Generation of replication-defective helper-free vectors based on simian immunodeficiency virus

A systematic study on generating simian immunodeficiency virus (SIV)-based vectors was carried out. The goal was to generate helper-free, replication-defective SIVmac-based vectors at high titers. The general approach was to cotransfect into human 293T cells a plasmid carrying the vector construct along with two helper plasmids that together expressed the SIVmac virion proteins. Initial vectors carried the bacterial beta-galactosidase gene (beta-gal). These vectors had a technical difficulty: "pseudotransduction" of beta-gal protein produced during the 293T cell transfections. As a result, infection of cultures with these vector stocks also resulted in passive transfer into, and X-gal staining of, cells that had not actually been infected by the vector. A second generation of vectors expressing the enhanced jellyfish green fluorescence protein (EGFP) was not subject to this artifact. A systematic study of the SIVmac-based EGFP vectors was carried out. Helper-free vector stocks were obtained when helper plasmids lacking the SIVmac packaging signals were used. By employing envelope helper plasmids derived from different SIVmac isolates, it was possible to generate SIVmac-based vectors pseudotyped with envelope proteins of different cell tropism. Optimization of vector and helper plasmid structures, transfection conditions, and infection procedures ultimately yielded vector titers in excess of 10(6)/ml.