Nucleic acid, pharmaceutical composition and conjugate containing nucleic acid, and uses thereof

Abstract

A nucleic acid, a pharmaceutical composition and conjugate containing the nucleic acid, and uses thereof. In particular, the present invention relates to an siRNA, having a sense strand and an antisense strand that are complementary to each other. Nucleotides in the siRNA are each independently a modified or unmodified nucleotide. The sense strand of the siRNA contains a sequence SEQ ID NO: 1, 21 or 23 or a sequence that differs by no more than four nucleotides therefrom, and the antisense strand of the siRNA contains a sequence SEQ ID NO: 2, 22 or 24 or a sequence that differs by no more than four nucleotides therefrom. The present invention further provides a nucleic acid group comprising the nucleic acid as a first siRNA, and a second siRNA which contains a sense strand containing a sequence as shown in SEQ ID NO: 5, 7 or 43 and an antisense strand containing a sequence as shown in SEQ ID NO: 6, 8 or 44, a pharmaceutical composition and conjugate containing the nucleic acid or the nucleic acid group, and uses thereof.

Description

Nucleic acid, pharmaceutical composition and conjugate containing the nucleic acid and use thereof Technical Field

[0001] The present invention relates to the fields of biomedicine and biotechnology, and in particular to nucleic acids, nucleic acid combinations, pharmaceutical compositions and conjugates containing the nucleic acids or nucleic acid combinations, and applications thereof. Background Art

[0002] Chronic hepatitis B (CHB) caused by hepatitis B virus (HBV) infection seriously endangers human health. Currently, the clinical treatment of chronic hepatitis B mainly includes two categories: interferon (IFN) and nucleoside (acid) analogues (NUCs). Among them, interferon can activate both innate and acquired immunity, and some patients can achieve HBsAg clearance within a limited course of treatment. However, there are problems such as poor patient tolerance, severe side effects, and low response rate. Studies have shown that the response rate of Asian B genotype to interferon is very low. Nucleoside (acid) analogues can effectively inhibit HBV viral replication, which is beneficial for restoring liver function and reducing the incidence of liver cancer. However, patients need to take medication for a long time or even lifelong, and are prone to drug resistance and rebound after discontinuation of medication, making it difficult to achieve HBsAg clearance.

[0003] With the goal of achieving a "functional cure" for chronic hepatitis B, research on how to achieve sustained hepatitis B surface antigen (HBsAg) negativity or sustained cccDNA silencing is currently a key focus in the field. Small interfering RNA (siRNA), based on the RNA interference (RNAi) mechanism, can sequence-specifically silence and degrade HBV transcripts, thereby reducing HBV antigen and cccDNA formation. Compared to traditional antiviral drugs, siRNAs offer unique advantages by potently and rapidly attacking viral transcripts and reducing HBsAg, facilitating the formation of an immune window.

[0004] HBV is a small, hepatotropic DNA virus with an approximately 3.2 kb genome consisting of a nicked, partially double-stranded circular DNA (rcDNA). It encodes four open reading frames (ORFs), including the hepatitis B surface antigen (HBsAg), viral core antigen (HBcAg), polymerase (Pol), and protein X (HBx). The rcDNA is released into the nucleus by the nucleocapsid, where it is repaired with the help of host DNA repair factors to form supercoiled, covalently closed circular DNA (cccDNA), which then binds to nucleosomes to form minichromosomes. Using the cccDNA as a template, pregenomic RNA (pgRNA) and multiple subgenomic RNAs are transcribed. Under the guidance of Pol and HBc, the pgRNA is packaged into viral core particles and reversely transcribed into rcDNA, which is then replenished into the nucleus to form the cccDNA reservoir.

[0005] In infected hepatocytes, multiple different forms of HBV-related mRNAs are present. Five major viral mRNAs are transcribed from cccDNA, with lengths of 3.5 kb, 3.5 kb, 2.4 kb, 2.1 kb, and 0.7 kb, respectively. All contain a polyadenylated tail (polyA) at their 3' ends, with overlapping sequences. The 3.5 kb mRNA serves as a template for core antigen and polymerase production, the 2.4 / 2.1 kb mRNAs encode preS1 / preS2 and S proteins, and the 0.7 kb mRNA encodes the X protein. These gene products play a crucial role in regulating viral gene transcription and can act as transactivators of host gene expression, promoting the development and progression of hepatocellular carcinoma. Furthermore, HBV integration-associated sequences (intHBV) also transcribe viral mRNAs. However, due to the linearization of the viral genome and the loss of some sequences after viral integration, the overlapping polyadenylated sequences at the original 3' end of the integration-derived sequence are lost. Studies have reported that siRNA targeting the common sequence of cccDNA-derived HBV RNA cannot effectively reduce the HBsAg level in HBeAg-negative patients because it cannot target the sequence derived from HBV integrated RNA.

[0006] Based on antigenic epitopes, HBV can be divided into four major serotypes (adr, adw, ayr, and ayw). Based on genomic sequence, the virus can also be classified into eight genotypes (A, B, C, D, E, F, G, and H). Genotype A is common in the Americas, Africa, India, and Western Europe. Genotypes B and C are found in Asia and the United States. Genotype D is most common in Southern Europe and India. Genotype E is found in West Africa and South Africa. Genotypes F and H are commonly found in Central and South America. Genotype G is commonly found in Europe and the United States. Due to the numerous HBV genotypes and the at least 8% sequence variation between each genotype, few siRNA therapeutics can comprehensively cover different HBV genotypes. Furthermore, siRNAs designed to target HBV consensus sequences have difficulty targeting HBV integration, while siRNAs designed to target HBsAg (or HBV integration) are ineffective against HBx. Therefore, there is an urgent clinical need for a small interfering nucleic acid (siRNA) drug that can broadly target multiple HBV genotypes and inhibit both HBsAg (HBV integration) and HBx.

[0007] Several studies have attempted to use RNAi for the treatment of HBV. However, single siRNA transfection often fails to provide sufficient gene silencing. Therefore, despite significant advances in RNAi technology, there remains a need for agents that can effectively inhibit HBV gene expression and treat diseases associated with HBV infection, such as liver disease and cancer.

[0008] The first siRNA drug to enter the clinic, ARC-520, consists of two siRNAs (si-74 and si-77) targeting regions near HBV DR1 and DR2 as active ingredients. However, the gene coverage of the two siRNAs is only 89%, and clinical studies have shown that they cannot target HBV integration.

[0009] For example, the siRNA molecular sequence in Biomike's authorized patent (CN101979554B) is the sense strand: 5'-GAUUGACGAUAAGGGAGANN-3', and the antisense strand: 5'-UCUCCCUUAUCGUCAAUCNN-3'. The conservation of its core sequence is only 20.1%, and it cannot target HBx.

[0010] The siRNA molecular sequence in the authorized patent (CN102140461B) of Suzhou Ruibo Biotechnology Co., Ltd. is the sense chain: 5'-CCUUGAGGAUACUUCAAAdTdT-3', and the antisense chain: 5'-UUUGAAGUAUGCCUCAAGGdTdT-3'. The conservation of its core sequence is only 78.3%, and it can simultaneously target the HBV consensus sequence (including HBx) and target HBV integration.

[0011] VIR-2218, developed by VIR, and AB-729, developed by Arbutus, both use a single siRNA to target the common HBV RNA sequence and can reduce HBsAg levels to the same extent in HBeAg-negative and HBeAg-positive patients, suggesting that they may simultaneously target HBV integration-derived sequences, but the sequence conservation of the above products is unknown.

[0012] Representatives include RG6346 developed by Roche and ALG-125755 developed by Aligos, which target the ORF region of HBsAg through a single siRNA but cannot target HBx; HBx is considered to be a key factor in the regulation of cccDNA activity and transcriptional reactivation.

[0013] Another reason for the difficulty in clinical application of siRNA drugs is their poor stability and susceptibility to nuclease degradation in vivo (particularly, after systemic administration, siRNA first enters the bloodstream, which is rich in endogenous nucleases). To overcome this obstacle, one approach is to chemically modify siRNA to improve its stability in the blood. However, the degradation process and mechanism of siRNA in the blood remain largely unknown in the field, and the selection of modification sites remains an empirical process that requires repeated experimental verification. Therefore, to achieve sufficient stability, siRNA must be subjected to extensive (even blind and redundant) chemical modifications. As a result, while these modifications may improve siRNA stability to a certain extent, excessive or inappropriate modifications inevitably lead to drawbacks such as increased cost, potential cytotoxicity, and compromised biological activity and specificity.

[0014] In addition, various aspects such as synthesis cost, synthesis efficiency, process convenience, siRNA stability, and siRNA activity should be considered to personalize, streamline, and optimize the modification of siRNA sequences in order to obtain better siRNAs. Considering that previous studies in this field have shown that different chemical modifications of siRNA sequences may have a "ripple effect," that is, adopting different modification strategies at only one site can fundamentally change the stability or biological activity of siRNA, completing the personalized design, streamlining, and optimization of the above-mentioned siRNA sequence modifications is not an easy task.

[0015] Therefore, developing an siRNA that is stable in the blood, has good biological activity, and has low cytotoxicity to inhibit HBV gene expression has become an urgent problem that needs to be solved.

[0016] Another difficulty in clinical siRNA drug development is the need to form pharmaceutical compositions using suitable carriers or conjugates with suitable conjugated molecules to further improve siRNA's blood stability, enhance its targeting, and address issues of siRNA delivery in vivo. For siRNAs intended to prevent and treat hepatitis B, carriers or conjugated molecules that can impart or enhance liver targeting will help the siRNA act within target cells, thereby increasing the efficiency of siRNA in inhibiting HBV gene expression and reducing potential side effects. There is also an urgent need in the art for such pharmaceutically acceptable carriers or conjugated molecules that can enhance the clinical application potential of siRNA, as well as siRNA pharmaceutical compositions or siRNA conjugates prepared using such pharmaceutically acceptable carriers or conjugated molecules.

[0017] Summary of the Invention

[0018] The present invention provides siRNA or siRNA combinations that can simultaneously target RNA from all HBV sources, including all HBV RNA derived from cccDNA and RNA derived from HBV integrated (intRNA), enabling broad targeting of multiple HBV genotypes (gene coverage >98%), thereby providing a formulation and therapy that can achieve HBsAg clearance. The siRNA of the present invention has strong specificity and conservatism, can widely target sequences of different clinical genotypes, and can compensate for the poor efficacy of currently under-developed nucleic acid interference drugs and the shortcomings of current hepatitis B treatment drugs, and is expected to become a new means of treating hepatitis B.

[0019] Considering that siRNA is unstable in blood and tissues and easily degraded by nucleases, modifications to the siRNA backbone can be used to improve siRNA stability. However, these chemical modifications only provide limited protection from nuclease degradation and may ultimately affect the activity of the siRNA. Therefore, a corresponding delivery system is also needed to ensure that siRNA can safely and efficiently cross the cell membrane. Due to the large molecular weight, high negative charge, and high water solubility of siRNA, it cannot successfully cross the cell membrane and reach the cell. Different chemical modifications are further introduced into the small interfering nucleic acid to achieve the purpose of improving the serum stability of the small interfering nucleic acid, reducing cytotoxicity, and retaining biological activity. The present invention also provides a high-efficiency siRNA pharmaceutical composition and conjugate targeting the HBV gene, which can effectively prevent and / or treat hepatitis B infection and hepatitis B infection-related diseases. Symptoms and diseases associated with hepatitis B infection include, but are not limited to, chronic liver disease / disorders, inflammation, fibrosis, proliferative disorders (including cancers such as hepatocellular carcinoma), hepatitis D virus (HDV) infection, and acute HBV infection. These symptoms and diseases are associated with chronic HBV infection and / or HDV infection.

[0020] The purpose of the present invention is to provide a highly effective small interfering nucleic acid, a small interfering nucleic acid group, and a pharmaceutical composition and conjugate containing the small interfering nucleic acid or nucleic acid group targeting the HBV gene. More preferably, a small interfering nucleic acid, a small interfering nucleic acid group, and a pharmaceutical composition and conjugate containing the small interfering nucleic acid or nucleic acid group are provided that can widely target multiple HBV genotypes and all sources of HBV RNA. The small interfering nucleic acid, the small interfering nucleic acid group, and the pharmaceutical composition and conjugate containing the small interfering nucleic acid or nucleic acid group can effectively prevent and / or treat hepatitis B infection and hepatitis B infection-related diseases.

[0021] The first aspect of the present invention provides a small interfering RNA (siRNA) having a complementary sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, characterized in that:

[0022] i) the sense strand of the siRNA comprises the sequence 5'-GCCGAUCCAUACUGCGGAA-3' (SEQ ID NO: 1), or a sequence that is not more than 4 nucleotides different therefrom, and the antisense strand of the siRNA comprises the sequence 5'-UUCCGCAGUAUGGAUCGGC-3' (SEQ ID NO: 2), or a sequence that is not more than 4 nucleotides different therefrom (e.g., not more than 3, not more than 2, not more than 1);

[0023] ii) the sense strand of the siRNA comprises the sequence 5'-GUUUACUAGUGCCAUUUGU-3' (SEQ ID NO: 21), or a sequence that differs therefrom by no more than 4 nucleotides (e.g., no more than 3, no more than 2, no more than 1), and the antisense strand of the siRNA comprises the sequence 5'-ACAAAUGGCACUAGUAAAC-3' (SEQ ID NO: 22), or a sequence that differs therefrom by no more than 4 nucleotides (e.g., no more than 3, no more than 2, no more than 1); or

[0024] iii) the sense strand of the siRNA comprises the sequence 5'-GGCUUUCAGUUAUAUGGAU-3' (SEQ ID NO: 23), or a sequence that is not more than 4 nucleotides different therefrom (e.g., not more than 3, not more than 2, not more than 1), and the antisense strand of the siRNA comprises the sequence 5'-AUCCAUAUAACUGAAAGCC-3' (SEQ ID NO: 24), or a sequence that is not more than 4 nucleotides different therefrom (e.g., not more than 3, not more than 2, not more than 1).

[0025] In some embodiments, the siRNA further comprises a 3' overhang consisting of 1 to 4 nucleotides in at least one of the sense and antisense strands. In some embodiments, the siRNA comprises a 3' overhang in both the sense and antisense strands. In some embodiments, the 3' overhang comprises 1 to 4 consecutive deoxythymidine nucleotides (dT), for example, 1 to 3, preferably 2 consecutive uracil nucleotides (U), or 1 to 4 consecutive nucleotides (e.g., 1 to 3, preferably 2 nucleotides) complementary to the target mRNA. In some embodiments, the sense strand of the siRNA comprises or consists of the sequence 5'-GCCGAUCCAUACUGCGGAAdTdT-3' (SEQ ID NO: 3), 5'-GUUUACUAGUGCCAUUUGUdTdT-3' (SEQ ID NO: 61), 5'-GGCUUUCAGUUAUAUGGAUdTdT-3' (SEQ ID NO: 63), and the antisense strand of the siRNA comprises or consists of the sequence 5'-UUCCGCAGUAUGGAUCGGCAGdTdT-3' (SEQ ID NO: 4), 5'-ACAAAUGGCACUAGUAAACUGdTdT-3' (SEQ ID NO: 62), 5'-AUCCAUAUAACUGAAAGCCAAdTdT-3' (SEQ ID NO: 64), wherein the lowercase letter d indicates that the nucleotide to the right of the letter d is a deoxyribonucleotide.

[0026] In some embodiments, the siRNA comprises one or more modified nucleotides and / or modified bonds. Preferably, the siRNA comprises one or more nucleotides in which the 2'-OH of the pentose is modified by methoxy and / or fluorine, such as adenine, guanine, cytosine and / or uracil nucleotides, preferably cytosine and / or uracil nucleotides. In some embodiments, except for the terminal deoxyribonucleotide (if present), all nucleotides of the siRNA are nucleotides in which the 2'-OH of the pentose is modified by methoxy and / or fluorine. In some embodiments, the siRNA comprises one or more modified bonds such as thiophosphate bonds. In some embodiments, the sense strand and / or antisense strand of the siRNA is preferably a sense strand connected to one or more (e.g., 1-5, e.g., 3) GalNac at the 5' end and / or the 3' end, preferably the 3' end. In some embodiments, the siRNA has the characteristics of good serum stability, low cytotoxicity and / or good biological activity, and can be used to inhibit hepatitis B virus gene expression. In some embodiments, by introducing specific modifications into the siRNA of the present invention, the shortcomings of existing small interfering nucleic acids for inhibiting hepatitis B virus gene expression, such as poor serum stability and poor biological activity, are overcome.

[0027] A second aspect of the present invention provides a small interfering RNA (siRNA) set comprising a first siRNA and a second siRNA, wherein the first siRNA targets PreS / S RNA and intHBV RNA, and the second siRNA targets all HBV cccDNA-derived RNAs including HBx. In some embodiments, the small interfering RNA (siRNA) set comprises:

[0028] i) a first siRNA, wherein the first siRNA is the siRNA according to the first aspect of the present invention; and

[0029] ii) a second siRNA having complementary sense and antisense strands, wherein:

[0030] a) the sense strand of the second siRNA comprises the sequence 5'-GUUCAUGUCCUACUGUUCA-3' (SEQ ID NO: 5), or a sequence that differs therefrom by no more than 4 nucleotides (e.g., no more than 3, no more than 2, no more than 1), and the antisense strand of the second siRNA comprises the sequence 5'-UGAACAGUAGGACAUGAAC-3' (SEQ ID NO: 6), or a sequence that differs therefrom by no more than 4 nucleotides (e.g., no more than 3, no more than 2, no more than 1);

[0031] b) the sense strand of the second siRNA comprises the sequence 5'-GCAUGGAGACCACCGUGAA-3' (SEQ ID NO: 7), or a sequence that differs therefrom by no more than 4 nucleotides (e.g., no more than 3, no more than 2, no more than 1), and the antisense strand of the second siRNA comprises the sequence 5'-UUCACGGUGGUCUCCAUGC-3' (SEQ ID NO: 8), or a sequence that differs therefrom by no more than 4 nucleotides (e.g., no more than 3, no more than 2, no more than 1); or

[0032] c) the sense strand of the second siRNA comprises the sequence 5'-GCGUCGCAGAAGAUCUCAA-3' (SEQ ID NO: 43), or a sequence that is not more than 4 nucleotides different therefrom (e.g., not more than 3, not more than 2, not more than 1), and the antisense strand of the second siRNA comprises the sequence 5'-UUGAGAUCUUCUGCGACGC-3' (SEQ ID NO: 44), or a sequence that is not more than 4 nucleotides different therefrom (e.g., not more than 3, not more than 2, not more than 1).

[0033] In some embodiments, the second siRNA further has a 3' overhang consisting of 1 to 4 nucleotides in at least one of the sense strand and the antisense strand;

[0034] Preferably, the second siRNA has 3' overhangs in both the sense and antisense strands;

[0035] More preferably, the 3' overhang is 1 to 4 consecutive, for example 1 to 3, preferably 2 deoxythymidine nucleotides dT, 1 to 4 consecutive, for example 1 to 3, preferably 2 uracil nucleotides U, or 1 to 4, for example 1 to 3, preferably 2 nucleotides complementary to the target mRNA;

[0036] More preferably, the sense strand of the second siRNA contains or consists of the sequence 5'-GUUCAUGUCCUACUGUUCAdTdT-3' (SEQ ID NO: 9), 5'-GCAUGGAGACCACCGUGAAdTdT-3' (SEQ ID NO: 11), 5'-GCGUCGCAGAAGAUCUCAAdTdT-3' (SEQ ID NO: 83), and the antisense strand of the second siRNA contains or consists of the sequence 5'-UGAACAGUAGGACAUGAACAAdTdT-3' (SEQ ID NO: 10), 5'-UUCACGGUGGUCUCCAUGCGAdTdT-3' (SEQ ID NO: 12), 5'-UUGAGAUCUUCUGCGACGCGGdTdT-3' (SEQ ID NO: 84).

[0037] In some embodiments, the molar ratio of the first siRNA to the second siRNA is 1:100 to 100:1, preferably 1:10 to 10:1. In some embodiments, the first siRNA and the second siRNA comprise one or more modified nucleotides and / or modified bonds, and preferably, the first siRNA and the second siRNA are any one of the siRNAs listed in Table 2 or Table 3a / Table 3b.

[0038] In some embodiments, the second siRNA comprises one or more modified nucleotides and / or modified bonds. Preferably, the second siRNA comprises one or more nucleotides in which the 2'-OH of the pentose is modified by methoxy and / or fluorine, such as adenine, guanine, cytosine and / or uracil nucleotides, preferably cytosine and / or uracil nucleotides. In some embodiments, except for the terminal deoxyribonucleotide (if present), all nucleotides of the second siRNA are nucleotides in which the 2'-OH of the pentose is modified by methoxy and / or fluorine. In some embodiments, the second siRNA comprises one or more modified bonds such as phosphorothioate bonds. In some embodiments, the sense strand and / or antisense strand of the second siRNA, preferably the sense strand, is connected to one or more (e.g., 1-5, e.g., 3) GalNac at the 5' end and / or the 3' end, preferably the 3' end.

[0039] In some embodiments, the first and second siRNAs in the siRNA set have good serum stability, low cytotoxicity, and / or good biological activity, and can be used to inhibit hepatitis B virus gene expression. In some embodiments, by introducing specific modifications into the siRNA of the present invention, the shortcomings of existing small interfering nucleic acids used to inhibit hepatitis B virus gene expression, such as poor serum stability and poor biological activity, are overcome.

[0040] The third aspect of the present invention provides a siRNA conjugate, which comprises the siRNA described in the first aspect of the present invention or the siRNA group described in the second aspect and a conjugated group conjugated to the siRNA. In some embodiments, the conjugated group is a pharmaceutically acceptable conjugated group. In some embodiments, the conjugated group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are covalently linked in sequence. In some embodiments, the linker is connected to the 3' end of the positive chain of the siNRA. In some embodiments, the targeting group is a ligand of the asialoglycoprotein receptor. In some embodiments, the targeting group is galactose or N-acetylgalactosamine. The inventors of the present disclosure have found that the siRNA conjugate of the present invention can specifically inhibit the expression of the HBV gene, and specifically target the liver, inhibit the expression of the HBV gene in the liver, and achieve the treatment or prevention of hepatitis B.

[0041] The fourth aspect of the present invention provides a pharmaceutical composition comprising the siRNA described in the first aspect of the present invention, the siRNA group described in the second aspect, and / or the siRNA conjugate described in the third aspect of the present invention, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier contains cationic lipids, auxiliary lipids, structural lipids and PEGylated lipids. In some embodiments, the cationic lipid is an amine-containing compound, such as an ionizable amine-containing compound and / or a biodegradable amine-containing compound, such as 8-[(2-hydroxyethyl)[6-oxo-6(undecyloxy)hexyl]amino]-octanoic acid 1-octylnonyl ester (SM-102). In some embodiments, the auxiliary lipid is phosphatidylcholine, an analog of phosphatidylcholine and / or a derivative of phosphatidylcholine. In some embodiments, the structural lipid is cholesterol, an analog of cholesterol and / or a derivative of cholesterol. In some embodiments, the PEGylated lipid is PEG-diacylglycerol (DAG), PEG dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer) or a mixture thereof, preferably polyethylene glycol-dimyristoylglycerol (PEG-DMG).

[0042] The fifth aspect of the present invention provides the use of the siRNA described in the first aspect of the present invention, the siRNA group described in the second aspect of the present invention, the siRNA conjugate described in the third aspect of the present invention and / or the pharmaceutical composition described in the fourth aspect of the present invention in the preparation of a medicament for treating and / or preventing hepatitis B.

[0043] The sixth aspect of the present invention provides a non-therapeutic method for inhibiting HBV gene expression in hepatocytes, which method comprises contacting the hepatocytes with an effective amount of the siRNA described in the first aspect of the present invention, the siRNA group described in the second aspect of the present invention, the siRNA conjugate described in the third aspect of the present invention and / or the pharmaceutical composition described in the fourth aspect of the present invention.

[0044] The seventh aspect of the present invention provides a kit comprising the siRNA described in the first aspect of the present invention, the siRNA group described in the second aspect of the present invention, the siRNA conjugate described in the third aspect of the present invention and / or the pharmaceutical composition described in the fourth aspect of the present invention.

[0045] The eighth aspect of the present invention provides a method for treating hepatitis B, which comprises administering an effective amount of the siRNA described in the first aspect of the present invention, the siRNA group described in the second aspect of the present invention, the siRNA conjugate described in the third aspect of the present invention and / or the pharmaceutical composition described in the fourth aspect of the present invention to a patient in need.

[0046] The ninth aspect of the present invention provides a method for inhibiting HBV gene expression in hepatitis cells infected with chronic HBV, the method comprising introducing an effective amount of the siRNA described in the first aspect of the present invention, the siRNA group described in the second aspect of the present invention, the siRNA conjugate described in the third aspect of the present invention and / or the pharmaceutical composition described in the fourth aspect of the present invention into the hepatitis cells infected with chronic HBV.

[0047] The tenth aspect of the present invention provides a method for screening the siRNA described in the first aspect of the present invention and / or the siRNA group described in the second aspect of the present invention, which comprises the following steps: 1) optionally, analyzing the conserved sequence of the HBV genome sequence; 2) designing siRNA that targets the HBV sequence but does not target the human genome sequence, and selecting siRNA located near the HBV conserved sequence region (i.e., targeting the HBV conserved sequence region, overlapping with the HBV conserved sequence region (partially or completely overlapping) or located within the conserved HBV conserved sequence region) as candidate siRNA, for example, by designing through siRNA design websites (including DSIR, siDirect, siDESIGN Venter, siRNA Target Finder); 3) using model cell lines for siRNA screening and verification, for example, the cell line models include HepG-HBV / EGFP cell lines containing EGFP green fluorescent protein as a reporter gene, HepAD38 (HBV replicon model), PLC / RCF / 5 (clinically screened liver cancer cell line containing HBV natural integration), HepG2-NTCP (HBV infection model), 5C-PHH (Science 2019), pHBV1.3 replicon plasmid system (four genotypes A / B / C / D), etc.; 4) the screened sequences are compared with the HBV genome sequence set in the hepatitis virus database, such as the HVDB hepatitis virus database, to obtain the conservation of their sequences (number of fully matched sequences / total number of HBV gene sequences × 100%); 5) sequences with good HBV inhibition rate and high conservation are selected; 6) optionally, these sequences are further combined, and the conservation of the sequence combinations is evaluated and analyzed in a model different from the model cell line in step 3; 7) optionally, sequence combinations that can achieve multiple targets (can simultaneously target HBV cccDNA-derived RNA and integrated HBV-derived RNA), are broad-spectrum (sequence conservation > 90%), and have good efficacy (target gene expression level inhibition rate > 90%) are selected.

[0048] In an eleventh aspect of the present invention, a method for screening an siRNA panel is provided, comprising the following steps: 1) selecting a first siRNA targeting PreS / S RNA and intHBV RNA, and selecting a second siRNA targeting all HBV cccDNA-derived RNAs, including HBx; 2) testing the combination in a model cell line, for example, the cell line model includes a HepG-HBV / EGFP cell line containing EGFP green fluorescent protein as a reporter gene, HepAD38 (HBV replicon model), PLC / RCF / 5 (clinically screened hepatocellular carcinoma cell line containing HBV natural integration), HepG2-NTCP (HBV infection model), 5C-PHH (Science 2019), pHBV1.3 replicon plasmid system (four genotypes A / B / C / D), etc. In some embodiments, the method includes, before step 2), performing a conservation analysis on the selected sequence combination (for example, by comparing it with a hepatitis virus database such as the HVDB hepatitis virus database to obtain the conservation of its sequence), and selecting a highly conservative sequence combination. In some embodiments, the method includes, before step 1), designing an siRNA that targets an HBV sequence but not a human genome sequence, and selecting an siRNA located near the HBV conserved sequence region (i.e., targeting a sequence covering the HBV conserved sequence region, overlapping (partially or completely overlapping) with the HBV conserved sequence region, or located within the conserved HBV conserved sequence region) as a candidate siRNA, for example, by designing through an siRNA design website (including DSIR, siDirect, siDESIGN Venter, siRNA Target Finder).

[0049] The following description and examples set forth embodiments of the present invention in detail. It is to be understood that the present invention is not limited to the specific embodiments described herein and therefore may be modified. It will be appreciated by those skilled in the art that there are many variations and modifications of the present invention, which are all included within the scope thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The above is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0051] FIG1 shows the siRNA screening process of the present invention.

[0052] FIG2 shows the target sites of different siRNAs designed in the present invention and their inhibitory effects on virological indicators in different HBV-related cell models.

[0053] FIG3 shows the interference results of different siRNAs designed in the present invention on different HBV genotypes (including four genotypes A / B / C / D) and PHH infection models.

[0054] FIG4 shows the IC values ​​of different siRNAs designed in the present invention in HepG2-NTCP infected cells 50 The values ​​and antiviral effects of different siRNA combinations in different HBV-related cell models were analyzed.

[0055] Figure 5 shows the inhibitory effect of the combination of si1260 and si1848 on virological indicators in different HBV-related cell models.

[0056] FIG6 shows the interference results of the combination of si1260 and si1848 on different HBV genotypes (including four genotypes A / B / C / D).

[0057] Figure 7 shows the duration of the antiviral effect of the combination of si1260 and si1848 in in vitro and in vivo models.

[0058] FIG8 shows the inhibitory effects on virological indicators, serum stability and cytotoxicity of si1260 and si1848 or a combination of the two with different chemical modification schemes.

[0059] Figure 9 shows the serum stability of si1260 and si1848 with different chemical modification schemes.

[0060] FIG10 shows the inhibition results of the positive drug ETV, LNP-siNC control group and exemplary LNP-siHBV on HBsAg, HBeAg and HBV DNA levels at different time points after administration at different dose levels and different dosing frequencies.

[0061] FIG11 shows the results of an exemplary LNP-siHBV single-dose safety evaluation.

[0062] FIG12 shows the results of an exemplary LNP-siHBV repeat-dose safety evaluation.

[0063] FIG13 shows the evaluation of off-target effects of exemplary LNP-siHBV in vitro and in vivo.

[0064] FIG14 shows the synthesis quality test results of exemplary siRNA conjugates of the present disclosure.

[0065] FIG15 shows the results of the inhibition of HBsAg, HBeAg and HBV DNA levels by exemplary siRNA conjugates and siRNA conjugate compositions disclosed herein in HBV-related model mice. DETAILED DESCRIPTION

[0066] Several aspects of the present invention are described below with reference to example applications for illustration. It should be understood that many specific details, relationships, and methods are set forth to provide a full understanding of the present invention. However, one of ordinary skill in the relevant art will readily recognize that the present invention can be practiced without one or more of these specific details or can be practiced with other methods.

[0067] The siRNA sequence combination provided by the present disclosure, the pharmaceutical composition containing the sequence combination and the conjugate have multiple targets, a broad spectrum, good stability, low off-target effects, high activity in inhibiting HBV mRNA, and / or can significantly treat and / or prevent hepatitis B.

[0068] The siRNA groups, pharmaceutical compositions, and conjugates provided by the present invention exhibit excellent inhibition rates of HBV mRNA target gene expression in hepatitis B model mice, effectively reducing hepatitis B surface antigen expression, and demonstrating a favorable therapeutic effect for chronic hepatitis B. In some embodiments, lipid nanoparticles composed of components such as cationic lipids, helper lipids, structural lipids, and PEGylated lipids are used as pharmaceutically acceptable carriers to form pharmaceutical compositions with the siRNA, siRNA groups, and / or siRNA conjugates of the present invention, thereby enabling specific delivery of the siRNA of the present invention to the liver.

[0069] The advantages of the present disclosure are that, compared with the disadvantage of traditional drugs that have limited coverage of different HBV genotypes, the siRNA group and pharmaceutical composition provided by the present invention can effectively act against multiple HBV genotypes at the same time; compared with the disadvantage of traditional drugs that cannot simultaneously target the common sequence of HBV cccDNA-derived mRNA and HBV integration, the siRNA group and pharmaceutical composition provided by the present invention can simultaneously target HBV genes from different sources, including mRNA from cccDNA and HBV integration.

[0070] The siRNA, siRNA group, pharmaceutical composition and conjugate provided by the present invention can achieve the effect of improving the stability of siRNA with very few site modifications by reducing the number of modified nucleotides. In in vitro experiments, the modified siRNA can maintain very high stability in serum for a long time and can achieve better therapeutic effects. Both in vitro cell experiments and in vivo animal experiments show very good activity in inhibiting HBV mRNA gene expression and significant inhibitory effect on hepatitis B surface antigen HBsAg.

[0071] In some embodiments, the siRNAs, siRNA panels, pharmaceutical compositions, and conjugates provided herein exhibit excellent target mRNA inhibitory activity in in vitro cell experiments. In some embodiments, the siRNAs, siRNA panels, pharmaceutical compositions, and conjugates provided herein exhibit at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% inhibition of target gene expression in HBV-related cell models.

[0072] In some embodiments, the siRNA, siRNA groups, pharmaceutical compositions and conjugates provided by the present disclosure may have higher stability and / or higher activity in vivo. In some embodiments, the siRNA, siRNA groups, pharmaceutical compositions and conjugates provided by the present disclosure show a target gene expression inhibition rate of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in HBV-related animal models. In some embodiments, the siRNA, siRNA groups, pharmaceutical compositions and conjugates provided by the present disclosure show significant inhibitory activity in the prcccDNA+pCMV-Cre high-pressure hydrodynamic model, and the inhibition rate of siRNA on the target gene expression level of HBV mRNA can reach 99.50%. In some embodiments, the siRNA pharmaceutical composition provided by the present disclosure shows significant inhibitory activity in the rAAV-HBV1.3 mouse hepatitis B chronic model, and the inhibition rate of siRNA on the target gene expression level of HBV mRNA can reach 99.77%. In some embodiments, the siRNA conjugates provided by the present disclosure show significant inhibitory activity in the rAAV-HBV1.3 mouse chronic hepatitis B model, and the inhibition rate of siRNA on the target gene expression level of HBV mRNA can reach 97.46%.

[0073] In some embodiments, the siRNA, siRNA groups, pharmaceutical compositions and conjugates provided by the present disclosure may have good safety in vivo. In some embodiments, the siRNA, siRNA groups, pharmaceutical compositions and conjugates provided by the present disclosure will not induce the production of immune factors in the short term after injection, and will not cause abnormal biochemical indicators related to liver and kidney function in mice, and will not lead to organ toxicity. In some embodiments, the siRNA, siRNA groups, pharmaceutical compositions and conjugates provided by the present disclosure will not cause abnormal biochemical indicators related to liver and kidney function in mice after repeated multiple administrations, and will not lead to organ toxicity. In some embodiments, the siRNA, siRNA groups, pharmaceutical compositions and conjugates provided by the present disclosure will not cause significant changes in the expression profile of HepG2 cells and / or mouse livers after repeated multiple administrations, and will not cause obvious off-target effects.

[0074] In some embodiments, the siRNA, siRNA groups, pharmaceutical compositions, and conjugates provided herein may have low immunogenicity in vivo. In some embodiments, the siRNA pharmaceutical compositions provided herein do not result in the production of anti-PEG antibodies after repeated multiple injections.

[0075] This shows that the siRNA, siRNA group, pharmaceutical composition and conjugate provided by the present disclosure can effectively inhibit the expression of HBV gene, can effectively treat and / or prevent hepatitis B symptoms, and have good application prospects.

[0076] The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" used herein are intended to include the plural forms. In addition, the open-ended expressions "comprise" and "include" are interpreted as also including unmentioned structural components or method steps, but it should be noted that such open-ended expressions also cover situations consisting only of the described components and method steps (i.e., covering situations where the closed-ended expression "consisting of..." is used).

[0077] As used throughout, ranges are used as a shorthand for describing each and every value within the range. Any value within a range, such as an integer value, can be selected as the endpoint of the range. For example, an integer in the range 19-26 represents the integers 19, 20, 21, 22, 23, 24, 25, and 26, and any subranges formed therefrom, such as 19-25, 20-24, 21-23, etc.

[0078] All scientific and technical terms mentioned in this specification have the same meanings as those commonly understood by those skilled in the art. In the event of a conflict, the definitions in this specification shall prevail. In order to make the description of the present invention easier to understand, some terms are explained below.

[0079] In the present invention, the terms "nucleic acid," "nucleic acid molecule," "oligonucleotide drug," and "nucleic acid drug" are used interchangeably to refer to any DNA, RNA, or DNA / RNA chimera, and may be an oligonucleotide or a polynucleotide, and may be unmodified RNA or DNA or modified RNA or DNA. The term includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded, double-stranded, or a mixture of single-stranded and double-stranded regions.

[0080] In the present invention, the terms "small interfering nucleic acid", "small interfering RNA" and "siRNA" are used interchangeably to refer to a short (usually 19 to 26 bp) double-stranded RNA (dsRNA) with a phosphorylated 5' end and a hydroxylated 3' end.

[0081] In some embodiments, the nucleic acid may comprise one or more modified nucleotides, modified bonds, and the like. Examples of modified bonds or internucleotide bonds include phosphorothioates, phosphorodithioates, and the like. In some embodiments, the nucleotide comprises a phosphorus derivative. The phosphorus derivative (or modified phosphate group) that can be attached to the sugar or sugar analog portion of the modified nucleotide of the present invention can be a monophosphate, a diphosphate, a triphosphate, an alkyl phosphate, an alkane phosphate, a phosphorothioate, and the like. The preparation of the above-mentioned phosphate analogs, and their incorporation into nucleotides, modified nucleotides, and nucleic acids is also known per se and need not be described herein.

[0082] In the present invention, the term "sequence conservation" refers to the percentage of HBV genome sequences that can be completely matched by the siRNA 19-mer core sequence among the 11,185 HBV genome sequences in the HBV database.

[0083] For nucleic acid modification

[0084] (1) Nucleic acid structure

[0085] In some embodiments, nucleic acid of the present invention comprises one or more modifications (e.g., base modification, backbone modification, etc.) to provide new or enhanced features (e.g., improved stability) to nucleic acid. As known in the art, nucleoside is a base-sugar combination. The base portion of a nucleoside is generally a heterocyclic base. The two most common categories of such heterocyclic bases are purine and pyrimidine. Nucleotide is a nucleoside that also includes a phosphate group covalently attached to the sugar portion of a nucleoside. For those nucleosides including pentofuranosyl, the phosphate group can be attached to the 2', 3' or 5' hydroxyl portion of the sugar. In forming nucleic acid, the phosphate group covalently attaches adjacent nucleosides to form a linear polymeric compound. Conversely, each end of a linear polymeric compound can further be connected to form a cyclic compound, but linear compounds are generally suitable. In addition, linear compounds can have internal nucleotide base complementarity and can therefore fold in order to produce a completely or partially double-stranded compound. In nucleic acid, the phosphate group is generally referred to as the internucleoside backbone forming nucleic acid. The normal bond or backbone of RNA and DNA is a 3' to 5' phosphodiester bond.

[0086] (2) Modified backbone and modified internucleoside linkage

[0087] Examples of suitable nucleic acids containing modifications include nucleic acids containing modified backbones or non-natural internucleoside linkages. Nucleic acids (having modified backbones) include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

[0088] The nucleic acid wherein containing the modification that is fit to of phosphorus atom for example comprises, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphate comprise 3 '-alkylene phosphate, 5 '-alkylene phosphate and chiral phosphate, phosphonate, phosphoramidate comprises 3 '-amino phosphoramidate and aminoalkyl phosphoramidate, diamino phosphoroate, thionyl phosphoramidate, thionyl phosphate, thionyl phosphotriester, selenophosphate and boron phosphate with normal 3 '-5 ' key, these 2 '-5 ' connect analog and have those of reverse polarity, wherein between one or more Nucleotides, key is 3 ' to 3 ', 5 ' to 5 ' or 2 ' to 2 ' key.The nucleic acid that is fit to with reverse polarity comprises single 3 ' to 3 ' key at the key place between most 3 ' Nucleotides, can be the single inversion nucleoside residue of abase (core base loses or it is replaced by hydroxyl).Also comprise various salts (for example as potassium or sodium), mixed salts and free acid form.

[0089] In some embodiments, the nucleic acids of the invention comprise one or more phosphorothioate bonds and / or heteroatom internucleoside bonds, specifically -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- (referred to as a methylene(methylimino) or MMI backbone), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -ON(CH3)-CH2-CH2- (wherein the natural phosphodiester internucleoside bond is represented as -OP(=O)(OH)-O-CH2-).

[0090] Other backbone modifications also comprise the nucleic acid of for example morpholino backbone structure.For example, in some embodiments, nucleic acid of the present invention comprises 6 yuan of morpholino rings that substitute ribose ring.In some of these embodiments, key substitutes phosphodiester bond between diaminophosphoroester or other non-phosphodiester nucleoside.

[0091] Suitable modified polynucleotide backbones that do not include phosphorus atoms have backbones formed by bonds between short-chain alkyl or cycloalkyl nucleosides, mixed heteroatoms and bonds between alkyl or cycloalkyl nucleosides, or bonds between one or more short-chain heteroatoms or heterocyclic nucleosides. These include those with: morpholino bonds (partially formed from the sugar moiety of nucleosides); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formyl acetyl and thioformyl acetyl backbones; methylene formyl acetyl and thioformyl acetyl backbones; ribose acetyl (riboacetyl) backbones; backbones containing olefins; sulfamic acid ester backbones; methylene imino and methylene hydrazine backbones; sulfonic acid ester and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S, and CH2 components.

[0092] Another backbone modification includes locked nucleic acid (LNA), in which the 2'-hydroxyl is connected to the 4' carbon atom of the sugar ring to form a 2'-C, 4'-C-oxymethylene bond, thereby forming a bicyclic sugar moiety. The chain can be a methylene (-CH2-) (a group bridging the 2' oxygen atom and the 4' carbon atom), where n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456). LNA and LNA analogs exhibit very high duplex thermal stability (T m = +3°C to +10°C), stability towards 3'-exonucleolytic degradation and good solubility properties.

[0093] The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil as well as their oligomerization and nucleic acid recognition properties have been described in the prior art (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630).

[0094] (3) Modified sugar moiety

[0095] The nucleic acids of the present invention may also include one or more substituted sugar moieties. Suitable polynucleotides contain sugar substituents selected from the group consisting of: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl groups. Particularly suitable is O((CH2) n O) m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON((CH2)n CH3)2, wherein n and m are 1 to about 10. Other suitable polynucleotides contain sugar substituents selected from the group consisting of C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of nucleic acids, or groups for improving the pharmacodynamic properties of nucleic acids, and other substituents of similar nature. Suitable modifications include 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., alkoxyalkoxy. Another suitable modification includes 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the Examples below; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2.

[0096] Other suitable sugar substituents include methoxy (—O—CH 3 ), aminopropoxy (—OCH 2 CH 2 CH 2 NH 2 ), allyl (—CH 2 —CH═CH 2 ), —O-allyl (—O—CH 2 —CH═CH 2 ), and fluorine (F). The 2′-sugar substituent can be in the arabinose (upper) position or the ribose (lower) position. A suitable 2′-arabinose modification is 2′-F. Similar modifications can also be made at other positions on the oligomeric compound, specifically at the 3′ position of the sugar on the 3′-terminal nucleoside or in a 2′-5′ linked nucleic acid and at the 5′ position of the 5′-terminal nucleotide. The oligomeric compound can also have a sugar mimetic such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

[0097] (4) Base modification and substitution

[0098] The nucleic acids of the present invention may also include modifications or substitutions of nucleobases (often referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives and other alkyl derivatives of adenine and guanine, 2-propyl derivatives and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, 2-methyl ... uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo (specifically 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Additional modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).

[0099] Heterocyclic base moieties can also include those in which purine or pyrimidine bases are replaced by other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Additional nucleobases include those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, Kroschwitz, JI, ed., John Wiley & Sons, 1990, those disclosed in Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed in Sanghvi, YS, Chapter 15, Antisense Research and Applications, pp. 289-302, Crooke, ST, and Lebleu, B., eds., CRC Press, 1993. Some of these nucleobases are useful for increasing the binding affinity of oligomeric compounds. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6°C-1.2°C (Sanghvi et al., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are suitable base substitutions, for example, when combined with 2'-O-methoxyethyl sugar modifications.

[0100] (5) Conjugate

[0101] Another possible modification of nucleic acid of the present invention relates to one or more parts or conjugate chemistry that will enhance the activity of nucleic acid, cell distribution or cellular absorption and be connected to polynucleotide.These parts or conjugates can comprise the conjugate group of the functional group of covalent binding such as primary hydroxyl or secondary hydroxyl.Conjugate groups include but are not limited to intercalating agent, reporter molecule, polyamine, polyamide, polyethylene glycol, polyether, the group that strengthens oligomer pharmacodynamic property and the group that strengthens oligomer pharmacokinetic property.Suitable conjugate groups include but are not limited to N-acetylgalactosamine (GalNAc), cholesterol, lipid, phospholipid, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and dye.The group that strengthens pharmacodynamic property comprises the group that improves absorption, strengthens resistance to degradation and / or strengthens sequence-specific hybridization with target nucleic acid.The group that strengthens pharmacokinetic property comprises the group that improves absorption, distribution, metabolism or excretion of nucleic acid of the present invention.

[0102] Conjugate moieties include, but are not limited to, N-acetylgalactosamine (GalNAc) moieties (e.g., 1 to 3 GalNAcs), lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-racemic glycerol or 1,2-di-O-hexadecyl-racemic glycero-3-H-triethylammonium phosphate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

[0103] As used herein, "unmodified" or "natural" nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleotides include nucleotides that occur only rarely or transiently in natural nucleic acids, for example, hypoxanthine, 6-methyladenine, 5-Me pyrimidine, particularly 5-methylcytosine (also known as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentiobiosyl HMC, as well as synthetic nucleotides, for example, 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6-diaminopurine. "Universal" bases known in the art, for example, inosine, may be included.

[0104] In the present invention, the terms "conjugated" and "linked" are used interchangeably to refer to connection through a chemical bond.

[0105] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in this field, which is incorporated herein by reference. The use of such media and agents is well known in the art. Unless any conventional media or agents are incompatible with the materials provided herein, their use in the compositions is contemplated.

[0106] 1. siRNA of the present invention

[0107] The siRNA of the present invention was screened and obtained via the following approach (see FIG1 ):

[0108] 1) First, we analyzed conserved sequences within the HBV genome. We downloaded a collection of 11,185 HBV genome sequences from the Hepatitis Virus Database (HVDB) (http: / / s2as02.genes.nig.ac.jp / index.html). We then performed a global alignment using the MEME (Motif-based sequence analysis tools) website (https: / / meme-suite.org / meme / index.html) to analyze conserved sequence regions (motifs) within the HBV genome sequence collection.

[0109] 2) Use siRNA design websites (including DSIR, siDirect, siDESIGN Venter, siRNA Target Finder) to design siRNAs that target HBV sequences but not human genome sequences, and select siRNAs located near the conserved sequence region of HBV as candidate siRNAs. The sequence design of the 19mer siRNA used for HBV RNAi is designed according to multiple HBV cDNA target sequences. The set parameters include that the length of the siRNA is 19 base sequences and the GC ratio is between 35-50. A total of 20 qualified siRNAs were selected, covering the entire HBV genome, targeting the conserved sequence regions in the HBV genome sequence set, and having no homology with known human genes and gene expression fragments. The candidate sequences in the present invention are shown in the following table (without adding protective bases such as TT, UU, UA):

[0110] Table 1 siRNA sequences

[0111] 3) Using the in-house original cell model HepG-HBV / EGFP cell line (this model can be found in Min Wu et al., Establishment of Cre-mediated HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral screening assays, Antiviral Research, 2018, 152, 45-52; and Feng Li et al., Minicircle HBV cccDNA with a Gaussia luciferase reporter for investigating HBV cccDNA biology and developing cccDNA-targeting drugs, Scientific Reports, 6, 36483.) for siRNA screening and validation. This model contains EGFP green fluorescent protein as a reporter gene, which can significantly improve the efficiency of siRNA screening and express HBsAg derived from cccDNA. Screening and validation were also carried out in HepAD38 (HBV replicon model), PLC / RCF / 5 (a clinically screened liver cancer cell line containing naturally integrated HBV, which can express HBsAg derived from integrated HBV), HepG2-NTCP (HBV infection model), 5C-PHH (in-house constructed, Science 2019), and pHBV1.3 replicon plasmid system (four genotypes A / B / C / D).

[0112] 4) The screened sequences were then compared with a set of 11,185 HBV genome sequences downloaded from the HVDB hepatitis virus database to determine their sequence conservation (number of fully matched sequences / total number of HBV gene sequences × 100%). Sequences with good inhibition rates and high conservation were selected for further combination (e.g., any combination of any siRNA in Table 3a with any siRNA in Table 3b, any combination of any two siRNAs in Table 4, especially combinations of siRNAs targeting HBsAg and HBV integration with siRNAs targeting the consensus sequence of HBx and HBV mRNA, and particularly the combinations in Table 11). The conservation of the sequence combinations was evaluated and analyzed in different models. Sequence combinations that achieved multi-target, broad-spectrum, and good efficacy were selected for subsequent studies.

[0113] The above screening strategy can effectively improve the efficiency of siRNA sequence screening. Compared with the previous siRNA screening, which often required hundreds of sequences (Molecular therapy, 2013, 21(5), 973-985), the strategy used in the present invention can obtain better target sequences with less work. Compared with the first clinical siRNA drug targeting HBV (ARC-520), the genotype coverage of the obtained siRNA was significantly improved (89% vs. 98%), and it can simultaneously target HBV cccDNA and HBV integration source sequences (ARC-520 cannot target HBV integration).

[0114] The siRNA or siRNA group targeting any one or multiple groups of RNA interference targets of HBV provided in the present invention, the encoding plasmid, recombinant viral vector and cell line containing or introducing the encoding nucleic acid sequence of the siRNA or siRNA group of any one or multiple groups of RNA interference targets of the present invention, and the therapeutic drug containing the siRNA or siRNA group of any one or multiple groups of RNA interference targets of the present invention, all fall within the scope of protection of the present invention.

[0115] In a preferred embodiment, two or more of the above siRNA molecules can be combined together to target different regions of the HBV genome transcript, thereby achieving the purpose of inhibiting the expression of RNA genes from all sources of HBV and widely targeting different HBV genotypes.

[0116] The present invention provides siRNAs, siRNA panels, siRNA pharmaceutical compositions, and conjugates targeting HBV. These siRNAs, siRNA panels, siRNA pharmaceutical compositions, and conjugates can be used to treat diseases, such as liver diseases or malignancies and / or cancers associated with HBV expression, or other conditions resulting from hepatitis B infection. The siRNAs can be administered alone or in combination with other siRNA molecules or conventional agents for treating HBV-related diseases or conditions.

[0117] 2. Modified siRNA of the present invention

[0118] The siRNA of the present invention contains a nucleotide group as a basic structural unit, and the nucleotide group contains a phosphate group, a ribose group and a base. The siRNA of the present invention may also include one or more modified nucleotide groups. Nucleotide group modification can be used to improve in vitro or in vivo characteristics, such as stability, activity, toxicity, immune response (such as prevention of interferon response, inflammation or proinflammatory cytokine response or stimulation of Toll-like receptor response) and / or bioavailability. The modified nucleotide group will not cause the function of the siRNA to inhibit HBV gene expression to be significantly weakened or lost. There are currently a variety of ways that can be used to modify siRNA in the art, including backbone modification (such as phosphate group modification), ribose group modification and base modification.

[0119] Chemically modified siRNA molecules with improved RNAi activity and / or stability compared to corresponding unmodified siRNA molecules are described herein. Various chemically modified siRNA motifs disclosed herein provide the ability to maintain RNAi activity substantially similar to that of the pre-modified or minimally modified active siRNA while providing nuclease resistance and pharmacokinetic properties suitable for use in therapeutic applications.

[0120] In various embodiments, the siRNA molecules comprise modifications wherein any one, multiple, or all nucleotides present in the sense strand and / or antisense strand are modified, some nucleotides are modified, or all nucleotides are modified nucleotides. In some embodiments, the siRNA molecules are partially modified by chemical modifications. In other embodiments, the siRNA molecules of the present invention are fully modified by chemical modifications.

[0121] In some embodiments, the siRNA of the invention is selected from si-1260, si-1606, si1848, si-675, si-726, and si-2413, and combinations thereof.

[0122] The sequence of si1260 is:

[0123] Sense strand: 5′-GCCGAUCCAUACUGCGGAAdTdT-3′;

[0124] Antisense strand: 5′-UUCCGCAGUAUGGAUCGGCdTdT-3′.

[0125] The sequence of si1606 is:

[0126] Sense strand: 5′-GCAUGGAGACCACCGUGAAdTdT-3′;

[0127] Antisense strand: 5′-UUCACGGUGGUCUCCAUGCGAdTdT-3′.

[0128] The sequence of si1848 is:

[0129] Sense strand: 5′-GUUCAUGUCCUACUGUUCAdTdT-3′;

[0130] Antisense strand: 5′-UGAACAGUAGGACAUGAACdTdT-3′.

[0131] The sequence of si-675 is:

[0132] Sense strand: 5′-GUUUACUAGUGCCAUUUGUdTdT-3′;

[0133] Antisense strand: 5′-ACAAAUGGCACUAGUAAACUGdTdT-3′.

[0134] The sequence of si-726 is:

[0135] Sense strand: 5′-GGCUUUCAGUUAUAUGGAUdTdT-3′;

[0136] Antisense strand: 5′-AUCCAUAUAACUGAAAGCCAAdTdT-3′.

[0137] The sequence of si-2413 is:

[0138] Sense strand: 5′-GCGUCGCAGAAGAUCUCAAdTdT-3′;

[0139] Antisense strand: 5′-UUGAGAUCUUCUGCGACGCGGdTdT-3′.

[0140] The capital letters C, G, U, and A represent the base composition of the nucleotides, respectively.

[0141] Preferably, in order to enhance the stability of the siRNA double strand, at least one of the single strands in the sense and antisense strands of the siRNA is further connected to the 3' end with 1 to 4 nucleotides, thereby forming at least one 3' overhang consisting of 1 to 4 nucleotides after complementary pairing of the sense and antisense strands, or referred to as an overhanging end. Preferably, the 3' end of each single strand in the sense and antisense strands of the siRNA is connected to 1 to 4 nucleotides. More preferably, the 3' overhang is a continuous 1-4, for example, 2, thymine deoxyribonucleotides (i.e., dT) or a continuous 1-4, for example, 2, uracil ribonucleotides (i.e., U).

[0142] In order to further improve the stability of siRNA, at least a portion of the phosphate groups in the phosphate-sugar backbone of the siRNA is a phosphate group with a modifying group, and at least a portion of the ribose groups in the phosphate-sugar backbone of the siRNA is a ribose group with a modifying group. The phosphate group and ribose group with a modifying group can be various phosphate groups and ribose groups with a modifying group that have the above-mentioned effects. Preferably, the ribose group with a modifying group is a 2'-methoxyribose group in which the 2'-hydroxyl group is replaced by a methoxy group or a 2'-fluororibose group in which the 2'-hydroxyl group is replaced by a fluorine group, and the phosphate group with a modifying group is a thiophosphate group in which one of the oxygen atoms in the phosphodiester bond in the phosphate group is replaced by a sulfur atom; the structure of the thiophosphate group is shown in the following formula:

[0143] In some embodiments, in the siRNA of the present invention, the modified sense strand and antisense strand are selected from the sequences shown in Table 2 below:

[0144] Table 2 Chemically modified siRNA sequence list

[0145] Note: Capital letters C, G, U, A and T represent the base composition of nucleotides; lowercase letter d indicates that the nucleotide to the right of the letter d is a deoxyribonucleotide; lowercase letter m indicates that the 2'-hydroxyl group of the ribose group of the nucleotide to the left of the letter m is replaced by a methoxy group; lowercase letter f indicates that the 2'-hydroxyl group of the ribose group of the nucleotide to the left of the letter f is replaced by a fluorine group; lowercase letter s indicates that the phosphodiester bond between two nucleotides is replaced by a phosphorothioate diester bond.

[0146] It is well known to those skilled in the art that the siRNA of the present invention can be obtained by conventional siRNA preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis). Solid phase synthesis is commercially available on a custom basis. Modified nucleotide groups can be introduced into the siRNA of the present invention by using correspondingly modified nucleotide monomers. Methods for preparing correspondingly modified nucleotide monomers and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.

[0147] 3. siRNA Pharmaceutical Compositions of the Present Invention

[0148] The present invention provides a small interfering nucleic acid pharmaceutical composition, wherein the pharmaceutical composition contains the siRNA, siRNA set or modified siRNA described in the present invention. In some embodiments, the small interfering nucleic acid pharmaceutical composition contains two small interfering nucleic acids, namely a first siRNA such as si1260 and a second siRNA such as si1848 or si1606, as active ingredients.

[0149] In the small interfering nucleic acid pharmaceutical composition of the present invention, the combination of the first siRNA and the second siRNA in any different ratios can achieve the purpose of the present invention. Preferably, the molar ratio of the first siRNA to the second siRNA is 1:100 to 100:1. In order to achieve better therapeutic effects and synergistic effects, according to a specific embodiment of the present invention, the molar ratio of the first siRNA to the second siRNA can be 1:10 to 10:1, 1:5 to 5:1 or 1:2 to 2:1. In some embodiments, the sense chain and antisense chain of the si1260 can be naked nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2; the sense chain and antisense chain of the si1606 can be naked nucleotide sequences as shown in SEQ ID NO: 7 and SEQ ID NO: 8; and / or the sense chain and antisense chain of the si1848 can be naked nucleotide sequences as shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0150] In some embodiments, in order to enhance the stability of the siRNA double strand, the 3' end of at least one of the sense and antisense strands of the first siRNA and the sense and antisense strands of the second siRNA is further connected to 1 to 4 nucleotides, thereby forming a 3' overhang consisting of at least 1 to 4 nucleotides after complementary pairing of the sense and antisense strands. Preferably, the 3' end of each of the sense and antisense strands of the first siRNA and the sense and antisense strands of the second siRNA is connected to a 3' overhang. More preferably, the 3' overhang is two consecutive thymine deoxyribonucleotides dTdT or two consecutive uracil ribonucleotides UU; alternatively, in order to increase the affinity of the siRNA antisense strand to the target mRNA, the 3' overhang is complementary to the nucleotide at the corresponding position of the target mRNA.

[0151] In order to further improve the stability of the first siRNA and / or the second siRNA, at least a portion of the phosphate groups in the phosphate-sugar backbone of the first siRNA and / or the second siRNA is a phosphate group with a modifying group, and at least a portion of the ribose groups in the phosphate-sugar backbone of the first siRNA and / or the second siRNA is a ribose group with a modifying group. The phosphate group and ribose group with a modifying group can be various phosphate groups and ribose groups with modifying groups that have the above-mentioned effects. Preferably, the ribose group with a modifying group is a 2'-methoxyribose group in which the 2'-hydroxyl group is replaced by a methoxy group or a 2'-fluororibose group in which the 2'-hydroxyl group is replaced by a fluorine group, and the phosphate group with a modifying group is a thiophosphate group in which one of the oxygen atoms in the phosphodiester bond in the phosphate group is replaced by a sulfur atom; the structure of the thiophosphate group is shown below:

[0152] According to a specific embodiment of the present invention, in the small interfering nucleic acid pharmaceutical composition provided by the present invention, the sense chain of the si1260 is a nucleotide sequence as shown in 5'-GCmCmGAUmCmCmAUmACmUmGCmGGAAdTdT-3', and the antisense chain is a nucleotide sequence as shown in 5'-UUCCGCAGUmAUGGAUmCGGCdTdT-3'; the sense chain of the si1848 is a nucleotide sequence as shown in 5'-GUmUmCmAUmGUmCmCmUmACmUmGUmUmCmAdTdT-3', and the antisense chain is a nucleotide sequence as shown in 5'-UGAACAGUmAGGACAUmGAACdTdT-3'. The capital letters C, G, U, A, and T represent the base composition of the nucleotide. A lowercase letter d indicates that the nucleotide to the right of the letter is a deoxyribonucleotide. A lowercase letter m indicates that the ribose group of the nucleotide to the left of the letter m is a 2'-methoxyribose group, where the 2'-hydroxyl group is replaced by a methoxy group. For example, in si1260, the "Cm" formed by the second capital letter C at the 5' end of the sense strand and the m to its right indicates a nucleotide containing a cytosine base and a methoxy group replacing the 2'-hydroxyl group of the ribose group.

[0153] It is well known to those skilled in the art that the first siRNA and / or the second siRNA of the present invention can be obtained by conventional siRNA preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis). Among them, solid phase synthesis already has commercial custom services. It is also well known to those skilled in the art that modified nucleotide groups can be introduced into the first siRNA and / or the second siRNA of the present invention by using nucleotide monomers with corresponding modifications. The method for preparing nucleotide monomers with corresponding modifications is well known to those skilled in the art, and commercial monomers are also available on the market.

[0154] The small interfering nucleic acid pharmaceutical composition of the present invention contains, in addition to the first siRNA and / or the second siRNA as active ingredients, a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, magnetic nanoparticles (e.g., Fe3O4, Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyaminoamine (PAMAM) dendrimer, poly (L-lysine, PLL), chitosan, 1,2-dioleoyl-3-thrimethylammonium-propane (DOTAP), poly (D / L-lactic / glycolic acid) copolymer (PLGA), poly (2-aminoethylethylene phosphate) One or more of poly(2-dimethylaminoethyl methacrylate), ...

[0155] The small interfering nucleic acid pharmaceutical composition of the present invention may also contain other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds conventionally used in the art. For example, other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator. The pH buffer may be a tris (hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5-8.5 and / or a phosphate buffer with a pH of 5.5-8.5, preferably a phosphate buffer with a pH of 5.5-8.5. The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The protective agent may be present in an amount of 0.01-30% by weight based on the total weight of the small interfering nucleic acid pharmaceutical composition. The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The amount of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmoles / kg. The amount of the osmotic pressure regulator can be easily determined by a person skilled in the art based on the desired osmotic pressure.

[0156] In some embodiments of the small interfering nucleic acid pharmaceutical composition of the present invention, the small interfering nucleic acid pharmaceutical composition can be a liquid preparation, such as an injection; or it can be a lyophilized powder injection, which is mixed with a liquid excipient during administration to form a liquid preparation. The liquid preparation can be, but is not limited to, for subcutaneous, intramuscular, or intravenous administration, and can also be, but is not limited to, administration by spray to the lungs, or by spray through the lungs to other organs and tissues (such as the liver). Preferably, the small interfering nucleic acid pharmaceutical composition is for intravenous administration.

[0157] In a preferred embodiment of the small interfering nucleic acid pharmaceutical composition of the present invention, the small interfering nucleic acid pharmaceutical composition can be in the form of a lipid nanoparticle formulation. In a more preferred embodiment of the small interfering nucleic acid pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier used in the lipid nanoparticle formulation comprises a cationic lipid, a helper lipid, a structural lipid and / or a PEGylated lipid. Among them, the cationic lipids, helper lipids, structural lipids and pegylated lipids can be selected from the cationic lipid compounds described in CN102119217B, CN112789031B, WO2018170336A1, CN106659803A, WO2021222304A1, WO2022221440A1, US 10,898,574B, US 10,702,600B, CN109331176B, WO2022152109A2, CN114206827A (which are incorporated herein by reference in their entirety), etc., or one or more of their pharmaceutically acceptable salts or derivatives, helper lipids, structural lipids and pegylated lipids.

[0158] Specifically, the cationic lipid may be a compound described in CN102119217B, CN112789031B, WO2018170336A1, CN106659803A, WO2021222304A1, WO2022221440A1, US 10,898,574B, US 10,702,600B, CN109331176B, WO2022152109A2, CN114206827A, etc. and / or a pharmaceutically acceptable salt thereof. In one embodiment, the cationic lipid may be, but is not limited to, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-dilinoleyl-4-(3-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-Dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-Dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K3-DMA), 2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-Dilinoleyl-4-N-methylpiperazinyl-[1,3]-dioxolane (DLin-K3-DMA), 2,2-Dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K3-DMA), 2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6 ...4-(4-dimethylaminobutyl)-[1,3]-dioxolane n-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyl-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyl-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl -3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazin-o)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethyl N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Cho l), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-carboxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-formylamino)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate (DOSPA), dioctadecylaminoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybut-4-oxy)-1-(cis, cis-9,12-octadecadienyloxy)propane (CLinDMA), 2- [5'-(cholest-5-en-3-β-oxy)-3'-oxapentyloxy)-3-dimethyl-1-(cis, cis-9',1-2'-octadienyloxy) propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP) or a mixture thereof. In some embodiments of the small interfering nucleic acid pharmaceutical composition of the present invention, the cationic lipid is 8-[(2-hydroxyethyl)[6-oxo-6(undecyloxy)hexyl]amino]-octanoic acid 1-octylnonyl ester (SM-102).

[0159] Preferably, the helper lipid may be one or more anionic lipids and / or neutral lipids. In certain embodiments, the helper lipid may include one of the following neutral lipid components: (1) cholesterol, cholesterol analogs and / or cholesterol derivatives; (2) phospholipids; or (3) a mixture of phospholipids and cholesterol or its analogs or derivatives. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof. Phospholipid can be a neutral lipid, which includes, but is not limited to, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, di-elaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg yolk phosphatidylcholine (EPC) and mixtures thereof. In some preferred embodiments, phospholipid is DPPC, DSPC or mixtures thereof. In some embodiments, the helper lipid can be a phospholipid.

[0160] The structured lipid may be cholesterol, an analog of cholesterol and / or a derivative of cholesterol.

[0161] The PEGylated lipid is one or more PEG lipids selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. Preferably, the PEGylated lipid can be 1,2-dimyristoyl-rac-glycerol-3-polyethylene glycol-2000 (PEG 2000 -DMG).

[0162] In a more preferred embodiment of the small interfering nucleic acid pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier used simultaneously comprises the four components of the cationic lipid, helper lipid, structural lipid and pegylated lipid described above, and the lipid mixture formed by these four components. In this preferred embodiment, the molar ratio between the cationic lipid, helper lipid, structural lipid and pegylated lipid in the pharmaceutical composition is (19.7-80): (1-40): (0.3-50): (0.1-40). More preferably, the molar ratio between the cationic lipid, helper lipid, structural lipid and pegylated lipid in the small interfering nucleic acid pharmaceutical composition is (50-70): (5-20): (30-60): (0.5-20).

[0163] The lipid nanoparticle particles formed by si1260 and si1848 and the above-mentioned lipid mixture have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, more typically, the average diameter of the lipid nanoparticle particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm or about 70 nm to about 90 nm, for example, the average diameter of the lipid nanoparticle particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.

[0164] In the small interfering nucleic acid pharmaceutical composition in the form of a lipid nanoparticle formulation, the weight ratio of the total weight of si1260 and si1848 to all lipids (e.g., cationic lipids, helper lipids, structural lipids, pegylated lipids) (weight / weight ratio) is in the range of from about 1:1 to about 1:50, from about 1:1 to about 1:40, from about 1:3 to about 1:30, from about 1:4 to about 1:20, from about 1:5 to about 1:18, for example, the weight ratio of the total weight of the first siRNA and the second siRNA to all lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 or 1:18.

[0165] The pharmaceutical composition provided by the present invention can exist independently when each component is sold, and can exist in the form of a liquid preparation when used. The pharmaceutical composition provided by the present invention containing the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods. According to one embodiment of the present invention, the pharmaceutical composition provided by the present invention and the pharmaceutical composition formed by the above-mentioned lipid mixture can be prepared according to the description method in CN102119217B, CN112789031B, WO2018170336A1, CN106659803A, WO2021222304A1, WO2022221440A1, US 10,898,574B, US 10,702,600B, CN109331176B, WO2022152109A2, CN114206827A, etc.; More preferably, it can be prepared according to the following method:

[0166] The cationic lipid, helper lipid, structural lipid, and PEGylated lipid are suspended in alcohol in the above molar ratios and mixed to obtain a lipid solution; the alcohol is used in an amount such that the total mass concentration of the obtained lipid solution is 0.2-25 mg / mL, preferably 1-10 mg / mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid at around room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, tert-butyl alcohol, isopropyl alcohol, dimethyl sulfoxide, glycerol, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400, preferably ethanol.

[0167] The first siRNA and the second siRNA are dissolved in a buffered saline solution to obtain an siRNA aqueous solution. The concentration of the buffered saline solution is 1-500 mM, preferably 1-200 mM. The pH of the buffered saline solution is adjusted to 4.0-6.2, preferably 5.0-5.2. The amount of buffered saline solution used is such that the concentration of the siRNA does not exceed 2 mg / mL, preferably 0.2-0.4 mg / mL. The buffer salt is selected from one or more soluble acetates and soluble citrates, preferably sodium acetate and / or potassium acetate.

[0168] The lipid solution and the siRNA aqueous solution are mixed, and the resulting mixture is incubated at 4-60° C. for at least 2 minutes, preferably 5-30 minutes, to obtain an incubated lipid nanoparticle formulation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1:(1-9), preferably 1:3.

[0169] The incubated lipid nanoparticle preparation is concentrated or diluted, impurities are removed, and sterilization is performed to obtain the pharmaceutical composition provided by the present invention, whose physicochemical parameters are as follows: pH value of 6.5-8, encapsulation efficiency of not less than 80%, particle size of 40-200 nm, polydispersity index of not more than 0.30, and osmotic pressure of 250-400 mOsm / kg; preferably, pH value of 7.2-7.6, encapsulation efficiency of not less than 90%, particle size of 60-100 nm, polydispersity index of not more than 0.20, and osmotic pressure of 300-400 mOsm / kg.

[0170] The concentration or dilution can be performed before, after, or simultaneously with the removal of impurities. Various existing methods can be used to remove impurities, such as using a tangential flow system, a hollow fiber column, and ultrafiltration at 100 kDa, with the ultrafiltration exchange solution being phosphate buffered saline (PBS) at pH 7.4. Various existing methods can be used for sterilization, preferably by filtration on a 0.22 μm filter.

[0171] IV. siRNA Combinations and siRNA Conjugates of the Present Invention

[0172] In one aspect, the present disclosure provides siRNA panels and siRNA conjugates of the present invention that are capable of inhibiting HBV gene expression.

[0173] The siRNA disclosed herein contains a nucleotide group as a basic structural unit. It is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be described in detail here.

[0174] The siRNA disclosed herein contains a sense strand and an antisense strand, and the sense strand and antisense strand are of the same or different lengths. The sense strand is 19-23 nucleotides long, and the antisense strand is 19-26 nucleotides long. Thus, the ratio of the length of the sense and antisense strands of the siRNA provided by the present disclosure can be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 21, 21 / 23, or 23 / 25. The siRNA set of the present invention comprises a first siRNA and a second siRNA.

[0175] First siRNA

[0176] According to the present disclosure, the siRNA may be the first siRNA of the present invention. The first siRNA contains a sense strand and an antisense strand, and each nucleotide in the first siRNA is independently a modified or unmodified nucleotide. In some embodiments, the first siRNA targets PreS / S RNA and intHBV RNA. In some embodiments, the first siRNA is any siRNA in Table 3a and Table 4. In some embodiments, the sense strand of the siRNA contains the sequence 5'-GCCGAUCCAUACUGCGGAA-3' (SEQ ID NO: 1) or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides therefrom, and the antisense strand of the siRNA contains the sequence 5'-UUCCGCAGUAUGGAUCGGC-3' (SEQ ID NO: 2) or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides therefrom. In some embodiments, the sense strand of the siRNA contains the sequence 5'-GUUUACUAGUGCCAUUUGU-3' (SEQ ID NO: 21) or a sequence that is not more than 4 nucleotides different therefrom (e.g., not more than 3, not more than 2, not more than 1), and the antisense strand of the siRNA contains the sequence 5'-ACAAAUGGCACUAGUAAAC-3' (SEQ ID NO: 22) or a sequence that is not more than 4 nucleotides different therefrom (e.g., not more than 3, not more than 2, not more than 1). In some embodiments, the sense strand of the siRNA contains the sequence 5'-GGCUUUCAGUUAUAUGGAU-3' (SEQ ID NO: 23) or a sequence that is not more than 4 nucleotides different therefrom (e.g., not more than 3, not more than 2, not more than 1), and the antisense strand of the siRNA contains the sequence 5'-AUCCAUAUAACUGAAAGCC-3' (SEQ ID NO: 24) or a sequence that is not more than 4 nucleotides different therefrom (e.g., not more than 3, not more than 2, not more than 1).

[0177] In some embodiments, the sense strand comprises only the sequence 5'-GCCGAUCCAUACUGCGGAA-3' (SEQ ID NO: 1), 5'-GUUUACUAGUGCCAUUUGU-3' (SEQ ID NO: 21), or 5'-GGCUUUCAGUUAUAUGGAU-3' (SEQ ID NO: 23), and the antisense strand comprises only the sequence 5'-UUCCGCAGUAUGGAUCGGC-3' (SEQ ID NO: 2), 5'-ACAAAUGGCACUAGUAAAC-3' (SEQ ID NO: 22), or 5'-AUCCAUAUAACUGAAAGCC-3' (SEQ ID NO: 24).

[0178] In some embodiments, the positive strand of the siRNA has no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO: 1, 21 or 23, and / or the antisense strand of the siRNA has no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO: 2, 22 or 24.

[0179] In some embodiments, the nucleotide difference between the antisense strand of the siRNA and the nucleotide sequence shown in SEQ ID NO: 2 includes a difference at the first nucleotide at the 5' end, and the first nucleotide at the 5' end is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at the first nucleotide at the 5' end, and the first nucleotide at the 5' end is selected from A, C or G. In some embodiments, the first nucleotide at the 3' end of the sense strand of the siRNA is a nucleotide complementary to the first nucleotide at the 5' end of the antisense strand of the siRNA. These nucleotide differences do not significantly reduce the target mRNA inhibition ability of the siRNA, and these siRNAs containing nucleotide differences are also within the scope of protection of the present disclosure.

[0180] In some embodiments, the sense strand of the siRNA and the antisense strand of the siRNA are substantially reverse complementary, essentially reverse complementary, or completely reverse complementary. Substantially reverse complementary means that there are no more than three base mismatches between the two nucleotide sequences; substantially reverse complementary means that there are no more than one base mismatch between the two nucleotide sequences; and completely reverse complementary means that there are no mismatches between the two nucleotide sequences.

[0181] In some embodiments, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV are each 1-4 nucleotides in length. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are identical or complementary to the target mRNA. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary. In some embodiments, the nucleotide sequence III is connected to the 5' end of the sense strand, and the nucleotide sequence IV is connected to the 3' end of the antisense strand. In some embodiments, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the nucleotide sequence at the corresponding position of the target mRNA, for example, the nucleotide at the corresponding position of the target mRNA refers to a nucleotide sequence in the target mRNA that is adjacent to the 5' end of the target sequence targeted by the nucleotide sequence represented by, for example, SEQ ID NO: 1, 21 or 23, and has the same length as the nucleotide sequence IV.

[0182] In some embodiments, for the si-1260, a) the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A; in this case, the length ratio of the sense chain to the antisense chain is 20 / 20; or, b) the length of the nucleotide sequence III and IV are both 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is CU, and the base composition of the nucleotide sequence IV is AG; in this case, the length ratio of the sense chain to the antisense chain is 21 / 21; or c) nucleotide sequences III and IV are both 3 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is UCU, and the base composition of nucleotide sequence IV is AGA; in this case, the length ratio of the sense strand to the antisense strand is 22 / 22; or d) nucleotide sequences III and IV are both 4 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is CUCU, and the base composition of nucleotide sequence IV is AGAG; in this case, the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the nucleotide sequences III and nucleotide sequence IV are both 2 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is CU, and the base composition of nucleotide sequence IV is AG; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21.

[0183] In some embodiments, for the si-675, a) the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is U; in this case, the length ratio of the sense chain to the antisense chain is 20 / 20; or, b) the length of the nucleotide sequence III and IV are both 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is CA, and the base composition of the nucleotide sequence IV is UG; in this case, the length ratio of the sense chain to the antisense chain is 21 / 21; or c) nucleotide sequences III and IV are both 3 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is UCA, and the base composition of nucleotide sequence IV is UGA; in this case, the length ratio of the sense strand to the antisense strand is 22 / 22; or d) nucleotide sequences III and IV are both 4 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is CUCA, and the base composition of nucleotide sequence IV is UGAG; in this case, the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the nucleotide sequences III and nucleotide sequence IV are both 2 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is CA, and the base composition of nucleotide sequence IV is UG; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21.

[0184] In some embodiments, for the si-726, a) the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A; in this case, the length ratio of the sense chain to the antisense chain is 20 / 20; or, b) the length of the nucleotide sequence III and IV are both 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is UU, and the base composition of the nucleotide sequence IV is AA; in this case, the length ratio of the sense chain to the antisense chain is 21 / 21; or c) nucleotide sequences III and IV are both 3 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is UUU, and the base composition of nucleotide sequence IV is AAA; in this case, the length ratio of the sense strand to the antisense strand is 22 / 22; or d) nucleotide sequences III and IV are both 4 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is GUUU, and the base composition of nucleotide sequence IV is AAAC; in this case, the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the nucleotide sequences III and nucleotide sequence IV are both 2 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is UU, and the base composition of nucleotide sequence IV is AA; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21.

[0185] In some embodiments, nucleotide sequence III and nucleotide sequence IV are the same length and are completely reverse complementary, so that when the base of nucleotide sequence III is given, the base of nucleotide sequence IV is also determined.

[0186] In some embodiments, the sense strand and the antisense strand are different in length, and the antisense strand further contains a nucleotide sequence V having a length of 1 to 3 nucleotides, which is connected to the 3' end of the antisense strand to form a 3' overhanging end of the antisense strand. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided herein can be 19 / 20, 19 / 21, 19 / 22, 20 / 21, 20 / 22, 20 / 23, 21 / 22, 21 / 23, 21 / 24, 22 / 23, 22 / 24, 22 / 25, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the length of the nucleotide V is 2 nucleotides, thus, the length ratio of the sense strand and the antisense strand of the siRNA provided herein can be 19 / 21, 21 / 23 or 23 / 25.

[0187] Each nucleotide in the nucleotide sequence V can be any nucleotide. In some embodiments, the nucleotide sequence V is two consecutive thymidine deoxyribonucleotides (dTdT) or two consecutive uracil ribonucleotides (UU); in some embodiments, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA.

[0188] In some embodiments, the sense strand of the si1260 is a nucleotide sequence as shown in 5'-GCCGAUCCAUACUGCGGAA-3' (SEQ ID NO: 1), and the antisense strand is a nucleotide sequence as shown in 5'-UUCCGCAGUAUGGAUCGGCAG-3' (SEQ ID NO: 47); or the sense strand of the si1260 is a nucleotide sequence as shown in 5'-CUGCCGAUCCAUACUGCGGAA-3' (SEQ ID NO: 48), and the antisense strand is a nucleotide sequence as shown in 5'-UUCCGCAGUAUGGAUCGGCAGAG-3' (SEQ ID NO: 49).

[0189] As previously described, the nucleotides in the siRNAs of the present disclosure are each independently modified or unmodified. In some embodiments, the nucleotides in the siRNAs of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNAs of the present disclosure are modified nucleotides, and these modifications to the nucleotide groups do not significantly weaken or abolish the ability of the siRNA conjugates of the present disclosure to inhibit hepatitis B virus gene expression.

[0190] In some embodiments, the siRNA provided by the present disclosure contains at least one modified nucleotide; in some embodiments, at least one nucleotide in the sense strand or antisense strand of the siRNA provided by the present disclosure is a modified nucleotide, and / or at least one phosphate group is a phosphate group having a modified group.

[0191] In some embodiments, all nucleotides in the siRNA described herein are modified nucleotides.

[0192] In some embodiments, in the direction from the 5' end to the 3' end, the 7th, 9th, 10th and 11th nucleotides in the siRNA sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the siRNA sense strand are methoxy-modified nucleotides, and in the direction from the 5' end to the 3' end, the 2nd, 6th, 8th, 9th, 14th and 16th nucleotides in the siRNA antisense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the siRNA antisense strand are methoxy-modified nucleotides.

[0193] In some embodiments, the sense strand of si1260 is a nucleotide sequence as shown in 5'-CmUmGmCmCmGmAfUmCfCfAfUmAmCmUmGmCmGmGmAmAm-3', and the antisense strand is a nucleotide sequence as shown in 5'-UmUfCmCmGmCfAmGfUfAmUmGmGmAfUmCfGmGmCmAmGmAmGm-3'. In which, capital letters C, G, U, A, and T represent the base composition of the nucleotide; the lowercase letter m indicates that the ribose group of the nucleotide to the left of the letter m is a 2'-methoxyribose group formed by replacing the 2'-hydroxyl group with a methoxy group; the lowercase letter f indicates that the ribose group of the nucleotide to the left of the letter f is a 2'-fluororibose group formed by replacing the 2'-hydroxyl group with a fluorine group.

[0194] In some embodiments, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA provided herein are phosphate groups with a modified group. In some embodiments, the phosphate group with a modified group is a phosphorothioate group in which at least one oxygen atom in the phosphodiester bond of the phosphate group is replaced by a sulfur atom; in some embodiments, the structure of the phosphorothioate group is shown below:

[0195] These modifications can stabilize the double-stranded structure of siRNA and maintain high specificity and affinity of base pairing.

[0196] In some embodiments, in the siRNA provided herein, the phosphorothioate linkage is present at least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination thereof. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 5' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 3' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at at least one of the following positions:

[0197] between the first and second nucleotides at the 5' end of the sense strand;

[0198] between the second and third nucleotides at the 5' end of the sense strand;

[0199] between the first and second nucleotides at the 3' end of the sense strand;

[0200] between the second and third nucleotides at the 3' end of the sense strand;

[0201] between the first and second nucleotides at the 5' end of the antisense strand;

[0202] between the second and third nucleotides at the 5' end of the antisense strand;

[0203] between the first and second nucleotides at the 3' end of the antisense strand; and

[0204] between the second and third nucleotides at the 3' end of the antisense strand.

[0205] In some embodiments, the sense strand of si1260 is a nucleotide sequence as shown in 5'-CmsUmsGmCmCmGmAfUmCfCfAfUmAmCmUmGmCmGmGmAmAm-3', and the antisense strand is a nucleotide sequence as shown in 5'-UmsUfsCmCmGmCfAmGfUfAmUmGmGmAfUmCfGmGmCmAmGmsAmsGm-3'. Capital letters C, G, U, A, and T represent the base composition of the nucleotide; a lowercase letter m indicates that the ribose group of the nucleotide to the left of the letter m is a 2'-methoxyribose group in which the 2'-hydroxyl group is replaced by a methoxy group; a lowercase letter f indicates that the ribose group of the nucleotide to the left of the letter f is a 2'-fluororibose group in which the 2'-hydroxyl group is replaced by a fluorine group; and a lowercase letter s indicates that the two nucleotides to the left of the letter s are connected by a phosphorothioate group.

[0206] In some embodiments, the 5'-terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide. Commonly used 5'-phosphate nucleotides or 5'-phosphate analog-modified nucleotides are well known to those skilled in the art. For example, a 5'-phosphate nucleotide may have the following structure:

[0207] Wherein, R is selected from H, OH, methoxy, and fluorine; Base represents a nucleic acid base, which is selected from A, U, C, G, or T.

[0208] Or nucleotides with 5'-phosphate analog modifications as disclosed by Anastasia Khvorova, Jonathan K Watts in The chemical evolution of oligonucleotide therapies of clinical utility. Nature biotechnology, 35(3), 238-248.

[0209] In some embodiments, the sense strand of si1260 is a nucleotide sequence as shown in 5'-CmsUmsGmCmCmGmAfUmCfCfAfUmAmCmUmGmCmGmGmAmAm-3', and the antisense strand is a nucleotide sequence as shown in 5'-P1UmsUfsCmCmGmCfAmGfUfAmUmGmGmAfUmCfGmGmCmAmGmsAmsGm-3'. Among them, the capital letters C, G, U, A and T represent the base composition of the nucleotide; the lowercase letter m indicates that the ribose group of the nucleotide to the left of the letter m is a 2'-methoxyribose group in which the 2'-hydroxyl group is replaced by a methoxy group; the lowercase letter f indicates that the ribose group of the nucleotide to the left of the letter f is a 2'-fluororibose group in which the 2'-hydroxyl group is replaced by a fluorine group; the lowercase letter s indicates that the two nucleotides to the left and right of the letter s are connected by a thiophosphate group; P1 indicates that the nucleotide to the right of the letter P is a 5'-phosphate nucleotide or a nucleotide modified by a 5'-phosphate analogue.

[0210] In some embodiments, the si1260 is any one listed in Table 3a.

[0211] Second siRNA

[0212] According to the present disclosure, the siRNA may be a second siRNA. The second siRNA comprises a sense strand and an antisense strand, and each nucleotide in the second siRNA is independently a modified or unmodified nucleotide. In some embodiments, the second siRNA targets all HBV cccDNA-derived RNAs, including HBx. In some embodiments, the second siRNA is any of the siRNAs listed in Table 3b and Table 4. In some embodiments, the sense strand of the second siRNA comprises the sequence 5'-GUUCAUGUCCUACUGUUCA-3' (SEQ ID NO: 5), 5'-GCAUGGAGACCACCGUGAA-3' (SEQ ID NO: 7), 5'-GCGUCGCAGAAGAUCUCAA-3' (SEQ ID NO: 43), or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom, and the antisense strand of the second siRNA comprises the sequence 5'-UGAACAGUAGGACAUGAAC-3' (SEQ ID NO: 6), 5'-UUCACGGUGGUCUCCAUGC-3' (SEQ ID NO: 8), 5'-UUGAGAUCUUCUGCGACGC-3' (SEQ ID NO: 44), or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom.

[0213] In some embodiments, the sense strand comprises only the sequence 5'-GUUCAUGUCCUACUGUUCA-3' (SEQ ID NO: 5), 5'-GCAUGGAGACCACCGUGAA-3' (SEQ ID NO: 7), or 5'-GCGUCGCAGAAGAUCUCAA-3' (SEQ ID NO: 43), and the antisense strand comprises only the sequence 5'-UGAACAGUAGGACAUGAAC-3' (SEQ ID NO: 6), 5'-UUCACGGUGGUCUCCAUGC-3' (SEQ ID NO: 8), or 5'-UUGAGAUCUUCUGCGACGC-3' (SEQ ID NO: 44).

[0214] In some embodiments, the positive strand of the siRNA has no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO: 5, 7 or 43, and / or the antisense strand of the siRNA has no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO: 6, 8 or 44.

[0215] In some embodiments, the nucleotide difference between the antisense strand of the siRNA and the nucleotide sequence shown in SEQ ID NO: 6, 8 or 44 includes a difference at the first nucleotide at the 5' end, and the first nucleotide at the 5' end is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at the first nucleotide at the 5' end, and the first nucleotide at the 5' end is selected from A, C or G. In some embodiments, the first nucleotide at the 3' end of the sense strand of the siRNA is a nucleotide complementary to the first nucleotide at the 5' end of the antisense strand of the siRNA. These nucleotide differences do not significantly reduce the target mRNA inhibition ability of the siRNA, and these siRNAs containing nucleotide differences are also within the scope of protection of the present disclosure.

[0216] In some embodiments, the sense strand of the siRNA and the antisense strand of the siRNA are substantially reverse complementary, essentially reverse complementary, or perfectly reverse complementary.

[0217] In some embodiments, the sense strand further comprises a nucleotide sequence III, and the antisense strand further comprises a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV are each 1-4 nucleotides in length. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are identical or complementary to the target mRNA. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary. In some embodiments, the nucleotide sequence III is connected to the 5' end of the sense strand, and the nucleotide sequence IV is connected to the 3' end of the antisense strand. In some embodiments, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to the nucleotide sequence at the corresponding position of the target mRNA, for example, the nucleotide at the corresponding position of the target mRNA refers to a nucleotide sequence in the target mRNA that is adjacent to the 5' end of the target sequence targeted by the nucleotide sequence represented by, for example, SEQ ID NO: 5, 7 or 43, and has the same length as the nucleotide sequence IV.

[0218] In some embodiments, for the si-1848, a) the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A; in this case, the length ratio of the sense strand to the antisense strand is 20 / 20; or, b) the length of the nucleotide sequence III and IV are both 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is UU, and the base composition of the nucleotide sequence IV is AA; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21; or c) nucleotide sequences III and IV are both 3 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is CUU, and the base composition of nucleotide sequence IV is AAG; in this case, the length ratio of the sense strand to the antisense strand is 22 / 22; or d) nucleotide sequences III and IV are both 4 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is UCUU, and the base composition of nucleotide sequence IV is AAGA; in this case, the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the nucleotide sequences III and nucleotide sequence IV are both 2 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is UU, and the base composition of nucleotide sequence IV is AA; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21.

[0219] In some embodiments, for the si-1606, a) the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is C, and the base of the nucleotide sequence IV is G; in this case, the length ratio of the sense chain to the antisense chain is 20 / 20; or, b) the length of the nucleotide sequence III and IV are both 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is UC, and the base composition of the nucleotide sequence IV is GA; in this case, the length ratio of the sense chain to the antisense chain is 21 / 21; or c) nucleotide sequences III and IV are both 3 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is GUC, and the base composition of nucleotide sequence IV is GAC; in this case, the length ratio of the sense strand to the antisense strand is 22 / 22; or d) nucleotide sequences III and IV are both 4 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is CGUC, and the base composition of nucleotide sequence IV is GACG; in this case, the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the nucleotide sequences III and nucleotide sequence IV are both 2 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is UC, and the base composition of nucleotide sequence IV is GA; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21.

[0220] In some embodiments, for the si-2413, a) the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is C, and the base of the nucleotide sequence IV is G; in this case, the length ratio of the sense strand to the antisense strand is 20 / 20; or, b) the length of the nucleotide sequence III and IV are both 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is CC, and the base composition of the nucleotide sequence IV is GG; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21; or c) nucleotide sequences III and IV are both 3 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is GCC, and the base composition of nucleotide sequence IV is GGC; in this case, the length ratio of the sense strand to the antisense strand is 22 / 22; or d) nucleotide sequences III and IV are both 4 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is CGCC, and the base composition of nucleotide sequence IV is GGCG; in this case, the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the nucleotide sequences III and nucleotide sequence IV are both 2 nucleotides in length, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is CC, and the base composition of nucleotide sequence IV is GG; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21.

[0221] In some embodiments, nucleotide sequence III and nucleotide sequence IV are the same length and are completely reverse complementary, so that when the base of nucleotide sequence III is given, the base of nucleotide sequence IV is also determined.

[0222] In some embodiments, the sense strand and the antisense strand are different in length, and the antisense strand further contains a nucleotide sequence V having a length of 1 to 3 nucleotides, which is connected to the 3' end of the antisense strand to form a 3' overhanging end of the antisense strand. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided herein can be 19 / 20, 19 / 21, 19 / 22, 20 / 21, 20 / 22, 20 / 23, 21 / 22, 21 / 23, 21 / 24, 22 / 23, 22 / 24, 22 / 25, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the length of the nucleotide V is 2 nucleotides, thus, the length ratio of the sense strand and the antisense strand of the siRNA provided herein can be 19 / 21, 21 / 23 or 23 / 25.

[0223] Each nucleotide in the nucleotide sequence V can be any nucleotide. In some embodiments, the nucleotide sequence V is two consecutive thymidine deoxyribonucleotides (dTdT) or two consecutive uracil nucleotides (UU); in some embodiments, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA.

[0224] In some embodiments, the sense strand of si1848 is a nucleotide sequence as shown in 5'-GUUCAUGUCCUACUGUUCA-3' (SEQ ID NO: 5), and the antisense strand is a nucleotide sequence as shown in 5'-UGAACAGUAGGACAUGAACAA-3' (SEQ ID NO: 50); or the sense strand of si1848 is a nucleotide sequence as shown in 5'-UUGUUCAUGUCCUACUGUUCA-3' (SEQ ID NO: 51), and the antisense strand is a nucleotide sequence as shown in 5'-UGAACAGUAGGACAUGAACAAGA-3' (SEQ ID NO: 52).

[0225] As previously described, the nucleotides in the siRNAs of the present disclosure are each independently modified or unmodified. In some embodiments, the nucleotides in the siRNAs of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNAs of the present disclosure are modified nucleotides, and these modifications to the nucleotide groups do not significantly weaken or abolish the ability of the siRNA conjugates of the present disclosure to inhibit hepatitis B virus gene expression.

[0226] In some embodiments, the siRNA provided by the present disclosure contains at least one modified nucleotide; in some embodiments, at least one nucleotide in the sense strand or antisense strand of the siRNA provided by the present disclosure is a modified nucleotide, and / or at least one phosphate group is a phosphate group having a modified group.

[0227] In some embodiments, all nucleotides in the siRNA described herein are modified nucleotides.

[0228] In some embodiments, in the direction from the 5' end to the 3' end, the 7th, 9th, 10th and 11th nucleotides in the siRNA sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the siRNA sense strand are methoxy-modified nucleotides, and in the direction from the 5' end to the 3' end, the 2nd, 6th, 8th, 9th, 14th and 16th nucleotides in the siRNA antisense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the siRNA antisense strand are methoxy-modified nucleotides.

[0229] In some embodiments, the sense strand of si1848 is a nucleotide sequence as shown in 5'-UmUmGmUmUmCmAfUmGfUfCfCmUmAmCmUmGmUmUmCmAm-3', and the antisense strand is a nucleotide sequence as shown in 5'-UmGfAmAmCmAfGmUfAfGmGmAmCmAfUmGfAmAmCmAmAmGmAm-3'. Capital letters C, G, U, A, and T represent the base composition of the nucleotide; a lowercase letter m indicates that the ribose group of the nucleotide to the left of the letter m is a 2'-methoxyribose group in which the 2'-hydroxyl group is replaced by a methoxy group; and a lowercase letter f indicates that the ribose group of the nucleotide to the left of the letter f is a 2'-fluororibose group in which the 2'-hydroxyl group is replaced by a fluorine group.

[0230] In some embodiments, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA provided herein are phosphate groups with a modified group. In some embodiments, the phosphate group with a modified group is a phosphorothioate group in which at least one oxygen atom in the phosphodiester bond of the phosphate group is replaced by a sulfur atom; in some embodiments, the structure of the phosphorothioate group is shown below:

[0231] These modifications can stabilize the double-stranded structure of siRNA and maintain high specificity and affinity of base pairing.

[0232] In some embodiments, in the siRNA provided herein, the phosphorothioate linkage is present at least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination thereof. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 5' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 3' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at at least one of the following positions:

[0233] between the first and second nucleotides at the 5' end of the sense strand;

[0234] between the second and third nucleotides at the 5' end of the sense strand;

[0235] between the first and second nucleotides at the 3' end of the sense strand;

[0236] between the second and third nucleotides at the 3' end of the sense strand;

[0237] between the first and second nucleotides at the 5' end of the antisense strand;

[0238] between the second and third nucleotides at the 5' end of the antisense strand;

[0239] between the first and second nucleotides at the 3' end of the antisense strand; and

[0240] between the second and third nucleotides at the 3' end of the antisense strand.

[0241] In some embodiments, the sense strand of si1848 is a nucleotide sequence such as 5'-UmsUmsGmUmUmCmAfUmGfUfCfCmUmAmCmUmGmUmUmCmAm-3', and the antisense strand is a nucleotide sequence such as 5'-UmsGfsAmAmCmAfGmUfAfGmGmAmCmAfUmGfAmAmCmAmAmsGmsAm-3'. Capital letters C, G, U, A, and T represent the base composition of the nucleotides; a lowercase letter m indicates that the ribose group of the nucleotide to the left of the letter m is a 2'-methoxyribose group in which the 2'-hydroxyl group is replaced by a methoxy group; a lowercase letter f indicates that the ribose group of the nucleotide to the left of the letter f is a 2'-fluororibose group in which the 2'-hydroxyl group is replaced by a fluorine group; and a lowercase letter s indicates that the two nucleotides to the left of the letter s are connected by a phosphorothioate group.

[0242] In some embodiments, the 5'-terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide. Commonly used 5'-phosphate nucleotides or 5'-phosphate analog-modified nucleotides are well known to those skilled in the art. For example, a 5'-phosphate nucleotide may have the following structure:

[0243] Wherein, R is selected from H, OH, methoxy, and fluorine; Base represents a nucleic acid base, which is selected from A, U, C, G, or T.

[0244] Or nucleotides with 5'-phosphate analog modifications as disclosed by Anastasia Khvorova, Jonathan K Watts in The chemical evolution of oligonucleotide therapies of clinical utility. Nature biotechnology, 35(3), 238-248.

[0245] In some embodiments, the sense strand of si1848 is a nucleotide sequence as shown in 5'-UmsUmsGmUmUmCmAfUmGfUfCfCmUmAmCmUmGmUmUmCmAm-3', and the antisense strand is a nucleotide sequence as shown in 5'-P1UmsGfsAmAmCmAfGmUfAfGmGmAmCmAfUmGfAmAmCmAmAmsGmsAm-3'. Among them, the capital letters C, G, U, A and T represent the base composition of the nucleotide; the lowercase letter m indicates that the ribose group of the nucleotide to the left of the letter m is a 2'-methoxyribose group in which the 2'-hydroxyl group is replaced by a methoxy group; the lowercase letter f indicates that the ribose group of the nucleotide to the left of the letter f is a 2'-fluororibose group in which the 2'-hydroxyl group is replaced by a fluorine group; the lowercase letter s indicates that the two nucleotides to the left and right of the letter s are connected by a thiophosphate group; P1 indicates that the nucleotide to the right of the letter P is a 5'-phosphate nucleotide or a nucleotide modified by a 5'-phosphate analogue.

[0246] In some embodiments, the si1848 is any one listed in Table 3b.

[0247] The inventors of the present disclosure unexpectedly discovered that the siRNA conjugates disclosed herein exhibit significantly improved plasma stability and reduced off-target effects while also demonstrating no significant reduction in HBV mRNA silencing activity. In some embodiments, the siRNAs disclosed herein can be selected from the siRNAs listed in Tables 3a-3b, or any combination thereof. siRNA conjugates containing these siRNAs exhibit even higher HBV mRNA silencing activity.

[0248] Table 3a The first siRNA sequence of the present disclosure

[0249] Table 3b Second siRNA sequences disclosed herein

[0250] Note: Capital letters C, G, U, and A represent the base composition of a nucleotide; lowercase letter m indicates that the nucleotide to the left of m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide to the left of f is a fluorinated-modified nucleotide; lowercase letter s indicates that the nucleotides to the left and right of the letter are linked by a phosphorothioate group; P1 indicates that the nucleotide to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide. In some embodiments, P1 is VP, Ps, or P, indicating a specific modification, wherein the letter combination VP indicates that the nucleotide to the right of the letter combination VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP)-modified nucleotide, the letter combination Ps indicates that the nucleotide to the right of the letter combination Ps is a phosphorothioate-modified nucleotide, and the capital letter P indicates that the nucleotide to the right of the letter P is a 5'-phosphate nucleotide.

[0251] The siRNA provided by the present disclosure can be obtained by conventional siRNA preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis has commercial customization services. Modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using methods with corresponding modified nucleotide monomers. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.

[0252] The present disclosure provides an siRNA conjugate, which contains the above-mentioned siRNA and a conjugation group conjugated to the siRNA.

[0253] In general, the conjugated group comprises at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA, the linker and the targeting group are connected in sequence. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The siRNA molecule can be non-covalently or covalently conjugated to the conjugated group, for example, it can be covalently conjugated to the conjugated group. The conjugation site of siRNA and conjugated group can be at the 3' end or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and conjugated group is at the 3' end of the siRNA sense strand.

[0254] In some embodiments, the conjugate group can be connected to the phosphate group, 2'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugate group can also be connected to the 3'-hydroxyl group, in which case the nucleotides are connected by a 2'-5' phosphodiester bond. When the conjugate group is connected to the end of the siRNA chain, the conjugate group is usually connected to the phosphate group of the nucleotide; when the conjugate group is connected to the internal sequence of the siRNA, the conjugate group is usually connected to the ribose sugar ring or the base. Various connection methods can be referred to in the literature: Muthiah Manoharan et al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosid es elicit ro bust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7.

[0255] In some embodiments, the siRNA and conjugated group can be linked via acid-labile or reducible chemical bonds. These bonds can degrade in the acidic environment of endosomes, freeing the siRNA. For non-degradable conjugations, the conjugated group can be attached to the sense strand of the siRNA to minimize the effect of conjugation on siRNA activity.

[0256] In some embodiments, the pharmaceutically acceptable targeting group can be a ligand conventionally used in the field of siRNA administration, such as WO2009082607A2; Kolhatkar et al., Curr Drug Discov Technol. 20118:197-206; Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci U SA. 2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133 (all of which are herein incorporated by reference in their entirety).

[0257] In some embodiments, the pharmaceutically acceptable targeting group can be selected from one or more of the ligands formed by the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acid, vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by hepatocytes, such as asialoglycoproteins, asialosugar residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.

[0258] In some embodiments, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to an asialoglycoprotein receptor (ASGPR) on the liver surface. The types of these ligands are well known to those skilled in the art, and their function is generally to bind to specific receptors on the surface of target cells, mediating the delivery of siRNA connected to the ligand to the target cells.

[0259] In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently an asialoglycoprotein, such as asialo serum mucin (ASOR) or asialo fetuin (ASF). In some embodiments, the ligand is a sugar or a sugar derivative.

[0260] In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or a monosaccharide derivative. In some embodiments, each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.

[0261] In some embodiments, each of the ligands can be independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucose, β-D-glucose, Sugar, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine , 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2, 3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside heptaside ethyl ester, 2,5-anhydro-D-allose nitrile, ribose, D-ribose, D-4-thioribose, L-ribose or L-4-thioribose. Other selections of the ligand can be found in, for example, the description of CN105378082A, the entire disclosure of which is incorporated herein by reference.

[0262] In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent herein refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate after the siRNA molecule forms the siRNA conjugate with the conjugated group containing the galactose or N-acetylgalactosamine molecule as the targeting group. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described in the present disclosure is conjugated to a conjugated group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described herein is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0263] The targeting group can be connected to the siRNA molecule via a suitable linker. Those skilled in the art can select a suitable linker based on the specific type of the targeting group. These linkers, the type of targeting group, and the mode of connection to the siRNA can be found in the disclosure of WO2015006740A2, which is incorporated herein by reference in its entirety.

[0264] In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be a structure as shown below:

[0265] The siRNA conjugates described above can be synthesized using methods well-described in the prior art. For example, WO2015006740A2 describes in detail methods for preparing various siRNA conjugates. The siRNA conjugates disclosed herein can be obtained using methods well known to those skilled in the art. For example, methods for preparing siRNA conjugates are described in WO2014025805A1, and methods for preparing siRNA conjugates are described by Rajeev et al. in ChemBioChem 2015, 16, 903-908.

[0266] The inventors of the present disclosure unexpectedly discovered that the siRNA conjugates disclosed herein exhibit significantly improved plasma stability and reduced off-target effects while also demonstrating no significant reduction in HBV mRNA silencing activity. In some embodiments, the siRNA disclosed herein may be one of the siRNAs listed in Tables 3a-3b. siRNA conjugates containing these siRNAs exhibit even higher HBV mRNA silencing activity.

[0267] The siRNA conjugates disclosed herein may also be used in combination with other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds conventionally used in the art. For details, please refer to the above description of the pharmaceutical composition disclosed herein.

[0268] 5. Kit of the present invention

[0269] The present disclosure provides a kit comprising at least one of the siRNA, siRNA set, pharmaceutical composition, and siRNA conjugate of the present disclosure.

[0270] When a siRNA group is included, the first siRNA and the second siRNA may be present alone or as a mixture of the two siRNAs.

[0271] In some embodiments, the kits described herein can provide the modified siRNA in one container. In some embodiments, the kits described herein can include one container providing a pharmaceutically acceptable excipient.

[0272] In some embodiments, the kit may further comprise other ingredients, such as stabilizers or preservatives. In some embodiments, the kits described herein may comprise at least one other therapeutic agent in a container other than the container in which the modified siRNA described herein is provided. In some embodiments, the kit may comprise instructions for mixing the modified siRNA with a pharmaceutically acceptable carrier and / or excipients or other ingredients (if any).

[0273] In the kit of the present disclosure, the siRNA and pharmaceutically acceptable carrier and / or adjuvant and the siRNA, pharmaceutical composition and / or siRNA conjugate, and / or pharmaceutically acceptable adjuvant can be provided in any form, such as liquid form, dried form or lyophilized form. In some embodiments, the siRNA and pharmaceutically acceptable carrier and / or adjuvant and the pharmaceutical composition and / or siRNA conjugate and optional pharmaceutically acceptable adjuvant are substantially pure and / or sterile. In some embodiments, sterile water can be provided in the kit of the present disclosure.

[0274] 6. Uses of the siRNA, pharmaceutical composition, and siRNA conjugates of the present invention

[0275] The present invention also provides the use of the siRNA, siRNA set, pharmaceutical composition, and conjugate described above in the preparation of a medicament for preventing and / or treating hepatitis B. The present invention also provides a method for preventing and / or treating hepatitis B, comprising administering the siRNA, siRNA set, pharmaceutical composition, and conjugate to a patient in need thereof, thereby achieving the purpose of treating hepatitis B through the mechanism of RNA interference.

[0276] As used herein, the term "administration / administration" refers to placing the siRNA, siRNA set, pharmaceutical composition and / or siRNA conjugate of the present invention into the body of a subject by a method or approach that at least partially localizes the siRNA, siRNA set, pharmaceutical composition and / or siRNA conjugate of the present invention to a desired site to produce a desired effect. Routes of administration suitable for the methods of the present invention include local administration and systemic administration. In general, local administration results in the delivery of the siRNA, siRNA set, pharmaceutical composition and / or siRNA conjugate of the present invention to a specific site compared to the entire body of the subject; while systemic administration results in the delivery of the siRNA, siRNA set, pharmaceutical composition and / or siRNA conjugate of the present invention to essentially the entire body of the subject. Considering that the present invention is intended to provide means for preventing and / or treating hepatitis B, a mode of administration that is capable of delivering the drug specifically to the liver is preferred.

[0277] The drug may be administered to a subject by any suitable route known in the art, including but not limited to oral or parenteral routes, including intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including buccal administration and sublingual administration). The frequency of administration may be once or more daily, weekly, every two weeks, every three weeks, every month, every two months, every three months, every six months, or every year.

[0278] The dosage of the siRNA, siRNA group, pharmaceutical composition or siRNA conjugate described in the present disclosure can be a conventional dosage in the art, which can be determined based on various parameters, especially the age, weight and sex of the subject. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determining LD 50 (the dose that is lethal to 50% of the population) and ED 50 (In quantitative response, it refers to the dose that can cause 50% of the maximum response intensity; in qualitative response, it refers to the dose that causes 50% of the experimental subjects to have a positive reaction.) The dose ratio between toxicity and efficacy is the therapeutic index, which can be expressed as LD50. 50 / ED 50The therapeutic index of the siRNA or pharmaceutical composition is preferably expressed as a ratio of 1:1 to 2:1. siRNA or pharmaceutical composition that exhibits a high therapeutic index is preferred. A range of human dosage can be derived based on data obtained from cell culture assays and animal studies.

[0279] When administering the siRNA, siRNA group, pharmaceutical composition, and / or siRNA conjugate described in the present disclosure, for example, to male or female C57BL / 6J mice aged 6-12 weeks and weighing 18-25 g, based on the amount of siRNA in the siRNA, pharmaceutical composition or siRNA conjugate: (i) for a pharmaceutical composition formed by siRNA and a pharmaceutically acceptable carrier, the amount of siRNA can be 0.001-50 mg / kg body weight, in some embodiments, 0.01-10 mg / kg body weight, in some embodiments, 0.05-5 mg / kg body weight, and in some embodiments, 0.1-3 mg / kg body weight. (ii) For siRNA conjugates formed by siRNA and pharmaceutically acceptable conjugated molecules, the amount of siRNA used can be 0.001-100 mg / kg body weight, in some embodiments, 0.01-50 mg / kg body weight, in some embodiments, 0.05-20 mg / kg body weight, in other embodiments, 0.1-15 mg / kg body weight, and in other embodiments, 0.1-10 mg / kg body weight.

[0280] In some embodiments, the present disclosure provides a method for inhibiting HBV gene expression in hepatocytes, the method comprising contacting an effective amount of the siRNA, siRNA group, pharmaceutical composition and / or siRNA conjugate disclosed herein with the hepatocytes, introducing the siRNA, siRNA group, pharmaceutical composition and / or siRNA conjugate disclosed herein into the hepatocytes, and achieving the purpose of inhibiting HBV gene expression in the hepatocytes through the mechanism of RNA interference. In some embodiments, the hepatocytes are hepatocytes infected with HBV. In some embodiments, the hepatocytes can be selected from liver cancer cell lines such as SMMC-7721, HepG2, Huh7, or isolated primary liver cells.

[0281] The method provided by the present disclosure is used to suppress HBV gene expression in cells. The siRNA dosage in the siRNA, siRNA groups, pharmaceutical compositions and / or siRNA conjugates of the modification provided is generally such an amount: it is enough to reduce the expression of the target gene and result in an extracellular concentration of 1pM to 1 μM, or 0.01nM to 100nM, or 0.05nM to 50nM or 0.05nM to about 5nM at the target cell surface. The amount required to reach the local concentration will vary with various factors, including delivery method, delivery site, the number of cell layers between the delivery site and the target cell or tissue, delivery route (local or systemic), etc. The concentration at the delivery site can be significantly higher than the concentration at the surface of the target cell or tissue.

[0282] The following specific embodiments illustrate the solutions of the present invention, but are not intended to limit the technical solutions of the present invention. Those skilled in the art should understand that the invention can still be modified or replaced by equivalents, and any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of protection of the present invention.

[0283] Example

[0284] Unless otherwise stated, the materials used in the examples herein were commercially available, and the various specific experimental methods used to perform the experiments were conventional experimental methods in the art or followed the steps and conditions recommended by the manufacturer, and can be routinely determined by those skilled in the art as needed.

[0285] Preparation Example 1: siRNA sequence screening and synthesis

[0286] The siRNA target region of the present invention and its position in the HBV genome are shown in Figure 2A. The siRNA targets the HBV conserved sequence and covers the entire HBV genome. After sequence alignment, the sequence of the above siRNA does not have a homologous sequence greater than 16 bases in length with the genome sequence of humans or mice. According to the siRNA design principles, Guangzhou Ruibo Biotechnology Co., Ltd. (RiboBio) or Shanghai Jima Pharmaceutical Technology Co., Ltd. (GenePharma) directly synthesizes siRNA oligomers. The distribution of the target sequence and target region of siRNA on HBV RNA is shown in Figure 2A; the chemically synthesized siRNA sequences are shown in Table 4.

[0287] Table 4 Chemically synthesized siRNA sequence list

[0288] The siRNA was prepared by annealing equimolar amounts of the sense and antisense strands. Chemically synthesized siRNA was pre-dissolved in RNase-free ddH₂O to prepare a 20 μM stock solution, aliquoted, and stored at -80°C to avoid repeated freeze-thaw cycles. siRNA was transfected alone using Lipofectamine RNAiMAX (Thermo Fisher (Invitrogen), Catalog No. 13778-150), while siRNA and DNA were co-transfected using Lipofectamine 3000 (Thermo Fisher (Invitrogen), Catalog No. L3000075).

[0289] Preparation Example 2: Design and Synthesis of Chemically Modified siRNA

[0290] Guangzhou RiboBio and Shanghai GenePharma were commissioned to synthesize small interfering nucleic acids with modifications as shown in Table 5, A1-A11 and / or B1-B11. An equimolar mixture of the sense and antisense strands was dissolved in an annealing salt solution and then conventionally annealed to form siRNA duplexes.

[0291] Table 5 Chemically modified siRNA sequence list

[0292] Note: Capital letters C, G, U, A and T represent the base composition of nucleotides; lowercase letter d indicates that the nucleotide to the right of the letter d is a deoxyribonucleotide; lowercase letter m indicates that the 2'-hydroxyl group of the ribose group of the nucleotide to the left of the letter m is replaced by a methoxy group; lowercase letter f indicates that the 2'-hydroxyl group of the ribose group of the nucleotide to the left of the letter f is replaced by a fluorine group; lowercase letter s indicates that the phosphodiester bond between two nucleotides is replaced by a phosphorothioate diester bond.

[0293] Preparation Example 3: Preparation and Characterization of Lipid Nanoparticles (LNPs)

[0294] Four lipid compounds (i.e., cationic lipid, helper lipid, structural lipid, and PEGylated lipid) were suspended in ethanol at a molar ratio of 50:10:38.5:3.05 and mixed, resulting in a total mass concentration of 2.0286 mg / mL. The synthesized siRNAs listed in Tables 4 or 5 were dissolved in 6.25 mM sodium acetate (pH 5.2) to a concentration of 0.048 mg / mL and a weight ratio of RNA to LNP of 1:14. (For single-use si1260 and si1848, the siRNA concentration was 0.0483 mg / mL; for combined use of si1260 and si1848, the two siRNAs were mixed in equal proportions, resulting in a concentration of 0.024 mg / mL for each.) The LNP-siRNA formulation was rapidly mixed at a volume ratio of 1:3 between the lipid ethanol solution and the siRNA sodium acetate aqueous solution. Table 6 shows the specific composition of the lipid nanoparticle preparation obtained after mixing.

[0295] Table 6 Composition of LNP-siRNA lipid nanoparticle formulations

[0296] The lipid nanoparticle preparation obtained after mixing (i.e., the composition of cationic lipid, auxiliary lipid, structural lipid and PEGylated lipid with siRNA) was incubated at room temperature (18-25°C) for 5 minutes. Ultrafiltration was performed using an Ultra-15 centrifugal filter device (100 kDa, 15 mL, Catalog No. UFC9100, Millipore). The ultrafiltration exchange solution was PBS, pH 7.4. During ultrafiltration, the preparation was concentrated or diluted to the desired siRNA concentration. The ultrafiltered preparation was sterilized by filtration using a 0.22 μm filter. The resulting pharmaceutical composition, LNP-siRNA, was stored at 4°C until use and tested for relevant physicochemical properties. The test results are shown in Table 7.

[0297] Table 7 Physicochemical parameters of the pharmaceutical composition

[0298] The encapsulation efficiency was detected by RiboGreen method, and the reagents used (Quanti-iT TM RNA Reagent and Kit (Cat. No. R11490) was purchased from Thermo Fisher (Invitrogen). The fluorescence intensity of siRNA in the sample was measured according to the instructions, and the encapsulation efficiency was calculated according to the following formula:

[0299] Encapsulation efficiency = [(fluorescence intensity of the Triton-treated group - fluorescence intensity of the non-Triton-treated group) / fluorescence intensity of the Triton-treated group] × 100%.

[0300] Other physical and chemical parameters are measured using conventional techniques well known to those skilled in the art.

[0301] Preparation Example 4: Design, Synthesis and Characterization of GalNac-siRNA

[0302] The siRNA conjugates of the present disclosure shown in Table 8 were synthesized: L96-si1260-2A2S and L96-si1848-2A2S. These siRNA conjugates contained siRNAs having the sense and antisense strand sequences corresponding to the respective siRNA conjugates in Table 8. The preparation methods differed only in that the sense and antisense strands were synthesized according to the siRNA sense and antisense strand sequences corresponding to the siRNA conjugates in Table 8, respectively.

[0303] After preparation, the molecular weights of the prepared siRNA conjugates were measured. The measured values ​​were consistent with the theoretical values, demonstrating that the synthesized siRNA conjugates were the designed double-stranded nucleic acid sequences with L96 conjugated molecules. The siRNAs contained in the siRNA conjugates had the sequences corresponding to the siRNA conjugates L96-si1260-2A2S and / or L96-si1848-2A2S shown in Table 8.

[0304] Table 8 siRNA conjugates

[0305] Among them, the capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; the lowercase letter s indicates that the two nucleotides to the left and right of the letter s are connected by a thiophosphate group; VP indicates that the nucleotide adjacent to the right of the VP is a 5'-vinyl phosphate-modified nucleotide.

[0306] After the siRNA or siRNA conjugate of the present disclosure is prepared, it is lyophilized to a solid powder and stored for future use. When used, it can be redissolved in a solution of the desired concentration using, for example, water for injection, normal saline (NS), phosphate buffer (PB), or phosphate buffered saline (PBS).

[0307] Experimental Example 1: siRNA sequence screening

[0308] HepG2-HBV / EGFP is a stable cell line constructed in-house using the Sleepy Beauty transposon system to stably integrate multiple copies of the HBV genome (serotype: ayw; genotype: D; GenBank accession number: V01460.1) with an EGFP reporter gene. Normally, it does not express EGFP fluorescent protein. However, after transduction with an adenovirus expressing Cre recombinase (Ad-Cre), it cleaves and circularizes recombinant cccDNA, which expresses EGFP fluorescent protein. This system can be used to indicate the presence and transcription of cccDNA. HepG2-HBV / EGFP cells were cultured in DMEM (Corning, catalog number 10-013-CV) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, catalog number 04-001-1A-US) at 37°C in a 5% CO2 incubator. HepG2-HBV / EGFP cells were plated at 10 5 Cells were seeded into 96-well cell culture plates at a density of 100 cells / well and allowed to adhere overnight in a 37°C, 5% CO2 incubator. After the cells grew to 80-90% confluency, Ad-Cre was added for treatment, and fresh culture medium was replaced after 6 hours. Three days after Ad-Cre transduction, chemically synthesized siRNA was transfected into the cells using Lipofectamine RNAiMAX transfection reagent, and si-NC was used as a negative transfection control. Fresh culture medium was replaced 6 hours after transfection. Three days after siRNA transfection, ELISA kit (Hepatitis B Virus Surface Antigen Detection Kit (Chemiluminescence), Antu Biological Company, Catalog No. 30030301CL01) was used to detect the HBsAg antigen level in the supernatant, and the fully automated high-content imaging analysis system PE Operetta (Perkin Elmer) was used to detect the expression of EGFP green fluorescent protein in the cells.

[0309] HepAD38 is a stable cell line that integrates 1.1 copies of the HBV genome (serotype: ayw, genotype: D). This cell line can regulate the transcription of the integrated HBV genome into HBV pgRNA and subgenomic RNA through tet-off elements, and translate and express various viral proteins. This cell line can also support viral replication and the assembly of complete viral particles. HepAD38 cells were cultured in DMEM medium containing 10% FBS and doxycycline (Sigma, product number D9891). HepAD38 cells were cultured at 10 5Cells were seeded into 96-well cell culture plates at a density of 100 cells / well and allowed to adhere overnight in a 37°C, 5% CO2 incubator. Culture medium was then replaced with DOX-free medium. Four days after plating, siRNA was transfected into the cells using Lipofectamine RNAiMAX transfection reagent, with si-NC serving as a negative transfection control. Six hours after transfection, fresh medium was replaced. Three days after siRNA transfection, cells were harvested and intracellular viral protein HBc and HBx levels were assessed by Western blotting, and intracellular viral core particle DNA levels were assessed by Southern blotting.

[0310] HepG2-NTCP cells are a cell line that stably overexpresses the HBV-specific receptor NTCP and can support HBV infection and replication throughout its lifecycle. HepG2-NTCP cells were cultured in complete medium supplemented with 2% DMSO (Sigma, Cat. No. D2650) upon plating. After cell attachment, HepG2-NTCP cells were infected with concentrated HBV supernatant from HepAD38 cells (MOI 200) in complete medium supplemented with 2% DMSO and 4% PEG8000 (Sigma, Cat. No. 89510). Sixteen hours after infection, the supernatant was removed, the cells were washed three to six times with PBS, and then cultured in complete medium supplemented with 2% DMSO. On day four post-infection, siRNA was transfected into the cells using Lipofectamine RNAiMAX transfection reagent. si-NC served as a negative transfection control. Fresh medium was replaced six hours after transfection. Three days after siRNA transfection, cells were harvested and the HBeAg antigen level in the supernatant was detected using an ELISA kit (Hepatitis B virus e antigen detection kit (chemiluminescence method), Antu Biotechnology Co., Ltd., Catalog No. 30030303CL01). Total cellular mRNA was extracted using TRIzol reagent (Ambion, Catalog No. 15596018) and reverse transcribed into cDNA using PrimeScriptTM RT reagent Kit with gDNA Eraser Kit (Takara, Catalog No. RR047A). Real-time fluorescence quantitative PCR (Q-PCR) was performed ( Premix Ex Taq™ (Tli RNaseH Plus, Bulk, Takara, Cat. No. RR420L) was used to detect intracellular HBV pgRNA, HBV total RNA, and the housekeeping gene GAPDH mRNA levels. The primers used for Q-PCR are shown in Table 9.

[0311] Table 9 Q-PCR primer sequence list

[0312] PLC / PRF / 5 cells are a clinically isolated and constructed human hepatocellular carcinoma cell line containing HBV DNA integration. They can be used to evaluate the interference effect of siRNA targeting mRNA derived from HBV integrated DNA (intHBV). PLC / PRF / 5 cells are cultured in RIPM 1640 (Corning, Cat. No. 10-040-CV) medium containing 10% FBS. PLC / PRF / 5 cells are cultured at 10 5 Cells were seeded into 96-well cell culture plates at a density of 100 cells / well. After the cells adhered overnight, siRNA was transfected into the cells using Lipofectamine RNAiMAX transfection reagent, with si-NC serving as a negative transfection control. Fresh culture medium was replaced 6 hours after transfection. Three days after siRNA transfection, cells were harvested, and HBsAg levels in the supernatant were assayed using an ELISA kit (Antu Biotechnology).

[0313] Follow the transfection reagent instructions for siRNA transfection. For one well of a 12-well plate, mix 2.5 μL of siRNA (20 μM) (50 pmol) with 50 μL of Opti-MEM (Gibco, Cat. No. 31985-070) and let it sit at room temperature for 5 minutes. Simultaneously, mix 5 μL of Lipofectamine RNAiMAX transfection reagent with 50 μL of Opti-MEM and let it sit at room temperature for 5 minutes. Combine the two solutions and let it sit at room temperature for 5-10 minutes. Slowly add the transfection reagent dropwise to the wells of the plate to a final concentration of 50 nM. Replace the culture medium with fresh medium 6 hours after transfection.

[0314] As shown in Figure 2B, the experimental results showed that all 20 designed and synthesized siRNAs inhibited EGFP green fluorescent protein expression in HepG2-HBV / EGFP cells to varying degrees. Considering that EGFP is expressed by HBV pgRNA, these results suggest that these siRNAs effectively interfere with HBV pgRNA levels. Compared with the control cells transfected with si-NC, siRNAs si-220 to si-1912 significantly reduced HBsAg expression, with inhibition rates exceeding 80%. However, siRNAs si-2303 to si-2831 failed to reduce HBsAg expression. Comparing the distribution of siRNA target sequences and target regions on HBV RNA in Figure 2A, si-2303 to si-2831 exclusively targeted HBV pgRNA and failed to target the PreS / S mRNA encoding HBsAg. This result is consistent with theoretical expectations.

[0315] All of the above siRNAs effectively inhibited HBV e antigen (HBeAg) secretion in the HepG2-NTCP infection system and significantly suppressed intracellular HBV pgRNA levels. Among them, si-220 to si-1912 significantly suppressed intracellular HBV total RNA levels, while si-2303 to si-2831 failed to reduce HBV total RNA levels. Comparing the distribution of siRNA target sequences and target regions on HBV RNA in Figure 2A, it can be seen that si-2303 to si-2831 only target HBV pgRNA, not PreS / S mRNA and X mRNA, and therefore failed to reduce total HBV RNA levels. This result is consistent with theoretical expectations (Figure 2C).

[0316] All of the above siRNAs inhibited HBc and HBx expression in HepAD38 cells to varying degrees, with si-675 to si-1606 showing the most significant inhibitory effects. Considering that si-675 to si-1260 do not directly target the 0.7 kb X mRNA, it is speculated that this is due to interference with HBV pgRNA, leading to decreased viral replication. Southern blot analysis also showed that all of the above siRNAs significantly inhibited viral replication in HepAD38 cells (Figure 2D). si-220 to si-1260 significantly inhibited HBsAg antigen secretion in the supernatant of PLC / PRF / 5 cells, while si-1606 to si-2831 failed to inhibit HBV surface antigen levels, suggesting that these siRNAs do not target HBV integrated (intHBV)-derived mRNA sequences (Figure 2E).

[0317] These results suggest that among the 20 siRNAs, siRNAs si-220 to si-1260 effectively inhibited the expression of HBV cccDNA and HBV integration-derived HBsAg antigens. However, based on the distribution of their target sequences and target regions on HBV RNA, these siRNAs were unable to effectively target X mRNA. SiRNAs si-1606 to si-2831 were also unable to target HBV integration. Based on the distribution of their target sequences and target regions on HBV RNA, siRNAs si1606 to si1912 simultaneously targeted the consensus sequence of HBV cccDNA-derived mRNAs, while siRNAs si-2303 to si-2831 only targeted HBV pgRNA, failing to target PreS / S mRNA and X mRNA, and failing to target HBV integration. Therefore, a combination of two or more siRNAs is necessary to achieve simultaneous targeting of HBV cccDNA and all HBV-related mRNAs derived from HBV integration.

[0318] Experimental Example 2: Broad-spectrum screening of siRNA sequences

[0319] HepG2 cells were transfected with a replicon plasmid (pHBV1.3) carrying 1.3 copies of the HBV genome sequence of four different genotypes, A, B, C, and D, to verify the targeted interference effect of the above siRNA on different HBV genotypes. HepG2 cells (purchased from ATCC) were cultured in DMEM medium containing 10% FBS. HepG2 cells were plated at 10 5 Cells were seeded into 96-well cell culture plates at a density of 100 cells / well. After adherence, pHBV1.3 plasmid and siRNA were co-transfected into the cells using Lipofectamine 3000 transfection preparation. si-NC was used as a negative control. On the third day after transfection, cell culture supernatants were collected and the levels of HBV antigen in the supernatants were detected using an ELISA kit (Antu Biotechnology). Total cellular RNA was extracted and reverse transcribed into cDNA using the PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara, Cat. No. RR047A). Q-PCR was used to detect the levels of HBV pgRNA, total RNA, and housekeeping gene GAPDH mRNA in the cells.

[0320] As shown in Figures 3A-D, the experimental results show that among the above-mentioned siRNAs, si-220, si-675, si-726, si-1260, si-1606, si-1848, si-1912, and si-2413 have good interference effects on different HBV genotypes, including the four genotypes A / B / C / D, and have a good interference effect in the human primary hepatocyte HBV infection model (Figure 3E).

[0321] The conservation of the aforementioned siRNAs targeting different HBV genotypes was further analyzed. The proportion of 19-mer sequences that fully matched the siRNA target sequence in 11,185 HBV genomes was used as an indicator of conservation. The proportion of 17-mer sequences that fully matched nucleotides 2-18 of the siRNA target sequence was also calculated. The results, as shown in Table 10 below, show that although si-255 showed strong conservation (19-mer: 94.14%; 17-mer: 98.42%), its interference effect was suboptimal in previous pHBV1.3 transfection models and human primary hepatocyte HBV infection models. While si-1912 interfered with all HBV cccDNA-derived RNA consensus sequences and significantly suppressed HBV antigen and HBV RNA levels, its conservation was less than ideal (19-mer: 32.01%; 17-mer: 37.66%). Finally, we selected siRNAs with high conservation (>70%) and good interference effects for subsequent studies, including si-675 (19-mer: 90.43%; 17-mer: 91.01%), si-726 (19-mer: 77.24%; 17-mer: 77.56%), si-1260 (19-mer: 91.42%; 17-mer: 92.96%), si-1606 (19-mer: 79.32%; 17-mer: 79.77%), si-1848 (19-mer: 71.94%; 17-mer: 72.00%), and si-2413 (19-mer: 90.02%; 17-mer: 90.36%). The conservation of two ARC-520 siRNAs was also calculated, including si74 (19-mer: 71.49%; 17-mer: 85.86%) and si77 (19-mer: 82.22%; 17-mer: 86.03%).

[0322] Table 10 Conservation analysis of siRNA targeting HBV genome sequences

[0323] Experimental Example 3: siRNA sequence combination screening

[0324] Previous studies have shown that none of the aforementioned single siRNAs can simultaneously target the consensus sequences of HBV integration-derived and all HBV cccDNA-derived RNAs. Among them, si-675, si-726, and si-1260 target all HBV RNAs and HBV integration-derived (intHBV) sequences except HBx; si-1606 and si-1848 target all HBV RNAs, including HBx, but not sequences derived from intHBV; and si-2413 only targets interfering PreC RNA / pgRNA. Therefore, a combination of two siRNAs is necessary to achieve targeting of all forms of HBV cccDNA and HBV integration-derived RNA while also targeting a wider range of HBV gene sequences. The proportion of sequences targeted by the two siRNA combinations across different HBV genotypes was further calculated. As shown in Table 11 below, ARC-520 (si74 + si77) targets less than 90% (19-mer) of HBV gene sequences. Combinations with >95% conserved (19-mer) sequences will be selected for further validation.

[0325] Table 11 Conservation analysis of siRNA combinations targeting HBV genome sequences

[0326] HepG2 cells were transfected with 1.3 copies of the replicon plasmid of different genotype HBV genome sequence (pHBV1.3) to verify the targeted interference effect of the above siRNA. HepG2 cells were cultured in DMEM medium containing 10% FBS. 5 Cells were seeded into 96-well cell culture plates at a density of 100 cells / well. After adherence, pHBV1.3 plasmid and siRNA were co-transfected into the cells using Lipofectamine 3000 transfection preparation. si-NC was used as a negative control. On the third day after transfection, cell culture supernatants were collected and the levels of HBV antigen in the supernatants were detected using an ELISA kit (Antu Biotechnology). Total cellular RNA was extracted and reverse transcribed into cDNA using the PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara, Cat. No. RR047A). Q-PCR was used to detect the levels of HBV pgRNA, total RNA, and housekeeping gene GAPDH mRNA in the cells.

[0327] The siRNA concentration gradient was further transfected and its IC50 value was calculated. The results are shown in Figure 4: si-1260 (IC 50 0.2084 nM) and si-77 (IC 50 0.1235nM) has a lower IC 50Value, si-726 (IC 50 is 12.11nM), si-1606 (IC 50 12.19 nM) and si-1848 (IC 50 11.54) had a moderate inhibitory effect, while si-675 (IC 50 30.68 nM) and si74 (IC 50 The inhibitory effect of the siRNA was 31.03 nM, which was poorer than that of other siRNAs (Figure 4A). Two of the above siRNAs were further combined, and the siRNA sequence group with a sequence conservation of >90% was selected for antiviral effect verification. The concentration ratio of the two siRNAs was 1:1 (the concentration of each single siRNA was 25 nM, and the concentration of the two siRNA sequence group was 50 nM). The antiviral effect was compared with that of a single siRNA (single siRNA concentrations of 25 nM and 50 nM). The test was carried out in the pHBV1.3 transfection model (genotype D), HepAD38 stable transfectant, PLC / PRF / 5 virus-integrated cell line, and HepG2-NTCP virus infection model. Results showed that combining a siRNA targeting both PreS / S RNA and intHBV RNA (si-675, si-726, si-1260) with a siRNA targeting all HBV cccDNA-derived RNAs, including HBx (si-1606, si-1848), simultaneously targeted all HBV RNAs and intHBV RNA. These combinations demonstrated greater than 90% conservation (except for si-726 + si-1848), outperforming ARC-520 (i.e., si74 + si77, which demonstrated less than 90% conservation and failed to target intHBV RNA). The si-1260 + si-1606 (19-mer: 98.03%) and si-1260 + si-1848 (19-mer: 98.55%) combinations demonstrated superior inhibitory efficacy, targeting >98% of the HBV genome sequence (Figures 4B-G).

[0328] In summary, current results demonstrate that combining a single siRNA targeting PreS / S RNA and intHBV RNA (si-675, si-726, si-1260) with a single siRNA targeting all HBV cccDNA-derived RNAs, including HBx (si-1606, si-1848), can achieve simultaneous targeting of all HBV RNAs and intHBV RNA with high conservation. The optimal siRNA combinations, si-1260 + si-1606 (19-mer: 98.03%) or si-1260 + si-1848 (19-mer: 98.55%), simultaneously targeted all HBV RNA forms, including HBx, and intHBV RNA, achieving simultaneous targeting of >98% of HBV genotypes. Compared to ARC-520 (19-mer: 89.82%), which does not target intHBV RNA, these two siRNA combinations offer significant advantages in terms of sequence conservation and targeting of diverse HBV RNA forms. In subsequent experiments, si-1260+si-1848 were selected for further study.

[0329] Experimental Example 4: Verification of the Broad-Spectrum and Multi-Target Effects of siRNA Sequence Combinations

[0330] 1. Multi-target effect evaluation

[0331] HepG2-HBV / EGFP cells were cultured at 5×10 5 Cells were seeded into 24-well cell culture plates at a density of 100 cells / well and allowed to adhere overnight in a 37°C, 5% CO2 incubator. After cells reached 80-90% confluency, Ad-Cre was added for treatment, and fresh medium was replaced 6 hours later. Three days after Ad-Cre transduction, cells were transfected with lipid nanoparticles (LNP-siRNA, prepared according to the method described in Preparation Example 3) encapsulating a test siRNA combination (subsequently referred to as si1260 + si1848). Alternatively, chemically synthesized test siRNA combinations were transfected into cells using Lipofectamine RNAiMAX transfection reagent (RNAiMAX-siRNA). The siRNA transfection procedure followed the transfection reagent manufacturer's instructions. Three days after siRNA transfection, HBV antigen levels in the supernatant were assessed using an ELISA kit (Antu Biotechnology), and intracellular EGFP expression was detected using the PE Operetta (Perkin Elmer) automated high-content imaging analysis system (Figures 5A and B).

[0332] HepAD38 cells were cultured in DMEM medium containing 10% FBS and doxycycline. 5 Cells were seeded into 24-well cell culture plates at a density of 100 cells / well and allowed to adhere overnight in a 37°C, 5% CO2 incubator. The culture medium was then replaced with one that did not contain doxycycline. On the fourth day after plating, lipid nanoparticle formulations (LNP-siRNA) encapsulating the test siRNA combination were transfected into the cells, or the test siRNA combination was transfected into the cells using Lipofectamine RNAiMAX transfection reagent (RNAiMAX-siRNA). Fresh culture medium was replaced 6 hours after transfection. Three days after siRNA transfection, cells were harvested, and the HBV antigen level in the supernatant was detected using an ELISA kit (Antu Biotechnology). The levels of intracellular viral proteins HBc and HBx were detected using Western Blot, and the grayscale values ​​of the bands were statistically analyzed (Figures 5C and D).

[0333] PLC / PRF / 5 cells were plated at 5×10 5 Cells were seeded at a density of 100 cells / well in 24-well cell culture plates. The day after plating, cells were transfected with lipid nanoparticles encapsulating the test siRNA combination (LNP-siRNA) or with Lipofectamine RNAiMAX transfection reagent (RNAiMAX-siRNA). Fresh culture medium was replaced 6 hours after transfection. Three days after siRNA transfection, HBsAg levels in the supernatant were measured using an ELISA kit (Antu Biotechnology) (Figure 5E).

[0334] 2. Broad-spectrum evaluation

[0335] HepG2 cells were cultured in DMEM medium containing 10% FBS. HepG2 cells were plated at 5×10 5 Cells were seeded into 24-well cell culture plates at a density of 100 cells / well. The day after plating, HepG2 cells were transfected with cloned plasmids carrying four different HBV genotypes, A, B, C, and D, to achieve HBV expression and replication. The day after transfection, the test siRNA combination (LNP-siRNA and RNAiMAX-siRNA) was transfected into the cells using the method described in Experimental Example 1. Fresh culture medium was replaced 6 hours after siRNA transfection. Three days after siRNA transfection, HBsAg and HBeAg antigen levels in the supernatant were measured using an ELISA kit (Antu Biotechnology) (Figure 6).

[0336] 3. In vivo and in vitro effectiveness evaluation

[0337] 5C-PHH cells are human primary hepatocytes (PHH) that were constructed in-house and cultured in a medium supplemented with five compounds. They can achieve long-term in vitro culture of PHH and support long-term HBV infection and replication (reference: Long-term functional maintenance of primary human hepatocytes in vitro, Science (New York, NY), 2019, 364(6438), 399-402). 5C-PHH cells were cultured at 2.5×10 5 The cells were inoculated into 24-well cell culture plates at a density of 100 cells / well. The concentrated HBV virus was resuspended in a culture medium containing 4% PEG 8000 the day after plating and incubated with the cells. After 20 hours, the cells were washed 5-6 times with pre-cooled PBS and replaced with fresh culture medium supplemented with the five compounds. On the 7th day after infection, the control siRNA (si-NC) or the test siRNA combination (si-HBV) was transfected into the cells using the method described in Experimental Example 1, and fresh culture medium was replaced 6 hours after siRNA transfection. The cell culture supernatant was collected every 2-3 days and the HBsAg and HBeAg antigen levels in the supernatant were detected using an ELISA kit (Antu Biotechnology), and the HBV DNA content in the supernatant was detected using an HBV nucleic acid quantitative detection kit (PCR-fluorescent probe method) (Shengxiang Biotechnology Co., Ltd.) (Figure 7A).

[0338] The prcccDNA / pCMV-Cre high-pressure hydrodynamic model refers to the injection of the prcccDNA plasmid and the pCMV-Cre plasmid into the mouse's tail vein within 5 to 8 seconds at a total volume of 10% of the mouse's body weight. The prcccDNA plasmid is cut and circularized in the mouse liver under the action of Cre recombinase to form recombinant cccDNA, thereby constructing a recombinant HBV cccDNA mouse model, which can support HBV viral transcription and replication and express various HBV proteins (Reference: Recombinant covalently closed circular DNA of hepatitis B virus induces long-term viral persistence with chronic hepatitis in a mouse model, Hepatology, 2018, 67(1), 56-70). Control siRNA or test siRNA combination was co-administered with the prcccDNA / pCMV-Cre plasmid system (4 μg each) into C57BL / 6 mice via high-pressure tail vein injection. Blood was collected on days 2, 4, and 7 after tail vein injection to detect serum HBV antigen levels. Some mice were sacrificed on days 2 and 7 after tail vein injection to detect intrahepatic HBc protein levels ( Figure 7C ).

[0339] The experimental results showed that the test siRNA combination could effectively inhibit the expression of HBV antigen in HepG2-HBV / EGFP cells, HepAD38 cells, and PLC / PRF / 5 cells in a dose-dependent manner (Figure 5A-D). The test siRNA combination could also dose-dependently inhibit the expression of EGFP fluorescent protein in HepG2-HBV / EGFP cells, and could dose-dependently inhibit the expression of HBc and HBx proteins in HepAD38 cells (Figure 5E).

[0340] The experimental results showed that the tested siRNA combination effectively inhibited HBV antigen expression in HepG2 cells transfected with cloned plasmids of four different HBV genotypes, A, B, C, and D, in a dose-dependent manner. This result demonstrates that the tested siRNA combination has broad coverage across multiple HBV genotypes (Figure 6).

[0341] Experimental results showed that compared with control siRNA, the test siRNA combination potently suppressed HBsAg and HBeAg antigen levels in the culture supernatant of HBV-infected 5C-PHH cells, and this suppression persisted until day 28 of infection (Figures 7A-B). Compared with control siRNA, the test siRNA combination potently suppressed HBsAg and HBeAg antigen levels in the serum of mice induced by prcccDNA / pCMV-Cre high-pressure hydrodynamics in a dose-dependent manner and significantly suppressed intrahepatic HBc antigen levels; however, this inhibitory effect gradually weakened by day 4 and completely disappeared by day 7 (Figures 7C-E). These results suggest that although the test siRNA combination exerts a long-lasting antiviral effect in vitro, unmodified naked test siRNA is easily degraded by nucleases in vivo, making it difficult to maintain a long-term antiviral effect. Therefore, subsequent experiments will utilize modified siRNA combinations and lipid nanoparticle formulations (LNPs) encapsulating the modified test siRNA combination.

[0342] Experimental Example 5: Screening of siRNA sequence chemical modification schemes

[0343] 1. Serum stability test

[0344] Synthesize small interfering nucleic acids with specific chemical modifications as shown in Table 5 of Preparation Example 2. 20 μL of 20 μM small interfering nucleic acid was added to 20 μL of fetal bovine serum and 160 μL of 1× PBS buffer solution (pH 7.4). Samples were taken after incubation at 37°C for a certain period of time, namely 0, 2, 4, 6, 8, 12, 24, and 48 hours. 20 μL of sample was taken each time and quickly frozen in liquid nitrogen to immediately terminate the action of serum nucleases on the small interfering nucleic acid. The sample was then frozen in a -80°C freezer until use. A 2 wt% gel electrophoresis (containing Gel-Red nucleic acid dye) was prepared, 4 μL of 6× loading buffer was mixed with the serum-incubated sample of the small interfering nucleic acid, and then 10 μL was loaded and electrophoresed at a constant voltage of 120 V for 20 minutes. After the electrophoresis, the gel was imaged and photographed (Figure 9).

[0345] 2. Cytotoxicity assay

[0346] HepG2 cells were cultured in DMEM medium containing 10% FBS. 5Cells were seeded into 96-well cell culture plates at a density of 100 cells / well and allowed to adhere overnight in a 37°C, 5% CO2 incubator. siRNA was transfected into the cells using Lipofectamine RNAiMAX transfection reagent, and a blank transfection reagent was used as a negative transfection control. Fresh culture medium was replaced 6 hours after transfection. Three days after siRNA transfection, the cell culture supernatant was replaced with a culture medium containing 10% CCK-8 detection reagent (Cell Counting Kit-8 Cell Proliferation Toxicity Detection Kit, DOJINDO, Cat. No. CK04), and the cells were incubated in a 37°C, 5% CO2 incubator for another 20 minutes. The absorbance value A of each well at a wavelength of 540 nm was measured using a microplate reader, and the cell viability was calculated according to the following formula (Figure 8).

[0347] Cell survival rate (%) = (average A value of experimental group / average A value of control group) × 100%

[0348] 3. Validity testing

[0349] HepG2-HBV / EGFP cells were cultured at 5×10 5 Cells were seeded into 24-well cell culture plates at a density of 100 cells / well and allowed to adhere overnight in a 37°C, 5% CO2 incubator. After cells reached 80-90% confluency, Ad-Cre was added for treatment, and fresh medium was replaced 6 hours later. Three days after Ad-Cre transduction, a test siRNA combination with specific chemical modifications (si-HBV-2m) was transfected into the cells using Lipofectamine RNAiMAX transfection reagent. A specific chemically modified si-NC (si-NC-2m, sense strand: 5'-UmUmCmUmCmCmGAACmGUmGUmCmACmGUmdTdT-3', antisense strand: 5'-ACGUmGACACGUUmCGGAGAAdTdT-3') was used as a negative transfection control. Fresh medium was replaced 6 hours after transfection. Three days after siRNA transfection, HBV antigen levels in the supernatant were measured using an ELISA kit (Antu Biotechnology) (Figure 8).

[0350] The experimental results are shown in Figures 8E and 9. Compared with the unmodified samples, the serum stability of si1260 and si1848 with different chemical modification schemes was significantly increased, and they could be stably present in serum for more than 48 hours. The modified si1260 and si1848 were both safe and had no obvious inhibitory effect on the cell growth curve (Figures 8B, D). Compared with the control cells transfected with si1260 and si1848 without chemical modification, si1260 and si1848 with the above different modification schemes could retain most of the activity of inhibiting HBV target gene expression, and could significantly reduce the expression levels of HBsAg and HBeAg antigens, and were concentration-dependent (Figures 8A, C). By co-encapsulating the modified si1260 and si1848 into LNP (LNP-siHBV-2m) and transfecting HepG2-HBV / EGFP cells, the results showed that compared with the control siNC (LNP-siNC-2m), LNP-siHBV-2m could effectively inhibit the expression of HBV antigen and EGFP reporter protein, and its IC 50 As low as 1.517 nM ( FIG8F , G ).

[0351] In summary, compared to unmodified small interfering nucleic acids, the small interfering nucleic acids provided by the present invention have significantly improved stability, lower cytotoxicity, and can maintain substantially the same biological activity as the unmodified small interfering nucleic acids. Thus, the present invention achieves the goal of increasing the serum stability of modified small interfering nucleic acids by introducing only a small amount of modification, thereby reducing the potential cytotoxicity of the modified small interfering nucleic acid molecules and the impact of the modification on the biological activity of the small interfering nucleic acid.

[0352] Experimental Example 6: In vivo effectiveness evaluation of LNP-siHBV

[0353] 1. Evaluation of the antiviral effect of a single dose of LNP-siHBV

[0354] In this experimental example, a lipid nanoparticle (LNP) formulation (prepared according to the method described in Preparation Example 3) was used to encapsulate a specifically chemically modified test siRNA combination (a combination of si1260-2m and si1848-2m, the corresponding sequences were synthesized according to the description in Preparation Example 2, the ratio of the two was 1:1, and was recorded as siHBV-2m or siHBV). The antiviral effect of this nucleic acid lipid nanoparticle formulation (abbreviated as LNP-siHBV) in a hepatitis B model mouse was evaluated. An LNP formulation (LNP-siNC) encapsulating the modified control siRNA (siNC-2m) described in Experimental Example 5 was used as a negative control at a dose of 1 mg / kg; entecavir (ETV) (MedChemExpress, Catalog No. HY-13623) was used as a positive control. The LNP formulation was administered by tail vein injection, and the positive drug ETV was administered by gavage at a dose of 1 mg / kg, with a dosing frequency of once daily (Figures 10A-D).

[0355] rAAV-HBV1.3 mouse model: rAAV-HBV1.3 model mice were prepared according to the literature (A mouse model for HBV immunotolerance and immunotherapy. Cell Mol Immunol, 2014, 11(1), 71-78). rAAV8-HBV1.3, type D (ayw) virus (purchased from Beijing Wujiahe Molecular Medicine Research Institute Co., Ltd., 1×10 12 viral genome (vg) / mL, Catalog No. AMV-002), diluted with sterile PBS to 1.25×10 11 vg / mL, and each mouse was injected with 200 μL of diluted rAAV8-HBV1.3 (i.e., each mouse was injected with 2.5×10 10 On days 14 to 28 after virus injection, all mice were bled via orbital bleeding for serum detection of HBsAg and HBV DNA.

[0356] First, the inhibitory effects of single-dose administration of different doses of LNP-siHBV on HBV-related indicators in hepatitis B model mice at different time points were tested. After the animal model was successfully established, the hepatitis B model mice were randomly divided into groups (8 per group) according to the HBV antigen expression level, namely: PBS control group, LNP-siNC (1 mg / kg) control group, LNP-siHBV (1 mg / kg) high-dose group, LNP-siHBV (0.3 mg / kg) medium-dose group, LNP-siHBV (0.1 mg / kg) low-dose group and ETV (1 mg / kg) positive drug group. The drug dosage for the animals was calculated based on body weight. The LNP-siNC (1 mg / kg) control group, the LNP-siHBV (1 mg / kg) high-dose group, the LNP-siHBV (0.3 mg / kg) medium-dose group, and the LNP-siHBV (0.1 mg / kg) low-dose group were all administered via tail vein injection as a single dose. The ETV (1 mg / kg) positive drug group was administered daily by gavage at a dose of 1 mg / kg. Blood was collected from the orbital venous plexus of the mice before dosing (day 0) and on days 7, 14, 21, and 28 after dosing, and the supernatant was collected after centrifugation. 2.5 μL of serum was diluted 50-fold with 122.5 μL of sterile water. Serum HBV antigen levels at each time point were measured using a Hepatitis B Virus Surface Antigen Detection Kit (Chemiluminescence Method) (Antu Biotechnology Co., Ltd., Catalog No. 30030301CL01) and a Hepatitis B Virus e Antigen Detection Kit (Chemiluminescence Method) (Antu Biotechnology Co., Ltd., Catalog No. 30030303CL01). Serum HBV DNA levels were measured using an HBV Nucleic Acid Quantification Detection Kit (PCR-Fluorescent Probe Method) (Shengxiang Biotechnology Co., Ltd.) according to the kit instructions. On day 28, the animals were sacrificed, and a portion of the liver tissue was preserved in 10% formalin for further pathological observation. The remaining liver tissue was quickly frozen in liquid nitrogen and stored at −80°C until further use.

[0357] The HBsAg content is expressed as the number of HBsAg equivalents (UI) per milliliter (mL) of serum.

[0358] The HBeAg content is expressed as the number of HBeAg equivalents (PEUI) per milliliter (mL) of serum.

[0359] The HBV DNA content is expressed as the number of equivalents (UI) of HBV DNA per milliliter (mL) of serum.

[0360] The HBsAg expression inhibition rate was calculated according to the following formula:

[0361] HBsAg expression inhibition rate = log10 (HBsAg content in the test group / HBsAg content in the PBS control group)

[0362] The HBeAg expression inhibition rate was calculated according to the following formula:

[0363] HBeAg expression inhibition rate = log10 (HBeAg content in the test group / HBeAg content in the PBS control group)

[0364] The HBV DNA expression inhibition rate was calculated according to the following formula:

[0365] HBV DNA expression inhibition rate = log10 (HBV DNA content in the test group / HBV DNA content in the PBS control group)

[0366] The control group is a control group of mice administered with PBS in this experiment, and each test group is a drug-administered group of mice administered with different drug compositions.

[0367] 2. Evaluation of the antiviral effect of multiple doses of LNP-siHBV

[0368] The inhibitory effects of multiple doses of LNP-siHBV at different doses and frequencies on HBV-related indicators in hepatitis B model mice at different time points were further tested. After the animal model was successfully established, the hepatitis B model mice were randomly divided into groups (8 per group) according to the HBV antigen expression level, namely: PBS control group, LNP-siHBV (1 mg / kg Q1W) high-dose once a week group, LNP-siHBV (1 mg / kg Q2W) high-dose once every two weeks group, LNP-siHBV (0.3 mg / kg Q1W) medium-dose once a week group, and LNP-siHBV (0.3 mg / kg Q2W) medium-dose once every two weeks group. The drug dose was calculated based on the body weight of the animals, and all groups were administered by tail vein injection. Blood was collected from the mice's orbital venous plexus every week before, during, and after dosing, and the supernatant was collected after centrifugation. The serum HBV antigen levels at each time point were detected using the Hepatitis B virus surface antigen detection kit (chemiluminescence method) (Antu Biological Company, Cat. No. 30030301CL01) and the Hepatitis B virus e antigen detection kit (chemiluminescence method) (Antu Biological Company, Cat. No. 30030303CL01). The serum HBV DNA level was detected using the HBV nucleic acid quantitative detection kit (PCR-fluorescent probe method) (Shengxiang Biotechnology Co., Ltd.) according to the kit instructions. On the 49th day, the animals were sacrificed and the animals were grossly dissected. The heart, liver, spleen, lung, and kidney of the mice were observed and preserved in 10% formalin for further pathological observation. The liver was collected, and the liver tissue was aliquoted and quickly frozen in liquid nitrogen and stored at -80°C for later use (Figure 10E-I).

[0369] The results are shown in Figure 10. The experimental results showed that at different time points after administration, the positive drug ETV control group significantly suppressed HBV DNA levels but had no inhibitory effect on HBV antigen. Compared with the LNP-siNC control group, LNP-siHBV dose-dependently suppressed serum HBsAg, HBeAg, and HBV DNA levels. The high-dose LNP-siHBV (1 mg / kg) group demonstrated an inhibition rate of 2.65 log10 (IU / mL) for serum HBsAg, 1.82 log10 (PEIU / mL) for serum HBeAg, and 2.29 log10 (IU / mL) for serum HBV DNA on day 7 after administration. Furthermore, the inhibitory effect of the high-dose LNP-siHBV (1 mg / kg) group was maintained for more than 14 days (inhibition rate >1 log10 (IU / mL)) (Figures 10A-D). At the same time, the results of multiple-dose experimental administration showed that high-dose weekly administration of LNP-siHBV (1 mg / kg Q1W) could maintain extremely low levels of HBsAg, HBeAg antigens, and HBV DNA in mouse serum. After drug withdrawal, the inhibitory effect could be maintained for more than 14 days (inhibition rate >1log10 (IU / mL)), suggesting that it has good clinical application potential (Figure 10E-I).

[0370] Experimental Example 7: Safety evaluation of single and repeated administration of LNP-siHBV

[0371] 1. Safety evaluation of single-dose administration

[0372] Normal C57BL / 6 mice were randomly divided into five groups (n=4 per group): a PBS control group, an LNP-siNC (1 mg / kg) control group, an LNP-siHBV (1 mg / kg) dose group, an LNP-siHBV (2.5 mg / kg) dose group, an LPS (5 mg / kg) positive control group, and a polyI:C (10 mg / kg) positive control group. The LNP-siNC (1 mg / kg) control group, the LNP-siHBV (1 mg / kg) dose group, and the LNP-siHBV (2.5 mg / kg) dose group were all administered via tail vein injection. The LPS (5 mg / kg) (Biyuntian Biotechnology Co., Ltd., Catalog No. S1732) positive control group and the polyI:C (10 mg / kg) (MedChemExpress, Catalog No. HY-107202) positive control group were administered via intraperitoneal injection. The drug dose for all animals was calculated based on body weight, and the volume of the formulation was adjusted with 1× PBS as needed before administration to accommodate the animal's needs. Blood was collected from the orbital venous plexus of mice at 3, 24, and 48 hours after administration. The blood was allowed to clot at room temperature for 2 hours and then centrifuged to collect serum. ELISA kits were used to detect the expression levels of TNF-α (Absin, Catalog No. abs520010-96T), IL-6 (Absin, Catalog No. abs520004-96T), and IFN-γ (Absin, Catalog No. abs520007-96T) cytokines in the serum. Biochemical detection kits (Nanjing Jiancheng Bioengineering Institute) were used to detect the levels of liver and kidney function-related biochemical indicators in the serum, including alanine aminotransferase (ALT) (C009-201), aspartate aminotransferase (AST) (C010-2-1), uric acid (UA) (C012-2-1), and creatinine (CREA) (C011-2-1) (according to the kit instructions). At 48 hours after administration, the mice were killed and their hearts, livers, spleens, lungs, and kidneys were collected for observation, fixed with 10% formalin, and subjected to pathological observation ( FIG11 ).

[0373] 2. Safety evaluation of multiple-dose administration

[0374] For mice treated with multiple doses of the drug in Experimental Example 6, serum levels of liver and kidney function-related biochemical indicators, including ALT, AST, UA, and CREA, were measured using biochemical assay kits (Nanjing Jiancheng Bioengineering Institute). Anti-PEG antibody levels in the serum were measured using ELISA. At the conclusion of the experiment, the animals were sacrificed and subjected to gross autopsy. The heart, liver, spleen, lungs, and kidneys were removed for examination and preserved in 10% formalin for further pathological observation (Figure 12).

[0375] Anti-PEG antibodies in serum are detected as follows:

[0376] i. Add 100 μL of 10 μg / mL PEG-DMG in ethanol (2 μg / well) to the medium-binding ELISA plate and evaporate overnight in a clean bench or fume hood.

[0377] ii. Wash three times with 200 μL of PBS solution containing 0.1% Tween-20, each time for 5 minutes;

[0378] iii. Add 200 μL of 3% bovine serum albumin (BSA) in PBS and block at 37°C for 2 hours. Discard the blocking solution and wash three times with 200 μL of 0.1% Tween-20 in PBS.

[0379] iv. Add serial dilutions of control mouse serum or test mouse serum (initial dilution 10-fold followed by 4-fold serial dilutions), incubate at 37°C for 1 hour, and then wash three times with 200 μL of PBS solution containing 0.1% Tween-20;

[0380] v. Add 100 μL of horseradish peroxidase (HRP)-labeled goat anti-mouse IgM / IgG antibody (1:5000 / 1:500), incubate at 37°C for 1 hour, and then wash four times with 200 μL of PBS solution containing 0.1% Tween-20;

[0381] vi. Add 100 μL of TMB solution and incubate at room temperature for 10 min (IgM) / 40 min (IgG). Add 100 μL of 0.18 M H2SO4 solution to terminate the reaction and measure the absorbance at a wavelength of 450 nm.

[0382] 3. Off-target effect detection in vivo and in vitro

[0383] HepG2 cells were cultured at a rate of 5×10 5 Cells were seeded into 24-well cell culture plates at a density of 100 cells / well. The day after plating, cells were treated with LNP-siHBV or LNP-siNC at a final siRNA concentration of 50 nM. A control group consisted of untreated HepG2 cells. After a 16-hour incubation, the supernatant was discarded, the cells were washed twice with PBS, and total RNA was collected for transcriptome sequencing (Figure 13).

[0384] Normal C57BL / 6 mice were injected with PBS, LNP-siNC (1 mg / kg) and LNP-siHBV (1 mg / kg) via the tail vein. 48 hours after administration, the mice were killed and their liver tissues were obtained for transcriptome sequencing.

[0385] The experimental results are shown in Figures 11 and 12. The experimental results show that LNP-siHBV has low immunogenicity. After a single dose, it does not induce significant TNF-α, IL-6, and IFN-γ cytokine production (Figures 11A-D). After repeated administration, it does not induce a significant increase in the level of anti-PEG antibodies produced by the body (Figures 12A-B); LNP-siHBV has good safety. Both single and repeated administrations do not cause abnormal biochemical indicators related to liver and kidney function (Figures 11E-H, Figures 12C-D), and single and repeated administrations do not cause obvious lesions in various organs of mice (Figures 11I, Figure 12E). According to the transcriptome sequencing analysis results in HepG2 cells and C57BL / 6 mice in Figure 13, LNP-siHBV treatment does not cause significant changes in the host cell transcriptional profile, and only a small number of genes are differentially expressed, suggesting that the RNAi preparation does not cause significant off-target effects (Figures 13A-D). The above results indicate that the LNP-siHBV preparation has good safety and has the potential for further clinical development and application.

[0386] Experimental Example 8: Evaluation of the effectiveness of GalNac-siHBV conjugate

[0387] The siRNA conjugate composition provided by the present disclosure ( FIG. 15A ) (referred to as GalNac-siHBV, which is composed of GalNac-si1260 and GalNac-si1848 mixed in a 1:1 ratio) was subjected to activity assay in hepatitis B model mice; GalNac-si1260 and GalNac-si1848 were prepared according to the method described in Preparation Example 3, and their molecular weights were detected by LC / MS, the purity of the siRNA single strand was detected by HPLC (purity>90%), and the purity of the siRNA double-stranded conjugate was detected by SEC (purity>90%). The results of the SEC test are shown in FIG14 , which show that the purity of GalNac-si1260 is 99.7% and the purity of GalNac-si1848 is 97.04%.

[0388] Two chronic hepatitis B mouse models were used for the experiment. One was a laboratory-built chronic recombinant cccDNA mouse model, namely the rAAV-rcccDNA / rAAV-Cre mouse model: pAAV2-rcccDNA and pAAV2-Cre plasmids were co-transfected with helper plasmids and AAV8 capsid plasmids into HEK293T cells respectively. Three days later, the cells and viruses in the supernatant were collected and concentrated and purified. pAAV2-rcccDNA and pAAV2-Cre plasmids were used for gradient dilution and fluorescence quantitative PCR detection and calculation of viral titers. The rAAV-rcccDNA and rAAV-Cre recombinant viruses were diluted to 2.5×1011 vg / mL and mixed in equal volumes at a ratio of 1:1, and each mouse was injected with 200 μL of diluted rAAV-rcccDNA / rAAV-Cre (i.e., each mouse was injected with 2.5×10 10 vgrAAV-rcccDNA and 2.5×10 10 vgrAAV-Cre). From day 14 to 28 after virus injection, all mice were bled via orbital sampling for serum detection of HBsAg and HBV DNA. The model mice were grouped according to the antigen level and given GalNac-si1260 (2.5 mg / kg), GalNac-si1848 (2.5 mg / kg) and GalNac-siHBV (GalNac-si1260 and GalNac-si1848 were mixed in a 1:1 mass ratio, each dose was 1.25 mg / kg, and the total dose was 2.5 mg / kg). Blood was collected before administration (day 0) and on day 7 after administration to detect the levels of HBsAg and HBeAg antigens in the serum (Figure 15B-C).

[0389] Another model is the rAAV-HBV1.3 mouse model, which was prepared according to the method described in Experimental Example 6. From day 14 to 28 after virus injection, all mice were bled by orbital sampling for serum detection of HBsAg and HBV DNA. According to the HBV antigen expression level, the hepatitis B model mice were randomly divided into groups (5 per group): PBS control group, LNP-siHBV (1 mg / kg) control group, ETV (1 mg / kg) positive drug group, GalNac-siHBV (2.5 mg / kg) high-dose group, GalNac-siHBV (1 mg / kg) medium-dose group and GalNac-siHBV (0.2 mg / kg) low-dose group. LNP / siHBV (abbreviated as LNP-siHBV) as described in Preparation Example 3 and Experimental Example 6 was used as a control; entecavir (ETV) was used as a positive control. The LNP-siHBV formulation was administered once via tail vein injection at a dose of 1 mg / kg. The positive drug ETV was administered via oral gavage at a dose of 1 mg / kg, with a dosing frequency of once daily. The siRNA conjugate was administered once via subcutaneous injection at doses of 0.2 mg / kg, 1 mg / kg, and 2.5 mg / kg. Blood was collected before dosing (day 0) and weekly thereafter to measure HBsAg, HBeAg, and HBV DNA levels (Figures 15D-E).

[0390] As shown in Figure 15 , the siRNA conjugates and siRNA conjugate compositions disclosed herein exhibited significant inhibitory effects on HBV antigens and HBV DNA in both rAAV-rcccDNA / rAAV-Cre and rAAV-HBV1.3 mouse models. At a dose of 2.5 mg / kg, the inhibition rate of HBV target gene expression remained as high as 86% one month after administration. This suggests that the siRNA conjugates and siRNA conjugate compositions disclosed herein have promising application prospects in treating HBV-related diseases, particularly pathological conditions or diseases caused by hepatitis B virus infection.

[0391] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art can make some simple modifications, equivalent changes or modifications based on the technical content disclosed above, which all fall within the scope of protection of the present invention.

[0392] It should also be noted that the various technical features described in the above embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0393] In addition, the various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A small interfering RNA (siRNA) group, characterized in that Include: i) a first siRNA, wherein the first siRNA has a complementary sense strand and an antisense strand, and each nucleotide in the first siRNA is independently a modified or unmodified nucleotide, wherein: a) the sense strand of the first siRNA contains the sequence 5'-GCCGAUCCAUACUGCGGAA-3' (SEQ ID NO: 1) or a sequence that is not more than 4 nucleotides different therefrom, and the antisense strand of the first siRNA contains the sequence 5'-UUCCGCAGUAUGGAUCGGC-3' (SEQ ID NO: 2) or a sequence that is not more than 4 nucleotides different therefrom (e.g., not more than 3, not more than 2, not more than 1); b) the sense strand of the first siRNA contains the sequence 5'-GUUUACUAGUGCCAUUUGU-3' (SEQ ID NO: 21) or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom, and the antisense strand of the first siRNA contains the sequence 5'-ACAAAUGGCACUAGUAAAC-3' (SEQ ID NO: 22) or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom; or c) the sense strand of the first siRNA contains the sequence 5'-GGCUUUCAGUUAUAUGGAU-3' (SEQ ID NO: 23) or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom, and the antisense strand of the first siRNA contains the sequence 5'-AUCCAUAUAACUGAAAGCC-3' (SEQ ID NO: 24) or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom; and ii) a second siRNA, wherein the second siRNA has a complementary sense strand and an antisense strand, and each nucleotide in the second siRNA is independently a modified or unmodified nucleotide, wherein: a) the sense strand of the second siRNA contains the sequence 5'-GUUCAUGUCCUACUGUUCA-3' (SEQ ID NO: 5) or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom, and the antisense strand of the second siRNA contains the sequence 5'-UGAACAGUAGGACAUGAAC-3' (SEQ ID NO: 6) or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom; b) the sense strand of the second siRNA contains the sequence 5'-GCAUGGAGACCACCGUGAA-3' (SEQ ID NO: 7) or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom, and the antisense strand of the second siRNA contains the sequence 5'-UUCACGGUGGUCUCCAUGC-3' (SEQ ID NO: 8) or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom; or c) the sense strand of the second siRNA contains the sequence 5'-GCGUCGCAGAAGAUCUCAA-3' (SEQ ID NO: 43) or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom, and the antisense strand of the second siRNA contains the sequence 5'-UUGAGAUCUUCUGCGACGC-3' (SEQ ID NO: 44) or a sequence that is not more than 4 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom.

2. The small interfering RNA (siRNA) set according to claim 1, characterized in that: The first siRNA further has a 3' overhang consisting of 1 to 4 nucleotides in at least one of the sense strand and the antisense strand; Preferably, the first siRNA has 3' overhangs in both the sense and antisense strands; More preferably, the 3' overhang is 1 to 4, such as 1 to 3, preferably 2 consecutive deoxythymidine nucleotides dT, 1 to 4, such as 1 to 3, preferably 2 consecutive uracil nucleotides U, or 1 to 4, such as 1 to 3, preferably 2 nucleotides complementary to the target mRNA; More preferably, the sense strand of the first siRNA contains or consists of the sequence 5'-GCCGAUCCAUACUGCGGAAdTdT-3' (SEQ ID NO: 3), 5'-GUUUACUAGUGCCAUUUGUdTdT-3' (SEQ ID NO: 61), 5'-GGCUUUCAGUUAUAUGGAUdTdT-3' (SEQ ID NO: 63), and the siRNA The antisense strand contains or consists of the sequence 5'-UUCCGCAGUAUGGAUCGGCAGdTdT-3' (SEQ ID NO: 4), 5'-ACAAAUGGCACUAGUAAACUGdTdT-3' (SEQ ID NO: 62), 5'-AUCCAUAUAACUGAAAGCCAAdTdT-3' (SEQ ID NO: 64).

3. The small interfering RNA (siRNA) set according to claim 1 or 2, characterized in that: The second siRNA further has a 3' overhang consisting of 1 to 4 nucleotides in at least one of the sense strand and the antisense strand.

4. The small interfering RNA (siRNA) set according to claim 3, characterized in that: The second siRNA has 3' overhangs in both the sense strand and the antisense strand; Preferably, the 3' overhang is 1 to 4, such as 1 to 3, preferably 2 consecutive deoxythymidine nucleotides dT, 1 to 4, such as 1 to 3, preferably 2 consecutive uracil nucleotides U, or 1 to 4, such as 1 to 3, preferably 2 nucleotides complementary to the target mRNA; More preferably, the sense strand of the second siRNA contains or consists of the sequence 5'-GUUCAUGUCCUACUGUUCAdTdT-3' (SEQ ID NO: 9), 5'-GCAUGGAGACCACCGUGAAdTdT-3' (SEQ ID NO: 11), 5'-GCGUCGCAGAAGAUCUCAAdTdT-3' (SEQ ID NO: 83), and the antisense strand of the second siRNA contains or consists of the sequence 5'-UGAACAGUAGGACAUGAACAAdTdT-3' (SEQ ID NO: 10), 5'-UUCACGGUGGUCUCCAUGCGAdTdT-3' (SEQ ID NO: 12), 5'-UUGAGAUCUUCUGCGACGCGGdTdT-3' (SEQ ID NO: 84).

5. The small interfering RNA (siRNA) set according to claim 1 or 2, characterized in that: The molar ratio of the first siRNA to the second siRNA is 1:100 to 100:1, preferably 1:10 to 10:1; and / or. The first siRNA and the second siRNA contain one or more modified nucleotides and / or modified bonds. Preferably, the first siRNA and the second siRNA are any one of the siRNAs in Table 2 or Table 3a / Table 3b.

6. A siRNA conjugate, characterized in that Comprising a siRNA set according to any one of claims 1 to 5 and a conjugated group conjugated to the siRNA; Preferably, the conjugated group is a pharmaceutically acceptable conjugated group, the conjugated group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are covalently linked in sequence; More preferably, the linker is connected to the 3' end of the sense strand of the siNRA; More preferably, the targeting group is a ligand of the asialoglycoprotein receptor; More preferably, the targeting group is galactose or N-acetylgalactosamine.

7. A pharmaceutical composition, characterized in that Comprising the siRNA group according to any one of claims 1 to 5, and / or the siRNA conjugate according to claim 6, and a pharmaceutically acceptable carrier; Preferably, the pharmaceutically acceptable carrier contains cationic lipids, helper lipids, structural lipids and PEGylated lipids; More preferably, the cationic lipid is an amine-containing compound, such as an ionizable amine-containing compound and / or a biodegradable amine-containing compound, such as 8-[(2-hydroxyethyl)[6-oxo-6(undecanyloxy)hexyl]amino]-octanoic acid 1-octylnonyl ester (SM-102); the auxiliary lipid is phosphatidylcholine, an analog of phosphatidylcholine and / or a derivative of phosphatidylcholine; the structural lipid is cholesterol, an analog of cholesterol and / or a derivative of cholesterol; the pegylated lipid is PEG-diacylglycerol (DAG), PEG dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer) or a mixture thereof, preferably polyethylene glycol-dimyristoylglycerol (PEG-DMG); 8. Use of the siRNA group according to any one of claims 1 to 5, the siRNA conjugate according to claim 6 and / or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating and / or preventing hepatitis B.

9. A non-therapeutic method for inhibiting HBV gene expression in hepatocytes, the method comprising contacting the hepatocytes with an effective amount of the siRNA group according to any one of claims 1 to 5, the siRNA conjugate according to claim 6 and / or the pharmaceutical composition according to claim 7. 10 . A kit comprising the siRNA set according to any one of claims 1 to 5 , the siRNA conjugate according to claim 6 , and / or the pharmaceutical composition according to claim 7 .

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