Directed Evolution of a Murine Model of Hepatitis C Virus Replication

丙型肝炎病毒复制小鼠模型的定向进化

基本信息

批准号：
7825454
负责人：
Priscilla Li-ning Yang
金额：
$ 25.43万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-05 至 2012-02-29
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Hepatitis C Virus (HCV) is an enveloped, plus-stranded RNA virus with a high mutation rate that replicates in the cytoplasm of hepatocytes. HCV causes acute and chronic hepatitis and is the leading cause of hepatocellular carcinoma (HCC) in the United States. Most patients fail to clear the virus despite mounting polyclonal and multi-specific humoral and cellular immune responses against all of the HCV proteins. Despite its significance as a human pathogen, the mechanisms responsible for HCV persistence and immune evasion are not understood, and current therapies for the treatment of chronic HCV are lacking. There is neither a cure for chronic HCV infection nor is there a vaccine that protects against infection. Progress in these areas has been severely hindered by the absence of suitable research models that permit the study of HCV infection, replication, immunobiology, and pathogenesis. The long term goal of this work is to elucidate the molecular and cellular mechanisms leading to HCV clearance versus HCV persistence and pathogenesis in the host. The focus of this study is the development of a murine model of HCV replication. The strategy of directed evolution, taken from the disciplines of chemical and synthetic biology, will be used to develop HCV replicons that have been adapted in vivo to the murine liver. Past unsuccessful attempts to develop an HCV mouse model by generating transgenic mice bearing an HCV genomic cDNA and by transfecting mouse livers with in vitro-transcribed HCV (+)- strand RNA have utilized "wild-type" HCV genomes isolated from natural infections or HCV variants adapted and isolated in cell culture through the use of a selectable marker. In no case has the HCV genome been "optimized" to function in the murine liver. This study will use mutagenic PCR and DNA shuffling to form artificial quasispecies of HCV replicons in vitro. These pools of HCV variants will be delivered to the murine liver by hydrodynamic injection. The phenomenon of liver repopulation will then be used to amplify a sub-population of hepatocytes in which HCV is replicating. HCV species replicating in the liver will be isolated and characterized. The system developed will permit the study of HCV's intracellular interactions with its host in authentic hepatocytes within the context of the liver and will permit the dissection of the interactions between HCV and the host immune system. In addition, a mouse model of HCV replication will provide a valuable tool in evaluating the efficacy of potential antiviral therapeutics.

描述（申请人提供）：丙型肝炎病毒（HCV）是一种有包膜的正链RNA病毒，具有高突变率，在肝细胞的细胞质中复制。 HCV 引起急性和慢性肝炎，并且是美国肝细胞癌 (HCC) 的主要原因。尽管针对所有 HCV 蛋白产生了多克隆、多特异性体液和细胞免疫反应，但大多数患者仍无法清除病毒。尽管它作为人类病原体具有重要意义，但导致 HCV 持续存在和免疫逃避的机制尚不清楚，并且目前缺乏治疗慢性 HCV 的疗法。慢性丙型肝炎病毒感染无法治愈，也没有预防感染的疫苗。由于缺乏合适的研究模型来研究 HCV 感染、复制、免疫生物学和发病机制，这些领域的进展受到严重阻碍。这项工作的长期目标是阐明导致 HCV 清除与 HCV 在宿主体内持续存在和发病机制的分子和细胞机制。本研究的重点是开发 HCV 复制的小鼠模型。取自化学和合成生物学学科的定向进化策略将用于开发已在体内适应小鼠肝脏的 HCV 复制子。过去通过产生携带HCV基因组cDNA的转基因小鼠以及通过用体外转录的HCV(+)-链RNA转染小鼠肝脏来开发HCV小鼠模型的失败尝试利用了从自然感染或HCV中分离的“野生型”HCV基因组通过使用选择标记在细胞培养物中适应和分离的变体。 HCV 基因组在任何情况下都没有被“优化”以在小鼠肝脏中发挥作用。本研究将使用诱变 PCR 和 DNA 改组在体外形成 HCV 复制子的人工准种。这些 HCV 变体库将通过流体动力注射输送至小鼠肝脏。然后，肝脏再生现象将被用来扩增丙型肝炎病毒正在其中复制的肝细胞亚群。将分离并表征在肝脏中复制的 HCV 种类。所开发的系统将允许在肝脏背景下的真实肝细胞中研究HCV与其宿主的细胞内相互作用，并将允许剖析HCV与宿主免疫系统之间的相互作用。此外，HCV复制的小鼠模型将为评估潜在抗病毒疗法的功效提供有价值的工具。