Factor VIII is the plasma protein that is functionally deficient in the clinical bleeding disorder hemophilia A. Patients require frequent infusions of factor VIII in order to maintain hemostasis. Factor VIII is synthesized in the hepatocyte and is secreted into the circulation where it is stabilized by non-covalent interactions with von Willebrand factor (vWF). Isolation of the factor VIII gene identified its domain structure of Al-A2-B-A3-Cl-C2 and enabled production of recombinant-derived factor VIII. Recombinant-derived factor VIII has minimized potential therapeutic complications associated with plasma-derived factor VIII, although significant limitations remain. Thus, the ultimate treatment for Hemophilia A would be a genetic cure for the disease. However, important questions need to be answered before gene therapy for hemophilia A becomes a reality. The proposed studies focus on three fundamental aspects that regulate factor VIII expression and activity. First, factor VIII expression in cultured mammalian cells is limited due to both inefficient rnRNA expression and protein secretion. The newly synthesized factor VIII is retained in the lumen of the endoplasmic reticulum (ER) in a complex with the immunoglobulin binding protein BiP and is inefficiently secreted. Recent results indicate that the limited factor VIII mRNA accumulation is coupled with factor VIII protein synthesis. The proposed studies will develop a hepatocyte-targeted adenoviral-based factor VIII gene delivery system to elucidate if similar limitations in factor VIll expression occur in vivo. Second, in the circulation, the ratio of vWF to factor VIII is tightly maintained at 50:1, although the mechanism responsible is not understood. We will identify the molecular mechanisms by which vWF regulates factor VIII levels using vWF-'knock-out' and transgenic mice. Finally, factor VIII activity is regulated by proteolytic activation and inactivation. The inactivation of factor VIII in vitro results from dissociation of the A2-domain peptide. Porcine factor VIll has increased specific activity and reduced A2-domain dissociation compared to human factor VIII. We have engineered a novel porcine/human hybrid factor VIII that has increased in vitro procoagulant activity. We propose to evaluate the hemostatic efficacy of this molecule in a hemophiliac dog model. The proposed studies will provide important insight into mechanisms that regulate fact6r VIII expression and activity in vitro and in vivo and the information obtained will be essential in considering avenues for ene therapy for hemophilia A.
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