Correction of a Murine Model of Von Willebrand Disease by Gene Transfer.

Abstract Von Willebrand disease (VWD) is the most common inherited bleeding disorder affecting 1 to 3% of the U.S. population. It is caused by defects in expression and processing of Von Willebrand factor (VWF), a blood glycoprotein required for normal hemostasis, that mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet glycoproteins and to constituents of exposed connective tissue. Since it is established that 1–5% of normal levels of VWF is sufficient to ameliorate the bleeding phenotype, the possibility that corrective levels of circulating VWF could be produced using a gene transfer strategy was investigated. However, since the normal sites of VWF production, endothelial cells, megakaryocytes and platelets, are difficult to target with traditional gene transfer methodologies, we explored the strategy of using gene transfer to the liver, an easily transduced ectopic site for VWF production. The study employed a murine “knockout” model of VWD created by disrupting the naturally occurring VWF gene (VWF-KO mice) which exhibit defects in hemostasis with a highly prolonged bleeding time and spontaneous bleeding events closely mimicking severe human VWD. Transected-tail wounds in untreated VWF-KO mice will bleed to death unless cauterized, whereas similar wounds in WT mice stop bleeding in <3 min. To investigate the impact of VWF gene transfer on this phenotype, a plasmid containing the complete murine VWF cDNA under the control of a cytomegalovirus immediate/early promoter/enhancer, was delivered to the VWF-KO mice intravenously. An identical plasmid containing the cDNA for green fluorescent protein (GFP) and saline were used as controls. Bleeding time after wounding was determined 24, 48 or 72 hr after transfer of 10 to 250 μg of plasmid. Bleeding time was normalized by 48 hr after delivery of 50 μg or more of VWF cDNA, but not by GFP cDNA. Western analysis of plasma from animals receiving VWF cDNA, but not GFP cDNA, revealed high-molecular weight VWF multimers similar to the pattern observed in wild type mice. These data suggest that it may be possible to correct VWD using gene therapy delivered to tissues other than the naturally occurring sites of VWF manufacture, and that the liver is capable of expressing, processing and secreting VWF in a physiologically active form.