Inhibition of hepatitis C by RNA Lassos

描述（由研究者提供）：本申请的目的是开发针对丙型肝炎RNA病毒（HCV）的特异性RNA Lasso抑制剂，并在细胞培养和动物模型中证明其疗效。RNA Lassos是专有的反义核酶，其不切割RNA靶标，而是与其形成拓扑连接并阻断基因表达。将在表达含有两种不同荧光蛋白的双顺反子RNA的细胞中进行初始功能检测，所述荧光蛋白可通过流式细胞术容易地监测[一种顺反子的基因表达依赖于HCV内部核糖体进入位点（IRES），另一种是Cap依赖性的]。Lassos的效果将由两种荧光蛋白的活性的相对比率确定。将使用流体动力学递送系统和全动物成像系统进一步分析最有效的抑制剂在小鼠中有效阻断报告基因（HCV-荧光素酶构建体）表达的能力。在高压条件下将抗HCV RNA Lassos与HCV IRES报告载体共注射至小鼠尾静脉中，并测定肝脏中的抑制量。HCV Lasso RNA抑制剂最初将设计为半随机分子（文库方法），其将在有利于特异性和强结合的条件下选择。将对在严格条件下存活的那些Lassos进行克隆和测序，并在组织培养模型和小鼠中进行评价之前，在凝胶迁移率变动试验和体外翻译系统中作为固定序列分子进行进一步测试。

DESCRIPTION (provided by investigator): The goal of this application is to develop specific RNA-based Lasso inhibitors against the hepatitis C RNA virus(HCV) and demonstrate efficacy in cell culture and animal models. RNA Lassos are proprietary antisense ribozymes that do not cleave an RNA target but rather form a topological linkage with it and block gene expression. Initial functional testing will be performed in cells expressing a bicistronic RNA containing two distinct fluorescent proteins that can be readily monitored by flow cytometry [gene expression of one cistron is dependent on the HCV internal ribosome entry site (IRES) and the other is Cap dependent]. The effect of the Lassos will be determined by the relative ratio of the activity of the two fluorescent proteins. The most effective inhibitors will be further analyzed for their ability to efficiently block expression of a reporter (HCV-luciferase construct) in mice using a hydrodynamic delivery system and whole-animal imaging system. Anti-HCV RNA Lassos will be co-injected with an HCV IRES reporter vector into mouse-tail veins under conditions of high pressure and the amount of inhibition in the liver determined. The HCV Lasso RNA inhibitors will be initially designed as semi-random molecules (library approach) that will be selected under conditions that favor specific and strong binding. Those Lassos surviving stringent conditions will be cloned and sequenced and further tested as fixed sequence molecules in a gel mobility shift assay and an in vitro translation system before evaluation in a tissue culture model and mice.