Platelet-Targeted Expression of Human BDD-FVIII Reduces Bleeding in Canine Hemophilia A.

Abstract 691 Introduction: The goal of our study was to develop a clinically relevant strategy for platelet-targeted gene therapy of the commonly inherited bleeding disorder, Hemophilia A. We hypothesized that adult dogs (25 kg) affected with Hemophilia A could serve as a relevant “large animal” model to test if FVIII could be synthesized and sequestered within platelets derived from lentivirus-transduced hematopoietic stem cells. This approach is novel because it should permit the regulated release of FVIII from activated platelet progeny directly at the wound site as a physiological hemostatic response to bleeding challenges. Methods: cG-CSF/cSCF mobilized peripheral blood stem cells (PBSC) were immuno-selected for CD34 antigen from an apheresis product, transduced with a lentivirus vector under the transcriptional control of platelet-specific integrin αaIIb gene promoter driving expression of human BDD-FVIII. The PBSC (3 × 10 6/kg) were then autologously transplanted into animals that were preconditioned with Bulsulfan (5-10 mg/kg i.v.). After transplant, the dogs received oral cyclosporine to maintain levels at 200–400 ng/ml for 90 days and MMF at 8mg/kg for 35 days. Results: Two transplant recipients underwent periodic testing for incorporation and expression of the FVIII transgene as well as immune tolerance and phenotypic correction of Hemophilia A. PCR analysis detected the lentivirus vector within genomic DNA isolated from circulating peripheral blood leukocytes for more than one year after transplant. Immunofluorescence confocal microscopy showed synthesis of FVIII within tissue cultured canine megakaryocytes and circulating peripheral blood platelets. Chromogenic analysis of platelets isolated from transplanted dogs demonstrated the presence of a biologically active form of FVIII (FVIII:C) at approximately 5 mU/ml/1×108 platelets from 20 weeks through greater than one year after PBSC transplant. In contrast, FVIII:C was not detected within the plasma of these animals. This result coupled with the immunomodulation drugs may help to explain why the dogs failed to develop inhibitory antibodies to human FVIII. Following successful gene transfer and engraftment, both animals showed signs of clinical improvement of Hemophilia A: one dog has had one bleed per year for 2 years (vs expected 5–6/year) and the second dog has had no bleeds for 8 months following transplantation. In addition, one dog showed significant recovery from prolonged (months) history of gastrointestinal bleeding. These data are consistent with our previous results demonstrating synthesis, trafficking and storage of FVIII within αa-granules of human megakaryoyctes in vitro and platelets of the murine “small animal” model for Hemophilia A. Conclusions: This outcome raises the possibility of developing a protocol for delivering a locally inducible secretory pool of FVIII in platelets of patients with Hemophilia A following autologous transplantation of FVIII-transduced hematopoietic stem cells. Disclosures: Montgomery: GTI Diagnostics: Consultancy; Baxter: Consultancy; AstraZeneca: Consultancy; Bayer: Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees. Wilcox:Amgen, INC: Research Reagents, canine growth factors: cG-CSF and cSCF.