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（由申请人提供）：丙型肝炎病毒（HCV）是一种包裹的，链的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，是美国肝细胞癌（HCC）的主要原因。尽管对所有HCV蛋白进行了多克隆和多克隆和多特异性的体液和细胞免疫反应，但大多数患者仍未清除病毒。尽管它是人类病原体的重要性，但尚不清楚负责HCV持久性和免疫逃避的机制，并且缺乏当前治疗慢性HCV的疗法。既没有治愈慢性HCV感染的方法，也没有防止感染的疫苗。由于缺乏允许研究HCV感染，复制，免疫生物学和发病机理的研究模型，这些领域的进展受到了严重阻碍。这项工作的长期目标是阐明导致HCV清除率与宿主中HCV持久性和发病机理的分子和细胞机制。这项研究的重点是开发HCV复制模型。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鼠肝脏的HCV复制子。过去的失败尝试通过生成带有HCV基因组cDNA的转基因小鼠和通过体外转录的HCV（+）RNA转染小鼠肝脏通过将“野生型” HCV基因组分离出来，从自然或HCV变体中隔离，并且通过适应性的适应性隔离。在任何情况下，HCV基因组都没有被“优化”以在鼠肝脏中发挥作用。这项研究将使用诱变的PCR和DNA改组在体外形成HCV复制子的人工准菜。这些HCV变体池将通过流体动力注射传递到鼠肝脏。然后，将使用肝回生量的现象来扩增HCV复制的肝细胞的亚群。在肝脏中复制的HCV物种将被分离和表征。开发的系统将允许研究HCV在肝脏中与真实肝细胞中其宿主的细胞内相互作用，并将允许解剖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宝贵的工具。