An antisense oligonucleotide for reducing hbv gene expression and use thereof

Abstract

The present disclosure provides an antisense oligonucleotide for reducing HBV gene expression and use thereof. The antisense oligonucleotide comprises a 5' wing, a central DNA region and a 3' wing; the antisense oligonucleotide satisfies one or more of the following conditions: (i) the nucleotide sequence of the 5' wing comprises the nucleotide sequence shown in any one of SEQ ID NOs: 181-186 or a modified sequence thereof; (ii) the nucleotide sequence of the central DNA region comprises the nucleotide sequence shown in any one of SEQ ID NOs: 13-18 or a modified sequence thereof; (iii) the nucleotide sequence of the 3' wing comprises the nucleotide sequence shown in any one of SEQ ID NOs: 189-194 or a modified sequence thereof. The antisense oligonucleotide of the present disclosure and / or the modified product thereof can significantly reduce the levels of HBsAg, HBeAg and HBV DNA in serum.

Description

Technical Field

[0001] This disclosure relates to the field of nucleic acid drug technology, 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

[0002] Antisense oligonucleotides (ASOs) are short, single-stranded (typically 15-30 nucleotides) DNA or RNA molecules that specifically bind to target DNA and / or target mRNA, thereby regulating the expression of target genes. ASOs are generally classified into two main types: ribonuclease-dependent and sterically hindered. In the case of DNA-based ASOs binding to target mRNA, ribonuclease-dependent ASOs recognize RNA-DNA hybrid double strands through endogenous RNase H enzymes and catalyze the degradation of mRNA; sterically hindered ASOs prevent the translation of target mRNA through a locating mechanism, thereby regulating gene expression.

[0003] The latest generation of ribonuclease-specific active nucleotide exchange (ASO) typically employs a "gapmer" layout. The inner region is DNA (the "gap"), which recruits RNase H enzymes. The flanking regions are chemically modified RNA, which enhances stability and promotes binding to target sequences. Notably, RNase H is present in both the cytoplasm and nucleus; therefore, this type of ASO is particularly suitable for targeting nuclear transcripts (such as immature pre-mRNA and long non-coding RNA (lncRNA)) that are difficult for other nucleic acid drugs (such as siRNA drugs) to penetrate.

[0004] In natural oligonucleotides, nucleotides are linked by phosphodiester bonds. Under physiological conditions, they are particularly sensitive to nucleases. Therefore, natural, unmodified, and structurally unaltered oligonucleotides are easily degraded by nucleases in vivo, resulting in poor biological activity and drug-like properties. Thus, it is necessary to modify oligonucleotides to improve their stability to nucleases, their affinity for RNA, and to promote endocytosis and tissue targeting, thereby effectively regulating the expression of target genes.

[0005] The basic structure of a nucleotide can generally be divided into four parts: the base, the ribose, the phosphate backbone, and the terminal. Examples of known modifications to these four parts are as follows: 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.

[0006] 2) Ribose modification: This mainly involves the modification and substitution of groups at specific positions on the ribose ring. Ribose modification includes, but is not limited to, 2'-position modification, 4'-position modification, 5'-position modification, and isomerization modification. The most common 2'-position modifications are 2'-OMe (2'-methoxy) modification and 2'-F (2'-fluoro) modification. Compared to natural oligonucleotides, oligonucleotides modified with both 2'-OMe and 2'-F exhibit higher Tm values, stronger serum stability, and better activity.

[0007] 3) Modification of the phosphate backbone: The main modification methods include, but are not limited to, modification of thiophosphates; modification via methyl phosphates, selenophosphates, methylboryl phosphates, dithiophosphates, and by replacing the bridging oxygen atoms in the phosphate diester bond linkage with sulfur atoms; and replacing the phosphate ester groups between nucleosides entirely with groups that do not contain phosphorus atoms, such as replacing P atoms with C, S, and N atoms, thereby forming guanidine, S-methylthiourea, or nitrate esters, etc.

[0008] 4) End modification: including but not limited to covalently linking specific groups to the 5' end and / or 3' end of the sense chain, phosphorylation modification of the 5' end of the antisense chain, etc.

[0009] Hepatitis B virus (HBV) is a double-stranded virus that infects only humans and non-human primates, primarily replicating in the liver. HBV infection is a major health problem worldwide, and long-term HBV infection carries a high probability of developing liver fibrosis, cirrhosis, and liver cancer.

[0010] 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. The current rate of functional cure through clinical medication is extremely low; therefore, it is necessary to develop drugs that downregulate HBsAg expression to achieve functional cure. Summary of the Invention

[0011] To address the issue of extremely low functional cure rates in existing clinical medications for HBV infection, this disclosure provides an antisense oligonucleotide that reduces HBV gene expression and its application. Based on the HBV genome sequence, this disclosure designs a series of unique antisense oligonucleotide base sequences and modifies these base sequences using different modification patterns to prepare corresponding antisense oligonucleotide modifiers.

[0012] This disclosure identifies antisense oligonucleotide modifiers that significantly reduce HBV gene expression by modifying the designed antisense oligonucleotide base sequence.

[0013] This disclosure solves the above-mentioned technical problems through the following technical means: This disclosure provides an antisense oligonucleotide comprising a 5' flanking region, a central DNA region, and a 3' flanking region; the antisense oligonucleotide satisfies one or more of the following conditions: (i) The nucleotide sequence of the 5' flanking region comprises the nucleotide sequence shown in any one of SEQ ID NO: 181-186 or a modified sequence thereof, or comprises a modified sequence of SEQ ID NO: 179 or 180; (ii) The nucleotide sequence of the central DNA region comprises the nucleotide sequence shown in any one of SEQ ID NO:13-18 or a modified sequence thereof, or comprises a modified sequence of SEQ ID NO:11 or 12; (iii) The nucleotide sequence of the 3' flanking region comprises the nucleotide sequence shown in any one of SEQ ID NO: 189-194 or a modified sequence thereof, or a modified sequence comprising SEQ ID NO: 187 or 188.

[0014] In some embodiments of this disclosure, the modification of the modified sequence is selected from any or a combination of at least two of the following: 3'-terminal deoxy-thymidine nucleotide, 2'-O-methyl modified nucleotide, 2'-fluorine modified nucleotide, 2'-deoxy-modified nucleotide, locked nucleotide, unlocked nucleotide, configuration-restricted nucleotide, restricted ethyl nucleotide, 5'-methylated modified cytosine nucleotide, baseless nucleotide, 2'-amino-modified nucleotide, 2'-O-allyl-modified nucleotide, 2'-C- Alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinonucleotides, aminophosphates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing oxophosphate groups, nucleotides containing methylphosphate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimics.

[0015] In some embodiments of this disclosure, the modification is selected from any one or a combination of at least two of the following: 2'-O-methoxyethyl modified nucleotides, 2,4'-restricted ethyl modified nucleotides, 2,4'-locked nucleotide modified nucleotides, 5'-methylated cytidine-3'-phosphate, 5'-methylated-2'-O-methoxyethyl cytidine-3'-phosphate, 5'-methylated-2,4'-restricted ethyl cytidine-3'-phosphate, 5'-methylated-2,4'-locked nucleotide cytidine-3'-phosphate, 3',5'-thiophosphodiester bonds, and 3',5'-oxophosphodiester bonds.

[0016] In some embodiments of this disclosure, the modifications of the modification sequence in (i) are selected from any group in the following table:

[0017]

[0018] The modifications in the modification sequence in (ii) are selected from any one of the groups in the table below:

[0019] The modifications in (iii) are selected from any one of the groups in the table below:

[0020]

[0021] Where x represents a nucleotide modified with 2'-O-methoxyethyl, and the specific nucleotide type is consistent with the basic sequence; cEt represents a nucleotide modified with 2,4'-restricted ethyl, and the specific nucleotide type is consistent with the basic sequence; + represents a nucleotide modified with 2,4'-locked nucleotide, and the specific nucleotide type is consistent with the basic sequence; mC represents a 5'-methylated cytidine-3'-phosphate; mc represents a 5'-methylated-2'-O-methoxyethyl cytidine-3'-phosphate; mc(cEt) represents a 5'-methylated-2,4'-restricted ethyl cytidine-3'-phosphate; +mc represents a 5'-methylated-2,4'-locked nucleotide cytidine-3'-phosphate; # indicates a 3',5'-thiophosphodiester bond; # indicates a 3',5'-oxophosphodiester bond; empty or "-" indicates that the position is unmodified; the numbers in the columns representing each modification in the table indicate the nucleotide position of that modification in the corresponding base sequence.

[0022] In some embodiments of this disclosure, the sequence of the antisense oligonucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NO: 1-8; or, the modified sequence is selected from any of the modified sequences in the following table:

[0023]

[0024]

[0025]

[0026]

[0027] Where x represents a nucleotide modified with 2'-O-methoxyethyl, and the specific nucleotide type is consistent with the basic sequence; cEt represents a nucleotide modified with 2,4'-restricted ethyl, and the specific nucleotide type is consistent with the basic sequence; + represents a nucleotide modified with 2,4'-locked nucleotide, and the specific nucleotide type is consistent with the basic sequence; mC represents a 5'-methylated cytidine-3'-phosphate; mc represents a 5'-methylated-2'-O-methoxyethyl cytidine-3'-phosphate; mc(cEt) represents a 5'-methylated-2,4'-restricted ethyl cytidine-3'-phosphate; +mc represents a 5'-methylated-2,4'-locked nucleotide cytidine-3'-phosphate; # indicates a 3',5'-thiophosphodiester bond; # indicates a 3',5'-oxophosphodiester bond; empty or "-" indicates that the position is unmodified; the numbers in the columns representing each modification in the table indicate the nucleotide position of that modification in the corresponding base sequence.

[0028] A second aspect of this disclosure provides a pharmaceutical composition comprising an antisense oligonucleotide as described in the first aspect of this disclosure, and a pharmaceutically acceptable carrier thereof.

[0029] A third aspect of this disclosure provides a kit comprising a box A, wherein box A comprises one or both of the antisense oligonucleotides as described in the first aspect of this disclosure or the pharmaceutical compositions as described in the second aspect of this disclosure.

[0030] In some preferred embodiments of this disclosure, 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 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.

[0031] This fourth aspect of the disclosure provides the use of antisense oligonucleotides as described in the first aspect of the disclosure or pharmaceutical compositions as described in the second aspect of the disclosure in the preparation of medicaments for treating HBV infection-related diseases.

[0032] In some embodiments of this disclosure, 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 diseases related to co-infection with HBV and HDV.

[0033] The fifth aspect of this disclosure provides 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 oligonucleotides as described in the first aspect of this disclosure, the pharmaceutical composition as described in the second aspect of this disclosure, and the kit as described in the third aspect of this disclosure.

[0034] In some preferred embodiments of this disclosure, the method is for non-therapeutic purposes.

[0035] The beneficial effects achieved by this disclosure include at least one of the following: (1) The antisense oligonucleotides and / or their modifications disclosed herein have significant inhibitory effects on HBsAg, which are significantly better than at least one ginseng.

[0036] (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.

[0037] (3) The antisense oligonucleotide modified by the modification mode of this disclosure has significantly improved HBV inhibitory activity compared with antisense oligonucleotides and / or antisense oligonucleotide modified by prior art with sequences that are relatively close. Attached Figure Description

[0038] Figure 1 The sequences C1582DL-E0F3, C1582CL-E2G3, C1582C-E2G3, C1582E-E1L3-M12, C1582EL-E1L3-M12 and C1582DL-E0G3 inhibited HBsAg levels.

[0039] Figure 2 The sequences C1582DL-E0F3, C1582CL-E2G3, C1582C-E2G3, C1582E-E1L3-M12, C1582EL-E1L3-M12 and C1582DL-E0G3 inhibit HBV DNA levels.

[0040] Figure 3 The sequences C1582DL-E0F3, C1582CL-E2G3, C1582C-E2G3, C1582E-E1L3-M12, C1582EL-E1L3-M12 and C1582DL-E0G3 inhibit HBeAg levels.

[0041] Figure 4 The effects of sequences C1582DL-E0F3, C1582CL-E2G3, C1582C-E2G3, C1582E-E1L3-M12, C1582EL-E1L3-M12 and C1582DL-E0G3 on ALT.

[0042] Figure 5 The effects of sequences C1582DL-E0F3, C1582CL-E2G3, C1582C-E2G3, C1582E-E1L3-M12, C1582EL-E1L3-M12 and C1582DL-E0G3 on mouse body weight.

[0043] Figure 6 The study aimed to determine HBsAg levels after sequential administration of C207S-DV32PG101, C1582DL-E0F3, and C1582EL-E1L3-M12.

[0044] Figure 7 The study aimed to determine HBV DNA levels after sequential administration of C207S-DV32PG101, C1582DL-E0F3, and C1582EL-E1L3-M12.

[0045] Figure 8 HBeAg levels after sequential administration of C207S-DV32PG101, C1582DL-E0F3, and C1582EL-E1L3-M12.

[0046] Figure 9 The ALT levels were determined after sequential administration of C207S-DV32PG101, C1582DL-E0F3, and C1582EL-E1L3-M12.

[0047] Figure 10 The body weight of mice after sequential administration of C207S-DV32PG101, C1582DL-E0F3 and C1582EL-E1L3-M12 was measured.

[0048] Figure 11 Following sequential administration of C207S-DV32PG101, C1582DL-E0F3, and C1582EL-E1L3-M12, IFN-γ spots were formed in mouse spleen cells upon antigen stimulation.

[0049] Figure 12 Following sequential administration of C207S-DV32PG101, C1582DL-E0F3, and C1582EL-E1L3-M12, TNF-α spots were formed in mouse spleen cells upon antigen stimulation.

[0050] Figure 13 Following sequential administration of C207S-DV32PG101, C1582DL-E0F3, and C1582EL-E1L3-M12, IL-2 spots were formed in mouse spleen cells upon antigen stimulation.

[0051] Figure 14 Following sequential administration of C207S-DV32PG101, C1582DL-E0F3, and C1582EL-E1L3-M12, the cell frequency (%) of HBsAg-specific B cells was determined by flow cytometry. Detailed Implementation

[0052] 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.

[0053] 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.

[0054] The term “including” is used here to refer to the phrase “including but not limited to” and is used interchangeably with it.

[0055] 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.

[0056] As used herein, the term “about” or “approximately” when applied to one or more target values ​​means a value similar to the reference value. In some embodiments, unless otherwise stated or otherwise apparent from the context, the term “approximately” or “about” means a range of values ​​falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in any direction (unless such a number would exceed 100% of the possible value).

[0057] 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.

[0058] “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.

[0059] 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).

[0060] 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.

[0061] 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.

[0062] 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.

[0063] A "gapmer" refers to a chimeric antisense compound in which an inner region containing multiple nucleotides supporting RNase H cleavage is located between an outer region containing one or more nucleotides, wherein the nucleotides constituting the inner region may be chemically different from the one or more nucleotides constituting the outer region. The inner region may be referred to as a "gap," and each of the 5' and 3' outer regions may be referred to as a "wing."

[0064] 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.

[0065] 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.

[0066] 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).

[0067] "Reducing HBV antigen expression" includes reducing the expression of HBsAg, HBeAg, and / or HBcAg proteins.

[0068] "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 partially reducing 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%.

[0069] 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.

[0070] 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.

[0071] 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).

[0072] 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.

[0073] 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.

[0074] "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.

[0075] 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.

[0076] In this article, unless otherwise specified, any nucleotide position referring to the base sequence, ASO modifier, ASO conjugate, modification position, etc., means the 5' to 3' orientation.

[0077] This disclosure provides an antisense oligonucleotide comprising a 5' flanking region, a central DNA region, and a 3' flanking region; the antisense oligonucleotide satisfies one or more of the following conditions: (i) The nucleotide sequence of the 5' flanking region comprises the nucleotide sequence shown in any one of SEQ ID NO: 181-186 or a modified sequence thereof, or comprises a modified sequence of SEQ ID NO: 179 or 180; (ii) The nucleotide sequence of the central DNA region comprises the nucleotide sequence shown in any one of SEQ ID NO:13-18 or a modified sequence thereof, or comprises a modified sequence of SEQ ID NO:11 or 12; (iii) The nucleotide sequence of the 3' flanking region comprises the nucleotide sequence shown in any one of SEQ ID NO: 189-194 or a modified sequence thereof, or a modified sequence comprising SEQ ID NO: 187 or 188.

[0078] In some embodiments of this disclosure, the modification of the modified sequence is selected from any or a combination of at least two of the following: 3'-terminal deoxy-thymidine nucleotide, 2'-O-methyl modified nucleotide, 2'-fluorine modified nucleotide, 2'-deoxy-modified nucleotide, locked nucleotide, unlocked nucleotide, configuration-restricted nucleotide, restricted ethyl nucleotide, 5'-methylated modified cytosine nucleotide, baseless nucleotide, 2'-amino-modified nucleotide, 2'-O-allyl-modified nucleotide, 2'-C- Alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinonucleotides, aminophosphates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing oxophosphate groups, nucleotides containing methylphosphate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimics.

[0079] In some embodiments of this disclosure, the modification is selected from any one or a combination of at least two of the following: 2'-O-methoxyethyl modified nucleotides, 2,4'-restricted ethyl modified nucleotides, 2,4'-locked nucleotide modified nucleotides, 5'-methylated cytidine-3'-phosphate, 5'-methylated-2'-O-methoxyethyl cytidine-3'-phosphate, 5'-methylated-2,4'-restricted ethyl cytidine-3'-phosphate, 5'-methylated-2,4'-locked nucleotide cytidine-3'-phosphate, 3',5'-thiophosphodiester bonds, and 3',5'-oxophosphodiester bonds.

[0080] In some embodiments of this disclosure, the modifications of the modification sequence in (i) are selected from any group in the following table:

[0081]

[0082] The modifications in the modification sequence in (ii) are selected from any one of the groups in the table below:

[0083] The modifications in (iii) are selected from any one of the groups in the table below:

[0084]

[0085] Where x represents a nucleotide modified with 2'-O-methoxyethyl, and the specific nucleotide type is consistent with the basic sequence; cEt represents a nucleotide modified with 2,4'-restricted ethyl, and the specific nucleotide type is consistent with the basic sequence; + represents a nucleotide modified with 2,4'-locked nucleotide, and the specific nucleotide type is consistent with the basic sequence; mC represents a 5'-methylated cytidine-3'-phosphate; mc represents a 5'-methylated-2'-O-methoxyethyl cytidine-3'-phosphate; mc(cEt) represents a 5'-methylated-2,4'-restricted ethyl cytidine-3'-phosphate; +mc represents a 5'-methylated-2,4'-locked nucleotide cytidine-3'-phosphate; # indicates a 3',5'-thiophosphodiester bond; # indicates a 3',5'-oxophosphodiester bond; empty or "-" indicates no modification; the numbers in the columns representing each modification in the table indicate the nucleotide position of that modification in the corresponding base sequence.

[0086] In some embodiments of this disclosure, the sequence of the antisense oligonucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NO: 1-8; or, the modified sequence is selected from any of the modified sequences in the following table:

[0087]

[0088]

[0089]

[0090]

[0091] Where x represents a nucleotide modified with 2'-O-methoxyethyl, and the specific nucleotide type is consistent with the basic sequence; cEt represents a nucleotide modified with 2,4'-restricted ethyl, and the specific nucleotide type is consistent with the basic sequence; + represents a nucleotide modified with 2,4'-locked nucleotide, and the specific nucleotide type is consistent with the basic sequence; mC represents a 5'-methylated cytidine-3'-phosphate; mc represents a 5'-methylated-2'-O-methoxyethyl cytidine-3'-phosphate; mc(cEt) represents a 5'-methylated-2,4'-restricted ethyl cytidine-3'-phosphate; +mc represents a 5'-methylated-2,4'-locked nucleotide cytidine-3'-phosphate; # indicates a 3',5'-thiophosphodiester bond; # indicates a 3',5'-oxophosphodiester bond; empty or "-" indicates that the position is unmodified; the numbers in the columns representing each modification in the table indicate the nucleotide position of that modification in the corresponding base sequence.

[0092] A second aspect of this disclosure provides a pharmaceutical composition comprising an antisense oligonucleotide as described in the first aspect of this disclosure, and a pharmaceutically acceptable carrier thereof.

[0093] A third aspect of this disclosure provides a kit comprising a box A, wherein box A comprises one or both of the antisense oligonucleotides as described in the first aspect of this disclosure or the pharmaceutical compositions as described in the second aspect of this disclosure.

[0094] In some preferred embodiments of this disclosure, 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 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.

[0095] This fourth aspect of the disclosure provides the use of antisense oligonucleotides as described in the first aspect of the disclosure or pharmaceutical compositions as described in the second aspect of the disclosure in the preparation of medicaments for treating HBV infection-related diseases.

[0096] In some embodiments of this disclosure, 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 diseases related to co-infection with HBV and HDV.

[0097] The fifth aspect of this disclosure provides 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 oligonucleotides as described in the first aspect of this disclosure, the pharmaceutical composition as described in the second aspect of this disclosure, and the kit as described in the third aspect of this disclosure.

[0098] In some preferred embodiments of this disclosure, the method is for non-therapeutic purposes.

[0099] 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.

[0100] 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.

[0101] 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, 1 Administer at doses of 8.5, 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] Antisense oligonucleotides, such as the ASO disclosed herein, can be completely encapsulated in lipid formulations (e.g., LNPs or other nucleic acid-lipid particles).

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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 stilbene-2,2'-disulfonic acid can reduce the recovery of partially thiophosphated dsRNA from liver tissue.

[0113] 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.).

[0114] 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.

[0115] 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.

[0116] 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.

[0117] The dosage forms, carrier compounds, drug carriers, excipients, etc. of the above compositions are described in U.S. Patent US10125369B2, which is incorporated herein by reference.

[0118] 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 HBVASO to a patient with a heterozygous HBV genotype.

[0119] 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.

[0120] 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.

[0121] 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).

[0122] 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.

[0123] 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.

[0124] The nucleotide abbreviations and modification codes used in this article are as follows: A = Deoxyadenosine-3'-phosphate a=2'-O-methoxyethyl adenosine-3'-phosphate a(cEt) = 2,4'-restricted ethyl adenosine-3'-phosphate +a=2,4'-locked adenosine-3'-phosphate ester G = Deoxyguanosine-3'-phosphate g = 2'-O-methoxyethylguanosine-3'-phosphate g(cEt) = 2,4'-restricted ethylguanosine-3'-phosphate +g=2,4'-guanosine-3'-phosphate C=Deoxycytidine-3'-phosphate mC=5'-methylcytidine-3'-phosphate mc = 5'-methylated-2'-O-methoxyethylcytidine-3'-phosphate mc(cEt) = 5'-methylated-2,4'-restricted ethylcytidine-3'-phosphate +mc=5'-methylated-2,4'-cytidine-3'-phosphate T = deoxythymidine-3'-phosphate t = 2'-O-methoxyethylthymidine-3'-phosphate t(cEt) = 2,4'-restricted ethylthymidine-3'-phosphate +t=2,4'-locked thymidine-3'-phosphate =3',5'-thiophosphate diester bond #=3',5'-oxophosphodiester bond x represents a nucleotide modified with 2'-O-methoxyethyl, including any one of a, g, and t, with the specific nucleotide type consistent with the basic sequence.

[0125] Nucleotides modified with +=2,4'-locked nucleotides, including any one of +a, +g, and +t, with the specific nucleotide type consistent with the basic sequence.

[0126] Nucleotides with cEt=2,4'-restricted ethyl modification, including any one of a(cEt), g(cEt) and t(cEt), with the specific nucleotide type consistent with the basic sequence.

[0127] Example

[0128] Example 1: The inhibitory effect of unmodified ASO (basic sequence) on HBV gene

[0129] In this embodiment, eight unmodified ASOs were designed targeting the HBV gene sequence NC_003977.2 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 the unmodified ASOs with better inhibitory effects were screened out.

[0130] 1. Synthesis of unmodified ASO sequences

[0131] 1.1 Instruments and reagents: The Qingke 192 P model DNA / RNA automated synthesizer, whose solid support is a universal carrier of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (Cytiva).

[0132] 1.2 Synthesis Method: Solutions of the following nucleotide monomers 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), and DMT-T phosphorus amide monomer (Formula 4).

[0133]

[0134] The specific steps are as follows: A solid support is loaded into the designated position of the synthesizer, and after several synthesis cycles, the product with all hydroxyl groups protected is obtained; the synthesis cycle includes (1) deprotection, (2) coupling, (3) oxidation and (4) hydroxyl protection: (1) Deprotection The DMT protecting group was removed using a 3% dichloroacetic acid toluene solution as a deprotecting agent, followed by washing with acetonitrile.

[0135] (2) Coupling

[0136] Each nucleotide monomer was coupled using a 0.25 M acetonitrile solution of 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.

[0137] (3) Oxidation

[0138] Oxidation was performed using a 0.05 M iodine pyridine / water (90 / 10) solution as the oxidant, followed by rinsing with acetonitrile.

[0139] (4) Hydroxyl protection

[0140] Hydroxyl protection was performed using a 10% tetrahydrofuran solution of acetic anhydride (CAP A) and tetrahydrofuran / pyridine / aziridine-methylimidazolium 4 / 10 / 16 (v / v / v) (CAP B) as hydroxyl protecting agents, followed by rinsing with acetonitrile.

[0141] Repeat the above steps, synthesizing each nucleotide in the specific order of each sequence, so that the solid support carries the ASO product with hydroxyl protected.

[0142] (5) Use a 3% dichloroacetic acid toluene solution as a deprotection agent to remove the DMT protecting group of the last nucleotide, and then wash with acetonitrile.

[0143] (6) Ammonolysis and purification

[0144] The solid support was transferred to the reactor, concentrated ammonia (25-28%) was added, and the mixture was kept at 60°C for 12 h for ammonolysis. The system was then cooled to room temperature, 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.

[0145] 2. Experimental Materials

[0146] 2.1 Test Substances: The unmodified ASO base sequences listed in Table 1 were all synthesized according to the above method. The positive control sequence is ISIS505358 from patent US8642752B2, and the corresponding unmodified ASO ID in this embodiment is APC-B.

[0147] Table 1. Unmodified ASO base sequences

[0148] 2.2 Cells: HepG2.2.15 cells (Shanghai WuXi AppTec Co., Ltd.)

[0149] 2.3 Drug solvent: sterile enzyme-free water, Opti-MEM (gibco).

[0150] 3. Experimental Methods

[0151] 3.1 Cell Culture

[0152] Subculture: HepG2.2.15 cells were subcultured in DMEM / F12 medium containing 10% fetal bovine serum, 370 μg / ml GENETICN, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin. The cells were incubated at 37°C in a cell culture incubator containing 5% CO2, and subcultured every three days. After digestion with 0.25% trypsin, the cells were centrifuged at 800 r / min for 3 min, the supernatant was discarded, and fresh medium was added for further subculturing.

[0153] 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.

[0154] 3.2 Cell transfection

[0155] Transfection reagent preparation: Mix Lipofectamine RNAiMAX and Opti-MEM at a volume ratio of 2:98 and vortex to mix.

[0156] 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.

[0157] 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.

[0158] Add the prepared transfection complex to each 96-well cell culture plate (15 μL per well, 3 replicates per sample). The ASO screening concentration is 1 nM. For the transfection control group, add 15 μL of the prepared transfection reagent to each well, 3 replicates. Then add 135 μL of cell suspension (containing 2.25 × 10⁻⁶ cells per well) to each well. 4 (1 cell), mix thoroughly, and then incubate in a 37°C, 5% CO2 cell culture incubator.

[0159] 3.3 Detection of HBsAg and HBeAg in cell supernatant

[0160] 1) Collection of cell supernatant

[0161] The medium was changed on day 3 after cell transfection, and the cell supernatant was collected for the detection of HBsAg and HBeAg levels.

[0162] 2) Quantitative detection of HBsAg and HBeAg

[0163] The concentrations of HBsAg and HBeAg were detected using the Hepatitis B virus e antigen detection kit (Antu Bio) and the Hepatitis B virus surface antigen detection kit (Antu Bio). The specific operating steps are as follows: a. Allow the kit and test samples to return to room temperature.

[0164] b. Add 50 μL each of the test sample, standard, negative control, and positive control to a well plate.

[0165] c. Add 50 μL of enzyme conjugate to each well. Mix thoroughly and incubate at 37°C for 60 min.

[0166] 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.

[0167] 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.

[0168] f. Measure the luminescence value on an ELISA reader.

[0169] 3.4 Calculate the inhibition rate

[0170] The HBsAg inhibition rate and HBeAg inhibition rate were calculated using the following formulas: HBsAg inhibition rate (%) = (1 - HBsAg expression level in sample / HBsAg expression level in control group in the same plate) × 100%.

[0171] HBeAg inhibition rate (%) = (1 - HBeAg expression level in sample / HBeAg expression level in control group in the same plate) × 100%.

[0172] 4. Experimental Results

[0173] Experimental results showed that 1582RL, 1584L2, 1582C, 1582D, 1582E and 1584C had significantly better inhibitory effects on HBsAg and HBeAg than Yangshen APC-B (Table 2).

[0174] Table 2. Inhibition rate results of ASO on HBsAg and HBeAg

[0175] Among them, the asterisks in the table above The meaning of the number is as commonly understood in the field, namely, the level of statistical significance. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This means p < 0.0001.

[0176] Example 2: Synthesis of LNA-modified ASO compounds

[0177] A total of 8 LNA-modified ASO sequences were designed, as shown in Table 3. The positive parameter sequence is ISIS505358 from US8642752B2, numbered APC-1.

[0178] 1. Instruments and reagents

[0179] The Qingke 192 P model DNA / RNA automated synthesizer uses cross-linked polystyrene beads as its solid-phase carrier, model Primer support 5G Unylinker 350 (Cytiva).

[0180] 2. Synthesis Method

[0181] 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).

[0182] The specific steps are as follows: A solid support is loaded into the designated position of the synthesizer, and the corresponding hydroxyl-protected product is obtained after several synthesis cycles; the synthesis cycle includes (1) deprotection, (2) coupling, (3) sulfidation and (4) hydroxyl protection: (1) Deprotection The DMT protecting group was removed using a 3% dichloroacetic acid toluene solution as a deprotecting agent, followed by washing with acetonitrile.

[0183] (2) Coupling

[0184] Each nucleotide monomer was coupled using a 0.25 M acetonitrile solution of 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.

[0185] (3) Sulfidation

[0186] The sulfidation was carried out using a pyridine solution of 3% hydroxanthin as the sulfiding agent, followed by rinsing with acetonitrile.

[0187] (4) Hydroxyl protection

[0188] Hydroxyl protection was performed using a 10% tetrahydrofuran solution of acetic anhydride (CAP A) and tetrahydrofuran / pyridine / aziridine-methylimidazolium 4 / 10 / 16 (v / v / v) (CAP B) as hydroxyl protecting agents, followed by rinsing with acetonitrile.

[0189] Repeat the above steps, synthesizing each nucleotide in the specific order of each sequence, so that the solid support carries the ASO product with hydroxyl protected.

[0190] (5) Use a 3% dichloroacetic acid toluene solution as a deprotection agent to remove the DMT protecting group of the last nucleotide, and then wash with acetonitrile.

[0191] (6) Ammonolysis and purification

[0192] The solid support was transferred to the reactor, concentrated ammonia (25-28%) was added, and the mixture was kept at 60°C for 12 h and then cooled to room temperature. The mixture was then filtered and 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.

[0193] The LNA-modified ASO derivatives shown in Table 3 below were synthesized sequentially using the methods described above.

[0194] Table 3 LNA-modified ASO modifiers

[0195] Note: The numbers in the columns representing each modification in the table indicate the nucleotide position of that modification in the corresponding base sequence.

[0196] Example 3: Inhibitory effect of LNA-modified ASO derivatives on HBsAg and HBeAg

[0197] The LNA-modified ASO modifiers synthesized in Example 2 were transfected into HepG2.2.15 cells via lipid nanoparticles (LNPs), and the inhibitory effects of each ASO modifier on HBsAg and HBeAg were detected by ELISA.

[0198] 1. Experimental Materials

[0199] 1.1 Test substance: LNA-modified ASO derivatives in Table 3 (synthesized according to the method in Example 2).

[0200] 1.2 Cells: HepG2.2.15 cells.

[0201] 1.3 Drug solvent: sterile enzyme-free water, Opti-MEM (gibco).

[0202] 2. Experimental Methods

[0203] Referring to Example 1, the ASO screening concentration was 1 nM.

[0204] 3. Experimental Results

[0205] Experimental results showed that B1582RL-GP01+m, B1584L2-GP01+m, 1582C-GP1+m, 1582D-GP1+m, 1582E-GP1+m, and 1584C-GP1+m all exhibited significantly better inhibitory effects on HBsAg and HBeAg than the positive control sequence APC-1 (Table 4).

[0206] Table 4. Inhibition rates of LNA-modified ASO derivatives against HBsAg and HBeAg.

[0207] Among them, the asterisks in the table above The meaning of the number is as commonly understood in the field, namely, the level of statistical significance. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This means p < 0.0001.

[0208] Example 4: Effects of ASO modifiers with different modification modes of cEt and / or MOE combination on cellular Caspase 3 / 7 activity

[0209] In this embodiment, eight unmodified ASO base sequences—1582RL, 1584L2, 1582C, 1582D, 1582E, 1582F, 1582G, and 1584C—were combined with cEt and MOE modifications using the modification pattern designed in this disclosure, resulting in 136 ASO modifiers with combined cEt and MOE modifications. After transfection into HepG2 cells via lipid nanoparticles (LNPs), the effect of each ASO modifier on the activity of cellular Caspase 3 / 7 was detected using a Caspase-Glo 3 / 7 kit.

[0210] 1. Experimental Materials

[0211] 1.1 Test Substances: The ASO modifiers listed in Table 5 are ASO modifiers obtained by combining cEt and / or MOE modifications of 8 basic ASO sequences (SEQ ID NO: 1~8) using different modification modes disclosed in this invention. The positive control sequence is ISIS505358 in patent US8642752B2, and the corresponding ASO modifier ID in this embodiment is APC-1.

[0212] Table 5. ASO modifiers obtained by combining cEt and / or MOE with different modification modes

[0213]

[0214]

[0215]

[0216]

[0217]

[0218] Note: The numbers in the columns representing each modification in the table indicate the nucleotide position of that modification in the corresponding base sequence.

[0219] 1.2 Synthesis of ASO Modifiers: ASO-modified compounds were synthesized according to Example 1, wherein MOE or cEt monomers were used in the synthesis of MOE and / 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:

[0220] When synthesizing sequences containing 5' methylated C and 5' methylated MOE modified C, 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:

[0221] 1.3 Cells: HepG2 cells

[0222] Drug solvent: sterile enzyme-free water, Opti-MEM (gibco).

[0223] 2. Experimental Methods

[0224] 2.1 Cell Culture

[0225] Passaging: HepG2 cells were passaged in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and placed in a cell culture incubator at 37°C with 5% CO2. Cells were passaged every three days. After digestion with 0.25% trypsin, the cells were centrifuged at 800 r / min for 3 min, the supernatant was discarded, and fresh medium was added for further passage.

[0226] Plate culture: HepG2 cells were plated and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0227] 2.2 Cell transfection

[0228] Transfection reagent preparation: Mix Lipofectamine RNAiMAX and Opti-MEM at a volume ratio of 0.3:4.7 and vortex to mix.

[0229] 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.

[0230] 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.

[0231] Add the prepared transfection complex to each 96-well cell culture plate (10 μL per well, 3 replicates per sample). The final ASO concentration is 30 nM. For the transfection control group, add 10 μL of the prepared transfection reagent to each well, 3 replicates. Then add 90 μL of cell suspension (containing 2.25 × 10⁻⁶ cells per well) to each well. 4 (1 cell), mix thoroughly. Add 3 wells containing only culture medium as a blank control. Incubate the culture plate at 37°C in a 5% CO2 cell culture incubator for 24 h.

[0232] 2.3 Detection of Caspase 3 / 7 activity in cells

[0233] 1) Before the experiment, equilibrate the Caspase-Glo® 3 / 7 buffer and Caspase-Glo® 3 / 7 lyophilized substrate to room temperature. Pour the Caspase-Glo® 3 / 7 buffer into the brown bottle containing the Caspase-Glo® 3 / 7 substrate. Mix by rotating or inverting until the substrate is completely dissolved, forming the Caspase-Glo® 3 / 7 reagent.

[0234] 2) Remove the 96-well plate containing the cultured cells from the incubator and allow it to equilibrate to room temperature.

[0235] 3) Add 100 μL of Caspase-Glo® 3 / 7 reagent to each well of the 96-well plate.

[0236] 4) Gently mix the contents of the micropores for 30 seconds using a plate shaker at 300-500 rpm. Incubate at room temperature for 30 minutes.

[0237] 5) Measure the chemiluminescence value on an ELISA reader.

[0238] 2.4 Data Processing

[0239] The formula for calculating the Caspase 3 / 7 activity percentage is as follows: Caspase 3 / 7 activity percentage (%) = (Chemical emission value of experimental group - luminescence value of blank group) / (Chemical emission value of transfected control group in the same plate - luminescence value of blank group) × 100%.

[0240] 3. Experimental Results

[0241] Experimental results showed that at higher transfection concentrations, such as 30 nM, 15 sequences C1584A-A3A3-O23O23, C1582A-A3A3-O23O23, C1584A-A3A3-O23O2, C1582A-A3A3-O23O2, C1584C-A3A3, C1582D-F3E0, C1584C-E2G3, C1584C-F3E0, C1584A-F2F2, C1584C-E1G3, C1582A-F2F2, C1584C-E3E0, C1584A-E1E3-O23O2, C1582D-E3E0-M, and C1584A-E1E3 could significantly induce cellular caspase. The percentage of Caspase 3 / 7 activity was 1.5 times higher than that of the Yangshen sequence APC-1, while the other sequences showed lower Caspase 3 / 7 activation ability, indicating that these sequences had good cell tolerance (Table 6). The percentage of Caspase 3 / 7 activity induced by C1582D-E2G3, C1582D-E3E0, C1584A-E1F2, C1584C-F2E0, C1584C-E0F3, C1582E-F2N2, C1582E-G2F2, C1582E-E0J3, and C1584C-E0H3 was significantly higher than that induced by the Yangshen sequence APC-1.

[0242] Table 6. Relative Caspase 3 / 7 activities of each ASO modifier

[0243]

[0244]

[0245] Among them, the asterisks in the table above The meaning of the number is as commonly understood in the field, namely, the level of statistical significance. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This means p < 0.0001.

[0246] Example 5: Inhibitory effects of ASO modifiers modified with different cEt and / or MOE combinations on HBsAg and HBeAg.

[0247] In this embodiment, the unmodified ASO base sequences 1582RL, 1584L2, 1582C, 1582D, 1582E, 1582F, 1582G, and 1584C were modified using the modification modifiers disclosed herein with cEt and / or MOE combinations, resulting in 121 ASO modifiers with cEt and / or MOE combinations. After transfection into HepG2.2.15 cells via lipid nanoparticles (LNPs), the inhibitory effects of each ASO modifier on HBsAg and HBeAg were detected using ELISA.

[0248] 1. Experimental Materials

[0249] 1.1 Test Substances: ASO modifiers numbered 1-2 in Table 7 and ASO modifiers numbered 2-6, 10, 12-15, 21-36, 38, 41-121, 123, 126-133, and 136 in Table 5. The first positive ion ASO is ISIS505358 in patent US8642752B2, with the corresponding ASO ID APC-1.

[0250] Table 7. ASO modifiers obtained by combining cEt and / or MOE with different modification modes

[0251] Note: The numbers in the columns representing each modification in the table indicate the nucleotide position of that modification in the corresponding base sequence.

[0252] 1.2 Synthesis of ASO Modifiers: ASO modifiers were synthesized according to the method in Example 4.

[0253] 1.3 Cells: HepG2.2.15 cells

[0254] 1.4 Drug solvent: sterile enzyme-free water, Opti-MEM (gibco)

[0255] 2. Experimental Methods

[0256] Referring to Example 1, the screening concentration was 1 nM.

[0257] 3. Experimental Results

[0258] The experimental results showed that 70 modifiers had a better inhibitory effect on HBsAg than Yangshen APC-1 (Table 8), and 73 ASO modifiers had a better inhibitory effect on HBeAg than Yangshen APC-1 (Table 9).

[0259] Table 8. Inhibition rate of ASO-modified HBsAg (%)

[0260]

[0261] Table 9. Inhibition rate of ASO-modified HBeAg (%)

[0262]

[0263] Among them, the asterisks in the table above The meaning of the number is as commonly understood in the field, namely, the level of statistical significance. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This means p < 0.0001.

[0264] Example 6: Comparison of the inhibitory effects of unmodified or modified ASO on the HBV gene in the prior art.

[0265] 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 1582RL, 1582C, 1582D, 1582E, 1582F, 1582G, and 1584C, as well as ASO with the modified pattern disclosed in this invention.

[0266] 1. Experimental Materials

[0267] 1.1 Test substance: (1) Prior art disclosed sequences SEQ ID NO: 9 and SEQ ID NO: 10 (AGGTGAAGCGAAGTGC) and the patent disclosed sequences SEQ ID NO: 1-8.

[0268] (2) The ASO modifier modified by LNA using the modification mode of this disclosure in Example 2.

[0269] (3) The ASO modifiers modified by combining cEt and MOE using the modification mode of this disclosure in Example 5.

[0270] 1.2 Cells: HepG2.2.15 cells

[0271] 1.3 Drug solvent: sterile enzyme-free water, Opti-MEM (gibco)

[0272] 2. Experimental Methods

[0273] Referring to Example 1, the concentration for the single-concentration screening experiment was set to 1 nM.

[0274] 3. Experimental Results

[0275] Experimental results show that the inhibition rates of HBsAg and HBeAg by the disclosed 1582RL, 1584L2, 1582C, 1582D, 1582E, and 1584C are significantly better than those of the sequence-similar P1583 (SEQ ID NO: 10) and APC-B. After cEt modification and cEt and MOE combination modification, the inhibition rates are further significantly improved.

[0276] Table 10 Comparison of HBsAg inhibition rates between prior art and the sequences disclosed herein.

[0277] Table 11 Comparison of HBeAg inhibition rates between prior art and the sequences disclosed herein

[0278] Example 7: Inhibitory effect of ASO modified by the modification pattern disclosed herein on HBsAg, HBeAg and HBV DNA in mouse serum.

[0279] In this embodiment, some basic ASO sequences 1582C, 1582D, and 1582E were selected, and these basic sequences were modified with cEt and / or LNA to obtain ASO modifiers with cEt and MOE, and LNA and MOE combined modifications (see Table 12 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 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 modifiers on HBsAg, HBeAg, and HBV DNA in serum at different time points.

[0280] 1. Experimental Materials

[0281] Test drug: In this embodiment, the ASO modifiers modified by cEt, cEt and MOE combined modification, and LNA and MOE combined modification are ASO modifier C1582E-E1L3-M12 with serial number 72 in Table 5, and ASO modifiers with serial numbers 1 to 5 in the table below. The first positive ASO is ISIS505358 in patent US8642752B2, and the corresponding ASO ID is APC-1.

[0282] Table 12 ASO Modifiers in Animal Experiments

[0283] Note: The numbers in the columns representing each modification in the table indicate the nucleotide position of that modification in the corresponding base sequence.

[0284] Preparation of test drug: Drug solvent: PBS buffer Preparation conditions: sterile environment Storage conditions: Prepare fresh for immediate use; store remaining samples at -80℃.

[0285] 2. Experimental Methods

[0286] 2.1 AAV-HBV mouse modeling

[0287] SPF-grade male C57BL / 6 mice were purchased from Guangdong Zhiyuan Biomedical Technology Co., Ltd. They were acclimatized in a barrier facility for 7 days and observed routinely. After confirming the mice were healthy and without abnormalities, modeling was performed. Mice were injected intravenously with rAAV-HBV1.3-merWT replicon (Wuhan Shumi Brain Science Technology Co., Ltd., batch number: ayw1-P4-230516), 1×10⁻⁶ per mouse. 10 vg / 100μL. Blood was collected, centrifuged, and plasma was collected at weeks 4 and 5 (D-14 and D-7) after modeling, and HBV DNA, HBsAg, and HBeAg were measured.

[0288] 2.2 Animal grouping and administration

[0289] Experimental date definition: The day on which the animal is given the solvent or test drug is defined as day 0 (D 0).

[0290] Mice with successful modeling were randomly divided into groups of 6 mice each, based on indicators at week 5 (D-7) of modeling. Statistical differences in HBsAg, HBV DNA, HBeAg, and body weight were ensured among the groups. After grouping, the ASO modifier was administered subcutaneously at week 6 (D0). The dosage was 20 mg / kg, administered on D0 / 3 / 7 / 15 / 21. The solvent control group received an equal volume of PBS.

[0291] 2.3 Observation and Indicator Monitoring

[0292] (1) General observation

[0293] During the modeling and experimental periods, the animals were observed and their observation records were kept daily. The observations included: whether the animals were dead or near death, their feed and water intake, external injuries, feces, appearance and coat, mental state, and activity level.

[0294] (2) Weight

[0295] Adaptation period: Weigh and record the animal upon receipt, and weigh and record the animal on the day the adaptation period ends.

[0296] 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.

[0297] (3) Measurement of serum HBsAg, HBeAg, HBV DNA, HBsAb, and ALT levels

[0298] Blood samples of 200 μL were collected from the inner canthus of the eye of animals 4 weeks (D-14), 5 weeks (D-7), before drug administration (D-0), and weekly after drug administration. The blood was anticoagulated in EDTA-K2 anticoagulant tubes and collected after centrifugation at 1000 g for 10 min. 20 μL of plasma was added to 980 μL of PBS, vortexed, and used to detect HBsAg, HBV DNA, HBeAg, and ALT levels. Another 15 μL of plasma was added to 210 μL of PBS, vortexed, and used to detect HBsAb levels. All processed samples were tested by Guangzhou Huayin Medical Laboratory Center Co., Ltd., and any remaining plasma was stored at -80℃.

[0299] 2.4 Data Processing and Statistical Analysis

[0300] 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. 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.

[0301] 3. Experimental Results

[0302] The experimental results are shown in Figures 1-5The results showed that sequences C1582DL-E0F3, C1582CL-E2G3, C1582E-E1L3-M12, and C1582EL-E1L3-M12 were comparable to APC-1 in inhibiting HBsAg levels; sequences C1582DL-E0F3 and C1582CL-E2G3 were superior to APC-1 in inhibiting HBV DNA levels, and sequence C1582EL-E1L3-M12 was comparable to APC-1 in inhibiting HBV DNA levels; sequences C1582DL-E0F3 and C1582C-E2G3 were comparable to APC-1 in inhibiting HBeAg levels, and sequences C1582CL-E2G3, C1582E-E1L3-M12, and C1582EL-E1L3-M12 were superior to APC-1 in inhibiting HBeAg levels. Except for C1582DL-E0F3, which showed a transient and slight increase in ALT on day 27 after drug administration, no other sequences showed a significant increase in ALT, indicating good safety. The weight of mice in each sequence group increased steadily.

[0303] Example 8: The inhibitory effect of sequential administration of anti-hepatitis B siRNA and ASO modified by the modification pattern disclosed herein on HBsAg, HBeAg and HBV DNA in mouse serum and its effect on host immune reconstitution.

[0304] This embodiment exemplarily selects some sequences, such as sequences C1582DL-E0F3 and C1582EL-E1L3-M12 from Example 7, as shown in Table 12. The inhibitory effects of sequential administration of anti-hepatitis B siRNA and these ASO sequences on serum HBsAg, HBeAg, and HBV DNA at different time points, and their influence on the reconstruction of adaptive immune function in mice, were evaluated using an AAV-HBV mouse model.

[0305] 1. Experimental Materials

[0306] Test drug: The sequences C1582DL-E0F3 and C1582EL-E1L3-M12 in Example 7, and the sequence C207S-DV32PG101 in patent CN119799705B.

[0307] Preparation of test drug: Drug solvent: PBS buffer Preparation conditions: sterile environment Storage conditions: Prepare fresh for immediate use; store remaining samples at -80℃.

[0308] 2. Experimental Methods

[0309] Same as Example 7, grouping and administration details are shown in Table 13.

[0310] Table 13 Group Setup and Dosing Details

[0311] Note: sc: subcutaneous injection.

[0312] 2.3 Observation and Indicator Monitoring

[0313] (1) General observation

[0314] During the modeling and experimental periods, the animals were observed and their observation records were kept daily. The observations included: whether the animals were dead or near death, their feed and water intake, external injuries, feces, appearance and coat, mental state, and activity level.

[0315] (2) Weight

[0316] Adaptation period: Weigh and record the animal upon receipt, and weigh and record the animal on the day the adaptation period ends.

[0317] 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.

[0318] (3) Measurement of serum HBsAg, HBeAg, HBV DNA, HBsAb, and ALT levels

[0319] Blood samples of 200 μL were collected from the inner canthus of the eye of animals 4 weeks (D-14), 5 weeks (D-7), before drug administration (D-0), and weekly after drug administration. The blood was anticoagulated in EDTA-K2 anticoagulant tubes and collected after centrifugation at 1000 g for 10 min. 20 μL of plasma was added to 980 μL of PBS, vortexed, and used to detect HBsAg, HBV DNA, HBeAg, and ALT levels. Another 15 μL of plasma was added to 210 μL of PBS, vortexed, and used to detect HBsAb levels. All processed samples were tested by Guangzhou Huayin Medical Laboratory Center Co., Ltd., and any remaining plasma was stored at -80℃.

[0320] (4) Detection of hepatitis B antigen-specific T cells in mouse spleen using elispot assay

[0321] a. Isolation of spleen mononuclear cells

[0322] Spleen tissue was harvested from the animal on day 84 and cut into 1-2 mm pieces. 3 Tissue blocks of a certain size were filtered through a 70 μm filter membrane to collect cells. Cells were counted in each isolated cell sample. Each tissue sample should yield at least 1.5 × 10⁻⁶ cells. 7 Each cell.

[0323] b. Detection of Elispot IFN-γ, IL-2, and TNF-α in splenic mononuclear cells

[0324] A. Cell incubation: Splenic mononuclear cells were incubated under three conditions: solvent control (DMSO), HBsAg peptide library, and positive control. The peptide library concentration was 2 μg / well. Cells were seeded into ELISpot IFN-γ, IL-2, and TNF-α plates, respectively. The final volume per well was 100 μL, the medium was 1640, and the cells were incubated in a cell culture incubator for 20 h.

[0325] B. Biotin incubation: Empty the plate to remove cells, wash with PBS (200 μL / well, 5 times). Dilute the detection antibodies (biotin-conjugated antibodies against IFN-γ, IL-2, and TNF-α) with PBS-0.5% fetal bovine serum to 1 μg / mL. Add 100 μL / well and incubate at room temperature for 2 h.

[0326] C. Streptavidin incubation: Remove the liquid and wash with PBS, 200 μL / well, 5 times. Dilute streptavidin-ALP (1:1000) in PBS-0.5% fetal bovine serum and add to the wells, 100 μL / well. Incubate at room temperature for 1 h.

[0327] D. Washing the plate: Remove the plate and wash it 5 times with PBS, 200 μL / well, 5 times; E. Substrate: Add 100 μL of substrate solution per well (filtered through a 0.45 μm filter), observe for 3–7 min until obvious spots appear, then wash the plate. Store all plates at room temperature away from light.

[0328] F. Scan and count the number of spots in the ELISpot reader.

[0329] (5) Flow cytometry detection of hepatitis B antigen-specific B cells in mouse spleen

[0330] a. Cell preparation: Prepare 3 × 10 6 A single splenic mononuclear cell.

[0331] b. Flow cytometry detection

[0332] A. Washing solution preparation: Prepare PBS containing 1% BSA (the PBS used for washing below is PBS containing 1% BSA).

[0333] B. Add 1 μL L / D (100×Zombie NIR™ Fixable Viability) dye to each group of cells, incubate at room temperature in the dark for 20 min, wash with 0.5 ml PBS, centrifuge at 500g for 5 min, and discard the supernatant.

[0334] C.FC blocking: Prepare 100 μL of each cell suspension, add 0.5 μL of TruStain FcX™ PLUS (anti-mouse CD16 / 32), incubate at 4°C for 10 min (without washing), and then incubate with antibody.

[0335] D. Add 2.5 μL each of Anti-CD19, Anti-B220, Anti-CD38, and Anti-CD27 to each group of cells, add biotinylated HBsAg (final concentration 10 ng / mL), incubate for 30 min, centrifuge at 500g (model 14D) for 2 min, wash twice with PBS, and resuspend in 200 μL PBS.

[0336] E. Streptavidin binding: Add streptavidin (final concentration 20 ng / mL), incubate for 20 min, centrifuge at 500g (model 14D) for 2 min, wash twice with PBS, and resuspend in 400 μL PBS.

[0337] F. Flow cytometry analysis: HBsAg-specific MBC response levels were detected in CD19 and B220 double-positive cell populations with co-expression of CD38 and CD27.

[0338] G. Preparation of single staining tubes: Prepare single staining tubes for L / D, Anti-CD19, Anti-B220, Anti-CD38, Anti-CD27, and PE-CY7 streptavidin.

[0339] 2.4 Data Processing and Statistical Analysis

[0340] 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. 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.

[0341] 3. Experimental Results

[0342] The results of the pharmacodynamic experiments are shown in Figure 6-10The results showed that sequential administration of C1582DL-E0F3 and C1582EL-E1L3-M12 significantly reduced HBeAg levels. Sequential administration of C207S-DV32PG101, C1582DL-E0F3, and C1582EL-E1L3-M12 maintained low levels of HBsAg and HBV DNA, especially HBV DNA. In the non-sequential administration group, HBV DNA began to rebound at day 83. Based on previous experimental experience, the rebound of HBV DNA often indicates a rebound of HBsAg. Sequential administration of C1582DL-E0F3 showed a slight increase in ALT, and the mice's body weight increased steadily.

[0343] The results of the immunoassay are shown in Figures 11-14 The results showed that sequential administration of C1582DL-E0F3 and C1582EL-E1L3-M12 elicited a strong T-cell response in mouse spleen cells upon antigen stimulation, while the negative control group showed almost no spot formation, suggesting that the body produced an HBV antigen-specific T-cell immune response. Flow cytometry analysis of HBsAg-specific B cells showed that the proportion of HBsAg-specific B cells was significantly increased after sequential administration of C1582EL-E1L3-M12 compared to the PBS group.

[0344] In this disclosure, when the sequences shown in Tables 3, 5, 7, and 12 are inconsistent with the sequence appendix attached to the specification, the sequence appendix shall prevail.

[0345] While specific embodiments of this disclosure 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 this disclosure. Therefore, the scope of protection of this disclosure is defined by the appended claims.

[0346] Sequence Appendix

[0347]

[0348]

[0349]

[0350]

[0351] .

Claims

1. An antisense oligonucleotide, characterized in that, The antisense oligonucleotide comprises a 5' flanking region, a central DNA region, and a 3' flanking region; the antisense oligonucleotide satisfies the following conditions: (i) The nucleotide sequence of the 5' flanking region comprises the nucleotide sequence shown in any one of SEQ ID NO: 181-186 or a modified sequence thereof, or comprises a modified sequence of SEQ ID NO: 179 or 180; (ii) The nucleotide sequence of the central DNA region comprises the nucleotide sequence shown in any one of SEQ ID NO:13-18 or a modified sequence thereof, or comprises a modified sequence of SEQ ID NO:11 or 12; (iii) The nucleotide sequence of the 3' flanking region comprises the nucleotide sequence shown in any one of SEQ ID NO: 189-194 or a modified sequence thereof, or a modified sequence comprising SEQ ID NO: 187 or 188.

2. The antisense oligonucleotide as described in claim 1, characterized in that, The modification of the modified sequence is selected from any one or a combination of at least two of the following: 3'-terminal deoxy-thymidine nucleotide, 2'-O-methyl modified nucleotide, 2'-fluorine modified nucleotide, 2'-deoxy-modified nucleotide, locked nucleotide, unlocked nucleotide, configuration-restricted nucleotide, restricted ethyl nucleotide, 5'-methylated modified cytosine nucleotide, baseless nucleotide, 2'-amino-modified nucleotide, 2'-O-allyl-modified nucleotide, 2'-C-alkyl-modified nucleoside nucleotide. Nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinonucleotides, aminophosphates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing oxophosphate groups, nucleotides containing methylphosphate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimics.

3. The antisense oligonucleotide as described in claim 2, characterized in that, The modification is selected from any one or a combination of at least two of the following: 2'-O-methoxyethyl modified nucleotides, 2,4'-restricted ethyl modified nucleotides, 2,4'-locked nucleotide modified nucleotides, 5'-methylated cytidine-3'-phosphate, 5'-methylated-2'-O-methoxyethyl cytidine-3'-phosphate, 5'-methylated-2,4'-restricted ethyl cytidine-3'-phosphate, 5'-methylated-2,4'-locked nucleotide cytidine-3'-phosphate, 3',5'-thiophosphodiester bonds, and 3',5'-oxophosphodiester bonds.

4. The antisense oligonucleotide as described in claim 3, characterized in that, The modifications in the modification sequence in (i) are selected from any one of the groups in the table below: The modifications in the modification sequence in (ii) are selected from any one of the groups in the table below: The modifications in (iii) are selected from any one of the groups in the table below: Where x represents a nucleotide modified with 2'-O-methoxyethyl, and the specific nucleotide type is consistent with the basic sequence; cEt represents a nucleotide modified with 2,4'-restricted ethyl, and the specific nucleotide type is consistent with the basic sequence; + represents a nucleotide modified with 2,4'-locked nucleotide, and the specific nucleotide type is consistent with the basic sequence; mC represents a 5'-methylated cytidine-3'-phosphate; mc represents a 5'-methylated-2'-O-methoxyethyl cytidine-3'-phosphate; mc(cEt) represents a 5'-methylated-2,4'-restricted ethyl cytidine-3'-phosphate; +mc represents a 5'-methylated-2,4'-locked nucleotide cytidine-3'-phosphate; # indicates a 3',5'-thiophosphodiester bond; # indicates a 3',5'-oxophosphodiester bond; empty or "-" indicates that the position is unmodified; the numbers in the columns representing each modification in the table indicate the nucleotide position of that modification in the corresponding base sequence.

5. The antisense oligonucleotide as described in claim 4, characterized in that, The sequence of the antisense oligonucleotide comprises the nucleotide sequence shown in any one of SEQ ID NO: 3-8; or, the modified sequence is selected from any of the modified sequences in the following table: Where x represents a nucleotide modified with 2'-O-methoxyethyl, and the specific nucleotide type is consistent with the basic sequence; cEt represents a nucleotide modified with 2,4'-restricted ethyl, and the specific nucleotide type is consistent with the basic sequence; + represents a nucleotide modified with 2,4'-locked nucleotide, and the specific nucleotide type is consistent with the basic sequence; mC represents a 5'-methylated cytidine-3'-phosphate; mc represents a 5'-methylated-2'-O-methoxyethyl cytidine-3'-phosphate; mc(cEt) represents a 5'-methylated-2,4'-restricted ethyl cytidine-3'-phosphate; +mc represents a 5'-methylated-2,4'-locked nucleotide cytidine-3'-phosphate; # indicates a 3',5'-thiophosphodiester bond; # indicates a 3',5'-oxophosphodiester bond; empty or "-" indicates that the position is unmodified; the numbers in the columns representing each modification in the table indicate the nucleotide position of that modification in the corresponding base sequence.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an antisense oligonucleotide as described in any one of claims 1-5, and a pharmaceutically acceptable carrier thereof.

7. A medicine box set, characterized in that, The kit includes a box A, which comprises one or both of the antisense oligonucleotides as described in any one of claims 1-5 or the pharmaceutical composition as described in claim 6.

8. The pillbox as described in claim 7, characterized in that, The kit also includes a medicine box 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 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.

9. The use of the antisense oligonucleotide according to any one of claims 1-5 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating HBV infection-related diseases.

10. The application according to claim 9, 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.

11. 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 1-5, the pharmaceutical composition as described in claim 6, and the kit as described in claim 7; the method is for non-therapeutic purposes.

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