Isolation of Genetic Suppressor Elements Against Hepatitis C Virus

描述（由申请人提供）：丙型肝炎病毒（HCV）感染是一个主要的公共卫生问题，使感染者（全球约1.8亿）处于发展为肝硬化、肝细胞癌和肝功能衰竭的风险中。慢性丙型肝炎是肝移植的主要原因。目前标准的基于干扰素的治疗是一个昂贵且耗时的过程，治愈率只有50%左右，而且目前还没有抗丙型肝炎药物被批准用于丙型肝炎治疗。尽管hcv特异性蛋白酶和聚合酶作为药物靶点显示出前景，但病毒耐药性的迅速出现表明，为了有效治疗，需要额外的靶点和抗病毒药物组合。我们建议从HCV片段基因组文库中分离基因抑制元件（GSEs），这些基因抑制元件既可以作为有效的抗HCV治疗剂，也可以作为探针来识别和验证进一步药物筛选的新靶点。gse是源自基因或基因组的核酸或蛋白质/肽分子，通过多种机制作为特定生物功能的跨显性抑制剂，包括结合和阻断蛋白质活性的基本相互作用表面。为了从HCV基因组中鉴定出对HCV具有抑制活性的GSEs，我们将开发并实施一种新的基于功能的选择系统。简而言之，将片段化的HCV基因组传递到对HCV细胞病变作用敏感的肝癌衍生细胞系。含有HCV衍生基因片段的细胞将受到外源性细胞培养衍生的HCV （HCVcc）感染颗粒的细胞病变攻击，在这种HCV攻击中存活的细胞将富含GSEs，对HCV发挥抑制作用。这种选择过程的反复应用将导致鉴定出保护细胞免受HCV感染和细胞毒性的高效gse。初步研究将评估鉴定的GSEs对HCV介导的细胞毒性的保护作用程度，以及GSEs在HCV生命周期中发挥抗HCV作用的阶段。从已鉴定的GSE开始的未来研究有望揭示抗hcv GSE活性的分子基础，从而为抗hcv药物的开发提供新的线索。预计从本研究中鉴定的gse及其衍生的分子模拟物将通过多种机制抑制HCV感染/传播，包括抑制病毒与宿主细胞的相互作用，从而在多个方面为迫切寻求新的有效的HCV抗病毒药物做出贡献。公共卫生相关性：丙型肝炎病毒感染是一个严重的全球卫生问题，可导致许多使人衰弱的肝脏疾病。目前的丙型肝炎治疗方案耗时、昂贵，而且往往无效，因此迫切需要新的有效药物。从该研究中分离出的新型抗丙型肝炎基因抑制元件将作为丙型肝炎药物，并作为打开丙型肝炎抗病毒开发研究新途径的关键。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) infection is a major public health problem, putting infected individuals (~180 million worldwide) at risk of developing cirrhosis, hepatocellular carcinoma, and liver failure. Chronic hepatitis C is the leading cause for liver transplantation. Current standard interferon-based therapy, a costly and time-consuming process, has only a ~50% cure rate, and no anti-HCV drugs have yet been approved for hepatitis C therapy. Despite the promise shown by HCV-specific proteases and polymerases as drug targets, the rapid emergence of viral resistance indicates that additional targets and combinations of antivirals will be necessary for effective treatment. We propose to isolate genetic suppressor elements (GSEs) from a library comprising a fragmented HCV genome which could be used both as potent anti-HCV therapeutic agents and as probes to identify and validate new targets for further drug screening. GSEs are nucleic acid or protein/peptide molecules derived from a gene or genome that act as transdominant inhibitors of a particular biological function through a variety of mechanisms, which includes binding to and blocking essential interaction surfaces for protein activity. In order to identify GSEs from within the HCV genome that exert inhibitory activity against HCV, a novel function-based selection system will be developed and implemented. Briefly, a fragmented HCV genome will be delivered to a hepatoma derivative cell line that is sensitive to a cytopathic effect exerted by HCV. The cells containing the HCV-derived genetic fragments will be subjected to a cytopathic challenge by exogenously administered cell culture-derived HCV (HCVcc) infectious particles, and cells surviving this HCV challenge will be enriched in GSEs that exert an inhibitory effect against HCV. Iterative application of this selection procedure will result in the identification of highly potent GSEs that protect cells against HCV infection and cytotoxicity. Preliminary studies will evaluate the degree of the protective effect conferred by the identified GSEs against HCV-mediated cytotoxicity, and the stage in the HCV life cycle at which the anti-HCV effect of the GSEs is exerted. Future studies originating from the identified GSEs are expected to reveal the molecular basis of the anti-HCV GSE activity, thus providing new leads for anti-HCV drug development. It is expected that the GSEs identified from the proposed research, and molecular mimetics derived therefrom, will inhibit HCV infection/propagation through diverse mechanisms, including the inhibition of virus-host cell interactions, thus contributing to the urgent quest for new and effective HCV antivirals on multiple fronts. PUBLIC HEALTH RELEVANCE: Infection by hepatitis C virus is a serious global health problem that causes numerous debilitating liver conditions. The current treatment regime for hepatitis C is time-consuming, expensive, and often ineffective, creating an urgent need for new and effective drugs. Novel anti-hepatitis C genetic suppressor elements isolated from this research will serve both as hepatitis C drugs, and as keys to open doors to new avenues of research in hepatitis C antiviral development.