Nucleic acid, pharmaceutical composition containing nucleic acid, conjugate containing nucleic acid and application of nucleic acid

Abstract

The invention relates to a nucleic acid, a pharmaceutical composition containing the nucleic acid, a conjugate containing the nucleic acid and application of the nucleic acid. Specifically, the invention relates to siRNA which has complementary positive-sense strand and antisense strand, each nucleotide in the siRNA is independently modified or unmodified nucleotide, and the siRNA is characterized in that the positive-sense strand of the siRNA contains a sequence SEQ ID NO: 1, 21 or 23 or a sequence which is similar to the sequence SEQ ID NO: 1, 21 or 23 by four nucleotides, and the antisense strand of the siRNA contains a sequence SEQ ID NO: 2, 22 or 24 or a sequence which is similar to the sequence SEQ ID NO: 2, 22 or 24 by four nucleotides. The invention also provides a nucleic acid group comprising the nucleic acid as a first siRNA and a second siRNA comprising a sense strand comprising the sequence of SEQ ID NO: 5, 7 or 43 and an antisense strand comprising the sequence of SEQ ID NO: 6, 8 or 44, a pharmaceutical composition and a conjugate comprising the nucleic acid or the nucleic acid group, and applications thereof.

Description

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. At present, the drugs for the clinical treatment of chronic hepatitis B mainly include two categories: interferon (IFN) and nucleoside (acid) analogs (NUC). Among them, interferon can activate both innate immunity and acquired immunity, and some patients can achieve HBsAg clearance within a limited course of treatment, but there are problems such as poor patient tolerance, serious side effects and low response rate; studies have shown that the response rate of Asian B genotype to interferon is very low. Nucleoside (acid) analogs can effectively inhibit HBV viral replication, which is beneficial to restore liver function and reduce the incidence of liver cancer. However, patients need to take medication for a long time or even for life, and are prone to drug resistance and rebound after drug withdrawal, making it difficult to achieve HBsAg clearance.

[0003] With the goal of "functional cure" of chronic hepatitis B, studying how to achieve sustained negative conversion of hepatitis B surface antigen (HBsAg) or sustained silencing of cccDNA is the current focus of work in this field. Based on the mechanism of RNA interference (RNAi), small interfering RNA (siRNA) can target and silence HBV transcripts in a sequence-specific manner, thereby reducing the formation of HBV antigens and cccDNA. Compared with traditional antiviral drugs, it can effectively and quickly attack viral transcripts and reduce HBsAg, which is conducive to the formation of an immune window, and therefore has unique advantages.

[0004] HBV is a small hepatotropic DNA virus with a genome of about 3.2 kb. It is a relaxed circular double-stranded DNA (rcDNA) with a gap, encoding a total of 4 open reading frames (ORFs), including hepatitis B surface antigen (HBsAg), viral core antigen (HBcAg), polymerase (Pol) and X protein (HBx). The rcDNA is released into the nucleus through the nucleocapsid, and repaired to form a supercoiled covalently closed circular DNA (cccDNA) with the help of host DNA damage repair factors, and combined with nucleosomes to form a minichromosome structure. The pregenomic RNA (pgRNA) and multiple subgenomic RNAs are transcribed using cccDNA as a template. The pgRNA is packaged into viral core particles under the action of Pol and HBc and reversely transcribed into rcDNA, which can be replenished into the nucleus again to form a cccDNA storage pool (cccDNA pool).

[0005] There are many different forms of HBV-related mRNA in infected hepatocytes. Among them, five major viral mRNAs are transcribed using cccDNA as a template, with lengths of 3.5kb, 3.5kb, 2.4kb, 2.1kb and 0.7kb, respectively, and all contain a polyadenylated tail (polyA) at the 3' end, with overlapping sequences. The 3.5kb mRNA serves as a template for the production of core antigens and polymerases, the 2.4 / 2.1kb mRNA encodes preS1 / preS2 and S protein, and the 0.7kb mRNA encodes X protein, the gene product of which has an important regulatory effect on the transcription of viral genes, and can act as a trans-activator of host gene expression and promote the occurrence and development of liver cancer. In addition, the HBV integration-related sequence (intHBV) also transcribes viral mRNA. Since the viral genome is linearized and part of the sequence is lost after viral integration, the overlapping sequence of the original 3' end polyadenylation of the integration source sequence will be 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 4 major serotypes (adr, adw, ayr, ayw). Based on the genome sequence, the virus can also be classified into 8 genotypes (A, B, C, D, E, F, G, H). Among them, genotype A is common in America, Africa, India and Western Europe. Genotypes B and C exist in Asia and the United States. Genotype D is most common in Southern Europe and India. Genotype E exists 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 large number of HBV genotypes and at least 8% difference in sequence between each genotype, there is rarely a siRNA drug that can widely cover different HBV genotypes. In addition, it is difficult to target HBV integration with siRNA designed to target HBV consensus sequences, while siRNA designed to target HBsAg (or HBV integration) cannot effectively target HBx. Therefore, there is an urgent need for a small interfering nucleic acid drug that can widely target multiple HBV genotypes and has inhibitory effects on both HBsAg (HBV integration) and HBx in clinical practice.

[0007] Several studies have attempted to use RNAi for the treatment of HBV. However, single siRNA transfection often does not provide adequate gene silencing. Therefore, despite significant advances in the field of RNAi technology, there is still a need for agents that can effectively inhibit HBV gene expression and can treat diseases associated with HBV infection such as liver disease and cancer.

[0008] The first siRNA drug to enter clinical trials, ARC-520, consists of two siRNAs (si-74 and si-77) targeting the 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 the authorized patent of Biomike (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 positive 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 source sequences, but the sequence conservation of the above products is unknown.

[0012] Represented by RG6346 developed by Roche and ALG-125755 developed by Aligos, they 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 why siRNA drugs are difficult to enter the clinic is that siRNA has poor stability and is easily degraded by nucleases in the body (especially, after systemic administration in the body, siRNA will first enter the blood circulation system in the body, and the blood is rich in endogenous nucleases). To overcome this obstacle, one of the means is to chemically modify siRNA to improve the stability of siRNA in the blood. However, there is still a lack of sufficient understanding of the degradation process and mechanism of siRNA in the blood in this field, and the selection of modification sites is still an empirical process that must be verified through repeated experiments. Therefore, in order to obtain sufficient stability, a large amount of (even blind, redundant) chemical modifications have to be introduced into siRNA. As a result, although the stability of siRNA may be improved to a certain extent through modification, the introduction of excessive or inappropriate modifications will inevitably lead to disadvantages such as increased costs, potential cytotoxicity of the modified siRNA, and affected biological activity and specificity of the modified siRNA.

[0014] In addition, the modification of siRNA sequences should be personalized, streamlined and optimized from various aspects such as synthesis cost, synthesis efficiency, process convenience of synthesis, siRNA stability, and siRNA activity, in order to obtain better siRNA. Considering that previous studies in this field have shown that different chemical modifications of siRNA sequences may "affect the whole body", that is, adopting different modification strategies at only one site can fundamentally change the stability or biological activity of siRNA, it is not easy to complete the personalized design, simplification and optimization of the above-mentioned siRNA sequence modifications.

[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 the clinical development of siRNA drugs is that it is necessary to form a pharmaceutical composition through a suitable carrier or form a conjugate with a suitable conjugated molecule to further improve the blood stability of siRNA, improve its targeting, solve the problem of siRNA delivery in vivo, etc. For siRNA intended to prevent and treat hepatitis B, a carrier or conjugated molecule that can impart or improve liver targeting will help siRNA to act in target cells, thereby improving 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 improve the clinical application potential of siRNA, as well as siRNA pharmaceutical compositions or siRNA conjugates prepared using such pharmaceutically acceptable carriers or conjugated molecules. Summary of the invention

[0017] The present invention provides siRNA or a combination of siRNA, which can simultaneously target RNA from all sources of HBV, including all HBV RNA from cccDNA and RNA from HBV integration (intRNA), and can achieve extensive targeting of multiple genotypes of HBV (gene coverage>98%), thereby providing a preparation and therapy that can achieve HBsAg clearance. The siRNA of the present invention has strong specificity and conservatism, can widely target different clinical genotype sequences, and can make up for the poor efficacy of nucleic acid interference drugs under development at this stage and the shortcomings of current hepatitis B treatment drugs, and is expected to become a new means of treating hepatitis B.

[0018] Considering that siRNA is unstable in blood and tissues and is easily degraded by nucleases, in order to improve the stability of siRNA, the siRNA backbone can be modified, but these chemical modifications only provide limited protection from nuclease degradation and may ultimately affect the activity of siRNA. Therefore, a corresponding delivery system is also needed to ensure that siRNA passes through the cell membrane safely and efficiently. Since siRNA has a large molecular weight, carries a large amount of negative charge, and has high water solubility, it cannot smoothly pass through the cell membrane to reach the cell. Different chemical modifications are further introduced for the small interfering nucleic acid, so as 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 an efficient siRNA pharmaceutical composition and conjugate for 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 cancer, 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.

[0019] The object 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 that can widely target multiple HBV genotypes and HBV RNA from all sources are provided. 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.

[0020] The first aspect of the present invention provides a small interfering RNA (siRNA), which has a complementary sense strand and an antisense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide, characterized in that:

[0021] i) 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 nucleotides different 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 nucleotides different therefrom (e.g., not more than 3, not more than 2, not more than 1);

[0022] ii) 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 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom, 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 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom; or

[0023] iii) 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 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom, 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 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom.

[0024] In some embodiments, the siRNA further has a 3' overhang consisting of 1 to 4 nucleotides in at least one of the sense strand and the antisense strand. In some embodiments, the siRNA has a 3' overhang in both the sense strand and the antisense strand. In some embodiments, the 3' overhang is 1 to 4, for example 1 to 3, preferably 2 consecutive deoxythymidine nucleotides dT, 1 to 4, for example 1 to 3, preferably 2 consecutive uracil nucleotides U, or 1 to 4, for example 1 to 3, preferably 2 nucleotides complementary to the target mRNA. In some embodiments, the sense strand of the siRNA contains or consists of the sequence 5'-GCCGAUCCAUACUGCGGAAdTdT-3' (SEQ ID NO: 3), 5'-GUUUACUAGUGCCAUUUGUdTdT-3' (SEQID NO: 61), 5'-GGCUUUCAGUUAUAUGGAUdTdT-3' (SEQ ID NO: 63), and the antisense strand of the siRNA 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), wherein the lowercase letter d indicates that the nucleotide to the right of the letter d is a deoxyribonucleotide.

[0025] In some embodiments, the siRNA comprises one or more modified nucleotides and / or modified bonds. Preferably, the siRNA comprises one or more nucleotides such as adenine, guanine, cytosine and / or uracil nucleotides in which the 2'-OH of the pentose is modified by methoxy and / or fluorine, 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 preferably sense strand is connected to one or more (e.g., 1-5, for example, 3) GalNac at the 5' end and / or 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, specific modifications are introduced into the siRNA of the present invention to overcome the shortcomings of poor serum stability and poor biological activity of existing small interfering nucleic acids used to inhibit hepatitis B virus gene expression.

[0026] The second aspect of the present invention provides a small interfering RNA (siRNA) group, which comprises a first siRNA and a second siRNA, wherein the first siRNA targets PreS / S RNA and intHBV RNA, and the second siRNA targets RNA derived from all HBV cccDNA including HBx. In some embodiments, the small interfering RNA (siRNA) group comprises:

[0027] i) a first siRNA, wherein the first siRNA is the siRNA according to the first aspect of the present invention; and

[0028] ii) a second siRNA, the second siRNA having a complementary sense strand and an antisense strand, wherein:

[0029] 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;

[0030] 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

[0031] 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.

[0032] 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;

[0033] Preferably, the second siRNA has 3' overhangs in both the sense and antisense strands;

[0034] 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;

[0035] 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).

[0036] 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, preferably, the first siRNA and the second siRNA are any one of the siRNAs in Table 2 or Table 3a / Table 3b.

[0037] 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 thiophosphate 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, for example, 3) GalNac at the 5' end and / or 3' end, preferably the 3' end.

[0038] In some embodiments, the first and second siRNAs in the siRNA group have the characteristics of good serum stability, low cytotoxicity and / or good biological activity, and can be used to inhibit the expression of hepatitis B virus genes. In some embodiments, by introducing specific modifications into the siRNA of the present invention, the shortcomings of poor serum stability and poor biological activity of the existing small interfering nucleic acids used to inhibit the expression of hepatitis B virus genes are overcome.

[0039] 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 sense strand 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.

[0040] The fourth aspect of the present invention provides a pharmaceutical composition, which comprises 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-octyl nonyl 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).

[0041] 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 drug for treating and / or preventing hepatitis B.

[0042] The sixth aspect of the present invention provides a non-therapeutic method for inhibiting HBV gene expression in hepatocytes, the method comprising 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.

[0043] The seventh aspect of the present invention provides a kit, which contains 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 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.

[0045] 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.

[0046] 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 targeting the HBV sequence but not targeting the human genome sequence, and selecting siRNA located near the HBV conserved sequence region (i.e., targeting the sequence covering 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, 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 model includes HepG-HBV / EGFP cell line 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 of 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, a sequence combination that can achieve multiple targets (can simultaneously target HBV cccDNA-derived RNA and integrated HBV-derived RNA), is broad-spectrum (sequence conservation>90%), and has good efficacy (target gene expression level inhibition rate>90%).

[0047] The eleventh aspect of the present invention provides a method for screening siRNA groups, comprising the following steps: 1) selecting a first siRNA targeting PreS / S RNA and intHBV RNA, and selecting a second siRNA targeting RNA derived from all HBV cccDNAs 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 hepatoma cell line containing HBV natural integration), HepG2-NTCP (HBV infection model), 5C-PHH (Science 2019), pHBV1.3 replicon plasmid system (four genotypes of 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 a HVDB hepatitis virus database, obtaining the conservation of its sequence), and selecting a highly conservative sequence combination. In some embodiments, the method includes, before step 1), designing siRNA that targets HBV sequences but not human genome sequences, and selecting siRNA located near the HBV conserved sequence region (i.e., targeting sequences 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 candidate siRNAs, for example, by designing through siRNA design websites (including DSIR, siDirect, siDESIGNVenter, siRNA Target Finder).

[0048] 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 can 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

[0049] 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 implementation methods.

[0050] Figure 1 The siRNA screening process of the present invention is shown.

[0051] Figure 2 The target sites of different siRNAs designed in the present invention and their inhibitory effects on virological indicators in different HBV-related cell models are shown.

[0052] Figure 3 The interference results of different siRNAs designed by the present invention on different HBV genotypes (including four genotypes A / B / C / D) and PHH infection models are shown.

[0053] Figure 4 The IC values ​​of different siRNAs designed in the present invention in HepG2-NTCP infected cells are shown. 50 Values ​​and the antiviral effects of different siRNA combinations in different HBV-related cell models.

[0054] Figure 5 The inhibitory effect of the combination of si1260 and si1848 on virological indicators in different HBV-related cell models was shown.

[0055] Figure 6 The interference results of the combination of si1260 and si1848 on different HBV genotypes (including four genotypes A / B / C / D) are shown.

[0056] Figure 7 The duration of the antiviral effect of the combination of si1260 and si1848 in in vitro and in vivo models is shown.

[0057] Figure 8 The inhibitory effects on virological indicators, serum stability and cytotoxicity of si1260 and si1848 or the combination of the two with different chemical modification schemes are shown.

[0058] Fig. 9 The serum stabilities of si1260 and si1848 with different chemical modification schemes are shown.

[0059] Fig.10 The results of inhibition of HBsAg, HBeAg and HBV DNA levels by the positive drug ETV, LNP-siNC control group and exemplary LNP-siHBV at different time points after administration at different dose levels and different dosing frequencies are shown.

[0060] Fig.11 The results of an exemplary LNP-siHBV single-dose safety evaluation are shown.

[0061] Fig.12 Results of an exemplary LNP-siHBV repeat dosing safety evaluation are shown.

[0062] Fig.13 The off-target effect evaluation of exemplary LNP-siHBV in vivo and in vitro is shown.

[0063] Fig.14 Shown are the results of synthesis quality testing of exemplary siRNA conjugates of the present disclosure.

[0064] Fig.15 The exemplary siRNA conjugates and siRNA conjugate compositions disclosed herein are shown in HBV-related Results of inhibition of HBsAg, HBeAg and HBV DNA levels in model mice. DETAILED DESCRIPTION

[0065] 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 may be implemented without one or more specific details or may be implemented with other methods.

[0066] The siRNA sequence combination, pharmaceutical composition containing the sequence combination and conjugate provided by the present invention have multiple targets, broad spectrum, good stability, low off-target effect, high activity in inhibiting HBV mRNA, and / or can significantly treat and / or prevent hepatitis B.

[0067] The siRNA group, pharmaceutical composition and conjugate provided by the present invention show excellent HBV mRNA target gene expression inhibition rate in hepatitis B model mice, can effectively reduce the expression of hepatitis B surface antigen, and show a good therapeutic effect on chronic hepatitis B. In some embodiments, lipid nanoparticles formed by components such as cationic lipids, auxiliary lipids, structural lipids, and pegylated lipids are used as pharmaceutically acceptable carriers to form pharmaceutical compositions with the siRNA, siRNA group and / or siRNA conjugate of the present invention, and the siRNA of the present invention can be specifically delivered to the liver.

[0068] 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 on multiple HBV genotypes at the same time; compared with the disadvantage of traditional drugs that cannot simultaneously target the HBV cccDNA-derived mRNA common sequence 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.

[0069] 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. The modified siRNA in in vitro experiments 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.

[0070] In some embodiments, the siRNA, siRNA group, pharmaceutical composition and conjugate provided by the present disclosure show excellent target mRNA inhibition activity in in vitro cell experiments. In some embodiments, the siRNA, siRNA group, pharmaceutical composition and conjugate provided by the present disclosure show at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition rate of target gene expression in HBV-related cell models.

[0071] In some embodiments, the siRNA, siRNA group, pharmaceutical composition and conjugate provided by the present disclosure may have higher stability and / or higher activity in vivo. In some embodiments, the siRNA, siRNA group, pharmaceutical composition and conjugate provided by the present disclosure show at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition rate of target gene expression in HBV-related animal models. In some embodiments, the siRNA, siRNA group, pharmaceutical composition and conjugate provided by the present disclosure have a higher stability and / or higher activity in vivo. In some embodiments, the siRNA, siRNA group, pharmaceutical composition and conjugate provided by the present disclosure have a higher inhibition rate of target gene expression in HBV-related animal models. cccDNA+pCMV-Cre In the high-pressure hydrodynamic model, the siRNA showed obvious inhibitory activity, and the inhibition rate of the target gene expression level of HBV mRNA by siRNA can reach 99.50%. In some embodiments, the siRNA pharmaceutical composition provided by the present disclosure shows obvious inhibitory activity in the rAAV-HBV1.3 mouse hepatitis B chronic model, and the inhibition rate of the target gene expression level of HBV mRNA by siRNA can reach 99.77%. In some embodiments, the siRNA conjugate provided by the present disclosure shows obvious inhibitory activity in the rAAV-HBV1.3 mouse hepatitis B chronic model, and the inhibition rate of the target gene expression level of HBV mRNA by siRNA can reach 97.46%.

[0072] 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 cause 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 cause 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.

[0073] In some embodiments, the siRNA, siRNA group, pharmaceutical composition and conjugate provided by the present disclosure may have lower immunogenicity in vivo. In some embodiments, the siRNA pharmaceutical composition provided by the present disclosure does not lead to the production of anti-PEG antibodies after repeated multiple injections.

[0074] 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.

[0075] The terms used herein are intended only to describe specific embodiments 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 also intended to include plural forms. In addition, the open-ended expressions "including" and "comprising" are interpreted as also containing unmentioned structural components or method steps, but it should be noted that the open-ended expressions also cover situations consisting only of the described components and method steps (i.e., covering the situation of the closed expression "consisting of ...").

[0076] As used throughout, ranges are used as shorthand for describing each and all values ​​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 within a range of 19-26 represents integers 19, 20, 21, 22, 23, 24, 25, and 26 and any subranges formed by them, such as 19-25, 20-24, 21-23, etc.

[0077] All scientific and technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of 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.

[0078] 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 can be an oligonucleotide or a polynucleotide, and can be an unmodified RNA or DNA or a 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, and RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that can be single-stranded or double-stranded or a mixture of single-stranded and double-stranded regions.

[0079] 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.

[0080] 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.

[0081] In the present invention, the term "sequence conservation" refers to: among the 11,185 HBV genome sequences in the HBV database, the percentage of HBV genome sequences that can be completely matched by the siRNA 19-mer core sequence to the total number of HBV genome sequences.

[0082] For nucleic acid modification

[0083] (1) Nucleic acid structure

[0084] 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 furanose, the phosphate group can be attached to the 2', 3' or 5' hydroxyl moiety of the sugar. In forming nucleic acid, the phosphate group covalently attaches nucleosides adjacent to each other to form a linear polymer compound. In turn, each end of a linear polymer compound can be further 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 be folded in order to produce a completely or partially double-stranded compound. In nucleic acid, the phosphate group is generally referred to as the internucleoside skeleton forming nucleic acid. The normal bond or skeleton of RNA and DNA is a 3' to 5' phosphodiester bond.

[0085] (2) Modified backbone and modified internucleoside bonds

[0086] 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 phosphorus atoms in the backbone and those that do not have phosphorus atoms in the backbone.

[0087] The nucleic acid wherein containing the modification that is suitable for phosphorus atom comprises for example, 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'-aminophosphoramidate and aminoalkylphosphoramidate, diaminophosphorate, thionylphosphoramidate, thionylphosphoramidate, thionylphosphotriester, selenophosphate and borophosphate with normal 3'-5' key, these 2'-5' connect analog and have those of reverse polarity, wherein one or more internucleotide keys are 3' to 3', 5' to 5' or 2' to 2' keys.The nucleic acid suitable for having reverse polarity comprises single 3' to 3' key at the most 3' internucleotide key place, can be single reverse nucleoside residue without base (core base loses or it is replaced by hydroxyl).Also comprise various salts (such as potassium or sodium), mixed salt and free acid form.

[0088] In some embodiments, nucleic acids of the invention comprise one or more phosphorothioate bonds and / or heteroatom internucleoside bonds, specifically -CH 2 -NH-O-CH 2 -、-CH 2 -N(CH 3 )-O-CH2 -(called methylene (methylimino) or MMI skeleton), -CH 2 -ON(CH 3 )-CH 2 -、-CH 2 -N(CH 3 )-N(CH 3 )-CH 2 - and -ON(CH 3 )-CH 2 -CH 2 -(wherein the natural phosphodiester internucleoside bond is represented by -OP(=O)(OH)-O-CH 2 -).

[0089] Other backbone modifications also include 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.

[0090] Suitable modified polynucleotide backbones not including phosphorus atoms have backbones formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatoms or heterocyclic nucleoside bonds. 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; riboacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methylene imino and methylene hydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and those with mixed N, O, S and CH 2 Other skeletons of components.

[0091] Another backbone modification includes locked nucleic acids (LNA), in which the 2'-hydroxyl group is linked 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 (-CH 2 -) (a group bridging the 2' oxygen atom and the 4' carbon atom), wherein 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.

[0092] 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).

[0093] (3) Modified sugar moiety

[0094] The nucleic acids of the 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((CH 2 ) n O) m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 and O(CH 2 ) n ON((CH 2 ) n CH 3 ) 2 , wherein n and m are from 1 to about 10. Other suitable polynucleotides contain a sugar substituent selected from the group consisting of C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 、OCN、Cl、Br、CN、CF 3 、OCF 3 、SOCH 3 、SO 2 CH 3 、ONO 2 、NO 2 、N 3 NH 2, heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of nucleic acid or group for improving the pharmacodynamic properties of nucleic acid and other substituents with similar properties. Suitable modifications include 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , 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(CH 2 ) 2 ON(CH 3 ) 2 The 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-CH 2 -O-CH 2 -N(CH 3 ) 2 .

[0095] Other suitable sugar substituents include methoxy (—O—CH 3 ), aminopropyloxy (--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 may be in the arabinose (upper) position or the ribose (lower) position. Suitable 2'-arabinose modifications are 2'-F. Similar modifications may 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 the 5' position of the 5' terminal nucleotide. The oligomeric compound may also have a sugar mimetic such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

[0096] (4) Base modification and substitution

[0097] 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-CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, 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).

[0098] Heterocyclic base moieties may 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, John Wiley & Sons, 1990, Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., 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., ed., 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.

[0099] (5) Conjugate

[0100] Another possible modification of nucleic acid of the present invention relates to one or more parts or conjugates chemically connected to polynucleotides that enhance the activity, cell distribution or cell absorption of nucleic acid. These parts or conjugates may include covalently bound conjugate groups of functional groups such as primary hydroxyl or secondary hydroxyl. Conjugate groups include but are not limited to intercalators, reporters, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance oligomer pharmacodynamic properties and groups that enhance oligomer pharmacokinetic properties. Suitable conjugate groups include but are not limited to N-acetylgalactosamine (GalNAc), cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and dyes. Groups that enhance pharmacodynamic properties include groups that improve absorption, enhance resistance to degradation and / or strengthen sequence-specific hybridization with target nucleic acids. Groups that enhance pharmacokinetic properties include groups that improve absorption, distribution, metabolism or excretion of nucleic acid of the present invention.

[0101] The 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), bile acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Man oharan 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 dodecanediol 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 glycerol-3-H-triethylammonium phosphate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamines or polyethylene glycol chains (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).

[0102] As used herein, "unmodified" or "natural" nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleotides include nucleotides that are only rare or transient 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, and 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.

[0103] In the present invention, the terms "conjugated" and "linked" are used interchangeably and refer to connection through a chemical bond.

[0104] 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 drug 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.

[0105] 1. siRNA of the present invention

[0106] The siRNA of the present invention is obtained by screening through the following methods (see Figure 1 ):

[0107] 1) First, the conservative sequences of HBV genome sequences were analyzed. A collection of 11,185 HBV genome sequences was downloaded from the Hepatitis Virus Database (HVDB) (http: / / s2as02.genes.nig.ac.jp / index.html). A global alignment was performed on the MEME (Motif-based sequence analysis tools) website (https: / / meme-suite.org / meme / index.html) to analyze the conservative sequence regions (motifs) in the HBV genome sequence collection;

[0108] 2) Use siRNA design websites (including DSIR, siDirect, siDESIGN Venter, siRNA TargetFinder) 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 19mer sequence design of the siRNA used for HBV RNAi is designed according to multiple HBVcDNA 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 base parts such as TT, UU, UA, etc.):

[0109] Table 1 siRNA sequences

[0110]

[0111]

[0112] 3) The in-house original cell model HepG-HBV / EGFP cell line was used (the model can be referred to 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, 2018, 152, 45-52). reports,6,36483.) for siRNA screening and verification. The 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; and screening and verification were carried out in HepAD38 (HBV replicon model), PLC / RCF / 5 (clinically screened liver cancer cell line containing natural HBV integration, which can express HBsAg derived from integrated HBV), HepG2-NTCP (HBV infection model), 5C-PHH (in-house construction, Science 2019), and pHBV1.3 replicon plasmid system (four genotypes A / B / C / D).

[0113] 4) The screened sequences were then compared with a set of 11,185 HBV genome sequences downloaded from the HVDB hepatitis virus database to obtain the conservation of their sequences (number of fully matched sequences / total number of HBV gene sequences × 100%), and sequences with good inhibition rate and high conservation were selected for further combination (e.g., any combination of any siRNA in Table 3a with any siRNA in Table 3b, a combination of any two siRNAs in Table 4, especially a combination of siRNA targeting HBsAg and HBV integration and siRNA targeting the common sequence of HBx and HBV mRNA, especially the combination in Table 11), and the conservation of sequence combinations was evaluated and analyzed in different models; sequence combinations that can achieve multi-target, broad-spectrum, and good efficacy were selected for subsequent studies.

[0114] The above screening strategy can effectively improve the efficiency of siRNA sequence screening. Compared with the previous siRNA screening of 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 for HBV (ARC-520), the genotype coverage of the obtained siRNA has been significantly improved (89% vs. 98%), and it can simultaneously target HBV cccDNA and HBV integration source sequences (ARC-520 cannot target HBV integration).

[0115] The siRNA or siRNA group targeting any group of RNA interference targets 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 group of RNA interference targets or multiple groups of RNA interference targets of the present invention, and the therapeutic drug containing the siRNA or siRNA group of any group of RNA interference targets or multiple groups of RNA interference targets of the present invention, all fall within the protection scope of the present invention.

[0116] In a preferred embodiment, two or more of the above siRNA molecules can be combined together to target different regions of the HBV genome transcripts, respectively, to achieve the purpose of inhibiting the expression of RNA genes from all sources of HBV and widely targeting different genotypes of HBV.

[0117] The present invention provides siRNA, siRNA group, siRNA pharmaceutical composition and conjugate targeting HBV. The siRNA, siRNA group, siRNA pharmaceutical composition and conjugate can be used to treat diseases, such as liver diseases or malignant tumors and / or cancers related to HBV expression, or other conditions caused by hepatitis B infection. The siRNA can be administered alone or in combination with other siRNA molecules or conventional agents for treating HBV-related diseases or conditions.

[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 contain 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 Toll-like receptor response stimulation) and / or bioavailability. The modified nucleotide group will not cause the siRNA to significantly weaken or lose the function of inhibiting HBV gene expression. 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, a plurality of 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] Among them, the capital letters C, G, U and A represent the base composition of the nucleotide respectively.

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

[0142] In order to further improve the stability of siRNA, at least a part of the phosphate groups in the phosphate-sugar backbone of the siRNA is a phosphate group with a modified group, and at least a part of the ribose groups in the phosphate-sugar backbone of the siRNA is a ribose group with a modified group. The phosphate group and ribose group with a modified group can be various phosphate groups and ribose groups with modified groups that have the above-mentioned effects. Preferably, the ribose group with a modified 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 modified group is a thiophosphate group in which one oxygen atom 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]

[0144] 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:

[0145] Table 2 Chemically modified siRNA sequence list

[0146]

[0147]

[0148] 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.

[0149] 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). Among them, solid phase synthesis has commercial customization services. The modified nucleotide groups can be introduced into the siRNA of the present invention by using nucleotide monomers with corresponding modifications. Methods for preparing nucleotide monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.

[0150] 3. siRNA pharmaceutical composition of the present invention

[0151] The present invention provides a small interfering nucleic acid pharmaceutical composition, wherein the pharmaceutical composition contains the siRNA, siRNA group or modified siRNA of the present invention. In some embodiments, the small interfering nucleic acid pharmaceutical composition comprises two small interfering nucleic acids, i.e., a first siRNA such as si1260 and a second siRNA such as si1848 or si1606, as active ingredients.

[0152] 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 reflect 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 strand and antisense strand of the si1260 can be naked nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2; the sense strand and antisense strand of the si1606 can be naked nucleotide sequences as shown in SEQ ID NO: 7 and SEQ ID NO: 8; and / or the sense strand and antisense strand of the si1848 can be naked nucleotide sequences as shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0153] In some embodiments, in order to enhance the stability of the siRNA double strand, the 3' end of at least one of the sense strand and antisense strand of the first siRNA and the sense strand and antisense strand of the second siRNA is further connected with 1 to 4 nucleotides, so that after the sense strand and the antisense strand are complementary paired, a 3' overhang consisting of at least 1 to 4 nucleotides is formed, or an overhanging end. Preferably, the 3' end of each of the sense strand and antisense strand of the first siRNA and the sense strand and antisense strand of the second siRNA is connected with a 3' overhang. More preferably, the 3' overhang is 2 consecutive thymine deoxyribonucleotides dTdT or 2 consecutive uracil ribonucleotides UU; or, in order to improve 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.

[0154] In order to further improve the stability of the first siRNA and / or the second siRNA, at least a part of the phosphate groups in the phosphate-sugar backbone of the first siRNA and / or the second siRNA is a phosphate group with a modified group, and at least a part of the ribose groups in the phosphate-sugar backbone of the first siRNA and / or the second siRNA is a ribose group with a modified group. The phosphate group and ribose group with a modified group can be various phosphate groups and ribose groups with modified groups that have the above-mentioned effects. Preferably, the ribose group with a modified 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 modified group is a thiophosphate group in which one oxygen atom 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:

[0155]

[0156] 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'. Among them, the capital letters C, G, U, A and T represent the base composition of the nucleotide; the lowercase letter d indicates that the nucleotide to the right of the letter d is a deoxyribonucleotide; 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. For example, in si1260, "Cm" composed of the second capital letter C at the 5' end of the sense strand and the m to its right indicates a nucleotide in which the base is cytosine and the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0157] It is well known to those skilled in the art that the first siRNA and / or the second siRNA described in 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 customization 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 described in the present disclosure 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 there is also a commercial monomer supply on the market.

[0158] In the small interfering nucleic acid pharmaceutical composition of the present invention, in addition to the first siRNA and / or the second siRNA as active ingredients, a pharmaceutically acceptable carrier is optionally contained. The pharmaceutically acceptable carrier can be a carrier conventionally used in the field of siRNA administration, such as but not limited to magnetic nanoparticles (such as Fe 3 O 4 , Fe 2 O 3 ), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyaminoamine (PAMAM) dendrimer, poly (L-lysine), chitosan, 1,2-dioleoyl-3-thrimethylammonium-propane (DOTAP), poly (D / L-lactic / glycolic acid) copolymer (PLGA), poly (2-aminoethylethylene phosphate), poly (2-dimethylaminoethyl methacrylate), poly (2-dimethylaminoethyl methacrylate), and one or more of their derivatives. In the small interfering nucleic acid pharmaceutical composition of the present invention, there is no special requirement for the content of the pharmaceutically acceptable carrier. Generally, the weight ratio of the total weight of the first siRNA and the second siRNA contained in the small interfering nucleic acid pharmaceutical composition of the present invention to the pharmaceutically acceptable carrier is 1:(1-500), preferably 1:(1-50).

[0159] In the small interfering nucleic acid pharmaceutical composition of the present invention, other pharmaceutically acceptable excipients may also be included, and the excipients 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 value of 7.5-8.5 and / or a phosphate buffer with a pH value of 5.5-8.5, preferably a phosphate buffer with a pH value of 5.5-8.5. The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the small interfering nucleic acid pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight. The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmole / kg. According to the desired osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.

[0160] 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; it can also 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 to the lungs by spray, or administration to other organs and tissues (such as the liver) through the lungs by spray. Preferably, the small interfering nucleic acid pharmaceutical composition is for intravenous administration.

[0161] 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, an auxiliary lipid, a structural lipid and / or a pegylated lipid. Among them, the cationic lipids, auxiliary lipids, structural lipids and pegylated lipids can be selected from one or more of 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 their pharmaceutically acceptable salts or derivatives, auxiliary lipids, structural lipids and pegylated lipids.

[0162] 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-N-methylpiperazinyl-[1,3]-dioxolane (DLin-K-C3-DMA), 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-N-methylpiperazinyl-[1,3]-dioxolane (DLin-K 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-methylpiperazine)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 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-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-carboxamido)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-ene-3-β-oxy)-3'-oxapentyloxy)-3-dimethyl-1-(cis, cis-9',1-2'-octadecadienyloxy) 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). .

[0163] 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, an analog of cholesterol and / or a derivative of cholesterol; (2) a phospholipid; or (3) a mixture of a phospholipid 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 neutral lipid, it 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-antioleyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg yolk phosphatidylcholine (EPC) and its mixture. In some preferred embodiments, phospholipid is DPPC, DSPC or its mixture. In some embodiments, described helper lipid can be phospholipid.

[0164] The structured lipid may be cholesterol, an analog of cholesterol and / or a derivative of cholesterol.

[0165] 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).

[0166] In a more preferred embodiment of the small interfering nucleic acid pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier used simultaneously contains the four components of the cationic lipid, auxiliary 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, auxiliary 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, auxiliary lipid, structural lipid and pegylated lipid in the small interfering nucleic acid pharmaceutical composition is (50-70): (5-20): (30-60): (0.5-20).

[0167] The lipid nanoparticle particles formed by si1260 and si1848 and the above-mentioned lipid mixture have an average diameter of about 30nm to about 200nm, typically about 40nm to about 135nm, more typically, the average diameter of the lipid nanoparticle particles is about 50nm to about 120nm, about 50nm to about 100nm, about 60nm to about 90nm or about 70nm to about 90nm, 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 160nm.

[0168] 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, auxiliary 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.

[0169] The components of the pharmaceutical composition provided by the present invention can exist independently when 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:

[0170] The cationic lipid, auxiliary lipid, structural lipid and pegylated lipid are suspended in alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol used is 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 room temperature, for example, ethanol, propylene glycol, benzyl alcohol, tert-butyl alcohol, isopropanol, dimethyl sulfoxide, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, preferably one or more, preferably ethanol.

[0171] 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, and the pH of the buffered saline solution is adjusted to 4.0-6.2, preferably 5.0-5.2. The amount of the buffered saline solution is such that the concentration of the siRNA does not exceed 2 mg / mL, preferably 0.2-0.4 mg / mL. The buffered salt is selected from one or more of soluble acetate and soluble citrate, preferably sodium acetate and / or potassium acetate.

[0172] The lipid solution and the siRNA aqueous solution are mixed, and the mixed product is incubated at 4-60° C. for at least 2 minutes, preferably 5-30 minutes, to obtain an incubated lipid nanoparticle preparation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1:(1-9), preferably 1:3.

[0173] 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 pH value of 6.5-8, encapsulation efficiency of not less than 80%, particle size of 40-200nm, polydispersity index of not more than 0.30, and osmotic pressure of 250-400mOsm / kg; preferably, pH value of 7.2-7.6, encapsulation efficiency of not less than 90%, particle size of 60-100nm, polydispersity index of not more than 0.20, and osmotic pressure of 300-400mOsm / kg.

[0174] The concentration or dilution can be performed before, after or simultaneously with the removal of impurities. The impurity removal method can adopt various existing methods, such as using a tangential flow system, a hollow fiber column, ultrafiltration under 100KDa conditions, and the ultrafiltration exchange solution is a phosphate buffered saline (PBS) with a pH of 7.4. The sterilization method can adopt various existing methods, preferably filtration sterilization on a 0.22μm filter.

[0175] IV. siRNA Groups and siRNA Conjugates of the Present Invention

[0176] In one aspect, the present disclosure provides siRNA sets and siRNA conjugates of the present invention that are capable of inhibiting HBV gene expression.

[0177] 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.

[0178] 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 length ratio of the sense strand and the antisense strand 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 and the antisense strand of the siRNA is 19 / 21, 21 / 23 or 23 / 25. The siRNA group of the present invention comprises a first siRNA and a second siRNA.

[0179] First siRNA

[0180] 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 different 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 different 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 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom, 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 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom. 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 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom, 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 (e.g., not more than 3, not more than 2, not more than 1) nucleotides different therefrom.

[0181] 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).

[0182] In some embodiments, the sense 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.

[0183] 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.

[0184] In some embodiments, the sense strand of the siRNA and the antisense strand of the siRNA are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary. The substantially reverse complementary refers to no more than 3 base mismatches between the two nucleotide sequences; the substantially reverse complementary refers to no more than 1 base mismatch between the two nucleotide sequences; the completely reverse complementary refers to no mismatches between the two nucleotide sequences.

[0185] 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.

[0186] 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 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 CU, and the base composition of the nucleotide sequence IV is AG; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21; or c) the lengths of nucleotide sequences III and IV are both 3 nucleotides, 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) the lengths of nucleotide sequences III and IV are both 4 nucleotides, 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 lengths of nucleotide sequences III and IV are 2 nucleotides, 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.

[0187] 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 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 CA, and the base composition of the nucleotide sequence IV is UG; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21; or c) the length of nucleotide sequence III and IV are both 3 nucleotides, 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) the length of nucleotide sequence III and IV are both 4 nucleotides, 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 length of nucleotide sequence III and nucleotide sequence IV is 2 nucleotides, 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.

[0188] 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 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) the length of nucleotide sequence III and IV are both 3 nucleotides, 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) the length of nucleotide sequence III and IV are both 4 nucleotides, 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 length of nucleotide sequence III and nucleotide sequence IV is 2 nucleotides, 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.

[0189] In some embodiments, nucleotide sequence III and nucleotide sequence IV are of 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.

[0190] In some embodiments, the sense strand and the antisense strand are different in length, and the antisense strand further contains a nucleotide sequence V, the length of the nucleotide sequence V is 1 to 3 nucleotides, connected to the 3' end of the antisense strand, and constitutes the 3' overhang end of the antisense strand. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure 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 by the present disclosure can be 19 / 21, 21 / 23 or 23 / 25.

[0191] Each nucleotide in the nucleotide sequence V can be any nucleotide. In some embodiments, the nucleotide sequence V is 2 consecutive thymine deoxyribonucleotides (dTdT) or 2 consecutive uracil ribonucleotides (UU); in some embodiments, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA.

[0192] In some embodiments, the sense strand of the si1260 is a nucleotide sequence as shown in 5'-GCCGAUCCAUACUGCGGAA-3' (SEQID 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).

[0193] As mentioned above, the nucleotides in the siRNA disclosed herein are independently modified or unmodified nucleotides. In some embodiments, the nucleotides in the siRNA disclosed herein are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNA disclosed herein are modified nucleotides, and these modifications on the nucleotide groups will not significantly weaken or lose the function of the siRNA conjugate disclosed herein to inhibit the expression of the hepatitis B virus gene.

[0194] 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.

[0195] In some embodiments, all nucleotides in the siRNA described in the present disclosure are modified nucleotides.

[0196] 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 chain are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the siRNA sense chain 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 chain are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the siRNA antisense chain are methoxy-modified nucleotides.

[0197] 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'. Wherein, capital letters C, G, U, A and T represent the base composition of nucleotides; lowercase letter m represents that the ribose group of a nucleotide on the left side of the letter m is a 2'-methoxy ribose group formed by 2'-hydroxyl being replaced by methoxy group; lowercase letter f represents that the ribose group of a nucleotide on the left side of the letter f is a 2'-fluoro ribose group formed by 2'-hydroxyl being replaced by fluorine.

[0198] 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 strand and the antisense strand of the siRNA provided by the present disclosure is a phosphate group with a modified group. In some embodiments, the phosphate group with a modified group is a thiophosphate group in which at least one oxygen atom in the phosphodiester bond in the phosphate group is replaced by a sulfur atom; in some embodiments, the structure of the thiophosphate group is shown in the following formula:

[0199]

[0200] These modifications can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.

[0201] In some embodiments, in the siRNA provided by the present disclosure, the phosphorothioate linkage is present in 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 of the above. 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:

[0202] Between the first nucleotide and the second nucleotide at the 5' end of the sense strand;

[0203] Between the second nucleotide and the third nucleotide at the 5' end of the sense strand;

[0204] Between the first nucleotide and the second nucleotide at the 3' end of the sense strand;

[0205] Between the second nucleotide and the third nucleotide at the 3' end of the sense strand;

[0206] Between the first nucleotide and the second nucleotide at the 5' end of the antisense strand;

[0207] Between the second nucleotide and the third nucleotide at the 5' end of the antisense strand;

[0208] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0209] Between the second nucleotide and the third nucleotide at the 3' end of the antisense strand.

[0210] 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'. Wherein, capital letters C, G, U, A and T represent the base composition of nucleotides; lowercase letter m represents that the ribose group of a nucleotide on the left side of the letter m is a 2'-methoxy ribose group formed by 2'-hydroxyl replaced by methoxy; lowercase letter f represents that the ribose group of a nucleotide on the left side of the letter f is a 2'-fluoro ribose group formed by 2'-hydroxyl replaced by fluorine; lowercase letter s represents that the two nucleotides on the left and right of the letter s are connected by a thiophosphate group.

[0211] 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, the 5'-phosphate nucleotide may have the following structure:

[0212]

[0213] 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.

[0214] Or nucleotides modified with 5'-phosphate analogs as disclosed by Anastasia Khvorova, Jonathan K Watts in The chemical evolution of oligonucleotide therapeutics of clinical utility. Nature biotechnology, 35(3), 238-248.

[0215] 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, capital letters C, G, U, A and T represent the base composition of the nucleotide; lowercase letter m indicates that the ribose group of the nucleotide on the left side of the letter m is a 2'-methoxyribose group in which the 2'-hydroxyl group is replaced by a methoxy group; lowercase letter f indicates that the ribose group of the nucleotide on the left side of the letter f is a 2'-fluororibose group in which the 2'-hydroxyl group is replaced by a fluorine group; lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by a thiophosphate group; P1 indicates that the adjacent nucleotide on the right side of the letter P is a 5'-phosphate nucleotide or a nucleotide modified by a 5'-phosphate analogue.

[0216] In some embodiments, the si1260 is any one listed in Table 3a.

[0217] Second siRNA

[0218] According to the present disclosure, the siRNA may be a second siRNA. The second siRNA contains 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 RNA derived from all HBV cccDNAs including HBx. In some embodiments, the second siRNA is any siRNA in Table 3b and Table 4. In some embodiments, the sense strand of the second siRNA contains 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 contains 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.

[0219] 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).

[0220] In some embodiments, the sense 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.

[0221] 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.

[0222] 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.

[0223] 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: 5, 7 or 43, and has the same length as the nucleotide sequence IV.

[0224] 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) the lengths of nucleotide sequences III and IV are both 3 nucleotides, 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) the lengths of nucleotide sequences III and IV are both 4 nucleotides, 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 lengths of nucleotide sequences III and IV are 2 nucleotides, 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.

[0225] 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) the length of nucleotide sequence III and IV are both 3 nucleotides, 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) the length of nucleotide sequence III and IV are both 4 nucleotides, 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 length of nucleotide sequence III and nucleotide sequence IV is 2 nucleotides, 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.

[0226] 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) the length of nucleotide sequence III and IV are both 3 nucleotides, and in the direction 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) the length of nucleotide sequence III and IV are both 4 nucleotides, and in the direction 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 length of nucleotide sequence III and nucleotide sequence IV is 2 nucleotides, and in the direction 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.

[0227] In some embodiments, nucleotide sequence III and nucleotide sequence IV are of 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.

[0228] In some embodiments, the sense strand and the antisense strand are different in length, and the antisense strand further contains a nucleotide sequence V, the length of the nucleotide sequence V is 1 to 3 nucleotides, connected to the 3' end of the antisense strand, and constitutes the 3' overhang end of the antisense strand. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure 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 by the present disclosure can be 19 / 21, 21 / 23 or 23 / 25.

[0229] Each nucleotide in the nucleotide sequence V can be any nucleotide. In some embodiments, the nucleotide sequence V is 2 consecutive thymine deoxyribonucleotides (dTdT) or 2 consecutive uracil nucleotides (UU); in some embodiments, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA.

[0230] In some embodiments, the sense strand of si1848 is a nucleotide sequence as shown in 5'-GUUCAUGUCCUACUGUUCA-3' (SEQID 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).

[0231] As mentioned above, the nucleotides in the siRNA disclosed herein are independently modified or unmodified nucleotides. In some embodiments, the nucleotides in the siRNA disclosed herein are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNA disclosed herein are modified nucleotides, and these modifications on the nucleotide groups will not significantly weaken or lose the function of the siRNA conjugate disclosed herein to inhibit the expression of the hepatitis B virus gene.

[0232] 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.

[0233] In some embodiments, all nucleotides in the siRNA described in the present disclosure are modified nucleotides.

[0234] 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 chain are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the siRNA sense chain 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 chain are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the siRNA antisense chain are methoxy-modified nucleotides.

[0235] 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'. Wherein, capital letters C, G, U, A and T represent the base composition of nucleotides; lowercase letter m represents that the ribose group of a nucleotide on the left side of the letter m is a 2'-methoxy ribose group formed by 2'-hydroxyl being replaced by methoxy group; lowercase letter f represents that the ribose group of a nucleotide on the left side of the letter f is a 2'-fluoro ribose group formed by 2'-hydroxyl being replaced by fluorine.

[0236] 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 strand and the antisense strand of the siRNA provided by the present disclosure is a phosphate group with a modified group. In some embodiments, the phosphate group with a modified group is a thiophosphate group in which at least one oxygen atom in the phosphodiester bond in the phosphate group is replaced by a sulfur atom; in some embodiments, the structure of the thiophosphate group is shown in the following formula:

[0237]

[0238] These modifications can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.

[0239] In some embodiments, in the siRNA provided by the present disclosure, the phosphorothioate linkage is present in 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 of the above. 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:

[0240] Between the first nucleotide and the second nucleotide at the 5' end of the sense strand;

[0241] Between the second nucleotide and the third nucleotide at the 5' end of the sense strand;

[0242] Between the first nucleotide and the second nucleotide at the 3' end of the sense strand;

[0243] Between the second nucleotide and the third nucleotide at the 3' end of the sense strand;

[0244] Between the first nucleotide and the second nucleotide at the 5' end of the antisense strand;

[0245] Between the second nucleotide and the third nucleotide at the 5' end of the antisense strand;

[0246] between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and

[0247] Between the second nucleotide and the third nucleotide at the 3' end of the antisense strand.

[0248] 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'. Wherein, capital letters C, G, U, A and T represent the base composition of nucleotides; lowercase letter m represents that the ribose group of a nucleotide on the left side of the letter m is a 2'-methoxy ribose group formed by 2'-hydroxyl replaced by methoxy; lowercase letter f represents that the ribose group of a nucleotide on the left side of the letter f is a 2'-fluoro ribose group formed by 2'-hydroxyl replaced by fluorine; lowercase letter s represents that the two nucleotides on the left and right of the letter s are connected by a thiophosphate group.

[0249] 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, the 5'-phosphate nucleotide may have the following structure:

[0250]

[0251] 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.

[0252] Or nucleotides modified with 5'-phosphate analogs as disclosed by Anastasia Khvorova, Jonathan K Watts in The chemical evolution of oligonucleotide therapeutics of clinical utility. Nature biotechnology, 35(3), 238-248.

[0253] 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, capital letters C, G, U, A and T represent the base composition of the nucleotide; lowercase letter m indicates that the ribose group of the nucleotide on the left side of the letter m is a 2'-methoxyribose group in which the 2'-hydroxyl group is replaced by a methoxy group; lowercase letter f indicates that the ribose group of the nucleotide on the left side of the letter f is a 2'-fluororibose group in which the 2'-hydroxyl group is replaced by a fluorine group; lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by a thiophosphate group; P1 indicates that the adjacent nucleotide on the right side of the letter P is a 5'-phosphate nucleotide or a nucleotide modified by a 5'-phosphate analogue.

[0254] In some embodiments, the si1848 is any one listed in Table 3b.

[0255] The inventors of the present disclosure unexpectedly found that the siRNA conjugates of the present disclosure have significantly improved stability in plasma, low off-target effects, and also show HBV mRNA silencing activity that is not significantly reduced. In some embodiments, the siRNA of the present disclosure can be selected from the siRNAs shown in Tables 3a-3b or any combination thereof. The siRNA conjugates containing these siRNAs show higher HBV mRNA silencing activity.

[0256] Table 3a The first siRNA sequence of the present disclosure

[0257]

[0258]

[0259] Table 3b Second siRNA sequences disclosed herein

[0260]

[0261]

[0262] Note: Capital letters C, G, U, and A represent the base composition of nucleotides; lowercase letter m represents that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of letter f is a fluorinated-modified nucleotide; lowercase letter s represents that the two nucleotides on the left and right of the letter are connected by a thiophosphate group; P1 represents that the nucleotide adjacent 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 representing a specific modification, wherein the letter combination VP represents that the nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modified nucleotide, the letter combination Ps represents that the nucleotide adjacent to the right of the letter combination Ps is a thiophosphate modified nucleotide, and the capital letter P represents that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.

[0263] The siRNA provided in 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. The 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.

[0264] The present disclosure provides a siRNA conjugate, wherein the siRNA conjugate contains the above siRNA and a conjugation group conjugated to the siRNA.

[0265] 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, also at the 5' end of the antisense strand, and can also be in the internal sequence of siRNA. In some embodiments, the conjugation site of siRNA and conjugated group is at the 3' end of the siRNA sense strand.

[0266] 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 inhepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7.

[0267] In some embodiments, the siRNA and the conjugated group can be connected by acid-labile or reducible chemical bonds, which can be degraded in the acidic environment of the cell endosome, thereby making the siRNA free. For non-degradable conjugation methods, the conjugated group can be connected to the sense strand of the siRNA, thereby minimizing the effect of conjugation on the activity of the siRNA.

[0268] 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 incorporated herein by reference in their entirety).

[0269] 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 hepatic parenchymal cells, such as asialoglycoproteins, asialosugar residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.

[0270] In some embodiments, each ligand is independently selected from a ligand that can bind to a cell surface receptor. In some embodiments, at least one ligand is a ligand that can bind to a hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand that can bind to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand that can bind to a human hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand that can bind to a liver surface asialoglycoprotein receptor (ASGPR). The types of these ligands are well known to those skilled in the art, and their function is generally to bind to a specific receptor on the surface of the target cell, mediating the delivery of the siRNA connected to the ligand to the target cell.

[0271] In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to an asialoglycoprotein receptor (ASGPR) on the surface of a mammalian hepatocyte. 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 derivative of a sugar.

[0272] 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 the following sugars: 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.

[0273] 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-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 heptanoside 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 refer to, for example, the records of CN105378082A, and all of its disclosures are incorporated herein by reference.

[0274] 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 and the conjugated group containing the galactose or N-acetylgalactosamine molecule as the targeting group form the siRNA conjugate respectively: 1, 1: 2, 1: 3, or 1: 4. 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 in the present disclosure is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0275] The targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group. These linkers, the types of targeting groups and the connection mode with siRNA can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.

[0276] In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be a structure as shown below:

[0277]

[0278] The above-mentioned siRNA conjugates can be synthesized by methods that have been described in detail in the prior art. For example, WO2015006740A2 describes in detail the preparation methods of various siRNA conjugates. The siRNA conjugates of the present disclosure are obtained by methods well known to those skilled in the art. For example, the preparation method of siRNA conjugates is described in WO2014025805A1, and the preparation method of siRNA conjugates is described by Rajeev et al. in ChemBioChem 2015, 16, 903-908.

[0279] The inventors of the present disclosure unexpectedly discovered that the siRNA conjugates of the present disclosure have significantly improved stability in plasma, low off-target effects, and also exhibit HBV mRNA silencing activity that is not significantly reduced. In some embodiments, the siRNA of the present disclosure may be one of the siRNAs shown in Tables 3a-3b. The siRNA conjugates containing these siRNAs exhibit higher HBV mRNA silencing activity.

[0280] 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, see the above description of the pharmaceutical composition disclosed herein.

[0281] V. Kit of the present invention

[0282] The present disclosure provides a kit comprising at least one of the siRNA, siRNA set, pharmaceutical composition and siRNA conjugate of the present disclosure.

[0283] When a siRNA group is included, the first siRNA and the second siRNA may be present alone or in the form of a mixture of the two siRNAs.

[0284] 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.

[0285] In some embodiments, the kit may further include other ingredients, such as stabilizers or preservatives, etc. In some embodiments, the kit described herein may include 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 include instructions for mixing the modified siRNA with a pharmaceutically acceptable carrier and / or excipient or other ingredients (if any).

[0286] 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, dry 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.

[0287] VI. Uses of siRNA, pharmaceutical compositions and siRNA conjugates of the present invention

[0288] The present invention also provides the use of the siRNA, siRNA group, pharmaceutical composition and conjugate described above in the preparation of a drug 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 group, pharmaceutical composition and conjugate to a patient in need, and achieving the purpose of treating hepatitis B through the mechanism of RNA interference.

[0289] The term "administration / administration" as used herein refers to placing the siRNA, siRNA group, pharmaceutical composition and / or siRNA conjugate of the present disclosure into the subject's body by a method or approach that at least partially positions the siRNA, siRNA group, pharmaceutical composition and / or siRNA conjugate of the present disclosure at 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 more siRNA, siRNA group, pharmaceutical composition and / or siRNA conjugate of the present disclosure being delivered to a specific site compared to the entire body of the subject; while systemic administration results in the delivery of the siRNA, siRNA group, pharmaceutical composition and / or siRNA conjugate of the present disclosure 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 specifically delivering the drug to the liver is preferred.

[0290] 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 oral administration and sublingual administration). The administration frequency may be once or more every day, every week, every two weeks, every three weeks, every month, every two months, every three months, every six months, or every year.

[0291] 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 gender 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 LD 50 / ED 50The therapeutic index is preferably expressed as a ratio of 1:1 to 2:1. siRNA or pharmaceutical composition that exhibits a high therapeutic index is preferred. The range of human dosage can be derived based on data obtained from cell culture assays and animal studies.

[0292] When administering the siRNA, siRNA group, pharmaceutical composition, and / or siRNA conjugate described in the present disclosure, for example, for 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 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.

[0293] 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 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 hepatoma cell lines such as SMMC-7721, HepG2, Huh7, or isolated primary liver cells.

[0294] The method provided by the present disclosure is used to inhibit the expression of HBV genes in cells. The amount of siRNA used in the modified siRNA, siRNA group, pharmaceutical composition and / or siRNA conjugate provided is generally such an amount: it is sufficient to reduce the expression of the target gene and result in an extracellular concentration of 1pM to 1μM, or 0.01nM to 100nM, or 0.05nM to 50nM or 0.05nM to about 5nM at the surface of the target cell. The amount required to reach this local concentration will vary with various factors, including the delivery method, delivery site, the number of cell layers between the delivery site and the target cell or tissue, the 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.

[0295] The following specific embodiments are used to illustrate the scheme of the present invention, but they are not intended to limit the technical scheme 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 protection scope of the present invention.

[0296] Example

[0297] Unless otherwise stated, the materials used in the examples herein are all commercially available, and the various specific experimental methods used to perform the experiments are all conventional experimental methods in the art or follow the steps and conditions recommended by the manufacturer, and can be routinely determined by those skilled in the art as needed.

[0298] Preparation Example 1: siRNA sequence screening and synthesis

[0299] For the siRNA target region of the present invention and its location in the HBV genome, Figure 2 As shown in A, the siRNA targets the conserved sequence of HBV and covers the entire HBV genome. After sequence comparison, the sequence of the above siRNA does not have a homologous sequence longer than 16 bases with the genome sequence of humans or mice. According to the siRNA design principles, Guangzhou RiboBio Biotechnology Co., Ltd. (RiboBio) or Shanghai GenePharma 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 2 A; the chemically synthesized siRNA sequences are shown in Table 4.

[0300] Table 4 Chemically synthesized siRNA sequence list

[0301]

[0302]

[0303] The above siRNA is formed by annealing equimolar sense and antisense strands. The chemically synthesized siRNA was dissolved in RNase Free ddH2O in advance to prepare a 20 μM stock solution, which was aliquoted and frozen at -80°C to avoid repeated freezing and thawing. When siRNA was transfected alone, Lipofectamine RNAiMAX (Thermo Fisher (Invitrogen) Company, Catalog No. 13778-150) was used for transfection, and when siRNA was co-transfected with DNA, Lipofectamine 3000 (Thermo Fisher (Invitrogen) Company, Catalog No. L3000075) was used for transfection.

[0304] Preparation Example 2: Design and Synthesis of Chemically Modified siRNA

[0305] Guangzhou RiboBio and Shanghai GenePharma were commissioned to synthesize small interfering nucleic acids with modifications shown in one of A1-A11 and / or B1-B11 (Table 5). An equimolar mixture of sense and antisense strands was dissolved in an annealing salt solution, followed by conventional annealing to form siRNA double strands.

[0306] Table 5 Chemically modified siRNA sequence list

[0307]

[0308]

[0309] 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.

[0310] Preparation Example 3: Preparation and Characterization of Lipid Nanoparticles (LNP)

[0311] Four lipid compounds (i.e., cationic lipids, auxiliary lipids, structural lipids, and pegylated lipids) were suspended in ethanol at a molar ratio of 50:10:38.5:3.05 and mixed, and the total mass concentration of the four lipid compounds was 2.0286 mg / mL. The synthesized siRNA listed in Table 4 or Table 5 was dissolved in a 6.25 mM sodium acetate (pH 5.2) solution, so that the concentration of siRNA was 0.048 mg / mL, and the weight ratio of RNA to LNP was 1:14. (For the case of using si1260 and si1848 alone, the concentration of siRNA was 0.0483 mg / mL; for the case of using si1260 and si1848 in combination, the two siRNAs were mixed in equal proportions, i.e., the concentration of each siRNA was 0.024 mg / mL). The lipid ethanol solution and the siRNA sodium acetate aqueous solution were mixed very quickly at a volume ratio of 1:3 to obtain an LNP-siRNA preparation. Table 6 shows the specific composition of the lipid nanoparticle preparation obtained after mixing.

[0312] Table 6 Composition of LNP-siRNA lipid nanoparticle preparations

[0313]

[0314] 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), and the ultrafiltration exchange solution was PBS at pH 7.4. The preparation can be concentrated or diluted to the desired siRNA concentration while ultrafiltration. The ultrafiltered preparation was sterilized by filtration on a 0.22 μm filter. The resulting pharmaceutical composition LNP-siRNA was stored at 4° C. before use, and the relevant physicochemical properties were tested. The test results are shown in Table 7.

[0315] Table 7 Physicochemical parameters of the pharmaceutical composition

[0316]

[0317]

[0318] The encapsulation efficiency was detected by RiboGreen method, and the reagents used (Quanti-iT TM RNA Reagent and Kit) was purchased from Thermo Fisher (Invitrogen), catalog number R11490. The fluorescence intensity of siRNA in the sample was detected according to the instructions, and the encapsulation efficiency was calculated according to the following formula:

[0319] 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%.

[0320] Other physical and chemical parameters are measured using conventional techniques well known to those skilled in the art.

[0321] Preparation Example 4: Design, synthesis and characterization of GalNac-siRNA

[0322] The siRNA conjugates of the present disclosure shown in Table 8 were synthesized: L96-si1260-2A2S and L96-si1848-2A2S. The siRNA conjugates contained siRNAs having the sense and antisense strand sequences corresponding to the siRNA conjugates in Table 8. The difference in the preparation methods is that the sense and antisense strands are synthesized according to the sense and antisense strand sequences of the siRNA conjugates in Table 8, respectively.

[0323] After the preparation, the molecular weight of the prepared siRNA conjugates was detected, and the measured values ​​were consistent with the theoretical values, indicating that the synthesized siRNA conjugates were the double-stranded nucleic acid sequences with L96 conjugated molecules of the target design. The siRNA contained in the siRNA conjugates has the sequence corresponding to the siRNA conjugates L96-si1260-2A2S and / or L96-si1848-2A2S shown in Table 8.

[0324] Table 8 siRNA conjugates

[0325]

[0326] Among them, capital letters C, G, U, and A represent the base composition of the nucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorinated-modified nucleotide; lowercase letter s indicates that the two nucleotides on 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.

[0327] After the siRNA or siRNA conjugate of the present disclosure is prepared, it is lyophilized into a solid powder and stored for later use. When used, it can be redissolved into a solution of a desired concentration using, for example, water for injection, normal saline (NS), phosphate buffer (PB) or phosphate buffered saline (PBS).

[0328] Experimental Example 1: siRNA sequence screening

[0329] HepG2-HBV / EGFP is a stable cell line that was constructed in-house and stably integrated with multiple copies of the HBV genome (serotype: ayw; genotype: D; GenBank retrieval number: V01460.1) sequence with an EGFP reporter gene using the sleepy beauty transposon system. It does not express EGFP fluorescent protein under normal conditions. After transduction with an adenovirus expressing Cre recombinase (Ad-Cre), it can be cut and circularized to form recombinant cccDNA and express EGFP fluorescent protein. This system can be used to indicate the presence and transcription of cccDNA. HepG2-HBV / EGFP cells were cultured in DMEM medium (Corning, catalog number 10-013-CV) containing 10% fetal bovine serum FBS (Biological Industries, catalog number 04-001-1A-US) at 37°C and 5% CO2 incubator. HepG2-HBV / EGFP cells were cultured for 10 5 The cells were inoculated into 96-well cell culture plates at a density of 100 cells / well and adhered to the wall overnight in a 37°C, 5% CO2 incubator. After the cells grew to 80-90% confluence, 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, the ELISA kit (Hepatitis B virus surface antigen detection kit (chemiluminescence method), Antu Biological Company, catalog number 30030301CL01) was used to detect the HBsAg antigen level in the supernatant, and the fully automatic high-content imaging analysis system PE Operetta (Perkin Elmer) was used to detect the expression of EGFP green fluorescent protein in the cells.

[0330] HepAD38 is a stable cell line that integrates 1.1 copies of the HBV genome (serotype: ayw, genotype: D). The cell line can regulate the transcription of HBV pgRNA and subgenomic RNA from the integrated HBV genome through tet-off elements, and translate and express various viral proteins. The 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, catalog number D9891). HepAD38 cells were cultured at 10 5 The cells were inoculated into 96-well cell culture plates at a density of 100 / well and adhered to the wall overnight in a 37°C, 5% CO2 incubator. The culture medium was then replaced with a DOX-free medium. On the 4th day after plating, 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. Cells were collected 3 days after siRNA transfection, and the levels of intracellular viral proteins HBc and HBx were detected by Western Blot, and the levels of intracellular viral core particle DNA were detected by Southern Blot.

[0331] HepG2-NTCP cells are cell lines that stably overexpress the HBV-specific receptor NTCP, which can support HBV infection and replication throughout the life cycle. HepG2-NTCP cells were cultured in a complete medium containing 2% DMSO (Sigma, Cat. No. D2650) while being plated. After the cells adhered, HepG2-NTCP cells (MOI 200) were infected with HBV virus supernatant concentrated from the supernatant of HepAD38 cells. The infection solution was a complete medium containing 2% DMSO and 4% PEG8000 (Sigma, Cat. No. 89510). The supernatant was removed 16 hours after infection, washed 3 to 6 times with PBS, and then continued to be cultured with a complete medium containing 2% DMSO. On the 4th day after infection, siRNA was transfected into the cells using Lipofectamine RNAiMAX transfection reagent, with si-NC as a negative transfection control. Fresh medium was replaced 6 hours after transfection. Three days after siRNA transfection, cells were collected 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 cell 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) ( Premix Ex TaqTM (Tli RNaseH Plus, Bulk, Takara, Catalog No. RR420L) was used to detect the levels of intracellular HBV pgRNA, HBV total RNA and housekeeping gene GAPDH mRNA. The primers used for Q-PCR are shown in Table 9 below.

[0332] Table 9 Q-PCR primer sequence list

[0333]

[0334]

[0335] PLC / PRF / 5 cells are clinically isolated and constructed human liver cancer cell lines containing HBV DNA integration, which can be used to evaluate the interference effect of siRNA targeting HBV integrated DNA (intHBV) derived mRNA. PLC / PRF / 5 cells were cultured in RIPM 1640 (Corning, Cat. No. 10-040-CV) medium containing 10% FBS. PLC / PRF / 5 cells were cultured at 10 5The cells were inoculated 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, and si-NC was used as a negative transfection control. Fresh culture medium was replaced 6 hours after transfection. Cells were collected 3 days after siRNA transfection, and HBsAg antigen levels in the supernatant were detected using an ELISA kit (Antu Biotechnology).

[0336] The siRNA transfection process was performed according to the instructions of the transfection reagent. Taking one well of a 12-well plate as an example, 2.5 μL siRNA (20 μM) (50 pmol) was mixed with 50 μL Opti-MEM (Gibco, Cat. No. 31985-070) and allowed to stand at room temperature for 5 min; at the same time, 5 μL Lipofectamine RNAiMAX transfection reagent was mixed with 50 μL Opti-MEM and allowed to stand at room temperature for 5 min; the two solutions were mixed and allowed to stand at room temperature for 5 to 10 min. The transfection reagent was slowly added dropwise to the well plate to a final concentration of 50 nM. The medium was replaced with fresh medium 6 hours after transfection.

[0337] like Figure 2 As shown in B, the experimental results show that the 20 designed and synthesized siRNAs can inhibit the expression level of EGFP green fluorescent protein in HepG2-HBV / EGFP cells to varying degrees. Considering that EGFP is translated and expressed by HBV pgRNA, the results suggest that the above siRNAs can effectively interfere with the level of HBV pgRNA; compared with the control cells transfected with si-NC, si-220 to si-1912 in the above siRNAs can significantly reduce the expression level of HBsAg, and the inhibition rate is greater than 80%; while si-2303 to si-2831 in the above siRNAs cannot reduce the expression level of HBsAg. Figure 2 The distribution of the target sequences and target regions of siRNA in A on HBV RNA shows that si-2303 to si-2831 only target HBV pgRNA and cannot target PreS / SmRNA encoding HBsAg. This result is consistent with theoretical expectations.

[0338] The above siRNAs can effectively inhibit the secretion level of HBV e antigen (HBeAg) in the HepG2-NTCP infection system, and can significantly inhibit the intracellular HBV pgRNA level. Among them, si-220~si-1912 can significantly inhibit the intracellular HBV total RNA level, while si-2303~si-2831 cannot reduce the HBV total RNA level. Figure 2The distribution of the target sequences and target regions of siRNA in A on HBV RNA shows that si-2303 to si-2831 can only target HBV pgRNA, but not PreS / SmRNA and X mRNA, and therefore cannot reduce the overall HBV RNA level. This result is consistent with theoretical expectations ( Figure 2 C).

[0339] The above siRNAs can inhibit the expression levels of HBc and HBx in HepAD38 cells to varying degrees, among which si-675 to si-1606 have a more significant effect in inhibiting HBc and HBx. Considering that si-675 to si-1260 do not directly target and interfere with the 0.7kb XmRNA, it is speculated that it is caused by interfering with HBV pgRNA and causing a decrease in viral replication. Southern Blot test results also showed that the above siRNAs can significantly inhibit the viral replication level in HepAD38 cells ( Figure 2 D). si-220 to si-1260 can significantly inhibit the secretion of HBsAg antigen in the supernatant of PLC / PRF / 5 cells, while si-1606 to si-2831 cannot inhibit the level of HBV surface antigen, suggesting that the above siRNAs of si-1606 to si-2831 cannot target the mRNA sequence derived from HBV integration (intHBV) ( Figure 2 E).

[0340] The above results suggest that among the 20 siRNAs, siRNAs from si-220 to si-1260 can effectively inhibit the expression of HBV cccDNA and HBV integration-derived HBsAg antigens, but according to the distribution of their target sequences and target regions on HBV RNA, the above siRNAs from si-220 to si-1260 cannot effectively target X mRNA; siRNAs from si-1606 to si-2831 cannot target HBV integration; according to the distribution of their target sequences and target regions on HBV RNA, siRNAs from si1606 to si1912 can simultaneously target the common sequence of mRNA derived from HBV cccDNA, while siRNAs from si-2303 to si-2831 can only target HBV pgRNA, not PreS / S mRNA and X mRNA, and cannot target HBV integration. Therefore, it is necessary to combine 2 or more siRNAs to achieve simultaneous targeting of HBV cccDNA and all HBV-related mRNAs derived from HBV integration.

[0341] Experimental Example 2: Broad-spectrum screening of siRNA sequences

[0342] HepG2 cells were transfected with a replicon plasmid (pHBV1.3) carrying 1.3 copies of HBV genome sequences 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 cultured at 10 5 The cells were inoculated 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, the cell culture supernatant was collected, and the level of HBV antigen in the supernatant was detected using an ELISA kit (Antu Biotechnology). At the same time, total RNA in the cells was extracted, and the PrimeScriptTM RT reagentKit with gDNA Eraser (Takara, Cat. No. RR047A) kit was used to reverse transcribe RNA into cDNA, and Q-PCR was used to detect the levels of HBV pgRNA, total RNA and housekeeping gene GAPDH mRNA in the cells.

[0343] like Figure 3 As shown in AD, the experimental results show that among the above siRNAs, si-220, si-675, si-726, si-1260, si-1606, si-1848, si-1912, si-2413, etc. have good interference effects on different HBV genotypes including four genotypes A / B / C / D, and have good interference effects in the human primary hepatocyte HBV infection model ( Figure 3 E).

[0344] The conservation of the above siRNA targeting sequences of different HBV genotypes was further analyzed. The ratio of 19-mer sequences that can completely match the siRNA targeting sequence in 11,185 HBV genomes was used as an indicator to evaluate its conservation, and the proportion of 17-mer sequences that can completely match the nucleic acid at positions 2-18 of the siRNA targeting sequence was calculated. The results are shown in Table 10 below. Although si-255 is highly conservative (19-mer: 94.14%; 17-mer: 98.42%), the results in the previous pHBV1.3 transfection model and the human primary hepatocyte HBV infection model showed that its interference effect was not ideal; and although si-1912 can interfere with all HBV cccDNA-derived RNA common sequences and can well inhibit HBV antigens and HBV RNA levels, its conservation is not ideal (19-mer: 32.01%; 17-mer: 37.66%). Finally, we selected siRNAs with high conservation (>70%) and good interference effects for follow-up 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%).

[0345] Table 10 Conservative analysis of siRNA targeting HBV genome sequences

[0346] siRNA 19-mer complete coverage 19-mer complete coverage si-220 63.45％(7,097 / 11,185) 63.76％(7,131 / 11,185) si-255 94.14％(10,530 / 11,185) 98.42％(11,008 / 11,185) si-439 51.01％(5,705 / 11,185) 52.10％(5,827 / 11,185) si-469 51.77％(5.791 / 11,185) 51.93％(5,808 / 11,185) si-675 90.43％(10,115 / 11,185) 91.01％(10,179 / 11,185) si-726 77.24％(8,639 / 11,185) 77.56％(8,675 / 11,185) si-980 24.37％(2,726 / 11,185) 24.39％(2,728 / 11,185) si-981 23.95％(2,679 / 11,185) 24.39％(2,728 / 11,185) si-1260 91.42％(10,225 / 11,185) 92.96％(10,398 / 11,185) si-1606 79.32％(8,872 / 11,185) 79.77％(8,922 / 11,185) si-1848 71.94％(8,047 / 11,185) 72.00％(8,053 / 11,185) si-1857 71.95％(8,048 / 11,185) 72.21％(8,077 / 11,185) si-1903 2.825％(316 / 11,185) 2.834％(317 / 11,185) si-1912 32.01％(3,580 / 11,185) 37.66％(4,212 / 11,185) si-2303 6.285％(703 / 11,185) 6.688％(748 / 11,185) si-2330 13.71％(1,534 / 11,185) 13.72％(1,535 / 11,185) si-2332 13.72％(1,535 / 11,185) 13.80％(1,543 / 11,185) si-2390 74.04％(8,281 / 11,185) 74.30％(8,311 / 11,185) si-2413 90.02％(10,069 / 11,185) 90.36％(10,107 / 11,185) si-2831 21.26％(2,378 / 11,185) 21.31％(2,384 / 11,185) si74 71.49％(7,996 / 11,185) 85.86％(9,604 / 11,185) si77 82.22％(9,196 / 11,185) 86.03％(9,623 / 11,185)

[0347] Experimental Example 3: siRNA sequence combination screening

[0348] According to previous studies, none of the above single siRNAs can simultaneously target the common sequences of HBV integration sources and all HBV cccDNA-derived RNAs. Among them, si-675, si-726, and si-1260 can target all HBV RNAs and HBV integration (intHBV) source sequences except HBx; si-1606 and si-1848 can target all HBV RNAs including HBx but cannot target intHBV source sequences; si-2413 can only target interfering PreC RNA / pgRNA, so it is necessary to achieve targeting of all forms of HBV cccDNA and HBV integration-derived RNAs through a combination of two siRNAs while targeting more HBV gene sequences. The proportion of targeting different HBV genotype sequences after the combination of the two siRNAs was further calculated. As shown in Table 11 below: Among them, ARC-520 (si74+si77) can only target less than 90% (19-mer) of HBV gene sequences. A combination with a conservativeness (19-mer) > 95% was subsequently selected for further verification.

[0349] Table 11 Conservative analysis of the combined siRNA targeting HBV genome sequences

[0350]

[0351] HepG2 cells were transfected with 1.3 copies of the replicon plasmid (pHBV1.3) of different genotype HBV genome sequences to verify the targeted interference effect of the above siRNA. HepG2 cells were cultured in DMEM medium containing 10% FBS. 5 Cells were inoculated into 96-well cell culture plates at a density of 100 cells / well. After adherence, pHBV1.3 plasmid and siRNA were co-transfected into cells using Lipofectamine 3000 transfection preparation. si-NC was used as a negative control. On the third day after transfection, the cell culture supernatant was collected, and the level of HBV antigen in the supernatant was detected using an ELISA kit (Antu Biotechnology). At the same time, total RNA in the cells was extracted, and the RNA was reverse transcribed into cDNA using the PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara, Cat. No. RR047A). The levels of HBV pgRNA, total RNA, and housekeeping gene GAPDH mRNA in the cells were detected using Q-PCR.

[0352] The siRNAs with different concentrations were further transfected and their IC50 values ​​were calculated. Figure 4 As shown: Among them, si-1260 (IC 50 0.2084 nM) and si-77 (IC 500.1235nM) has a lower IC 50 Value, si-726 (IC 50 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 31.03nM) was poorer than that of other siRNAs ( Figure 4 A). Two of the above siRNAs were further combined, and the siRNA sequence group with a sequence conservation of >90% after the combination was selected for antiviral effect verification. The concentration ratio of the two siRNAs was 1:1 (the concentration of each single siRNA was 25nM, and the concentration of the two siRNA sequence group was 50nM), and the antiviral effect was synchronously compared with that of a single siRNA (the concentration of a single siRNA was 25nM and 50nM); the test was carried out in the pHBV1.3 transfection model (genotype D), HepAD38 stable transfection strain, PLC / PRF / 5 virus integrated cell line and HepG2-NTCP virus infection model. The results showed that by combining a siRNA targeting 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), it is possible to simultaneously target all HBV RNA and intHBV RNA, and the conservatism of the above combinations is >90% (except si-726+si-1848), which is better than ARC-520 (i.e. si74+si77, which has a conservatism of <90% and cannot target intHBV RNA). Among them, si-1260+si-1606 (19-mer: 98.03%) and si-1260+si-1848 (19-mer: 98.55%) have better inhibitory effects and can target >98% of HBV genome sequences ( Figure 4 BG).

[0353] To summarize the above, the current results show that by combining a siRNA targeting 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), it is possible to simultaneously target all HBV RNA and intHBV RNA with high conservation; the best combination is the combination of two siRNAs si-1260+si-1606 (19-mer: 98.03%) or si-1260+si-1848 (19-mer: 98.55%), which can simultaneously target all forms of HBV RNA and intHBV RNA including HBx, and can simultaneously target >98% of HBV genotype sequences. Compared with ARC-520 (19-mer: 89.82%), which cannot target intHBV RNA, these two siRNA combinations have significant advantages in sequence conservation and targeting various forms of HBV RNA. In subsequent experiments, si-1260+si-1848 were selected for further study.

[0354] Experimental Example 4: Verification of the broad-spectrum and multi-target effects of siRNA sequence combinations

[0355] 1. Multi-target effect evaluation

[0356] HepG2-HBV / EGFP cells were cultured at 5×10 5 Cells were inoculated into 24-well cell culture plates at a density of cells / well and allowed to adhere overnight in an incubator at 37°C and 5% CO2. After the cells grew to 80-90% confluence, Ad-Cre was added for treatment, and fresh culture medium was replaced after 6 hours. Three days after Ad-Cre transduction, the lipid nanoparticle preparation (LNP-siRNA, prepared according to the method described in Preparation Example 3) encapsulating the test siRNA combination (subsequent test siRNA combinations refer to si1260+si1848) was transfected into the cells; or the chemically synthesized test siRNA combination was transfected into the cells using Lipofectamine RNAiMAX transfection reagent (RNAiMAX-siRNA), and the siRNA transfection process was performed according to the instructions provided by the transfection reagent manufacturer. Three days after siRNA transfection, an ELISA kit (Antu Bio) was used to detect the level of HBV antigen in the supernatant, and the fully automatic high-content imaging analysis system PE Operetta (Perkin Elmer) was used to detect the expression of EGFP green fluorescent protein in the cells ( Figure 5 A and B).

[0357] HepAD38 cells were cultured in DMEM medium containing 10% FBS and doxycycline. HepAD38 cells were plated at 5×10 5 Cells were seeded into 24-well cell culture plates at a density of 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 4th day after plating, the lipid nanoparticle preparation (LNP-siRNA) encapsulating the test siRNA combination was 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, the cells were harvested, and the HBV antigen level in the supernatant was detected using an ELISA kit (Antu Biotechnology), and the levels of intracellular viral proteins HBc and HBx were detected using Western Blot, and the grayscale values ​​of the bands were statistically analyzed ( Figure 5 C and D).

[0358] PLC / PRF / 5 cells were plated at 5×10 5 The cells were inoculated into 24-well cell culture plates at a density of 100 cells / well. The lipid nanoparticle formulation (LNP-siRNA) encapsulating the test siRNA combination was transfected into the cells the day after plating, 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. ELISA kit (Antu Bio) was used to detect HBsAg levels in the supernatant 3 days after siRNA transfection ( Figure 5 E).

[0359] 2. Broad-spectrum evaluation

[0360] HepG2 cells were cultured in DMEM medium containing 10% FBS. HepG2 cells were plated at 5×10 5 The cells were inoculated into 24-well cell culture plates at a density of 100 cells / well. On the day after plating, cloned plasmids carrying four groups of different HBV genotypes A, B, C and D were transfected into HepG2 cells to achieve HBV expression and replication. On the day after transfection, the test siRNA combination (LNP-siRNA and RNAiMAX-siRNA) was transfected into the cells using the method in Experimental Example 1, and fresh culture medium was replaced 6 hours after siRNA transfection. ELISA kits (Antu Bio) were used to detect HBsAg and HBeAg antigen levels in the supernatant 3 days after siRNA transfection ( Figure 6 ).

[0361] 3. In vivo and in vitro effectiveness evaluation

[0362] 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 cells / well. On the day after plating, the concentrated HBV virus was resuspended in a culture medium containing 4% PEG 8000 and incubated with the cells. After 20 hours, the cells were washed 5 to 6 times with pre-cooled PBS and replaced with fresh culture medium to which 5 compounds were added. 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 in Experimental Example 1, and fresh culture medium was replaced 6 hours after siRNA transfection. The cell culture supernatant was collected every 2 to 3 days and the HBsAg and HBeAg antigen levels in the supernatant were detected using an ELISA kit (Antu Biotechnology), and the HBV nucleic acid quantitative detection kit (PCR-fluorescent probe method) (Shengxiang Biotechnology Co., Ltd.) was used to detect the HBV DNA content in the supernatant ( Figure 7 A).

[0363] The prcccDNA / pCMV-Cre high-pressure hydrodynamic model refers to the injection of the prcccDNA plasmid and the pCMV-Cre plasmid into the mouse through the tail vein within 5 to 8 seconds at a total volume of 10% of the mouse body weight. The prcccDNA plasmid is cut and circularized in the mouse liver under the action of Cre recombinase to form recombinant cccDNA, so as to construct 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). The control siRNA or test siRNA combination was co-injected with the prcccDNA / pCMV-Cre plasmid system (4 μg each) into C57BL / 6 mice by high-pressure tail vein injection. Blood samples were collected on days 2, 4, and 7 after tail vein injection to detect serum HBV antigen levels. Some mice were killed on days 2 and 7 after tail vein injection to detect intrahepatic HBc protein levels ( Figure 7 C).

[0364] The experimental results showed that the tested 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 5 AD); the tested siRNA combination can also dose-dependently inhibit the expression of EGFP fluorescent protein in HepG2-HBV / EGFP cells, and can dose-dependently inhibit the expression of HBc and HBx proteins in HepAD38 cells ( Figure 5 E).

[0365] The experimental results showed that the test siRNA combination can effectively inhibit the expression of HBV antigen in HepG2 cells transfected with cloned plasmids of four different HBV genotypes, A, B, C and D, and it is dose-dependent. This result shows that the test siRNA combination can widely cover multiple HBV genotypes ( Figure 6 ).

[0366] The experimental results showed that compared with the control siRNA, the test siRNA combination could effectively inhibit the HBsAg and HBeAg antigen levels in the culture supernatant of 5C-PHH cells infected with HBV, and the inhibition could be sustained until the 28th day of infection without rebound ( Figure 7AB). Compared with the control siRNA, the test siRNA combination can strongly inhibit the levels of HBsAg and HBeAg antigens in the serum of prcccDNA / pCMV-Cre high-pressure hydrodynamic model mice, with obvious dose dependence, and can significantly inhibit the level of HBc antigen in the liver; however, the inhibitory effect gradually weakened on the 4th day and completely disappeared on the 7th day ( Figure 7 CE). The results show that although the test siRNA combination can exert a long-lasting antiviral effect in vitro, the unmodified naked test siRNA is easily degraded by nucleases in vivo and is difficult to maintain a long-term antiviral effect. Therefore, subsequent experiments will be conducted using modified siRNA combinations and lipid nanoparticles (LNPs) encapsulating the modified test siRNA combination.

[0367] Experimental Example 5: Screening of siRNA sequence chemical modification schemes

[0368] 1. Serum stability test

[0369] Synthesize small interfering nucleic acids with specific chemical modifications as shown in Table 5 of Preparation Example 2. Add 20 μL of small interfering nucleic acid with a concentration of 20 μM to 20 μL of fetal bovine serum and 160 μL of 1×PBS buffer solution (pH 7.4), and incubate at 37°C for a certain period of time before sampling, which are 0, 2, 4, 6, 8, 12, 24 and 48 hours respectively; take 20 μL of sample each time for quick freezing in liquid nitrogen to immediately terminate the effect of serum nuclease on small interfering nucleic acid. The sample was then frozen in a -80°C refrigerator for later use. Prepare 2 wt% gel electrophoresis (containing Gel-Red nucleic acid dye), mix 4 μL of 6× loading buffer with the serum incubated sample of small interfering nucleic acid, then take 10 μL of sample, and electrophoresed at a constant voltage of 120V for 20 minutes. After the electrophoresis, gel imaging and photography were performed ( Fig. 9 ).

[0370] 2. Cytotoxicity assay

[0371] HepG2 cells were cultured in DMEM medium containing 10% FBS. 5Cells were inoculated into 96-well cell culture plates at a density of cells / well and allowed to adhere overnight in a 37°C, 5% CO2 incubator. Lipofectamine RNAiMAX transfection reagent was used to transfect siRNA into cells, and 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, catalog number CK04), and 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 an enzyme reader, and the cell survival rate was calculated according to the following formula ( Figure 8 ).

[0372] Cell survival rate (%) = (average A value of experimental group / average A value of control group) × 100%

[0373] 3. Effectiveness test

[0374] HepG2-HBV / EGFP cells were cultured at 5×10 5 The cells were inoculated into 24-well cell culture plates at a density of cells / well and allowed to adhere overnight in a 37°C, 5% CO2 incubator. After the cells grew to 80-90% confluence, Ad-Cre was added for treatment, and fresh culture medium was replaced after 6 hours. Three days after Ad-Cre transduction, the test siRNA combination with specific chemical modifications (si-HBV-2m) was transfected into the cells using Lipofectamine RNAiMAX transfection reagent, and the si-NC with specific chemical modifications (si-NC-2m, sense chain: 5'-UmUmCmUmCmCmGAACmGUmGUmCmACmGUmdTdT-3', antisense chain: 5'-ACGUmGACACGUUmCGGAGAAdTdT-3') was used as a negative transfection control. Fresh culture medium was replaced 6 hours after transfection. Three days after siRNA transfection, an ELISA kit (Antu Bio) was used to detect the HBV antigen level in the supernatant ( Figure 8 ).

[0375] The experimental results are as follows Figure 8 E. Fig. 9 As shown. Compared with the unmodified samples, the serum stability of si1260 and si1848 with different chemical modification schemes was significantly increased, and they could be stable 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 ( Figure 8B, D). Compared with the control cells transfected with si1260 and si1848 without chemical modification, the above-mentioned different modified si1260 and si1848 can retain most of the activity of inhibiting HBV target gene expression and significantly reduce the expression levels of HBsAg and HBeAg antigens in a concentration-dependent manner ( Figure 8 A, 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 1.517nM( Figure 8 F, G).

[0376] In summary, compared with unmodified small interfering nucleic acids, the stability of the small interfering nucleic acids provided by the present invention is significantly improved, while the cytotoxicity is low, and the biological activity substantially the same as that of the unmodified small interfering nucleic acids can be maintained. It can be seen that the present invention achieves the purpose of increasing the serum stability of the 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 effect of the modification on the biological activity of the small interfering nucleic acids.

[0377] Experimental Example 6: Evaluation of the in vivo effectiveness of LNP-siHBV

[0378] 1. Evaluation of the antiviral effect of a single dose of LNP-siHBV

[0379] In this experimental example, a lipid nanoparticle (LNP) preparation (prepared according to the method described in Preparation Example 3) was used to encapsulate a specific 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), and the antiviral effect of the nucleic acid lipid nanoparticle preparation (abbreviated as LNP-siHBV) in hepatitis B model mice was evaluated. The LNP preparation (LNP-siNC) encapsulating the modified control siRNA (siNC-2m) described in Experimental Example 5 was used as a negative control, and the dosage was 1 mg / kg; Entecavir (Entecavir, abbreviated as ETV) (MedChemExpress, Product No. HY-13623) was used as a positive control. The LNP preparation was administered by tail vein injection, and the positive drug ETV was administered by gavage, with a dosage of 1 mg / kg, and the frequency of administration was once a day ( Fig.10 AD).

[0380] rAAV-HBV1.3 mouse model: rAAV-HBV1.3 model mice were prepared according to the literature method (A mouse model for HBV immunotherapy 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 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 vg). From day 14 to day 28 after virus injection, all mice were bled by orbital sampling for serum detection of HBsAg and HBV DNA.

[0381] First, the inhibitory effect of single-dose administration of different doses of LNP-siHBV on HBV-related indicators in hepatitis B model mice at different time points was detected. After the animal model was successfully established, the hepatitis B model mice were randomly divided into groups (8 in each 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 animals used were dosed according to their body weight, including 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, which were all administered by tail vein injection once a day, and the ETV (1 mg / kg) positive drug group was administered by gavage every day at a dose of 1 mg / kg. Blood was collected from the orbital venous plexus of mice before administration (day 0) and on days 7, 14, 21, and 28 after administration, and the supernatant was collected after centrifugation. Take 2.5 μL of serum and add 122.5 μL of sterile water to dilute 50 times. The serum HBV antigen level at each time point was detected using the Hepatitis B virus surface antigen detection kit (chemiluminescence method) (Antu Biological Company, Catalog No. 30030301CL01) and the Hepatitis B virus e antigen detection kit (chemiluminescence method) (Antu Biological Company, Catalog No. 30030303CL01). The HBV nucleic acid quantitative detection kit (PCR-fluorescent probe method) (Shengxiang Biotechnology Co., Ltd.) was used to detect the level of serum HBV DNA according to the kit instructions. The animals were killed on the 28th day, and part 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 for later use.

[0382] The HBsAg content is expressed as the number of HBsAg equivalents (UI) per milliliter (mL) of serum.

[0383] The HBeAg content is expressed as the amount of HBeAg equivalent (PEUI) per milliliter (mL) of serum.

[0384] The HBV DNA content is expressed as the number of HBV DNA equivalents (UI) per milliliter (mL) of serum.

[0385] The HBsAg expression inhibition rate was calculated according to the following formula:

[0386] HBsAg expression inhibition rate = log10 (HBsAg content in test group / HBsAg content in PBS control group)

[0387] The HBeAg expression inhibition rate was calculated according to the following formula:

[0388] HBeAg expression inhibition rate = log10 (HBeAg content in test group / HBeAg content in PBS control group)

[0389] The HBV DNA expression inhibition rate was calculated according to the following formula:

[0390] HBV DNA expression inhibition rate = log10 (HBV DNA content in the test group / HBV DNA content in the PBS control group)

[0391] The control group is a control group of mice administered with PBS in this experiment, and each test group is a drug administration group of mice administered with different drug compositions.

[0392] 2. Evaluation of the antiviral effect of multiple doses of LNP-siHBV

[0393] The inhibitory effects of multiple doses of LNP-siHBV at different doses and different dosing 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 in each 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 of the animals used was calculated according to their body weight, and all groups were administered by tail vein injection. Blood was collected from the orbital venous plexus of the mice every week before (day 0), during and after dosing, and the supernatant was collected after centrifugation. The hepatitis B virus surface antigen detection kit (chemiluminescence method) (Antu Biological Company, catalog number 30030301CL01) and the hepatitis B virus e antigen detection kit (chemiluminescence method) (Antu Biological Company, catalog number 30030303CL01) were used to detect the serum HBV antigen level at each time point. The HBV nucleic acid quantitative detection kit (PCR-fluorescent probe method) (Shengxiang Biotechnology Co., Ltd.) was used to detect the level of serum HBV DNA according to the kit instructions. The animals were killed on the 49th day, 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 packaged and quickly frozen in liquid nitrogen and stored at -80°C for later use ( Fig.10 EI).

[0394] The results are as follows Fig.10As shown. The experimental results show that at different time points after administration, the positive drug ETV control group can significantly inhibit the HBV DNA level, but has no inhibitory effect on HBV antigen. Compared with the LNP-siNC control group, LNP-siHBV can dose-dependently inhibit the levels of HBsAg, HBeAg and HBV DNA in serum. Among them, the inhibition rate of LNP-siHBV (1 mg / kg) high-dose group on serum HBsAg on the 7th day after administration was 2.65log10 (IU / mL), the inhibition rate of serum HBeAg was 1.82log10 (PEIU / mL), and the inhibition rate of serum HBV DNA was 2.29log10 (IU / mL); and the inhibitory effect of the LNP-siHBV (1 mg / kg) high-dose group can be maintained for more than 14 days (inhibition rate> 1log10 (IU / mL)) ( Fig.10 AD). At the same time, the results of multiple-dose administration experiments showed that high-dose weekly administration of LNP-siHBV (1 mg / kg Q1W) can keep HBsAg, HBeAg antigen and HBV DNA in mouse serum at extremely low levels, and after drug withdrawal, its inhibitory effect can be maintained for more than 14 days (inhibition rate> 1log10 (IU / mL)), indicating that it has good clinical application potential ( Fig.10 EI).

[0395] Experimental Example 7: Safety evaluation of single and repeated administration of LNP-siHBV

[0396] 1. Safety evaluation of single-dose administration

[0397] Normal C57BL / 6 mice were randomly divided into 5 groups (4 mice in each group), namely: PBS control group, LNP-siNC (1 mg / kg) control group, LNP-siHBV (1 mg / kg) dose group, LNP-siHBV (2.5 mg / kg) dose group, LPS (5 mg / kg) positive control group and polyI:C (10 mg / kg) positive control group. Among them, the LNP-siNC (1 mg / kg) control group, LNP-siHBV (1 mg / kg) dose group, and LNP-siHBV (2.5 mg / kg) dose group were all administered by tail vein injection, and the LPS (5 mg / kg) (Biyuntian Biotechnology Co., Ltd., Product No. S1732) positive control group and polyI:C (10 mg / kg) (MedChemExpress, Product No. HY-107202) positive control group were administered by intraperitoneal injection. The dosage of all animals was calculated according to body weight, and the volume of the preparation was adjusted with 1×PBS as needed before administration to meet the needs of animal administration. Blood was collected from the orbital venous plexus of mice at 3, 24 and 48 hours after administration, and the blood was left to stand at room temperature for 2 hours to allow coagulation. The serum was collected by centrifugation, and 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 were detected using ELISA kits. Biochemical detection kits (Nanjing Jiancheng Bioengineering Institute) were used to detect the levels of biochemical indicators related to liver and kidney function in serum, including alanine aminotransferase (ALT) (Catalog No. C009-201), aspartate aminotransferase (AST) (Catalog No. C010-2-1), uric acid (UA) (Catalog No. C012-2-1) and creatinine (CREA) (Catalog No. C011-2-1) (operation was performed according to the instructions of the kit). The mice were killed 48 hours after administration and the heart, liver, spleen, lung and kidney were collected for observation. The organs were fixed with 10% formalin and pathological observation was performed ( Fig.11 ).

[0398] 2. Safety evaluation of multiple administration

[0399] For the mice treated with multiple doses of drug in Experimental Example 6, biochemical detection kits (Nanjing Jiancheng Bioengineering Institute) were used to detect the levels of liver and kidney function-related biochemical indicators including ALT, AST, UA and CREA in the mouse serum, and ELISA was used to detect the level of anti-PEG antibodies in the mouse serum. After the experiment, the animals were killed and grossly dissected, and the heart, liver, spleen, lung and kidney of the mice were taken for observation and preserved in 10% formalin for further pathological observation ( Fig.12 ).

[0400] The detection method of anti-PEG antibodies in serum is as follows:

[0401] i. Add 100 μL of 10 μg / mL PEG-DMG in ethanol solution (2 μg / well) to the medium binding ELISA plate and evaporate overnight in a clean bench or fume hood;

[0402] ii. Wash three times with 200 μL of PBS solution containing 0.1% Tween-20, 5 minutes each time;

[0403] iii. Add 200 μL of PBS solution containing 3% bovine serum albumin (BSA), block at 37 ° C for 2 hours, discard the blocking solution, and wash three times with 200 μL of PBS solution containing 0.1% Tween-20;

[0404] iv. Add gradient dilutions of control mouse serum or test mouse serum (initial dilution 10-fold and then 4-fold gradient dilution), incubate at 37°C for 1 hour, and wash three times with 200 μL of PBS solution containing 0.1% Tween-20;

[0405] 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 wash 4 times with 200 μL of PBS solution containing 0.1% Tween-20;

[0406] vi. Add 100 μL of TMB solution, 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 detect the absorbance at a wavelength of 450 nm.

[0407] 3. Off-target effect detection in vivo and in vitro

[0408] HepG2 cells were cultured at 5×10 5 The cells were inoculated into 24-well cell culture plates at a density of 100 cells / well. The cells were treated with LNP-siHBV or LNP-siNC the day after plating, with the final concentration of siRNA being 50 nM. The control group consisted of HepG2 cells that were not treated with drugs. After incubation for 16 hours, the supernatant was discarded, the cells were washed twice with PBS, and the total RNA of the cells was collected for transcriptome sequencing ( Fig.13 ).

[0409] Normal C57BL / 6 mice were injected with PBS, LNP-siNC (1 mg / kg) and LNP-siHBV (1 mg / kg) through the tail vein. 48 hours after administration, the mice were killed and the liver tissues were obtained for transcriptome sequencing.

[0410] The experimental results are as follows Fig.11 , Fig.12 The experimental results showed that LNP-siHBV had low immunogenicity and did not induce significant TNF-α, IL-6 and IFN-γ cytokine production after a single administration ( Fig.11 AD), and repeated administration does not induce a significant increase in the level of anti-PEG antibodies produced by the body ( Fig.12 AB); LNP-siHBV has good safety, and single or multiple administrations will not cause abnormalities in biochemical indicators related to liver and kidney function ( Fig.11 EH, Fig.12 CD), and single and multiple administrations did not cause obvious lesions in the organs of mice ( Fig.11 I, Fig.12 E) According to Fig.13 The results of transcriptome sequencing analysis in HepG2 cells and C57BL / 6 mice showed that LNP-siHBV treatment did not cause significant changes in the transcriptional profile of host cells, with only a small number of genes differentially expressed, suggesting that the RNAi agent did not cause significant off-target effects ( Fig.13 AD). The above results indicate that the LNP-siHBV preparation has good safety and has the potential for further clinical development and application.

[0411] Experimental Example 8: Evaluation of the effectiveness of GalNac-siHBV conjugate

[0412] The siRNA conjugate composition provided by the present disclosure ( Fig.15 A) (referred to as GalNac-siHBV, which is a mixture of GalNac-si1260 and GalNac-si1848 in a 1:1 ratio) was tested for activity 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 siRNA single strands was detected by HPLC (purity>90%), and the purity of siRNA double strand conjugates was detected by SEC (purity>90%). The results of SEC detection are as follows: Fig.14 As shown, the above results show that the purity of GalNac-si1260 is 99.7% and the purity of GalNac-si1848 is 97.04%.

[0413] Two chronic hepatitis B mouse models were used for the experiment. One was a chronic recombinant cccDNA mouse model built in the laboratory, namely the rAAV-rcccDNA / rAAV-Cre mouse model: pAAV2-rcccDNA and pAAV2-Cre plasmids were co-transfected with auxiliary 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. rAAV-rcccDNA and rAAV-Cre recombinant viruses were diluted to 2.5×10 11 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 the 14th to the 28th day after virus injection, all mice were bled by 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 the 7th day after administration to detect the levels of HBsAg and HBeAg antigens in the serum ( Fig.15 BC).

[0414] Another model is the rAAV-HBV1.3 mouse model, which was prepared according to the method described in Experimental Example 6. From the 14th to the 28th day after virus injection, all mice were bled by orbital sampling for serum detection of HBsAg and HBV DNA. The hepatitis B model mice were randomly divided into groups (5 in each group) according to the HBV antigen expression level, namely: 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. The LNP / siHB described in Preparation Example 3 and Experimental Example 6 was used. V (LNP-siHBV for short) As a control, entecavir (ETV) was used as a positive control. LNP-siHBVThe preparation was administered once by tail vein injection at a dose of 1 mg / kg; the positive drug ETV was administered by oral gavage at a dose of 1 mg / kg, and the administration frequency was once a day. The siRNA conjugate was administered once by subcutaneous injection at doses of 0.2 mg / kg, 1 mg / kg, and 2.5 mg / kg. Blood was collected before administration (day 0) and weekly after administration to detect HBsAg, HBeAg, and HBV DNA levels ( Fig.15 DE).

[0415] The results are as follows Fig.15 As shown, the siRNA conjugates and siRNA conjugate compositions disclosed in the present invention showed good inhibitory effects on HBV antigen and HBV DNA in rAAV-rcccDNA / rAAV-Cre mouse models and rAAV-HBV1.3 model mice; when 2.5 mg / kg was administered, the inhibition rate of HBV target gene expression was still as high as 86% one month after administration. It can be seen that the siRNA conjugates and siRNA conjugate compositions disclosed in the present invention have good application prospects for treating HBV-related diseases, especially pathological conditions or diseases caused by hepatitis B virus infection.

[0416] 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 may make some simple modifications, equivalent changes or modifications using the technical contents disclosed above, which all fall within the protection scope of the present invention.

[0417] It should also be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0418] In addition, 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 antisense strand of the siRNA 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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