Cytosine base editing inhibits hepatitis B virus replication and reduces HBsAg expression in vitro and in vivo

Area of Science:

• Molecular Biology
• Hepatology
• Gene Therapy

Background:

• Chronic hepatitis B virus (HBV) infection is a global health challenge with limited curative treatments.
• Persistent HBV covalently closed circular DNA (cccDNA) and integrated HBV DNA contribute to viral rebound and immune evasion.
• Hepatitis B surface antigen (HBsAg) expression from integrated HBV DNA impairs anti-HBV immune responses.

Purpose of the Study:

• To evaluate the efficacy of cytosine base editors (CBEs) in targeting and disabling HBV DNA.
• To assess the potential of base editing to reduce viral markers and protein expression in vitro and in vivo.

Main Methods:

• Cytosine base editors (CBEs) were designed to introduce stop codons in HBV HBs and Precore genes.
• mRNA encoding CBEs and guide RNAs were transfected into HBV-infected cells (HepG2-NTCP, primary human hepatocytes) and cell lines with integrated HBV DNA (PLC/PRF/5, HepG2.2.15).
• An HBV minicircle mouse model with lipid nanoparticulate delivery was used for in vivo evaluation.

Main Results:

• Robust editing of HBV cccDNA and sustained reduction of viral markers were observed in cell cultures.
• Base editing significantly reduced HBsAg expression from integrated HBV DNA in cell lines.
• In vivo studies showed >3log10 reduction in serum HBV DNA, >2log10 reduction in HBsAg, and HBsAg loss in 4/5 mice.

Conclusions:

• Base editing effectively targets and mutates both cccDNA and integrated HBV DNA.
• This approach abrogates HBV replication and silences viral protein expression, offering a promising therapeutic strategy.
• Gene editing via CBEs demonstrates significant antiviral efficacy for chronic hepatitis B.