Development of a PCR assay for quantitative detection of HBV cccDNA

HBV cccDNA定量PCR检测方法的建立 目前迫切需要一种有效的方法来检测B型肝炎病毒 (HBV)cccDNA在血液中。HBV感染影响全球近3.5亿人。慢性 乙型肝炎病毒感染是发展为严重肝病如肝硬化的主要危险因素 和肝细胞癌，其存活率很低。目前的治疗方法是有效的 抑制病毒复制然而，由于他们无法消除病毒， 为了消除质粒样附加体，共价闭合环状DNA（cccDNA）可以用作 作为传染性病毒不断产生的模板。这种cccDNA， 在宿主细胞核中，是由松弛的环状DNA（rcDNA）产生的， 在循环病毒体中发现并传输到宿主肝细胞中的单链DNA。的 需要去除cccDNA来治愈HBV感染，这促使药物发现小组将重点放在 致力于开发可以靶向和消除cccDNA的化合物。即使在目前的 在新药开发中，仍然缺乏定量的，特异的和可靠的 cccDNA测定，其对于检测血液中的cccDNA高度敏感，以及 其特异性足以检测不到rcDNA。该SBIR的目标是开发一种创新的 和敏感的cccDNA测定，适用于医生的常规检测， 慢性HBV感染患者，促进抗HBV药物的开发。我们设计了 我们的初步研究表明，特异性检测1,000个拷贝的 cccDNA和10，000个rcDNA拷贝的不可检测扩增。提出了两个目标 在第一阶段的申请中。目的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%。目的2是用肝活检进行检测验证 慢性B肝炎（CHB）患者外周血cccDNA的连续测定 接受治疗在第二阶段，我们将进一步开发和评估检测方法，以监测治疗效果。 功效

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.