Chronic infection with hepatitis C virus (HCV) is an important cause of life-threatening liver disease worldwide. Current treatments for HCV are poorly tolerable and incompletely effective, especially for HCV of certain subtypes (1a and 1b) that are prevalent in North America, Europe and Japan. Thus there are many pharmaceutical companies which are developing novel anti-HCV drugs. However, based on experience with other chronic viral infections such as HIV-1, antiviral drug resistance will likely be an important cause of failure of antiviral chemotherapy. Thus we anticipate the need for assays to measure susceptibility of patient-derived HCV to antiviral drugs. The goal of this project is to develop a rapid (<14 day), sensitive HCV drug susceptibility assay. The proposed assay will be performed using recombinant HCV replicons that contain a reporter gene (e.g. luciferase). A segment of each HCV replicon is derived from patient isolates and encodes a specific antiviral drug target (e.g. HCV protease/ helicase or polymerase). Permissive hepatic cell cultures are transfected with HCV replicon RNA and drug susceptibility/resistance is evaluated by comparing reporter gene expression levels in the presence and absence of antiviral drugs. The principal applications for the proposed assay are as follows. First, it can be used to aid in the discovery and development of first generation anti-HCV drugs by characterizing resistance in vitro. Second, it will be an important tool for monitoring the development of drug resistant HCV during the clinical evaluation of investigational anti-HCV agents. Third, it can be used to select optimal drug combinations for patients considering, undergoing, or failing anti-HCV drug therapy. Finally, it can be used to aid the discovery and development of second-generation drugs that are active against drug resistant strains, by screening new drug candidates against HCV replicon vector libraries. As a result of the first two potential applications, it is advantageous to develop the proposed assay before there is a perceived clinical need for it, which would result from the development of resistance following use of specific anti-HCV drugs in patients. ? ?
