De Novo Synthesis & Storage of Human Factor VIII In Platelets Reduces Bleeding In Canine Hemophilia A

The goal of our study was to develop a clinically relevant strategy for platelet-targeted gene therapy of the 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 Factor VIII (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 a site of vascular injury as a physiological hemostatic response to a severe intermittent bleeding challenge.

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 αIIb gene promoter driving expression of human BDD-FVIII. The PBSC (2 × 10⁶/kg) were then autologously transplanted (Tx) 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 8 mg/kg for 35 days.

Three Tx recipients underwent periodic testing for incorporation and expression of the FVIII transgene as well as immune tolerance and phenotypic correction of Hemophilia A. LAM PCR analysis detected and localized the lentivirus vector within genomic DNA isolated from circulating peripheral blood leukocytes from each animal. Immunofluorescence confocal microscopy detected FVIII within a subset of circulating peripheral blood platelets. Immune electron microscopic analysis revealed that the FVIII had been trafficked and stored within the platelet secretory α-granules. Chromogenic analysis of platelets isolated from dogs demonstrated that FVIII was present in its biologically active form (FVIII:C) at approximately 5 mU/ml/1×10⁸ platelets from 20 weeks through greater than two years after PBSC Tx. In contrast, FVIII:C was not detected within the plasma of these animals. This result coupled with the use immunomodulation drugs may help to explain why the dogs remained tolerant of human FVIII as indicated by our inability to detect inhibitory antibodies to FVIII. Following successful gene transfer and PBSC engraftment, all animals showed signs of clinical improvement of Hemophilia A: the longest Tx recipient had one bleed per year for 2 years (vs expected 5 – 6/year), the second dog has had no bleeds for twenty months following PBSC Tx and the third animal had no bleeding episodes for first seven months post Tx. In addition, the first Tx dog showed significant recovery from prolonged (months) history of chronic gastrointestinal bleeding. Remarkably, all three animals displayed improved whole blood clotting times. These data are consistent with our previous results demonstrating synthesis, trafficking, storage and regulated release of FVIII from α-granules of human megakaryoyctes in vitro and platelets of the murine “small animal” model for Hemophilia A.

The outcome of this work demonstrates a feasible strategy for controlling severe bleeding episodes within patients with Hemophilia A by providing a locally inducible secretory pool of FVIII in platelets derived from an autologous Tx of FVIII-transduced PBSC.

No relevant conflicts of interest to declare.