Transgenic Targeting of Human FVIII Expression to Endothelial Cells Corrects the Murine Hemophilia A Phenotype and Re-Establishes a Releasable Pool of FVIII Together with VWF.

The deficiency of Factor VIII (FVIII) leads to hemophilia A, a severe X-linked bleeding disorder. To date, it is still unknown with certainty where FVIII is synthesized and how the storage pool of desmopressin acetate (DDAVP)-releasable FVIII is established. In vitro studies performed by our laboratory have shown that FVIII will store with VWF in endothelial cells if FVIII expression is induced by transfection or transduction. This FVIII is released by agonist stimulation. Furthermore, our studies have demonstrated that FVIII is not taken up from culture media by cells in culture and transfused FVIII does not re-establish the releasable pool in vivo. In this study, we generated 2 transgenic mouse lines in which human B-domain deleted FVIII (hFVIII) expression is under control of the endothelial-specific Tie 2 promoter (Tie2F8). This transgene was then bred into FVIII-deficient mice so that FVIII expression was restricted to endothelium. Functional FVIII activity (FVIII:C) was quantitated by a chromogenic assay. FVIII antigen (FVIII:Ag) and VWF antigen (VWF:Ag) were quantitated by ELISA. RT-PCR was used to analyze transcription of the transgene. Phenotypic correction was assessed by tail clip survival test, electrically-induced femoral venous thrombus formation, and ferric chloride induced carotid thrombus formation studies. The results demonstrated that the levels of FVIII in our transgenic mice were 559 ± 161 mU/ml in a higher expression line and 186 ± 31 mU/ml in a lower expression line. Tie2F8 transgene transcription was most abundant in lung and heart tissues. All transgenic mice survived tail clipping and achieved hemostasis within 6 hours. Both arterial and venous thrombi formation models demonstrated significant improvement of hemostasis at sites of injury. Confocal microscopy demonstrated recombinant FVIII stored together with VWF in endothelial cells, with especially high expression in the outlet branches of the aorta. Furthermore, this endothelial-specific expressed FVIII can be co-released together with VWF by epinephrine. The ratios of plasma FVIII:C and VWF:Ag post-epinephrine treatment to pre-epinephrine were determined, and the mean ± SEM were 1.68 ± 0.13 and 2.08 ± 0.34, respectively, at 30 minutes post-epinephrine treatment. Our results demonstrate that targeting human FVIII expression to endothelial cells corrects the murine hemophilia A phenotype and re-establishes a releasable pool of FVIII together with VWF.