Chemoselection of Allogeneic HSC After Murine Neonatal Transplantation Without Myeloablation or Post-transplant Immunosuppression

Department of Medicine, University of California-San Francisco, San Francisco, California, USA.
Molecular Therapy (Impact Factor: 6.23). 08/2012; 20(11). DOI: 10.1038/mt.2012.136
Source: PubMed


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.

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Available from: Stanton L Gerson, Mar 23, 2014
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    • "Studies aimed at coupling selective stem cell enrichment to therapeutic gene expression have focused on the use of dual-gene vectors (Persons et al., 2003; Falahati et al., 2012; Richard et al., 2004; Trobridge et al., 2009). Many strategies have been employed to co-express two genes from the same vector, including the use of alternative splice sites, internal promoters, gene fusions, IRES elements , and ribosome slippage sites (Morgan et al., 1992; Sugimoto et al., 1994; de Felipe et al., 1999; Klump et al., 2001). "
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