g. The remaining three mice were left untreated. Three mice in the blank control group were also challenged in the same way.

[0753] 2.4 Observation and Indicator Monitoring

[0754] (1) General observation

[0755] During the modeling and experimental periods, the animals were observed and recorded daily. The observations included: whether they were dead or near death, their feed and water intake, external injuries, feces, appearance and coat, mental state, and activity level.

[0756] (2) Weight

[0757] Adaptation period: Weigh and record the animal upon receipt, and weigh and record the animal on the day the adaptation period ends.

[0758] Experimental period: Animals were weighed and recorded weekly during the experimental period. If medication or blood was to be taken on the same day, the animals were weighed before the procedure and before euthanasia.

[0759] (3) Measurement of serum HBsAg, HBeAg, HBV DNA, HBsAb, and ALT levels

[0760] Animals underwent blood collection at the inner canthus of the orbit 5 weeks (D-14), 6 weeks (D-7), before drug administration (D0), and weekly after drug administration. 200 μL of blood was collected and anticoagulated in EDTA-k2 anticoagulant tubes. Plasma was collected after centrifugation at 1000 g for 10 min. Mice challenged with the virus were challenged on D60, and blood was collected at D63, D67, D74, and D81. 200 μL of blood was collected and anticoagulated in EDTA-k2 anticoagulant tubes. Plasma was collected after centrifugation at 1000 g for 10 min. 20 μL of plasma was added to 980 μL of PBS, vortexed, and used to detect HBsAg, HBV DNA, HBeAg, and ALT levels. Another 15 μL of plasma was added to 210 μL of PBS, vortexed, and used to detect HBsAb levels. All processed samples were tested by Guangzhou Huayin Medical Testing Center Co., Ltd., and any remaining plasma was stored at -80℃.

[0761] 3. Experimental Results

[0762] Experimental results showed that, compared with the solvent control group and the negative control group, sequences B207S-DV32P, A1550-DV27P, B1572-DV27P, B1575-DV27P, B207S-DV29P, A1573-DV29P, B1575-DV29P, B418S-DV34Pd7B, and A416-DV27Pd7B, after being conjugated to conjugate G5, could be targeted and delivered to the liver, and significantly and persistently inhibited the levels of HBsAg, HBeAg, and HBV DNA in plasma.

[0763] Among them, sequences C207S-DV32PG5, C207S-DV29PG5, and C418S-DV34Pd7BG5 showed superior effects in reducing HBsAg, HBV DNA, and HBeAg, significantly better than the positive control sequence VIR-2218-GL. In addition, sequences C207S-DV32PG5 and C207S-DV29PG5 ultimately achieved HBsAg and HBV DNA seroconversion in all mice in the group and produced high levels of antibodies. In the group with sequence C418S-DV34Pd7BG5, 3 / 6 mice ultimately achieved HBsAg seroconversion and also produced high levels of antibodies.

[0764] The results of the secondary challenge experiment showed that the control group (mice that had not been infected with HBV before D56) developed HBsAg, HBeAg, and HBV DNA after challenge, indicating that the challenge method was effective. In the C207S-DV32PG5 challenge group, HBV DNA initially increased and then decreased to the detection limit after challenge, while serum HBsA antibody showed a significant increase, and HBsAg remained below the detection limit, proving the effectiveness of the challenge. Furthermore, hepatitis B mice treated with this small nucleic acid sequence were immune to reinfection with HBV. This demonstrates that hepatitis B mice treated with this small nucleic acid sequence are immune to reinfection with HBV.

[0765] (i) Changes in HBsAg, HBeAg, HBV DNA, HBsAb, and ALT levels in mice of each sequence group over time.

[0766] The changes in HBsAg, HBeAg, HBV DNA, HBsAb, ALT, and body weight levels in each group of mice over time are shown in Tables 51-59. Figures 1A-1F As shown in the table, sequences C207S-DV32PG5, C207S-DV29PG5, and C418S-DV34Pd7BG5 exhibited superior effects in reducing HBsAg, HBV DNA, and HBeAg, significantly outperforming the positive control sequence VIR-2218-GL. Furthermore, sequences C207S-DV32PG5 and C207S-DV29PG5 ultimately achieved HBsAg and HBV DNA seroconversion in all mice within their respective groups, and both produced high levels of antibodies. Specifically, in the C207S-DV32PG5 group, HBsAg and HBV DNA seroconversion was achieved in all mice on days 14 and 21 after administration, respectively, and this seroconversion continued until the experimental endpoint, indicating that the mice achieved functional cure. In the C418S-DV34Pd7BG5 group, 3 / 6 of the mice ultimately achieved HBsAg seroconversion and also produced high levels of antibodies. The above data demonstrate the superior efficacy of the C207S-DV32PG5, C207S-DV29PG5, and C418S-DV34Pd7BG5 sequences.

[0767] Table 51 Changes in plasma HBsAg levels in mice of different sequence groups

[0768]

[0769]

[0770] Note: "-" indicates that the test was stopped; " / " indicates that the C207S-DV32PG5 group was selected as the challenge test animals at D56 of drug administration. Compared with the PBS group, * indicates P < 0.05; ** indicates P < 0.01. Compared with the positive control group VIR-2218-GL group, # This indicates that P < 0.05; ## This indicates that P < 0.01.

[0771] Table 52 Changes in plasma HBV DNA levels in mice of different sequence groups

[0772]

[0773] Note: "-" indicates that the test was stopped; " / " indicates that the C207S-DV32PG5 group was selected as the challenge test animals at D56 of drug administration. Compared with the PBS group, * indicates P < 0.05; ** indicates P < 0.01. Compared with the positive control group VIR-2218-GL group, # This indicates that P < 0.05; ## This indicates that P < 0.01.

[0774] Table 53 Changes in plasma HBeAg levels in mice of different sequence groups

[0775]

[0776] Note: "-" indicates that the test was stopped; " / " indicates that the C207S-DV32PG5 group was selected as the challenge test animals at D56 of drug administration. Compared with the PBS group, * indicates P < 0.05; ** indicates P < 0.01. Compared with the positive control group VIR-2218-GL group, # This indicates that P < 0.05; ## This indicates that P < 0.01.

[0777] Table 54 Changes in the number of mice that became HBsAg negative in each sequence group

[0778]

[0779] Table 55 Changes in the number of mice that became HBV DNA negative in each sequence group

[0780]

[0781] Table 56 Changes in the number of mice producing antibodies in each sequence group

[0782]

[0783] Table 57 Changes in plasma HBsAb levels in mice of different sequence groups

[0784]

[0785] Table 58 Changes in plasma ALT levels in mice of different sequence groups

[0786]

[0787] Table 59 Changes in body weight of mice in each sequence group

[0788]

[0789] (ii) Protective effect of sequence CS7S-DV32PG5 against secondary challenge in mice

[0790] Mice in the CS7S-DV32PG5 group underwent high-pressure hydrodynamic injection of HBV plasmid via the tail vein on day 60 to simulate HBV secondary infection. Experimental results are shown below. Figures 2A-2F The results showed that the control group (mice that had not been infected with HBV before D56) developed HBsAg, HBeAg, and HBV DNA after high-pressure hydrodynamic injection of the HBV plasmid, indicating that the challenge method was effective. In the C207S-DV32PG5 challenge group, HBV DNA initially increased and then decreased to the detection limit after challenge, while serum HBsA antibody showed a significant increase, and HBsAg remained below the detection limit. These results demonstrate the effectiveness of the challenge and that hepatitis B mice treated with this small nucleic acid sequence were immune to reinfection with HBV.

[0791] Example 8: Inhibitory effect of the sequence (low dose) modified with the template of this disclosure on HBV in mice.

[0792] This embodiment exemplarily selects some sequences, such as sequences C207S-DV32PG5, C207S-DV29PG5, C418S-DV34Pd7BG5 and C1575-DV27PG5 in Example 7, as shown in Table 49. The in vivo efficacy of these RNAi agents after multiple administrations at low doses, such as 0.5 mg / kg, was analyzed using an AAV-HBV mouse model.

[0793] 1. Experimental Materials

[0794] The sequences C207S-DV32PG5, C207S-DV29PG5, C418S-DV34Pd7BG5, C1575-DV27PG5, and VIR-2218-GL in Example 7 are shown in Table 49. Also included is entecavir (ETV).

[0795] 2. Experimental Methods

[0796] Same as in Example 7, grouping and administration details are shown in Table 60.

[0797] Table 60 Group Setup and Dosing Details

[0798]

[0799] Note: sc: subcutaneous injection; ig: gavage.

[0800] 3. Experimental Results

[0801] The changes in HBsAg, HBeAg, HBV DNA, HBsAb, and ALT levels in each group of mice over time are shown in Tables 61-68. Figures 3A-3F Experimental results showed that sequences C207S-DV32PG5 and C207S-DV29PG5 were significantly more effective than the positive control sequence VIR-2218-GL in inhibiting HBsAg, HBV DNA, and HBeAg. C207S-DV32PG5 exhibited the best effect, reducing HBsAg by approximately 2.9 log10 on day 56, which was about 1.4 log10 higher than C207S-DV29PG5. Furthermore, on day 56, 3 mice achieved HBsAg seroconversion, and 4 mice developed antibodies. No mice in the C207S-DV29PG5 group showed seroconversion. These data further demonstrate the superior efficacy of sequence C207S-DV32PG5.

[0802] Table 61 Changes in plasma HBsAg levels in mice of different sequence groups

[0803]

[0804] Note: Compared with the PBS group, * indicates P < 0.05; ** indicates P < 0.01.

[0805] Table 62 Changes in plasma HBV DNA levels in mice of different sequence groups

[0806]

[0807] Note: Compared with the PBS group, * indicates P < 0.05; ** indicates P < 0.01.

[0808] Table 63 Changes in plasma HBeAg levels in mice of different sequence groups

[0809]

[0810] Note: Compared with the PBS group, * indicates P < 0.05; ** indicates P < 0.01.

[0811] Table 64 Number of mice with HBsAg seroconversion in each sequence group

[0812]

[0813] Table 65 Number of mice with negative HBV DNA in plasma of each sequence group

[0814]

[0815] Table 66 Number of mice producing HBsAb in each sequence group

[0816]

[0817] Table 67 Changes in plasma HBsAb levels in mice of different sequence groups

[0818]

[0819] Table 68 Changes in plasma ALT levels in mice of different sequence groups

[0820]

[0821] Example 9: Inhibition of HBV in mice using the ligand-modified sequence delivered according to this disclosure.

[0822] This embodiment exemplarily selects some sequences, including the basic sequences B418S and B207S, and modifies these sequences, for example, by using only template modification, or by using both template modification and off-target protection design, and conjugates different ligands for delivery. The in vivo efficacy of these RNAi agents is analyzed using an AAV-HBV mouse model.

[0823] 1. Experimental Materials

[0824] Test drug:

[0825] The sequences in Table 69, including those using only template modification, those using template modification and those designed to prevent off-target delivery, deliver ligands G5, G101, or G103 via a 3' end coupling to the positive strand of the sequence. Their structural formulas are as follows:

[0826] ,

[0827] , .

[0828] The method for conjugating oligonucleotides with ligands G5, G101, or G103 is described in Example 3 of patent application CN116854754A.

[0829] Oligonucleotides form conjugates with ligands G5, G101, or G103 as shown below:

[0830] ,

[0831] ,

[0832] 。

[0833] For the specific sequences of each sequence, see Table 69. In the sequence numbers, G5 indicates that the sequence is coupled with the delivery ligand G5, G101 indicates that the sequence is coupled with the delivery ligand G101, G103 indicates that the sequence is coupled with the delivery ligand G103, and GL indicates that the sequence is coupled with the delivery ligand L96. The structural formula of L96 is as follows:

[0834]

[0835] Table 69 siRNA sequences in animal experiments

[0836]

[0837]

[0838] Preparation of the test drug:

[0839] Same as Example 7.

[0840] Experimental animal information:

[0841] Species / strain: AAV-HBV mice

[0842] Grade: SPF

[0843] Sex: Male

[0844] Number: 82

[0845] Age: 5 weeks

[0846] Body weight: 19 - 24 g

[0847] Source: Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0848] Production license number: SCXK(Beijing)2021-0006

[0849] Experimental animal ethical review (IACUC):

[0850] After receiving the experimental animals, they were housed in Beijing Vitalstar Biotechnology Co., Ltd., with the use license number: SCXK(Beijing)2022-0013. This project has passed the review of the Experimental Animal Ethics Committee of Beijing Vitalstar Biotechnology Co., Ltd., with the IACUC number VST-SY-24062701. The test process was carried out strictly in accordance with the requirements of IACUC to ensure animal welfare.

[0851] Feeding and management:

[0852] The animals were housed in a negative pressure barrier environment, using plastic (polycarbonate) boxes (370×157×180mm) for IVC (in vitro cultivation). Because the experimental animals were male and aggressive, they were housed individually. Animal husbandry was handled by Beijing Vitonda Biotechnology Co., Ltd.

[0853] The use and testing of animal feed, bedding, and drinking water were all carried out in accordance with GB14925-2010 "Laboratory Animal Environment and Facilities". The control of the animal housing environment, including temperature, humidity, pressure difference, noise, illuminance, air exchange rate, and ammonia concentration, was temporarily carried out according to GB50447-2008 "Technical Specifications for the Construction of Laboratory Animal Facilities". The animal room temperature was controlled at 20-26℃ (daily temperature difference ≤4℃); relative humidity was controlled at 40-70%; artificial lighting was used, with a 12 / 12-hour day / night alternation. Compressed wood shavings bedding was purchased from Beijing Keao Xieli Feed Co., Ltd. (batch number: 23109613), and rat and mouse growth and reproduction feed was purchased from Beijing Keao Xieli Feed Co., Ltd. (batch number: 23103313). All records related to animal husbandry management and environmental control during the experiment were kept at Beijing Weitongda Biotechnology Co., Ltd.

[0854] 2. Experimental Methods

[0855] 2.1 AAV-HBV mouse model

[0856] Trial date definition: The day on which the animal is given the solvent or test drug is defined as day 0.

[0857] One hundred SPF-grade male C57BL / 6 mice were acclimatized in a barrier facility for 7 days and observed daily to ensure they were healthy and free of abnormalities before modeling. A mouse model of persistent HBV infection was established by intravenous injection of rAAV8-1.3HBV via the tail vein. The AAV virus injection dose was 1.00 × 10⁻⁶. 10 vg / animal, AAV virus diluted with sterile PBS to 5.00×10 10 The dose was vg / mL, and each mouse was injected with 200 μL. Four weeks after the injection, the serum levels of HBV DNA, HBeAg, and HBsAg were measured.

[0858] 2.2 Animal grouping and administration

[0859] Trial date definition: The day on which the animal is given the solvent or test drug is defined as day 0 (D 0).

[0860] On day -2, plasma was collected from all mice via submandibular blood collection. The collected blood samples were anticoagulated with EDTA, centrifuged at 5000 rpm for 10 min, and the supernatant was used for HBV modeling detection. On day 0, based on the test results from day -2, 72 animals were selected and divided into 12 groups of 6 animals each. Details of grouping and drug administration are shown in Table 70.

[0861] Table 70 Group Setup and Dosing Details

[0862]

[0863] Note: sc: subcutaneous injection.

[0864] 2.4 Observation and Indicator Monitoring

[0865] (1) General observation

[0866] During the modeling and experimental periods, the animals were observed and recorded daily. The observations included: whether they were dead or near death, their feed and water intake, external injuries, feces, appearance and coat, mental state, and activity level.

[0867] (2) Weight

[0868] Adaptation period: Weigh and record the animal upon receipt, and weigh and record the animal on the day the adaptation period ends.

[0869] Experimental period: Animals were weighed and recorded weekly during the experimental period. If medication or blood was to be taken on the same day, the animals were weighed before the procedure and before euthanasia.

[0870] (3) Measurement of serum HBsAg, HBeAg, HBV DNA and ALT levels

[0871] Plasma was collected from all animals via submandibular blood collection on days -2, 6, 13, 20, 27, and 34. Collected blood samples were anticoagulated with EDTA and centrifuged at 5000 rpm for 10 min to separate serum. Serum was then separated from blood samples, diluted with PBS, and sent for testing. For each sample, 10 μL of serum was taken and diluted to 500 μL (50-fold dilution) with PBS and sent to Beijing Dian Medical Laboratory Co., Ltd. for testing of serum HBV DNA, HBeAg, and HBsAg. For each sample, 30 μL of serum was taken and diluted to 120 μL (4-fold dilution) with PBS and sent to Beijing Dian Medical Laboratory Co., Ltd. for serum ALT testing. Remaining plasma was stored at -80℃.

[0872] 3. Experimental Results

[0873] The changes in HBsAg, HBeAg, HBV DNA, ALT levels, and body weight of mice in each group over time are shown in Tables 71-75. Figures 4A-4E The experimental results showed that all sequences were more effective than the Yangshen sequence VIR-2218-GL. Among them, C207S-DV29PG101, C207S-DV29PG103, C207S-DV32PG101, and C207S-DV32PG103 showed superior effects in reducing HBsAg, HBV DNA, and HBeAg, which were significantly better than the Yangshen sequence VIR-2218-GL. On day 28 after administration, they could still reduce HBsAg by more than 3 log10 and HBV DNA by more than 3 log10, and there was no rebound, demonstrating excellent anti-HBV efficacy.

[0874] Table 71 Changes in plasma HBsAg levels in mice of different sequence groups

[0875]

[0876] Table 72 Changes in plasma HBV DNA levels in mice of different sequence groups

[0877]

[0878] Table 73 Changes in plasma HBeAg levels in mice of different sequence groups

[0879]

[0880] Table 74 Changes in plasma ALT levels in mice of different sequence groups

[0881]

[0882] Table 75 Changes in body weight of mice in each sequence group

[0883]

Claims

1. A double stranded RNAi agent, characterized in that, The double stranded RNAi agent comprises an antisense strand and a sense strand complementary to the antisense strand forming a double stranded region, the nucleotide sequence of the antisense strand is set forth in SEQ ID NO: 13, or the nucleotide sequence of the antisense strand is a modified sequence of the sequence set forth in SEQ ID NO: 13; the nucleotide sequence of the sense strand is set forth in SEQ ID NO: 6, or the nucleotide sequence of the sense strand is a modified sequence of the sequence set forth in SEQ ID NO:

6.

2. The double stranded RNAi agent of claim 1, wherein, The sense strand and the antisense strand comprise at least one modified nucleotide.

3. The double stranded RNAi agent of claim 1, wherein The double stranded RNAi agent has a function of inhibiting the expression of HBV gene.

4. The double stranded RNAi agent of claim 2, wherein, The at least one modified nucleotide is selected from one or more of the group consisting of: a deoxy-nucleotide, a conformationally restricted nucleotide, an abasic nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a nucleotide comprising a phosphorothioate group, and a nucleotide comprising a methylphosphonate group.

5. The double stranded RNAi agent of claim 2, wherein The at least one modified nucleotide is selected from one or more of the group consisting of: 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 constrained ethyl nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-O-methoxyethyl-modified nucleotide, a morpholino nucleotide, a tetrahydropyranyl-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide comprising a 5'-phosphate, and a nucleotide comprising a 5'-phosphate mimic.

6. The double stranded RNAi agent of claim 1, wherein, The antisense strand of the double stranded RNAi agent has a 2-nucleotide 3' overhang.

7. The double stranded RNAi agent of claim 1, wherein The double stranded region of the double stranded RNAi agent is 20 pairs of nucleotides.

8. The double stranded RNAi agent of claim 1, wherein, The sense strand of the double stranded RNAi agent is 20 nucleotides in length and the antisense strand is 22 nucleotides in length.

9. The double stranded RNAi agent of claim 1, wherein, All modifications of the nucleotides on the sense strand and the antisense strand are chemical modifications of the 2' position of the ribose sugar of the nucleotides.

10. The double stranded RNAi agent of claim 9, wherein, The chemical modification of the 2' position of the ribose sugar of the nucleotides is selected from one or more of the group consisting of: 2'-methoxy, 2'-O-methoxyethyl, 2'-fluoro, 2'-benzyloxy, 2'-methylcarbonylamino, and 2'-pyridyloxy.

11. The double stranded RNAi agent of claim 10, wherein, The chemical modification of the 2' position of the ribose sugar of the nucleotides is 2'-methoxy or 2'-fluoro.

12. The double stranded RNAi agent of claim 1, wherein, The nucleotides are linked by 3',5'-phosphodiester bonds.

13. The double stranded RNAi agent of claim 12, wherein, The 3',5'-phosphodiester bonds comprise a phosphorothioate modification.

14. The double stranded RNAi agent of claim 1, wherein, The 5' position of the 5' terminal nucleotide sugar of the antisense strand is phosphorylated.

15. The double stranded RNAi agent of claim 14, wherein The phosphorylated 5' position comprises one or more of the group consisting of: a 5'-vinylphosphonate group, a 5'-methylphosphonate group, a 5'-C-methylphosphonate group, a 5'-phosphorothioate group, and a 5'-phosphate group, and has the structure: ； R is hydrogen, hydroxyl, amine, C 1-4 alkyl, aryl, C 1-4 alkoxy, C 1-4 alkylcarbonylamino, or halogen; The base is selected from any one of the group consisting of: adenine, guanine, cytosine, thymine, and uracil.

16. The double stranded RNAi agent of claim 1, wherein, The terminal nucleotides of the sequence are linked by 3', 5'-phosphodiester bonds containing a thio-modification and form chiral pure 3', 5'-thiophosphodiester bonds.

17. The double stranded RNAi agent of claim 16, wherein, The 5' terminal of the sense strand and the antisense strand contains 1-3 thio- linkages, and the 3' terminal of the antisense strand contains 1-3 thio-linkages.

18. The double stranded RNAi agent of claim 1, wherein, The antisense strand adopts one of the following modification patterns: ， ， The numbers 1-22 in the table represent the positions of the nucleotides on the antisense strand, respectively; The sense strand adopts one of the following modification patterns: ， The numbers 1-20 in the table represent the positions of the nucleotides on the sense strand, respectively; Wherein 2'-OMe is 2'-methoxy; 2'-F is 2'-fluoro; PS is phosphorothioate; EVP is 5'-vinyl-(E)-phosphonate; The modification pattern of the double-stranded RNAi agent is: The antisense strand adopts modification pattern A, and the sense strand adopts modification pattern a; The antisense strand adopts modification pattern A, and the sense strand adopts modification pattern b; The antisense strand adopts modification pattern B, and the sense strand adopts modification pattern a; The antisense strand adopts modification pattern B, and the sense strand adopts modification pattern b; The antisense strand adopts modification pattern C, and the sense strand adopts modification pattern b; or The antisense strand adopts modification pattern D, and the sense strand adopts modification pattern b.

19. The double stranded RNAi agent of claim 1, wherein, The second to eighth positions from the 5' end of the antisense strand adopt a modification group selected from one or more of UNA, GNA and DNA, and the structures of the UNA and GNA are: ； The base is selected from any one of adenine, guanine, cytosine, thymine and uracil.

20. The double stranded RNAi agent of any of claims 1-19, wherein, The double-stranded RNAi agent comprises any one of the following oligonucleotide duplexes selected from the pairing of the following sense strands and antisense strands: (1) the sequence of the sense strand is as shown in SEQ ID NO: 106; and the sequence of the antisense strand is as shown in SEQ ID NO: 213, 214, 215 or 216; (2) the sequence of the sense strand is as shown in SEQ ID NO: 107; and the sequence of the antisense strand is as shown in SEQ ID NO: 214 or 213; (3) the sequence of the sense strand is as shown in SEQ ID NO: 108; and the sequence of the antisense strand is as shown in SEQ ID NO: 217 or 218; (4) the sequence of the sense strand is as shown in SEQ ID NO: 109; and the sequence of the antisense strand is as shown in SEQ ID NO: 218; (5) the sequence of the sense strand is as shown in SEQ ID NO: 110; and the sequence of the antisense strand is as shown in SEQ ID NO: 218; and (6) the sequence of the sense strand is as shown in SEQ ID NO: 306; and the sequence of the antisense strand is as shown in SEQ ID NO:

321.

21. A conjugate, characterized in that, The conjugate comprises the double-stranded RNAi agent of any one of claims 1-20, and a ligand conjugated to the double-stranded RNAi agent.

22. The conjugate of claim 21, wherein, The ligand is conjugated to the 3'-terminal or 5'-terminal of the sense strand of the oligonucleotide.

23. The conjugate of claim 21, wherein, The ligand is one or more GalNAc attached using a bivalent or trivalent branched linker, or GalNAc attached using a monovalent linker.

24. The conjugate of claim 21, wherein, The ligand is: ， wherein X is hydrogen or a hydroxyl protecting group including acetyl, benzoyl, or isobutyryl; Y is an amine protecting group or H, the amine protecting group being formyl, acetyl, propionyl, n-butyryl, or isobutyryl; n is an integer between 0 and 20; q, r, and s are independently integers between 1 and 7.

25. The conjugate of claim 24, wherein, The ligand is: 。 26. The conjugate of claim 21, wherein, The ligand is: ， The ligand is: wherein X is oxygen, nitrogen, or sulfur; Y is alkyl or aryl; R2is hydrogen, amine, C 1-4 alkyl, aryl, C 1-4 alkoxy or halogen; A is -(CH2) a -, -(CH2CH2O) b -, -((CH2) c NHCO) d - or -((CH2) c CONH) d -, wherein 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 - wherein e is an integer from 0 to 7; R1 is oxygen or sulfur; X1is -(CH2) f - or -(CH2CH2O) f CH2- and f is an integer from 1 to 5; X2is -(CH2) g g is an integer from 1 to 6; L is -CONH- or -NHCO-; 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; 27. The conjugate of claim 26, wherein, n is an integer from 0 to 4. ， ， or 。 28. The conjugate of claim 27, wherein, The ligand is G4, G5, G6, or G7: ， ， ， or 。 29. The conjugate of claim 21, wherein, The conjugate has the structure shown below: , , , , or .

30. The conjugate of claim 29, wherein, wherein the ligand is G101, G102, G103, G105, or G106: ， ， ， or 。 31. The conjugate of claim 21, wherein, The conjugate has the structure shown below: The conjugate comprises any of the oligonucleotide duplexes selected from the group consisting of the following sense and antisense strand pairings:

32. The conjugate of any one of claims 21-31, wherein, the sequence of the sense strand is set forth in SEQ ID NO: 350, 351, or 352; and the sequence of the antisense strand is set forth in SEQ ID NO:

321.

33. A pharmaceutical composition comprising, The conjugate has a function of inhibiting the expression of HBV genes.

34. The pharmaceutical composition of claim 33, wherein The pharmaceutical composition comprises the double stranded RNAi agent of any one of claims 1-20 or the conjugate of any one of claims 21-32, and a pharmaceutically acceptable carrier.

35. The pharmaceutical composition of claim 34, wherein, The double stranded RNAi agent or the conjugate is administered in a non-buffered solution.

36. The pharmaceutical composition of claim 33, wherein The non-buffered solution is saline or water.

37. The pharmaceutical composition of claim 36, wherein, The double stranded RNAi agent or the conjugate is administered with a buffered solution.

38. The pharmaceutical composition of claim 37, wherein, The buffered solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

39. The pharmaceutical composition of claim 36, wherein, The buffered solution is a phosphate buffered saline.

40. The pharmaceutical composition of claim 39, wherein, The double stranded RNAi agent or the conjugate is formulated into a lipid formulation for delivery in a membrane molecular assembly.

41. The pharmaceutical composition of claim 40, wherein, The lipid formulation is a nucleic acid-lipid particle.

42. The pharmaceutical composition of claim 39, wherein The lipid formulation is a lipid nanoparticle.

43. The pharmaceutical composition of claim 39, wherein The mass / mass ratio of lipid to the double stranded RNAi agent or the conjugate is 1:1-50:

1.

44. The pharmaceutical composition of claim 39, wherein, The mass / mass ratio of lipid to the double stranded RNAi agent or the conjugate is 1:1-25:

1.

45. The pharmaceutical composition of claim 39, wherein the compound is of formula (I): ###00019### (I) or a pharmaceutically acceptable salt thereof. The mass / mass ratio of lipid to the double stranded RNAi agent or the conjugate is 3:1-15:

1.

46. The pharmaceutical composition of claim 39, wherein The mass / mass ratio of lipid to the double stranded RNAi agent or the conjugate is 4:1-10:

1.

47. The pharmaceutical composition of claim 39, wherein the compound is of formula (I): ###00019### (I) or a pharmaceutically acceptable salt thereof. The mass / mass ratio of lipid to the double stranded RNAi agent or the conjugate is 5:1-9:

1.

48. The pharmaceutical composition of claim 41, wherein, The mass / mass ratio of lipid to the double stranded RNAi agent or the conjugate is 6:1-9:

1. The lipid nanoparticle comprises a cationic lipid, a neutral lipid, a structural lipid, and a polymer-conjugated lipid.

49. The pharmaceutical composition of claim 48, wherein, The cationic lipid is a compound of structure (I), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, (I)， G1is C 1～6 alkylene; G2is C 2～8 alkylene; G3is C 1～3 alkylene; L1is C 6～15 straight-chain alkyl; L2is C 12～25 branched-chain alkyl.

50. The pharmaceutical composition of claim 49, wherein, The cationic lipid is YK-009 of structure (I-I): (I-I).

51. The pharmaceutical composition of claim 48, wherein, The cationic lipid is a compound of structure (II), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, (I), (II), G1is C 2~8 alkylene; G2is C 2~8 alkylene; L1is -C(O)O- or -OC(O)-; L2is -C(O)O- or -OC(O)-; R1is C 6~25 linear or branched alkyl; R2is C 6~25 linear or branched alkyl; G3is HO(CH2)2- or HO(CH2)3-; G4is HO(CH2)2- or HO(CH2)3-; L is (CH2)2- or -(CH2)3- or -(CH2)4-.

52. The pharmaceutical composition of claim 51, wherein, The cationic lipid is YK-401 of structure (II-I) or YK-402 of structure (II-II): (I-I), (I-I). (II-II).

53. The pharmaceutical composition of claim 48, wherein, The cationic lipid is a compound of structure (III), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, (III), G1is C 1~6 alkylene; G2is C 2~8 alkylene; R1is C 6~20 straight or branched chain alkyl; R2is C 12~25 branched chain alkyl; G3is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(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-.

54. The pharmaceutical composition of claim 53, wherein, The cationic lipid is YK-201 of structure (III-I) or YK-202 of structure (III-II): (III-I), (III-II).

55. The pharmaceutical composition of claim 48, wherein, The cationic lipid is a compound of structure (IV), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, (IV), G1is C 1~8 alkylene; G2is C 2~8 alkylene; R1is C 6~25 straight or branched chain alkyl; R2is C 12~25 straight or branched chain alkyl; G3is: HO(CH2)2N(R3)CH2CH(OH)CH2- wherein R3is -CH3or -CH2CH3or -CH2CH2OH.

56. The pharmaceutical composition of claim 55, wherein, The cationic lipid is YK-305 of structure (IV-I) or YK-310 of structure (IV-II): (IV-I), (IV-II).

57. The pharmaceutical composition of claim 48, wherein the compound is of formula (I): ###0010### (I) or a pharmaceutically acceptable salt thereof. The cationic lipid is a compound of structure (V), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, (V)， G 1 and G 2 each independently unsubstituted C6-C 10 alkylene; G 3 is unsubstituted C1-C 12 alkylene; R 1 and R 2 each independently C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is OR 5 , N, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 is C1-C 12 alkyl; and R 5 is H or C1-C6 alkyl.

58. The pharmaceutical composition of claim 57, wherein, The cationic lipid is ALC0315 of structure (V-I): (VI) 59. The pharmaceutical composition of claim 48, wherein, The cationic lipid is SM102 of structure (VI-I): (VI-I).

60. The pharmaceutical composition of claim 48, wherein, The cationic lipid is a compound DLIN-MC3-DMA of structure (VII), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, (VII).

61. The pharmaceutical composition of claim 48, wherein, The cationic lipid comprises one or more selected from YK-009 of structure (I-I), YK-401 of structure (II-I), YK-305 of structure (IV-I), ALC0315 of structure (V-I), SM102 of structure (VI-I), and DLIN-MC3-DMA of structure (VII), (I-I), (I-I), (II-I), (IV-I), (V-I), (VI-I), (VII).

62. The pharmaceutical composition of claim 48, wherein, The molar ratio of the cationic lipid to the neutral lipid is 1:1-10:

1.

63. The pharmaceutical composition of claim 48, wherein, The molar ratio of the cationic lipid to the structural lipid is 1:1-5:

1.

64. The pharmaceutical composition of claim 48, wherein, 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).

65. The pharmaceutical composition of claim 64, wherein, 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).

66. The pharmaceutical composition of claim 65, wherein, 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.

67. The pharmaceutical composition of claim 48, wherein the compound is of formula (I): ###0010### (I) or a pharmaceutically acceptable salt thereof. The neutral lipid comprises one or more selected from phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, and sterol.

68. The pharmaceutical composition of claim 67, wherein, The neutral lipid is selected from one or more of 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-didodecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2- cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3- phosphocholine, 1,2-dilinolenoyl-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-dilinolenoyl-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, dipalmitoyl phosphatidylglycerol, palmitoyloleoyl phosphatidyl ethanolamine, distearoyl- phosphatidyl-ethanolamine, dipalmitoyl phosphatidyl ethanolamine, dimyristyl phosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl- phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine.

69. The pharmaceutical composition of claim 68, wherein, The neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1,2- distearoyl-sn-glycero-3-phosphocholine.

70. The pharmaceutical composition of claim 48, wherein, The structural lipid is selected from one or more of cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol, and corticosteroid.

71. The pharmaceutical composition of claim 70, wherein, The structural lipid is cholesterol.

72. The pharmaceutical composition of claim 48, wherein The polymeric conjugated lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

73. The pharmaceutical composition of claim 72, wherein, The polymeric conjugated lipid is selected from one or more of: distearoylphosphatidyl ethanolamine polyethylene glycol 2000, dimyristoylglycerol-3-methoxy polyethylene glycol 2000, and methoxypolyethylene glycol bismyristylformamid.

74. A kit, comprising: The kit of parts comprises a kit A comprising one or more of the double stranded RNAi agent of any one of claims 1-20, the conjugate of any one of claims 21-32, or the pharmaceutical composition of any one of claims 33-73.

75. The kit of claim 74, wherein The kit of parts further comprises a kit B comprising one or both of: (1) another drug that reduces HBV gene expression or a composition comprising the drug that reduces HBV gene expression; (2) one or more of the group consisting of a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.

76. Use of the double stranded RNAi agent of any one of claims 1-20, the conjugate of any one of claims 21-32, or the pharmaceutical composition of any one of claims 33-73 in the manufacture of a medicament for preventing and / or treating a disease associated with HBV gene expression; The disease associated with HBV gene expression is chronic hepatitis B or acute hepatitis B.

77. A method for reducing HBV gene expression or inhibiting HBV replication for non-preventive and / or therapeutic purposes, characterized in that, The method comprises administering to a sample one or more of the double stranded RNAi agent of any one of claims 1-20, the conjugate of any one of claims 21-32, the pharmaceutical composition of any one of claims 33-73, and the kit of parts of claims 74 or 75.

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