In vitro selection of lentivirus vector-transduced human CD34(+) cells

Human CD34(+) cells with in vivo repopulating potential hold much promise a s a target for corrective gene transfer for numerous hematopoietic disorder s. However, the efficient introduction of exogenous genes into this small, quiescent population of cells continues to present a significant challenge. To circumvent the need for high initial transduction efficiency of human h ematopoietic cells, we investigated a dominant selection strategy using a v ariant of the DHFR gene (DHFRL22Y) For this purpose, we constructed a lenti virus-based bicistronic vector expressing EGFP and DHFRL22Y Here we demonst rate efficient in vitro selection and enrichment of lentivirus vector-trans duced human CD34(+) hematopoietic cells from fetal liver, umbilical cord bl ood, bone marrow, and peripheral blood after cytokine mobilization, Growth of transduced human CD34(+) cells in semisolid culture under selective pres sure resulted in enrichment of transduced progenitor cells to 99.5% (n = 14 ), Selection for DHFRL22Y+ cells after expansion of transduced progenitors in liquid culture resulted in a 7- to 13-fold increase in the percentage of marked cells. Thus we have shown that transduced human hematopoietic cells may be effectively enriched in vitro by dominant selection, suggesting tha t development of such strategies holds promise for future in vivo applicati on.