Novel Assays for measuring Integrase and RNase-H Inhibition

DESCRIPTION (provided by applicant): Currently approved drugs inhibit HIV-1 replication by interfering with the enzymatic activities of either protease (PR), reverse transcriptase (RT), or with the entry process. The ability of HIV to rapidly evolve drug resistance to approved PR, RT and entry inhibitors, together with toxicity problems of current antiretroviral regimens, requires the development of additional classes of antiviral drugs. As an essential enzyme for HIV-1 replication, integrase (IN) is an attractive antiviral target. Clinical evaluation (phase 1 and 2) of several IN inhibitors (INIs) is underway, with at least one phase 3 trial expected to begin in 2006. The ultimate goal of this project is to validate for clinical use rapid, sensitive phenotypic and genotypic assays to evaluate the susceptibility of HIV-1 isolates to inhibitors of all HIV-1 viral enzymes, with particular emphasis on IN inhibitors (INIs). Assay development will involve evaluation of two alternative vector systems (pol or RHIN) that produce virus particles containing HIV-1 pol proteins derived from patient isolates. These assays will be based on the technology underlying PhenoSense HIVTM, an existing cell-based antiviral drug susceptibility assay that evaluates inhibitors of protease (PR) and reverse transcriptase (RT). Specific aims for this project are: Aim 1: Select assay format for Clinical Validation (YEAR 1) A. Refine the RT-PCR amplification steps of the pol assay, with the objective being to replicate as closely as possible the performance characteristics of the existing, CLIA validated PhenoSense and GeneSeq assays for PIs and RTIs, and the RUO validated RHIN assays for INIs. B. Compare phenotypic and genotypic results from the single pol assay to those from the currently validated PhenoSense assay (susceptibility to PIs and RTIs, Replication Capacity), and from the RHIN assay developed during Phase I (susceptibility to INIs) C. Select either the pol or RHIN assays for pre-validation and eventual CLIA validation. Aim 2: Perform pre-validation studies (including RUO validation for pol assay if selected) for RHIN or pol phenotypic and genotypic assay (YEAR 2). Aim 3: Validate the performance of the RHIN or pol assay format in a commercial clinical laboratory (YEAR 3) A. Clinically validate the performance of the RHIN or pol phenotypic assay B. Clinically validate the performance of the RHIN or pol genotypic assay C. Obtain CLIA certification for validated genotypic and phenotypic assays The ability of HIV-1 to rapidly evolve resistance to approved drugs, together with toxicity problems of current antiretroviral regimens, requires the development of additional classes of antiviral drugs. As an essential enzyme for HIV-1 replication, integrase (IN) is an attractive antiviral target. Clinical evaluation of several IN inhibitors is underway. The ultimate goal of this project is to validate for clinical use rapid, sensitive phenotypic and genotypic assays to evaluate the susceptibility of HIV-1 isolates to inhibitors of all HIV-1 viral enzymes, with particular emphasis on IN inhibitors.