A platform using methylation-free HBV mini-circle DNA reporters for high-throughput screening of antiviral compounds

Background

HBV infection is a global health problem. Two billion people have been infected with HBV, 250 million developed chronic infections, and 600,000 die each year from HBV-related liver diseases, including liver cancer. Current therapies rarely achieved a cure due to the refractory nature of a stable chromatinized episomal DNA in the nucleus referred to as covalently closed circular DNA (cccDNA). To achieve a cure for HBV, cccDNA must be transcriptionally silenced or eradicated.

Technology

Researchers at Princeton have developed a novel strategy to screen for anti-HBV drugs. This novel screening method leverages reporter genes designed to monitor the activities of viral promoters that are active in chronic HBV patients; the mini-HBV reporters can be used to screen small molecules that interfere with the transcription or stability of cccDNA, without using or producing hazardous infectious virus.

Benefits

• Reporter genes monitor the activities of viral promoters that are active in chronic HBV patients (Traditional HBV reporters monitor the activities of promoters that may not be active in chronic HBV patients)
• Reporter DNA is free of DNA methylation (Traditional HBV reporters generated in E. coli contain DNA methylation that interferes with transcription and creates artifacts)
• Allows for screening of anti-HBV drugs while without the usage or production of hazardous infections

Applications

• High-throughput HBV drug screening
• Antiviral compound efficacy testing
• Pharmaceutical HBV research tool
• Reporter assay kit for HBV

Additional info

Experimental Verification

• Researchers have generated fluorescent protein- and luciferase-based HBV mini-circle reporters; they have also performed a substantial number of experiments to demonstrate that these reporters can be used to detect HBV activity with very high sensitivity, and without production of hazardous infectious virus
• Large quantities of mini-circle reporters free of DNA methylation can be generated; the reporters retain all key regulatory elements for viral transcription and produce key viral proteins similarly to authentic HBV in the HepG2 human hepatoma cell line
• Mini-circle reporters produce high amounts of fluorescent proteins and luciferase in HepG2 cell line

Inventors

• Alexander Ploss, Ph.D. 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. 
• Lei Wei, Ph.D. is an Assistant Professor at West Lake University in Hongzhou, China. Dr. Wei was previously a Postdoctoral Fellow in the Ploss Lab. Dr. Wei received his Ph.D. in Genetics and Molecular Biology from Memorial Sloan-Kettering Cancer Center and his Master’s degree in Structural Biology from Tsinghua University.