Designer Nucleases to Cure Chronic Hepatitis B

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。所有人发生肝细胞癌的风险都显著增加。尽管用核苷类似物治疗可以阻断病毒复制，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转录的模板在感染细胞中持续存在。到目前为止，缺乏一种功能性灭活甚至破坏感染肝细胞内CCC DNA的治疗策略。我们建议使用ccdna特异性的转录激活因子样效应核酸酶（TALENs）在病毒DNA复制所必需的区域切割和突变CCC DNA。因此，本应用的目的是为感染的肝细胞可以通过灭活和可能破坏CCC DNA来治愈HBV的想法获得原理证明。我们实验室的初步实验已经证明，CCC DNA确实容易被TALENs切割。本提案的目的是研究TALENs在修复反应中通过插入/删除碱基来功能性失活CCC DNA的潜力，甚至破坏CCC DNA，并确定基于TALENs的抗病毒治疗是否可以治愈感染细胞。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乙肝病毒（DHBV），该病毒允许病毒DNA复制的所有步骤，包括形成CCC DNA。此应用程序的目标将通过以下两个目标实现：目标1。DHBV特异性TALENs的构建与验证。本研究的目的是i)针对DNA复制所需的DHBV上的三个靶点构建TALENs， ii)测量每个TALENs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TALENs切割是否导致CCC DNA整合到染色体DNA中。目标2。目的：测定TALENs的抗病毒活性。本实验旨在研究以talen为基础的治疗方法对正在分裂的肝癌细胞和未分裂的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中CCC DNA水平的时间依赖性降低，并不仅确定CCC DNA是否可以在非分裂细胞中功能失活，而且确定它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.