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HBVccDNA的聚合酶链式反应方法的建立 迫切需要一种有效的方法来检测乙肝病毒，这种需求尚未得到满足 (乙肝病毒)ccDNA在血液中。全球有近3.5亿人感染了乙肝病毒。慢性 乙肝病毒感染是发展为严重肝病如肝硬变的主要危险因素。 以及存活率较低的肝细胞癌。目前的治疗方法是有效的 在抑制病毒复制方面。然而，由于他们无法消除病毒，他们并不能消除病毒。 为了消除质粒样的Episome，共价闭合的环状DNA(CccDNA)可以 作为持续产生传染性病毒的模板。这个cccDNA，它驻留在 在宿主核中，是由松弛的环状DNA(RcDNA)产生的，它是一种部分双核糖核酸。 在循环中的病毒粒子中发现的滞留的DNA，并传播到宿主肝细胞。这个 由于需要去除cccDNA来治疗乙肝病毒感染，这促使药物发现小组将重点放在 努力开发能够靶向和消除cccDNA的化合物。即使是正在进行的 新药的开发，还缺乏一种定量、特异、可靠的方法 一种检测血液中cccDNA的高度敏感的cccDNA分析方法，以及 具有足够的特异性，不会检测到rcDNA。这项SBIR的目标是开发一种创新的 和敏感的cccDNA检测，这将适合于医生的常规检测 有利于慢性乙肝患者抗病毒药物的开发。我们设计了 这样的检测，我们的初步研究表明，特异性检测到1000份拷贝的 10,000个拷贝的rcDNA未检测到扩增。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，该方法具有 0.1%的cccDNA对rcDNA的敏感性。目标2是通过肝活检进行化验验证 慢性乙型肝炎(CHB)患者外周血中CCDNA的连续检测 在接受治疗。在第二阶段，我们将进一步开发和评估该检测方法以监测治疗情况。 功效。