EX-VIVO TRANSDUCTION AND EXPANSION OF CD4(- PRELUDE TO A RIBOZYME GENE-THERAPY TRIAL() LYMPHOCYTES FROM HIV+ DONORS )

Preparations for a phase I trial of ex vivo, anti-HIV ribozyme gene th erapy have included optimization of transduction and expansion of CD4( +) lymphocytes from HIV-1 infected donors, using reagents suitable for production of cell products for human infusion We also determined whe ther transduction by the ribozyme vector would inhibit replication and spread of endogenous HIV-1, and result in preferential survival of ri bozyme-transduced CD4(+) cells during lymphocyte expansion. Transducti on efficiency, as estimated by DNA quantitative competitive (QC)-PCR, was similar for both control (LNL6) and ribozyme expressing (MJT) muri ne retroviral vectors (approximately 20%.) In the absence of antiviral agents, Cells transduced with MJT exhibited three-fold greater number s of CD4(+) cells 2 weeks after transduction than did LNL6 transduced cells. In addition, viral replication was delayed 2-3 weeks in MJT tra nsduced cultures. Both transduced cell populations expanded by 2-3 log s within 2 weeks. The clinical protocol involves infusion of both ribo zyme and control vector transduced cells, making identification of age nts capable of suppressing replication and spread of endogenous virus during ex vivo expansion necessary. The combination of nevirapine (100 nM) and CD4-PE40 (100 nM) completely suppressed endogenous virus repl ication in cultures transduced with either vector. At reduced concentr ations of nevirapine, virus replication was suppressed only in MJT tra nsduced cells.