Hepatitis C Virus and Hepatocellular Carcinoma

描述（由申请人提供）：丙型肝炎病毒感染原因 长期的肝脏炎症，经常导致慢性肝病 疾病、肝纤维化和肝细胞癌。治疗肝 肝癌和肝硬化是移植。因此，小说的发展 根除丙型肝炎的疗法可以挽救一些终末期肝脏 疾病和肝细胞癌。创新设计理念 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 可靠的细胞培养模型来复制HCV。最近，我们报道了 HCV在人肝细胞癌细胞系和淋巴母细胞中的表达 用全长HCV RNA转染后，我们证明 这两个转染的细胞系产生了感染性HCV颗粒， 超过2个月。此外，我们发现，我们可以将HCV传播给一个 静脉接种来自转染HepG 2的HCV后的黑猩猩 细胞此外，避免RNA中低水平HCV的潜在问题 转染模型，我们开发了一种诱导模型来研究HCV复制 并且通过生物化学方法表达而不使用RT-PCR。的基础上 这些初步结果，我们建议扩大我们的研究，并建立 用真正黑猩猩进行HCV基因型Ia感染性细胞培养 由国家卫生研究所的Jens Bukh制备的感染性克隆。它 我们的假设是，从一个细胞中有效转染全长HCV RNA， 黑猩猩感染性HCV克隆到HepG 2细胞中将导致 细胞培养中的HCV。用于la和lb的这种细胞培养系统的建立 HCV毒株应促进研究，以测试治疗潜力， 干扰素、核酶和蛋白酶抑制剂。非结构蛋白NS 3 具有多种酶活性（蛋白酶、解旋酶和NTR）， 对HCV复制很重要。我们构建了几种腺病毒 含有源自人的单链抗体片段， 核心、E2、NS 3和NS 4蛋白。此外，我们假设细胞内 靶向结构蛋白的单链抗体的表达应 抑制病毒形成和那些靶向非结构 蛋白质NS 3和NS 4可阻断蛋白酶、解旋酶和NTR活性， 从而干扰HCV蛋白的加工和复制。直接 为了验证我们的假设，我们制定了三个具体目标：1）研究 丙型肝炎病毒1a基因型在人肝细胞中复制、表达和形态发生 黑猩猩转染人肝癌细胞系HepG 2 感染性克隆，并确定复制水平是否可以 与有或无肝细胞癌的感染的人肝脏的结果相当 癌，并利用HCV细胞培养模型来鉴定治疗 干扰素、核酶和蛋白酶抑制剂可能阻断一种 丙型肝炎病毒复制周期的一个或多个步骤。2)研究分子 NS 3蛋白的蛋白酶、解旋酶和NTR结构域的生物学及其 使用诱导型表达系统在负链RNA合成中的作用， 利用T7 RNA聚合酶和转录质粒。我们建议设立 一个更强大的RNA复制和表达系统作为替代低 RNA转染模型中的HCV水平。3)以确定是否 单链抗体（scFv）的细胞内表达 结构蛋白，核心和E2，干扰病毒形态发生， 针对NS 3和NS 4蛋白的抗体可以抑制蛋白酶、解旋酶、 和HCV的NTR活性和复制。实现这些目标将 促进开发治疗慢性丙型肝炎感染的新疗法 并可降低发生肝硬化的潜在风险， 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.