929. Functional Potential of Human T Cells Following Lentiviral Suicide Gene Transduction
Authors: Taylor Mackey, Joanne Buddle, Christine Kinnon, Adrian Thrasher, Hubert Gaspar, Waseem Qasim
Journal: Molecular Therapy (2006)

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Clinical trials of retroviral T cell suicide gene therapy following allogeneic haematopoietic stem cell transplantation (HSCT) have shown that T cells causing graft versus host disease (GVHD) can be eliminated following the administration of the prodrug Ganciclovir (GCV). This strategy offers the possibility of harnessing powerful donor lymphocyte immunity in patients undergoing alternative donor HSCT (in both the HLA matched or mismatched settings) who would otherwise be at high risk of intractable GVHD. A number of limitations have been identified, including the detection of GCV resistant alternate splice variants, evidence of impaired anti-viral immunity, and immune response against gene-modified cells.

We have generated lentiviral vector systems capable of mediating gene transfer to T cells in the absence of T cell receptor or CD3 activation. The HIV-1 based, self-inactivating constructs encode a central polypurine tract (cPPT) element and an X-protein start site mutated woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). A dual suicide gene/selection marker encoding an enhanced HSVTK mutant (with corrected splice site) and truncated CD34 (tCD34) has been incorporated. Virus was pseudotyped with the Vesicular Stomatitis Virus (VSV-G) or a chimaeric RD114 envelope encoding the Molony leukaemia virus cytoplasmic tail . We compared transduction protocols using either full activation of T cells (anti-CD3/CD28 conjugated beads plus Interleukin-2) or pre-treatment of T cells with IL-2 or IL-7 cytokines alone or in combination. We show that the culture of T cells in media containing cytokines for 96h prior to exposure to viral supernatant renders cells permissive to lentiviral transduction. Transuction efficiency in IL-2 or IL-7 averaged 25% comapred to over 70% for fully activated cells.We observed reduced cell division on the basis of CFSE dye dilution in the cytokine stimulated cells, and flow cytometry confirms greater retention of CD45RA+ naive populations, with less skewing towards the CD45RO+CD27+ subgroups. T cells transduced in the presence of IL-7 retained the highest levels of viability. Preservation of virus specific responses was confirmed by tracking CMV specific T cells through the transduction procedure using tetramer staining. After enrichment on the basis of CD34 expression, transduced cells mediated intact responses against autologous dendritic cells pulsed with CMV. Similarly, proliferative responses against allogeneic dendritic cells were best preserved following IL-7 stimulation. Primary T cells, and virus specific T cells, specific for Adenovirus or CMV, were readily eliminated by exposure to GCV or the less toxic agent Aciclovir and there was no evidence splice variants resistant to prodrug action. The vectors offer the potential of high efficiency transduction and selection, efficient prodrug mediated elimination with preservation of T cell effector responses using cytokine pre-stimulation.