Suppression Of HBV Gene Expression Through Base Editing And Restoration Of HBV-specific T Cell Function By PD-L1 Antibody

Background and aims:Chronic hepatitis B（CHB）is a serious public health problem worldwide,accounting for about 650,000 deaths every year.Two categories of drugs have been approved to treat CHB:the interferon-alpha and nucleos（t）ide analogues.However,most of the patients could not achieve a functional cure,which is defined as loss of hepatitis B surface antigen（HBsAg）.Long-term and even lifetime therapy is needed for these patients.The major obstacle to HBV cure is the covalently closed circular DNA could not be eradicated by current antiviral drugs.Meanwhile,integrated HBV DNA in the host genome is another important source of HBsAg.Clustered regularly interspaced short palindromic repeat（CRISPR）/CRISPR-associated protein 9（Cas9）is a novel genome editing technology that could knock out genes through DNA double-strand break.Several in vitro and in vivo studies have proved that CRISPR/Cas9 could inhibit HBV replication and reduce the expression level of viral proteins.Nonetheless,repair of DNA double-strand break not only leads to small insertion and deletion（indel）but also induces large deletion and impairs genome stability.Since HBV integration occurs commonly in CHB patients,CRISPR/Cas9targeting HBV sequence raises concerns about safety problems.CRISPR base editors could precisely modify a single nucleotide in the genome which doesn’t lead to DNA double-strand break and has a higher safety profile.Cytidine base editor（CBE）could generate stop codons under the guidance of single guide RNA（sgRNA）.The feasibility and efficiency of CBE in silencing genes of integrated HBV within the genome of hepatocytes have not been investigated.The study aimed to determine the feasibility and efficiency of silencing HBV S gene through CBE,and evaluate possible off-target effects.T cell response plays a dominant role in the clearance of HBV infection.However,T cell exhaustion occurs during chronic HBV infection,leading to viral persistence.Exhausted HBV-specific T cells are characterized by upregulation of multiple inhibitory receptors including programmed death-1（PD-1）.Several studies have proved that blocking the binding of PD-1 and its ligand PD-L1 could enhance the function of HBV-specific T cells,promote clearance of viral infection and reduce the risk of viral rebound.Nonetheless,recent clinical trials show that only a small proportion of CHB patients demonstrated a significant reduction in serum HBsAg after PD-1/PD-L1 blockade therapy.Factors that affect the patient’s response to PD-1/PD-L1 blockade were undetermined.In this study,the improvement of HBV-specific T cell function after in vitro PD-L1 blockade in CHB patients was accessed to determine the clinical parameters that may affect the response to PD-L1antibody therapy.Methods:Conservative regions of the HBV S gene were identified through bioinformatic analysis.sgRNAs which could induce premature stop codons in S gene were designed in the conservative region.PLC/PRF/5 is a human hepatoma cell line with integrated HBV DNA.Using lentiviral transduction,a stable PLC/PRF/5 cell line expressing CBE was established（PLC/PRF/5-CBE）,which was infected with either empty lentiviral vectors or lentiviral vectors expressing nonspecific sgRNA or sgRNA targeting HBV S gene.The on-target base editing efficiency is evaluated through Sanger and next-generation sequencing（NGS）.HBs mRNA levels were determined by northern blot.Immunofluorescence and enzyme-linked immunosorbent assay were used to measure the intracellular and extracellular HBsAg expression.Possible off-target sites in human genome of sgRNA targeting S gene was predicted using the Cas-Offinder online tool.Off-target editing was accessed by Sanger sequencing.To identify factors that may affect CHB patients’response to PD-L1 blockade therapy,37 CHB patients in different clinical phases were recruited in this study.Peripheral blood mononuclear cells（PBMCs）were isolated from patients’blood through density-gradient centrifugation.The expression of PD-1 and lymphocyte activating gene 3（LAG-3）on total and HBV-specific CD8+T cells were measured by flow cytometry and MHC-peptide multimer staining.PBMCs were stimulated with HBV core peptide pool in the presence of anti-PD-L1（10mg/ml）or control Ig G（10mg/ml）.After five days,the numbers of interferon-γ（IFN-γ）secreting T cells were determined by enzyme-linked immunospot assay.Moreover,changes in expression levels of inhibitory receptors,including LAG-3,cytotoxic T lymphocyte antigen-4（CTLA-4）,T cell immunoglobulin mucin-3（Tim-3）,and CD244,were accessed through flow cytometry.Results:Using multiple sequence alignment,we found nucleotides 72 to 118 of S gene were highly conservative among different HBV genotypes.A sgRNA targeting this region was designed and named as gRNA＿S.gRNA＿S could convert the 30^thcodon of S gene from CAG（glutamine）to stop codon TAG.Sequence analysis demonstrated that 59%of S gene sequences could bind specifically with gRNA＿S.Subgroup analysis showed that 95%,93%,93%,9%,and 72%of S gene sequences of HBV genotypes B,C,F,G,and H could be targeted by gRNA＿S.A stable cell line PLC/PRF/5-CBE was successfully constructed in which high levels of CBE mRNA and protein expression could be detected.NGS sequencing was performed in PLC/PRF/5-CBE cells transduced with lentiviral vectors encoding gRNA＿S.57.5%of cells edited by gRNA＿S generated a premature stop codon TAG.The frequency of indel was 6.1%in gRNA＿S edited cells.In contrast,no nucleotide alterations were found in cells treated with non-specific sgRNA.Northern blot analysis revealed an82.5%reduction of HBs mRNA after gRNA＿S treatment.The decrease of 2.1 kb RNA was more profound than that of 2.4 kb RNA.Creation of stop codon led to a 92%reduction of HBsAg in the culture supernatant.Similarly,the proportion of HBsAg-positive cells dropped by 66.7%.To further increase the efficiency of S gene silencing,we used a dual sgRNA strategy.However,dual sgRNA system showed a lower base editing efficiency and capability in suppressing HBsAg expression.Sequencing result in 11 predicted off-targets sites did not reaveal off-target activity for gRNA＿S.Immunophenotyping analysis of CHB patients showed that the expression of PD-1 in HBV-specific CD8⁺T cells was significantly higher than in total CD8⁺T cells（55.0%vs17.9%）.PD-1 was coexpressed with LAG-3.On average,PD-L1 blockade in vitro led to a1.5-fold increase in the number of IFN-γproducing HBV-specific T cells.The improvement in the IFN-γproduction capacity of T cells was more prominent in inactive carriers and HBe Ag-negative CHB patients that in immune tolerant and HBe Ag-positive CHB patients.Multiple linear regression analysis revealed that HBe Ag status was the only factor that was correlated with PD-L1 blockade induced enhancement of IFN-γproduction（P=0.033）.HBe Ag-positive patients had greater improvement in IFN-γproduction than HBe Ag-negative patients（fold change in the number of IFN-γproducing T cells:1.79 vs1.14,P<0.001）.PD-L1 blockade in vitro led to the compensatory upregulation of LAG-3expression on T cells and did not affect the expression of CTLA-4,Tim-3,and CD244.Conclusion:CBE is an efficient approach to silence the HBV S gene,resulting in substantially reduced HBs mRNA,intracellular and extracellular HBsAg.CBE exhibits minimum off-target editing,suggesting its potential as a novel treatment strategy for CHB.PD-L1 antibody could enhance the IFN-γproduction capability of HBV-specific T cells in vitro,while the improvement of T cell function is mainly affected by HBe Ag status.