Directed Evolution of a Murine Model of Hepatitis C Virus Replication

描述（由申请方提供）：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，可在肝细胞质中复制。HCV引起急性和慢性肝炎，是美国肝细胞癌（HCC）的主要原因。尽管针对所有HCV蛋白的多克隆和多特异性体液和细胞免疫应答不断增加，但大多数患者未能清除病毒。尽管其作为人类病原体的重要性，但对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。目前既没有治愈慢性HCV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清除的分子和细胞机制与HCV在宿主中的持久性和发病机制。 本研究的重点是HCV复制的小鼠模型的发展。从化学和合成生物学的学科，定向进化的策略，将被用来开发HCV复制子，已在体内适应小鼠肝脏。过去通过产生携带HCV基因组cDNA的转基因小鼠和通过用体外转录的HCV（+）-链RNA转染小鼠肝脏来开发HCV小鼠模型的不成功的尝试利用了从天然感染分离的“野生型”HCV基因组或通过使用选择标记在细胞培养物中适应和分离的HCV变体。在任何情况下，HCV基因组都没有被“优化”以在鼠肝脏中发挥功能。本研究将利用诱变PCR和DNA改组技术在体外构建HCV复制子的人工准种。这些HCV变体池将通过流体动力学注射递送至鼠肝脏。然后将使用肝脏再增殖的现象来扩增HCV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。 开发的系统将允许研究HCV的细胞内相互作用与其主机在真正的肝细胞内的背景下，肝脏，并允许解剖HCV和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治疗的功效提供有价值的工具。

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.