SiRNA for targeted degradation of hepatitis B virus HBsAg and HBX mRNA

Abstract

The invention provides siRNA for targeted degradation of hepatitis B viruses HBsAg and HBX mRNA, and relates to the technical field of biological medicines. A positive-sense strand of the siRNA comprises a base sequence as shown in SEQ ID NO. 1, and an antisense strand of the siRNA comprises a base sequence as shown in SEQ ID NO. 2; or, the positive-sense strand comprises the base sequence as shown in SEQ ID NO. 3, and the antisense strand comprises the base sequence as shown in SEQ ID NO. 4. The siRNA and the siRNA conjugate provided by the invention can significantly inhibit expression of HBsAg and HBX by targeting HBV, which indicates that the siRNA and the siRNA conjugate can be used as effective components for preparing anti-HBV drugs and for treating hepatitis B caused by HBV.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a siRNA that targets and degrades hepatitis B virus HBsAg and HBX mRNA. Background Technology

[0002] Thanks to routine hepatitis B surface antigen (HBsAg) vaccination, the number of newly acquired hepatitis B virus (HBV) infections has significantly decreased. However, there are still over 300 million people worldwide with chronic HBV (CHB), posing a significant threat to human health and causing up to 900,000 deaths annually. Persistent HBV infection can lead to cirrhosis, liver failure, and hepatocellular carcinoma (HCC), which accounts for the majority of end-stage liver diseases.

[0003] Currently, nucleoside (acid) analogs (NAs) and pegylated interferon are the two clinically approved antiviral treatments for chronic HBV infection (CHB). Existing antiviral therapies can only achieve approximately 1%–10% HBsAg loss (HBsAg serological clearance) and cannot clear HBsAg or eliminate the persistent viral template library, i.e., covalently closed circular DNA (cccDNA), nor can they reduce the risk of developing HCC. Complete cure, i.e., complete elimination of cccDNA, remains a significant challenge and is unlikely to be achieved in the short term. With existing drugs, long-term functional cure—characterized by persistent undetectable circulating HBsAg and HBV DNA six months after treatment—appears achievable. Therefore, there is an urgent need to develop new and effective treatments to cure chronic HBV infection.

[0004] The HBV genome is approximately 3.2 kb in size and encodes seven proteins: large (L), medium (M), and small (S) HBV surface proteins (HBs), the X protein (HBX), the HBV core antigen (HBcAg), polymerase, and the HBV e antigen (HBeAg). The three HBV surface proteins are translated from different start codons and therefore share a common S domain at the C-terminus, but have different N-terminal extensions. Small HBsAg (SHBs) contains only the S domain, medium HBsAg proteins (MHBs) have a preS2 domain at the N-terminus, while large HBsAg (LHBs) additionally possess a preS1 domain. Compared to the S domain, the preS1 domain of LHBs has been proposed as the optimal immunogen for developing therapeutic vaccines. Through its interaction with the sodium taurocholic acid cotransport polypeptide (NTCP) (the receptor for HBV entry into cells), preS1 plays a crucial role in HBV entry. Studies have shown that monoclonal antibodies against preS1 (MA18 / 7 and KR127) can prevent HBV infection in vitro and in vivo. In CHB patients, immune tolerance to preS1 appears to be lower than that to HBsAg. Furthermore, HBcAg is crucial for viral particle generation and has been identified as a potential vaccine target in several studies. Patients with acute self-limiting infection have reported significantly higher levels of HBcAg-specific cytotoxic T lymphocytes compared to those with chronic infection. Protein X is a multifunctional protein that regulates viral gene expression and replication, and its expression level is much lower than other viral proteins. Because high protein abundance often leads to immune exhaustion, given the limited content of protein X, it is likely to be an optimal target for vaccination.

[0005] RNAi technology has attracted significant attention in the treatment of viral infectious diseases, with particularly active research into its application in the treatment of AIDS, hepatitis B, and hepatitis C. For example: Xalnesiran is an siRNA drug developed by Roche and Dicerna that can target conserved regions of the HBV genome to silence multiple HBV transcripts. BRII-835 (Elebsiran): This is an siRNA drug that targets the HBV genome. It inhibits the expression of HBV RNA through RNA interference, thereby reducing the levels of HBsAg and other viral antigens. This drug has shown good tolerability and a significant HBsAg reduction effect. RBD1016 injection: is a GalNAc-siRNA drug that targets the X gene of HBV. It can simultaneously inhibit HBV DNA replication, reduce cccDNA and integrate DNA-derived HBsAg. The efficacy can last for nearly six months and it has shown good safety and tolerability. BW-03: Targets the HBV genome through RNA interference mechanism, inhibiting viral replication and antigen expression; HRS-5635: Currently in Phase I clinical trials in China, it is undergoing safety, tolerability and pharmacokinetics studies for single-dose administration, as well as evaluation of its antiviral effects with multiple doses in patients with chronic hepatitis B. HT-101: Developed by Xingyao Kunze, the results of its Phase 1b and 1c studies were presented at the 2025 Asia-Pacific Association for the Study of the Liver (APAS). This was a randomized, double-blind, placebo-controlled, multi-dose escalation study in patients with chronic hepatitis B who had received nucleoside (nucleotide) reverse transcriptase inhibitor therapy for ≥6 months. The study showed that HT-101 administered at multiple doses (up to 400 mg) to patients with chronic hepatitis B demonstrated good safety and tolerability. HBsAg levels decreased over time in all HT-101 treatment groups, with a greater decrease in HBsAg at higher doses. A significant dose-dependent reduction in HBsAg levels relative to baseline was observed for up to 44 weeks after the last dose, including one subject achieving HBsAg serological clearance at week 48.

[0006] In summary, siRNA has shown promising application prospects in the treatment of hepatitis B, and several siRNA drugs in clinical trials have achieved certain results in reducing HBsAg levels, with some drugs also showing good safety and tolerability. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a siRNA that targets and degrades hepatitis B virus (HBsAg) and HBX mRNA. Based on the genomic sequences of hepatitis B and C viruses, this invention designs siRNAs that target HBV, effectively inhibiting the expression of HBsAg and HBX. Furthermore, the coupling compound formed by coupling the siRNA with GalNAc exhibits significant serological conversion in a hepatitis B model.

[0008] To achieve the above objectives, the specific technical solution of the present invention is as follows: In a first aspect, the present invention provides an siRNA that targets and degrades hepatitis B virus HBsAg and HBX mRNA, wherein the sense strand of the siRNA comprises the base sequence shown in SEQ ID NO. 1 and the antisense strand comprises the base sequence shown in SEQ ID NO. 2; or, the sense strand comprises the base sequence shown in SEQ ID NO. 3 and the antisense strand comprises the base sequence shown in SEQ ID NO. 4.

[0009] Furthermore, the sense and antisense strands of the siRNA contain modifying groups, including but not limited to at least one of thiophosphate modification, 2'-fluoro modification, and 2'-oxymethyl modification.

[0010] Furthermore, the modified siRNA has the following base sequence: Chain of Justice: 5'-mU mC mCmGmUmCfCmGfAfAfGmGmUmUmUmUmGmUmA-3'; Antisense chain: 5'-mU fA mCmAmAfAmAmCmCmUmUmCmGfGmAfCmG mG mAdTdT-3'; or, Chain of Justice: 5'-mG mU mAmAmAmGfAmGfAfGfGmUmGmCmGmCmCmCmC-3'; Antisense chain: 5'-mG fG mGmGmCfGmCmAmCmCmUmCmUfCmUfUmU mA mCdTdT-3'.

[0011] Secondly, the present invention provides an siRNA coupling compound that targets and degrades hepatitis B virus HBsAg and HBX mRNA, comprising the siRNA and a targeting ligand.

[0012] Furthermore, the targeting ligand includes GalNAc (N-acetyl-D-glucosamine). GalNAc is a natural amino sugar that is widely found in organisms and is an important component of glycoproteins and glycolipids. Here, GalNAc, as a targeting ligand, can specifically bind to lectins on the cell surface (such as the desialylate glycoprotein receptor on hepatocytes), thereby achieving precise delivery of siRNA to target cells.

[0013] Thirdly, the present invention provides the use of the siRNA or the siRNA coupling compound in the preparation of anti-hepatitis B virus drugs.

[0014] Fourthly, the present invention provides a drug for treating hepatitis B virus, wherein the active ingredient includes the siRNA or the siRNA coupling compound.

[0015] Compared with the prior art, the advantages of the present invention are: The siRNA and siRNA coupling products provided by the present invention can target HBV and significantly degrade HBsAg and HBX, indicating that the siRNA and siRNA coupling products can be used as active ingredients in the preparation of anti-HBV drugs and for the treatment of HBV-induced hepatitis B. Attached Figure Description

[0016] Figure 1 The results of qPCR detection of HBsAg and HBX mRNA degradation by unmodified siRNA; Figure 2 Experimental results for ELISA detection of HBsAg and HBX degradation by unmodified siRNA and GalNAc-siRNA coupling compound; Figure 3 The results of Western blotting were used to detect the degradation of HBX by unmodified siRNA and GalNAc-siRNA couplers. Figure 4 Experimental results on the degradation of HBsAg and HBV DNA load in hepatitis B mice by GalNAc-siRNA coupling compound. Detailed Implementation

[0017] To enable those skilled in the art to clearly and completely understand the technical solution of the present invention, the present invention will be further described in detail below with reference to embodiments. Obviously, the embodiments described herein are only for explaining the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0018] 1. Design and synthesis of siRNA 1.1 siRNA Design To design highly conserved siRNAs targeting HBV, this invention downloaded 11,185 HBV genome sequences from the Hepatitis Virus Database (HVDB) website (http: / / s2as02.genes.nig.ac.jp / index.html) and analyzed highly conserved motifs using a motif-based sequence analysis tool (MEME Suite 5.4.1, https: / / meme-suite.org / meme / tools / meme). Typical HBV sequences were input into siRNA design websites, including siDirect, siDESIGN Center, and DSIR. This invention screened out 46 siRNAs targeting conserved HBV motifs, distributed throughout the entire HBV genome. The siRNA design criteria of this invention are as follows: a. conforming to the basic design principles provided by the online software; b. limited homology with any known sequences in the human, mouse, and rat genomes.

[0019] Of the 46 siRNAs targeting conserved HBV motifs screened in this invention, two siRNAs (siRNA 19 and siRNA 41) can target and degrade HBsAg (sequence number: YP_009173869.1) and HBX (sequence number: YP_009173867.1). The nucleotide sequences of siRNA 19 and siRNA 41 are as follows: siRNA 19: Chain of Justice: 5'-CGACGGGACGUAGACAAAGdTdT-3' (see SEQ ID NO. 1); Antisense chain: 5'-CUUUGUCUACGUCCCGUCGdTdT-3' (see SEQ ID NO. 2); siRNA 41: Chain of Justice: 5'-GUAAAGAGAGGUGCGCCCCdTdT-3' (see SEQ ID NO. 3); Antisense chain: 5'-GGGGCGCACCUCUCUUUACdTdT-3' (see SEQ ID NO. 4).

[0020] 1.2 siRNA Modification This invention chemically modifies the nucleotide sequences of siRNA 19 and siRNA 41, and the resulting modified nucleotide sequences are as follows: After siRNA 19 modification: Chain of Justice: 5'-mU mC mCmGmUmCfCmGfAfAfGmGmUmUmUmUmGmUmA.-3'; Antisense chain: 5'-mU fA mCmAmAfAmAmCmCmUmUmCmGfGmAfCmG mG mAdTdT-3'; After siRNA 41 modification: Chain of Justice: 5'-mG mU mAmAmAmGfAmGfAfGfGmUmGmCmGmCmCmCmC-3'; Antisense chain: 5'-mG fG mGmGmCfGmCmAmCmCmUmCmUfCmUfUmU mA mCdTdT-3'; The chemical modification is well known to those skilled in the art, wherein the modification of the phosphodiester bond refers to the modification of the oxygen in the phosphodiester bond, including the modification of the thiophosphate ester group, i.e. As shown in equation (1), modification can stabilize the siRNA structure and maintain high specificity and high affinity for base pairing. (1) In addition, ribose modification refers to the modification of the 2'-OH in the pentose of nucleotides, that is, the introduction of certain substituents at the hydroxyl position of ribose, including 2'-fluoro modification or 2'-oxomethyl, f represents 2'-fluoro modification, as shown in formula (2); m represents 2'-oxomethyl modification, as shown in formula (3).

[0021] (2) (3) 1.3 Coupling of GlcNAc and siRNA This invention covalently links GlcNAc to the end (5' or 3' end) of siRNA via a "connector arm," as follows: The reagents and materials are as follows: Reagents: GlcNAc with a purity ≥98%, siRNA (purified by HPLC, without amino modification), linker arms (such as bifunctional cross-linking agents like SMCC and SPDP), activating reagents (EDC, NHS), anhydrous organic solvents (DMSO, DMF), and buffers (PBS, Tris-HCl). Materials: centrifuge tubes, dialysis bags (molecular weight cutoff 3-5 kDa), HPLC system, mass spectrometer, agarose gel electrophoresis equipment.

[0022] The steps are as follows: Step 1: Preparation of Activation and Modification Groups for GlcNAc Dissolve an appropriate amount of GlcNAc in anhydrous DMF, add excess linker (SMCC), and stir at room temperature for 2-4 hours. After the reaction, the fraction containing the "GlcNAc-linker arm" was purified by silica gel column chromatography and vacuum dried for later use (the linker arm must retain the active group that can react with siRNA, such as maleimide group).

[0023] Step 2: Activation of the siRNA terminus (if required) If the siRNA does not have an amino group at the end, amino modification is required first: dissolve the siRNA in PBS buffer (pH 7.4), add EDC and NHS, and activate at room temperature for 30 minutes to activate the carboxyl group at the end of the siRNA; If the siRNA already has amino modifications (such as 5'-amino-modified siRNA), this step can be skipped, and it can be directly dissolved in buffer for later use.

[0024] Step 3: Coupling reaction of GlcNAc and siRNA Dissolve the purified “GlcNAc-linker” in DMSO and slowly add it to the activated siRNA solution (GlcNAc-linker to siRNA molar ratio 3:1-5:1). Incubate at 4°C in the dark for 12-16 hours to ensure that the active group of the linker forms a stable covalent bond (such as an amide bond) with the amino group of the siRNA.

[0025] Step 4: Purification of the coupling products Dialysis method: Add the reaction solution to the dialysis bag and dialyze in PBS buffer for 24 hours (change the buffer 3 times) to remove unreacted free GlcNAc and connecting arms; Further purification was performed by HPLC (reversed-phase C18 column), the target peak (the retention time of the coupling product is different from that of free siRNA) was collected, and the pure product was obtained after freeze-drying.

[0026] Step 5: Product Identification Agarose gel electrophoresis: Comparing the migration rates of the coupled products and free siRNA, the molecular weight increases after coupling, and the migration is slower; Mass spectrometry analysis: The molecular weight of the product was detected to confirm that GlcNAc was successfully linked (the increase in molecular weight corresponds to the molecular weight of the GlcNAc+ linker arm).

[0027] 2. siRNA transfection Experimental objective: To deliver siRNA into in vitro cell lines using transfection reagents.

[0028] Experimental steps: (1) Cell preparation: On day 1, HepG2.2.15 was seeded at 100,000 cells per well in a 24-well plate with 500 µL of medium per well and cultured overnight; On day 2, (1) 100 nM siRNA was diluted with 50 µL of Opti-MEM and mixed by gently pipetting 3-5 times. (2) Invert and mix the transfection reagent, then dilute 2.0 µL of Lipofectamine with 50 µL of Opti-MEM. TM Mix 2000 transfection reagent by gently blowing and aspirating 3-5 times, and let stand at room temperature for 5 minutes. (3) Mix siRNA and transfection reagent, gently blow and aspirate 3-5 times to mix, and let stand at room temperature for 20 min; (4) Add the transfection complex to the 24-well cell plate, 100 µL / well, and mix well; (5) The cell plates were placed in a 37°C, 5% CO2 incubator and cultured for 48 h.

[0029] 3. qPCR method for detecting siRNA-degraded hepatitis B virus antigen mRNA Experimental objective: To detect the ability of unmodified siRNA to degrade HBV surface antigens HBsAg and HBX mRNA.

[0030] Experimental steps: (1) Extract total RNA from the cells from step 3.2 according to the instructions of the cell / tissue total RNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number RC101-01); (2) Take 500 ng of total RNA and synthesize cDNA using the FastKing cDNA first strand synthesis kit (Tiangen Biotech (Beijing) Co., Ltd., catalog number KR116); (3) Dilute the cDNA to 1 / 200 and use 2×SuperFast Universal SYBR Master Mix (Jiangsu Kangwei Century Biotechnology Co., Ltd., catalog number CW3888H) to detect the Ct value in a Roche LightCycler 96 qPCR instrument; (4) Calculate ΔCT value: For each sample, calculate the difference between the Ct value of the target gene and the Ct value of the internal reference gene in that sample (ΔCT = Ct(target gene) - Ct(mean value of reference gene). Calculate ΔCT for the control group: Similarly, calculate the average difference between the Ct values ​​of the target gene and the internal reference gene in the control sample (usually an untreated sample or other set standard sample); (5) Calculate the △△CT value: Calculate the difference between the △CT value of each test sample and the △CT value of the control group (△△CT = △CT(target gene) - △CT(control mean); (6) Calculation of relative expression levels: The expression level of the target gene in each sample relative to the control group was calculated using the formula 2^(-△△CT). The results are usually expressed as a fold change relative to the control group. The experimental results are shown in […]. Figure 1 .

[0031] 4. ELISA detection of HBsAg in cell supernatant Experimental objective: To detect the ability of unmodified siRNA and GalNAc-siRNA couplers to degrade hepatitis B surface antigen HBsAg.

[0032] Experimental steps: (1) Take the cell supernatant from step 3.2 and filter it through a 0.45 μm filter membrane; (2) The HBsAg content in the cell supernatant was detected according to the Hepatitis B virus surface antigen detection kit (chemiluminescence method) (Beijing Wantai Biological Pharmaceutical Co., Ltd.).

[0033] Experimental results are as follows Figure 2 As shown, Figure 2 Figures A and B show that siRNA 41 significantly reduced the levels of HBsAg mRNA and HBsAg in the cell supernatant of HepG2.2.15 cells; Figure 2 Figures C and D show that siRNA 41 and its GalNAc coupling compound significantly reduced the HBsAg content in the supernatant of HepG2.2.15 and PLC / PRF / 5 cells; Figure 2 E and F in the middle section showed that siRNA 41 and its GalNAc coupling compound significantly reduced the HBV DNA content in the supernatant of HepG2.2.15 and PLC / PRF / 5 cells.

[0034] 5. Western blot detection of siRNA-degraded HBX protein Experimental objective: To detect the ability of unmodified siRNA and GalNAc-siRNA coupling compound to degrade hepatitis B HBX protein.

[0035] Experimental steps: (1) Transfect siRNA according to the method in step 3.2; (2) The cells were washed with pre-cooled phosphate-buffered saline (PBS) and then lysed in RIPA buffer (Beyotime, China) containing a mixture of protease inhibitors and a mixture of phosphatase inhibitors (Roche) for 30 minutes. The lysate was centrifuged at 15,000 rpm for 5 minutes at 4°C, and the supernatant was mixed with sodium dodecyl sulfate (SDS) loading buffer. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Chemiluminescence detection was performed using an anti-HBX horseradish peroxidase (HRP) labeled antibody (Abcam, catalog number ab39716) and HRP labeled secondary antibody.

[0036] Experimental results are as follows Figure 3 As shown, Figure 3 The results from the study showed that siRNA 19 significantly reduced the HBX protein level in HepG2.2.15 cells; Figure 3 Studies 131.B and C showed that siRNA 19 significantly reduced HBX protein levels in HepG2.2.15 and PLC / PRF / 5 cells.

[0037] 6. GalNAc-siRNA coupling compound degrades HBsAg and HBV DNA load in hepatitis B mice. Experimental objective: To detect the HBsAg and HBV DNA load in hepatitis B mice degraded by siRNA.

[0038] Male C57BL / 6 mice were injected via tail vein with 1x10 11 GC mice were injected with AAV HBV1.3 virus. Blood samples were collected from the orbital venous plexus of mice on days 1, 3, 5, 7, and up to day 21 post-injection to detect changes in HBV surface antigen levels in the mouse serum. Mice that remained positive for HBV surface antigen for 21 consecutive days were selected as chronic hepatitis B model mice.

[0039] Mice with chronic hepatitis B were randomly divided into 5 groups, with 6 mice in each group. The mice in each group were subcutaneously injected with GalNAc-siRNA non-target, GalNAc-siRNA 19, GalNAc-siRNA 41, GalNAc-ALN-HBV, or GalNAc-HBX PC, respectively, at a dose of 1 mg / kg / mouse. Administering the medication once a week, blood was collected from the orbital venous plexus 2 days after administration, and serum was collected.

[0040] The HBsAg content in serum was detected using a Hepatitis B virus surface antigen detection kit (chemiluminescence method) (Beijing Wantai Biological Pharmacy Co., Ltd.), and the HBV DNA content in serum was detected using a Hepatitis B virus nucleic acid detection kit (PCR-fluorescent probe method) (Shanghai Kehua Bioengineering Co., Ltd.).

[0041] Experimental results are as follows Figure 4 As shown, Figure 4 Figures A and B show that weekly subcutaneous injections of GalNAc-siRNA 19 and GalNAc-siRNA 41 significantly reduced HBsAg and HBV DNA loads in mice with chronic hepatitis B.

[0042] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A siRNA targeting the degradation of hepatitis B virus HBsAg and HBX mRNA, characterized in that, The sense strand of the siRNA comprises the base sequence shown in SEQ ID NO. 1, and the antisense strand comprises the base sequence shown in SEQ ID NO. 2; or, the sense strand comprises the base sequence shown in SEQ ID NO. 3, and the antisense strand comprises the base sequence shown in SEQ ID NO.

4.

2. The siRNA targeting degradation of hepatitis B virus HBsAg and HBX mRNA according to claim 1, characterized in that, The sense strand and the antisense strand of the siRNA contain a modification group, which includes but is not limited to at least one of a phosphorothioate group, a 2'-fluoro modification, and a 2'-O-methyl modification. 3.The siRNA targeting and degrading HBsAg and HBX mRNA of hepatitis B virus according to claim 2, characterized in that, The modified siRNA has the following base sequence: Sense: 5'-mGmUmAmUmAmGmUmAmGmUmGmUmUmUmUmUmGmUmGmUmGmU-3' mC mCmGmUmCfCmGfAfAfGmGmUmUmUmUmGmUmA-3' Antisense: 5 '-mU fA mCmAmAfAmAmCmCmUmUmCmGfGmAfCmG mG mAdTdT-3 ' or, Sense: 5'-mG mU mAmAmAmGfAmGfAfGfGmUmGmCmGmCmCmCmC-3' Antisense: 5 '-mG fG mGmGmCfGmCmAmCmCmUmCmUfCmUfUmU mA mCdTdT-3'.

4. A siRNA conjugate targeting degradation of hepatitis B virus HBsAg and HBX mRNA, characterized in that, The siRNA and the targeting ligand.

5. The siRNA conjugate targeting degradation of hepatitis B virus HBsAg and HBX mRNA according to claim 4, characterized in that, The targeting ligand is GalNAc.

6. Use of the siRNA according to any one of claims 1-4 or the siRNA conjugate according to claim 5 in the preparation of a drug for resisting hepatitis B virus.

7. An anti-HBV agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The effective component comprises the siRNA according to any one of claims 1-4 or the siRNA conjugate according to claim 5.

Images