Novel assay to detect integrated HBV DNA in urine of chronic hepatitis B patients

检测慢性B型肝炎患者尿液中整合型HBV DNA的新方法 B型肝炎病毒（HBV）感染是一个全球性的公共卫生问题。疾病控制和预防中心估计， 100万美国人慢性感染HBV。HBV感染引起急性和慢性 肝炎，后者往往导致严重的肝脏疾病，如肝硬化和HBV相关 肝细胞癌（HBV-HCC）。HBV-HCC占全球HCC病例的50%。HBV是 一种DNA病毒，其部分基因组可以整合到宿主染色体中。虽然HBV DNA 通过核苷类似物、整合的病毒DNA和共价结合， 闭环HBV DNA（cccDNA）即使在长期抗病毒治疗后仍持续存在于肝细胞中。 持续的抗病毒治疗是必要的，以保持病毒复制受到抑制，因为它 在消除“残余”HBV DNA方面无效（即，整合HBV DNA和cccDNA）。 目前，治疗目标是实现功能性治愈，定义为清除B型肝炎 表面抗原（HBsAg），而不明确消除整合或cccDNA。无临床试验， 然而，目前能够识别这种“治愈”的人群。目前的想法是， 抗HBV治疗有效期延长后，整合的HBV DNA是残留HBV DNA的主要种类 在HBeAg阴性患者中，大多数HBsAg来源于此DNA。检测能力 慢性B型肝炎患者的DNA整合是一个迫切的未满足的需求。这个目标 应用是开发一种非侵入性尿液检测，可以检测和表征整合HBV DNA，并用于监测抗病毒治疗效果，并确定“治愈”的受试者谁可以 取消了终身治疗在第一阶段的可行性研究中，我们将改进我们目前的原型 HBV-宿主连接检测分析，并开发计算管道HBVJSeq，用于全面的 HBV整合和HBV遗传学分析（目的1）。在目标2中，我们将执行比较 分析尿中整合DNA的定量和定性特征， 血浆中HBsAg和HBV前基因组RNA（pgRNA）在B肝炎e抗原中的水平 （HBeAg）阴性患者。这些分析的结果将用于确定无细胞整合是否 HBV DNA可作为生物标志物用于HBeAg阴性患者的“治愈”鉴定 人群，启动第二阶段研究，将测试带到诊所进行疾病管理。

Novel assay to detect integrated HBV DNA in urine of chronic hepatitis B patients Hepatitis B virus (HBV) infection is a global public health concern. The CDC estimates that up to 2 million Americans are chronically infected with HBV. HBV infection causes acute and chronic hepatitis, the latter often leading to severe liver diseases such cirrhosis and HBV-associated hepatocellular carcinoma (HBV-HCC). HBV-HCC accounts for 50% of HCC cases worldwide. HBV is a DNA virus, and its partial genome can integrate into a host chromosome. Although HBV DNA replication can be suppressed effectively by nucleoside analogs, integrated viral DNA and covalently closed circular HBV DNA (cccDNA) persist in hepatocytes even after prolonged antiviral therapy. Continuous antiviral treatment is necessary to keep viral replication suppressed due to its ineffectiveness in eliminating the “remnant” HBV DNA (i.e., integrated HBV DNA and cccDNA) fully. Currently, the treatment goal is to achieve a functional cure, defined as the clearance of hepatitis B surface antigen (HBsAg), without definitively eliminating integrated or cccDNA. NO clinical test, however, is currently capable of identifying this “cured” population. The current thinking is that after prolonged effective anti-HBV therapy, integrated HBV DNA is the major species of residual HBV DNA in liver and most HBsAg in HBeAg-negative patients is derived from this DNA. The ability to detect integrated DNA in patients with chronic hepatitis B is an urgent unmet need. The goal of this application is to develop a noninvasive urine test that can detect and characterize integrated HBV DNA, and be used to monitor antiviral treatment efficacy and identify “cured” subjects who can be taken off the lifelong therapy. In the phase I feasibility study, we will improve our current prototype HBV-host junction detection assay and develop a computation pipeline, HBVJSeq, for comprehensive profiling of HBV integration and HBV genetics (Aim 1). In Aim 2, we will perform comparative analyses to correlate quantitative and qualitative characteristics of integrated DNA in urine and plasma with the levels of HBsAg and HBV pregenomic RNA (pgRNA) in hepatitis B e-antigen (HBeAg)-negative patients. Results of these analyses will be used to determine if cell-free integrated HBV DNA can be developed as a biomarker to identify “cured” patients in HBeAg-negative populations, warranting a phase II study to bring the test to the clinic for disease management.