We recently developed a novel gene therapy approach in which FVIII is targeted and stored in platelet a-granules. Using a transgenic mouse model, we showed that FVIII can be specifically expressed and stored together with its carrier protein, von Willebrand factor (VWF), in platelet a-granules when it is driven by the platelet-specific aIIb promoter (2bF8) and that platelet-FVIII can correct the murine hemophilia A phenotype even in the presence of high-titer inhibitors.

To apply this gene therapy to a clinically translatable protocol, we use lentiviral gene delivery to hematopoietic stem cells (HSCs) to introduce FVIII expression in platelets. We found that 2bF8 lentiviral gene delivery to HSCs can not only restore hemostasis, but also induce antigen-specific immune tolerance in hemophilia A mice. Our current work aims to 1) further optimize this approach; 2) dissect the potential underlying mechanisms by which immune tolerance is induced after platelet gene therapy; and 3) translate findings made in laboratory animals to human patients.

Although platelet-FIX does not maintain clinical efficacy in the face of inhibitors, targeting FIX expression to platelets is still an attractive potential strategy for gene therapy of hemophilia B. Indeed, our studies demonstrate that 2bF9 gene delivery to HSCs not only restores hemostasis, but also induces FIX-specific immune tolerance in hemophilia B mice. Our current work aims to optimize the FIX expression and develop a protocol for gene therapy of hemophilia B with pre-existing immunity.

In addition, we want to know how VWF impacts FVIII immune responses in hemophilia A. Our studies demonstrate that VWF is essential for maintaining platelet-FVIII efficacy in hemophilia A with inhibitors. VWF reduces inhibitor activation of FVIII, exerting a protective effect both in vitro and in vivo. VWF/FVIII association, plus the apparent ability of VWF to delay the time-dependent inactivation of FVIII by inhibitors, provides mechanisms by which platelet-derived FVIII maintains function even in the presence of inhibitors when FVIII is targeted to platelets for gene therapy of hemophilia A with inhibitors. Our current work aims to investigate the roles of VWF in FVIII immunity in hemophilia A.