Directed Evolution of a Murine Model of Hepatitis C Virus Replication

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是一种包膜的正链RNA病毒，具有高突变率，可在肝细胞的细胞质中复制。丙型肝炎病毒可引起急性和慢性肝炎，是美国肝细胞癌的主要原因。大多数患者未能清除病毒，尽管增加了针对所有丙型肝炎病毒蛋白的多克隆和多特异性体液和细胞免疫反应。尽管作为一种人类病原体，丙型肝炎病毒持续存在和免疫逃避的机制尚不清楚，目前治疗慢性丙型肝炎病毒的方法也很缺乏。既没有治愈慢性丙型肝炎病毒感染的方法，也没有预防感染的疫苗。由于缺乏合适的研究模型来研究丙型肝炎病毒的感染、复制、免疫生物学和发病机制，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丙型肝炎病毒清除的分子和细胞机制与丙型肝炎病毒在宿主中的持久性和致病机制。 本研究的重点是建立一种复制丙型肝炎病毒的小鼠模型。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丙型肝炎病毒复制子。过去，通过产生携带丙型肝炎病毒基因组cDNA的转基因小鼠和通过体外转录的丙型肝炎病毒链RNA转染小鼠肝脏来建立丙型肝炎病毒小鼠模型的尝试失败了，这些尝试利用了从自然感染中分离出来的野生型丙型肝炎病毒基因组或通过使用可选择标记在细胞培养中适应和分离的丙型肝炎病毒变异体。在任何情况下，丙型肝炎病毒基因组都没有被“优化”以在小鼠肝脏中发挥作用。本研究将利用诱变聚合酶链式反应和DNA改组技术，在体外形成丙型肝炎病毒复制子的人工准种。这些丙型肝炎病毒变异体池将通过流体动力注射传递到小鼠肝脏。然后，肝脏再繁殖现象将被用来放大在其中复制丙型肝炎病毒的肝细胞亚群。在肝脏中复制的丙型肝炎病毒种类将被分离和鉴定。 开发的系统将允许在肝脏的背景下研究丙型肝炎病毒与其宿主在真实肝细胞中的细胞内相互作用，并将允许剖析丙型肝炎病毒与宿主免疫系统之间的相互作用。此外，复制丙型肝炎病毒的小鼠模型将为评估潜在的抗病毒疗法的疗效提供一个有价值的工具。

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.