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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 transduced with an intron-based Moloney murine leukemia virus (Mo-MuLV) retroviral vector that contained the B domain-deleted human factor VIII (FVIIIdeltaB) cDNA. This FVIII-retroviral vector was pseudotyped with the gibbon ape leukemia virus envelope (GALV-env) to attain higher transduction efficiencies. Using optimized transduction methods, high in vitro FVIII expression levels 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 xenografting of 1.5-3 x 106 engineered BM stromal cells into the spleen of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice, human plasma FVIII levels rose to 13 +/- 4 ng/ml but declined to basal levels by 3 weeks postinjection because of promoter inactivation. About 10% of these stromal cells engrafted in the spleen and persisted for at least 4 months after transplantation in the absence of myeloablative conditioning. No human BM stromal cells could be detected in other organs. These findings indicate that retroviral vector-mediated gene therapy using engineered BM stromal cells may lead to therapeutic levels of FVIII in vivo and that long-term engraftment of human BM stromal cells was achieved in the absence of myeloablative conditioning and without neo-organs. Hence, BM stromal cells may be useful for gene therapy of hemophilia A, provided prolonged expression can be achieved by using alternative promoters.

Attempts to develop an ex vivo gene therapy strategy for hemophilia A, using either primary T cells or bone marrow (BM) stem/progenitor cells have been unsuccessful, due to the inability of these cell types to express coagulation factor VIII (FVIII). As an alternative, we evaluated the potential of BM-derived stromal cells which can be readily obtained and expanded in vitro. Human and murine BM stromal cells were transduced with an intron-based Moloney murine leukemia virus (MoMLV) retroviral vector expressing a B-domain-deleted human factor VIII cDNA (designated as MFG-FVIIIdeltaB). Transduction efficiencies were increased 10- to 15-fold by phosphate depletion and centrifugation, which obviated the need for selective enrichment of the transduced BM stromal cells. This resulted in high FVIII expression levels in transduced human (180 +/- 4 ng FVIII/10[6] cells per 24 hr) and mouse (900 +/- 130 ng FVIII/10[6] cells per 24 hr) BM stromal cells. Pseudotyping of the MFG-FVIIIdeltaB retroviral vectors with the gibbon ape leukemia virus envelope (GALV-env) resulted in significantly higher transduction efficiencies (100 +/- 20%) and FVIII expression levels (390 +/- 10 ng FVIII/10[6] cells per 24 hr) in transduced human BM stromal cells than with standard amphotropic vectors. This difference in transduction efficiency correlated with the higher titer of the GALV-env pseudotyped viral vectors and with the higher GALV receptor (GLVR-1) versus amphotropic receptor (GLVR-2) mRNA expression levels in human BM stromal cells. These findings demonstrate the potential of BM stromal cells for gene therapy in general and hemophilia A in particular.