The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely

Area of Science:

• Virology
• Molecular Biology
• Drug Discovery

Background:

• Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is a persistent, drug-resistant form of the virus.
• Developing effective anti-cccDNA drugs is crucial for eradicating HBV infection.

Purpose of the Study:

• To develop a convenient and scalable technology for producing recombined cccDNA (rcccDNA) as a surrogate for wild-type cccDNA (wtcccDNA).
• To validate the biological relevance and utility of rcccDNA in HBV research and drug development.

Main Methods:

• Utilized a minicircle DNA vector (MC)-based technology for producing rcccDNA.
• Characterized rcccDNA for structural and functional resemblance to wtcccDNA.
• Assessed rcccDNA's biological features, including nuclear entry, RNA/protein production, and virion generation.
• Employed CRISPR/Cas9 system to demonstrate rcccDNA cleavage in vitro.

Main Results:

• Successfully generated milligram quantities of rcccDNA using a convenient, lab-scale procedure.
• rcccDNA exhibited key biological characteristics of wtcccDNA, including nucleation, serving as a transcription template, and producing infectious virions.
• Demonstrated in vitro cleavage of rcccDNA by the CRISPR/Cas9 system, validating its potential as a drug target.

Conclusions:

• Developed a practical technology for large-scale rcccDNA production, serving as a valuable surrogate for wtcccDNA.
• This rcccDNA system provides a powerful tool for investigating HBV biology and advancing the development of novel anti-cccDNA therapeutics.
• The findings pave the way for new strategies to combat the global burden of Hepatitis B virus.