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HBV functional cure finding-Cytosine base editing inhibits Hepatitis B Virus replication and reduces HBsAg expression in vitro and in vivo
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Chronic Hepatitis B virus (HBV) infection remains a global health problem due to the lack of treatments that prevent viral rebound from HBV covalently closed circular (ccc)DNA. Additionally, HBV DNA integrates in the human genome serving as a source of hepatitis B surface antigen (HBsAg) expression, which impairs anti-HBV immune responses. Cytosine Base Editors (CBEs) enable precise conversion of a cytosine into a thymine within DNA. In this study, CBEs were utilized to introduce stop codons in HBV genes, HBs and Precore. Transfection with mRNA encoding a CBE and a combination of two guide RNAs led to robust cccDNA editing and sustained reduction of the viral markers in HBV infected HepG2-NTCP cells and primary human hepatocytes. Furthermore, base editing efficiently reduced HBsAg expression from HBV sequences integrated within the genome of PLC/PRF/5 and HepG2.2.15 cell lines. Finally, in the HBV minicircle mouse model, using lipid nanoparticulate delivery, we demonstrated antiviral efficacy of the base editing approach with a >3log10 reduction in serum HBV DNA and >2log10 reduction in HBsAg, and 4/5 mice showing HBsAg loss. Combined, these data indicate that base editing can introduce mutations in both cccDNA and integrated HBV DNA, abrogating HBV replication and silencing viral protein expression.
In this study, we have identified a combination of two guide RNAs (gRNAs), that, when paired with a CBE, inactivated both cccDNA and integrated HBV DNA in relevant HBV cell models, including HBV-infected HepG2-NTCP, HBV-infected primary hepatocytes (PHHs), as well as HepG2.2.15 and PLC/PRF/5 cell lines with artificially and naturally integrated HBV DNA, respectively. Furthermore, for the first time, we have shown durable antiviral efficacy, including HBsAg loss, in vivo in the HBVcircle mouse model using lipid nanoparticulate (LNP) delivery of base editing reagents (mRNA/gRNA). Combined with a thorough evaluation of gRNA-dependent off-target effects, this data advances our understanding of the potential of base editing to enable a functional cure for chronic HBV infection.
Taken together, our findings show that a nonviral vector can deliver cytosine base editing reagents capable of efficiently and irreversibly silencing cccDNA and integrated HBV DNA sequences in relevant in vitro and in vivo systems. These data improve our understanding of the potential of the base editing to cure HBV and contribute to our knowledge of the molecular mechanism of action by which base editing can serve as an effective antiviral.

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