SiRNA targeting the regulation of hbv gene expression and application thereof

Abstract

The present disclosure provides siRNA targeting the regulation of HBV gene expression and applications thereof. The double-stranded RNAi agent comprises an antisense strand and a sense strand complementary to the antisense strand to form a double-stranded region, wherein the nucleotide sequence of the antisense strand is set forth in SEQ ID NO: 11, or the nucleotide sequence of the antisense strand is a modified sequence of the sequence set forth in SEQ ID NO: 11. The results of cell and animal experiments show that the double-stranded RNAi agent provided by the present disclosure can significantly reduce the expression of one or more HBV genes, block the viral life cycle, and be used for the development of drugs for treating diseases related to HBV gene expression.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of nucleic acid modification, in particular, to siRNA targeting the regulation of HBV gene expression and application thereof. BACKGROUND

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

[0003] Oligonucleotides are short DNA or RNA molecules, oligomers, which can easily bind to their respective complementary oligonucleotides, DNA or RNA in a sequence-specific manner to form duplexes, or more rarely, hybrids. This basic property makes oligonucleotides widely used in gene detection, research and medicine. In nature, oligonucleotides are usually small RNA molecules that play a role in gene expression regulation, or intermediates derived from the degradation of larger nucleic acid molecules.

[0004] RNA interference (RNAi) is a natural defense mechanism against foreign genes. siRNA can down-regulate target genes by recognizing specific sequences and decomposing target mRNA.

[0005] The effective molecule of classical RNAi consists of a characteristic 19 + 2 nucleotide polymer structure (a double helix structure composed of 21 nucleotide RNA molecules and 19 corresponding nucleotide molecules, containing a 3' end overhang of 2 nucleotides). One strand of siRNA (guide or antisense strand) is complementary to the target mRNA transcript, and the other strand is designated as the passenger strand (or sense strand). The siRNA (antisense strand) guides the argonaute protein (AGO2) to the target transcript and becomes part of the RNA-induced silencing complex (RISC). The complete complementarity of siRNA (antisense strand) to the target causes the target transcript to be broken at the corresponding position of 10-11 points of the guide strand (antisense strand) under the catalysis of AGO2 protein.

[0006] Compared with small molecules and antibody drugs, siRNA has natural advantages because siRNA performs its function by completing Watson-Crick base pairing with mRNA, while small molecules and monoclonal antibody drugs need to recognize the complex spatial structure of specific proteins. Therefore, many diseases cannot be treated by small molecules and monoclonal antibodies because the target molecules have high activity and cannot recognize the molecular structure with affinity and binding specificity. The mechanism of siRNA drugs enables them to regulate the expression of target proteins at the genetic level, and has higher target specificity than small molecules or antibody drugs. Based on the principle of base complementary pairing, the treatment range of siRNA is wider, the design is simpler, and the development cycle is shorter.

[0007] In natural oligonucleotides, nucleotides are connected by phosphodiester bonds, which are particularly sensitive to nucleases under physiological conditions. Therefore, natural, unstructured, unmodified oligonucleotide drugs are easily degraded by nucleases in the body, have low activity, and are less likely to be developed into drugs. Chemical modification of oligonucleotide structure is an effective way to improve its activity, which can improve its stability to nucleases, affinity to RNA, and better promote endocytosis and tissue targeting, thereby effectively regulating the expression of target genes.

[0008] According to the basic structure of oligonucleotides, bases, sugar rings, phosphate backbones, and ends, chemical modification can be performed in four parts:

[0009] 1) Base modification: mainly divided into three forms of purine modification, pyrimidine modification and base replacement. Purine modification includes N6-methyladenosine, N1-methyladenosine, 7-methylguanosine modification; pyrimidine modification includes 3-methyluracil nucleoside, 5-methyluracil nucleoside, 5-methylcytosine nucleoside, N4-acetylcytosine, pseudouridine, thio-uracil nucleoside, propyne uracil nucleoside and dihydrouracil nucleoside.

[0010] 2) Sugar ring modification: mainly divided into sugar ring modification and replacement. Sugar ring modification includes 2'-modification, 4'-modification, 5'-modification, isomerization modification, and combination modification of these modifications. Among the 2'-modifications of siRNA, the most common are 2'-OMe (2'-methoxy) and 2'-F (2'-fluorine) modifications. Compared with natural siRNA, siRNA with 2'-OMe and 2'-F modifications has higher Tm value, stronger serum stability and better activity.

[0011] 3) Modification of the phosphate backbone: mainly modification of phosphorothioate; modification of methylphosphonate, selenophosphate, boronophosphate, dithiophosphate, and replacement of the bridging oxygen atom of the phosphodiester linkage with a sulfur atom; replacement of the phosphate group between nucleotides with a group containing no phosphorus atom, such as replacement of P atom with C, S and N atom, forming guanidyl, S-methyl thiourea, etc.

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

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

[0014] The current standard of care for chronic HBV infection is treatment with oral nucleos(t)ide analogues (NA) and injection of interferon alpha. NA inhibits HBV replication by inhibiting HBV DNA synthesis, and most patients require long-term treatment, with a high rate of virological relapse after discontinuation. Interferon alpha plays a dual role of immune regulation and antiviral through enhancing immune cell function, promoting cytokine expression, inducing interferon signaling, and encoding various antiviral proteins. Interferon alone is only effective in some patients, and the tolerance is relatively poor. The 2022 version of the 'Guidelines for the Prevention and Treatment of Chronic Hepatitis B' recommends nucleos(t)ide analogues including entecavir, tenofovir disoproxil fumarate, propofol tenofovir fumarate and emtricitabine, and alpha interferon including peginterferon alpha.

[0015] Currently, the goal of treatment for chronic HBV infection is to achieve functional cure, i.e., undetectable HBV DNA and HBV surface antigen (HBsAg) after discontinuation, with or without HBsAg seroconversion. The sustained reduction of HBsAg and seroconversion are very important for achieving functional cure, which is expected to alleviate liver inflammatory response, improve liver histopathology, reduce the incidence of end-stage liver disease, and prolong patient survival. Currently, it is difficult to achieve functional cure or the rate of functional cure is very low through clinical drugs, so it is necessary to further develop drugs that down-regulate HBsAg expression to achieve functional cure. SUMMARY

[0016] To solve the technical problem that there is no drug capable of more effectively inhibiting HBV gene expression in the prior art, the present disclosure provides siRNA targeting the regulation of HBV gene expression and applications thereof. The present disclosure designs a series of unique siRNA sequences by targeting the HBV genome sequence, and performs specific template modification on the siRNA sequences.

[0017] Generally, siRNA adopts 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) monomers for modification. However, even if only the above two monomers are considered for combination, there are 44 bases in the sense strand and the antisense strand of siRNA, i.e., there are 2 44 possible combinations. Moreover, the number of possible modification schemes is further increased by different end thio-modification layouts.

[0018] For the same siRNA sequence, the activity has a very large difference when different modification methods are used. For different siRNAs, the activity also has a very large difference when the same modification method is used. Although there are some modification principles for siRNA modification design, existing research has shown that the activity cannot be accurately predicted according to the modification method, i.e., there is no definite relationship between the modification method and the activity. Therefore, it is very difficult to screen a modification scheme with high activity from the numerous possible modification combinations.

[0019] The present disclosure screens some special modified sequences with significant inhibitory effect on HBV gene expression by chemically modifying the designed siRNA sequences.

[0020] In one aspect, the present disclosure provides a double-stranded RNAi agent, which comprises an antisense strand and a sense strand complementary to the antisense strand to form a double-stranded region, wherein the nucleotide sequence of the antisense strand is as shown in SEQ ID NO: 11, or the nucleotide sequence of the antisense strand is a modified sequence of the sequence as shown in SEQ ID NO: 11.

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

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

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

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

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

[0026] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining preferred embodiments of the present disclosure.

[0027] The reagents and raw materials used in the present disclosure are commercially available.

[0028] The positive progress effect of the present disclosure is that:

[0029] (1) The modified sequences, including the base sequence B1575, have a significant inhibitory effect on HBsAg, which is significantly better than the sequence of the scirpus.

[0030] (2) The modified sequence is conjugated with the GalNAc compound, which can be efficiently delivered to the liver of an animal and significantly inhibit the expression of HBV genes, thereby significantly reducing the levels of HBsAg, HBeAg and HBV DNA in serum.

[0031] (3) Compared with the sequence disclosed in the prior art, the sequences modified by the modified template of the present disclosure have significantly improved inhibitory activity on HBV.

[0032] (4) The present disclosure finds that siRNAs with similar sequences have very large differences in activity.

[0033] (5) The present disclosure also finds that different sequences have different sensitivities to each modified template, and it is uncertain which modified template is used to modify the siRNA sequence to have high activity. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1A Serum HBsAg levels of AAV-HBV model mice at 3 mg / kg level of candidate siRNA sequence.

[0035] Figure 1B Serum HBV DNA levels of AAV-HBV model mice at 3 mg / kg level of candidate siRNA sequence.

[0036] Figure 1C Serum HBeAg levels of AAV-HBV model mice at 3 mg / kg level of candidate siRNA sequence.

[0037] Figure 1D Serum HBsAb level of AAV-HBV model mice at 3 mg / kg level of candidate siRNA sequence.

[0038] Figure 1E Serum ALT level of AAV-HBV model mice at 3 mg / kg level of candidate siRNA sequence.

[0039] Figure 1F Body weight change of AAV-HBV model mice at 3 mg / kg level of candidate siRNA sequence.

[0040] Figure 2A Serum HBsAg level of mice after secondary challenge.

[0041] Figure 2B Serum HBV DNA level of mice after secondary challenge.

[0042] Figure 2C Serum HBeAg level of mice after secondary challenge.

[0043] Figure 2D Serum HBbAg level of mice after secondary challenge.

[0044] Figure 2E Serum ALT level of mice after secondary challenge.

[0045] Figure 2F Body weight change of mice after secondary challenge.

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

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

[0048] Figure 3C Serum HBeAg level of AAV-HBV model mice at 0.5 mg / kg level of candidate siRNA sequence.

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

[0050] Figure 3E Serum ALT level of AAV-HBV model mice at 0.5 mg / kg level of candidate siRNA sequence.

[0051] Figure 3FBody weight change of AAV-HBV model mice for candidate siRNA sequences coupled with different GalNAc at 0.5 mg / kg level.

[0052] Figure 4A HBsAg level of AAV-HBV model mice for candidate siRNA sequences coupled with different GalNAc.

[0053] Figure 4B HBV DNA level of AAV-HBV model mice for candidate siRNA sequences coupled with different GalNAc.

[0054] Figure 4C HBeAg level of AAV-HBV model mice for candidate siRNA sequences coupled with different GalNAc.

[0055] Figure 4D ALT level of AAV-HBV model mice for candidate siRNA sequences coupled with different GalNAc.

[0056] Figure 4E Body weight change of AAV-HBV model mice for candidate siRNA sequences coupled with different GalNAc. DETAILED DESCRIPTION

[0057] To enable a better understanding of the present disclosure, certain terms are defined first. Also, it should be noted that whenever a value or a range of values for a parameter are recited, it is intended that the intermediate values suitable for the same purpose are also part of the present disclosure.

[0058] The articles "a" and "an" as used herein mean one or more than one (i.e., at least one) of the grammatical article's object. By way of example, "an element" means one element or more than one element, e.g., multiple elements.

[0059] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".

[0060] The term "or" as used herein is used to mean, and is used interchangeably with, the term "and / or", unless context clearly indicates otherwise.

[0061] As used herein, the term "about" or "approximately," as applied to one or more target values, refers to a value similar to that of the referenced value. In certain embodiments, unless otherwise stated or otherwise clear from context, the term "approximately" or "about" means a range 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 referenced value in either direction (greater than or less than), unless such number would exceed 100% of the possible values.

[0062] As used herein, "HBV" refers to hepatitis B virus, including genotypes A, B, C, D, E, F, G, H, I, J, and subtypes thereof, and is not limited to a particular genotype.

[0063] "G," "C," "A," and "U" each generally represent a nucleotide comprising, respectively, guanine, cytosine, adenine, and uracil as the base. "T" and "dT" are used interchangeably herein and refer to a deoxyribonucleotide in which the nucleobase is thymine, such as deoxyribothymine, 2'-deoxythymidine, or thymidine. However, it will be understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can also refer to a modified nucleotide (as described further below) or an alternative substituent moiety. The skilled artisan will be well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide, including a nucleotide having such a substituent moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide comprising uracil, guanine, or adenine can be replaced in a nucleotide sequence of the disclosure by a nucleotide comprising, for example, inosine. Sequences comprising such substituent moieties are embodiments of the disclosure.

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

[0065] The term "small interfering ribonucleic acid" or "siRNA" refers to a small interfering ribonucleic acid RNAi molecule. It is a class of double-stranded RNA molecules, also known in the art as short interfering RNA or silencing RNA. siRNAs typically comprise a sense strand (also known as a passenger strand) and an antisense strand (also known as a guide strand), each strand being 17 to 30 nucleotides in length, typically 19 to 25 nucleotides in length, wherein the antisense strand is complementary (such as at least 95% complementary, such as fully complementary) to a target nucleic acid (suitably a mature mRNA sequence), and the sense strand is complementary to the antisense strand, such that the sense and antisense strands form a duplex or duplex region. The siRNA strands can form a blunt end duplex, or preferably, the 3' ends of the sense and antisense strands can form 3' overhangs, for example 1, 2 or 3 nucleosides, similar to the Dicer produced product, which can form a RISC substrate in vivo. Efficient extended versions of the Dicer substrate have been described in US 8349809 and US 8513207, incorporated herein by reference. In some embodiments, both the sense and antisense strands have 3' overhangs of 2 nucleotides. Thus, the duplex region can be, for example, 17 to 25 nucleotides in length, such as 21 to 23 nucleotides in length.

[0066] The term "antisense strand" refers to the strand of an RNAi (e.g., dsRNA) that includes a region of substantial complementarity to a target sequence. As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., a target sequence). When the region of complementarity is not perfectly complementary to the target sequence, mismatches can be in the internal region or the terminal region of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' terminus.

[0067] The term "sense strand" as used herein refers to the strand of an RNAi that includes a region of substantial complementarity to a region of an antisense strand (as that term is defined herein).

[0068] The term "inhibit", as used herein, can be used interchangeably with "reduce", "silence", "down-regulate", "suppress" and other similar terms, and includes inhibition at any level.

[0069] As used herein, the phrase "inhibiting expression of a gene" includes inhibiting expression of HBV DNA, HBV mRNA, HBsAg, HBeAg, Hepatitis B core antigen (HBcAg), and the like.

[0070] "Inhibiting expression of an HBV antigen" includes inhibiting expression of HBsAg, HBeAg, HBcAg proteins.

[0071] "Inhibiting expression of HBV genes" includes inhibition of HBV DNA, HBV mRNA, HBsAg, HBeAg, HBcAg at any level, for example at least partial inhibition of expression of HBV DNA, HBV mRNA, HBsAg, HBeAg, HBcAg, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0072] Expression of HBV genes can be assessed based on any variable level associated with HBV gene expression, for example, HBV DNA level, HBV mRNA level, HBV antigen protein level, HBV viral particle level. Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level utilized in the art, for example, a pre-dosing baseline level or a level determined from a similar untreated or control (e.g., buffer control or inert agent control) treated subject, cell, or sample.

[0073] As used herein, "patient" or "subject" is intended to include a human or non-human animal, preferably a mammal, for example a mouse. Most preferably, the subject or patient is a human.

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

[0075] As used herein, "therapeutically effective amount" is intended to include the amount of an RNAi agent that, when administered to a patient for treating a HBV-associated disease, is sufficient to effect treatment (e.g., by abrogating, ameliorating, or maintaining the existing disease or one or more symptoms of the disease). The "therapeutically effective amount" can vary depending on the RNAi agent, how the agent is administered, the disease and its severity, and the history, age, weight, family history, genetic makeup, stage of pathological processes mediated by HBV expression, type of previous or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

[0076] As used herein, "prophylactically effective amount" is intended to include the amount of an RNAi agent that, when administered to a subject who is not yet experiencing or displaying symptoms of a HBV-associated disease but who is at risk of developing the disease, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the progression of the disease or reducing the severity of the disease that develops later. The "prophylactically effective amount" can vary depending on the RNAi agent, how the agent is administered, the degree of risk of the disease, and the history, age, weight, family history, genetic makeup, type of previous or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

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

[0078] As used herein, the term "sample" includes similar fluids, cells, or tissues isolated from a subject, as well as a collection of fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, and the like. Tissue samples can include samples from a tissue, organ, or a localized region. For example, a sample can be derived from a particular organ, organ portion, or fluid or cells within these organs. In certain embodiments, a sample can be derived from the liver (e.g., the entire liver or certain segments of the liver, or certain types of cells in the liver, e.g., hepatocytes). In preferred embodiments, a "sample derived from a subject" refers to blood or plasma drawn from the subject. In other embodiments, a "sample derived from a subject" refers to liver tissue (or sub-components thereof) derived from the subject.

[0079] In one aspect, the present disclosure provides a double stranded RNAi agent comprising an antisense strand and a sense strand complementary to the antisense strand forming a double stranded region, the nucleotide sequence of the antisense strand is set forth in SEQ ID NO: 11, or the nucleotide sequence of the antisense strand is a modified sequence of the sequence set forth in SEQ ID NO: 11.

[0080] In some embodiments, the double stranded RNAi agent comprises an oligonucleotide duplex of a sense strand and an antisense strand pair: the sense strand has a sequence set forth in SEQ ID NO: 4, or a fragment thereof, or a modified sequence of the sequence or fragment thereof.

[0081] In some embodiments, the sense strand and the antisense strand comprise at least one modified nucleotide.

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

[0083] In some embodiments, at least one of the modified nucleotides is selected from one or more of the group consisting of: a deoxy-nucleotide, a 3'-terminal deoxy-thymine nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, and a nucleotide comprising a 5'-phosphate mimic.

[0084] In some embodiments, at least one strand of the double stranded RNAi agent comprises a 3' overhang of at least 2 nucleotides.

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

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

[0087] In some embodiments, all modifications of the nucleotides on the sense strand and the antisense strand comprise chemical modifications of the 2' position of the ribose of the nucleotides.

[0088] In some embodiments, the chemical modification of the 2' position of the ribose sugar of the nucleotides is selected from one or more of the group consisting of: 2'-methoxy, 2'-0-methoxyethyl, 2'-fluoro, 2'-benzyloxy, 2'-methylcarbonylamino, and 2'-pyridinomethoxy.

[0089] In some embodiments, the chemical modification of the 2' position of the ribose sugar of the nucleotides is 2'-methoxy or 2'-fluoro.

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

[0091] In some embodiments, the 3',5'-phosphodiester bonds comprise a phosphorothioate modification.

[0092] In some embodiments, the 5' position of the 5' terminal nucleotide sugar of the antisense strand is phosphorylated.

[0093] In some embodiments, the phosphorylated 5' position phosphorylation group comprises one or more selected from the group consisting of: 5'-vinylphosphonate group, 5'-methylphosphonate group, 5'-C-methylphosphonate group, 5'-phosphorothioate group, and 5'-phosphate group, having the structure:

[0094]

[0095] ;

[0096] R is hydrogen, hydroxyl, amine, C 1-4 alkyl, aryl, C 1-4 alkoxy, C 1-4 alkylcarbonylamino, or halogen;

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

[0098] In some embodiments, the sequence terminal nucleotides are linked by 3',5'-phosphodiester bonds comprising a phosphorothioate modification, and form chiral pure 3',5'-phosphorothioate bonds.

[0099] In some embodiments, the sense and antisense strands contain 1-3 phosphorothioate linkages at the 5' terminal, and 1-3 phosphorothioate linkages at the 3' terminal.

[0100] In some embodiments, the antisense strand employs one of the following table of modifications:

[0101]

[0102]

[0103]

[0104] and / or, the sense strand employs one of the modifications in the following table:

[0105]

[0106] where 2'-OMe is 2'-methoxy; 2'-F is 2'-fluoro; PS is a phosphorothioate backbone; and EVP is 5'-vinyl-(E)-phosphonate.

[0107] In some embodiments, the double stranded RNAi agent has the modification pattern:

[0108] The siRNA modification template with the antisense strand having Modification A and the sense strand having modification pattern a is designated DV27P;

[0109] The siRNA modification template with the antisense strand having Modification A and the sense strand having modification pattern b is designated DV29P;

[0110] The siRNA modification template with the antisense strand having Modification B and the sense strand having modification pattern a is designated DV26P;

[0111] The siRNA modification template with the antisense strand having Modification B and the sense strand having modification pattern b is designated DV28P;

[0112] The siRNA modification template with the antisense strand having Modification C and the sense strand having modification pattern b is designated DV32P;

[0113] The siRNA modification template with the antisense strand having Modification D and the sense strand having modification pattern b is designated DV34P;

[0114] The siRNA modification template with the antisense strand having Modification E and the sense strand having modification pattern a is designated DV25P; or,

[0115] The siRNA modification template with the antisense strand having Modification F and the sense strand having modification pattern b is designated DV33P.

[0116] In some embodiments, the antisense strand from the second to the eighth position from the 5' end employs a modification group selected from one or more of UNA, GNA, and DNA, the UNA and GNA having the structure:

[0117] ;

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

[0119] In some embodiments, the double stranded RNAi agent comprises any of the following oligonucleotide duplexes paired by a sense strand and an antisense strand:

[0120] (1) the sense strand has a sequence as shown in SEQ ID NO: 96; and the antisense strand has a sequence as shown in SEQ ID NO: 201, 202, 203, or 204;

[0121] (2) the sense strand has a sequence as shown in SEQ ID NO: 97; and the antisense strand has a sequence as shown in SEQ ID NO: 201 or 202;

[0122] (3) the sense strand has a sequence as shown in SEQ ID NO: 98; and the antisense strand has a sequence as shown in SEQ ID NO: 205 or 206;

[0123] (4) the sense strand has a sequence as shown in SEQ ID NO: 99; and the antisense strand has a sequence as shown in SEQ ID NO: 206; and,

[0124] (5) the sense strand has a sequence as shown in SEQ ID NO: 100; and the antisense strand has a sequence as shown in SEQ ID NO: 206.

[0125] The double stranded RNA (dsRNA) agents of the present disclosure (double stranded RNAi agents) can be optionally conjugated to one or more ligands. The ligand can be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. For example, the ligand can be conjugated to the sense strand. In preferred embodiments, the ligand is bound to the 3' end of the sense strand. In one preferred embodiment, the ligand is a GalNAc ligand.

[0126] In another aspect, the present disclosure provides a conjugate comprising a double stranded RNAi agent as previously described, and a ligand conjugated to the double stranded RNAi agent.

[0127] In some embodiments, the ligand is conjugated at the 3'-terminal or 5'-terminal end of the oligonucleotide sense strand.

[0128] In some embodiments, the ligand is one or more GalNAc derivatives attached using a bivalent or trivalent branched linker, or a GalNAc derivative attached using a monovalent linker.

[0129] In some embodiments, the ligand is:

[0130] ,

[0131] wherein X is hydrogen or a hydroxyl protecting group including acetyl, benzoyl or isobutyryl; Y is an amine protecting group or H, the amine protecting group being formyl, acetyl, propionyl, n-butyryl or isobutyryl; n is an integer between 0 and 20; q, r and s are independently integers between 1 and 7.

[0132] In some embodiments, the ligand is:

[0133] .

[0134] In some embodiments, the ligand is:

[0135] ,

[0136] wherein X is oxygen, nitrogen or sulfur;

[0137] Y is alkyl or aryl;

[0138] R1is oxygen or sulfur;

[0139] R2is hydrogen, amine, C 1-4 alkyl, aryl, C 1-4 alkoxy or halogen;

[0140] A is -(CH2) a -, -(CH2CH2O) b -, -((CH2) c NHCO) d - or -((CH2) c CONH) d - wherein a is an integer between 1 and 15, b is an integer between 1 and 7, c is an integer between 1 and 7, d is an integer between 1 and 5;

[0141] B is -(CH2) e - wherein e is an integer between 0 and 7;

[0142] L is -CONH- or -NHCO-;

[0143] X1is -(CH2) f - or -(CH2CH2O) f CH2- wherein f is an integer between 1 and 5;

[0144] X2is -(CH2) g - wherein g is an integer between 1 and 6;

[0145] Y1is 0 or 1;

[0146] Y2 is 0, 1 or 2;

[0147] Y3 is 1, 2 or 3;

[0148] m is an integer from 0 to 4;

[0149] n is an integer from 0 to 4.

[0150] In some embodiments, the ligand is G4, G5, G6 or G7:

[0151] ,

[0152] ,

[0153] , or

[0154] .

[0155] In some embodiments, the conjugate has the structure shown below:

[0156] ,

[0157] ,

[0158] ,

[0159] , or

[0160] .

[0161] In some embodiments, wherein the ligand is:

[0162]

[0163] wherein X is oxygen, nitrogen or sulfur;

[0164] Y is alkyl or aryl;

[0165] R1 is oxygen or sulfur;

[0166] R2 is hydrogen, amine, C 1-4 alkyl, aryl, C 1-4 alkoxy or halogen;

[0167] A is -(CH2) a -, -(CH2CH2O) b -, -((CH2) c NHCO) d - or -((CH2) c CONH) dwherein a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5;

[0168] B is -(CH2) e wherein e is an integer from 0 to 7;

[0169] L is -CONH- or -NHCO-;

[0170] X1is -(CH2) f - or -(CH2CH2O) f CH2-, and f is an integer from 1 to 5;

[0171] X2is -(CH2) g -, and g is an integer from 1 to 6;

[0172] Y1is 0 or 1;

[0173] Y2is 0, 1, or 2;

[0174] Y3is 1, 2, or 3;

[0175] m is an integer from 0 to 4;

[0176] n is an integer from 0 to 4.

[0177] In some embodiments, wherein the ligand is G101, G102, G103, G105, or G106:

[0178] , , , , or .

[0179] In some embodiments, the conjugate has a structure as shown below:

[0180] ,

[0181] ,

[0182] ,

[0183] , or

[0184] .

[0185] In some embodiments, the conjugate comprises any of the oligonucleotide duplexes selected from the following sense and antisense strand pairings:

[0186] the antisense strand has a sequence as set forth in SEQ ID NO: 201.

[0187] In some embodiments, the conjugate has a function of inhibiting expression of an HBV gene.

[0188] In another aspect, the disclosure provides a pharmaceutical composition comprising the aforementioned double-stranded RNAi agent or the aforementioned conjugate, and a pharmaceutically acceptable carrier.

[0189] In one embodiment, provided herein is a pharmaceutical composition comprising a double-stranded RNAi agent as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising a double-stranded RNAi agent can be used to treat a disease or disorder associated with expression or activity of an HBV gene, such as chronic hepatitis B. Such pharmaceutical compositions are formulated based on the delivery model. One example is a composition formulated for systemic administration by parenteral delivery, for example, by intravenous (IV) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma, for example, by infusion into the brain, for example, by continuous pump infusion.

[0190] Pharmaceutical compositions comprising a double-stranded RNAi agent of the disclosure can be, for example, a solution with or without a buffer or a composition containing a pharmaceutically acceptable carrier. Such compositions include, for example, aqueous or crystalline compositions, liposome formulations, micelle formulations, emulsions, and gene therapy vectors.

[0191] In the methods of the disclosure, the double-stranded RNAi agent can be administered in a solution. A free double-stranded RNAi agent can be administered in a non-buffered solution, for example, in physiological saline or in water. Alternatively, the free siRNA can also be administered in a suitable buffered solution. The buffered solution can include acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffered solution is phosphate buffered saline (PBS). The pH and the osmolality of the buffer comprising the double-stranded RNAi agent can be adjusted such that it is suitable for administration to a subject.

[0192] 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.

[0193] The pharmaceutical compositions disclosed herein can be administered at a dose sufficient to inhibit HBV gene expression. Typically, a suitable dose of the double-stranded RNAi agent of this disclosure is in the range of about 0.001 to about 200.0 mg per kilogram of body weight per day, and generally in the range of about 0.1 to 50 mg per kilogram of body weight per day. For example, double-stranded RNAi agents (e.g., dsRNA) can be administered at doses of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3... 3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7 5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 1 8, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or approximately 50 mg / kg.

[0194] The pharmaceutical composition can be administered once a day, or the double- stranded RNAi agent can be administered in two, three, or more sub-doses at appropriate intervals throughout the day, or even continuously with an implanted reservoir. In such instances, the total daily dose of the double-stranded RNAi agent is achieved by the administration of the sub-does. Dosage units can also be compounded for delivery over several days, for example using conventional sustained release formulations that provide for sustained release of the double-stranded RNAi agent over a period of several days. Sustained release formulations are well known in the art and are particularly useful for delivering agents to a particular site, and can be used with the agents of the disclosure. In this embodiment, the dosage unit comprises a corresponding plurality of daily doses.

[0195] In other embodiments, a single dose of the pharmaceutical composition can be sustained for a long duration, such that subsequent doses are administered 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 the disclosure, a single dose of the pharmaceutical composition of the disclosure is administered once a week. In other embodiments of the disclosure, a single dose of the pharmaceutical composition of the disclosure is administered once a month.

[0196] Those of skill in the art will appreciate 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 general health and / or age of the subject, and other existing diseases. In addition, treatment of a subject with a therapeutically effective dose of the composition can include a single treatment or a series of treatments. As described elsewhere herein, effective doses and in vivo half-lives of the various double-stranded RNAi agents encompassed by the disclosure can be estimated using conventional methods or based on in vivo testing using appropriate animal models.

[0197] The pharmaceutical compositions of the disclosure can be administered in a number of ways depending upon the treatment desired and the area to be treated. Administration can be local (e.g., through a skin patch); pulmonary; by inhalation or insufflation of powders or aerosols, including by nebulizers; intratracheal; intranasal; epidermal and transdermal, oral or parenteral. Parenteral administration includes subcutaneous, intravenous, intraarterial, subdermal, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via implantation devices; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular administration.

[0198] Double-stranded RNAi agents for use in the compositions and methods of the disclosure can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term "liposome" refers to a vesicle comprised of amphiphilic lipids arranged in at least one bilayer (e.g., one bilayer or multiple bilayers). Liposomes include unilamellar or multilamellar vesicles which have a membrane that is formed from a lipophilic material and an aqueous interior. The aqueous portion contains the double-stranded RNAi agent composition. The lipophilic material separates the aqueous interior from the aqueous exterior (although in some examples, it can not) which typically does not include the double-stranded RNAi agent composition. Liposomes are useful for transferring and delivering active ingredients to a site of action. Because the liposome membrane is structurally similar to a biological membrane, when a liposome is administered to a tissue, the liposome bilayer fuses with the bilayer of a cell membrane. As fusion of the liposome with the cell proceeds, the internal aqueous contents, including the double-stranded RNAi agent, are delivered into the cell, where the double-stranded RNAi agent can specifically bind to a target RNA and can mediate RNAi. In some cases, the liposomes are also specifically targeted, e.g., to direct the double-stranded RNAi agent to a particular cell type.

[0199] Liposomes comprising double-stranded RNAi agents can be prepared by a variety of methods. In one example, the lipid components of the liposome are dissolved in a detergent such that micelles are formed with the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The double-stranded RNAi agent formulation is then added to the micelles comprising the lipid components. The cationic groups on the lipids interact with the double-stranded RNAi agent and condense around the double-stranded RNAi agent to form liposomes. After condensation, the detergent is removed, e.g., by dialysis, to obtain a liposomal formulation of the double-stranded RNAi agent.

[0200] Double-stranded RNAi agents, e.g., dsRNAs of the disclosure, can be fully encapsulated in a lipid formulation, e.g., an LNP or other nucleic acid-lipid particle.

[0201] As used herein, the term "lipid nanoparticle (LNP)" refers to a stable nucleic acid-lipid particle. LNPs contain a cationic lipid, a non-cationic lipid, and a lipid that prevents the particle from aggregating (e.g., a PEG-lipid conjugate). LNPs are extremely useful for synthetic applications because they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., at sites physically separate from the site of administration).

[0202] In one embodiment, the ratio of lipid to drug (mass / mass ratio) (e.g., the ratio of lipid to dsRNA) will be in a range from about 1 : 1 to about 50: 1, from about 1 : 1 to about 25: 1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1.

[0203] In some embodiments, the double stranded RNAi agent or the conjugate is administered in a non-buffered solution.

[0204] In some embodiments, the non-buffered solution is saline or water.

[0205] In some embodiments, the double stranded RNAi agent or the conjugate is administered with a buffered solution.

[0206] In some embodiments, the buffered solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

[0207] In some embodiments, the buffered solution is a phosphate buffered saline.

[0208] In some embodiments, the double stranded RNAi agent or the conjugate is formulated into a lipid formulation for delivery in a membranous molecular assembly.

[0209] In some embodiments, the lipid formulation is a nucleic acid-lipid particle.

[0210] In some embodiments, the lipid formulation is a lipid nanoparticle.

[0211] In some embodiments, the mass / mass ratio of lipid to the double stranded RNAi agent or the conjugate is 1 : 1-50: 1, 1 : 1-25: 1, 3: 1-15: 1, 4: 1-10: 1, 5: 1-9: 1, or 6: 1-9: 1.

[0212] In some embodiments, the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a structural lipid, and a polymer-conjugated lipid.

[0213] In some embodiments, the cationic lipid is a compound of structure (I), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof,

[0214] (I),

[0215] G1is C 1～6 alkylene; G2is C 2～8 alkylene; G3is C 1～3 alkylene; L1is C6～15 linear alkyl; L2 is C 12～25 branched alkyl.

[0216] In some embodiments, the cationic lipid is YK-009 of structure (I-I) (see patent CN114044741B):

[0217] (I-I).

[0218] In some embodiments, the cationic lipid is a compound of structure (II), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof,

[0219] (II),

[0220] G1 is C 2~8 alkylene; G2 is C 2~8 alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6~25 linear or branched alkyl; R2 is C 6~25 linear or branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2- or -(CH2)3- or -(CH2)4-.

[0221] In some embodiments, the cationic lipid is YK-401 of structure (II-I) or YK-402 of structure (II-II) (see patent CN115784921B):

[0222] (II-I),

[0223] (II-II).

[0224] In some embodiments, the cationic lipid is a compound of structure (III), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof,

[0225] (III),

[0226] G1 is C 1~6 alkylene; G2 is C 2~8 alkylene; R1 is C 6~20 linear or branched alkyl; R2 is C 12~25branched alkyl; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2-, or CH3CH2NH(CH2)2-.

[0227] In some embodiments, the cationic lipid is YK-201 of structure (III-I) or YK-202 of structure (III-II) (see patent CN115677518B):

[0228] (III-I),

[0229] (III-II).

[0230] In some embodiments, the cationic lipid is a compound of structure (IV), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof,

[0231] (IV),

[0232] G1 is C 1~8 alkylene; G2 is C 2~8 alkylene; R1 is C 6~25 linear or branched alkyl; R2 is C 12~25 linear or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is -CH3 or -CH2CH3 or -CH2CH2OH .

[0233] In some embodiments, the cationic lipid is YK-305 of structure (IV-I) or YK-310 of structure (IV-II) (see patent CN115745820B):

[0234] (IV-I),

[0235] (IV-II).

[0236] In some embodiments, the cationic lipid is a compound of structure (V), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof,

[0237] (V),

[0238] G 1 and G 2 each independently unsubstituted C6-C 10 alkylene; G 3 is unsubstituted C1-C 12 alkylene; R 1 and R 2 each independently C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is OR 5 , N, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 is C1-C 12 alkyl; and R 5 is H or C1-C6 alkyl.

[0239] In some embodiments, the cationic lipid is ALC0315 of structure (V-I) (see patent CN108368028B):

[0240] (V-I).

[0241] In some embodiments, the cationic lipid is a compound of structure (VI), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof,

[0242] (VI),

[0243] R4is selected from -(CH2) n Q and -(CH2) n CHQR; Q is selected from the group consisting of -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R8, and a heterocycle; n is 1, 2, or 3.

[0244] In some embodiments, the cationic lipid is SM102 of structure (VI-I) (see patent CN110520409A):

[0245] (VI-I).

[0246] In some embodiments, the cationic lipid is compound DLIN-MC3-DMA of structure (VII) (see CN102625696B), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof,

[0247] (VII).

[0248] In some embodiments, the cationic lipid comprises one or more selected from YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA.

[0249] In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:1-10:1.

[0250] In some embodiments, the molar ratio of the cationic lipid to the structural lipid is 1:1-5:1.

[0251] In some embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-70):(0.5-5).

[0252] In some embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-45):(0.5-5).

[0253] In some embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 50:10:38.5:1.5 or 49:10:39.5:1.5.

[0254] In some embodiments, the neutral lipid comprises one or more selected from phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0255] In some embodiments, the neutral lipid is selected from one or more of: 1,2-dilinoleoyl- sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- ditridecandecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1- hexadecyl-sn-glycero-3-phosphocholine (CI 6 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn- glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- diphytany-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl- sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3- phospho-rac-(l-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1- stearoyl-2-oleoyl-stearoethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0256] In some embodiments, the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1,2-distearoyl-sn-glycero-3-phosphocholine.

[0257] In some embodiments, the structural lipid is selected from one or more of the following: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, a-tocopherol, and corticosteroids.

[0258] In some embodiments, the structural lipid is cholesterol.

[0259] In some embodiments, the polymeric conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0260] In some embodiments, the polymeric conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol bismyristyl acetyl amide (ALC-0159).

[0261] Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating liver disorders, such as liver cancer.

[0262] Pharmaceutical compositions of the present disclosure, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing into association the active ingredients with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

[0263] Pharmaceutical compositions of the present disclosure can be formulated in any of a number of possible dosage forms, including, but not limited to, tablets, capsules, gelcaps, liquid syrups, soft gels, suppositories, and enemas. Pharmaceutical compositions of the present disclosure can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions can further comprise substances that increase the viscosity of the suspension, such substances including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension can also contain stabilizers.

[0264] Certain pharmaceutical compositions of the disclosure also incorporate a carrier compound into the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid or its analog that is inert (i.e., not biologically active per se) but which is considered a nucleic acid in vivo processes, e.g., by degrading or otherwise facilitating removal of biologically active nucleic acids from circulation, to reduce bioavailability of biologically active nucleic acids. Co-administration of a nucleic acid and a carrier compound, typically with the latter in excess, can result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidney, or other extracirculatory reservoir, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, co-administration with a polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4' isothiocyanatostilbene-2,2'-disulfonic acid can reduce recovery of partially phosphorothioated dsRNA in liver tissue.

[0265] A "pharmaceutical carrier" or "excipient" in contrast to a carrier compound, is a pharmaceutically acceptable solvent, suspending, or other vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected with the aim of providing suitable bulk, consistency, etc., to the formulation when combined with the nucleic acid and other components of the particular pharmaceutical composition, as appropriate to the mode of administration desired. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, or dicalcium phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oil, 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.).

[0266] Pharmaceutically acceptable organic or inorganic excipients, which do not deleteriously react with the nucleic acid, suitable for non-parenteral administration can also be used to formulate pharmaceutical compositions of the disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidine, etc.

[0267] Formulations for topical administration of nucleic acids can include sterile or non-sterile aqueous solutions, non-aqueous solutions, or suspensions of the nucleic acid in a liquid or solid oil base. Such solutions also can include buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients, which do not deleteriously react with the nucleic acid, suitable for non-parenteral administration can be used.

[0268] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous parlecyl, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0269] The dosage forms, carrier compounds, pharmaceutical carriers, excipients, etc. of the above pharmaceutical composition are described in US Patent No. 10125369B2, which is incorporated herein by reference.

[0270] In another aspect, the present disclosure provides a kit-of-parts, which comprises a kit A comprising one or more of the aforementioned double-stranded RNAi agent, conjugate, or pharmaceutical composition.

[0271] In some embodiments, the kit-of-parts further comprises a kit B comprising one or both of:

[0272] (1) other HBV gene expression-reducing drugs or compositions comprising the HBV gene expression-reducing drugs;

[0273] (2) one or more of the group consisting of hormone agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0274] The "other HBV gene expression-reducing drugs or compositions comprising the HBV gene expression-reducing drugs" described herein refer to drugs not containing the double-stranded RNAi agent, conjugate, or pharmaceutical composition provided by the present disclosure.

[0275] In another aspect, the present disclosure provides the use of the aforementioned double-stranded RNAi agent, conjugate, or pharmaceutical composition in the preparation of a medicament for preventing and / or treating a HBV gene expression-related disease.

[0276] In some embodiments, the HBV gene expression-related disease is selected from the following disease types: chronic hepatitis B, liver fibrosis, liver cirrhosis, liver cancer, acute hepatitis B, and HBV / HDV co-infection-related diseases.

[0277] In another aspect, the present disclosure provides a method for reducing HBV gene expression or inhibiting HBV replication for non-prevention and / or treatment purposes, which comprises administering one or more of the aforementioned double-stranded RNAi agent, conjugate, pharmaceutical composition, and kit-of-parts to a sample.

[0278] The "non-prevention and / or treatment purposes" described herein refers to reducing HBV gene expression or inhibiting HBV replication in, for example, a laboratory for research purposes.

[0279] Nucleotide abbreviations used herein are as follows:

[0280] A = adenosine-3 '-phosphate

[0281] Am = 2'-methoxyadenosine-3 '-phosphate

[0282] Ams = 2'-methoxyadenosine-3 '-phosphorothioate

[0283] Af = 2'-fluoroadenosine-3 '-phosphate

[0284] Afs = 2'-fluoroadenosine-3 '-phosphorothioate

[0285] G = guanosine-3 '-phosphate

[0286] Gm = 2'-methoxyguanosine-3 '-phosphate

[0287] Gms = 2'-methoxyguanosine-3 '-phosphorothioate

[0288] Gf = 2'-fluoroguanosine-3 '-phosphate

[0289] Gfs = 2'-fluoroguanosine-3 '-phosphorothioate

[0290] C = cytidine-3 '-phosphate

[0291] Cm = 2'-methoxycytidine-3 '-phosphate

[0292] Cms = 2'-methoxycytidine-3 '-phosphorothioate

[0293] Cf = 2'-fluorocytidine-3 '-phosphate

[0294] Cfs = 2'-fluorocytidine-3 '-phosphorothioate

[0295] U = uridine-3 '-phosphate

[0296] Um = 2'-methoxyuridine-3 '-phosphate

[0297] Ums = 2'-methoxyuridine-3 '-phosphorothioate

[0298] Uf = 2'-fluorouridine-3 '-phosphate

[0299] Ufs = 2'-fluorouridine-3 '-phosphorothioate

[0300] AmsEVP = 5'-vinyl-(E)-phosphonate-2'-methoxyadenosine-3 '-phosphorothioate

[0301] UmsEVP = 5'-vinyl-(E)-phosphonate-2'-methoxyuridine-3'-phosphorothioate

[0302] A(gna) = adenosine-diol nucleic acid

[0303] C(gna) = cytidine-diol nucleic acid

[0304] G(gna) = guanosine-diol nucleic acid

[0305] T(gna) = thymidine-diol nucleic acid

[0306] U(gna) = uridine-diol nucleic acid

[0307] dA = deoxyadenosine-3'-phosphate

[0308] dG = deoxyguanosine-3'-phosphate

[0309] dC = deoxycytidine-3'-phosphate

[0310] dT = deoxythymidine-3'-phosphate

[0311] The following examples are intended to illustrate the present disclosure but not to limit the scope of the present disclosure. If not specifically mentioned, the technical means used in the examples are the conventional means well known to those skilled in the art, and the raw materials used are commercially available.

[0312] Example 1: Synthesis of small interfering oligonucleotide modified with DV29P template

[0313] Thirty-six siRNA base sequences were designed, VIR-2218 of Alnylam was used as a positive control, and the sequence in this example was numbered as APC-VIR; ANC-DV29P was used as a negative control. The base sequence was modified with DV29P template. The 7th, 9th, 11th, and 17th positions of the sense strand were 2'-F modified, and the remaining positions were 2'-OMe modified; the 2nd, 4th, 5th, 6th, 14th, and 16th positions of the antisense strand were 2'-F modified, and the remaining positions were 2'-OMe modified. In addition, there were two thio modifications at the 5' end of the sense strand; there were two thio modifications at the 5' end and 3' end of the antisense strand; and there was one EVP modification at the 5' end of the antisense strand. The siRNA sequence modified with DV29P template is shown in Table 1.

[0314] 1. Synthesis of A194-DV29P sequence modified with DV29P template

[0315] The base sequence of the small interfering ribonucleic acid numbered as A194-DV29P in Table 1 is:

[0316] Sense strand: 5'-UCGUGUUACAGGCGGGGUUUU-3' (SEQ ID NO: 15)

[0317] Sense: 5' - TCGAGACCGGAAAGAUGUUCUA - 3' (SEQ ID NO: 46)

[0318] The 7th, 9th, 11th and 17th positions of the sense strand are 2'-F modified, and the remaining positions are 2'-OMe modified. In addition, the 5' end of the sense strand has two thio modifications; the 5' and 3' ends of the antisense strand each have two thio modifications; and the 5' end of the antisense strand has one EVP modification.

[0319] Instrument and reagent: Genocept 192 P model DNA / RNA automatic synthesizer, its solid phase carrier is a general carrier of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (cytiva manufacturer).

[0320] Preparation method:

[0321] According to the monomer concentration of 0.15 M, the following nucleotide monomer solutions were prepared with acetonitrile: DMT-A-OMe phosphoramidite monomer (Formula 1), DMT-C-OMe phosphoramidite monomer (Formula 2), DMT-G-OMe phosphoramidite monomer (Formula 3) and DMT-U-OMe phosphoramidite monomer (Formula 4), DMT-A-F phosphoramidite monomer (Formula 5), DMT-C-F phosphoramidite monomer (Formula 6), DMT-G-F phosphoramidite monomer (Formula 7), DMT-U-F phosphoramidite monomer (Formula 8), vinyl-(E)-phosphonate-A-OMe phosphoramidite monomer (Formula 9), and vinyl-(E)-phosphonate-U-OMe phosphoramidite monomer (Formula 10).

[0322] Formula 1 Formula 2

[0323] Formula 3 Formula 4

[0324] Formula 5 Formula 6

[0325] Formula 7 Formula 8

[0326] Formula 9 Formula 10

[0327] The 7th base from the 5' end of the antisense strand of the APC-VIR sequence contains a glycol nucleic acid (GNA) modification, and the structure of the GNA monomer is as follows:

[0328] (S)-GNA-U (S)-GNA-C

[0329] (S)-GNA-A (S)-GNA-G

[0330] Preparation is carried out using the following steps:

[0331] (1) Deprotection

[0332] Using 3% dichloroacetic acid in toluene as deprotection reagent, the DMT protecting group of the last nucleotide is removed, followed by washing with acetonitrile.

[0333] (2) Coupling

[0334] Using 0.25 M 5-ethylthiotetrazole in acetonitrile as activating reagent, the acetonitrile solution of each nucleotide monomer is coupled, followed by washing with acetonitrile.

[0335] (3) Oxidation / Sulfurization

[0336] Oxidation: using 0.05 M iodine in pyridine / water (90 / 10) as oxidizing reagent, oxidation is carried out, followed by washing with acetonitrile.

[0337] Sulfurization: using 3% hydrogenated xanthate in pyridine as sulfurizing reagent, sulfurization is carried out, followed by washing with acetonitrile.

[0338] (4) Hydroxyl protection

[0339] Using 10% acetic anhydride in tetrahydrofuran (CAP A) and tetrahydrofuran / pyridine / n-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as hydroxyl protecting reagent, hydroxyl protection is carried out, followed by washing with acetonitrile.

[0340] The above operations are repeated and the cycle is repeated according to the set sequence to obtain the fully protected product.

[0341] (5) Using 3% dichloroacetic acid in toluene as deprotection reagent, the DMT protecting group of the last nucleotide is removed, followed by washing with acetonitrile.

[0342] (6) Aminolysis and purification

[0343] The solid phase carrier is transferred to a reactor, concentrated ammonia water (25-28%) is added, and after aminolysis at 60°C for 12 h, the system is reduced to room temperature, and the mixture is filtered, eluted with a mixed solution of purified water and ethanol, the filtrates are combined, passed through a chromatography column, concentrated, freeze-dried, and the product is obtained.

[0344] (7) Annealing

[0345] The purified sense and antisense strands were mixed at a ratio of 1:1, heated to 95°C and kept for 3 min, then slowly cooled to room temperature to form double-stranded.

[0346] A194-DV29P purity 90.52%; measured molecular weight: 14727.09.

[0347] 2. Synthesis of other sequences

[0348] The other sequences listed in Table 1 were synthesized according to the above method.

[0349] Table 1 DV29P template modification siRNA sequence

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358] Example 2: Inhibition of HBsAg and HBeAg by DV29P template modification sequences

[0359] After the DV29P template modification siRNA sequences synthesized in Example 1 were transfected into HepG2.2.15 cells by lipid nanoparticles (LNP), the inhibition of HBsAg and HBeAg by each sequence was detected using ELISA technology.

[0360] 1. Experimental materials

[0361] Test sample: DV29P template modification siRNA sequences listed in Table 1 (synthesized in Example 1).

[0362] Cell type: HepG2.2.15 cells

[0363] Drug vehicle: sterile enzyme-free water, gibco Opti-MEM (Thermo Fisher Scientific).

[0364] 2. Experimental method

[0365] The inhibition of HBsAg and HBeAg of HepG2.2.15 cell line by the sample was detected by ELISA.

[0366] 2.1 Cell culture

[0367] Subculture: HepG2.2.15 cells were subcultured in DMEM / F12 medium containing 10% fetal bovine serum, 370 μg / ml GENETICN, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin, and placed in a cell culture incubator at 37°C with 5% CO2, subcultured every three days. Subculture with 0.25% trypsin digestion, centrifuged at 800 r / min for 3 min, discard the supernatant, add fresh culture medium for subculture.

[0368] Plate culture: HepG2.2.15 cells were plated in DMEM / F12 medium containing 10% fetal bovine serum, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin.

[0369] 2.2 Cell transfection

[0370] Transfection mixture preparation: Lipofectamine TM RNAiMAX (Thermo Fisher Scientific) and Opti-MEM were mixed at a ratio of 2:98 and vortexed to mix.

[0371] Transfection complex preparation: 30 μL of siRNA solution diluted with Opti-MEM was added to 30 μL of transfection mixture at a ratio of 1:1 (v / v), vortexed to mix, and then incubated at room temperature for 15 min to obtain the transfection complex.

[0372] Transfection control reagent preparation: 15 μL of prepared transfection mixture was added to 15 μL of Opti-MEM. Vortex to mix, and incubate at room temperature for 15 min.

[0373] The prepared transfection complex was added to a 96-well cell culture plate (15 μL per well, 3 replicates per sequence), with a final siRNA concentration of 0.3 nM per well. 135 μL of cell suspension (containing 2.25×10 4 Using the cross method to mix, then placed in a 37°C, 5% CO2 cell culture incubator for culture.

[0374] 2.3 Detection of HBsAg and HBeAg in cell supernatant

[0375] 1) Cell supernatant collection

[0376] a. On the 3rd day after cell transfection, change the medium, discard the cell supernatant, and add 150 μL / well of fresh medium for continued culture.

[0377] b. On the 6th day after transfection, collect the cell supernatant for HBsAg and HBeAg content detection.

[0378] 2) HBsAg and HBeAg quantitative detection

[0379] The concentration of HBsAg and HBeAg was detected using the Hepatitis B Virus e Antigen Test Kit (Antibody CL0310) and the Hepatitis B Virus Surface Antigen Test Kit (Antibody CL0312). The specific operation steps are as follows:

[0380] a. The kit and the sample to be tested were recovered to room temperature.

[0381] b. The sample to be tested, standard, negative control, and positive sample were each added to the well plate, 50 μL each.

[0382] c. Add 50 μL of enzyme conjugate to each well. After mixing well, incubate at 37°C for 60 min.

[0383] d. Remove the liquid in the well plate and wash it with washing solution for 5 times. Finally, pat the well plate dry on the absorbent paper.

[0384] e. Mix the luminescent substrate A and B in equal proportions, add 50 μL / well, and react at room temperature for 3 min in the dark.

[0385] f. Measure the luminescence value on the enzyme label instrument.

[0386] 2.4 Data processing

[0387] The calculation formula of HBsAg and HBeAg inhibition rate is as follows:

[0388] HBsAg inhibition rate (%) = (1 - sample HBsAg expression amount / transfection control group HBsAg expression amount in the same plate) x 100%;

[0389] HBeAg inhibition rate (%) = (1 - sample HBeAg expression amount / transfection control group HBeAg expression amount in the same plate) x 100%.

[0390] HBsAg relative to the inhibition rate of the positive sample (the inhibition rate of the positive sample is set to 1) = sample HBsAg inhibition rate (%) / positive sample inhibition rate (%) in the same plate; it should be understood that the value greater than 1 indicates that the sequence has better inhibition effect on HBsAg than the positive sample, and the value less than 1 indicates that the sequence has lower inhibition efficiency on HBsAg than the positive sample.

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

[0392] 2.5 IC50 experiment

[0393] In this experiment, the concentration of each sequence was set from 10 nM, 4-fold dilution, a total of 8 concentration points (10 nM, 2.5 nM, 0.625 nM, 0.15625 nM, 39.06 pM, 9.77 pM, 2.44 pM and 0.61 pM), and the inhibition rate of each sequence at each concentration was determined, and a graph was drawn to calculate the IC50 concentration of each sequence and Bupleurum.

[0394] 2.6 Cytotoxicity experiment

[0395] When performing the IC50 experiment, after collecting the cell supernatant, the cell viability was determined by using the CellTiter-Glo ® (Promega) kit, and the method is briefly described as follows: the CellTiter-Glo reagent was mixed with the culture medium at 1:1, 100 μL was added to each well, and after incubation at room temperature for 10 min, the luminescence signal value was detected by using an enzyme-labeled instrument.

[0396] The cell viability calculation formula is: cell viability (%) = (sample signal value - blank control average value) / (transfection control average value - blank control average value) x 100%.

[0397] 3. Experimental results

[0398] The experimental results show that the sequences B207S-DV29P, B1572-DV29P, B1575-DV29P, B418S-DV29P, A206-DV29P, A416-DV29P, A1548-DV29P, A1550-DV29P and A1573-DV29P have a significantly better inhibition effect on HBsAg than other sequences, and have a significantly better inhibition effect on HBeAg than other sequences.

[0399] The inhibition rates of each sequence at a single concentration on the expression of HBsAg and HBeAg, and the inhibition rates relative to the sequence APC-VIR of Bupleurum are shown in Tables 3, 4, 5 and 6.

[0400] The single concentration screening experiment results show that some sequences modified by the template DV29P, including B207S-DV29P, B1572-DV29P, B1575-DV29P, B418S-DV29P, A206-DV29P, A416-DV29P, A1548-DV29P, A1550-DV29P, and A1573-DV29P (see Table 2 for specific sequences) have higher ranking of inhibition rates of HBsAg and HBeAg than that of the siRNA of the common sunflower.

[0401] The IC50 experiment and cytotoxicity results of each sequence are shown in Table 7. The IC50 experiment results show that the 11 candidate sequences modified by the design modification template DV29P have IC50 values of HBsAg in the range of 0.01-0.08 nM, which are better than 0.1777 nM of the common sunflower. For example, the IC50 values of A261-DV29P, A1573-DV29P, and A206-DV29P are 0.0169 nM, 0.0226 nM, and 0.0226 nM, respectively. These sequences can effectively inhibit the expression of HBsAg at a low concentration. The 11 candidate sequences have IC50 values of HBeAg in the range of 0.06-0.4 nM, which are better than 0.7685 nM of the common sunflower. For example, the IC50 values of A1573-DV29P, A206-DV29P, and A1550-DV29P are 0.0684 nM, 0.1105 nM, and 0.1202 nM, respectively. The cytotoxicity results show that among the 11 sequences, the cell viability of sequence A416-DV29P is 81.82% at the highest tested concentration of 10 nM, and the cell viability of the remaining sequences is greater than 90%, which does not exhibit obvious cytotoxicity, indicating that the sequences have a wide range of concentration selection.

[0402] The HBsAg inhibition effect is mainly considered, and the HBeAg inhibition effect is also considered. Based on the above results, 9 sequences are selected from the 11 sequences for further optimization design, and the sequences B207S-DV29P, B1572-DV29P, B1575-DV29P, B418S-DV29P, A206-DV29P, A416-DV29P, A1548-DV29P, A1550-DV29P, and A1573-DV29P can be used as candidate sequences.

[0403] Table 2 siRNA sequences with better inhibition rates of HBsAg and HBeAg than those of the common sunflower

[0404]

[0405]

[0406] (i) Inhibition of HBV gene expression at a single concentration after modification of each sequence with the DV29P template

[0407] (1) Sequences with better inhibitory effect on HBsAg than Xiangshen are shown in Table 3, including B207S-DV29P, B1575-DV29P, B1572-DV29P, B418S-DV29P, A261-DV29P, A206-DV29P, A1550-DV29P, A1573-DV29P, A1548-DV29P, and A416-DV29P. Among them, B207S-DV29P, B1575-DV29P, B1572-DV29P, and B418S-DV29P have an inhibition rate of more than 80% on HBsAg at a concentration of 0.3 nM. For example, B207S-DV29P has an inhibition rate of 93.37%, which is 23.59% higher than Xiangshen.

[0408] Table 3 DV29P-modified sequences with higher inhibition rate on HBsAg than Xiangshen

[0409]

[0410] (2) Sequences with worse inhibitory effect on HBsAg than Xiangshen are shown in Table 4, such as sequence B416S-DV29P, which has an inhibition rate of only 6.98% on HBsAg.

[0411] Table 4 DV29P-modified sequences with lower inhibition rate on HBsAg than Xiangshen

[0412]

[0413] (3) Sequences with better inhibitory effect on HBeAg than Xiangshen are shown in Table 5, among which B1575-DV29P, B207S-DV29P, B1572-DV29P, and B418S-DV29P rank the highest, with an inhibition rate 38.62%, 22.07%, 17.10%, and 13.24% higher than Xiangshen, respectively.

[0414] Table 5 DV29P-modified sequences with higher inhibition rate on HBeAg than Xiangshen

[0415]

[0416] (4) Sequences with worse inhibitory effect on HBeAg than Xiangshen are shown in Table 6, such as sequence A1520-DV29P, which has an inhibition rate of only 0.28% at a concentration of 0.3 nM.

[0417] Table 6 DV29P-modified sequences with lower inhibition rate on HBeAg than Xiangshen

[0418]

[0419] (ii) IC50 experiment and cytotoxicity results

[0420] The sequences with good performance in the single concentration experiment were selected, including B207S-DV29P, B1572-DV29P, B1575-DV29P, B418S-DV29P, A206-DV29P, A416-DV29P, A1548-DV29P, A1550-DV29P, and A1573-DV29P, a total of 9 sequences, to carry out IC50 experiment. The results show that the IC50 of these candidate sequences on HBsAg and HBeAg are all less than that of the Xiangshen, proving that the inhibition effect is better than that of the Xiangshen. For example, the IC50 values of A261-DV29P, A1573-DV29P, and A206-DV29P on HBsAg are 0.019 nM, 0.022 nM, and 0.024 nM, respectively, and the IC50 values of A1573-DV29P, B207S-DV29P, A206-DV29P, and A1550-DV29P on HBeAg are 0.079 nM, 0.093 nM, 0.115 nM, and 0.138 nM, respectively. The specific results are shown in Table 7.

[0421] The cytotoxicity results show that among these sequences, the cell viability of sequence A416-DV29P is 81.82% at the highest tested concentration of 10 nM, and the cell viability of the remaining sequences is greater than 90%, without showing obvious cytotoxicity, indicating that the sequences have a wide concentration selection range. The specific results are shown in Table 7.

[0422] Table 7 IC50 experiment on HBsAg and HBeAg

[0423]

[0424] Example 3: Inhibition effect of unmodified sequences on HBV gene

[0425] In this example, some unmodified sequences corresponding to the modified sequences in Example 1 were synthesized, and were transfected into HepG2.2.15 cells through lipid nanoparticles (LNP), and the inhibition effect of each sequence on HBsAg and HBeAg was detected by ELISA technology, to screen unmodified siRNA sequences with good inhibition effect.

[0426] 1. Experimental materials

[0427] Tested samples:

[0428] The unmodified small interfering ribonucleic acid sequences listed in Table 8. All sequences were synthesized according to the method in Example 1.

[0429] Table 8 Unmodified siRNA sequences

[0430]

[0431] Cell type: HepG2.2.15 cells

[0432] Drug solvent: sterile enzyme-free water, gibco Opti-MEM.

[0433] 2. Experimental method

[0434] Referring to Example 2, the screening concentration is set to 0.3 nM.

[0435] 3. Experimental results

[0436] The experimental results show that sequences B207S, B1572, B1575, B418S, A206, A416, A1548, A1550, and A1573 have significantly better inhibitory effects on HBsAg than other sequences and significantly better inhibitory effects on HBeAg than other sequences.

[0437] The inhibitory rates of each sequence on the expression of HBsAg and HBeAg at a single concentration are better than those of the sequence of the common sage, as shown in Table 9, including B207S, B1575, B418S, A1573, B1572, A261, A206, A1550, A416, and A1548, wherein the inhibitory rates of B207S on HBsAg and HBeAg are 77.02% and 40.74%, respectively, which are significantly better than the inhibitory rates of 61.45% and 20.85% of the sequence APC of the common sage.

[0438] Table 9 Basic sequences with higher inhibitory rates on HBsAg and HBeAg than the common sage

[0439]

[0440] Example 4: Inhibitory effects of sequences modified using different templates on HBsAg and HBeAg

[0441] This example is modified by using 8 modification templates DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P and DV34P designed by the present disclosure to modify 15 sequences A205, B208, B207S, A261, B262, B264, A1550, B1556, B1560, B1575, A1573, B1572, B418S, A416, B416S, and existing disclosed templates DV30P, DV31P, DV21P, DV22P to modify, a total of 154 sequences. After transfection into HepG2.2.15 cells by lipid nanoparticles (LNP), the inhibitory effect of each sequence on HBsAg and HBeAg was detected by ELISA technology.

[0442] 1. Experimental materials

[0443] Test samples: sequences modified by 15 siRNA base sequences using different templates (DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, DV34P, DV30P, DV31P, DV21P, DV22P) listed in Table 11 (synthesis method refers to Example 1).

[0444] Cell type: HepG2.2.15 cells

[0445] Drug vehicle: sterile enzyme-free water, gibco Opti-MEM

[0446] The modification principle of the modification templates of the present disclosure is as follows:

[0447] When the length of the antisense strand is 23 nucleotides, the antisense strand uses one or more combinations of the modification methods shown in Table 10-1 as follows:

[0448] Table 10-1 Antisense strand modification method

[0449]

[0450]

[0451]

[0452] When the length of the antisense strand is 22 nucleotides, the antisense strand uses one or more combinations of the modification methods shown in Table 10-2 as follows:

[0453] Table 10-2 Antisense strand modification method

[0454]

[0455]

[0456] When the length of the sense strand is 21 nucleotides, the sense strand adopts one or a combination of two of the following modification patterns shown in Table 10-3:

[0457] Table 10-3 Modification patterns of sense strand

[0458]

[0459] When the length of the sense strand is 20 nucleotides, the sense strand adopts one or a combination of two of the following modification patterns shown in Table 10-4:

[0460] Table 10-4 Modification patterns of sense strand

[0461]

[0462] In the above Tables 10-1 to 10-4, 2'-OMe is 2'-methoxy; 2'-F is 2'-fluoro; PS is phosphorothioate backbone; EVP is 5'-vinyl-(E)-phosphonate;

[0463] The siRNA modified template in which the antisense strand adopts Modification A and the sense strand adopts modification pattern a is named as DV27P;

[0464] The siRNA modified template in which the antisense strand adopts Modification A and the sense strand adopts modification pattern b is named as DV29P;

[0465] The siRNA modified template in which the antisense strand adopts Modification B and the sense strand adopts modification pattern a is named as DV26P;

[0466] The siRNA modified template in which the antisense strand adopts Modification B and the sense strand adopts modification pattern b is named as DV28P;

[0467] The siRNA modified template in which the antisense strand adopts Modification C and the sense strand adopts modification pattern b is named as DV32P;

[0468] The siRNA modified template in which the antisense strand adopts Modification D and the sense strand adopts modification pattern b is named as DV34P;

[0469] The siRNA modified template in which the antisense strand adopts Modification E and the sense strand adopts modification pattern a is named as DV25P;

[0470] The siRNA modified template in which the antisense strand adopts Modification F and the sense strand adopts modification pattern b is named as DV33P.

[0471] The synthesis method of each sequence is the same as in Example 1.

[0472] Table 11 Sequences modified with different modification templates

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504] 2. Experimental method

[0505] Referring to Example 2, the single concentration screening experiment concentration is set to 0.3 nM.

[0506] 3. Experimental results

[0507] The experimental results show that the sequences B207S-DV32P, A1550-DV27P, B1572-DV27P, B1575-DV27P, B207S-DV29P, A1573-DV29P, B1575-DV29P, B418S-DV34P, and A416-DV27P have significantly better inhibitory effect on HBsAg than the sequence of the common sunflower, and have significantly better inhibitory effect on HBeAg than the sequence of the common sunflower.

[0508] The inhibition rates of each sequence on the expression of HBsAg and HBeAg at a single concentration and relative to the sequence APC-VIR of the common sunflower are shown in Tables 12-41. The comprehensive ranking is represented as the ranking of the inhibition rate of the sequence relative to the common sunflower.

[0509] The single concentration screening experiment results show that some basic sequences are modified by the modification templates DV25P-29P and DV32P-34P designed by the present disclosure, and have better inhibitory effect on HBsAg and HBeAg than the sequence of Xanthium. Among them, the modified sequences designed by the basic sequences B207S and B1575 are ranked first in the inhibition rate of HBsAg, which proves the sensitivity of the target site. Among them, the sequences B207S-DV29P, B207S-DV32P, B1575-DV29P, B1575-DV26P and B1575-DV27P have an inhibition rate of HBsAg of more than 90%. For example, the sequences B207S-DV29P and B1575-DV29P have an inhibition rate of HBsAg of 92.11% and 91.78%, respectively. Among them, the modified sequences designed by the basic sequences B1575 and A1573 are ranked first in the inhibition rate of HBeAg, which proves the sensitivity of the target site. Among them, the sequences B1575-DV29P, B1575-DV26P, B1575-DV27P and B1575-DV32P have an inhibition rate of HBeAg of more than 60%.

[0510] The activity difference is very large after the same siRNA sequence is modified by different modification templates. For example, the inhibition rate of HBsAg of the basic sequence B1556 modified by the modification template DV27P of the present disclosure is 48.63% higher than that of the modification template DV28P of the present disclosure, which is significantly improved.

[0511] Although the basic sequences are similar, the sensitivity to each modification template is different. For example, the inhibition rate of HBsAg and HBeAg of the basic sequence B1572 modified by the modification template DV27P designed by the present disclosure is the highest, while the inhibition rate of HBsAg and HBeAg of the basic sequences A1573 and B1575 modified by the template DV29P is the highest. Therefore, it is uncertain which modification template is used to modify the siRNA sequence to have high activity.

[0512] The IC50 experiment and cytotoxicity results of each sequence are shown in Table 42. The experimental results show that the IC50 values of the 11 sequences for HBsAg are between 0.05 nM and 0.15 nM, all less than 0.3362 nM of the Gynostemma pentaphyllum sequence, proving that the inhibition effect is better than that of Gynostemma pentaphyllum. For example, the IC50 values of B1572-DV27P, B1550-DV27P and B207S-DV32P for HBsAg are 0.0538 nM, 0.0570 nM and 0.0572 nM, respectively. Except for sequence B261-DV26P, the IC50 values of the remaining 10 sequences for HBeAg are between 0.2 nM and 0.85 nM, all less than 1.89 nM of the Gynostemma pentaphyllum sequence, proving that the inhibition effect is better than that of Gynostemma pentaphyllum. For example, the IC50 values of sequences B1550-DV27P, B1572-DV27P and B1575-DV26P for HBeAg are 0.2073 nM, 0.2143 nM and 0.2923 nM, respectively. The cytotoxicity results show that the cell viability of the 11 sequences is greater than 90% at the highest tested concentration of 10 nM, without showing obvious cytotoxicity, indicating that the sequences have a wide concentration selection range.

[0513] In summary, the above results, 10 sequences B207S-DV29P, B207S-DV32P, B1575-DV29P, B1575-DV26P, B1575-DV27P, B1572-DV27P, B1573-DV29P, B1550-DV27P, B416-DV27P and B418S-DV34P can be used as candidate sequences.

[0514] (i) Inhibition of HBsAg and HBeAg by each base sequence after template modification

[0515] (1) Base sequence A1550

[0516] Table 12 Inhibition rate of base sequence A1550 after template modification for HBsAg

[0517]

[0518] Table 13 Inhibition rate of base sequence A1550 after template modification for HBeAg

[0519]

[0520] The basic sequence A1550 is modified by the modification templates DV25P-29P, DV32P-34P designed in the present disclosure, and compared with the modification by the modification templates disclosed in the prior art, the inhibition rates of HBsAg and HBeAg are both improved. For example, after modification by the templates DV26P and DV27P, the inhibition rates of B1550-DV26P and B1550-DV27P on HBsAg are increased by 25.36% and 25.51% respectively, and the inhibition rates on HBeAg are increased by 24.77% and 26.55% respectively. The specific results are shown in Tables 12 and 13.

[0521] (2) Basic sequence B1556

[0522] Table 14 Inhibition rate of HBsAg after modification of the basic sequence B1556 by a template

[0523]

[0524] Table 15 Inhibition rate of HBeAg after modification of the basic sequence B1556 by a template

[0525]

[0526] After modification of the basic sequence B1556 by the modification templates DV26P-29P, DV32P, and DV34P designed in the present disclosure, compared with the modification by the modification templates disclosed in the prior art, after modification by the templates DV26P and DV27P, the inhibition rates of B1556-DV26P and B1556-DV27P on HBsAg are increased by 37.46% and 47.61% respectively, and the inhibition rates on HBeAg are increased by 25.29% and 31.73% respectively. The specific results are shown in Tables 14 and 15.

[0527] (3) Basic sequence B1560

[0528] Table 16 Inhibition rate of HBsAg after modification of the basic sequence B1560 by a template

[0529]

[0530] Table 17 Inhibition rate of HBeAg after modification of the basic sequence B1560 by a template

[0531]

[0532] The base sequence B1560 is modified by the modification template DV26P-29P, DV32P, and DV34P designed by the present disclosure. Compared with the modification templates disclosed in the prior art, the inhibition rates of B1560-DV26P and B1560-DV27P on HBsAg are increased by 35.82% and 43.60% respectively, and the inhibition rates on HBeAg are increased by 20.84% and 28.54% respectively. The specific results are shown in Tables 16 and 17.

[0533] Therefore, it can be seen that:

[0534] 1) For the base sequences A1550, B1556, and B1560, the modification templates DV25P-29P and DV32P-34P designed by the present disclosure are significantly better than the modification templates disclosed in the prior art in terms of the inhibition of HBsAg and HBeAg. The modification templates DV26P and DV27P are the best and significantly better than the modification templates disclosed in the prior art in terms of the inhibition of HBsAg and HBeAg.

[0535] 2) The activities of the same siRNA sequence modified by different modification templates are very different. For example, the base sequence B1556 modified by the modification template DV27P designed by the present disclosure is significantly better than the base sequence B1556 modified by the modification template DV28P designed by the present disclosure in terms of the inhibition of HBsAg, with an increase of 48.63%.

[0536] 3) The activities of siRNAs with similar sequences are very different. For example, the inhibition rate of A1550-DV29P on HBsAg is increased by 40.86% compared with A1556-DV29P.

[0537] (4) Base sequence B1572

[0538] Table 18 Inhibition rates of base sequence B1572 modified by templates on HBsAg

[0539]

[0540] Table 19 Inhibition rates of base sequence B1572 modified by templates on HBeAg

[0541]

[0542] The basic sequence B1572 is modified by the modification templates DV26P-29P, DV32P and DV34P designed by the present disclosure. Compared with the modification templates disclosed in the prior art, the inhibition rates of B1572-DV26P and B1572-DV27P on HBsAg are increased by 26.84% and 27.59% respectively, and the inhibition rates on HBeAg are increased by 32.02% and 36.74% respectively after modification by the templates DV26P and DV27P. The specific results are shown in Tables 18 and 19.

[0543] (5) Basic sequence A1573

[0544] Table 20 Inhibition rate of basic sequence A1573 after modification by templates on HBsAg

[0545]

[0546] Table 21 Inhibition rate of basic sequence A1573 after modification by templates on HBeAg

[0547]

[0548] The basic sequence A1573 is modified by the modification templates DV26P-29P, DV32P and DV34P designed by the present disclosure. Compared with the modification templates disclosed in the prior art, the inhibition rates of A1573-DV29P on HBsAg and HBeAg are increased by 16.08% and 32.68% respectively after modification by the template DV29P. The specific results are shown in Tables 20 and 21.

[0549] (6) Basic sequence B1575

[0550] Table 22 Inhibition rate of basic sequence B1575 after modification by templates on HBsAg

[0551]

[0552] Table 23 Inhibition rate of basic sequence B1575 after modification by templates on HBeAg

[0553]

[0554] The basic sequence B1575 is modified by the modification templates DV26P-29P, DV32P and DV34P designed by the present disclosure. Compared with the modification templates disclosed in the prior art, the inhibition rates on HBsAg and HBeAg are significantly increased. For example, the inhibition rates of B1575-DV29P on HBsAg and HBeAg are increased by 31.19% and 46.17% respectively after modification by the template DV29P. The specific results are shown in Tables 22 and 23.

[0555] Therefore, it can be seen that:

[0556] 1) For the base sequences B1572, A1573, B1575, the inhibition of HBsAg and HBeAg after modification by the modification templates DV26P-29P, DV32P, DV34P designed by the present disclosure is significantly better than that by the modification templates disclosed in the prior art. For the base sequence B1572, the inhibition of HBsAg and HBeAg after modification by the modification templates DV26P and DV27P is the highest, and is significantly better than that by the modification templates disclosed in the prior art. For the base sequences A1573 and B1575, the inhibition of HBsAg and HBeAg after modification by the modification template DV29P is the highest, and is significantly better than that by the modification templates disclosed in the prior art.

[0557] 2) The activity difference is very large after modification of the same siRNA sequence by different modification templates. For example, the inhibition rate of HBsAg of the base sequence B1575 modified by the modification template DV29P designed by the present disclosure is increased by 29.99% compared with that of the base sequence modified by the modification template DV28P, which is significantly improved.

[0558] 3) The sensitivity of siRNAs with similar sequences to each modification template is also different. For example, the inhibition rate of HBsAg and HBeAg of the base sequence B1572 modified by the modification template DV27P designed by the present disclosure is the highest, while the inhibition rate of HBsAg and HBeAg of the base sequences A1573 and B1575 modified by the modification template DV29P is the highest.

[0559] (7) Base sequence B205

[0560] Table 24 Inhibition rate of HBsAg of base sequence B205 modified by templates

[0561]

[0562] Table 25 Inhibition rate of HBeAg of base sequence B205 modified by templates

[0563]

[0564] The base sequence B205 is modified by the modification templates DV26P-29P, DV32P, and DV34P designed by the present disclosure. Compared with the modification templates disclosed in the prior art, the inhibition rates of HBsAg and HBeAg are significantly improved. For example, after modification by the template DV28P, the inhibition rates of HBsAg and HBeAg of B205-DV28P are increased by 36.55% and 25.36%, respectively. The specific results are shown in Tables 24 and 25.

[0565] (8) Basic sequence B207S

[0566] Table 26 Inhibition rate of HBsAg after modification of basic sequence B207S using template modification

[0567]

[0568] Table 27 Inhibition rate of HBeAg after modification of basic sequence B207S using template modification

[0569]

[0570] Compared with the prior art modification templates, the modification templates DV25P-29P and DV32P-34P designed by the present disclosure can significantly improve the inhibition rates of HBsAg and HBeAg. For example, after modification using templates DV29P and DV32P, the inhibition rates of B207S-DV29P for HBsAg and HBeAg are increased by 35.37% and 26.71%, respectively, and the inhibition rates of B207S-DV32P for HBsAg and HBeAg are increased by 34.98% and 18.95%, respectively. The specific results are shown in Table 26 and Table 27.

[0571] (9) Basic sequence B208

[0572] Table 28 Inhibition rate of HBsAg after modification of basic sequence B208 using template modification

[0573]

[0574] Table 29 Inhibition rate of HBeAg after modification of basic sequence B208 using template modification

[0575]

[0576] Compared with the prior art modification templates, the modification templates DV26P-29P, DV32P, and DV34P designed by the present disclosure can significantly improve the inhibition rates of HBsAg and HBeAg. For example, after modification using template DV26P, the inhibition rates of B208-DV26P for HBsAg and HBeAg are increased by 37.63% and 19.38%, respectively. The specific results are shown in Table 28 and Table 29.

[0577] Therefore, it can be known that:

[0578] 1) For the base sequences B205, B207S, and B208, the inhibition of HBsAg and HBeAg by the modified templates DV25P-29P and DV32P-34P designed according to the present disclosure is significantly better than that of the prior art disclosed modified templates. For the base sequence B205, the inhibition of HBsAg and HBeAg by the modified template DV28P designed according to the present disclosure is the highest, and is significantly better than that of the prior art disclosed modified templates. For the base sequence B207S, the inhibition of HBsAg and HBeAg by the modified template DV29P designed according to the present disclosure is the highest, and is significantly better than that of the prior art disclosed modified templates. For the base sequence B208, the inhibition of HBsAg and HBeAg by the modified template DV26P designed according to the present disclosure is the highest, and is significantly better than that of the prior art disclosed modified templates.

[0579] 2) The activity of the same siRNA sequence modified by different modified templates is very different. For example, the inhibition of HBsAg by the modified sequence B208 modified by the modified template DV26P according to the present disclosure is 21.96% higher than that of the modified sequence B208 modified by the modified template DV29P according to the present disclosure, which is significantly improved.

[0580] 3) The activity of siRNA with similar sequences is very different. For example, the inhibition of HBsAg by the modified sequence B207S-DV29P is 45.84% higher than that of the modified sequence B208-DV29P, and 23.77% higher than that of the modified sequence B205-DV29P.

[0581] 4) Therefore, it can be seen that although the base sequences are similar, the sensitivity to each modified template is different. For example, the inhibition of HBsAg and HBeAg by the base sequence B205 modified by the modified template DV28P according to the present disclosure is the highest, which is 78.48% and 35.95%, respectively; the inhibition of HBsAg and HBeAg by the base sequence B207S modified by the modified template DV29P is the highest, which is 92.11% and 55.18%, respectively; and the inhibition of HBsAg and HBeAg by the base sequence B208 modified by the modified template DV26P is the highest, which is 68.23% and 41.45%, respectively.

[0582] (10) Base sequence A261

[0583] Table 30 Inhibition rate of HBsAg by base sequence A261 modified by templates

[0584]

[0585] Table 31 Inhibition rate of HBeAg by base sequence A261 modified by templates

[0586]

[0587] The basic sequence A261 is modified by the modification templates DV26P-29P, DV32P and DV34P designed by the present disclosure. Compared with the modification templates disclosed in the prior art, the inhibition rates of HBsAg and HBeAg can be significantly improved. For example, after modification by the template DV26P, the inhibition rates of B261-DV26P on HBsAg and HBeAg are increased by 16.82% and 11.89%, respectively. The specific results are shown in Tables 30 and 31.

[0588] (11) Basic sequence B262

[0589] Table 32 Inhibition rate of HBsAg after modification of the basic sequence B262 by a template

[0590]

[0591] Table 33 Inhibition rate of HBeAg after modification of the basic sequence B262 by a template

[0592]

[0593] The basic sequence B262 is modified by the modification templates DV26P-29P, DV32P and DV34P designed by the present disclosure. Compared with the modification templates disclosed in the prior art, the inhibition rates of HBsAg and HBeAg can be significantly improved. For example, after modification by the template DV26P, the inhibition rates of B261-DV26P on HBsAg and HBeAg are increased by 16.82% and 11.89%, respectively. The specific results are shown in Tables 30 and 31.

[0594] (12) Basic sequence B264

[0595] Table 34 Inhibition rate of HBsAg after modification of the basic sequence B264 by a template

[0596]

[0597] Table 35 Inhibition rate of HBeAg after modification of the basic sequence B264 by a template

[0598]

[0599] The basic sequence B264 is modified by the modification template DV26P-29P, DV32P and DV34P designed by the present disclosure, and compared with the modification templates disclosed in the prior art, the inhibition rates of HBsAg and HBeAg are significantly improved. For example, after modification by the template DV29P, the inhibition rates of B264-DV29P on HBsAg and HBeAg are increased by 35.22% and 13.12%, respectively. The specific results are shown in Tables 34 and 35.

[0600] Therefore, it can be seen that:

[0601] 1) For the basic sequences A261, B262 and B264, after modification by the modification templates DV26P-29P, DV32P and DV34P designed by the present disclosure, the inhibition effects on HBsAg and HBeAg are significantly better than those of the modification templates disclosed in the prior art. For the basic sequence A261, after modification by the modification template DV26P designed by the present disclosure, the inhibition rate of the sequence on HBsAg is the highest, and after modification by the template DV27P, the inhibition rate of the sequence on HBeAg is the highest, which is significantly better than that of the modification template disclosed in the prior art. For the basic sequence B262, after modification by the modification template DV28P designed by the present disclosure, the inhibition rates of the sequence on HBsAg and HBeAg are the highest, which are significantly better than those of the modification templates disclosed in the prior art. For the basic sequence B264, after modification by the modification template DV29P designed by the present disclosure, the inhibition rates of the sequence on HBsAg and HBeAg are the highest, which are significantly better than those of the modification templates disclosed in the prior art.

[0602] 2) After modification by different modification templates, the activities of the same siRNA sequence are quite different. For example, after modification by the modification template DV27P designed by the present disclosure, the inhibition rate of the sequence B264-DV27P on HBsAg is reduced by 20.16% compared with that of B264-DV29P, which is significantly reduced.

[0603] 3) The activities of siRNAs with similar sequences are quite different. For example, the inhibition rate of A261-DV29P on HBsAg is increased by 40.21% compared with that of B262-DV29P, and is increased by 30.46% compared with that of B264-DV29P.

[0604] 4) Therefore, it can be seen that although the basic sequences are similar, the sensitivities to the modification templates are different. For example, after modification by the modification template DV28P designed by the present disclosure, the inhibition rates of the sequence B262 on HBsAg and HBeAg are the highest, and after modification by the template DV29P, the inhibition rates of the sequence B264 on HBsAg and HBeAg are the highest.

[0605] (13) Basic sequence A416

[0606] Table 36 Inhibition rate of HBsAg after modification of base sequence A416 using template

[0607]

[0608] Table 37 Inhibition rate of HBeAg after modification of base sequence A416 using template

[0609]

[0610] The base sequence A416 is modified using the modification template DV26P-29P, DV32P, and DV34P designed according to the present disclosure, and the inhibition rates of HBsAg and HBeAg are significantly improved compared with the modification templates disclosed in the prior art. For example, the inhibition rates of HBsAg and HBeAg are increased by 29.19% and 33.83% respectively after modification with DV27P. The specific results are shown in Table 36 and Table 37.

[0611] (14) Base sequence B416S

[0612] Table 38 Inhibition rate of HBsAg after modification of base sequence B416S using template

[0613]

[0614] Table 39 Inhibition rate of HBeAg after modification of base sequence B416S using template

[0615]

[0616] The base sequence B416S is modified using the modification template DV26P-29P, DV32P, and DV34P designed according to the present disclosure, and the inhibition rates of HBsAg and HBeAg are significantly lower than those of the modification templates disclosed in the prior art, which are less than 25%. The specific results are shown in Table 38 and Table 39.

[0617] (15) Base sequence B418S

[0618] Table 40 Inhibition rate of HBsAg after modification of base sequence B418S using template

[0619]

[0620] Table 41 Inhibition rate of HBeAg after modification of base sequence B418S using template

[0621]

[0622] The basic sequence B418S is modified by the modification template DV26P-29P, DV32P and DV34P designed by the present disclosure. Compared with the modification templates disclosed in the prior art, the inhibition rates of HBsAg and HBeAg are significantly improved. For example, the inhibition rates of HBsAg and HBeAg are increased by 25.62% and 26.91% respectively after modification by the DV34P template. The specific results are shown in Tables 40 and 41.

[0623] Therefore, it can be seen that:

[0624] 1) For the basic sequences A416 and B418S, the inhibition effects of HBsAg and HBeAg after modification by the modification templates DV26P-29P, DV32P and DV34P designed by the present disclosure are significantly better than those of the modification templates disclosed in the prior art. For the basic sequence A416, the inhibition rates of HBsAg and HBeAg are the highest after modification by the modification template DV27P designed by the present disclosure. For the basic sequence B418S, the inhibition rates of HBsAg and HBeAg are the highest after modification by the DV34P template. For the basic sequence B416S, the inhibition rates of HBsAg and HBeAg are all lower than 25% after modification by the modification templates DV26P-28P, DV32P and DV34P designed by the present disclosure and the modification templates disclosed in the prior art.

[0625] 2) The activities of the same siRNA sequence modified by different modification templates are very different. For example, the inhibition rate of HBsAg of the sequence modified by the modification template DV27P designed by the present disclosure is increased by 16.79% compared with that of the sequence modified by the DV32P template.

[0626] 3) The activities of siRNAs with similar sequences are very different. For example, the inhibition rate of HBsAg of the sequence A416-DV29P is increased by 72.41% compared with that of the sequence B416S-DV29P.

[0627] 4) Therefore, it can be seen that although the basic sequences are similar, the sensitivities to the modification templates are different. For example, the inhibition rates of HBsAg and HBeAg are the highest after modification of the basic sequence A416 by the modification template DV27P designed by the present disclosure, and the inhibition rates of HBsAg and HBeAg are the highest after modification of the basic sequence B418S by the DV34P template.

[0628] (ii) IC50 experiment and cytotoxicity results

[0629] The advantage sequences in the single concentration screening experiment were selected, including B207S-DV29P, B207S-DV32P, B1575-DV29P, B1575-DV26P, B1575-DV27P, B1572-DV27P, B1573-DV29P, B1550-DV27P, B261-DV26P, B416-DV27P and B418S-DV34P, and one positive sequence APC-VIR, a total of 12 sequences, to carry out IC50 experiment to determine the IC50 concentration of each sequence.

[0630] Table 42 IC50 experiment results of each modified sequence

[0631]

[0632] The IC50 experiment results show that the IC50 values of the 11 sequences to HBsAg are between 0.05 nM and 0.15 nM, all less than 0.3362 nM of the positive sequence, proving that the inhibition effect is better than that of the positive sequence. For example, the IC50 values of sequences B1572-DV27P, B1550-DV27P and B207S-DV32P to HBsAg are 0.0538 nM, 0.0570 nM and 0.0572 nM respectively, indicating that these sequences can effectively inhibit the expression of HBsAg at low concentrations. Except for sequence B261-DV26P, the IC50 values of the remaining 10 sequences to HBeAg are between 0.2 nM and 0.85 nM, all less than 1.89 nM of the positive sequence, proving that the inhibition effect is better than that of the positive sequence. For example, the IC50 values of sequences B1550-DV27P, B1572-DV27P and B1575-DV26P to HBeAg are 0.2073 nM, 0.2143 nM and 0.2923 nM respectively, indicating that these sequences can effectively inhibit the expression of HBeAg at low concentrations. The experimental results are shown in Table 42.

[0633] The cytotoxicity results show that the cell viability of the 11 sequences at the highest tested concentration of 10 nM is greater than 90%, without showing obvious cytotoxicity, indicating that the sequences have a wide concentration selection range. The specific results are shown in Table 42.

[0634] Example 5: Comparison of the inhibition of HBV genes by the sequences disclosed in the prior art

[0635] The present embodiment compares the inhibition efficiency of the HBV gene of the unmodified sequence disclosed in the prior art, which is the same as or similar to the base sequence B207S, B1575, B1550, B1572, A416, B1573, B418S of the present disclosure, with the sequence modified by the prior disclosed modification templates DV30P, DV31P, DV21P, DV22P and the sequence modified by the modification templates DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P and DV34P of the present disclosure.

[0636] 1. Experimental materials

[0637] Test samples:

[0638] (1) Prior art disclosed sequences, see Table 43.

[0639] Table 43 Prior art disclosed sequences

[0640]

[0641] Note: The disclosed sequences in the above patent applications are all unmodified sequence RNA.

[0642] (2) siRNA sequences modified by the modification templates DV25P, DV26P, DV27P, DV28P, DV29P, DV32P, DV33P, DV34P, DV30P, DV31P, DV21P, DV22P and the corresponding unmodified base sequences in Example 4.

[0643] Cell type: HepG2.2.15 cells

[0644] Drug vehicle: sterile enzyme-free water, gibco Opti-MEM

[0645] 2. Experimental method

[0646] Referring to Example 2, the single concentration screening experiment concentration is set to 0.3 nM.

[0647] 3. Experimental results

[0648] The experimental results show that the unmodified base sequences B207S, A1550, B1572, B1575, A1573, B418S, A416 of the present disclosure have significantly better inhibition rates on HBsAg and HBeAg than the sequences similar to P206, P413, P1551 and APC, and after modification by the templates, the inhibition rates are further significantly improved.

[0649] Table 44 and Table 45 respectively show the comparison of the inhibition rates of the sequences similar to the prior art disclosed and the sequences of the present disclosure on HBsAg and HBeAg.

[0650] As can be seen from Table 43, compared with the prior art sequence P206, the disclosed sequence B207S is moved back by 1 base and the length is reduced by 1 base, and the inhibition rates of B207S on HBsAg and HBeAg are increased by 15.57% and 11.69% respectively compared with P206, and after the B207S is modified by the disclosed template, the inhibition efficiency is further improved, for example, after the B207S is modified by DV32P, the inhibition rates of B207S-DV32P on HBsAg and HBeAg are increased by 28.92% and 20.55% respectively, and B207S-DV32P is significantly better than the prior art modification templates DV30P, DV31P, DV21P and DV22P.

[0651] Compared with the prior art sequence P413, the disclosed sequence A416 is moved back by 3 bases, and the inhibition rates of A416 on HBsAg and HBeAg are increased by 5.95% and 7.42% respectively compared with P413, and after the A416 is modified by the disclosed template, the inhibition efficiency is further improved, for example, after the B416 is modified by DV27P, the inhibition rates of B416-DV27P on HBsAg and HBeAg are increased by 24.16% and 21.26% respectively, and B416-DV27P is significantly better than the prior art modification templates DV30P, DV31P, DV21P and DV22P.

[0652] Compared with the prior art sequence P1551, the disclosed sequence A1550 is moved forward by 1 base, and the inhibition rates of A1550 on HBsAg and HBeAg are increased by 7.98% and 9.03% respectively compared with P1551, and after the A1550 is modified by the disclosed template, the inhibition efficiency is further improved, for example, after the B1550 is modified by DV27P, the inhibition rates of B1550-DV27P on HBsAg and HBeAg are increased by 23.96% and 26.06% respectively, and B1550-DV27P is significantly better than the prior art modification templates DV30P, DV31P, DV21P and DV22P.

[0653] Compared with the prior art sequence APC, the disclosed sequences B1575, B1573 and B1572 are respectively moved forward by 4, 6 and 7 bases, and the length is all extended by 2 bases. Compared with APC, the inhibition rates of B1575, B1573 and B1572 on HBsAg are respectively increased by 13.46%, 10.80% and 9.94%. After modification by the disclosed template, the inhibition efficiency is further improved. For example, the sequence B1575-DV29P has the most significant improvement effect, with an increase of 29.28%, and the inhibition rate on HBeAg is increased by 24.25%. The sequence B1575-DV26P has an increase of 27.18% on the inhibition rate of HBsAg, and an increase of 16.55% on the inhibition rate of HBeAg, which is significantly better than the prior art modification templates DV30P, DV31P, DV21P and DV22P.

[0654] Table 44 Comparison of inhibition rates of prior art and disclosed sequences on HBsAg

[0655]

[0656]

[0657] Table 45 Comparison of inhibition rates of prior art and disclosed sequences on HBeAg

[0658]

[0659]

[0660] Example 6: Inhibition of HBV and off-target genes by sequences with specific off-target prevention design

[0661] In the actual application of siRNA, there are many cases of non-target mRNA expression being inhibited only partially complementary to the guide strand (antisense strand). The research of Arima Company shows that the hepatotoxicity of n-acetylgalactosamine (GalNAc) conjugated siRNA is mainly due to the off-target effect caused by gene inhibition on the wrong target through a mechanism similar to microRNA (miRNA) recognition.

[0662] To solve this problem, hot unstable nucleotides such as glycol nucleic acid (GNA), unlocked nucleic acid (UNA), DNA, etc. can be modified at positions 6 and 7 of the antisense strand seed region to disturb the seed region of the antisense strand, thereby affecting the binding of siRNA and non-target mRNA through the seed region recognition mode, and further significantly reducing the off-target effect and alleviating hepatotoxicity. Arima Company has adopted a method of placing a GNA-modified nucleotide at position 7 of the antisense strand of siRNA in the latest fifth-generation template design to reduce the off-target effect of siRNA.

[0663] In this embodiment, the sequences B207S, B1575, A1550, B1572, A1573, A261, A416, and B418S were used for off-target and anti-off-target research. In order to evaluate the off-target of each sequence, the inhibition efficiency of each sequence on target genes and off-target genes in cell experiments was compared by qPCR.

[0664] In order to reduce the off-target effect of the sequence, the sequences B418S-DV34P and B416-DV27P with potential off-target effect were modified with DNA or GNA anti-off-target at positions 6 and 7 of the antisense strand, and the activity and off-target effect were compared with the sequence without anti-off-target modification.

[0665] 1. Experimental materials

[0666] Test samples: template modified sequences and corresponding sequences with anti-off-target design listed in Table 46 (synthesis method refers to Example 1). d67B in the sequence number indicates that the sequence is modified with d67B anti-off-target, d7B indicates that the sequence is modified with d7B anti-off-target, and + indicates that the sequence is modified with GNA anti-off-target.

[0667] Cell type: HepG2.2.15 cells

[0668] Drug vehicle: sterile enzyme-free water, gibco Opti-MEM.

[0669] Table 46 Template modification and anti-off-target design sequence

[0670]

[0671] 2. Experimental method

[0672] The inhibition of HBV and off-target genes of the sample on HepG2.2.15 cells was detected by qRT-PCR method.

[0673] 2.1 Cell culture

[0674] Subculture: HepG2.2.15 cells were subcultured in DMEM / F12 medium containing 10% fetal bovine serum, 370 μg / ml GENETICN, 1% L-glutamine, 1% non-essential amino acids, and 1% penicillin-streptomycin, and placed in a cell incubator at 37°C with 5% CO2. Subculture every three days. Subculture with 0.25% trypsin digestion, centrifuge at 800 r / min for 3 min, discard the supernatant, and subculture with fresh medium.

[0675] Plating: HepG2.2.15 cells were plated in DMEM / F12 medium containing 10% fetal bovine serum, 1% L-glutamine, 1% non-essential amino acids, 1% penicillin-streptomycin.

[0676] 2.2 Cell transfection

[0677] Transfection reagent preparation: Lipofectamine RNAiMAX and Opti-MEM were mixed at a ratio of 2:98 and vortexed to mix well.

[0678] Transfection complex preparation: 30 μL of siRNA solution diluted in Opti-MEM was added to 30 μL of transfection mixture at a ratio of 1:1 (v / v), vortexed to mix well, and then incubated at room temperature for 15 min to obtain the transfection complex.

[0679] Transfection control reagent preparation: The prepared transfection mixture was added to 15 μL of Opti-MEM. Vortexed to mix well, and then incubated at room temperature for 15 min.

[0680] The prepared transfection complex was added to a 96-well cell culture plate (15 μL per well, 3 replicate wells per sequence), with a final siRNA concentration of 0.065, 0.14, and 0.28 nM per well. Three concentrations were set for each siRNA, and three replicate wells were set for each concentration. 135 μL of cell suspension (containing 2.25 x 10 4 After mixing using the cross method, the plate was incubated in a 37°C, 5% CO2 cell incubator.

[0681] 2.3 RNA extraction and reverse transcription

[0682] After transfection for 48 h, the culture medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using the RNeasy® 96 Kit (QIAGEN-74182) according to the kit instructions. Subsequently, RNA was reverse transcribed to cDNA using the HiScript III RT SuperMix for qPCR (Vazyme) according to the instructions.

[0683] 2.4 RT-qPCR

[0684] HBV and off-target gene cDNA were detected by qPCR, and GAPDH cDNA was detected as an internal control. 8 μL of prepared qPCR reaction solution and 2 μL of sample cDNA were added in a 384-well plate. The SYBR qPCR reaction program was as follows: 50 °C for 2 min, 95 °C for 2 min, then enter the cycle mode, 95 °C for 5 s, then 60 °C for 30 s, for a total of 40 cycles; finally, the melting curve was heated at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s.

[0685] 2.5 Data analysis

[0686] The CT value of each sample was used to calculate the RNA expression level of the target gene in the sample, and the calculation was performed by the ΔΔCT relative quantification method. The relative expression of the target gene was expressed by 2 -ΔΔCT

[0687] The calculation formula is as follows:

[0688] ΔCT = average CT value of target gene - average CT value of GAPDH;

[0689] ΔΔCT = ΔCT (experimental group) - ΔCT (transfection control group);

[0690] Relative expression of target gene mRNA = 2 -ΔΔCT

[0691] Gene expression inhibition rate (%) = (1 - sample mRNA expression / transfection control mRNA expression) x 100%

[0692] 3. Experimental results

[0693] The experimental results show that sequences B207S, B1575, A1550, B1572, A1573, and A261 have no off-target effect. Sequences B418S-DV34P and A416-DV27P have significantly reduced inhibition effect on off-target genes after adopting the off-target prevention design, while not affecting the inhibition effect of the target gene HBV. It is shown that the off-target prevention design does not affect the inhibition effect of the template modification sequence of the present disclosure on the HBV gene, but can significantly inhibit the off-target effect.

[0694] (i) qPCR analysis of the potential off-target effect and off-target prevention effect of each sequence

[0695] ​(1) The inhibition efficiency of each sequence on target genes and off-target genes is shown in Table 47. The results show that the inhibition of sequence A416 on gene SLCO2B1 has obvious concentration dependence, and the maximum inhibition rate is 40.05%; the inhibition of sequence B418S on gene SLC41A2 has a certain concentration dependence, and the maximum inhibition rate is 24.13%; the inhibition rates of the remaining sequences B207S, A1550, and A416 on off-target genes are all less than 20%, indicating no off-target effect. The two sequences A416 and BS18S have more potential off-target genes, so the sequences A416 and BS18S have potential off-target effects.

[0696] Table 47 Inhibition effect of each sequence on off-target genes and target genes

[0697]

[0698] (2) The sequences A416 and BS18S were designed with d7B and d67B off-target prevention. The inhibition effect of off-target prevention design sequences and non-off-target prevention design sequences on off-target genes and target genes is shown in Table 48. The results show that after d7B modification, the IC50 of sequence C416-DV27Pd7B on off-target gene SLCO2B1 is increased by 5.69 times, and the maximum inhibition rate is reduced by 9.54%; the maximum inhibition rate of sequence C418S-DV34Pd7B on off-target gene SLC41A2 is reduced by 16.91%, which is significantly better than d67B and GNA modification. At the same time, the IC50 of sequence C418S-DV34Pd7B on target gene HBV is 0.22 nM, which is close to sequence B418S-DV34P without off-target prevention modification. The maximum inhibition rates on target genes are both about 81%, indicating that after d7B modification, the inhibition of sequence C418S-DV34Pd7B on target genes is not affected.

[0699] Table 48 Inhibition effect of off-target prevention modification sequences on off-target genes and target genes

[0700]

[0701] Example 7: Inhibition of HBV in mice by sequences modified by the template modification of the present disclosure (high dose)

[0702] Some sequences were selected in this example, including base sequences B418S, A416, B1550, B207S, B1575, B1572, A1573, which were modified, for example, only using template modification, using template modification and off-target prevention design, and all coupled with GalNAc ligand. The in vivo efficacy of these RNAi agents was analyzed in an AAV-HBV mouse model after repeated administration at a high dose, such as 3 mg / kg. This mouse model can continuously produce HBV viral particles and HBV antigens and is serum negative for more than 1 year after infection with recombinant adeno-associated virus (AAV) carrying a replicable HBV genome, which reproduces some of the immunological characteristics of clinical patients with chronic hepatitis B. Therefore, this model is also used to evaluate new immune-based therapies and antiviral treatments. In this example, the AAV-HBV mouse model was used to detect the inhibitory effect of each sequence on serum HBsAg, HBeAg, and HBV DNA at different time points, and the effect of each sequence on the reconstruction of adaptive immune function in mice at a specific time point.

[0703] 1. Experimental materials

[0704] Test drug:

[0705] Each sequence in Table 49, including only template modification sequences, template modification and off-target prevention design sequences, and sequences with GalNAc ligand G5 coupled to the 3' end of the sense strand, have the following structure:

[0706]

[0707] The coupling method of oligonucleotide and ligand G5 is referred to the preparation method in Example 3 of patent application CN116854754A.

[0708] The oligonucleotide and ligand G5 form the conjugate as shown below:

[0709]

[0710] The specific sequences of each sequence are shown in Table 49. G5 in the sequence number indicates that the sequence is coupled with GalNAc ligand G5, and GL indicates that the sequence is coupled with GalNAc ligand L96.

[0711] The structure of L96 is as follows:

[0712]

[0713] Table 49 siRNA sequences used in animal experiments

[0714]

[0715]

[0716] Test article preparation:

[0717] Vehicle: PBS buffer

[0718] Preparation condition: sterile environment

[0719] Identification method: The prepared administration preparation is identified by a label, and the outer package indicates the project number, name, concentration, quantity, preparation date, preparer, and storage condition.

[0720] Storage condition: use fresh, and store the remaining sample at -80℃.

[0721] Information of experimental animals:

[0722] Species / strain: AAV-HBV mice

[0723] Grade: SPF

[0724] Gender: male

[0725] Number: 100

[0726] Age: 7 weeks

[0727] Weight: 19-24 g

[0728] Source: Guangdong Ziru Biological Medicine Technology Co., Ltd.

[0729] Production license number: SCXK (Yue) 2021-0057

[0730] Ethical review of experimental animals (IACUC):

[0731] After receiving the experimental animals, they were bred in Guangzhou Jennio Biological Technology Co., Ltd., and the license number was JENNIO (Jinbin) 2019-0002. This project has been reviewed by the Experimental Animal Ethics Committee of Guangzhou Jennio Biological Technology Co., Ltd., and the IACUC number is JENNIO-IACUC-2024-A005. The experimental process was strictly carried out in accordance with the requirements of IACUC, and the animal welfare was guaranteed.

[0732] Breeding and management:

[0733] Breeding conditions: After receiving the experimental animals, they were bred in Guangzhou Jennio Biological Technology Co., Ltd. They were bred in breeding cages with specifications of length × width × height = 29.0 cm × 18.5 cm × 13.0 cm. The temperature range was set to 20-26℃, the humidity range was set to 40%-70%, automatic lighting, and 12 hours of light and dark alternation.

[0734] The standard for the rearing environment conditions refers to the national standard of the People's Republic of China GB14925-2010.

[0735] The animals were allowed to eat and drink freely. The feed was the irradiation sterilization experimental mouse maintenance feed, which was provided by Jiangsu Cooperation Pharmaceutical and Biological Engineering Co., Ltd., and the production license number was Su Feed Certificate (2019) 01008. The feed nutrient component detection referred to the national standard of the People's Republic of China GB14924.3-2010, and the pollutant content detection referred to the national standard of the People's Republic of China GB14924.2-2001, and the detection report of each batch was provided by the feed supplier. The drinking water was reverse osmosis water, which was stored in a drinking water bottle, and the drinking water detection referred to the national standard of the People's Republic of China GB5750-2006, and was detected by a third-party testing institution once a year.

[0736] The animal bedding was corn cob bedding, which was provided by Guangzhou Saibainuo Biological Technology Co., Ltd., and the animal bedding production license number was SCXK (Jing) 2019-0004. The bedding pollutant content detection referred to the national standard of the People's Republic of China GB14924.2-2001, and the detection report of each batch was provided by the bedding supplier.

[0737] The animal rearing cages and bedding were replaced at least once a week, and all the animal rearing cages and bedding were disinfected by a pulsating vacuum sterilizer at high pressure before being used in the barrier environment; the animal rearing cages were cleaned, disinfected and wiped at least once a week.

[0738] The animal rearing observation room was cleaned and disinfected every day, including flat shelves, floors, table tops, etc.

[0739] The disinfectant used in the barrier environment included 6.67% new jieer solution, 0.5% 84 disinfectant, 75% disinfectant, and 0.08% baidu killer, which were used in rotation and could not be mixed.

[0740] 2. Experimental method

[0741] 2.1 AAV-HBV mouse modeling

[0742] The test date was defined as day 0 (day 0) on the day when the animals were administered with the solvent or the test drug.

[0743] 100 SPF C57BL / 6 male mice were adaptively fed in the barrier facility for 7 days, and daily observation was performed. After confirming that the mice were healthy and had no abnormalities, modeling was performed. The mice were injected with rAAV8-1.3HBV (Guangzhou Pai Zhen Biological Technology Co., Ltd., name: AAV8 [HBV-D, ayw] (D#2012), batch number: HBV101-6) through the tail vein, 1×10 11GC / 100 μL. Blood samples were collected from animals at weeks 5 and 6 (D-14, D-7) after modeling, centrifuged and plasma was collected for HBV DNA, HBsAg and HBeAg content testing.

[0744] 2.2 Animal grouping and dosing

[0745] Test date definition: The day when animals are dosed with vehicle or test drug is defined as day 0 (D 0).

[0746] Grouping was performed based on the week 6 (D-7) indicators after modeling. From the successfully modeled animals, 72 animals were selected and randomly divided into 12 groups according to the HBsAg indicator, with 6 animals in each group. The average HBsAg value was ensured to be at the same level among the groups, and there was no statistical difference in the HBV DNA and HBeAg indicators among the groups. Dosing was performed at week 7 (D 0) after modeling after grouping. The grouping and dosing details are shown in Table 50.

[0747] Table 50. Group setting and dosing details

[0748]

[0749] Note: s.c.: subcutaneous injection.

[0750] 2.3 Second challenge experiment

[0751] At D 60, 3 mice in the C207S-DV32PG5 group were subjected to tail vein hydrodynamic injection of 8 μg pAAV-HBV1.2 plasmid (Fenghui Biotechnology) at a volume of 100 μL / g for challenge, and the remaining 3 mice were not treated. Another 3 mice in the blank group were also subjected to the same challenge operation as controls.

[0752] 2.4 Observation and indicator monitoring

[0753] (1) General observation

[0754] During the modeling period and the test period, animals were observed and observation records were made every day. The observation contents included: whether death, near-death, feed and water intake, trauma, feces, appearance and fur, mental state, activity state, etc.

[0755] (2) Body weight

[0756] Acclimation period: The animals were weighed and recorded upon receipt, and the animals were weighed and recorded on the last day of the acclimation period.

[0757] Test period: The animals were weighed and recorded every week during the test period. If dosing or blood collection was required on the same day, the animals were weighed before the operation and before being sacrificed.

[0758] (3) Serum HBsAg, HBeAg, HBV DNA, HBsAb, ALT level determination

[0759] Animals were taken blood from the orbital inner canthus 200 μL into EDTA-k2 anticoagulation tubes at 5 weeks after modeling (D-14), 6 weeks (D-7), before administration (D 0), and once a week after administration. The blood was centrifuged at 1000 g for 10 min, and the plasma was collected. The mice subjected to challenge were subjected to challenge at D60, and blood was taken from the orbital inner canthus 200 μL into EDTA-k2 anticoagulation tubes at D63, D67, D74, and D81, respectively. The blood was centrifuged at 1000 g for 10 min, and the plasma was collected. 20 μL of plasma was added to 980 μL of PBS, vortexed, and used to detect HBsAg, HBV DNA, HBeAg, and ALT indicators; 15 μL of plasma was added to 210 μL of PBS, vortexed, and used to detect the HBsAb indicator. All treated samples were detected by Guangzhou Huayin Medical Test Center Co., Ltd., and the remaining plasma was stored at -80°C.

[0760] 3. Experimental results

[0761] The experimental results show that, compared with the solvent control group and the negative control group, sequences B207S-DV32P, A1550-DV27P, B1572-DV27P, B1575-DV27P, B207S-DV29P, A1573-DV29P, B1575-DV29P, B418S-DV34Pd7B, and A416-DV27Pd7B can be targeted to deliver to the liver after coupling with conjugate G5, and significantly inhibit the levels of HBsAg, HBeAg, and HBV DNA in plasma.

[0762] Among them, sequences C207S-DV32PG5, C207S-DV29PG5, and C418S-DV34Pd7BG5 exhibit superior HBsAg, HBV DNA, and HBeAg reduction effects, which are significantly better than the positive control sequence VIR-2218-GL. In addition, sequences C207S-DV32PG5 and C207S-DV29PG5 ultimately achieve HBsAg and HBV DNA negative conversion in all mice in the group, and produce relatively high levels of antibodies. Sequence C418S-DV34Pd7BG5 ultimately achieves HBsAg negative conversion in 3 / 6 mice in the group, and also produces relatively high levels of antibodies.

[0763] The results of the secondary challenge experiment show that the blank group (not infected with HBV virus before D56) has HBsAg, HBeAg and HBV DNA in the body after challenge, indicating that the challenge method is effective. The HBV DNA of the mice in the C207S-DV32PG5 challenge group first increased and then decreased to the detection limit after challenge, and the serum HBsA antibody also increased significantly, and the HBsAg remained below the detection limit, proving that the challenge is effective, and the hepatitis B mice treated with the small nucleic acid sequence can resist the re-attack of HBV virus. It is proved that the hepatitis B mice treated with the small nucleic acid sequence can resist the re-attack of HBV virus.

[0764] (i) Changes of HBsAg, HBeAg, HBV DNA, HBsAb, ALT levels of mice in each sequence group over time

[0765] Changes of HBsAg, HBeAg, HBV DNA, HBsAb, ALT and body weight of mice in each group over time are shown in Tables 51-59 and Figures 1A-1F As can be seen from the table, sequences C207S-DV32PG5, C207S-DV29PG5 and C418S-DV34Pd7BG5 exhibit superior effects of reducing HBsAg, HBV DNA and HBeAg, which are significantly better than the sequence VIR-2218-GL. In addition, sequences C207S-DV32PG5 and C207S-DV29PG5 ultimately achieve HBsAg and HBV DNA negative conversion of all mice in the group, and produce high levels of antibodies. Among them, the C207S-DV32PG5 group achieves HBsAg and HBV DNA negative conversion of all mice in the group at 14 days and 21 days after administration, respectively, and continues until the end of the experiment. It can be considered that the mice have achieved functional cure. Three out of six mice in the C418S-DV34Pd7BG5 group ultimately achieved HBsAg negative conversion, and also produced high levels of antibodies. The above data prove the superiority of C207S-DV32PG5, C207S-DV29PG5 and C418S-DV34Pd7BG5 sequences in terms of efficacy.

[0766] Table 51 Changes of plasma HBsAg levels of mice in each sequence group

[0767]

[0768]

[0769] Note: "-" means stop testing; " / " means C207S-DV32PG5 group is selected as challenge test animals at D56 of administration. * means P<0.05; ** means P<0.01 compared with PBS group. Compared with positive control group VIR-2218-GL group, # P<0.05; ## P<0.01.

[0770] Table 52 Changes of plasma HBV DNA levels of mice in each sequence group

[0771]

[0772] Note: "-" means stop testing; " / " means C207S-DV32PG5 group is selected as challenge test animals at D56 of administration. * means P<0.05; ** means P<0.01 compared with PBS group. Compared with positive control group VIR-2218-GL group, # P<0.05; ## P<0.01.

[0773] Table 53 Changes of plasma HBeAg levels of mice in each sequence group

[0774]

[0775] Note: "-" means stop testing; " / " means C207S-DV32PG5 group is selected as challenge test animals at D56 of administration. * means P<0.05; ** means P<0.01 compared with PBS group. Compared with positive control group VIR-2218-GL group, # P<0.05; ## P<0.01.

[0776] Table 54 Changes of HBsAg negative mice numbers of mice in each sequence group

[0777]

[0778] Table 55 Changes of HBV DNA negative mice numbers of mice in each sequence group

[0779]

[0780] Table 56 Changes of antibody producing mice numbers of mice in each sequence group

[0781]

[0782] Table 57 Changes of plasma HBsAb levels of mice in each sequence group

[0783]

[0784] Table 58 Changes of ALT levels in plasma of mice in each sequence group

[0785]

[0786] Table 59 Changes of body weight of mice in each sequence group

[0787]

[0788] (ii) Protective effect of sequence CS7S-DV32PG5 on secondary infection of mice

[0789] The mice in the CS7S-DV32PG5 group were injected with HBV plasmid through tail vein by high-pressure hydrodynamic injection on D60 to simulate the secondary infection of HBV. The experimental results are shown in Table 61. Figures 2A-2F As shown, the blank group (not infected with HBV virus before D56) had HBsAg, HBeAg and HBV DNA in vivo after the high-pressure hydrodynamic injection of HBV plasmid, indicating that the mode of infection was effective. The HBV DNA of the mice in the C207S-DV32PG5 infection group increased first and then decreased to the detection limit, and the serum HBsA antibody increased significantly, and the HBsAg remained below the detection limit. This result proves that the infection is effective, and the hepatitis B mice treated with the sequence can resist the re-infection of HBV virus.

[0790] Example 8: Inhibition of HBV in mice by sequences modified by the template modification of the present disclosure (low dose)

[0791] In this example, some sequences were exemplarily selected, such as the sequences C207S-DV32PG5, C207S-DV29PG5, C418S-DV34Pd7BG5 and C1575-DV27PG5 in Example 7, see Table 49, and the in vivo efficacy of these RNAi agents after multiple administrations at a low dose, such as 0.5 mg / kg, was analyzed by using the AAV-HBV mouse model.

[0792] 1. Experimental materials

[0793] The sequences C207S-DV32PG5, C207S-DV29PG5, C418S-DV34Pd7BG5, C1575-DV27PG5 and VIR-2218-GL in Example 7, see Table 49, and entecavir (ETV) were used.

[0794] 2. Experimental methods

[0795] The grouping and administration details are shown in Table 60.

[0796] Table 60 Group setting and administration details table

[0797]

[0798] Note: s.c.: subcutaneous injection; i.g.: intragastric administration.

[0799] 3. Experimental results

[0800] The changes of HBsAg, HBeAg, HBV DNA, HBsAb, ALT levels of mice in each group over time are shown in Tables 61-68 and Figures 3A-3F The experimental results show that the sequences C207S-DV32PG5 and C207S-DV29PG5 have a significantly better effect than the sequence VIR-2218-GL in inhibiting HBsAg, HBV DNA and HBeAg, and the sequence C207S-DV32PG5 has the best effect, reducing HBsAg by about 2.9 log10 on the 56th day, which is about 1.4 log10 higher than C207S-DV29PG5, and on the 56th day, 3 mice in the C207S-DV32PG5 group had HBsAg turned negative, and 4 mice produced antibodies, while there were no negative mice in the C207S-DV29PG5 group. The above data again prove the superiority of the sequence C207S-DV32PG5 in terms of drug efficacy.

[0801] Table 61 Changes of HBsAg levels in mice plasma of each sequence group

[0802]

[0803] Note: compared with the PBS group, * indicates P<0.05; ** indicates P<0.01.

[0804] Table 62 Changes of HBV DNA levels in mice plasma of each sequence group

[0805]

[0806] Note: compared with the PBS group, * indicates P<0.05; ** indicates P<0.01

[0807] Table 63 Changes of HBeAg levels in mice plasma of each sequence group

[0808]

[0809] Note: compared with the PBS group, * indicates P<0.05; ** indicates P<0.01.

[0810] Table 64 Number of mice with HBsAg turned negative in each sequence group

[0811]

[0812] Table 65 Number of mice in each sequence group with plasma HBV DNA negative conversion

[0813]

[0814] Table 66 Number of mice in each sequence group with HBsAb production

[0815]

[0816] Table 67 Plasma HBsAb level changes of mice in each sequence group

[0817]

[0818] Table 68 Plasma ALT level changes of mice in each sequence group

[0819]

[0820] Example 9: Inhibition of HBV in mice using ligand-modified sequences delivered using the present disclosure

[0821] This example exemplarily selected some sequences, including base sequences B418S, B207S, modified these sequences, for example, only using template modification, using template modification and off-target prevention design, and coupling to deliver different ligands. The in vivo efficacy of these RNAi agents was analyzed using the AAV-HBV mouse model.

[0822] 1. Experimental materials

[0823] Test drugs:

[0824] The sequences in Table 69, including only template-modified sequences, template-modified and off-target prevention designed sequences, and the 3' end of the sense strand of these sequences coupled to deliver ligands G5, G101 or G103, have the following structures:

[0825] ,

[0826] , .

[0827] The coupling method of oligonucleotides and ligands G5, G101 or G103 is referred to the preparation method in Example 3 of patent application CN116854754A.

[0828] The oligonucleotides and ligands G5, G101 or G103 form conjugates as shown below:

[0829] ,

[0830] ,

[0831] .

[0832] The specific sequences of each sequence are shown in Table 69. G5 in the sequence number indicates that the sequence is coupled with delivery ligand G5, G101 indicates that the sequence is coupled with delivery ligand G101, G103 indicates that the sequence is coupled with delivery ligand G103, and GL indicates that the sequence is coupled with delivery ligand L96. The structural formula of L96 is as follows:

[0833]

[0834] Table 69 siRNA sequences used in animal experiments

[0835]

[0836]

[0837] Preparation of test drugs:

[0838] The same as Example 7.

[0839] Information of experimental animals:

[0840] Species / strain: AAV-HBV mice

[0841] Grade: SPF

[0842] Gender: male

[0843] Number: 82

[0844] Age: 5 weeks

[0845] Weight: 19-24 g

[0846] Source: Beijing Vivotec Animal Technology Co., Ltd.

[0847] Production license number: SCXK (Jing) 2021-0006

[0848] Experimental animal ethics review (IACUC):

[0849] After receiving the experimental animals, they were bred in Beijing Vivotek Biotechnology Co., Ltd. The use license number is SCXK (Jing) 2022-0013. This project has been reviewed by the Experimental Animal Ethics Committee of Beijing Vivotek Biotechnology Co., Ltd., and the IACUC number is VST-SY-24062701. The experimental process is strictly in accordance with the requirements of IACUC, and the animal welfare is guaranteed.

[0850] Breeding and management:

[0851] Animals were housed in a negative pressure barrier environment, using plastic (polycarbonate) boxes (volume 370 x 157 x 180 mm) IVC housing, due to the experimental animals for male fighting, so a single cage feeding. The animal feeding by Beijing Vivotec Biotech Co., Ltd. was responsible.

[0852] The use, testing of animal feed, bedding and drinking water were in accordance with GB14925-2010 "Experimental animal environment and facilities" specification. The control of animal housing environment, including temperature, humidity, pressure difference, noise, illumination, air changes, ammonia concentration, etc. was temporarily in accordance with GB50447-2008 "Technical code for construction of experimental animal facilities" was executed. The temperature control of animal room was 20~26℃ (daily temperature difference ≤4℃); the relative humidity control was 40~70%; artificial lighting was used for illumination, 12 / 12 hours of day and night light and dark alternation. Compressed shaving bedding was purchased from Beijing Kaohe Cooperation Feed Co., Ltd. (batch number: 23109613), and mouse growth and reproduction feed was purchased from Beijing Kaohe Cooperation Feed Co., Ltd. (batch number: 23103313). The related records of animal feeding management and environmental control during the experiment were kept in Beijing Vivotec Biotech Co., Ltd.

[0853] 2. Experimental method

[0854] 2.1 AAV-HBV mouse modeling

[0855] Test date definition: the day when the animals were given the solvent or the test drug was defined as day 0 (day 0).

[0856] 100 SPF C57BL / 6 male mice, adaptive feeding in barrier facilities for 7 days, daily observation, and confirmation of healthy mice without abnormalities before modeling. Persistent HBV infected mouse animal models were prepared by tail intravenous injection of rAAV8-1.3HBV. The injection dose of AAV virus was 1.00 x 10 10 vg / each, and the AAV virus was diluted with sterile PBS to 5.00 x 10 10 vg / mL, 200 μL per mouse, and the content of HBV DNA, HBeAg and HBsAg in serum was detected 4 weeks after virus injection.

[0857] 2.2 Animal grouping and drug administration

[0858] Test date definition: the day when the animals were given the solvent or the test drug was defined as day 0 (D 0).

[0859] On Day -2, all mice were submandibularly bled to collect plasma. The collected blood sample was anticoagulated with EDTA, centrifuged at 5000 rpm for 10 min, and the supernatant was used for HBV modeling detection. On Day 0, 72 animals were selected according to the detection results on Day -2, and were grouped into 12 groups, 6 animals in each group. The grouping and administration details are shown in Table 70.

[0860] Table 70 Grouping and administration details table

[0861]

[0862] Note: s.c.: subcutaneous injection.

[0863] 2.4 Observation and index monitoring

[0864] (1) General observation

[0865] During the modeling period and the experimental period, the animals were observed and observation records were made every day. The observation contents included: whether death, near-death, feed and water intake, trauma, feces, appearance and fur, mental state, activity state, etc.

[0866] (2) Body weight

[0867] Adaptation period: the animals were weighed and recorded when received, and the animals were weighed and recorded on the last day of the adaptation period.

[0868] Experimental period: the animals were weighed and recorded every week during the experimental period. If administration or blood collection was required on the same day, the animals were weighed before the operation and before being sacrificed.

[0869] (3) Determination of HBsAg, HBeAg, HBV DNA, and ALT levels in serum

[0870] All animals were submandibularly bled on Days -2, 6, 13, 20, 27, and 34 to collect plasma. The collected blood sample was anticoagulated with EDTA, centrifuged at 5000 rpm for 10 min, and the serum was separated. After blood collection, the serum was separated, diluted with PBS solution, and then sent for detection. 10 μL of serum was taken from each sample, diluted to 500 μL with PBS solution (dilution 50 times), and sent to Beijing Di'an Medical Laboratory Co., Ltd. for detection of serum HBV DNA, HBeAg, and HBsAg. 30 μL of serum was taken from each sample, diluted to 120 μL with PBS solution (dilution 4 times), and sent to Beijing Di'an Medical Laboratory Co., Ltd. for detection of serum ALT. The remaining plasma was stored at -80°C.

[0871] 3. Experimental results

[0872] The changes of HBsAg, HBeAg, HBV DNA, and ALT levels in serum and body weight of mice in each group over time are shown in Tables 71-75 andFigures 4A-4E The experimental results show that the efficacy of all sequences is better than that of the sequence VIR-2218-GL, wherein C207S-DV29PG101, C207S-DV29PG103, C207S-DV32PG101, and C207S-DV32PG103 exhibit superior effects of reducing HBsAg, HBV DNA, and HBeAg, which are significantly better than those of the sequence VIR-2218-GL, and at day 28 after administration, HBsAg is reduced by more than 3 log10, HBV DNA is reduced by more than 3 log10, and rebound has not yet occurred, which shows excellent anti-HBV efficacy.

[0873] Table 71 Change in plasma HBsAg level of mice in each sequence group

[0874]

[0875] Table 72 Change in plasma HBV DNA level of mice in each sequence group

[0876]

[0877] Table 73 Change in plasma HBeAg level of mice in each sequence group

[0878]

[0879] Table 74 Change in plasma ALT level of mice in each sequence group

[0880]

[0881] Table 75 Change in body weight of mice in each sequence group

[0882]

Claims

1. A double-stranded RNAi agent, characterized in that, The double-stranded RNAi agent comprises an antisense strand and a sense strand complementary to the antisense strand forming the double-stranded region, wherein the nucleotide sequence of the antisense strand is as shown in SEQ ID NO: 11, or the nucleotide sequence of the antisense strand is a modified sequence of the sequence shown in SEQ ID NO:

11. The double-stranded RNAi agent comprises an oligonucleotide duplex consisting of a pair of sense and antisense strands: The nucleotide sequence of the positive strand is as shown in SEQ ID NO: 4, or the nucleotide sequence of the positive strand is a modified sequence of the sequence shown in SEQ ID NO:

4.

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

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

4. The double-stranded RNAi agent according to claim 2, characterized in that, At least one of the modified nucleotides is a configuration-restricted nucleotide and / or a nucleotide containing a non-natural base.

5. The double-stranded RNAi agent according to claim 2, characterized in that, At least one of the modified nucleotides is selected from one or more of the group consisting of: deoxy-nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, locked nucleotides, unlocked nucleotides, restricted ethyl nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, aminophosphates, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphate groups, and nucleotides containing 5'-phosphate groups.

6. The double-stranded RNAi agent according to claim 5, characterized in that, The deoxy-nucleotide is a 3'-terminal deoxy-thymidine nucleotide and / or a 2'-deoxy-modified nucleotide.

7. The double-stranded RNAi agent according to claim 1, characterized in that, The antisense strand of the double-stranded RNAi agent has a 3' overhang of 2 nucleotides.

8. The double-stranded RNAi agent according to claim 1, characterized in that, The double-stranded region of the double-stranded RNAi agent consists of 21 pairs of nucleotides.

9. The double-stranded RNAi agent according to claim 1, characterized in that, The double-stranded RNAi agent has a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides.

10. The double-stranded RNAi agent according to claim 1, characterized in that, All modifications contained in the nucleotides on the sense and antisense strands are chemical modifications at the 2' position of the nucleotide ribose.

11. The double-stranded RNAi agent according to claim 10, characterized in that, The chemical modification at the 2' position of the nucleotide ribose is selected from one or more of the group consisting of: 2'-methoxy, 2'-O-methoxyethyl, 2'-fluoro, 2'-benzyloxy, 2'-methylcarbonylamino, and 2'-pyridinemethoxy.

12. The double-stranded RNAi agent according to claim 11, characterized in that, The 2' position of the nucleotide ribose is chemically modified with 2'-methoxy or 2'-fluoro.

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

14. The double-stranded RNAi agent according to claim 13, characterized in that, The 3',5'-phosphodiester bond contains a thiolated modification.

15. The double-stranded RNAi agent according to claim 1, characterized in that, Phosphorylation of the 5' carbon atom of the antisense strand 5'-terminal nucleotide glycoside.

16. The double-stranded RNAi agent according to claim 15, characterized in that, The phosphorylated 5'-phosphorylation group comprises one or more selected from: 5'-vinylphosphonate group, 5'-methylphosphonate group, 5'-C-methylphosphate group, 5'-thiophosphate group, and 5'-phosphate group, with the following structure: ， ， ， ， ； R represents hydrogen, hydroxyl group, amino group, C 1-4 Alkyl, aromatic, C 1-4 Alkoxy, C 1-4 Alkyl carbonyl amino or halogen; The bases are selected from any one of adenine, guanine, cytosine, thymine, and uracil.

17. The double-stranded RNAi agent according to claim 1, characterized in that, The nucleotides at the end of the sequence are linked by 3',5'-phosphodiester bonds containing thiomodification, forming chiral pure 3',5'-thiophosphodiester bonds.

18. The double-stranded RNAi agent according to claim 17, characterized in that, The 5' ends of the sense chain and antisense chain contain 1-3 thioligases, and the 3' ends of the antisense chain contain 1-3 thioligases.

19. The double-stranded RNAi agent according to claim 1, characterized in that, The antisense chain is modified in one of the following table formats: ， ； And / or, the chain of justice is modified in one of the following table formats: ； Wherein 2'-OMe is 2'-methoxy; 2'-F is 2'-fluoro; PS is the thiophosphate skeleton; and EVP is 5'-vinyl-(E)-phosphonate.

20. The double-stranded RNAi agent according to claim 19, characterized in that, The modification method of the double-stranded RNAi agent is as follows: The antisense chain uses modification method A, and the justice chain uses modification method a; The antisense chain uses modification method A, and the justice chain uses modification method b; The antisense chain uses modification method B, and the justice chain uses modification method a; The antisense chain uses modification method B, and the justice chain uses modification method b. The antisense chain uses modification method C, and the justice chain uses modification method b; The antisense chain uses modification method D, and the justice chain uses modification method b; The antisense chain uses modification E, and the justice chain uses modification a; or, The antisense chain uses modification method F, and the justice chain uses modification method b.

21. The double-stranded RNAi agent according to claim 1, characterized in that, The antisense strand employs a modifying group at positions 2 to 8 starting from the 5' end. This modifying group is selected from one or more of UNA, GNA, and DNA. The structures of UNA and GNA are as follows: ； The bases are selected from any one of adenine, guanine, cytosine, thymine, and uracil.

22. The double-stranded RNAi agent according to any one of claims 1-21, characterized in that, The double-stranded RNAi agent comprises any oligonucleotide duplex selected from the following sense and antisense strand pairings: (1) The nucleotide sequence of the sense strand is shown in SEQ ID NO: 96; and the nucleotide sequence of the antisense strand is shown in SEQ ID NO: 201, 202, 203 or 204; (2) The nucleotide sequence of the sense strand is shown in SEQ ID NO: 97; and the nucleotide sequence of the antisense strand is shown in SEQ ID NO: 201 or 202; (3) The nucleotide sequence of the sense strand is shown in SEQ ID NO: 98; and the nucleotide sequence of the antisense strand is shown in SEQ ID NO: 205 or 206; (4) The nucleotide sequence of the sense strand is shown in SEQ ID NO: 99; and the nucleotide sequence of the antisense strand is shown in SEQ ID NO: 206; and, (5) The nucleotide sequence of the sense strand is shown in SEQ ID NO: 100; and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:

206.

23. A conjugate, characterized in that, The conjugate comprises a double-stranded RNAi agent as described in any one of claims 1-22, and a ligand conjugated to the double-stranded RNAi agent.

24. The conjugate according to claim 23, characterized in that, The ligand is conjugated to the 3'-end or 5'-end of the sense strand of the oligonucleotide.

25. The conjugate according to claim 23, characterized in that, The ligand is one or more GalNAc derivatives attached using divalent or trivalent branched bonds, or GalNAc derivatives attached using monovalent bonds.

26. The conjugate according to claim 23, characterized in that, The ligand is: ， Wherein, X is hydrogen or a hydroxyl protecting group, the hydroxyl protecting group including acetyl, benzoyl or isobutyryl; Y is an amine protecting group or H, the amine protecting group being formyl, acetyl, propionyl, n-butyryl or isobutyryl; n is an integer between 0 and 20; q, r and s are independently integers between 1 and 7.

27. The conjugate according to claim 26, characterized in that, The ligand is: 。 28. The conjugate according to claim 23, characterized in that, The ligand is: ， Where X is oxygen, nitrogen, or sulfur; Y is an alkyl or aromatic group; R1 is oxygen or sulfur; R2 is hydrogen, amino group, or C. 1-4 Alkyl, aromatic, C 1-4 Alkoxy or halogen; A is -(CH2) a -、-(CH2CH2O) b -、-((CH2) c NHCO) d -or-((CH2) c CONH) d - where a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5; B is -(CH2) e -, where e is an integer between 0 and 7; L is either -CONH- or -NHCO-; X1 is -(CH2) f -or-(CH2CH2O) f CH2-, f is an integer from 1 to 5; X2 is -(CH2) g -, g is an integer from 1 to 6; Y1 is 0 or 1; Y2 is 0, 1, or 2; Y3 is 1, 2, or 3; m is an integer between 0 and 4; n is an integer between 0 and 4.

29. The conjugate according to claim 28, characterized in that, The ligand is G4, G5, G6, or G7: ， ， ,or 。 30. The conjugate according to claim 29, characterized in that, The conjugate has the following structure: ， ， ， ,or 。 31. The conjugate according to claim 23, characterized in that, The ligands are: ， Where X is oxygen, nitrogen, or sulfur; Y is an alkyl or aromatic group; R1 is oxygen or sulfur; R2 is hydrogen, amino group, or C. 1-4 Alkyl, aromatic, C 1-4 Alkoxy or halogen; A is -(CH2) a -、-(CH2CH2O) b -、-((CH2) c NHCO) d -or-((CH2) c CONH) d - where a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5; B is -(CH2) e -, where e is an integer between 0 and 7; L is either -CONH- or -NHCO-; X1 is -(CH2) f -or-(CH2CH2O) f CH2-, f is an integer from 1 to 5; X2 is -(CH2) g - g is an integer from 1 to 6; Y1 is either 0 or 1; Y2 is 0, 1, or 2; Y3 is 1, 2, or 3; m is an integer between 0 and 4; n is an integer between 0 and 4.

32. The conjugate according to claim 31, characterized in that, The ligands are G101, G102, G103, G105, or G106: ， ， ， ， or .

33. The conjugate according to claim 32, characterized in that, The conjugate has the following structure: ， ， ， ,or 。 34. The conjugate according to claim 23, characterized in that, The conjugate comprises any oligonucleotide duplex selected from the following sense and antisense strand pairings: The nucleotide sequence of the sense strand is shown in SEQ ID NO: 338 or 341; and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:

201.

35. The conjugate according to any one of claims 23-34, characterized in that, The conjugate has the function of inhibiting HBV gene expression.

36. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a double-stranded RNAi agent as described in any one of claims 1-22 or a conjugate as described in any one of claims 23-35, and a pharmaceutically acceptable carrier.

37. The pharmaceutical composition of claim 36, characterized in that, The double-stranded RNAi agent or the conjugate is administered in a non-buffered solution.

38. The pharmaceutical composition of claim 37, characterized in that, The non-buffered solution is salt water or water.

39. The pharmaceutical composition according to claim 36, characterized in that, The double-stranded RNAi agent or the conjugate was administered with a buffer solution.

40. The pharmaceutical composition of claim 39, characterized in that, The buffer solution includes acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate, or any combination thereof.

41. The pharmaceutical composition according to claim 40, characterized in that, The buffer solution is a phosphate buffer salt.

42. The pharmaceutical composition according to claim 39, characterized in that, The double-stranded RNAi agent or the conjugate is formulated into a lipid formulation for delivery in a membrane-bound molecular assembly.

43. The pharmaceutical composition according to claim 42, characterized in that, The lipid formulation is a nucleic acid-lipid particle.

44. The pharmaceutical composition according to claim 43, characterized in that, The lipid formulation is lipid nanoparticles.

45. The pharmaceutical composition according to claim 42, characterized in that, The mass / mass ratio of lipid to the double-stranded RNAi agent or the conjugate is 1:1 to 50:

1.

46. ​​The pharmaceutical composition according to claim 42, characterized in that, The mass / mass ratio of lipid to the double-stranded RNAi agent or the conjugate is 1:1 to 25:

1.

47. The pharmaceutical composition according to claim 42, characterized in that, The mass / mass ratio of lipid to the double-stranded RNAi agent or the conjugate is 3:1 to 15:

1.

48. The pharmaceutical composition according to claim 42, characterized in that, The mass / mass ratio of lipid to the double-stranded RNAi agent or the conjugate is 4:1 to 10:

1.

49. The pharmaceutical composition according to claim 42, characterized in that, The mass / mass ratio of lipid to the double-stranded RNAi agent or the conjugate is 5:1 to 9:

1.

50. The pharmaceutical composition according to claim 42, characterized in that, The mass / mass ratio of lipid to the double-stranded RNAi agent or the conjugate is 6:1 to 9:

1.

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

52. The pharmaceutical composition according to claim 51, characterized in that, The cationic lipid is a compound of formula (I) or a solvate thereof. (I)， G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; G3 is C 1～3 Alkylene; L1 is C 6～15 Straight-chain alkyl; L2 is C 12～25 Branched alkyl groups.

53. The pharmaceutical composition according to claim 52, characterized in that, The cationic lipid is an N-oxide, pharmaceutically acceptable salt, or stereoisomer of a compound with the structure of formula (I).

54. The pharmaceutical composition according to claim 52 or 53, characterized in that, The cationic lipid is YK-009 with the structure of formula (II): (I-I)。 55. The pharmaceutical composition according to claim 51, characterized in that, The cationic lipid is a compound of formula (II) or a solvate thereof. (II), G1 is C 2~8 Alkylene; G2 is C 2~8 Alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6~25 Straight-chain or branched alkyl; R2 is C 6~25 Straight-chain or branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2- or -(CH2)3- or -(CH2)4-.

56. The pharmaceutical composition according to claim 55, characterized in that, The cationic lipid is an N-oxide, pharmaceutically acceptable salt, or stereoisomer of a compound with the structure of formula (II).

57. The pharmaceutical composition according to claim 55 or 56, characterized in that, The cationic lipid is YK-401 with formula (II-I) or YK-402 with formula (II-II): (II-I), (II-II).

58. The pharmaceutical composition according to claim 51, characterized in that, The cationic lipid is a compound of formula (III) or a solvate thereof. (III), G1 is C 1~6 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~20 Straight-chain or branched alkyl; R2 is C 12~25 Branched alkyl; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(C H2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-.

59. The pharmaceutical composition of claim 58, characterized in that, The cationic lipid is an N-oxide, pharmaceutically acceptable salt, or stereoisomer of a compound with the structure of formula (III).

60. The pharmaceutical composition according to claim 58 or 59, characterized in that, The cationic lipid is YK-201 with formula (III-I) or YK-202 with formula (III-II): (III-I), (III-II)。 61. The pharmaceutical composition according to claim 51, characterized in that, The cationic lipid is a compound of formula (IV) or a solvate thereof. (IV), G1 is C 1~8 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~25 Straight-chain or branched alkyl; R2 is C 12~25 Straight-chain or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3 or -CH2CH3 or -CH2CH2OH.

62. The pharmaceutical composition according to claim 61, characterized in that, The cationic lipid is an N-oxide, pharmaceutically acceptable salt, or stereoisomer of a compound with the structure of formula (IV).

63. The pharmaceutical composition according to claim 61 or 62, characterized in that, The cationic lipid is YK-305 with formula (IV-I) or YK-310 with formula (IV-II): (IV-I), (IV-II)。 64. The pharmaceutical composition according to claim 51, characterized in that, The cationic lipid is a compound of formula (V) or a solvate thereof. (V)， G 1 and G 2 Each is independently unsubstituted C6-C 10 Alkylene; G 3 For unsubstituted C1-C 12 Alkylene; R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl; R 3 OR 5 N, -C (=O) OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 ;R 4 For C1-C 12 hydrocarbon group; and R 5 It is an H or C1-C6 hydrocarbon group.

65. The pharmaceutical composition according to claim 64, characterized in that, The cationic lipid is an N-oxide, pharmaceutically acceptable salt, or stereoisomer of a compound with the structure of formula (V).

66. The pharmaceutical composition according to claim 64 or 65, characterized in that, The cationic lipid is ALC0315 of formula (VI): (VI) 67. The pharmaceutical composition of claim 51, characterized in that, The cationic lipid is a compound of formula (VI) or a solvate thereof. (WE), R4 is selected from -(CH2). n Q and -(CH2) n CHQR; Q is selected from the following groups: -OR, -OH, -O(CH2). n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R8 and heterocycles; n is 1, 2 or 3.

68. The pharmaceutical composition of claim 67, characterized in that, The cationic lipid is an N-oxide, pharmaceutically acceptable salt, or stereoisomer of a compound with the structure of formula (VI).

69. The pharmaceutical composition according to claim 67 or 68, characterized in that, The cationic lipid is SM102 with the structure of formula (VI-I): (VI-I).

70. The pharmaceutical composition according to claim 51, characterized in that, The cationic lipid is a compound of formula (VII) DLIN-MC3-DMA or its solvates. (VII)。 71. The pharmaceutical composition according to claim 70, characterized in that, The cationic lipid is an N-oxide, pharmaceutically acceptable salt, or stereoisomer of a compound with the structure of formula (VII).

72. The pharmaceutical composition according to claim 51, characterized in that, The cationic lipids include one or more selected from YK-009, YK-401, YK-305, ALC0315, SM102 and DLIN-MC3-DMA.

73. The pharmaceutical composition according to claim 51, characterized in that, The molar ratio of the cationic lipid to the neutral lipid is 1:1 to 10:

1.

74. The pharmaceutical composition according to claim 51, characterized in that, The molar ratio of the cationic lipid to the structural lipid is 1:1 to 5:

1.

75. The pharmaceutical composition according to claim 51, characterized in that, The molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25–65):(5–25):(25–70):(0.5–5).

76. The pharmaceutical composition according to claim 75, characterized in that, The molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25–65):(5–25):(25–45):(0.5–5).

77. The pharmaceutical composition of claim 76, characterized in that, The molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 50:10:38.5:1.5 or 49:10:39.5:1.

5.

78. The pharmaceutical composition of claim 51, characterized in that, The neutral lipids include one or more selected from phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, and sterols.

79. The pharmaceutical composition of claim 78, characterized in that, The neutral lipid is selected from one or more of the following: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-distearateoyl-sn-glycerol-3-phosphate choline, 1,2-diundecanoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline Base, 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl)-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-Diphylanoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1- Sodium glycerol, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, and mixtures thereof.

80. The pharmaceutical composition of claim 79, characterized in that, The neutral lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine and / or 1,2-distearate-sn-glycerol-3-phosphate choline.

81. The pharmaceutical composition according to claim 51, characterized in that, The structural lipids are selected from one or more of the following: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

82. The pharmaceutical composition according to claim 81, characterized in that, The structural lipid is cholesterol.

83. The pharmaceutical composition according to claim 51, characterized in that, The polymer conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

84. The pharmaceutical composition according to claim 83, characterized in that, The polymeric conjugated lipid is selected from one or more of the following: distearylphosphatidylethanolamine polyethylene glycol 2000, dimyristoylglycerol-3-methoxy polyethylene glycol 2000, and methoxy polyethylene glycol bistetradecylacetamide.

85. A medicine box set, characterized in that, The kit includes a box A, which comprises one or more of the double-stranded RNAi agent as described in any one of claims 1-22, the conjugate as described in any one of claims 23-35, or the pharmaceutical composition as described in any one of claims 36-84.

86. The medicine box according to claim 85, characterized in that, The kit also includes a medicine box B, which contains one or two of the following: (1) Other drugs that reduce HBV gene expression or compositions containing said drugs that reduce HBV gene expression; (2) One or more of the group consisting of diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, inhibitors of inhibitory molecules and vaccines.

87. The pillbox according to claim 86, characterized in that, The (2) is one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, cytokines and co-stimulatory molecules activators.

88. The use of the double-stranded RNAi agent as described in any one of claims 1-22, the conjugate as described in any one of claims 23-35, or the pharmaceutical composition as described in any one of claims 36-84 in the preparation of a medicament for the prevention and / or treatment of HBV gene expression-related diseases; The HBV gene expression-related diseases are: chronic hepatitis B or acute hepatitis B.

89. A method for reducing HBV gene expression or inhibiting HBV replication in vitro for non-preventive and / or therapeutic purposes, characterized in that, The method includes applying to a sample one or more of the following: a double-stranded RNAi agent as described in any one of claims 1-22, a conjugate as described in any one of claims 23-35, a pharmaceutical composition as described in any one of claims 36-84, and a kit as described in any one of claims 85-87.

Images