PROJECT SUMMARY The development of inhibitory antibodies against factor VIII (FVIII), which is referred to as FVIII inhibitors, is not only a significant complication of protein replacement therapy but also a major concern in gene therapy of hemophilia A. Generation of such inhibitors in protein replacement therapy might potentially preclude gene therapy for hemophilia A, or results in a failure in gene therapy. Our long-term goals are to formulate innovative safe and effective approaches for gene therapy of hemophilia A, including hemophilia A with inhibitors. We hypothesize that targeting the production of FVIII to platelets that activate at the site where FVIII is needed could overcome the presence of inhibitors. We further hypothesize that targeting FVIII expression in platelets that will undergo apoptosis when aged will modulate the immune response to the neo-protein, promoting immune tolerance to FVIII. We have developed a clinically translatable gene therapy protocol for hemophilia A using lentiviral gene delivery of the FVIII expression cassette under the control of the platelet-specific αIIb promoter (2bF8) to hematopoietic stem cells (HSCs) via transduction followed by transplantation, resulting in FVIII expression in platelets. Our previous studies have demonstrated that 2bF8 lentiviral gene delivery to HSCs can effectively introduce sustained therapeutic levels of platelet-FVIII expression and induce FVIII- specific immune tolerance in hemophilia A mice even with pre-existing anti-FVIII immunity when sufficient preconditioning is employed. In the current application, we propose three specific aims to further pursuit of our long-term goal. In Aim 1, we will investigate how platelet biological activities impact platelet-specific FVIII gene therapy of hemophilia A. In Aim 2, we will dissect the underlying mechanisms of how immune responses can be modified in platelet gene therapy. In Aim 3, we will evaluate the safety issue of the long-term genotoxicity in lentivirus-mediated platelet gene therapy of hemophiliacs. Through these studies, we expect to develop a safe, curative platelet gene therapy protocol that will not only restore hemostasis but also induce immune tolerance for all hemophilia A patients, including patients with pre-existing anti-FVIII immunity. These studies will have a significant impact on the hemophilia A community. Furthermore, these studies will also help us to understand the biological properties of platelets and FVIII and their interaction in the blood coagulation cascade.