Use of pglyrp2 gene and related drug thereof

Abstract

Use of PGLYRP2 gene and a related drug thereof. The present invention relates to the field of biopharmaceuticals, and in particular, to use of the PGLYRP2 gene and the related drug thereof. The present invention aims to solve the problem that existing drugs can only inhibit the replication of hepatitis B virus and cannot eliminate viral cccDNA. The PGLYRP2 gene is used for preparing a drug for treating a hepatitis B virus infection. The PGLYRP2 gene is used for preparing a drug for eliminating hepatitis B virus cccDNA. Also provided is a gene therapy vector for eliminating hepatitis B virus. The gene therapy vector comprises a promoter, an intron, an enhancer, and a PGLYRP2 gene encoding region. The HBV virus inhibition function and the cccDNA-eliminating effect of the PGLYRP2 protein are defined, so that the elimination of HBV in liver cells and a mouse liver can be significantly promoted.

Description

Uses of PGLYRP2 gene and related drugs Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to uses of the PGLYRP2 gene and related drugs. Background Art

[0002] Hepatitis B virus (HBV) is the pathogen that causes hepatitis B (abbreviated as HBV), which belongs to the genus Hepadnavirus. HBV mainly infects human liver cells, and its infection can significantly increase the incidence of cirrhosis and liver cancer. For HBV-infected patients, the main clinical treatments are interferon α and nucleoside analogs, or polyclonal antibodies against its surface antigens. Patent CN101906417A discloses a recombinant adeno-associated virus gene therapy vector, which uses gene recombination technology to clone shRNA that has an inhibitory effect on hepatitis B virus into the backbone plasmid of the adeno-associated virus vector, and co-transfect the packaging cells with the auxiliary plasmid to obtain the recombinant adeno-associated virus. Although the recombinant adeno-associated virus can effectively inhibit the replication and expression of hepatitis B virus, it can only slow down the progression of the disease, and is prone to repeated treatment with poor results.

[0003] After chronic infection with hepatitis B virus, covalently closed HBV cccDNA will be produced in the nucleus of liver cells. cccDNA is very stable and has a long half-life. It is difficult for nucleoside (acid) analogs and interferon α to clear cccDNA, and the disease is prone to rebound after drug withdrawal. Therefore, the cure of clinical chronic hepatitis B requires the clearance of viral cccDNA in the liver. Although existing drugs can effectively inhibit the replication and expression of hepatitis B virus, they cannot clear viral cccDNA and can only slow down the progression of the disease, making it easy to relapse after drug withdrawal.

[0004] Summary of the Invention

[0005] The present invention aims to solve the problem that existing drugs can only inhibit the replication of hepatitis B virus but cannot eliminate viral cccDNA, and provides the use of PGLYRP2 gene and related drugs.

[0006] The present invention provides use of the PGLYRP2 gene in preparing a medicine for treating hepatitis B virus infection.

[0007] The present invention further provides the use of the PGLYRP2 gene in preparing a drug for clearing hepatitis B virus cccDNA.

[0008] The invention provides a gene therapy vector for eliminating hepatitis B virus, comprising a promoter, an intron, an enhancer and a PGLYRP2 gene coding region.

[0009] The vector backbone is a viral vector or a non-viral vector.

[0010] The viral vector is an adeno-associated viral vector, a lentiviral vector, a retroviral vector, an adenoviral vector, a herpes simplex viral vector, a Sendai viral vector or a bocaviral vector. The adeno-associated viral vector is an AAV2, AAV5, AAV7, AAV8, AAV9 or AAV-DJ type adeno-associated viral vector.

[0011] The non-viral vector is a liposome nanoparticle, a polymer nanoparticle, an exosome, a polypeptide complex, an mRNA vector, iTOP or a Feldan shuttle.

[0012] The promoter is a truncated version M of the PGLYRP2 gene promoter, and its nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing.

[0013] The enhancers are CMV enhancer and HBV EnII element. The nucleotide sequence of the CMV enhancer is shown in SEQ ID NO: 2 in the sequence listing, and the nucleotide sequence of the HBV EnII element is shown in SEQ ID NO: 3 in the sequence listing.

[0014] The nucleotide sequence of the PGLYRP2 gene coding region is shown in SEQ ID NO: 4 in the sequence listing.

[0015] The present invention provides a pharmaceutical composition for eliminating hepatitis B virus, comprising the above-mentioned gene therapy vector.

[0016] Beneficial effects of the present invention:

[0017] The present invention clarifies the HBV virus inhibitory function and cccDNA clearance effect of PGLYRP2 protein, which can significantly promote the clearance of HBV in hepatocytes and mouse livers. The adeno-associated virus gene therapy vector of the present invention can significantly clear the viral cccDNA of hepatocytes in vitro within 9 days and significantly clear the viral surface antigen in mouse serum within 6 weeks.

[0018] The present invention further prepares a gene therapy vector for eliminating hepatitis B virus, wherein the adeno-associated virus gene therapy vector includes a gene therapy delivery vector pAAV-EnII-EnCMV-M. The gene therapy delivery vector pAAV-EnII-EnCMV-M constructed by the present invention has the characteristics of long-term expression and liver tissue-specific expression in mice, and has a self-feedback mechanism, which gives the gene therapy product better safety.

[0019] The present invention has great potential to achieve a complete cure of hepatitis B. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 shows the results of PGLYRP2 protein inhibiting intracellular HBV DNA levels;

[0021] Figure 2 shows the results of PGLYRP2 protein inhibiting the level of HBV pgRNA in cells;

[0022] FIG3 shows the results of PGLYRP2 protein inhibiting the level of extracellular HBV DNA;

[0023] FIG4 shows the results of PGLYRP2 protein inhibiting the level of extracellular HBsAg;

[0024] FIG5 shows the results of PGLYRP2 protein inhibiting intracellular cccDNA levels;

[0025] FIG6 shows the correlation between PGLYRP2 protein and HBV infection degree in HBV-positive liver tissue;

[0026] Figure 7 is an enlarged view of Figure 6;

[0027] FIG8 is a correlation curve between PGLYRP2 protein and HBV infection degree in HBV-positive liver tissue;

[0028] FIG9 is a diagram of the HBV promoter-Luciferase model and the results of the luciferase activity comparison of the inhibitory effects of human hPGLYRP2 and mouse mPGLYRP2 on hepatitis B virus replication;

[0029] FIG10 is a schematic diagram of the gene therapy vector pAAV-EnII-EnCMV-M-PGLYRP2 recombinant;

[0030] Figure 11 shows the positive regulatory effect of PGLYRP2 protein on the backbone vector pAAV-EnCMV-M;

[0031] FIG12 shows the self-feedback regulatory effect of PGLYRP2 protein on the gene therapy delivery vector pAAV-EnII-EnCMV-M;

[0032] Figure 13 shows the difference between C57 / BL6 wild type and PGLYRP2 - / - Schematic diagram of AAV / hPGLYRP2 injection and HBV infection model in mice, as well as the results of serum HBV DNA detection in mice 6 weeks after the experiment;

[0033] FIG14 shows the detection results of HBs levels in mouse serum after 6 weeks of HBV infection model experiment;

[0034] Figure 15 shows the detection results of viral cccDNA in mice after 6 weeks of the mouse HBV infection model experiment. DETAILED DESCRIPTION

[0035] The following embodiments of the present invention are described in detail. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation plans and specific operating processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0036] The present invention first constructs the PGLYRP2 gene into the lentiviral vector pLVSIN, packages the lentivirus and infects the hepatocyte HepAD38 cells to form a HepAD38 / PGLYRP2 stable strain. After tetracycline (tet-) is withdrawn from the culture medium, the in vitro inhibitory effect of PGLYRP2 protein on HBV virus is analyzed.

[0037] In the mouse in vivo experiment, the human hPGLYRP2 gene was constructed into the highly hepatotropic adeno-associated virus vector pAAV2 / 8, and the virus was packaged and injected into the mouse tail vein to achieve PGLYRP2 - / - Human hPGLYRP2 protein was exogenously expressed in mouse hepatocytes, and the in vivo inhibitory effect of human PGLYRP2 protein on HBV virus was analyzed.

[0038] The PGLYRP2 gene is constitutively expressed at a high level in normal human hepatocytes, but is expressed at a low level in hepatocytes with a high hepatitis B virus index. The present invention uses a viral expression vector carrying the PGLYRP2 gene to target hepatocytes for expressing PGLYRP2 protein, thereby restoring the moderately high level of PGLYRP2 protein in virus-infected hepatocytes and achieving the effect of exogenously expressing PGLYRP2 protein to inhibit hepatitis B virus replication.

[0039] Example 1: Analysis of the inhibitory effect of PGLYRP2 protein on HBV virus

[0040] To test the inhibitory effect of PGLYRP2 protein on HBV, a lentiviral expression construct for stably transfected PGLYRP2 cell lines was first constructed. The PGLYRP2 gene was amplified using human cDNA as a template using the upstream primer hPGLYRP2 XhoI_F: 5'-CTCGAGATGGCCCAGGGCGTGCTC-3' and the downstream primer hPGLYRP2NotI_R: 5'-GCGGCCGCCTGCAGGTCGGTGGCGGGCA-3'. The empty lentiviral vector pLVSIN (purchased from TAKARA) was then double-digested with the restriction endonucleases XhoI and NotI, and the PGLYRP2 gene was ligated to generate the lentiviral expression construct pLVSIN / PGLYRP2.

[0041] HEK293T cells were transfected with the lentiviral packaging plasmid and the recombinant vector at a ratio of GAG:VSVA:pLVSIN / PGLYRP2 = 9:1:10. Virus was harvested 72 hours after transfection and used to infect HepAD38 hepatocytes. Stable transfected cell lines were selected for HepAD38 / PGLYRP2-positive cells using 1.5 μg / ml puromycin. After tetracycline (tet-) was removed from the culture medium of the stably transfected cell lines, the cells were cultured for 9 days. Genomic DNA was extracted from the cells using a genomic DNA extraction kit (TIANGEN, DP304-03), and RNA was extracted using Trizol. HBsAg levels in the culture medium were measured using ELISA (Sangon Biotech, D711407). The inhibitory effect of PGLYRP2 protein on HBV virulence was analyzed.

[0042] Results: Compared with the control HepAD38 / Con cells, the intracellular HBV DNA levels (Figure 1), intracellular pgRNA levels (Figure 2), extracellular HBV DNA levels (Figure 3), and extracellular HBsAg levels (Figure 4) of the PGLYRP2-overexpressing stably transfected cell line HepAD38 / PGLYRP2 were significantly reduced, indicating that PGLYRP2 protein has a clear inhibitory effect on HBV virus. The results of PGLYRP2 protein inhibiting intracellular cccDNA levels are shown in Figure 5. Compared with the control HepAD38 / Con cells, the intracellular cccDNA levels of the PGLYRP2-overexpressing stably transfected cell line HepAD38 / PGLYRP2 were significantly downregulated, indicating that PGLYRP2 protein clearly inhibits intracellular cccDNA levels.

[0043] Example 2: Correlation analysis between PGLYRP2 protein level and HBV infection degree in liver tissue of HBV positive patients

[0044] In this example, immunohistochemistry was used to detect the expression levels of PGLYRP2 protein and hepatitis B virus core antigen HBc in the liver tissues of 17 HBV-positive patients. Rabbit anti-human PGLYRP2 protein antibody (#NBP2-32042, Novus) was used at a concentration of 1:100, and mouse anti-HBc (#orb99015, Biorbyt) was used at a concentration of 1:50 to incubate the immunohistochemical slides.

[0045] Results: PGLYRP2 and HBc protein levels in tissues were detected using anti-PGLYRP2 and anti-HBc antibodies, respectively. Immunohistochemical images are shown in Figures 6 and 7. The correlation curve between PGLYRP2 protein expression and HBV infection severity in HBV-positive liver tissue is shown in Figure 8. A significant negative correlation was found between PGLYRP2 protein expression and the hepatitis B virus core antigen (HBc) in liver tissues of HBV-positive patients. PGLYRP2 protein levels were significantly higher in liver tissues with low HBc expression compared with liver tissues with high HBc expression (n = 17, p < 0.001, R = -0.773, 95% CI: -0.917 to -0.449), suggesting that PGLYRP2 protein expression is significantly reduced in liver tissues with high HBV infection severity.

[0046] Example 3: Comparison of the inhibitory effects of human hPGLYRP2 and mouse mPGLYRP2 on hepatitis B virus replication

[0047] The inhibitory effects of human hPGLYRP2 and mouse mPGLYRP2 on HBV replication were analyzed using the HBV promoter-luciferase dual-luciferase reporter gene assay system. HEK293 cells were cotransfected with 100 ng of HBV promoter-luciferase, 400 ng of the pLVSIN Con vector, pLVSIN / hPGLYRP2, or pLVSIN / mPGLYRP2, and 50 ng of the Renilla luciferase reporter plasmid. Sixteen hours after transfection, the cells were lysed, and luciferase activity was measured using a CytoFluorplate 4000 Luminescence Microplate Reader (ABI, CA).

[0048] Results: Compared with the pLVSIN Con vector group, human hPGLYRP2 significantly inhibited the luciferase activity of the HBV promoter-Luciferase (p<0.001), and mouse hPGLYRP2 significantly inhibited the luciferase activity of the HBV promoter-Luciferase (p<0.05). Human hPGLYRP2 had a significantly stronger inhibitory effect on the luciferase activity of the HBV promoter-Luciferase than mouse hPGLYRP2 (p<0.05). These results indicate that both human hPGLYRP2 and mouse mPGLYRP2 have significant inhibitory effects on HBV replication, with human hPGLYRP2 exhibiting a greater inhibitory effect than mouse mPGLYRP2 (Figure 9).

[0049] Example 4: Construction of gene therapy vector pAAV-EnII-EnCMV-M / PGLYRP2 recombinant

[0050] The present invention uses the gene therapy delivery vector pAAV-EnII-EnCMV-M as a backbone vector; human cDNA is used as a template to amplify the PGLYRP2 gene, the PGLYRP2 gene sequence of which is shown in SEQ ID NO: 4. The vector pAAV-EnII-EnCMV-M is then double-digested with restriction endonucleases ClaI and BamHI, and the PGLYRP2 gene coding fragment is ligated to obtain the pAAV-EnII-EnCMV-M-PGLYRP2 recombinant, a schematic diagram of which is shown in Figure 10.

[0051] The primers for amplifying the PGLYRP2 gene are as follows:

[0052] Upstream primer PGLYRP2 ClaI_F: 5′-ATCGATATGGCCCAGGGCGTGCTC-3′, downstream primer hPGLYRP2 BamHI_R: 5′-GGATCCCTGCAGGTCGGTGGCGGGCA-3′.

[0053] The gene therapy delivery vector pAAV-EnII-EnCMV-M is constructed as follows:

[0054] 1. Using MluI-M F and NcoI-M R as primers and plasmid PGL4-PGLYRP2 (1--2005) as template, the truncated M of the PGLYRP2 gene promoter was amplified.

[0055] The upstream primer MluI-M F: 5'-CGACGCGTCGGTGGCGCATGCCTGTAACCTGA-3', the downstream primer NcoI-M R: 5'-CATGCCATGGCATGGATTTCAAGCCACCAGCAGTAGCTG-3'. The nucleotide sequence of the PGLYRP2 gene promoter truncation M is shown in SEQ ID NO: 1 in the sequence listing.

[0056] The plasmid PGL4-PGLYRP2 (1--2005) has been disclosed in the article Hepatology. 2020 May; 71(5): 1626-1642.

[0057] 2. HBV EnII element was amplified using primers MluI-HBV-EnII F and SacI-HBV-EnII R and plasmid pBB4.5-HBV1.2, genotype C as a template. The nucleotide sequence of the HBV EnII element is shown in SEQ ID NO: 3 in the sequence listing.

[0058] Upstream primer MluI-HBV-EnII F: 5′-CGACGCGTCGTCCTGCCCAAGGTCTTACATAA-3′, downstream primer SacI-HBV-EnII R: 5′-CGAGCTCGCAGCTCCTCCCAGTCCTTAAAC-3′.

[0059] The plasmid pBB4.5-HBV1.2, genotype C was donated by others and has been published in the article Emerg Microbes Infect. 2022 Dec; 11(1): 1356-1370. The AAV-MCS empty vector was purchased from Miaoling Plasmid Platform (P0244, containing CMV promoter).

[0060] 3. Use restriction endonucleases MluI and NcoI to double-digest the AAV-MCS empty vector, remove the CMV promoter, retain the CMV enhancer, and connect the PGLYRP2 gene promoter truncation M to obtain pAAV-EnCMV-M. The nucleotide sequence of the CMV enhancer EnCMV is shown in SEQ ID NO: 2 in the sequence listing; then use restriction endonucleases MluI and SacI to double-digest pAAV-EnCMV-M, and connect the HBV EnII element to finally construct the pAAV-EnII-EnCMV-M vector.

[0061] Example 5: Analysis of the hepatocyte-specific expression and self-feedback regulation of PGLYRP2 protein on the gene therapy delivery vector pAAV-EnII-EnCMV-M

[0062] The gene therapy delivery vector pAAV-EnII-EnCMV-M was double-digested with restriction endonucleases BamHI and HindIII, and then ligated with the Luciferase gene sequence to generate the pAAV-EnII-EnCMV-M-Luciferase recombinant. The nucleotide sequence of the Luciferase gene is shown in SEQ ID NO: 5 in the sequence listing.

[0063] First, the gene therapy delivery vectors pAAV-EnII-EnCMV-M-Luciferase and pAAV-EnCMV-M-Luciferase were shaken overnight and plasmids were extracted (according to the kit instructions, Omega, D6943-02). The obtained plasmid was purified as follows: 1 / 10 volume of sodium acetate (3M, pH5.2) and 7 / 10 volume of isopropanol were added to the extracted plasmid, mixed and placed at room temperature for 5 minutes, and centrifuged at 12000rpm for 10 minutes. At this time, white DNA precipitate was visible, and 1mL of 70% ethanol was added, centrifuged at 12000rpm for 10 minutes, the supernatant was discarded, and after drying the residual ethanol, about 500μL of saline was added to dissolve the DNA, and the concentration was measured and marked. The plasmid pLVSIN-PGLYRP2 used for co-transfection has been disclosed in the article Hepatology.2020May;71(5):1626-1642. Huh7 cells cultured in 12-well cell culture plates were divided into two groups, designated Group 1 and Group 2. Each group consisted of three experimental wells and one control well. Each well in Group 1 was transfected with 0.5 μg of pAAV-EnII-EnCMV-M-Luciferase, while each well in Group 2 was transfected with 0.5 μg of pAAV-EnCMV-M-Luciferase. The control wells in each group were transfected with 0.5 μg of pLVSIN empty plasmid (purchased from TAKARA). The experimental wells in each group were transfected with 0.1 μg, 0.2 μg, and 0.4 μg of pLVSIN-PGLYRP2 plasmid, respectively. After 24 hours, luciferase intensity was detected using a luciferase reporter gene kit.

[0064] Results: As shown in Figure 11, PGLYRP2 protein promoted the protein expression activity of the backbone vector pAAV-EnCMV-M-Luciferase in a dose-dependent manner, and low-dose PGLYRP2 protein had a certain expression-promoting effect on the backbone vector; considering that PGLYRP2 protein has the characteristics of hepatocyte-specific expression, it is suggested that the vector backbone has the ability of hepatocyte-specific expression.

[0065] On the basis of the backbone vector pAAV-EnCMV-M, the PGLYRP2 protein negative regulatory element EnII was added to form the complete gene therapy delivery vector pAAV-EnII-EnCMV-M. The expression results in hepatocytes showed that under the condition of low expression of PGLYRP2 protein, the expression regulation of PGLYRP2 protein on the gene therapy delivery vector pAAV-EnII-EnCMV-M was mainly positive regulation; while the high expression of PGLYRP2 protein, due to its inhibitory effect on EnII, significantly reduced the protein expression level of the gene therapy delivery vector, and played a negative regulatory role on the gene therapy delivery vector. Therefore, PGLYRP2 protein has a bidirectional feedback regulatory effect on the regulation of the gene therapy delivery vector pAAV-EnII-EnCMV-M. Given that this bidirectional regulatory mechanism is derived from PGLYRP2 protein, PGLYRP2 promoter M, and PGLYRP2 regulatory elements, it is called self-feedback regulation (as shown in Figure 12).

[0066] Example 6: Analysis of the Hepatitis B Virus Clearance Effect of Adeno-Associated Virus Gene Therapy Vector in Mice

[0067] Preparation of pAAV-EnII-EnCMV-M / PGLYRP2 virus solution:

[0068] HEK293T cells were transfected with phelper (Addgene, #112867), RC8 (Addgene, #112864), and pAAV-EnII-EnCMV-M-PGLYRP2 plasmids at a molar ratio of 1:1:1. After transfection, the cells were cultured in a 37°C, 5% CO2 incubator. Cells and culture supernatants were collected after 72 hours, and the cell suspension was repeatedly frozen and thawed three times at -80°C and room temperature. The suspension was then passed through a 15%-60% iodixanol density gradient ultracentrifugation (36300 rpm, 16°C for 3 h). After ultracentrifugation, the virus solution between 40% iodixanol and 60% iodixanol was aspirated and finally concentrated using a 100 KDa ultrafiltration tube (Millipore).

[0069] Six weeks before the mouse experiment, PGLYRP2 - / - C57BL / 6J mice were injected with pAAV-EnII-EnCMV-M / PGLYRP2 virus solution (1×10 11 copies / mouse), and 1×10 11All groups of mice were injected with AAV / 1.2*HBV virus via the tail vein. Four weeks after model establishment, HBV-positive mice were screened for subsequent studies. HBV viral titers (HBV DNA copy number) in mouse blood were measured 1-6 weeks after virus injection. Six weeks after virus injection, mouse blood was collected and the serum level of HBs in each group of mice was measured using ELISA (Sangon Biotech, D711407). Six weeks after virus injection, mouse livers were harvested and extrachromosomal free DNA was extracted using the Hirt method. HBV cccDNA was then treated with ExoI / ExoIII / T5 exonucleases. The purified HBV cccDNA was then used to detect the content of HBV cccDNA using real-time quantitative PCR. The primers used were cccDNA primer_F and cccDNA primer_R.

[0070] cccDNA primer_F: 5'-GTTCTGTGCCTTCTCATCTGC-3',

[0071] cccDNA primer_R: 5'-ACAAGAGATGATTAGGCAGAGG-3'.

[0072] Results: In the HBV mouse model, HBV virus titers (HBV DNA copy numbers) in the mouse blood were found 1-6 weeks after virus injection. Figure 13 shows the red curve ■ indicating mPGLYRP2. - / - , the blue curve ● represents WT (mPGLYRP2), and the black curve ▲ represents hPGLYRP2 (mPGLYRP2 - / - / hPGLYRP2), compared with mPGLYRP2 - / - Group, WT (mPGLYRP2) and hPGLYRP2 (mPGLYRP2 - / - The HBV DNA copy number in the serum of mice in the hPGLYRP2 / hPGLYRP2 group was significantly decreased. - / - The HBV DNA copy number in the serum of mice in the WT (mPGLYRP2) group decreased more significantly than that in the WT (mPGLYRP2) group, indicating that the adeno-associated virus gene therapy vector of the present invention can significantly inhibit viral replication, and the adeno-associated virus vector carrying hPGLYRP2 has more advantages than the adeno-associated virus vector carrying the mPGLYRP2 gene.

[0073] Six weeks after virus injection, relative to mPGLYRP2 - / - group, WT (mPGLYRP2) group and hPGLYRP2 (mPGLYRP2 - / -The viral HBsAg content in the blood of mice in the hPGLYRP2 / hPGLYRP2 group was significantly reduced. - / - The viral HBsAg content in the blood of mice in the WT (mPGLYRP2) group was more significantly reduced than that in the WT (hPGLYRP2) group, indicating that both hPGLYRP2 and mPGLYRP2 proteins have significant effects on the clearance of viral surface antigens in vivo, among which the clearance effect of hPGLYRP2 protein is more significant (Figure 14).

[0074] Six weeks after virus injection, the HBV cccDNA content in the mouse liver was detected by real-time quantitative PCR and found that the HBV cccDNA content in the mouse liver was significantly higher than that in the mPGLYRP2 - / - group, WT (mPGLYRP2) group and hPGLYRP2 (mPGLYRP2 - / - The viral cccDNA content in the liver of mice in the hPGLYRP2 / hPGLYRP2 group was significantly decreased, among which the hPGLYRP2 (mPGLYRP2 - / - The viral cccDNA content in the blood of mice in the WT (mPGLYRP2) group was more significantly reduced than that in the WT (hPGLYRP2) group, indicating that both hPGLYRP2 and mPGLYRP2 proteins have a significant effect on the clearance of viral cccDNA in the body, among which the clearance effect of hPGLYRP2 protein is more significant (Figure 15).

[0075] Current treatment strategies for chronic hepatitis B can effectively inhibit HBV replication, but achieving a complete cure is difficult because free cccDNA in the patient's liver persists in the cell nucleus for a long time, making it difficult for drugs to clear it, and discontinuation of medication often leads to relapse. Therefore, the long-term presence of cccDNA in infected hepatocytes is a key factor in the persistence of viral infection and a major obstacle to the cure of hepatitis B. The present invention has clearly demonstrated its ability to clear hepatitis B virus cccDNA in both cell and mouse experiments. As a potential drug capable of clearing the cccDNA reservoir in the body, the present invention will bring hope for a complete cure for hepatitis B.

Claims

1. Application of PGLYRP2 gene in the preparation of drugs for treating hepatitis B virus infection.

2. Application of PGLYRP2 gene in the preparation of drugs for eliminating hepatitis B virus cccDNA.

3. A gene therapy vector for eliminating hepatitis B virus, characterized in that: The gene therapy vector comprises a promoter, an intron, an enhancer and a PGLYRP2 gene coding region.

4. The gene therapy vector according to claim 3, characterized in that The vector backbone is a viral vector or a non-viral vector.

5. The gene therapy vector according to claim 4, characterized in that: The viral vector is an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a herpes simplex viral vector, a Sendai viral vector or a boca viral vector; the adeno-associated viral vector is an AAV2, AAV5, AAV7, AAV8, AAV9 or AAV-DJ type adeno-associated viral vector.

6. The gene therapy vector according to claim 4, characterized in that The non-viral vector is liposome nanoparticles, polymer nanoparticles, exosomes, polypeptide complexes, mRNA vectors, iTOP or Feldan shuttle.

7. The gene therapy vector according to claim 3, characterized in that: The promoter is a truncated version M of the PGLYRP2 gene promoter, and its nucleotide sequence is shown in SEQ ID NO: 1 in the sequence table.

8. The gene therapy vector according to claim 3, characterized in that: The enhancers are CMV enhancer and HBV EnII element. The nucleotide sequence of CMV enhancer is shown in SEQ ID NO: 2 in the sequence list, and the nucleotide sequence of HBV EnII element is shown in SEQ ID NO: 3 in the sequence list.

9. The gene therapy vector according to claim 3, characterized in that: The nucleotide sequence of the PGLYRP2 gene coding region is shown in SEQ ID NO: 4 in the sequence listing.

10. A pharmaceutical composition for eliminating hepatitis B virus, characterized in that: The pharmaceutical composition contains the gene therapy vector as described in any one of claims 3 to 9.

Images