Efficacy and Safety of Continuous Expression of An Improved Variant of FVIIa On Murine Hemostasis with or without Inhibitors to Human Factor IX.

Abstract 2466 Poster Board II-443 Recombinant human activated Factor VII (rhFVIIa) is extensively used in the management of hemophilic inhibitor patients. As an alternative, using an adeno-associated viral (AAV) vector as a delivery vehicle for an engineered transgene for activated canine FVII (cFVIIa), our large animal studies in hemophilia A and B dogs demonstrated effective, long-term hemostasis and elimination of the bleeding episodes. However, the relatively high vector doses required for efficacy underscores the need for improvements in the delivery vector and/or the transgene itself. To address this, we decided to investigate whether changes in the catalytic domain of FVIIa can improve its coagulant activity, thus resulting in lowering of the effective vector dose. Using the murine version of our engineered FVIIa (mFVIIa) as a backbone, we generated a variant with four amino-acid substitutions in the catalytic domain of murine FVIIa (L305V/A314E/K337A/I374Y, mFVIIa-VEAY), based on an existing enhanced activity human FVIIa variant. As we have previously described (Amer. Soc. Hematol. Meeting 2006, #3276; Amer. Soc. Gene Cell Ther. Meeting 2008, #127), purified mFVIIa-VEAY exhibited 6-7 fold higher intrinsic coagulant activity than mFVIIa. The hemostatic properties relative to mFVIIa were determined following AAV-mediated gene delivery in hemophilia A (HA) mice. Administration of AAV-mFVIIa-VEAY at 40-100 fold lower vector dose (1.2 - 3E10 vector genomes [vg]/mouse) than AAV-mFVIIa (1.2E12 vg/mouse, High dose) resulted in ∼10-fold lower expression of the mFVIIa-VEAY (based on RNA transcript levels), but was sufficient to result in long-term (>12 weeks) normalization of the hemophilic activated partial thromboplastin time (aPTT), similar to that seen with high dose AAV-mFVIIa (P>0.05). In addition, it improved hemostasis similar to that seen with AAV-mFVIIa following in vivo hemostatic challenges (tail clip assay and ferric chloride carotid artery injury). As an extension of these studies, we have now investigated the ability of low-dose mFVIIa-VEAY gene delivery to improve hemostasis in the presence of inhibitory antibodies to human Factor IX (hFIX). Using an adjuvant, we developed a protocol for generating persistent (>3 months, ongoing) inhibitory antibodies to human Factor IX (hFIX) in hemophilia B (HB) mice. Using the low dose of 3E10 vg/mouse, we administered AAV-mFVIIa-VEAY in HB mice with high titer (7-30 Bethesda units [BU]) to hFIX. In agreement with our previous results, following gene transfer, we observed normalization of the hemophilic aPTT at the expression plateau (4 weeks [ongoing]; P>0.05 vs. wildtype mice; P<0.05 vs. untreated HA mice with similar titers of inhibitory antibodies to hFIX). However, despite the clear efficacy observed in hemophilia mice following low-dose mFVIIa-VEAY gene delivery, our previous observations with mFVIIa-VEAY overexpression (following a 1.2E12 vg/mouse of administered AAV [High dose]) in HA mice indicated a 70% reduction in survival within 6 weeks post vector administration. This coincided with a time-dependent increase in plasma thrombin-antithrombin levels that peaked at 4 weeks post AAV infusion (∼70 ng/ml) and was not observed in untreated HA mice (∼ 20ng/ml, P>0.05), HA mice treated with low-dose mFVIIa-VEAY (∼40ng/ml, P>0.05) or HA mice treated with AAV-mFVIIa (∼35ng/ml, P<0.05). Further extending these studies, histological examination from organs of deceased mice revealed thrombi in the heart as well as gross loss of lung structure. Immunofluoresence microscopy demostrated fibrin deposition in the lung parenchyma, suggesting a compromise of the lung vascular bed in the deceased mice. The identical experiment using hemostatically normal mice resulted in 100% mortality within 6 weeks with similar histological findings compared to HA mice following high dose AAV-mFVIIa-VEAY administration. Overall, our results using a high activity mFVIIa variant, demonstrate similar efficacy to mFVIIa but at a substantially reduced vector dose/expression, in a gene transfer setting for hemophilia with or without inhibitors. This variant thus serves as a potential candidate that can lower the effective vector dose in FVIIa gene-based studies in large animal models of hemophilia. However, the increased mortality observed in mice expressing high levels of mFVIIa-VEAY, warrants further investigation into the long-term safety of coagulation proteases with enhanced activity. Disclosures: High: Novo Nordisk: Grant Review Panel.