The Important Role of Von Willebrand Factor In Platelet-Derived FVIII Gene Therapy of Murine Hemophilia A In the Presence of Inhibitors

Abstract 2201 von Willebrand factor (VWF) is a carrier protein for FVIII and protects FVIII from protease degradation. Our previous studies have demonstrated that targeting FVIII expression to platelets results in FVIII storage together with VWF in platelet α-granules and that platelet-FVIII (2bF8) corrects murine hemophilia A phenotype even in the presence of high titer anti-FVIII inhibitory antibodies. While ectopic expression of FIX in platelets is also trafficked and stored in α-granules and corrects the bleeding diathesis in hemophilia B mice, the efficacy of platelet-FIX is limited in the face of anti-FIX inhibitors, possibly due to the lack of protective carrier protein like VWF for FVIII. In the current study, we wanted to explore the role of VWF in platelet-derived FVIII gene therapy of murine hemophilia A with inhibitory antibodies. We immunized transgenic mice in which 2bF8 transgene was on a FVIII and VWF double knockout background (2bF8 tg+/− FVIII null VWF null ), with recombinant human B-domain deleted FVIII (rhFVIII) to induce inhibitory antibody development. Inhibitor titer was determined by Bethesda assay and phenotypic correction was assessed using tail clip survival tests. The results demonstrated that only 18% (n = 11) of VWF-deficient animals with inhibitor titers between 3 and 8000 BU/ml survived the tail clip challenge. In contrast, 82% (n = 22) of immunized 2bF8 tg+/− FVIII null transgenic mice, which had normal VWF levels, survived tail clipping with inhibitor titers of 10 – 50,000 BU/ml (P tg+/− VWF null FVIII null mice (n = 12) without inhibitors survived tail clipping and no (n = 6) VWF null FVIII null mice survived this challenge. Since VWF is synthesized by endothelial cells and megakaryocytes and distributes in plasma and platelets in peripheral blood, we further investigated the effect of each compartment of VWF (plasma-VWF vs. platelet-VWF) in platelet-FVIII gene therapy of hemophilia A with inhibitors. To address the effect of plasma-VWF, FVIII null mice were immunized with rhFVIII to induce inhibitor development and then they received bone marrow transplantation (BMT) from 2bF8 tg+/− FVIII null VWF null mice. To study the effect of platelet-VWF, VWF null FVIII null mice were immunized followed by BMT from 2bF8 tg+/− FVIII null VWF +/+ mice. After at least 6-weeks of BM constitution, mice were analyzed. Viable BMT engraftment in recipients was confirmed by PCR and platelet lysate FVIII activity assay. The levels of VWF in plasma and platelets were quantitated by ELISA. The phenotypic correction was assessed by the tail clip survival test. In the group with plasma-VWF, 5 of 12 (42%) mice survived tail clipping with inhibitor titers between 22 – 1200 BU/ml. In the group with platelet-VWF, 6 of 12 (50%) mice survived tail clipping with inhibitor titers of 5 – 810 BU/ml. As controls, all recipients (6 mice in each group) without inhibitors survived the tail clip challenge. To further investigate the dose effect of inhibitors on platelet-FVIII gene therapy of animals that only have plasma-VWF, we infused varied levels of inhibitory plasma from immunized VWF null FVIII null mice into FVIII null mice that received BMT from 2bF8 tg+/− FVIII null VWF null , followed by tail clip survival tests. Four of 6 mice with 2.5 BU/ml of inhibitors, 2 of 6 mice with an inhibitor titer of 25 BU/ml, and 1 of 6 mice with an inhibitor titer of 250 BU/ml survived tail clipping. Taken together, in this acute inhibitor model 7/18 (39%) mice with inhibitors between 2.5 – 250 BU/ml survived tail clipping. This survival rate is significantly lower than the group with normal VWF (P Disclosures: No relevant conflicts of interest to declare.