84. Assessing Hemostatic Efficacy of Continuous Expression of Factor VIIa after AAV-Mediated, Liver-Directed Gene Transfer in Hemophilia A and B Mice

We have shown that portal vein infusion of an AAV vector expressing an engineered secreted activated murine factor VIIa (mFVIIa) results in long-term phenotypic correction of murine hemophilia B (HB) mice, as judged by shortening of the prothrombin time (PT), activated partial thromboplastin time (aPTT), and the tail-clip bleeding time (J Clin Invest. 2004; 113: 1025). We sought to compare endpoints for hemostasis in hemophilia A and B mice infused with AAV-mFVIIa, analyzing VIIa levels, the tail-clip assay, the FeCl3-carotid artery model, and real-time imaging of clot formation utilizing the cremasteric muscle arteriole. Twenty-two (N=22) Hemophilia A (HA) and forty-eight (N=48) HB mice were injected with AAV-mFVIIa via the portal vein at doses ranging from 1.0|[times]|10^8 to 4.8|[times]|10^12 vg/mouse. Based on a PT assay we observed a dose response with circulating VIIa levels ranging from 11 ng/ml for the untreated and the lowest dose to 1000 ng/ml for the higher doses. In HB mice (N=15) injected at 1.2|[times]|10^12 vg/mouse, the mean PT shortened from 23 to 17 sec, and the aPTT from 86 to 43 seconds, 8 weeks post vector injection. Measurement of the blood loss post tail-clip was documented as a statistically significant reduction in blood loss in HB AAV-mFVIIa-treated mice (N=5) relative to the untreated HB mice (N=10) * [P-value 25ug/kg), animals developed complete, albeit transient, occlusion, but neither FVIIa protein (>90ug/kg) nor AAV-mFVIIa mice exhibited formation of an occlusive thrombus in HB mice. Similar results have been obtained in hemophilia A mice. Together these data suggest that 1) AAV-mFVIIa is functional in supporting hemostasis as judged by in vitro coagulation assays and by some but not all in vivo hemostasis challenges; and 2) results in these hemostasis assays may vary as a function of the diameter of the vessel that is injured. Which of these endpoints most faithfully reflects hemostasis in humans is not yet clear, but the major morbidity of hemophilia is from joint bleeds which likely begin as bleeding in the microcirculation. Our studies provide clear evidence of improvement of in vivo hemostasis and support continuous expression of FVIIa as an alternative strategy for the treatment of hemophilia.