Recombinant DNA synthesis methods for generating recombinantly HBV genome replication intermediate

Background

Hepatitis B virus (HBV) causes chronic infection in at least 257 million people who are at risk of developing severe liver disease, including fibrosis, cirrhosis, and liver cancer (hepatocellular carcinoma, HCC). Although a prophylactic vaccine for HBV exists, less than 40% of the world is vaccinated, putting them at risk for contracting HBV. For individuals with chronic HBV, there is no cure. The standard of care consists of nucleos(t)ide infection, which must be taken for life and almost never cures chronic infection.

Covalently closed circular DNA (cccDNA) is a unique form of the HBV genome that persists in the nucleus of infected liver cells and therefore is a key factor in the persistence of chronic HBV infections. It is extremely stable and resistant to current antiviral therapies, making it a clear obstacle to achieving a complete cure for chronic HBV. Studying cccDNA is challenging due to technical limitations in detection, its low abundance in cells, and difficulties in developing suitable research models.

Technology

Princeton researchers have invented a new process which can be applied to recombinantly produce critical replication intermediates of HBV (e.g., cccDNA) as well as intermediates of other viruses. These intermediates can be used in biochemical, cell culture, and in vivo assays to preclinically test the efficacy of antiviral drug candidates.

Benefits

• More accurate resemblance to the intermediates of a real HBV infection (Current methods to produce in vitro cccDNA rely on T4 ligation, which generates concatemers that are NOT detected in a real HBV infection)
• Maintenance of open reading frames (Current methods may rely on Cre-LoxP recombination, which leaves a LoxP sequence in the cccDNA, disrupting at least one of the open reading frames)
• Requires small amounts of cccDNA 
• Can easily produce rcDNA molecules with different plus strand lengths
• Broad applicability of method to produce ssDNA constituting genomic DNA of other viruses 

Applications

• Generation of recombinant hepatitis B virus (HBV) intermediates: covalently closed circular DNA (RcccDNA) and relaxed circular DNA (RrcDNA) 
• Usage of recombinant HBV intermediates in biochemical, cell culture, and in vivo assays for preclinical testing of antiviral drug candidates
• Generation of covalently closed circular DNA of other viruses (e.g., HPV, polyomavirus) 
• Synthesis of genomes of ssDNA viruses in vitro 
• Generation of reporters for monitoring DNA damage (e.g., molecules that resemble the origin of replication, protein DNA adducts) 

Additional info

Experimental Validation

• The cccDNA was verified by T5 digestion and plasmid sequencing (Plasmidsaurus)
• The rcDNA motility was verified by agarose gel electrophoresis
• Both molecules were extensively tested in vitro and in mice, and shown to be suitable for initiating the entire viral life cycle 

Relevant Publications (exact invention has not been publicly disclosed yet)

• Gomez-Moreno, Andoni, and Alexander Ploss. (2024) 2024. “Mechanisms of Hepatitis B Virus CccDNA and Minichromosome Formation and HBV Gene Transcription.”. Viruses 16 (4): 609. doi:10.3390/v16040609.
• Gomez-Moreno, Andoni, Jinchao Guo, Heidi Temple, and Alexander Ploss. (2024) 2024. “Formation and Transcriptional Regulation of Hepatitis B Virus Covalently Closed Circular DNA.”. Journal of Hepatology. doi:10.1016/j.jhep.2024.03.008.

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. 
• Andoni Gomez-Moreno, Ph.D. is a Postdoctoral Fellow in the Ploss Lab. Dr. Gomez-Moreno completed his Ph.D. at the National center for Biotechnology (CNB-CSIC) in Madrid, Spain and received his Master’s degree in Virology from Complutense University in Madrid, Spain.