Work

Suicide gene therapy for human T cell mediated graft versus host disease in a murine xenograft model

Year: 2005

Type: article

Abstract: Graft-versus-host disease (GvHD) remains a major cause of morbidity and mortality following allogeneic bone marrow transplantation. We developed a novel chimeric suicide gene in which the extracellular and transmembrane domains of human CD34 were fused in frame to the entire coding region of HSV-tk (CD34-tk). In this study, we developed a new model for xenogenic GvHD using immunodeficient NOD SCID β2M null mice (β2) and evaluated the ability of ganciclovir (GCV) to prevent GvHD after infusion of CD34-tk-modified human T cells (Td). Sublethally irradiated (250 cGy) β2 mice injected retro-orbitally (ro) with human PBMCs (107T cells; n=11) or human T cells (HuT; 107; n=14) had 19% HuT engraftment after 2–3 weeks with an OS of 75% and 25%, respectively, at 5 weeks (p=0.006). These mice lost >20% of their body weight and had extensive HuT infiltration in the spleen, liver, lung and kidney. On day 10 and before the start of any clinical signs of GvHD, mice that went on to develop lethal GvHD had a 10–15 fold increase in the expression of the HuT activation markers CD25, CD30, and CD69 in blood (p<0.0005) and tissues, and a 128-fold increase in serum levels of IFNγ (5150 pg/ml vs 40 pg/ml) compared to mice that did not develop GVHD. To generate CD34-tk-modified human T cells, PBMCs were activated with anti-CD3 and anti-CD28 mAbs immobilized on magnetic beads (Xcyte™ Dynabeads®) in the presence of IL-2 (50 U/mL). After 2 days, cells were incubated with 293 GPG-derived VSV-G pseudotyped CD34-tk oncoretroviral supernatants for 6h at 37C. Td cells were then expanded for 2 more days and isolated by MACS (Miltineyi Biotech; Td >60%). Td cells were purified to >94% by CD34 immunomagnetic selection using a VarioMACS magnetic cell separator. Naive T cells (n=2), activated non-Td T cells (Act; n=3) and Td and selected T cells (n=6) were then injected ro into 250 cGy conditioned β2 mice. Animals receiving Td cells were then either left untreated or treated with GCV (1 mg/day, ip) from days 1–7. Mice that received naive T cells died of lethal GvHD on day 15. Mice that received Act or Td HuT had an average engraftment of 16% and 34%, respectively, at 3 weeks post-infusion but did not develop GvHD. We were able to demonstrate that Td T cells could be efficiently eliminated in vivo by treatment with GCV. 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.

Source: Transplantation and Cellular Therapy

DOI: 10.1016/j.bbmt.2004.12.135