title: Retroviral modification of mesenchymal stem cells for gene therapy of hemophilia.
abstract: Mesenchymal stem cells (MSCs) are a promising target for the delivery of secreted proteins due to their ease of isolation, expansion, and genetic modification. The bleeding disorder hemophilia A results from the deficiency of a secreted blood clotting factor termed factor VIII (fVIII). Hemophilia A could be cured by gene-transfer-based procedures targeting virtually any cell type, including MSCs. Here, we describe methods for retroviral modification of MSCs incorporating a high-expression porcine (HEP)-fVIII transgene and a murine model of hemophilia A. MSCs were isolated from bone marrow of hemophilia A mice, expanded, and transduced ex vivo. Genetically modified MSCs secreted high levels of HEP-fVIII into the conditioned medium. HEP-fVIII was purified from the conditioned medium and demonstrated to have a specific activity, relative electrophoretic mobility, and proteolytic activation pattern similar to HEP-fVIII produced by other commercial cell lines. Collectively, these data support the concept that MSCs can be utilized as a cellular vehicle for successful gene-transfer-based therapy of hemophilia A and other disorders resulting from the deficiency of a secreted protein.
authors.affiliation: Department of Pediatrics, Emory University, Atlanta, GA, USA.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Doering
mesh_terms: D000818:Animals; D002469:Cell Separation; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics* / Q000627:therapeutic use; D015316:Genetic Therapy / Q000379:methods*; D006467:Hemophilia A / Q000235:genetics* / Q000628:therapy*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D051379:Mice; D012190:Retroviridae / Q000235:genetics*; D013552:Swine; D014161:Transduction, Genetic
chemical_list: D005169:Factor VIII