474. Transduction of Minimally Stimulated Hematopoietic Progenitor Cells with a Foamy Virus Vector That Expresses MGMTP140K

Abstract

Top of pageAbstract The ability to deliver genetic sequences that encode therapeutic proteins to hematopoietic stem and progenitor cells (HSC) in the absence of cell cycle progression provides an opportunity to transduce polyclonal populations of stem cells without loss of long-term function. We constructed a foamy virus vector MD9-P140K-EGFP that co-expresses a mutant form of O6-methylguanine DNA methyltransferase (MGMTP140K) and the enhanced green fluorescent protein (EGFP) to study the outcome of transducing noncycling populations of HSC derived from human and mouse sources. We also compared in parallel transduction of HSC with an oncoretrovirus vector, SF1-P140K-EGFP, using standard transduction protocols. In the human studies, CD34+ cells derived from Granulocyte-Colony Stimulating Factor-mobilized peripheral blood (MPB) were used since MPB is difficult to transduce with oncoretrovirus vectors. We demonstrated previously that although MPB could be selected in vivo by expression of MGMTP140K using an oncoretrovirus vector and drug treatment, the number of selected HSC was significantly lower than that obtained when using UCB HSC presumably due to inefficient gene transfer. (Pollok et al, Hum. Gene Ther. 14:1703). Cell cycle analysis indicated that 98-99% of the MPB were in G0/G1 prior to transduction. MPB HSC were transduced on Retronectin-coated plates with the MD9-P140K-EGFP vector in the presence of G-CSF, Stem Cell Factor (SCF), and Thrombopoietin (TPO) or SCF, TPO, and Flt3-ligand at a multiplicity of infection (MOI) ranging from 10-20. Transduction on Retronectin-coated plates increased the transduction efficiency and also prevented detrimental cell fusion. In colony-forming unit (CFU) assays, similar numbers of CFU were present in transduced and nontransduced cultures, indicating that no gross toxicity occurred during the transduction. The MD9 vector preferentially transduced the committed progenitor pool regardless of the cytokine combination used. While the bulk transduction efficiencies ranged from 3%-5%, the CFU transduction efficiency ranged from 40%-70% in lineage-restricted progenitors (n=5). Consistent with our previous studies, transduction of HSC with the SF1 oncoretrovirus vector resulted in similar bulk and CFU transduction efficiencies (55%-60%). MGMT DNA repair activity in EGFP-positive CFU derived from foamy virus MD9-transduced HSC exhibited 50%-70% higher level of activity compared to EGFP-positive CFU derived from oncoretrovirus SF1-transduced HSC. In mouse studies, bone marrow from C57BL/6 mice was lineage depleted to enrich for noncycling HSC. In MD9 transductions, no significant toxicity was observed up to a MOI of 40. Bulk transduction efficiency ranged from 12%-25% and the CFU transduction efficiency was 55%-57% (n=3).Transduction with the oncoretrovirus vector resulted in similar bulk and CFU transduction efficiencies (55%-60%). Current studies are now using these conditions to evaluate in vivo the selection of minimally stimulated HSC transduced with the MD9-P140K-EFGP foamy virus vector.