A method for scarless production of recombinant viral DNA intermediates for Hepatitis B Virus research and therapeutic development

Background

HBV exhibits wide genetic diversity with at least 9 genotypes (GTs), which differ in terms of prevalence, geographic distribution, natural history, disease progression, and treatment outcome. However, differences in HBV replicative capacity, gene expression, and infective capability across different GTs remain incompletely understood.

Technology

Princeton researchers have constructed infectious clones covering the major HBV GTs (A-E) and characterized in detail their replicative capacity in cell lines, primary hepatocytes, and (humanized) mice. The patient-derived HBV infectious clones replicate robustly in both vitro and in vivo and have broad utility as genetic tools that can mechanistically dissect intergenotypic differences in antiviral immunity and pathogenesis and aid in HBV drug development and screening.

Benefits

• Enables scarless, biologically relevant production of HBV covalently closed circular DNA (cccDNA) and relaxed circular DNA (rcDNA) that initiate full viral life cycle in vitro and in vivo
• Requires minimal template DNA from diverse sources, enhancing flexibility and reducing material needs
• Produces replication intermediates free of bacterial sequence scars and concatemer artifacts, improving authenticity for research
• Versatile and adaptable method applicable to multiple hepadnaviruses and other single-stranded DNA viruses
• Supports scalable production suitable for preclinical antiviral drug testing and virology research
• Facilitates molecular and functional studies of HBV replication, persistence, and therapeutic targeting
• Enables in vitro repair studies of rcDNA to cccDNA conversion, advancing understanding of viral genome maturation

Applications

Broad utility as genetic tool capable of dissecting intergenotypic differences in antiviral immunity and pathogenesis:

• Preclinical antiviral drug screening
• HBV research reagent supply 
• Diagnostic assay development
• Gene therapy vector engineering

Additional info

Publication and Experimental Validation

Liu, Y., Park, D., Cafiero, T. R., Bram, Y., Chandar, V., Tseng, A., Gertje, H. P., Crossland, N. A., Su, L., Schwartz, R. E., & Ploss, A. (2022). Molecular clones of genetically distinct hepatitis B virus genotypes reveal distinct host and drug treatment responses. JHEP reports : innovation in hepatology, 4(9), 100535. https://doi.org/10.1016/j.jhepr.2022.100535

Inventors

• Professor Alexander Ploss completed his Ph.D. in Immunology at Memorial Sloan-Kettering Cancer Center and Cornell University and postdoctoral training at The Rockefeller University in New York City. Dr. Ploss is the endowed Harry C. Wiess Professor in the Life Science and Molecular Biology, member of the executive committee of the Center Health and Wellbeing and Co-Director of the Program in Global Health and Health Policy at Princeton University. He is also a full member of the Cancer Institute of New Jersey. In support and recognition of his work Dr. Ploss received numerous awards including the including the Astella’s Young Investigator Award from the Infectious Disease Society of America, the Liver Scholar Award from the American Liver Foundation, the Löffler-Frosch Prize from the German Society of Virology, Merck Irving Sigal Memorial Award from the American Society for Microbiology, and the Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease Award. Dr. Ploss is also an elected Fellow of the American Academy of Microbiology and the American Association for the Advancement of Science. 
• Yongzhen Liu, Ph.D. is an Assistant Professor at Nanjing University in Nanjing, China. Dr. Liu was previously a Postdoctoral Fellow in the Ploss Lab. Dr. Liu received his Ph.D. from the Department of Microbiology and Infectious Disease Center at Peking University Health Science Center in Beijing, China, studying HBV and HCC.