title: 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-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.
authors.affiliation: Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology-University of Leuven, Belgium.
authors.forename: M K
authors.initials: MK
authors.lastname: Chuah
authors.forename: H
authors.initials: H
authors.lastname: Brems
authors.forename: V
authors.initials: V
authors.lastname: Vanslembrouck
authors.forename: D
authors.initials: D
authors.lastname: Collen
authors.forename: T
authors.initials: T
authors.lastname: VandenDriessche
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D005169:Factor VIII / Q000235:genetics*; D015870:Gene Expression; D015752:Genes, env; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006467:Hemophilia A / Q000628:therapy*; D006801:Humans; D017989:Leukemia Virus, Gibbon Ape; D051379:Mice; D008979:Moloney murine leukemia virus; D011991:Receptors, Virus / Q000235:genetics; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism*
chemical_list: D011991:Receptors, Virus; C067981:leukemia virus receptor, gibbon ape; D005169:Factor VIII