Bone Marrow Stromal Cells as Targets for Gene Therapy of Hemophilia A

Abstract

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-FVIIIΔB). 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/10⁶ cells per 24 hr) and mouse (900 ± 130 ng FVIII/10⁶ cells per 24 hr) BM stromal cells. Pseudotyping of the MFG-FVIIIΔB 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/10⁶ 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.

Overview summary

This study demonstrates that bone marrow (BM) stromal cells may be used to achieve high levels of blood coagulation factor VIII expression in vitro after transduction with amphotropic Moloney murine leukemia virus (MoMLV) and gibbon ape leukemia virus envelope (GALV-env) pseudotyped retroviral vectors using optimized transduction protocols. Therefore, BM stroma may be useful as a target for gene therapy of hemophilia A.