Transduction of hematopoietic stem cells using lentiviral vectors in models of drug resistance gene therapy: Determinants of transduction and selection

Correction of hematologic disorders by gene therapy has been hampered by the inability to transduce sufficient numbers of hematopoietic stem cells (HSC) to exert a phenotypic change. This has partially been due to the biology of the current class of gene therapy vectors based on oncoretroviruses, which cannot efficiently transduce nondividing cells. Drug resistance gene therapy strategies centering around the DNA repair gene, P140K MGMT, have thus been devised as a way to increase the proportion of gene modified HSC in vivo, since modified cells are resistant to BG/BCNU treatment. This study thereby describes use of a new class of viral vector based on a lentivirus for transduction of P140K MGMT to human HSC for the purpose of in vivo selection. The transduction culturing conditions resulting in high gene transfer levels and their effect on HSC survival are described. The viral protein, vpr, in combination with SDF-1α are analyzed for their ability to increase transduction. The in vitro resistance to BG/BCNU conferred by P140K MGMT transduction is defined as well as demonstration of selection of human HSC in a NOD/SCID mouse model. Novel tools are used to analyze clonality of repopulation and the relationship of expression to integration copies. SarCNU is proposed as an alternative selection agent. In toto, these results mark an advance in drug resistance gene therapy using lentiviral vectors and suggest future emphasis to be placed on clinical investigation.