Designer Nucleases to Cure Chronic Hepatitis B

设计核酸酶可治愈慢性乙型肝炎

• 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
• 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

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）：B型肝炎病毒导致全球约3.5亿人慢性感染。所有人患肝细胞癌的风险都显著增加。虽然病毒复制可以通过核苷类似物治疗来阻断，但感染的肝细胞不能治愈，因为共价闭合环状DNA（CCC DNA），病毒RNA转录的模板持续存在于感染的细胞中。到目前为止，还缺乏一种治疗策略来功能性地抑制或甚至破坏感染肝细胞内的CCC DNA。我们建议使用转录激活因子样效应核酸酶（TALEN）的CCC DNA特异性切割和突变的CCC DNA在病毒DNA复制所必需的区域。因此，本申请的目的是获得通过CCC DNA的失活和可能的破坏可以治愈感染的肝细胞的HBV的想法的原理证据。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通过在修复反应期间插入/缺失碱基来功能性地破坏CCC DNA的潜力，或者甚至破坏CCC DNA，并确定基于TALEN的抗病毒治疗是否可以治愈感染的细胞。作为HBV的模型，我们将使用在肝癌细胞和原代肝细胞培养物中表达的鸭B肝炎病毒（DHBV），这些培养物允许病毒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实现：目标1。DHBV特异性TALEN的构建和验证。该目的的目的是i）构建针对DNA复制所需的DHBV上的三个靶标的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。目标二。确定TALEN的抗病毒活性。本研究的目的是研究基于TALEN的治疗对分裂肝癌细胞和非分裂原代鸭肝细胞感染的影响。我们将研究TALEN治疗是否会导致每个细胞CCC DNA水平的时间依赖性降低，并确定CCC DNA是否可以在非分裂细胞中功能失活，以及是否可以在不需要细胞死亡的情况下被破坏。此外，我们将确定TALEN治疗是否可以保护肝细胞免受从头感染。