Ex Vivo Gene Therapy for Hemophilia A That Enhances Safe Delivery and Sustained In Vivo FVIII Expression From Lentivirally-engineered Endothelial Progenitors

¹ Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada K7L 3N6
² Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada K7L 3N6; Department of Pediatrics, Nara Medical University, Kashihara City, Nara, 634-8522, Japan
³ Vascular Biology Center and Division of Hematology Oncology Transplantation, University of Minnesota Medical School, Minneapolis, MN 55455, USA
⁴ Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada H3T 1E2

^* To whom correspondence should be addressed. E-mail: lillicrap{at}cliff.path.queensu.ca.

	Abstract

Novel therapeutic strategies for hemophilia must be at least as effective as current treatments and demonstrate long-term safety. To date, several small clinical trials of hemophilia gene transfer have failed to show the promise of pre-clinical evaluations. Therefore, we wanted to develop and evaluate the feasibility of a novel ex vivo gene transfer strategy whereby cells derived from progenitor cells are engineered to express factor VIII (FVIII) and then implanted subcutaneously to act as a depot for FVIII expression. Circulating blood outgrowth endothelial cells (BOECs) were isolated from canine and murine blood, and transduced with a lentiviral vector encoding the canine FVIII transgene. To enhance safety, these cells were implanted subcutaneously in a Matrigel^TM scaffold and the efficacy of this strategy was compared to intravenous delivery of engineered BOECs in non-hemophilic NOD/SCID mice. Therapeutic levels of FVIII persisted for 15 weeks and these levels of stable expression were extended to 20 weeks when the CMV promoter was replaced with the thrombomodulin regulatory element. Subsequent studies in immunocompetent hemophilic mice, pre-treated with tolerizing doses of FVIII or with transient immunosuppression, showed therapeutic FVIII expression for 27 weeks before the eventual return to baseline levels. This loss of transgene expression appears to be due to the disappearance of the implanted cells. The animals treated with either of the two tolerizing regimens did not develop anti-FVIII antibodies. Biodistribution analysis demonstrated that BOECs were retained inside the subcutaneous implants. These results indicate, for the first time, that genetically-modified endothelial progenitor cells implanted in a subcutaneous scaffold can provide sustained therapeutic levels of FVIII and are a promising and safe treatment modality for hemophilia A.

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