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. ? ? ?
| Dallas, Anne; Ilves, Heini; Ma, Han et al. (2014) Inhibition of hepatitis C virus in chimeric mice by short synthetic hairpin RNAs: sequence analysis of surviving virus shows added selective pressure of combination therapy. J Virol 88:4647-56 |
| Vlassov, Alexander V; Korba, Brent; Farrar, Kristine et al. (2007) shRNAs targeting hepatitis C: effects of sequence and structural features, and comparision with siRNA. Oligonucleotides 17:223-36 |
| Wang, Qian; Contag, Christopher H; Ilves, Heini et al. (2005) Small hairpin RNAs efficiently inhibit hepatitis C IRES-mediated gene expression in human tissue culture cells and a mouse model. Mol Ther 12:562-8 |
