Chromatin Accessibility-Guided Targeting Identifies Structurally Constrained Regions in HBV cccDNA and Suppresses...

Area of Science:

• Virology
• Epigenetics
• Molecular Biology

Background:

• Covalently closed circular DNA (cccDNA) is the stable hepatitis B virus (HBV) genome form, crucial for viral replication and a key obstacle to HBV eradication.
• Understanding cccDNA's interaction with host cell machinery, particularly chromatin, is vital for developing effective therapies.

Purpose of the Study:

• To map chromatin accessibility patterns of HBV cccDNA in infected hepatocytes at single-molecule resolution.
• To identify specific cccDNA regions vulnerable to therapeutic targeting based on chromatin accessibility.
• To evaluate the efficacy of targeting these regions using a CRISPR/Cas9-based approach.

Main Methods:

• Single-molecule resolution analysis of chromatin accessibility on HBV cccDNA in HBV-infected hepatocytes.
• CRISPR/Cas9 gene editing to target identified accessibility-associated regions on cccDNA.
• Quantification of HBV DNA-related signals, including cccDNA fractions and total HBV DNA, to assess therapeutic impact.

Main Results:

• Most HBV cccDNA copies showed limited chromatin accessibility around nucleotides 800-1000, overlapping key viral gene regions.
• A subset of cccDNA displayed detectable accessibility at this site, indicating heterogeneous chromatin states.
• Targeting the accessibility-associated region with CRISPR/Cas9 led to significant reductions in HBV DNA levels and cccDNA enrichment.

Conclusions:

• Chromatin accessibility profiling can identify novel, vulnerable regions within HBV cccDNA for therapeutic intervention.
• This study provides a proof-of-concept for an accessibility-informed strategy to target and reduce HBV.
• Further research into chromatin-associated vulnerabilities of HBV cccDNA is warranted for advancing HBV cure strategies.