Platelet-Targeted Expression of Coagulation Factor VIII (FVIII) Shows Efficacy for Using the Dog as a Large Animal Model for Gene Therapy of Hemophilia a

In order to develop a large animal model for platelet-targeted gene therapy of Hemophilia A, we transduced normal canine bone marrow with a lentivirus vector under the transcriptional control of integrin αIIb gene promoter driving expression of human BDD-FVIII. Bone marrow was collected from normal dogs and transduced in the presence of recombinant canine c-SCF, c-IL-6, human h-TPO, and h-flt-3/flk-2 ligand differentiation factor. Immunofluorescence analysis using antibodies against human FVIII, and the megakaryocyte-specific marker integrin αIIb revealed synthesis of FVIII within tissue cultured megakaryocytes derived from lentivirus transduced bone marrow cells. This result appeared similar to synthesis, trafficking and storage of FVIII trafficked to α-granules of the murine “small animal” model for hemophilia A and human megakaryoyctes in vitro. To examine the effect of FVIII expression in platelets, in vivo, FVIII-transduced canine bone marrow was xenotransplanted into immune-compromised “NOD-SCID” mice treated with a sublethal dose (350 cGy) of irradiation. Flow cytometric analysis using antibodies specific for canine glycoproteins Ibα and integrin αIibβ3 revealed that circulating canine platelets comprised approximately 20–30% of the total platelet population in whole blood isolated from the mice within four weeks after xenotransplant. Immunofluorescence confocal microscopy detected a punctuate staining for FVIII that co-localized with a platelet α–granule protein, P-selectin, within a subpopulation of canine platelets isolated from murine whole blood. In contrast, FVIII was not detected in platelets from control samples. In addition, chromogenic analysis of platelets isolated from mice transplanted with FVIII-transduced bone marrow demonstrated the presence of a biologically active form of FVIII, FVIII:C at 2 mU/ml/lysate of 1×108 platelets. These results indicate that canine megakaryocytes are able to synthesize and store a biologically active form of FVIII that can be retained within progeny platelets. We speculate that dogs affected with hemophilia A should serve as an ideal “large animal” model to test if FVIII can undergo regulated release from platelets following physiologic hemostatic response to vessel injury. This raises the possibility of developing a locally inducible secretory pool of FVIII in platelets of patients with Hemophilia A following autologous transplantation of FVIII-transduced hematopoietic stem cells.