Production of SIV Vectors for Gene Delivery

Abstract

INTRODUCTIONThe acquisition of vectors suitable for in vivo gene delivery has been a recurrent theme in gene therapy research. Several challenging hurdles must be overcome to reach this goal. First, the vectors must be prepared at high titers. Second, to avoid inactivation, the gene-transfer vectors should not be recognized by the host immune system. Third, the vectors should be able to replicate and to express a transgene in cells that are nonproliferating or slowly proliferating, a predominant situation in vivo. Lentiviral vectors derived from simian immunodeficiency virus (SIV) are similar to those derived from human immunodeficiency virus (HIV-1 or HIV-2) in that they can insert transgenes in nonproliferating cells. It is becoming clear, however, that SIV vectors perform better than HIV-1 vectors in simian cells, and thus may be a valid alternative to HIV-1-based vectors in the early phases of the clinical testing of lentiviral vectors in nonhuman primate models. This protocol describes the production of SIV vector particles and the assay used to measure transduction efficiency. Included also are methods for detecting replication-competent retroviruses (RCRs) and testing the putative mobilization of the green fluorescent protein (GFP)-containing SIV vector. The percentage of GFP-positive cells is determined by fluorescence-activated cell sorting (FACS). These safety assays make use of sMAGI target cells that are permissive to SIVmac251 replication and harbor a stably integrated lacZ transcription unit driven by the SIV/HIV tat-inducible HIV-1 long terminal repeat (LTR).