Abstract
T cell suicide gene therapy is designed to permit the use of donor T cells for their powerful anti-leukaemic and anti-viral effects following haematopoietic stem cell transplantation. In the event of graft versus host disease (GVHD) such cells can be eliminated through the administration of suicide-gene dependent prodrugs. Though the feasibility of the strategy has been demonstrated in clinical trials, there has been a lower incidence of GVHD than anticipated and evidence of impaired anti-viral immunity. Current vectors under clinical investigation are based on retroviral delivery systems and require T cells to be actively dividing at the time of vector exposure to for successful transduction. The process of ex-vivo activation to induce mitogenesis has been shown to impair subsequent functional potential, and even under optimized conditions using combinations of anti-CD3 and anti-CD28 antibodies, T cells become prone to exhaustion. It has been previously reported that lentiviral vectors permit T cell transduction without the need for mitogenic stimulation if used in combination with certain cytokines such as Interleukin-2 (IL-2) or Interleukin-7 (IL-7). Thus we have adapted a self-inactivating lentiviral vector system based on HIV-1 and encoding strong promoter elements derived from the long terminal repeat (LTR) regions of Spleen Focus forming virus (SFFV) for the delivery of suicide genes to T cells. The Woodchuck hepatitis virus post-transcriptional regulatory element and the central polypurine tract element of HIV-1 have been incorporated to enhance transduction efficiency and increase transgene expression. T cells cultured in the presence of either IL-2 or IL-7 alone, or in combination could be transduced at between 20–30% efficiency following a single round of exposure to virus pseudotyped with the vesicular stomatitis virus envelope or the feline endogenous leukaemia virus envelope. By the end of the procedure there were minimal changes in T cell surface phenotype, preservation of niave/memory subsets and retention of virus specific populations as quantified by tetramer staining of Cytomegalovirus (CMV) specific T cells. Functional analysis indicated preservation of proliferative responses to autologous dendritic cells pulsed with CMV antigen. In addition, strong responses in mixed lymphocyte culture indicated intact allo-reactive responses. Cells transduced to express a fusion construct encoding a variant herpes simplex thymidine kinase fused to the truncated CD34 selection marker could be enriched to >95% purity by a single round of anti-CD34 magnetic bead selection. The functional integrity of the suicide gene was confirmed by elimination of enriched cells following exposure to the prodrugs Gancilcovir and Aciclovir. In conclusion, non-dividing T cells transduced with suicide gene/selection transgenes using lentiviral constructs, retain phenotypic characteristics and functional responsiveness.
Combination gene therapy for HIV using a conditional suicidal gene with CCR5 knockout