PROJECT SUMMARY/ABSTRACT Patients with severe hemophilia A (PWHA) have a significant deficiency (<1% normal) in coagulation factor VIII (FVIII) that frequently causes recurrent spontaneous bleeding episodes leading to significant morbidity and mortality. Conventional therapy for HA employing the infusion of donor plasma FVIII cryoprecipitate product can be complicated by blood-borne transmission of viral illnesses (including HIV & hepatitis C). Use of recombinant FVIII (rFVIII) products has largely replaced the use of human-derived FVIII (because rFVIII prevents risk of viral transmission); however, plasma FVIII remains a valuable resource for HA throughout the world. Prophylactic therapy with FVIII requires the intravenous administration of FVIII 2-3 times weekly throughout a patient's lifetime. Unfortunately, recurrent intravenous access, low compliance, break-through bleeding, and joint-damage can occur despite FVIII prophylaxis. Additionally, ≈30% of HA develop allo-antibodies to FVIII replacement products that inhibit its ability to restore hemostasis. Thus, treatment of bleeding in these patients involves the administration of a costly “bypass” agent therapy (i.e., FVIII with immune suppression and/or FVIIa). Success has been achieved by inducing immune tolerization to FVIII in ≈60% of HA with inhibitory antibodies by several rigorous infusions of FVIII (often in the setting of prophylaxis with bypassing agents) although treatment for FVIII inhibitors remains a critical issue for HA patients. Due to its monogenic nature, HA is an ideal candidate for gene replacement therapy with the potential for correction of HA. Promising approaches include the targeted expression of human FVIII to the liver by intravenous infusion of naked DNA, the generation of a novel adeno-associated viral (AAV) vector equipped with less immunogenic coat proteins, and vectors incorporating small active forms of FVIII (that conform to the 4.4 kb packaging capacity of AAV). However, these strategies exclude individuals with 1) inhibitory antibodies to FVIII (≈30% PWHA), 2) pre-existing antibodies to the AAV (≈40% humans) and 3) chronic liver damage. To address this problem of considerable clinical significance, we propose a first-in-human phase I clinical trial employing a hematopoietic stem cell (HSC) gene therapy strategy that utilizes a lentiviral gene transfer vector encoding human FVIII under the transcriptional control of the megakaryocyte-specific ITGA2B gene promoter that targets expression of the FVIII gene in megakaryocytes causing ectopic synthesis, storage and regulated-release of factor VIII from α-granules of activated platelets precisely at the site of vascular injury. This proposal is supported by pre-clinical studies that showed platelet FVIII safely and efficiently improved hemostasis in murine and canine models of HA without the development of inhibitory antibodies to FVIII and even in the presence of pre-existing inhibitory antibodies to FVIII in mice. In summary, the proposed trial should reduce the risk of severe bleeding in PWHA with inhibitory antibodies to FVIII for whom current strategies employing factor bypassing agents, tolerizing therapy, and other genetic therapies are inadequate. 1

PROJECT NARRATIVE Hemophilia A (HA) is an inherited bleeding disorder affecting ≈1:10,000 people worldwide due to molecular-genetic defects on the X-chromosome that cause a deficiency of coagulation factor VIII (FVIII). HA is currently treated with a continuous infusion of plasma protein replacement therapy that is ineffective in ≈30% of HA patients who make inhibitory antibodies (PWIA) that completely neutralize plasma FVIII. To address this serious public health issue, we propose to utilize a novel strategy in a Phase I Clinical Trial for long-term correction of severe HA in PWIA with hematopoietic stem cell gene therapy that produces genetically engineered human blood platelets that synthesize, store, and secrete FVIII directly at the site of a vascular injury to repair blood vessels without alerting the immune system. 1