ABSTRACT: Hemophilia A, which results in defective or deficient factor VIII (FVIII) protein, is one of the genetic diseases that has been addressed through gene therapy trials. FVIII synthesis does not occur in normal megakaryocytes. In hemophilia patients who have inhibitors to FVIII activity, megakaryocytes could be a protected site of FVIII synthesis and subsequent release. Since von Willebrand factor (VWF) is a carrier protein for FVIII, we hypothesize that by directing FVIII synthesis to megakaryocytes, it would traffick together with VWF to storage in megakaryocyte α-granules and the platelets derived from these cells. Such synthesis would establish a protected, releasable α-granule pool of FVIII together with VWF. When platelets are activated in a region of local vascular damage, FVIII and VWF could potentially be released together to provide improved local hemostatic effectiveness. To direct FVIII expression to the megakaryocyte lineage, we designed a FVIII expression cassette where the human B-domain deleted FVIII cDNA was placed under the control of the megakaryocytic/platelet-specific glycoprotein IIb (αIIb) promoter. We demonstrated by means of a functional FVIII activity assay that the biosynthesis of FVIII occurred normally in Dami cells transfected with FVIII. FVIII production was higher when driven by the αIIb promoter compared to the CMV promoter, and was increased about 8-fold following PMA treatment of the transfected Dami cells. Immunofluorescence staining of the transfected cells showed that FVIII stored together with VWF in the granules. The data indicate that the megakaryocytic compartment of hematopoietic cells may represent a potential target of gene therapy for hemophilia A—especially in those patients who have developed inhibitors to plasma FVIII.
Molecular Genetics and Metabolism.