Hepatitis C Virus and Hepatocellular Carcinoma

描述(申请人提供)：丙型肝炎病毒感染原因 肝脏的长期炎症，经常导致慢性肝脏 疾病、肝纤维化和肝细胞癌。肝的治疗 癌症和肝硬变就是移植。因此，小说的发展 根除丙型肝炎的治疗可以挽救一些终末期肝脏 疾病和肝细胞癌。创新的设计理念 抗病毒疗法，这将阻止病毒复制和基础研究 病毒复制的几个方面，由于缺乏 复制丙型肝炎病毒的可靠细胞培养模型。最近，我们报告了复制 丙型肝炎病毒在人肝细胞癌细胞系和淋巴母细胞系中的表达 将丙型肝炎病毒全长RNA导入细胞系。我们演示了 这两个细胞系产生了具有感染性的丙型肝炎病毒颗粒 两个多月。此外，我们还证明了我们可以将丙型肝炎病毒传播给 静脉接种转染型丙型肝炎病毒后的黑猩猩 细胞。此外，避免核糖核酸中低水平的丙型肝炎病毒的潜在问题 模型，我们开发了一个可诱导模型来研究丙型肝炎病毒复制。 不使用RT-PCR，通过生化手段进行表达。在…的基础上 这些初步结果，我们建议扩展我们的研究，并建立 利用一只真正的黑猩猩进行1a型丙型肝炎病毒感染细胞培养 Jens Bukh在国家卫生研究所准备的感染性克隆。它 我们的假设是有效地从一个 黑猩猩感染性丙型肝炎病毒克隆到HepG2细胞将导致复制 细胞培养中的丙型肝炎病毒。乳酸菌和乳酸菌培养体系的建立 丙型肝炎病毒株应有助于测试治疗潜力的研究 干扰素、核酶和蛋白酶抑制剂。非结构蛋白NS3 具有多种酶活性(蛋白酶、解旋酶和NTPase) 对丙型肝炎病毒复制很重要。我们产生了几个腺病毒载体 含有来自人类的单链抗体片段，该抗体片段是 核心、E2、NS3和NS4蛋白。此外，我们假设细胞内 针对结构蛋白的单链抗体的表达应 抑制病毒形成和针对非结构蛋白的抗体 蛋白质NS3和NS4可以阻止蛋白酶、解旋酶和NTPase的活性，并 从而干扰了丙型肝炎病毒蛋白的加工和复制。直接 验证我们的假设，我们制定了三个具体目标：1)研究 丙型肝炎病毒1a亚型的复制、表达和形态发生 黑猩猩基因转染人肝癌细胞株(HepG2)的研究 感染性克隆并确定复制水平是否可以 与有或无肝细胞感染的人肝脏相当 并利用丙型肝炎病毒细胞培养模型来确定治疗方法 干扰素、核酶和蛋白酶抑制剂可能阻断其中一种的可能性 或更多步骤的丙型肝炎复制周期。2)研究分子 NS3蛋白的蛋白酶、解旋酶和NTPase结构域及其生物学 使用可诱导表达系统在负链RNA合成中的作用 利用T7RNA聚合酶和转录质粒。我们建议建立 一个更强大的RNA复制和表达系统，作为Low的替代 RNA转染模型中丙型肝炎病毒的水平。3)确定是否 靶向单链抗体(ScFv)的细胞内表达 结构蛋白，核心和E2，干扰病毒的形态发生和 针对NS3和NS4蛋白的抗体可以抑制蛋白酶、解旋酶 和NTPase活性与丙型肝炎病毒复制。实现这些目标将 促进治疗慢性丙型肝炎的新疗法的开发 可能会降低发展为肝硬变的潜在风险 肝细胞癌。

DESCRIPTION (provided by applicant): Hepatitis C virus infection causes long-term inflammation of the liver which frequently leads to chronic liver disease, liver fibrosis and hepatocellular carcinoma. Treatment for liver cancer and liver cirrhosis is transplantation. Therefore, development of novel therapies to eradicate hepatitis C can save some of these end stage liver diseases and hepatocellular carcinomas. Concepts of designing innovative anti-viral therapies, which will stop virus replication and basic studies on several aspects of virus replication, have been hampered due to the lack of a reliable cell culture model to replicate HCV. Recently, we reported replication of HCV in a human hepatocellular carcinoma cell line and in a lymphoblastoid cell line after transfecting them with full-length HCV RNA. We demonstrated that these two transfected cell lines produced infectious HCV particles for over 2 months. In addition, we showed that we could transmit HCV to a chimpanzee after intravenous inoculation of HCV derived from transfected HepG2 cells. Furthermore, avoid potential problems of low levels of HCV in the RNA transfection models, we developed an inducible model to study HCV replication and expression by biochemical means without the use of RT-PCR. On the basis of these preliminary results, we propose to extend our studies and establish infectious cell culture for HCV genotype la using a bonafide chimpanzee infectious clone prepared by Jens Bukh at the National Institute of Health. It is our hypothesis that efficient transfection of full-length HCV RNA from a chimpanzee infectious HCV clone to HepG2 cells will result in replication of HCV in cell culture. Establishment of such cell culture systems for la and lb strains of HCV should facilitate studies to test therapeutic potential of interferons, ribozymes and protease inhibitors. The non-structural protein NS3 possess multiple enzyme activities (protease, helicase and NTPase) which appear to be important for HCV replication. We generated several adenoviral constructs containing single-chain antibody fragments derived from human which target core, E2, NS3 and NS4 proteins. Furthermore, we postulate that intracellular expression of single-chain antibodies targeted to structural proteins should inhibit virus formation and those antibodies targeted to non-structural proteins, NS3 and NS4, may block protease, helicase and NTPase activities and thus interfere with the processing of HCV proteins and replication. To directly test our hypotheses, we have developed three Specific Aims: 1) To study replication, expression and morphogenesis of hepatitis C virus genotype la in human hepatocellular carcinoma cell line (HepG2) transfected with chimpanzee infectious clone and determine whether the level of replication can be comparable to that of infected human liver with or without hepatocellular carcinoma and utilize the HCV cell culture models to identify therapeutic potential of interferons, ribozymes and protease inhibitors that may block one or more steps of hepatitis C replication cycle. 2) To study the molecular biology of the protease, helicase and NTPase domains of the NS3 protein and its role in negative strand RNA synthesis using an inducible expression system that utilizes T7 RNA polymerase and a transcription plasmid. We propose to establish a more robust RNA replication and expression system as an alternative to low levels of HCV in the RNA transfection model. 3) To determine whether intracellular expression of single-chain antibodies (scFv) targeted to structural proteins, core and E2, interfere with virus morphogenesis and antibodies targeted to NS3 and NS4 proteins could inhibit protease, helicase and NTPase activities and replication of HCV. Accomplishment of these aims will promote development of novel therapies to treat chronic hepatitis C infection and may reduce the potential risk of developing liver cirrhosis and hepatocellular carcinoma.