EVALUATION OF PCR AND ELISA ASSAYS FOR SCREENING CLINICAL-TRIAL SUBJECTS FOR REPLICATION-COMPETENT RETROVIRUS

Gene delivery via murine-based recombinant retroviral vectors is curre ntly widely used in gene therapy clinical trials. The vectors are engi neered to be replication defective by replacing the structural and non structural genes of a cloned infectious retrovirus with a therapeutic gene of interest. The retroviral particles are currently generated in packaging cell lines, which supply all retroviral proteins in trans. R ecombination between short homologous regions of the retroviral vector and packaging cell line elements can theoretically generate replicati on-competent retrovirus (RCR) and hence the Food and Drug Administrati on (FDA) requires the monitoring of clinical trial subjects for the pr esence of RCR. Sensitive polymerase chain reaction (PCR) assays have b een used for the detection of murine leukemia virus (MLV) nucleotide s equences in peripheral blood mononuclear cells (PBMCs). A novel serolo gical enzyme-linked immunosorbent assay (ELISA) for the detection of a nti-MLV specific immunoglobulin (Ig) has been developed to be used as an alternative to the PCR assay. Both assays were used to monitor huma n immunodeficiency virus (HIV)-positive clinical trial sub jects who h ad received multiple injections of HIV-IT (V), a retroviral vector enc oding HIV-1 IIIBenv/rev. Western blot analysis and an in vitro vector neutralization assay were used to characterize further a subset of ser um samples tested by ELISA. Results show no evidence of RCR infection in clinical trial subjects. PCR and ELISA assays are discussed in term s of their advantages and limitations as routine screening assays for RCR. The PCR assay is our current choice for monitoring clinical trial subjects receiving direct administration of vector, and the ELISA is our choice for those receiving ex vivo treatment regimens.