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。目前的标准干扰素治疗是一个昂贵和耗时的过程，只有~50%的治愈率，并且还没有抗HCV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希望，但病毒耐药性的迅速出现表明，其他靶点和抗病毒药物的组合对于有效治疗是必要的。我们建议从包含片段化的HCV基因组的文库中分离遗传抑制元件（GSE），所述片段化的HCV基因组可用作有效的抗HCV治疗剂和探针以鉴定和验证用于进一步药物筛选的新靶点。GSE是来源于基因或基因组的核酸或蛋白质/肽分子，其通过多种机制充当特定生物功能的反式显性抑制剂，所述机制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基因组中鉴定对HCV发挥抑制活性的GSE，将开发和实施一种新的基于功能的选择系统。简而言之，片段化的HCV基因组将被递送至对HCV所施加的细胞病变效应敏感的肝癌衍生细胞系。含有HCV衍生的遗传片段的细胞将通过外源施用的细胞培养物衍生的HCV（HCV-HCV）感染性颗粒进行致细胞病变的攻击，并且在该HCV攻击中存活的细胞将富含对HCV发挥抑制作用的GSE。反复应用该选择程序将导致鉴定出保护细胞免受HCV感染和细胞毒性的高效GSE。初步研究将评估所鉴定的GSE对HCV介导的细胞毒性的保护作用的程度，以及GSE发挥抗HCV作用的HCV生命周期阶段。对GSE的进一步研究有望揭示其抗HCV活性的分子基础，为抗HCV药物的开发提供新的思路。预计从拟议的研究中鉴定的GSE和由此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-宿主细胞相互作用，从而有助于在多个方面迫切寻求新的有效的HCV抗病毒药物。公共卫生相关性：丙型肝炎病毒感染是一个严重的全球性健康问题，可导致许多使人衰弱的肝脏疾病。目前丙型肝炎的治疗方案耗时，昂贵，而且往往无效，迫切需要新的有效药物。从这项研究中分离出的新型抗丙型肝炎基因抑制因子既可以作为丙型肝炎药物，也可以作为打开丙型肝炎抗病毒开发研究新途径的钥匙。

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.