The Impact of GPIba on the Efficacy of Platelet-Targeted FVIII Gene Therapy in Hemophilia a with Pre-Existing Anti-FVIII Immunity

Abstract Platelets play fundamental roles in hemostasis through surface proteins, e.g. GPIb, and storage proteins, such as von Willebrand factor (VWF). Our previous studies have demonstrated that FVIII ectopically targeted to platelets under control of the platelet-specific aIIb promoter (2bF8) is therapeutic in hemophilia A mice even in the presence of anti-FVIII inhibitory antibodies (inhibitors). Our recent studies have shown that VWF is essential in platelet-targeted FVIII gene therapy of hemophilia A with inhibitors. Without VWF, the clinical efficacy of platelet-FVIII was aborted. In primary hemostasis, platelets adhere to the vessel wall through the binding of GPIb to VWF. Thus, we wanted to explore whether GPIb is critical for maintaining the clinical efficacy of platelet-FVIII gene therapy of hemophilia A in the presence of inhibitors. To address this question, recipient FVIIInull (F8null) mice were immunized with recombinant FVIII (rhF8) to induce anti-FVIII inhibitory antibody development (the inhibitor model). Hematopoietic stem cells (HSCs) from GPIba knockout (Ibnull) mice were transduced with 2bF8 lentivirus and transplanted into rhF8-primed F8null mice under 1100 cGy total body irradiation (2bF8-Ibnull/F8null). As an inhibitor model control (2bF8-F8null/F8null), HSCs from F8null mice were transduced with 2bF8 and transplanted into rhF8-primed recipients under the same preconditioning. After bone marrow transplantation and reconstitution, animals were assessed for transgene expression and phenotypic correction. PCR analysis showed that the 2bF8 transgene was detected in all of the transduced recipients, demonstrating the viability of 2bF8-transduced engraftment. The level of functional platelet-FVIII expression as determined by a Chromogenic assay was 6.37 ± 5.62 mU/108 platelets (n = 6), which appeared to be higher than the level obtained in the inhibitor control group (1.97 ± 0.96 mU/108 platelets, n = 5), but there was no significant difference between these two groups. Since the size of Ibnull platelets is larger and the platelet number is lower than those in F8null mice, we converted FVIII levels into mU/ml whole blood and the level was 10.36 ± 5.33 mU/ml in 2bF8-Ibnull/F8null mice, which is not significantly different from the 2bF8-F8null/F8null mice (9.04 ± 3.38 mU/ml). When the tail bleeding test was used to grade phenotypic correction of the F8null coagulation defect, all transduced recipients in both groups survived beyond a 6-hour tail clipping test and the remaining hemoglobin level in the 2bF8-Ibnull/ F8null group was 46.4 ± 8.5%, which is not significantly different from the 2bF8-F8null/F8null group (48.3 ± 9.7%). The remaining hemoglobin levels in both groups were significantly higher than in the untransduced F8null control, in which only 5 of 12 animals survived beyond the 6-hour tail clipping test with a remaining hemoglobin level of 34.8 ± 7%. Thus, our data suggest that a lack of platelet GPIba does not negate the clinical efficacy of platelet FVIII gene therapy in hemophilia A in the presence of inhibitors. Disclosures Montgomery: Baxter: Consultancy; Bayer: Consultancy; CSL: Consultancy; Biogen: Consultancy; Octapharma: Consultancy; Grifols: Consultancy. Shi: BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..