In Vivo Suicide Gene Therapy of Human T Lymphocytes To Prevent Graft Versus Host Disease in a Murine Xenograft Model.

Graft-versus-host disease (GvHD) remains a major cause of morbidity and mortality following allogeneic stem cell transplantation. One approach to preserve the beneficial graft-versus-leukemia while minimizing GvHD is to genetically modify donor T cells with a herpes simplex virus thymidine kinase (HSV-tk) suicide gene that can selectively phosphorylate the prodrug ganciclovir (GCV) resulting in T cell suicide should GvHD develop. We recently developed a new chimeric suicide gene by fusing HSV-tk to the extracellular and transmembrane domains of human CD34 (ΔCD34-tk;

ΔCD34-tk-modified cells can be rapidly and efficiently selected using a well-established and clinically approved CD34 immunoselection technique ensuring coordinated expression of the CD34 epitope and tk suicide gene in transduced affinity purified human T cells (HuT). We have developed a new model for xenogeneic GvHD induced by HuT in NOD-SCID-β2M null mice (β2 mice). β2 mice lack macrophage activity, T, B and NK cells. In this model 79% (11/14) of the β2 mice conditioned with 250 cGy and injected retro-orbitaly (ro) with 10⁷ HuT developed lethal GvHD, with loss of pretransplant body weight (>20%), decreased activity, hunched posture, ruffled fur, accompanied by HuT expansion and infiltration of blood and multiple tissues. In this study, we evaluated the ability of GCV to prevent GvHD after infusion of CD34-tk-modified HuT in this xenogeneic model. Human PBMCs cells were activated with anti-CD3 and anti-CD28 mAbs immobilized on magnetic beads (CD3/CD28 beads) in the presence of IL-2 (50 U/mL). Two days post-activation, cells were incubated with the 293 GPG-derived VSV-G pseudotyped CD34-tk oncoretroviral supernatants for 6 h at 37°C. Transduced (Td) cells were then expanded in media containing IL-2 and CD3/CD28 beads and isolated by MACS (Miltineyi Biotech) on day 4 postactivation (transduction efficiency >60%). Td cells were purified to >94% by CD34 immunomagnetic selection using a VarioMACS magnetic cell separator. Naive (unmanipulated) T cells (n=2), naive PBMC (n=3), activated non Td T cells (Activated; n=3) and CD34-tk transduced T cells (Td; n=6) were then injected (10⁷/mouse) ro into 250 cGy conditioned β2 mice. Animals receiving Td cells were then either left untreated or treated with GCV (1 mg/day, intraperitoneal) from days 1–7 post- HuT cell injection. Mice that received naive T cells died of lethal GvHD on days 14 and 16. Although we observed an average of 15.5% and 33.6% HuT engraftment in the Activated and Td groups respectively, and increased expression of CD25, CD30, and CD69 in the peripheral blood of mice 4 weeks post-infusion, we observed no GvHD (mice maintained their pretransplant body weight and did not developed any signs of GvHD). However, we were able to demonstrate that Td T cells could be efficiently eliminated in vivo by treatment with ganciclovir. Animals treated with ganciclovir from days 1–7 had less than 1% engraftment at 4 weeks post-infusion. This selective elimination of the Td T cells was not sustained however, as evidenced by the 3.5% HuT engraftment on blood (5.4% of these HuT were CD34+) and 18% HuT in the spleen (24% CD34+) at week 10 post-infusion. In conclusion, this xenograft model provides a unique opportunity for preclinical testing of the CD34-tk/GCV suicide gene system as well as other methods of GvHD control.