Designer Nucleases to Cure Chronic Hepatitis B

描述(申请人提供)：乙肝病毒导致全球约3.5亿人慢性感染。所有患者患肝细胞癌的风险都显著增加。虽然核苷类似物可以阻止病毒复制，但感染的肝细胞无法治愈，因为共价闭合环状DNA(CCC DNA)是病毒RNA转录的模板，在感染细胞中持续存在。到目前为止，还没有一种治疗策略可以在功能上灭活甚至破坏受感染的肝细胞内的CCC DNA。我们建议使用针对CCC DNA的转录激活器样效应核酸酶(TALEN)来切割和突变对于病毒DNA复制至关重要的区域的CCC DNA。因此，这项申请的目标是获得原理证据的想法，感染的肝细胞可以治愈的乙肝病毒，通过灭活，并可能破坏的ccDNA。我们实验室的初步实验已经证明，CCC DNA确实容易被TALEN切割。这项建议的目的是研究TALEN在修复反应中通过插入/删除碱基来功能性灭活CCC DNA的可能性，甚至破坏CCC DNA的可能性，并确定基于TALEN的抗病毒治疗是否能够治愈感染的细胞。作为乙肝病毒的模型，我们将使用在肝癌细胞和原代肝细胞培养中表达的鸭乙型肝炎病毒(DHBV)，这些细胞允许病毒DNA复制的所有步骤，包括形成CCC DNA。这项应用的目标将通过以下两个目标来实现：目标1.构建和验证针对DHBV的TALEN。本研究的目的是：1)构建针对DHBVDNA复制所需的三个靶点的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切割是否导致CCC DNA整合到染色体DNA中。目的2.测定TALENS的抗病毒活性。本研究的目的是研究TALEN治疗对原代培养鸭肝细胞分裂和未分裂的感染的影响。我们将调查TALEN治疗是否会导致每个细胞的CCC DNA水平随时间而下降，并不仅确定在未分裂的细胞中CCC DNA是否可以功能失活，而且是否可以在不需要细胞死亡的情况下将其摧毁。此外，我们将确定TALEN治疗是否可以保护肝细胞免受新生感染。

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.