Designer Nucleases to Cure Chronic Hepatitis B

设计核酸酶可治愈慢性乙型肝炎

基本信息

批准号：
8608995
负责人：
Christoph Seeger
金额：
$ 26.78万
依托单位：
RESEARCH INST OF FOX CHASE CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-02-01 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8608995
关键词：
Adenoviruses Animals Antiviral Agents Antiviral Therapy Cell Death Cell Line Cell division Cells Chickens Chronic Chronic Hepatitis B Circular DNA Cleaved cell DNA DNA Maintenance DNA biosynthesis Duck Hepatitis B Virus Ducks Escape Mutant Exhibits Future Gene Mutation Genetic Transcription Goals Hepatitis B Virus Hepatocyte Immune response Infection Interphase Cell Investigation Laboratories Lead Measures Mitosis Modeling Mutate Patients Primary carcinoma of the liver cells Public Health RNA-Directed DNA Polymerase Reaction Risk Safety Site Staging Therapeutic Time Transcription Coactivator Validation Viral Virus Virus Replication Woodchuck Hepatitis B Virus Zinc Fingers base entecavir hepatoma cell insertion/deletion mutation nuclease nucleoside analog preference public health relevance repaired research study theories viral DNA viral RNA

项目摘要

DESCRIPTION (provided by applicant): Hepatitis B virus causes chronic infections in approximately 350 million people worldwide. All are at significantly increased risk of developing hepatocellular carcinoma. Although virus replication can be blocked by therapy with nucleoside analogs, infected hepatocytes are not cured, because covalently closed circular DNA (CCC DNA), the template for transcription of viral RNAs persists in infected cells. So far, a therapeuti strategy to functionally inactivate or even destroy CCC DNA within infected hepatocytes is lacking. We propose to use transcription activator-like effector nucleases (TALENs) specific for CCC DNA to cleave and mutate CCC DNA in regions essential for viral DNA replication. Hence, the goal of this application is to obtain proof of principle for the idea that an infected hepatocye can be cured of HBV through inactivation and possibly destruction of CCC DNA. Preliminary experiments from our laboratory already demonstrated that CCC DNA is indeed susceptible to cleavage by TALENs. The purpose of this proposal is to investigate the potential of TALENs to functionally inactivate CCC DNA by insertion/deletion of bases during the repair reaction, or to even destroy CCC DNA and to determine whether TALEN-based antiviral therapy could lead to the cure of infected cells. As a model for HBV, we will be using duck hepatitis B virus (DHBV) expressed in hepatoma cells and primary hepatocyte cultures that permit all steps of viral DNA replication including the formation of CCC DNA. The goals of this application will be achieved through the following two aims: Aim 1. Construction and validation of TALENs specific for DHBV. The purpose of this aim is i) to construct TALENs against three targets on DHBV required for DNA replication, ii) to measure the ability of each TALEN to cleave CCC DNA, iii) to determine the effects of cleavage on CCC DNA stability, and iv) to investigate whether cleavage by TALENs leads to integration of CCC DNA into chromosomal DNA. Aim 2. To determine the antiviral activity of TALENs. The purpose of this aim is to study the effect of TALEN-based therapy on infection of dividing hepatoma cells and non-dividing primary duck hepatocytes. We will investigate whether TALEN therapy leads to a time-dependent reduction in CCC DNA levels per cell and determine not only if CCC DNA can be functionally inactivated in non-dividing cells, but also if it can be destroyed without a need for cell death. Furthermore, we will determine whether TALEN therapy can protect hepatocytes from de novo infection.

描述（由申请人提供）：乙型肝炎病毒导致全球约 3.5 亿人慢性感染。所有这些人患肝细胞癌的风险都显着增加。虽然核苷类似物治疗可以阻断病毒复制，但受感染的肝细胞无法治愈，因为病毒 RNA 转录模板共价闭合环状 DNA (CCC DNA) 仍然存在于受感染的细胞中。迄今为止，尚缺乏功能性失活甚至破坏受感染肝细胞内 CCC DNA 的治疗策略。我们建议使用 CCC DNA 特异性的转录激活剂样效应核酸酶 (TALEN) 在病毒 DNA 复制必需的区域中切割和突变 CCC DNA。因此，本申请的目的是获得原理证明，证明感染的肝细胞可以通过 CCC DNA 的灭活和可能的破坏来治愈 HBV。我们实验室的初步实验已经证明 CCC DNA 确实容易被 TALEN 切割。该提案的目的是研究 TALEN 在修复反应过程中通过碱基插入/删除来功能性灭活 CCC DNA 的潜力，甚至破坏 CCC DNA 的潜力，并确定基于 TALEN 的抗病毒疗法是否可以治愈感染者细胞。作为 HBV 模型，我们将使用在肝癌细胞和原代肝细胞培养物中表达的鸭乙型肝炎病毒 (DHBV)，其允许病毒 DNA 复制的所有步骤，包括 CCC DNA 的形成。本申请的目标将通过以下两个目标来实现：目标 1. 构建和验证 DHBV 特异性的 TALEN。该目标的目的是 i) 针对 DNA 复制所需的 DHBV 的三个靶标构建 TALEN，ii) 测量每个 TALEN 切割 CCC DNA 的能力，iii) 确定切割对 CCC DNA 稳定性的影响，以及 iv ) 研究 TALEN 的切割是否导致 CCC DNA 整合到染色体 DNA 中。目标 2. 确定 TALEN 的抗病毒活性。本研究的目的是研究基于 TALEN 的疗法对分裂型肝癌细胞和非分裂型原代鸭肝细胞感染的影响。我们将研究 TALEN 疗法是否会导致每个细胞的 CCC DNA 水平随时间依赖性降低，并不仅确定 CCC DNA 是否可以在非分裂细胞中功能性失活，还确定是否可以在不需要细胞死亡的情况下将其破坏。此外，我们将确定 TALEN 疗法是否可以保护肝细胞免受新发感染。