Critical molecular interactions of VWF and FVIII

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Hemophilia A has been widely studied, and gene therapy has been a clinical approach carried out in animal models and some human clinical trials. Since standard approaches for treating hemophilia A have yet to be successful, alternative novel strategies are thought to be needed. One such approach has been developed in this grant during the past funding cycle, and has focused on expressing FVIII in megakaryo¿¿ cytes and endothelial cells. The natural interactions between VWF and FVIII might facilitate the expression of FVIII and make use of these interactions clinically. This grant has three aims that explore the interactions between FVIII and VWF, and how this could lead to strategies to optimize gene therapy for hemophilia. Even more exciting, this approach might be advantageous for hemophilic patients who have developed treatment-altering inhibitory antibodies. Aim 1 will study the local and systemic effect of VWF complexes with FVIII in the presence of specific FVIII inhibitory antibodies, and determine the safety of delivering FVIII in platelets. Aim 2 will study the delivery of FVIII in platelets, and determine its efficacy in a large animal model of hemophilia. Aim 3 will study a gene therapy approach in which FVIII is synthesized in and stored by endothelial cells, and whether endothelial cells normally synthesize FVIII. These models will dissect the contribution of VWF to the therapeutic efficacy of FVIII in the presence of inhibitory antibodies. Since the murine and canine models are less severe clinical models than the human disorder, and because dog platelets normally lack VWF. another animal model with platelet VWF is needed. The ovine model of hemophilia provides just such a model characterized by clinical severe bleeding, normal VWF in its platelets like humans, and most uniquely, the fetal ovine model has been demonstrated to permit long-term, life-long chimeric expression of human hematopoietic cells where human megakaryocytes/platelets, transduced to express and store FVIII, can be studied for safety and clinical efficacy. These studies will provide much additional critical safety and efficacy on this novel approach to gene therapy for hemophilia and provide a pre-clinical model of highest relevance before considering human trials. RELEVANCE (See instructions): This grant addresses a novel strategy to carry out gene therapy of hemophilia, a severe clinical bleeding disorder. While most studies have targeted liver cells, our unique approach will targets the expressed FVIII to endothelial cells or megakaryocytes - cells that normally synthesize and store VWF. These approaches may not only treat patients with hemophilia, but also treat those patients that have high-titer inhibitory antibodies that would normally mitigate against traditional replacement therapy, or even traditional gene therapy approaches to replace FVIII in plasma.