Chemoselection of allogeneic HSC after murine neonatal transplantation without myeloablation or post-transplant immunosuppression

Abstract

The feasibility of allogeneic transplantation, without myeloablation or post-transplant immunosuppression, was tested using in vivo chemoselection of allogeneic hematopoietic stem cells (HSCs) after transduction with a novel tricistronic lentiviral vector (MGMT P140K -2A-GFP-IRES-TK (MAGIT)). This vector contains P140K-O 6 -methylguanine-methyltransferase (MGMT P140K), HSV-thymidine kinase (TK HSV), and enhanced green fluorescent protein (eGFP) enabling (i) in vivo chemoselection of HSC by conferring resistance to benzylguanine (BG), an inhibitor of endogenous MGMT, and to chloroethylating agents such as 1,3-bis(2-chloroethyl)nitrosourea (BCNU) and, (ii) depletion of proliferating cells such as malignant clones or transduced donor T cells mediating graft versus host disease (GVHD), by expression of the suicide gene TK HSV and Ganciclovir (GCV) administration. Non-myeloablative transplantation of transduced, syngeneic, lineage-depleted (Lin ) BM in neonates resulted in 0.67% GFP mononuclear cells in peripheral blood. BG/BCNU chemoselection, 4 and 8 weeks post-transplant, produced 50-fold donor cell enrichment. Transplantation and chemoselection of major histocompatibility complex (MHC)-mismatched MAGIT-transduced Lin BM also produced similar expansion for >40 weeks. The efficacy of this allotransplant approach was validated in Hbb th3 heterozygous mice by correction of β-thalassemia intermedia, without toxicity or GVHD. Negative selection, by administration of GCV resulted in donor cell depletion without graft ablation, as re-expansion of donor cells was achieved with BG/BCNU treatment. These studies show promise for developing non-ablative allotransplant approaches using in vivo positive/negative selection. © The American Society of Gene & Cell Therapy.