Long-term persistence of human bone marrow stromal cells transduced with factor VIII-retroviral vectors and transient production of therapeutic levels of human factor VIII in nonmyeloablated immunodeficient mice

The potential of using bone marrow (BM)-derived human stromal cells for ex vivo gene therapy of hemophilia A was evaluated. BM stromal cells were tran sduced with an intron-based Moloney murine leukemia virus (Mo-MuLV) retrovi ral vector that contained the B domain-deleted human factor Vm (FVIII Delta B) cDNA. This FVIII-retroviral vector was pseudotyped with the gibbon ape leukemia virus envelope (GALV-env) to attain higher transduction efficienci es, Using optimized transduction methods, high in vitro FVIII expression le vels of 700 to 2500 mU of FVIII/10(6) cells per 24 hr were achieved without selective enrichment of the transduced BM stromal cells. After xenograftin g of 1.5-3 x 10(6) engineered BM stromal cells into the spleen of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice, human plasma FVII I levels rose to 13 +/- 4 ng/ml but declined to basal levels by 3 weeks pos tinjection because of promoter inactivation. About 10% of these stromal cel ls engrafted in the spleen and persisted for at least 4 months after transp lantation in the absence of myeloablative conditioning, No human BM stromal cells could be detected in other organs. These findings indicate that retr oviral vector-mediated gene therapy using engineered BM stromal cells may l ead to therapeutic levels of FVIII in vivo and that long-term engraftment o f human BM stromal cells was achieved in the absence of myeloablative condi tioning and without neo-organs. Hence, BM stromal cells may be useful for g ene therapy of hemophilia A, provided prolonged expression can be achieved by using alternative promoters.