Development of a PCR assay for quantitative detection of HBV cccDNA

Development of a PCR assay for quantitative detection of HBV cccDNA There is an urgent and unmet need for an effective method to detect hepatitis B virus (HBV) cccDNA in the blood. HBV infection affects nearly 350 million people worldwide. Chronic HBV infection is a major risk factor for the development of severe liver diseases such as cirrhosis and hepatocellular carcinoma, which has a poor survival rate. Current therapies are effective at suppressing viral replication. However, they do not eliminate the virus due to their inability to eliminate the plasmid-like episome, covalently closed circular DNA (cccDNA) which can serve as a template for the continuous generation of infectious viruses. This cccDNA, which resides in the host nucleus, is generated from relaxed circular DNA (rcDNA), a partially double- stranded DNA found in circulating virions and transmitted into the host hepatocytes. The need to remove cccDNA to cure HBV infection has prompted drug discovery groups to focus efforts on developing compounds that can target and eliminate cccDNA. Even with the ongoing development of novel drugs, there is still an absence of a quantitative, specific, and reliable cccDNA assay that is both highly sensitive for the detection of cccDNA in the blood, as well as being specific enough to not detect rcDNA. The goal of this SBIR is to develop an innovative and sensitive cccDNA assay that would be suitable for routine testing by physicians to manage patients with chronic HBV infection and facilitate anti-HBV drug development. We have designed such an assay, and our preliminary studies have indicated specific detection of 1,000 copies of cccDNA and no detectable amplification of 10,000 copies of rcDNA. Two aims are proposed in this phase I application. Aim 1 is to develop an innovative quantitative PCR assay that has a sensitivity of 0.1% of cccDNA to rcDNA. Aim 2 is to perform assay validation with liver biopsies and serial measurement of cccDNA from peripheral blood of chronic hepatitis B (CHB) patients on therapy. In phase II, we will further develop and evaluate the assay to monitor therapeutic efficacy.