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A major goal in allogeneic bone marrow transplantation is to preserve the anticancer effects of donor T-cells, while avoiding their deleterious graft-versus-host disease (GVHD)-inducing effects. Donor T lymphocytes can be engineered with inducible suicide genes, such as thymidine kinase (Tk). In case of GVHD, systemic administration of the Tk-activatable pro-drug, ganciclovir, will activate the suicide mechanism in the gene-modified, in vivo alloreactive T-lymphocytes, and abrogate GVHD. However, an efficient selection system is required to eliminate non-Tk-expressing T-lymphocytes before administration to the patient. We have recently described a new selection strategy for gene-transduced hematopoietic stem cells, based on the retroviral vector-mediated overexpression of the heavy subunit of gamma-glutamyl cysteine synthetase (gamma-GCSh) and the gamma-GCSh inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to apply the gamma-GCSh selection strategy to gene-transduced T-lymphocytes.

Two SF91-based bicistronic retroviral vectors, co-expressing gamma-GCSh and the fluorescent marker, eGFP, or gamma-GCSh and Tk, were assembled. T-lymphocytes were isolated from buffy coats from human volunteers by a Ficoll gradient, followed by magnetic concentration employing CD3/CD28 Dynabeads. Retroviral transduction was performed on Retronectin at a multiplicity of infection of 1 or less.

After activation with CD3/CD28 beads in the presence of IL-2, we transduced human T-cells twice on Retronectin with SF91/gamma-GCSh-Tk at a MOI of 1. Exposure of SF91/gamma-GCSh-eGFP-transduced T-cells to BSO resulted in a dose-dependent increase in the percentage of eGFP+ cells. Using a MOI of 1, after exposure to 750 μM BSO for three days, over 95% of selected cells were eGFP+. In a second set of experiments, T-cells were transduced twice on Retronectin with SF91/gamma-GCSh-Tk at the same MOI of 1, and selected by exposure to BSO. The addition of ganciclovir 24 h later resulted in a profound cytotoxic effect, as determined by the MTS/PMS assay, with only 9 and 2% of the cells still viable after exposure to 5 and 10 μM ganciclovir, respectively, in the presence of BSO. To be able to fully control GVHD, all T cells must be eliminated by ganciclovir when required, and thus the presence of HSV-Tk-negative T cells should be kept to a minimum. Although our results suggest that further investigations and improvements of the gamma-GCSh-Tk bicistronic vectors are required to achieve complete ganciclovir-mediated elimination of selected T-lymphocytes, they nonetheless prove that (i) it is possible to obtain more than 95% selection of T-lymphocytes transduced with a bicistronic vector coding for gamma-GCSh and a fluorescent marker; (ii) BSO can select gamma-GCSh-Tk-transduced T-cells, rendering them more susceptible to ganciclovir-mediated elimination.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA