Antisense oligonucleotide for reducing HBV gene expression and use thereof

Abstract

An antisense oligonucleotide and the use thereof. A series of ASOs are designed on the basis of the genome sequence of a hepatitis B virus (HBV), and are modified by means of using a specific modification mode. Cell and animal experiment results show that some modified ASOs obtained by means of using the specific modification mode can significantly reduce the expression of one or more HBV genes and block the life cycle of viruses, and therefore can be used to develop drugs for treating HBV infection-related diseases.

Description

An antisense oligonucleotide that reduces HBV gene expression and its application

[0001] This application claims priority to Chinese patent application No. 2024109094046, filed on July 8, 2024. Technical Field

[0002] This disclosure relates to the field of nucleic acid modification technology, and more specifically, to an antisense oligonucleotide that reduces HBV gene expression and its application in the preparation of drugs for diseases related to HBV gene expression. Background Technology

[0003] 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.

[0004] Oligonucleotides are a class of short DNA or RNA molecules, oligomers, that readily bind in a sequence-specific manner to their respective complementary oligonucleotides, DNA, or RNA to form 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.

[0005] ASOs are synthetic, small (approximately 18–30 nucleotides) single-stranded nucleotide polymers with diverse chemical structures. ASOs can regulate gene expression through a variety of mechanisms. They can be classified into two main categories: ribonuclease-dependent and sterically hindered. Gene expression is regulated either by binding to their associated target mRNA via endogenous RNase H enzymes that recognize RNA-DNA hybrid double strands and catalyze the degradation of mRNA, or by using steric occupancy mechanisms to prevent the translation of target mRNA.

[0006] The latest generation of ribonuclease-based ASOs typically employs a "gapmer" layout, where the central DNA base "gap" is surrounded by chemically modified RNA bases. The flanking portions promote affinity for the target sequence. Notably, RNase H is active in both the cytoplasm and nucleus; therefore, these ASOs can also be used to target nuclear transcripts (such as immature pre-mRNA and long non-coding RNA (lncRNA)) that are difficult for other technologies (such as siRNA) to access.

[0007] ASOs have a natural advantage over small molecule and antibody drugs because they perform their 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 ASO drugs allows them to regulate the expression of target proteins at the gene level, exhibiting target specificity compared to small molecule or antibody drugs. Their mechanism based on the principle of complementary base pairing also makes ASOs have a wider therapeutic range, simpler design, and shorter development cycles.

[0008] In natural oligonucleotides, adjacent 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.

[0009] Based on the basic structure of nucleotides—bases, sugar rings, and phosphate backbones—these three parts can be chemically modified.

[0010] 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.

[0011] 2) Glycocycle Modification: This mainly involves glycocycle modification and substitution. Glycocycle modifications include 2'-modification, 4'-modification, 5'-modification, isomerization, and combinations of these modifications. The most common modifications in ASOs are 2'-MOE (2'-O-methoxyethyl), 2,4'-LNA (2,4'-locked nucleotide), and 2,4'-cEt (2,4'-restricted ethyl). Compared to natural ASOs, modified ASOs exhibit higher Tm values, stronger serum stability, and better activity.

[0012] 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 atoms, S atoms and / or N atoms to form guanidino, S-methylthiourea, etc.

[0013] Hepatitis B virus (HBV) is a double-stranded hepatotropic virus that infects only humans and non-human primates. It primarily replicates in the liver and can be transmitted through mother-to-child transmission, blood (including minor skin and mucous membrane injuries), and sexual contact. Currently, 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] This disclosure designs a series of unique antisense oligonucleotide base sequences based on the HBV genome sequence, and modifies these antisense oligonucleotide base sequences with different modification patterns to prepare corresponding antisense oligonucleotide modifiers.

[0017] Antisense oligonucleotides with the same base sequence but different modification methods exhibit significantly different activities; similarly, antisense oligonucleotides with different base sequences but the same modification method also show significantly different activities. Although some modification principles exist for antisense oligonucleotide modification design, existing research indicates 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 patterns from countless possible combinations is extremely difficult.

[0018] This disclosure identifies antisense oligonucleotide modifiers that significantly reduce HBV gene expression by chemically modifying the designed antisense oligonucleotide base sequence.

[0019] On the one hand, this disclosure provides antisense oligonucleotides for reducing HBV gene expression, said antisense oligonucleotides consisting of 10 to 30 consecutive nucleotides and having a nucleotide sequence or fragment thereof represented by any one of SEQ ID NO:1-89 or a modified sequence thereof.

[0020] In another aspect, this disclosure also provides a pharmaceutical composition comprising an antisense oligonucleotide as described in this disclosure and a pharmaceutically acceptable carrier thereof.

[0021] In another aspect, this disclosure also provides the use of antisense oligonucleotides as described herein or pharmaceutical compositions as described herein in the preparation of medicaments for treating HBV infection-related diseases.

[0022] In another aspect, this disclosure also provides a kit containing a kit A, which includes one or both of the antisense oligonucleotides as described in this disclosure or pharmaceutical compositions as described in this disclosure.

[0023] In another aspect, this disclosure also provides a kit containing a kit A, said kit A comprising one or both of the antisense oligonucleotides as described in this disclosure and / or pharmaceutical compositions as described in this disclosure.

[0024] On the other hand, this disclosure also provides methods for reducing HBV gene expression or inhibiting HBV replication.

[0025] The beneficial effects achieved by this disclosure include at least one of the following:

[0026] (1) The antisense oligonucleotides and / or their modifications disclosed herein have significant inhibitory effects on HBsAg, which are significantly superior to at least one ginseng (e.g., GSK836 and BPC-A3A3).

[0027] (2) The antisense oligonucleotides and / or their modifications disclosed herein can be delivered to the liver of animals by subcutaneous administration and significantly reduce HBV gene expression and significantly reduce serum HBsAg, HBeAg and HBV DNA levels.

[0028] (3) The antisense oligonucleotide modified by the modification mode of this disclosure has significantly improved HBV inhibitory activity compared with the antisense oligonucleotides and / or antisense oligonucleotide modified by the prior art.

[0029] (4) This disclosure found that antisense oligonucleotides with similar sequences have very different activities.

[0030] (5) This disclosure also found that different sequences are sensitive to different modification modes, and it is uncertain which modification mode can be used to modify the ASO sequence to achieve high activity. Attached Figure Description

[0031] Figure 1 shows the effect of different ASO sequences on plasma HBsAg in AAV-HBV mice.

[0032] Figure 2 shows the effect of different ASO sequences on plasma HBV DNA in AAV-HBV mice.

[0033] Figure 3 shows the effect of different ASO sequences on plasma HBeAg in AAV-HBV mice.

[0034] Figure 4 shows the effect of different ASO sequences on plasma HBsAb in AAV-HBV mice.

[0035] Figure 5 shows the effect of different ASO sequences on the body weight of AAV-HBV mice. Detailed Implementation

[0036] 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.

[0037] 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.

[0038] The term “including” is used here to refer to the phrase “including but not limited to” and is used interchangeably with it.

[0039] 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.

[0040] As used herein, the term “about” or “approximately” when applied to one or more target values ​​refers to a value similar to the reference value. In some embodiments, unless otherwise stated or otherwise apparent from the context, the term “approximately” or “about” refers to a range of values ​​falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in any direction (unless such a number would exceed 100% of the possible value).

[0041] 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.

[0042] “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 detailed further 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 substitution moieties are suitable for the oligonucleotides, oligonucleotide modifications, pharmaceutical compositions, and methods of this disclosure.

[0043] The terms “ASO” and “antisense oligonucleotide” are used interchangeably herein and refer to oligonucleotide sequences that, as defined herein, can bind to target mRNA in a sequence complementary manner and mediate targeted cleavage of mRNA via the RNase H pathway. ASO regulates, for example, the inhibition of HBV expression in cells such as those of a subject (e.g., a mammalian subject).

[0044] The terms “modified ASO”, “modified antisense oligonucleotide”, “ASO modifier” and “antisense oligonucleotide modifier” are used interchangeably in this document and refer to an antisense oligonucleotide containing at least one modified nucleotide.

[0045] In this article, in some cases, "ASO", "antisense oligonucleotide", "modified ASO", "modified antisense oligonucleotide", "ASO modifier" and "antisense oligonucleotide modifier" may appear as "ASO sequence", "antisense oligonucleotide sequence", "modified ASO sequence", "modified antisense oligonucleotide sequence", "ASO modifier sequence" and "antisense oligonucleotide modifier sequence", respectively. Those skilled in the art should know that their technical meanings are matched and clear.

[0046] In this article, "base sequence" refers to an antisense oligonucleotide that does not contain any modified nucleotides. In some cases, it may also refer to the corresponding nucleotide sequence of the antisense oligonucleotide. In this article, "base sequence," "antisense oligonucleotide base sequence," "unmodified ASO," and "ASO base sequence" are used interchangeably.

[0047] In this disclosure, "other drugs that reduce HBV gene expression" means drugs that do not contain the ASO and / or ASO modifiers of this disclosure.

[0048] The term “reduction” as used in this article may be used interchangeably with “reduction,” “silence,” “downsizing,” “suppression,” “inhibition,” and other similar terms, and includes any level of reduction.

[0049] As used in this article, the phrase “reducing HBV gene expression” includes reducing the expression of HBV DNA, HBV mRNA, HBsAg, hepatitis B e antigen (HBeAg) and / or hepatitis B core antigen (HBcAg).

[0050] "Reducing HBV antigen expression" includes reducing the expression of HBsAg, HBeAg, and / or HBcAg proteins.

[0051] "Reduced HBV gene expression" includes any level of reduction in the expression of HBV DNA, HBV mRNA, HBsAg, HBeAg, and / or HBcAg, for example, at least a partial reduction in the expression of HBV DNA, HBV mRNA, HBsAg, HBeAg, and / or HBcAg, such as a reduction 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%.

[0052] HBV gene expression can be assessed based on the levels of any variables associated with HBV gene expression, such as HBV DNA levels, HBV mRNA levels, HBV antigen protein levels, and HBV viral particle levels. A reduction can be assessed by a decrease 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 control or inert agent control) subjects, cells, or samples.

[0053] 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.

[0054] As used herein, “HBV infection-related disease” is intended to include any disease associated with the HBV gene or protein. Such disease 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 infection-related diseases include HBV infection-related hepatitis, such as chronic hepatitis B, liver fibrosis, cirrhosis, liver cancer, acute hepatitis B, and diseases associated with co-infection with HBV and hepatitis D virus (HDV).

[0055] As used herein, “therapeutic effective dose” is intended to include the amount of antisense oligonucleotide that, when administered to a patient for the treatment of an HBV infection-related disease, is sufficient to achieve therapeutic effect on the disease (e.g., by attenuating, improving, or maintaining the existing disease or symptoms of one or more diseases). This “therapeutic effective dose” can vary depending on the antisense oligonucleotide, how the agent is administered, the disease and its severity, and medical history, age, weight, family history, genetic makeup, stage of the pathological process mediated by HBV expression, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0056] As used herein, a “preventive effective dose” is defined as the amount of an antisense oligonucleotide 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 infection-related disease but may be susceptible to it. Improvement of the disease includes slowing its progression or reducing the severity of subsequent disease development. This “preventive effective dose” can vary depending on the antisense oligonucleotide, how the agent 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.

[0057] "Therapeutic effective amount" or "preventive effective amount" also includes the amount of antisense oligonucleotide that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The antisense oligonucleotide 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.

[0058] 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 a subject. Examples of biological fluids include blood, serum and serous fluids, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs, or localized areas. 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 a portion 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.

[0059] In one aspect, this disclosure provides an antisense oligonucleotide composed of 10 to 30 consecutive nucleotides and comprising a nucleotide sequence or fragment thereof shown in any of SEQ ID NO:1-89, wherein the antisense oligonucleotide optionally comprises at least one modified nucleotide.

[0060] In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence or fragment thereof shown in any of SEQ ID NO:1-7, or a modified sequence thereof.

[0061] In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence or fragment thereof shown in any of SEQ ID NO:2, 6 and 7, or a modified sequence thereof.

[0062] In some implementations, the antisense oligonucleotide can reduce HBV gene expression.

[0063] In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence having 1-3 nucleotide substitutions, additions, or deletions compared to the nucleotide sequence shown in any of SEQ ID NO:1-89.

[0064] In some embodiments, the at least one modified nucleotide is selected from any one or a combination of two or more of the following: 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'-phosphate groups, and nucleotides containing 5'-phosphate mimics.

[0065] In some embodiments, the antisense oligonucleotide has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

[0066] In some implementations, the antisense oligonucleotide is 10-30 nucleotides in length.

[0067] In some implementations, the antisense oligonucleotide is 13-25 nucleotides in length.

[0068] In some implementations, the antisense oligonucleotide is 13-20 nucleotides in length.

[0069] In some implementations, the antisense oligonucleotide is 16 nucleotides in length.

[0070] In some embodiments, the modification of the nucleotide is a chemical modification at the 2' position of the nucleotide ribose.

[0071] In some embodiments, the chemical modification at the 2' position of the nucleotide ribose is selected from any one or a combination of two or more of the following: 2'-methoxy modification, 2'-O-methoxyethyl modification, 2'-fluoro modification, 2'-benzyloxy modification, 2'-methylcarbonylamino modification, 2'-pyridinemethoxy modification, 2,4'-restricted nucleotide modification, and 2,4'-restricted ethyl nucleotide modification.

[0072] In some embodiments, the chemical modification at the 2' position of the nucleotide ribose is a 2,4'-locked nucleotide modification.

[0073] In some embodiments, the chemical modification at the 2' position of the nucleotide ribose of a portion of the antisense oligonucleotide is a 2,4'-restricted ethyl nucleotide modification.

[0074] In some embodiments, the nucleotides are linked by 3',5'-phosphodiester bonds.

[0075] In some embodiments, the 3',5'-phosphodiester bond comprises a thiolated modification.

[0076] In some embodiments, the 3',5'-phosphodiester bonds all contain thiomodification and form chiral pure 3',5'-thiophosphodiester bonds.

[0077] In some embodiments, the antisense oligonucleotide comprises: a spacer region composed of consecutive nucleotides; a 5' flanking region composed of consecutive nucleotides; a 3' flanking region composed of consecutive nucleotides; the spacer region being located between the 5' flanking region and the 3' flanking region, and the 5' flanking region and the 3' flanking region comprising modified nucleotides.

[0078] In some embodiments, the spacer region consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nucleotides.

[0079] In some embodiments, the antisense oligonucleotide has any or both of the following modifications:

[0080] A3: Indicates that the first, second and third nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides or nucleotides modified with 2,4'-restricted ethyl.

[0081] A2: Indicates that the first and second nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides, or nucleotides modified with 2,4'-restricted ethyl.

[0082] B3: Indicates that the first, second and fourth nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides or nucleotides modified with 2,4'-restricted ethyl.

[0083] C3: indicates that the first, third and fourth nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides or nucleotides modified with 2,4'-restricted ethyl.

[0084] B2: Indicates that the first and third nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides, or nucleotides modified with 2,4'-restricted ethyl.

[0085] D3: Indicates that the first, third and fifth nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides or nucleotides modified with 2,4'-restricted ethyl.

[0086] E2: indicates that the first and fourth nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides, or nucleotides modified with 2,4'-restricted ethyl.

[0087] A1: Indicates that the first nucleotide at the 5' end and / or 3' end of the antisense oligonucleotide is a nucleotide modified with 2'-O-methoxyethyl, 2,4'-locked nucleotide, or 2,4'-restricted ethyl.

[0088] In some embodiments, the antisense oligonucleotide has any of the following modification modes:

[0089] A3A3: This indicates that the 5' end of the antisense oligonucleotide has a modification pattern A3, and the 3' end of the antisense oligonucleotide has a modification pattern A3;

[0090] A3A2: This indicates that the 5' end of the antisense oligonucleotide has modification A3, and the 3' end of the antisense oligonucleotide has modification A2;

[0091] A3B2: This indicates that the 5' end of the antisense oligonucleotide has modification A3, and the 3' end of the antisense oligonucleotide has modification B2;

[0092] A2A2: This indicates that the 5' end of the antisense oligonucleotide has modification A2, and the 3' end of the antisense oligonucleotide has modification A2.

[0093] A2B2: This indicates that the 5' end of the antisense oligonucleotide has modification A3, and the 3' end of the antisense oligonucleotide has modification B2;

[0094] B3A3: This indicates that the 5' end of the antisense oligonucleotide has modification type B3, and the 3' end of the antisense oligonucleotide has modification type A3;

[0095] B3A2: This indicates that the 5' end of the antisense oligonucleotide has modification type B3, and the 3' end of the antisense oligonucleotide has modification type A2;

[0096] B3A1: This indicates that the 5' end of the antisense oligonucleotide has modification type B3, and the 3' end of the antisense oligonucleotide has modification type A1;

[0097] B3B2: This indicates that the 5' end of the antisense oligonucleotide has modification type B3, and the 3' end of the antisense oligonucleotide has modification type B2;

[0098] C3A3: This indicates that the 5' end of the antisense oligonucleotide has a modification of type C3, and the 3' end of the antisense oligonucleotide has a modification of type A3;

[0099] C3A2: This indicates that the 5' end of the antisense oligonucleotide has modification C3, and the 3' end of the antisense oligonucleotide has modification A2;

[0100] C3A1: This indicates that the 5' end of the antisense oligonucleotide has modification C3, and the 3' end of the antisense oligonucleotide has modification A1;

[0101] C3B2: This indicates that the 5' end of the antisense oligonucleotide has modification C3, and the 3' end of the antisense oligonucleotide has modification B2;

[0102] B2B2: This indicates that the 5' end of the antisense oligonucleotide is modified by method B2, and the 3' end of the antisense oligonucleotide is modified by method B2.

[0103] D3A3: This indicates that the 5' end of the antisense oligonucleotide has a modification pattern D3, and the 3' end of the antisense oligonucleotide has a modification pattern A3;

[0104] D3A2: This indicates that the 5' end of the antisense oligonucleotide has modification mode D3, and the 3' end of the antisense oligonucleotide has modification mode A2;

[0105] D3A1: This indicates that the 5' end of the antisense oligonucleotide has modification mode D3, and the 3' end of the antisense oligonucleotide has modification mode A1;

[0106] D3B2: This indicates that the 5' end of the antisense oligonucleotide has modification mode D3, and the 3' end of the antisense oligonucleotide has modification mode B2;

[0107] E2A2: This indicates that the 5' end of the antisense oligonucleotide has a modification pattern E2, and the 3' end of the antisense oligonucleotide has a modification pattern A2;

[0108] E2B2: This indicates that the 5' end of the antisense oligonucleotide has a modification pattern E2, and the 3' end of the antisense oligonucleotide has a modification pattern B2.

[0109] In some implementations, the phosphodiester bonds between the nucleotides of the antisense oligonucleotide are all thio-modified phosphodiester bonds.

[0110] In some embodiments, the antisense oligonucleotide comprises any one of the group consisting of oligonucleotides with the sequences shown below: SEQ ID NO: 93-102 and 179-276.

[0111] In some embodiments, the antisense oligonucleotide is selected from the group consisting of the following nucleotide sequences: SEQ ID NO: 93-102 and 179-276.

[0112] In some embodiments, the antisense oligonucleotide comprises any one of the group consisting of oligonucleotides with the sequences shown below: SEQ ID NO: 179, 193, 194, 195, 219, 231, 243, 252, 254 and 265.

[0113] This disclosure also provides a pharmaceutical composition comprising an antisense oligonucleotide as described in this disclosure, and a pharmaceutically acceptable carrier thereof.

[0114] In one embodiment, pharmaceutical compositions comprising an antisense oligonucleotide as described herein and a pharmaceutically acceptable carrier thereof are provided herein. Pharmaceutical compositions containing antisense oligonucleotides 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.

[0115] Pharmaceutical compositions comprising the antisense oligonucleotides 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.

[0116] This disclosure also provides a kit containing a box A, which includes one or both of the antisense oligonucleotides or pharmaceutical compositions as described in this disclosure.

[0117] This disclosure also provides a kit containing a box A, which includes one or two of the antisense oligonucleotides and / or pharmaceutical compositions as described in this disclosure.

[0118] Preferably, the kit further includes a pillbox B, which contains one or both of the following (1) and (2):

[0119] (1) Other drugs that reduce HBV gene expression or compositions containing said drugs that reduce HBV gene expression;

[0120] (2) Any one or a combination of two 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.

[0121] This disclosure also provides the use of antisense oligonucleotides as described herein or pharmaceutical compositions as described herein in the preparation of medicaments for treating HBV infection-related diseases.

[0122] In some implementations, the HBV infection-related disease is selected from any one or a combination of two or more of the following diseases: chronic hepatitis B, liver fibrosis, cirrhosis, liver cancer, acute hepatitis B, and HBV / HDV co-infection-related diseases.

[0123] This disclosure also provides a method for reducing HBV gene expression or inhibiting HBV replication, the method comprising administering to a sample any one or a combination of two or more of the antisense oligonucleotides as described in this disclosure, pharmaceutical compositions as described in this disclosure, and kits as described in this disclosure; preferably, the method is for non-therapeutic purposes.

[0124] In the method of this disclosure, the antisense oligonucleotide can be administered in a solution. A free antisense oligonucleotide can be administered in a non-buffered solution, such as in physiological saline or water. Alternatively, the free ASO 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 osmolality of the buffer containing the antisense oligonucleotide can be adjusted to make it suitable for administration to the subject.

[0125] 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.

[0126] The pharmaceutical compositions disclosed herein can be administered at doses sufficient to reduce HBV gene expression. Typically, suitable doses of the antisense oligonucleotides of this disclosure range from about 0.001 to about 200.0 mg per kilogram of body weight per day, and generally from about 0.1 to 50 mg per kilogram of body weight per day. For example, the antisense oligonucleotides 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, 18, 18.5, 19 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or approximately 50 mg / kg.

[0127] The pharmaceutical composition can be administered once daily, or the antisense oligonucleotide 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 antisense oligonucleotide 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 antisense oligonucleotide 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.

[0128] 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, two single doses of the pharmaceutical composition of this disclosure are given in the first week, followed by one single dose of the pharmaceutical composition of this disclosure every week. In other embodiments of this disclosure, one single dose of the pharmaceutical composition of this disclosure is given every week.

[0129] 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, previous treatments, 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 antisense oligonucleotides covered by this disclosure can be estimated using conventional methods or based on in vivo testing using suitable animal models.

[0130] 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.

[0131] The antisense oligonucleotides used in the compositions and methods of this disclosure can be formulated for delivery in membrane-bound molecular assemblages, 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 antisense oligonucleotide composition. The lipophilic material separates the aqueous interior from an aqueous exterior that typically does not include the antisense oligonucleotide 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 interior contents, including the antisense oligonucleotide, are delivered into the cell, wherein the antisense oligonucleotide can specifically bind to a target mRNA and can mediate its degradation. In some cases, these liposomes are also specifically targeted, for example, to direct the antisense oligonucleotide to a specific cell type.

[0132] Liposomes containing antisense oligonucleotides can be prepared by a variety of 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 antisense oligonucleotide formulation is then added to micelles containing the lipid component. The cationic groups on the lipid interact with the antisense oligonucleotide and condense around it to form liposomes. After condensation, the detergent is removed, for example by dialysis, to obtain the liposome formulation of the antisense oligonucleotide.

[0133] Antisense oligonucleotides, such as the ASO disclosed herein, can be completely encapsulated in lipid formulations (e.g., LNPs or other nucleic acid-lipid particles).

[0134] 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.

[0135] The pharmaceutical formulations disclosed herein (which can be conveniently presented in unit dosage forms) can be prepared using 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 formulations are prepared by 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.

[0136] The 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 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.

[0137] Some compositions of this disclosure 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 biologically active nucleic acids by degrading them or promoting their removal from circulation. Co-administration of nucleic acids and carrier compounds (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, polycytidine, or 4-acetamido-4'-isothiocyanate 2,2'-disulfonic acid can reduce the recovery of partially thiophosphated dsRNA from liver tissue.

[0138] 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.).

[0139] Pharmaceutically acceptable organic or inorganic excipients that are suitable for non-parenteral administration, do not react toxically with nucleic acids, and are suitable for formulation of the compositions disclosed herein may also be used. Suitable pharmaceutically acceptable carriers 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.

[0140] 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.

[0141] 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.

[0142] The dosage form, carrier compound, drug carrier, excipient, etc. of the above composition are described in U.S. Patent US10125369B2, which is incorporated herein by reference.

[0143] This disclosure also provides methods for treating or preventing diseases and conditions that can be regulated by downregulating HBV gene expression. For example, the antisense oligonucleotides described herein can be used to treat HBV infection-related diseases including HBV infection-associated hepatitis, such as chronic hepatitis B, acute hepatitis B, and diseases associated with HBV / hepatitis D virus (HDV) co-infection. These methods include administering a therapeutically effective or preventatively effective amount of one of the antisense oligonucleotides or compositions of this disclosure to the subject. In some embodiments, the method includes administering a therapeutic amount of HBV ASO to a patient with a heterozygous HBV genotype.

[0144] The antisense oligonucleotides disclosed herein can be administered to a subject using any administration method known in the art, including (but not limited to) subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination thereof. In a preferred embodiment, these agents are administered subcutaneously.

[0145] In another embodiment, ASO is administered in combination with another therapeutic agent. ASO and the other therapeutic agent may be administered in combination in the same composition, for example, parenterally, or the other therapeutic agent may be administered as part of a separate composition or by another method described herein.

[0146] Other examples of therapeutic agents include those known to be used to treat hepatitis B. For instance, other drugs for treating chronic hepatitis B are selected from nucleoside (acid) analogs (such as entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, and tenofovir alafenamide), and alpha interferon (such as pegylated alpha interferon).

[0147] In one embodiment, an antisense oligonucleotide is given to the patient, followed by another therapeutic agent (or vice versa). In another embodiment, the antisense oligonucleotide and another therapeutic agent are given simultaneously.

[0148] 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.

[0149] The nucleotide abbreviations and modification codes used in this article are as follows: A = deoxyadenosine-3'-phosphate; A(MOE) = 2'-O-methoxyethyladenosine-3'-phosphate; a = 2,4'-restricted ethyladenosine-3'-phosphate; +a = 2,4'-locked nucleotide adenosine-3'-phosphate; G = deoxyguanosine-3'-phosphate; G(MOE) = 2'-O-methoxyethylguanosine-3'-phosphate; g = 2,4'-restricted ethylguanosine-3'-phosphate; +g = 2,4'-locked nucleotide guanosine-3'-phosphate; C = deoxycytidine-3'-phosphate; C(MOE) = 2'-O-methoxyethylcytidine-3'-phosphate; c =5'-methylated-2,4'-restricted ethyl cytidine-3'-phosphate + c =5'-methylated-2,4'-deoxynucleotide cytidine-3'-phosphate T = deoxythymidine-3'-phosphate T(MOE) = 2'-O-methoxyethyl thymidine-3'-phosphate t = 2,4'-restricted ethyl thymidine-3'-phosphate + t = 2,4'-deoxynucleotide thymidine-3'-phosphate * = 3',5'-thiophosphodiester bond mC = 5'-methylated cytidine-3'-phosphate mC(MOE) = 5'-methylated-2'-O-methoxyethyl cytidine-3'-phosphate

[0150] Example

[0151] Example 1: Synthesis of LNA-modified ASO compounds

[0152] A total of 84 basic ASO sequences were designed (SEQ ID NO: 1-10, 14-87 as shown in Table 1). To improve the inhibition efficiency and stability of the sequences, LNA modification was performed on them. In this embodiment, the 5' and 3' nucleotides of the LNA-modified ASO derivatives are LNA-modified nucleotides, while the remaining positions are deoxynucleoside-3'-phosphate esters. The phosphodiester bonds between the nucleotides of the ASO derivatives are all thio-modified phosphodiester bonds. The Yangshen sequence is SSO-1 from the literature Mol Ther Nucleic Acids. 2018 Jun 1; 11:441-454, which is numbered APC-GP01 after LNA modification in this embodiment (as shown in Table 1).

[0153] 1. Synthesis of LNA-modified ASO compound A221-GP01

[0154] The base sequence corresponding to the ASO modifier numbered A221-GP01 in Table 1 is as follows:

[0155] 5'-GTGAGGATTCTTGTCA-3'(SEQ ID NO:14)

[0156] The nucleotides at positions 1-3 of the 5' and 3' ends are LNA-modified nucleotides, while the remaining positions are deoxynucleoside-3'-phosphate esters. The phosphodiester bonds between the ASO-modified nucleotides are all thio-modified phosphodiester bonds.

[0157] Instruments and reagents: Qingke 192P model DNA / RNA automated synthesizer, with cross-linked polystyrene beads as the solid carrier, model Primer support 5G Unylinker 350 (Cytiva).

[0158] Synthesis method:

[0159] The following nucleotide monomer solutions were prepared with acetonitrile at a monomer concentration of 0.15 M: DMT-A phosphorus amide monomer (Formula 1), DMT-C phosphorus amide monomer (Formula 2), DMT-G phosphorus amide monomer (Formula 3), DMT-T phosphorus amide monomer (Formula 4), DMT-A-LNA phosphorus amide monomer (Formula 5), ​​DMT-5' methylated C-LNA phosphorus amide monomer (Formula 6), DMT-G-LNA phosphorus amide monomer (Formula 7), and DMT-T-LNA phosphorus amide monomer (Formula 8).

[0160] The specific steps are as follows:

[0161] (1) Deprotection

[0162] The DMT protecting group was removed using a toluene solution of 3% dichloroacetic acid as a deprotecting agent, followed by washing with acetonitrile.

[0163] (2) Coupling

[0164] Each nucleotide monomer was coupled using a 0.25M acetonitrile solution of 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.

[0165] (3) Sulfurization

[0166] Vulcanization: Vulcanization was carried out using a pyridine solution of 3% hydroxanthin as the sulfiding agent, followed by rinsing with acetonitrile.

[0167] (4) Hydroxyl protection

[0168] Hydroxyl protection was performed using a 10% tetrahydrofuran solution of acetic anhydride (CAP A) and tetrahydrofuran / pyridine / aziridine 4 / 10 / 16 (v / v / v) (CAP B) as the hydroxyl protecting agent, followed by rinsing with acetonitrile.

[0169] Repeat the above steps, synthesizing each nucleotide in the specific order of each sequence to obtain a vector containing a fully protected ASO structure.

[0170] (5) Use a toluene solution of 3% dichloroacetic acid as a deprotecting agent to remove the DMT protecting group of the last nucleotide, and then wash with acetonitrile.

[0171] (6) Ammonolysis and purification

[0172] The solid support was transferred to the reactor, concentrated ammonia (25-28%) was added, and the mixture was kept at 60°C for 12 hours. After cooling the system to room temperature, the mixture was filtered, washed with a mixture of purified water and ethanol, the filtrates were combined, passed through a chromatography column, concentrated, and lyophilized to obtain the ASO-modified product.

[0173] A221-GP01 purity: 91.47%; measured molecular weight: 5349.43

[0174] 2. Synthesis of other ASO modifiers

[0175] Other ASO modifiers were synthesized according to the synthesis method described in "1" above. For a detailed list of ASO modifiers, please refer to Table 1.

[0176] Table 1. LNA-modified ASO modifiers

[0177] Example 2: Inhibitory effect of LNA-modified ASO on HBsAg and HBeAg

[0178] The LNA-modified ASO modifiers synthesized in Example 1 were transfected into HepG2.2.15 cells (Shanghai WuXi AppTec New Drug Development Co., Ltd.) via lipid nanoparticles (LNP). The inhibitory effects of each ASO modifier on HBsAg and HBeAg were then detected by ELISA.

[0179] 1. Experimental Materials

[0180] Sample: LNA-modified ASO derivatives in Table 1 (synthesized according to the method in Example 1).

[0181] Cells: HepG2.2.15 cells

[0182] Drug solvent: sterile enzyme-free water, Opti-MEM (gibco).

[0183] 2. Experimental Methods

[0184] The inhibitory effect of the samples on HBsAg and HBeAg in the HepG2.2.15 cell line was detected by ELISA.

[0185] 2.1 Cell Culture

[0186] 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, and placed in a cell culture incubator at 37°C with 5% CO2. Subcultures were performed by digestion with 0.25% trypsin, centrifuged at 800 rpm for 3 min, and the supernatant was discarded. Fresh medium was then added for further subculturing.

[0187] 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.

[0188] 2.2 Cell transfection

[0189] Transfection reagent preparation: Mix Lipofectamine RNAiMAX and Opti-MEM at a volume ratio of 2:98 and vortex to mix.

[0190] Preparation of transfection complex: Take 30 μL of ASO modified solution diluted with Opti-MEM at a ratio of 1:1 (v / v), add 30 μL of transfection reagent, vortex mix, and let stand at room temperature for 15 min to obtain transfection complex.

[0191] Transfection control group transfection reagent preparation: Add 30 μL of transfection reagent to 30 μL of Opti-MEM. Vortex mix and let stand at room temperature for 15 min.

[0192] Add the prepared transfection complex to each 96-well cell culture plate (15 μL per well, 3 replicates per sample). In the single-concentration screening experiment, the final concentration of ASO was 1 nM. For the transfection control group, add 15 μL of the prepared transfection reagent to each well, with 3 replicates. Then add 135 μL of cell suspension (containing 2.25 × 10⁻⁶ cells per well) to each well. 4 Mix thoroughly and then incubate at 37°C in a 5% CO2 cell culture incubator.

[0193] 2.3 Detection of HBsAg and HBeAg in cell supernatant

[0194] 1) Collection of cell supernatant

[0195] a. Change the medium on day 3 after cell transfection, collect the cell supernatant for HBsAg and HBeAg content detection, and add 150 μL / well of fresh medium to continue culturing.

[0196] b. Collect cell supernatant on day 6 post-transfection for quantitative detection of HBsAg and HBeAg.

[0197] 2) Quantitative detection of HBsAg and HBeAg

[0198] The concentrations of HBsAg and HBeAg were detected using a hepatitis B virus e antigen detection kit and a hepatitis B virus surface antigen detection kit. The specific operating steps are as follows:

[0199] a. Allow the kit and test samples to return to room temperature.

[0200] b. Add 50 μL each of the test sample, standard, negative control, and positive control to the well plate.

[0201] c. Add 50 μL of enzyme conjugate to each well. Mix thoroughly and incubate at 37°C for 60 min.

[0202] 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.

[0203] 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.

[0204] f. Measure the luminescence value on an ELISA reader.

[0205] 2.4 Data Processing

[0206] The formulas for calculating the inhibition rates of HBsAg and HBeAg are as follows:

[0207] HBsAg inhibition rate (%) = (1 - HBsAg expression level in sample / HBsAg expression level in transfected control group in the same plate) × 100%;

[0208] HBeAg inhibition rate (%) = (1 - HBeAg expression level in sample / HBeAg expression level in control group in the same plate) × 100%.

[0209] 2.5EC 50 experiment

[0210] In this experiment, the concentrations of each ASO modifier were set as follows: 30 nM as the starting concentration, followed by a 3-fold serial dilution to prepare eight concentrations: 30 nM, 10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.12 nM, 0.04 nM, and 0.01 nM. The inhibition rates of each sample at each concentration were measured, plotted, and the EC50 of each sample and the *Codonopsis pilosula* were calculated. 50 concentration.

[0211] 2.6 Cytotoxicity assay

[0212] In EC 50 During the experiment, after collecting the cell supernatant, cell viability was determined using the CellTiter-Glo reagent kit. The method was as follows: CellTiter-Glo reagent was mixed with culture medium at a 1:1 ratio, 100 μL was added to each well, and the mixture was incubated at room temperature for 10 min. The luminescence signal value was then detected using a microplate reader.

[0213] The formula for calculating cell viability is:

[0214] Cell viability (%) = (sample signal value – mean value of blank control) / (mean value of transfection control – mean value of blank control) × 100%.

[0215] The formula for calculating the index is:

[0216] HBsAg Selection Index = CC 50 / HBsAg EC 50 ;

[0217] HBeAg Selection Index = CC 50 / HBsAg EC 50 .

[0218] 3. Experimental Results

[0219] Experimental results showed that sequences B1584L2-GP01, B1558R2-GP01, A1584-GP01, B1529RL-GP01, and B1582RL-GP01 exhibited significantly better inhibitory effects on HBsAg and HBeAg than the positive control sequence APC-GP01. In particular, sequences B1584L2-GP01 and B1582RL-GP01 showed significantly better inhibitory effects on the ECGs of HBsAg and HBeAg. 50 The lowest value indicates its effectiveness, and these two sequences also have the highest selectivity for HBsAg, indicating a wider action window. Furthermore, among the sequences targeting the HBV S region, sequences A229-GP01, A230-GP01, and A304-GP01 are significantly more effective than other sequences.

[0220] The inhibition rates of each sequence on HBsAg and HBeAg expression at single concentrations are shown in Table 2. EC50 of each sequence... 50 The experimental and cytotoxic results are shown in Table 3.

[0221] Table 2. Inhibitory effects of single-concentration LNA-modified ASO sequences on HBsAg and HBeAg.

[0222] Table 3. Inhibitory effects and cytotoxicity of multiple concentrations of LNA-modified ASO sequences on HBsAg and HBeAg.

[0223] Example 3: Inhibitory effect of unmodified ASO (basic sequence) on HBV gene

[0224] In this embodiment, based on the unmodified ASOs corresponding to the ASO modifiers in Example 1, some unmodified ASOs were selected and transfected into HepG2.2.15 cells via lipid nanoparticles (LNPs). The inhibitory effects of each unmodified ASO on HBsAg and HBeAg were detected by ELISA, and unmodified ASOs with better inhibitory effects were screened out.

[0225] 1. Experimental Materials

[0226] sample:

[0227] Table 4 lists the unmodified ASOs. All were synthesized according to the method in Example 1, except that the sulfidation in step (3) was replaced by oxidation, specifically oxidation using a 0.05M iodine pyridine / water (90 / 10) solution as the oxidant, followed by rinsing with acetonitrile. Therefore, the phosphodiester bonds between the nucleotides in the synthesized unmodified ASOs were all oxo-modified phosphodiester bonds.

[0228] Table 4 Unmodified ASO

[0229] Cells: HepG2.2.15 cells

[0230] Drug solvent: sterile enzyme-free water, Opti-MEM (gibco).

[0231] 2. Experimental Methods

[0232] Refer to Example 2.

[0233] 3. Experimental Results

[0234] Experimental results showed that the basic sequences B1584L2, B1558R2, A1558, A1559, B1529RL, B1582RL, and A229 exhibited significantly better inhibitory effects on HBsAg and HBeAg than other sequences, and were also significantly better than Yangshen PC and GSK836-B. Among them, the basic sequences B1584L2, B1558R2, and B1582RL showed the most significant inhibitory effects. The inhibition rates of HBsAg and HBeAg expression by each sequence are shown in Table 5.

[0235] Table 5 shows the basic sequences that exhibit higher inhibition rates of HBsAg and HBeAg than those of *Yangshen*.

[0236] Example 4: Inhibitory effects of ASO modifiers with different cEt modification modes on HBsAg and HBeAg

[0237] In this embodiment, 13 unmodified ASO base sequences (1558R2, 1584L2, 1558, 1559, 1529RL, 229, 1582RL, 1592, 1592L, 229R, 230, 304, and 304L) were modified with cEt using the modification pattern designed in this disclosure, resulting in 182 cEt-modified ASO derivatives. After transfection into HepG2.2.15 cells via lipid nanoparticles (LNPs), the inhibitory effects of each ASO derivative on HBsAg and HBeAg were detected using ELISA.

[0238] 1. Experimental Materials

[0239] 1) Samples: The ASO modifiers listed in Table 6 are ASO modifiers obtained by cEt modification of 13 ASO base sequences (SEQ ID NO: 1-13) using different modification modes disclosed in this invention. The positive reference sequence is ISIS50535 in patent US8642752B2, and the corresponding ASO modifier ID in this embodiment is GSK836. Another positive reference sequence is SSO-1 in the literature Mol Ther Nucleic Acids. 2018 Jun 1; 11:441-454, and the modified ASO modifier ID in this embodiment is BPC-A3A3.

[0240] Table 6. ASO modifiers obtained by cEt modification under different modification modes.

[0241] 2) Synthesis of ASO modifiers:

[0242] ASO-modified compounds were synthesized according to Example 1, wherein MOE or cEt monomers were used in the synthesis of MOE-modified or cEt-modified ASO-modified compounds. For example, the structures of DMT-A-MOE phosphorus amide monomer (Formula 9), DMT-C-MOE phosphorus amide monomer (Formula 10), DMT-G-MOE phosphorus amide monomer (Formula 11), DMT-T-MOE phosphorus amide monomer (Formula 12), DMT-A-cEt phosphorus amide monomer (Formula 13), DMT-5' methylated C-cEt phosphorus amide monomer (Formula 14), DMT-G-cEt phosphorus amide monomer (Formula 15), and DMT-T-cEt phosphorus amide monomer (Formula 16) are shown below:

[0243] Yangshen GSK836 contains a 5' methylated C and a 5' methylated and MOE-modified C. In its synthesis, DMT-5' methylated C phosphorous amide monomer (Formula 17) and DMT-5' methylated C-MOE phosphorous amide monomer (Formula 18) are used, and their structures are shown in the following examples:

[0244] Cells: HepG2.2.15 cells

[0245] Drug solvent: sterile, enzyme-free water, Opti-MEM (gibco)

[0246] In this embodiment, the ASO modifier has any of the following modification modes disclosed herein: A3A3, A3A2, A3B2, A2A2, A2B2, B3A3, B3A2, B3A1, A3B2, C3A3, C3A2, C3A1, C3B2, B2B2, D3A3, D3A2, D3A1, D3B2, E2A2, and E2B2; and the remaining positions of the antisense oligonucleotide are all deoxynucleoside-3'-phosphate esters.

[0247] In this embodiment, the phosphodiester bonds between the nucleotides of the ASO-modified compounds are all thio-modified phosphodiester bonds.

[0248] 2. Experimental Methods

[0249] Referring to Example 2, the concentration for the single-concentration screening experiment was set to 1 nM.

[0250] 3. Experimental Results

[0251] Experimental results showed that the basic sequences B1582RL, B1584L2, A229, A1559, B1529RL, B1558R2, and A1558, after modification with different cEt modification modes, exhibited significantly higher inhibition rates against HBsAg and HBeAg than the Yangshen sequence GSK836. Among them, seven sequences—B1558-C3A2, B1558R2-A3A3, B1558R2-B3A2, B1584L2-A3A3, B1590-A3A3, B1558R2-B3B2, and B1558R2-D3A2—achieved inhibition rates exceeding 90% against HBsAg and HBeAg after three days of cell treatment. A total of 32 sequences, including B1... 584L2-A3A2, B1559-B3A2, B1529RL-A3A3, B1529RL-B3B2, B1584-A3A3, B1559-A 3A2, B1584L2-A2B2, B1584L2-B3A3, B1558R2-C3B2, B1529RL-B3A2, B1584L2-A3B 2. B1558-C3B2, B1584L2-A2A2, B1529RL-A3B2, B1584L2-C3A3, B1558R2-B2B2, B 1558-A3A2, B1559-C3A2, B1558R2-C3A3, B1558R2-A3B2, B1558R2-D3B2, B1592-A The inhibition rates of sequences B1558R2-C3A2, B1559-A3A3, B1529RL-C3B2, B1529RL-C3A2, B1529-A3A3, B1592-C3B2, B1558-B2A2, B1558-C3A1, B1592-B2B2, and B1592-A3B2 were between 80% and 90%. Furthermore, among sequences targeting the HBV S region, sequences B229-A3B2 and B229-A2B2 showed significantly better efficacy than other sequences. ECG of HBsAg by sequences B1558R2-A3A3, B1558R2-B3A2, B1584L2-A3A3, B1558-C3A2, B1559-B3A2, B1529RL-B3B2, B1584L2-A3A2, B1584L2-A3B2, B229-A3B2, B229-A2B2, B1582RL-A3A3, and B1582RL-A3B2 50 Significantly lower than Yangshen GSK836, except for B1582RL-A3A3 and B1582RL-A3B2, the remaining sequences showed lower ECG values ​​for HBsAg. 50 All levels were lower than those of Yangshen BPC-A3A3, indicating its superior efficacy.

[0252] The inhibition rates of HBsAg and HBeAg expression by each sequence at single concentrations and their relative increases compared to the Yangshen sequence are shown in Tables 7-8. EC50 of each sequence... 50 The experimental and cytotoxic results are shown in Table 9.

[0253] Table 7. Inhibitory effect of cEt-modified ASO sequence on HBsAg.

[0254] Table 8. Inhibitory effect of cEt-modified ASO sequence on HBeAg.

[0255] Table 9. EC5 values ​​of cEt-modified sequences inhibiting HBsAg and HBeAg. 50 experiment

[0256] Example 5: Comparison of the inhibitory effect of unmodified ASO on HBV gene disclosed in the prior art.

[0257] This embodiment compares the HBV gene inhibition efficiency of unmodified ASO disclosed in the prior art with unmodified ASO with the same or similar base sequences 1558R2, 1584L2, 1558, 1559, 1529RL, 229, and 1582RL disclosed in this invention, as well as ASO modified with LNA or cEt according to the modification pattern disclosed in this invention.

[0258] 1. Experimental Materials

[0259] sample:

[0260] (1) Prior art disclosure sequence, see Table 10 below.

[0261] Table 10 Prior Art Disclosure Sequence and This Patent Disclosure Sequence Note: The disclosed sequences in the above patent applications are all unmodified ASOs.

[0262] (2) The ASO modifier modified by LNA using the modification pattern disclosed in Example 2.

[0263] (3) The ASO modifier modified by cEt using the modification mode of this disclosure in Example 4.

[0264] Cells: HepG2.2.15 cells

[0265] Drug solvent: sterile, enzyme-free water, Opti-MEM (gibco)

[0266] 2. Experimental Methods

[0267] Referring to Example 2, the concentration for the single-concentration screening experiment was set to 1 nM.

[0268] 3. Experimental Results

[0269] Experimental results show that the B1529RL, B1582RL, B1584L2, A1558, B1558R2, A1559 and A229 disclosed in this paper have significantly better inhibition rates against HBsAg and HBeAg than those of P1529, P1583, GSK836-B, P1556 and P229 with similar sequences. After cEt modification, the inhibition rate is further significantly improved.

[0270] Tables 11 and 12 show the comparison of the inhibition rates of HBsAg and HBeAg by sequences that are similar to those disclosed in the prior art and those disclosed in this invention.

[0271] As can be seen from Tables 11-12, compared with the prior art disclosed sequence P1529, the disclosed sequence B1529RL has one additional base on each side. Compared with P1529, the inhibition rates of B1529RL against HBsAg and HBeAg are increased by 5.50% and 5.22%, respectively. After B1529RL is modified with the template disclosed in this paper, the inhibition efficiency is further improved. For example, after modification with B3B2, the inhibition rates of B1529RL-B3B2 against HBsAg and HBeAg are increased by 47.72% and 32.91%, respectively.

[0272] Compared with the prior art disclosed sequence P1583, the disclosed sequence B1582RL has two additional bases on the left side. Compared with P1583, B1582RL increases the inhibition rate of HBsAg and HBeAg by 3.71% and 5.93%, respectively. After B1582RL is modified with the template disclosed in this paper, the inhibition efficiency is further improved. For example, after modification with A3A3, the inhibition rate of B1582RL-A3A3 against HBsAg and HBeAg is increased by 22.14% and 22.06%, respectively.

[0273] Compared with the prior art disclosed sequence GSK836-B, the disclosed sequence B1584L2 has one additional base on the left and three bases reduced on the right. Compared with GSK836-B, B1584L2 increases the inhibition rate of HBsAg and HBeAg by 13.85% and 20.11%, respectively. After B1584L2 is modified with the template disclosed in this paper, the inhibition efficiency is further improved. For example, after modification with A3A3, the inhibition rate of B1584L2-A3A3 against HBsAg and HBeAg is increased by 57.25% and 44.90%, respectively.

[0274] Compared with the prior art disclosed sequence P1556, the disclosed sequence A1558 is shortened by 2 bases on each side, sequence B1558R2 is shortened by 2 bases on the left side, and sequence A1559 is shortened by 3 bases on the left side and 1 base on the right side. Compared with P1556, the inhibition rates of A1558, B1558R2, and A1559 against HBsAg are increased by 5.58%, 13.29%, and 2.12%, respectively. After modification with the template disclosed in this paper, the inhibition efficiency is further improved. For example, the enhancement effect of sequence B1559-B3A2 is the most significant, with an increase of 58.43%, while the inhibition rate against HBeAg is increased by 45.90%. Sequence B1558R2-A3A3 increases the inhibition rate against HBsAg by 49.06% and the inhibition rate against HBeAg by 41.94%.

[0275] Compared with the prior art disclosed sequence P229, the disclosed sequence A229 has two bases shortened on each side. Compared with P229, the inhibition rates of A229 against HBsAg and HBeAg are increased by 1.94% and 2.73%, respectively. After A229 is modified with the template disclosed in this paper, the inhibition efficiency is further improved. For example, after modification with A3B2, the inhibition rates of A229-A3B2 against HBsAg and HBeAg are increased by 59.26% and 60.27%, respectively.

[0276] Table 11 Comparison of HBsAg inhibition rates between prior art and the sequences disclosed herein

[0277] Table 12 Comparison of HBeAg inhibition rates between prior art and the sequences disclosed herein

[0278] Example 6: Inhibitory effect of ASO modified by the modification pattern of this disclosure on HBsAg, HBeAg and HBV DNA in mouse serum.

[0279] In this embodiment, several basic ASO sequences, 1558R2, 1584L2, 1558, 1559, 1529RL, 229, and 1582RL, were selected and cEt modified to obtain cEt-modified ASO products (see Table 13 below). The AAV-HBV mouse model, after infection with recombinant adeno-associated virus (AAV) carrying a replicative 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 new immuno-based therapies and antiviral treatments. In this embodiment, the AAV-HBV mouse model was used to detect the inhibitory effects of the above-mentioned ASO products on HBsAg, HBeAg, and HBV DNA in serum at different time points.

[0280] 1. Experimental Materials

[0281] Test drug:

[0282] Table 13 lists the cEt-modified ASO modifiers in this embodiment.

[0283] Table 13 ASO Modifiers in Animal Experiments

[0284] Preparation of test drug:

[0285] Drug solvent: PBS buffer

[0286] Preparation conditions: sterile environment

[0287] Labeling method: The prepared drug formulation shall be labeled, and the outer packaging shall indicate the topic number, name, concentration, quantity, preparation date, preparer, and storage conditions;

[0288] Storage conditions: Prepare fresh for immediate use; store remaining samples at -80℃.

[0289] Laboratory animal information:

[0290] Species / Strain: AAV-HBV mouse

[0291] Rating: SPF

[0292] Sex: Male

[0293] Quantity: 100

[0294] Age: 7 weeks

[0295] Weight: 19-24g

[0296] Source: Guangdong Zhiyuan Biomedical Technology Co., Ltd.

[0297] Production License Number: SCXK(Guangdong)2021-0057

[0298] Institutional Animal Care and Use Committee (IACUC) review:

[0299] After being received, the experimental animals were housed in Guangzhou Jennio Biotech Co., Ltd. The license number for use is JENNIO(Tianjin Binhai)2019-0002. This project has passed the review of the Experimental Animal Ethics Committee of Guangzhou Jennio Biotech Co., Ltd. The IACUC number is JENNIO-IACUC-2024-A005. The experiment process was carried out strictly in accordance with the requirements of IACUC to ensure animal welfare.

[0300] Feeding and management:

[0301] Feeding conditions: After being received, the experimental animals were housed in Guangzhou Jennio Biotech Co., Ltd. They were housed in cages with the cage specifications of length × width × height = 29.0 cm × 18.5 cm × 13.0 cm; the set temperature range is 20-26°C, the set humidity range is 40%-70%, with automatic lighting and a 12-hour light-dark cycle.

[0302] The standards for feeding environmental conditions refer to the national standard of the People's Republic of China GB14925-2010.

[0303] The animals had free access to food and water. The feed was irradiated and sterilized maintenance feed for experimental rats, 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 a test report for each batch. The drinking water was 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.

[0304] The animal bedding was corncob bedding, provided by Guangzhou Saibenuo Biotech Co., Ltd. The production license number for animal bedding is SCXK(Beijing)2019-0004. The detection of pollutant content in the bedding refers to the national standard of the People's Republic of China GB14924.2-2001, and the bedding supplier provides a test report for each batch. [[ID=2A]]

[0305] The animal cages and bedding were replaced at least once a week. All animal cages and bedding were sterilized by high pressure in a pulsating vacuum sterilizer before entering the barrier environment for use; the animal cage racks were cleaned and disinfected by wiping at least once a week.

[0306] The animal feeding and observation room was cleaned and disinfected every day, including the plate racks, floors, desks, etc.

[0307] The disinfectants used in the barrier environment include: 6.67% benzalkonium chloride solution, 0.5% 84 disinfectant, 75% disinfectant, and 0.08% virucidal solution. These four disinfectants should be used in rotation and should not be mixed.

[0308] 2. Experimental Methods

[0309] 2.1 AAV-HBV mouse modeling

[0310] Two hundred and twenty SPF-grade male C57BL / 6 mice were acclimatized in a barrier facility for seven days and observed daily to ensure they were healthy and free of abnormalities before modeling. The mice were randomly divided into two batches. One batch of 180 mice was injected intravenously via tail vein with rAAV8-1.3HBV (Guangzhou Paizhen Biotechnology Co., Ltd., name: AAV8[HBV-D,ayw](D#2012), batch number: HBV101-6), with each mouse receiving 1×10⁻⁶ HBV. 11 GC / 100μL was used as the low baseline group; the remaining 40 mice were injected via tail vein with rAAV-HBV1.3-mer WT replicon (Wuhan Shumi Brain Science Technology Co., Ltd., batch number: ayw1-P4-230516), 1×10⁻⁶ per mouse. 10 Animals were tested at a concentration of 100 μL (vg / 100 μL) as the high baseline group. Blood was collected from the animals at weeks 4 and 5 (D-14 and D-7) after modeling, centrifuged, and plasma was collected to detect HBV DNA, HBsAg, and HBeAg. Successful modeling was defined as HBV DNA > 10 μL. 5 IU / mL and fluctuation not exceeding 2log 10 HBsAg fluctuation does not exceed 1 log 10 .

[0311] 2.2 Animal grouping and administration

[0312] Trial date definition: The day on which the animal is given the solvent or test drug is defined as day 0 (D 0).

[0313] Animals were grouped based on indicators at week 5 (D-7) of modeling. Seventy-eight animals with successful modeling were randomly divided into 13 groups of six. Statistical differences in HBsAg, HBV DNA, HBeAg, and body weight were ensured among the groups. Drug administration was initiated at week 6 (D-0) after grouping. Details of grouping and drug administration are shown in Table 14.

[0314] Table 14 Grouping and Dosing Details Note: sc: subcutaneous injection.

[0315] 2.3 Observation and Indicator Monitoring

[0316] (1) General observation

[0317] 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.

[0318] (2)Weight

[0319] Adaptation period: Weigh and record the animal upon receipt, and weigh and record the animal on the day the adaptation period ends.

[0320] 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.

[0321] (3) Measurement of serum HBsAg, HBeAg, HBV DNA, and HBsAb levels

[0322] Animals underwent blood collection at the inner canthus of the orbit 4 weeks (D-14), 5 weeks (D-7), before drug administration (D-0), and weekly after drug administration. 200 μL of blood was collected and anticoagulated in EDTA-k2 anticoagulant tubes. Plasma was collected after centrifugation at 1000g for 10 min. Twelve mice in two challenge groups underwent challenge on drug administration on D60. Blood samples of 200 μL were collected from the inner canthus of the orbit on D63, D67, D74, and D81, and anticoagulated in EDTA-k2 anticoagulant tubes. Plasma was collected after centrifugation at 1000g for 10 min. 20 μL of plasma was added to 980 μL of PBS, vortexed, and used to detect HBsAg, HBV DNA, and HBeAg 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℃.

[0323] 2.4 Data Processing and Statistical Analysis

[0324] Experimental data are expressed as mean ± standard deviation (Mean ± SD) and analyzed using GraphPad Prism 8.3 software. Data conformed to a normal distribution and homogeneity of variance, and were tested using one-way ANOVA. When variances were unequal, Welch ANOVA was used for testing. When data did not conform to a normal distribution, the non-parametric Kruskal-Wallis H test was used. A p-value < 0.05 was considered statistically significant.

[0325] 3. Experimental Results

[0326] The changes in HBsAg, HBeAg, HBV DNA, and HBsAb levels in each group of mice over time are shown in Tables 15-21 and Figures 1-4. The results indicate that sequences B1582RL-A3A3, B1584L2-A3A2, B1584L2-A3A3, and B229-A2B2 were significantly more effective than the Yangshen sequence GSK836 in inhibiting HBsAg, HBV DNA, and HBeAg. Among these, B1582RL-A3A3 showed the best effect, achieving HBsAg seroconversion in all mice from day 28 after administration and maintaining this effect until the experimental endpoint, with a reduction of 2.86 log [value missing]. 10 IU / mL, from day 42, achieved HBV DNA negativity in all mice and maintained it until the experimental endpoint, with a reduction of 2.72 log. 10 The concentration was 1 IU / mL, and antibodies were induced in all mice, while the maximum reduction in HBsAg by the positive control GSK836 was 1.39 log. 10 The level was IU / mL, and it began to rebound after day 28, returning to baseline levels by day 49.

[0327] Table 15 Changes in the number of mice that became HBsAg negative in each sequence group Note: "-" indicates that the detection has stopped.

[0328] Table 16 Changes in the number of mice that became HBV DNA-negative in each sequence group Note: "-" indicates that the detection has stopped.

[0329] Table 17 Changes in the number of mice producing antibodies in each sequence group. Note: "-" indicates that the detection has stopped.

[0330] The body weight of the mice is shown in Figure 5. The results indicate that the body weight of the mice in each group increased steadily, and there was no statistically significant difference between the groups.

[0331] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An antisense oligonucleotide, characterized in that, The antisense oligonucleotide consists of 10 to 30 consecutive nucleotides and contains the nucleotide sequence or fragment thereof shown in any of SEQ ID NO:1-89, wherein the antisense oligonucleotide optionally contains at least one modified nucleotide.

2. The antisense oligonucleotide according to claim 1, characterized in that, The antisense oligonucleotide comprises a nucleotide sequence or fragment thereof shown in any of SEQ ID NO:1-7, or a modified sequence thereof.

3. The antisense oligonucleotide according to claim 1 or 2, characterized in that, The antisense oligonucleotide contains a nucleotide sequence that has 1-3 nucleotide substitutions, additions, or deletions compared to the nucleotide sequence shown in any of SEQ ID NO:1-89.

4. The antisense oligonucleotide according to any one of claims 1-3, characterized in that, The at least one modified nucleotide is selected from any one or a combination of two or more of the following: 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'-phosphate groups, and nucleotides containing 5'-phosphate mimics.

5. The antisense oligonucleotide according to any one of claims 1-4, characterized in that, The nucleotide contains a chemical modification at the 2' position of the nucleotide ribose.

6. The antisense oligonucleotide according to claim 5, characterized in that, The chemical modification at the 2' position of the nucleotide ribose is selected from any one or a combination of two or more of the following: 2'-methoxy modification, 2'-O-methoxyethyl modification, 2'-fluoro modification, 2'-benzyloxy modification, 2'-methylcarbonylamino modification, 2'-pyridinemethoxy modification, 2,4'-restricted nucleotide modification, and 2,4'-restricted ethyl nucleotide modification.

7. The antisense oligonucleotide according to any one of claims 1-6, characterized in that, The nucleotides are linked by 3',5'-phosphodiester bonds.

8. The antisense oligonucleotide according to claim 7, characterized in that, The 3',5'-phosphodiester bond contains a thiolated modification.

9. The antisense oligonucleotide according to claim 7, characterized in that, All of the 3',5'-phosphodiester bonds contain thiomodification and form chiral pure 3',5'-thiophosphodiester bonds.

10. The antisense oligonucleotide according to any one of claims 1-9, characterized in that, The antisense oligonucleotide has any or both of the following modifications: A3: Indicates that the first, second and third nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides or nucleotides modified with 2,4'-restricted ethyl. A2: Indicates that the first and second nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides, or nucleotides modified with 2,4'-restricted ethyl. B3: Indicates that the first, second and fourth nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides or nucleotides modified with 2,4'-restricted ethyl. C3: indicates that the first, third and fourth nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides or nucleotides modified with 2,4'-restricted ethyl. B2: Indicates that the first and third nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides, or nucleotides modified with 2,4'-restricted ethyl. D3: Indicates that the first, third and fifth nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides or nucleotides modified with 2,4'-restricted ethyl. E2: indicates that the first and fourth nucleotides at the 5' end and / or 3' end of the antisense oligonucleotide are nucleotides modified with 2'-O-methoxyethyl, nucleotides modified with 2,4'-locked nucleotides, or nucleotides modified with 2,4'-restricted ethyl. A1: Indicates that the first nucleotide at the 5' end and / or 3' end of the antisense oligonucleotide is a nucleotide modified with 2'-O-methoxyethyl, 2,4'-locked nucleotide, or 2,4'-restricted ethyl.

11. The antisense oligonucleotide according to claim 10, characterized in that, The antisense oligonucleotide has any of the following modification patterns: A3A3: This indicates that the 5' end of the antisense oligonucleotide has a modification pattern A3, and the 3' end of the antisense oligonucleotide has a modification pattern A3; A3A2: This indicates that the 5' end of the antisense oligonucleotide has modification A3, and the 3' end of the antisense oligonucleotide has modification A2; A3B2: This indicates that the 5' end of the antisense oligonucleotide has modification A3, and the 3' end of the antisense oligonucleotide has modification B2; A2A2: This indicates that the 5' end of the antisense oligonucleotide has modification A2, and the 3' end of the antisense oligonucleotide has modification A2. A2B2: This indicates that the 5' end of the antisense oligonucleotide has modification A3, and the 3' end of the antisense oligonucleotide has modification B2; B3A3: This indicates that the 5' end of the antisense oligonucleotide has modification type B3, and the 3' end of the antisense oligonucleotide has modification type A3; B3A2: This indicates that the 5' end of the antisense oligonucleotide has modification type B3, and the 3' end of the antisense oligonucleotide has modification type A2; B3A1: This indicates that the 5' end of the antisense oligonucleotide has modification type B3, and the 3' end of the antisense oligonucleotide has modification type A1; B3B2: This indicates that the 5' end of the antisense oligonucleotide has modification type B3, and the 3' end of the antisense oligonucleotide has modification type B2; C3A3: This indicates that the 5' end of the antisense oligonucleotide has a modification of type C3, and the 3' end of the antisense oligonucleotide has a modification of type A3; C3A2: This indicates that the 5' end of the antisense oligonucleotide has modification C3, and the 3' end of the antisense oligonucleotide has modification A2; C3A1: This indicates that the 5' end of the antisense oligonucleotide has modification C3, and the 3' end of the antisense oligonucleotide has modification A1; C3B2: This indicates that the 5' end of the antisense oligonucleotide has modification C3, and the 3' end of the antisense oligonucleotide has modification B2; B2B2: This indicates that the 5' end of the antisense oligonucleotide is modified by method B2, and the 3' end of the antisense oligonucleotide is modified by method B2. D3A3: This indicates that the 5' end of the antisense oligonucleotide has a modification pattern D3, and the 3' end of the antisense oligonucleotide has a modification pattern A3; D3A2: This indicates that the 5' end of the antisense oligonucleotide has modification mode D3, and the 3' end of the antisense oligonucleotide has modification mode A2; D3A1: This indicates that the 5' end of the antisense oligonucleotide has modification mode D3, and the 3' end of the antisense oligonucleotide has modification mode A1; D3B2: This indicates that the 5' end of the antisense oligonucleotide has modification mode D3, and the 3' end of the antisense oligonucleotide has modification mode B2; E2A2: This indicates that the 5' end of the antisense oligonucleotide has a modification pattern E2, and the 3' end of the antisense oligonucleotide has a modification pattern A2; E2B2: This indicates that the 5' end of the antisense oligonucleotide has a modification pattern E2, and the 3' end of the antisense oligonucleotide has a modification pattern B2; Furthermore, the remaining positions of the antisense oligonucleotide are all deoxynucleoside-3'-phosphate esters.

12. The antisense oligonucleotide according to claim 10 or 11, characterized in that, The antisense oligonucleotides are selected from the group consisting of the following nucleotide sequences: SEQ ID NO: 93-102 and 179-276.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an antisense oligonucleotide as described in any one of claims 1-12, and a pharmaceutically acceptable carrier thereof.

14. A medicine box set, characterized in that, The kit includes a box A, which comprises one or both of the antisense oligonucleotide as described in any one of claims 1-12 or the pharmaceutical composition as described in claim 13. Preferably, the kit further includes a pillbox B, which contains one or both of the following (1) and (2): (1) Other drugs that reduce HBV gene expression or compositions containing said drugs that reduce HBV gene expression; (2) Any one or a combination of two 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.

15. The use of the antisense oligonucleotide according to any one of claims 1-12 or the pharmaceutical composition according to claim 13 in the preparation of a medicament for treating HBV infection-related diseases.

16. The application according to claim 15, characterized in that, The HBV infection-related diseases are selected from any one or a combination of two or more of the following groups: chronic hepatitis B, liver fibrosis, cirrhosis, liver cancer, acute hepatitis B, and diseases related to co-infection with HBV and HDV.

17. A method for reducing HBV gene expression or inhibiting HBV replication, characterized in that, The method comprises administering to a sample any one or a combination of two or more of the antisense oligonucleotide as described in any one of claims 1-12, the pharmaceutical composition as described in claim 13, and the kit as described in claim 14; preferably, the method is for non-therapeutic purposes.

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