Development of an assay for quantitative detection of cccDNA This SBIR application is in response to the Funding Opportunity Announcement (FOA) PA-15- 077 New Technologies for Viral Hepatitis SBIR (R43/R44), more specifically to #7 Develop genetic tests that might help in patient management of viral hepatitis, including tests that assess the risk of complications as well as likelihood of success of specific therapies. There is an urgent and unmet need for an effective method to detect hepatitis B virus (HBV) cccDNA in serum. HBV infection affects nearly 350 million people worldwide, and chronic infection is a major risk factor for development of cirrhosis and hepatocellular carcinoma, which has a poor survival rate. Current therapies are effective at suppressing viral replication, although they do not eliminate the virus due to the persistent viral replication intermediate known as covalently closed circular DNA (cccDNA) that serves as a template for generation of progeny virus. This plasmid-like episome that resides in the host nucleus is produced 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. However, the absence of a quantitative reliable cccDNA assay that is highly sensitive, obtainable from serum, and not contaminated by detection of the highly analogous rcDNA is needed to facilitate the drug development and to monitoring cccDNA level in the blood of HBV infected patient under anti-viral therapy with repeated liver biopsies The goal of this project is to develop an innovative sensitive cccDNA assay that overcomes these challenges and would be suitable for routine testing by physicians. We have designed such an assay and our preliminary studies have indicated specific detection of 103 copies of cccDNA and no detectable amplification of 104 copies of rcDNA. Two aims are proposed in this phase I application. Aim 1 is to develop this prototype assay to be quantitative and sensitivity of 0.1% of cccDNA to rcDNA. Aim 2 is to demonstrate the detection of cccDNA in serum of patients with HBV infection and correlate this detection with HBV surface antigen (HBsAg). In phase 2 we will further develop and evaluate the assay for monitoring of therapeutic efficacy and extent of liver damage.
Public Health Relevance Statement: PUBLIC HEALTH RELEVENCE: HBV infection places nearly 350 million people globally at risk for development of cirrhosis and subsequent hepatocellular carcinoma (HCC), which is the third leading cause of cancer worldwide and has one of the highest recurrence rates. A cure for HBV is now recognized as a treatment that can completely eliminate cccDNA. Development of such therapies will require suitable companion diagnostics to monitor the persistence of cccDNA. The goal of this phase 1 project is to develop a highly sensitive cccDNA assay that can accurately detect cccDNA from a serum sample.
Project Terms: Affect; anti-hepatitis B; Antiviral Therapy; Archives; assay development; Biological Assay; Biotechnology; bisulfite; Blood; Cancer Etiology; Cell Culture Techniques; Cell Nucleus; Chronic; Chronic Hepatitis B; Chronic Hepatitis C; Circular DNA; Cirrhosis; Clinical; companion diagnostics; cost; Data; design; Detection; Development; Diagnostic; Disease; DNA; DNA Sequence; drug development; Drug Discovery Groups; ds-DNA; Episome; Funding Opportunities; Generations; Genetic screening method; Goals; Hepatitis B; Hepatitis B Surface Antigens; Hepatitis B Virus; Hepatocyte; HepG2; Infection; innovation; intrahepatic; Laboratories; Liver; liver biopsy; Liver Cirrhosis; liver injury; Malignant neoplasm of liver; Measurement; Measures; Methods; Modification; Molecular; Monitor; Multi-Institutional Clinical Trial; new technology; novel; novel strategies; Patients; Performance; peripheral blood; Phase; phase 1 study; Physicians; plasmid DNA; Plasmids; Ploidies; Primary carcinoma of the liver cells; prototype; Public Health; Recurrence; response; Risk; Risk Factors; Sample Size; Sampling; Serological; serological marker; Serum; Small Business Innovation Research Grant; success; Surface Antigens; Survival Rate; Technology; Testing; Time; Tissues; Treatment Efficacy; Viral; viral DNA; Viral hepatitis; Virion; Virus; Virus Diseases; Virus Replication
