Lentiviral vector transduction of hematopoietic stem cells that mediate long-term reconstitution of lethally irradiated mice

Lentiviral vectors efficiently transduce human CD34(+) cells that mediate l ong-term engraftment of nonobese diabetic/severe combined immunodeficient m ice, However, hematopoiesis in these animals is abnormal, Typically, 95% of the human cells in peripheral blood are B lymphocytes. To determine whethe r lentiviral vectors efficiently transduce stem cells that maintain normal hematopoiesis in vivo, we isolated Sca-1(+)c-Kit(+)Lin(-) bone marrow cells from mice without 5-fluorouracil treatment, and transduced these cells in the absence of cytokine stimulation with a novel lentiviral vector containi ng a GFP (green flourescent protein) reporter gene. These cells were transp lanted into lethally irradiated C57Bl/6 mice, In fully reconstituted animal s, GFP espression was observed in 8.0% of peripheral blood mononuclear cell s for 20 weeks posttransplantation, Lineage analysis demonstrated that a si milar percentage (approximately 8.0%) of GFP-positive cells was detected in peripheral blood B cells, T cells, granulocytes and monocytes, bone marrow erythroid precursor cells, splenic B cells? and thymic T cells. In seconda ry transplant recipients, up to 20% of some lineages expressed GFP, Our res ults suggest that quiescent, hematopoietic stem cells are efficiently trans duced by lentiviral vectors without impairing self-renewal and normal Linea ge specification in vivo. Efficient gene delivery into murine stem cells wi th lentiviral vectors will allow direct tests of genetic therapies in mouse models of hematopoietic diseases such as sickle cell anemia and thalassemi a, in which corrected cells may have a selective survival advantage.