Development of a PCR assay for quantitative detection of HBV cccDNA

定量检测 HBV cccDNA 的 PCR 检测方法的开发

• 批准号: 10602051
• 负责人: Selena Lin
• 金额: $ 30.65万
• 依托单位: JBS SCIENCE, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-07 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10602051
• 关键词: Acute Hepatitis; Affect; Antiviral Therapy; Archives; Biological Assay; Biological Markers; Biopsy Specimen; Biotechnology; Blood; Cell Culture Techniques; Cell Nucleus; Chronic Hepatitis; Chronic Hepatitis B; Chronic Hepatitis C; Circular DNA; Cirrhosis; Clinical; DNA; DNA Sequence; DNA biosynthesis; Data; Detection; Development; Diagnostic; Disease; Drug Discovery Groups; Episome; Generations; Goals; HepG2; Hepatitis B; Hepatitis B Virus; Hepatitis B e Antigens; Hepatocyte; Laboratories; Liver; Liver diseases; Malignant neoplasm of liver; Marketing; Measurement; Measures; Methods; Modification; Monitor; Multi-site clinical study; Patients; Performance; Persons; Phase; Physicians; Plasma; Plasmids; Ploidies; Primary carcinoma of the liver cells; Privatization; Public Health; Regimen; Relaxation; Research; Risk; Risk Factors; Sample Size; Sampling; Science; Serum; Small Business Innovation Research Grant; Surface Antigens; Survival Rate; Technology; Testing; Time; Treatment Efficacy; United States National Institutes of Health; Validation; Viral; Virion; Virus; Virus Diseases; Virus Replication; anti-hepatitis B; assay development; bisulfite; chronic liver disease; cost; design; detection assay; detection sensitivity; diagnostic technologies; drug development; ds-DNA; innovation; intrahepatic; liver biopsy; manufacture; novel; novel strategies; novel therapeutics; nucleoside analog; peripheral blood; pgRNA; phase 1 study; plasmid DNA; prospective; repository; research clinical testing; serological marker; transmission process; viral DNA

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.

定量检测 HBV cccDNA 的 PCR 检测方法的开发 迫切需要一种有效的方法来检测乙型肝炎病毒，但这一需求尚未得到满足 (HBV) 血液中的 cccDNA。 HBV 感染影响全球近 3.5 亿人。慢性的 乙型肝炎病毒感染是发生肝硬化等严重肝脏疾病的主要危险因素 和肝细胞癌，其生存率很低。目前的治疗方法是有效的 在抑制病毒复制方面。然而，由于他们的无能，他们并没有消灭病毒。 消除质粒样附加体、共价闭合环状 DNA (cccDNA) 作为不断产生传染性病毒的模板。这个 cccDNA 位于 在宿主细胞核中，由松弛的环状 DNA (rcDNA) 产生，这是一种部分双 在循环病毒体中发现的链状DNA并传输到宿主肝细胞中。这 需要去除 cccDNA 来治愈 HBV 感染，这促使药物研发小组关注 致力于开发能够靶向并消除 cccDNA 的化合物。即使持续进行 新药开发仍缺乏定量、特异、可靠的方法 cccDNA 检测对血液中的 cccDNA 检测高度敏感，并且 足够特异性，不会检测到 rcDNA。该 SBIR 的目标是开发一种创新型 和敏感的 cccDNA 检测，适合医生进行常规检测以管理 慢性乙型肝炎患者，并促进抗乙型肝炎药物的开发。我们设计了 我们的初步研究表明可以特异性检测到 1,000 个拷贝 cccDNA 且未检测到 10,000 个 rcDNA 拷贝的扩增。提出了两个目标 在这个第一阶段的应用中。目标 1 是开发一种创新的定量 PCR 检测方法， cccDNA 对 rcDNA 的敏感性为 0.1%。目标 2 是通过肝活检进行检测验证 慢性乙型肝炎（CHB）患者外周血cccDNA的连续测定 治疗上。在第二阶段，我们将进一步开发和评估监测治疗效果的检测方法 功效。