By Robert Flaumenhaft, MD, PhD

2009-09-01

Dr. Flaumenhaft indicated no relevant conflicts of interest.

Margaritis P, Roy E, Aljamali MN, et al. Successful treatment of canine hemophilia by continuous expression of canine FVIIa. Blood. 2009;113:3682-89.

The formation of inhibitory antibodies to factor VIII or IX following therapy with either plasma-derived or recombinant factors remains a challenging problem in hemophilia management. Recombinant human activated factor VIIa (rhFVIIa) could potentially provide secondary prophylaxis as a bypass agent in hemophilia complicated by inhibitor formation.¹ Its short half-life (2.7 hours) and expense, however, limit this approach. Continuous expression of FVIIa using gene therapy would obviate the need for multiple, costly rhFVIIa infusions. To this end, Margaritis and colleagues, from Kathy High’s group at Children’s Hospital of Philadelphia, have developed a FVII transgene that is intracellularly processed and secreted as FVIIa.² Building on previous success in mice,^2,3 Margaritis et al. have now performed FVIIa gene transfer in dogs with hemophilia.

Outbred canine models of both hemophilia A and B have proven predictive value in the evaluation of gene therapy for hemophilia. To assess the feasibility of gene transfer of FVIIa, investigators infused a type 8 adeno-associated virus vector expressing canine FVIIa (cFVIIa) into three dogs with hemophilia A and one dog with hemophilia B. Efficacy of therapy was assessed by measuring circulating cFVIIa levels, whole-blood clotting time, and PT, as well as by thromboelastography. The dog with hemophilia B received the smallest vector dose and demonstrated little detectable change in cFVIIa levels. Dogs with hemophilia A received progressively higher doses of vector and demonstrated sustained cFVIIa levels between 1.3 and 2.6 μg/ml. A stable reduction in whole-blood clotting times and PT, as well as a near normalization of thromboelastography reaction times, was observed. More importantly, no spontaneous bleeding was observed in more than 45 months of cumulative observation for the dogs with hemophilia A (21 spontaneous bleeds were expected) or more than the 34-month observation period for the dog with hemophilia B (15 spontaneous bleeds were expected). The observation that no spontaneous bleeds occurred in the dog with hemophilia B was particularly interesting, since this dog had cFVIIa expression below detectable levels. Aware that FVIIa expression levels of 2 μg/ml or more in mice were associated with early mortality and fibrin deposition in the heart and lungs,³ investigators screened dogs for indicators of thrombosis. Thrombin-antithrombin (TAT) complex levels, Ddimer, and fibrinogen levels remained within normal limits after several months of treatment. Liver and kidney function tests were also essentially normal. These results suggest that the cFVIIa gene transfer strategy was both effective and safe.

Hemophilias A and B are suitable candidates for gene therapy because they result from single, known genetic defects and since expression of a fraction of normal levels has a marked clinical benefit. Yet, 20 percent to 30 percent of individuals with severe hemophilia A and ~5 percent of individuals with severe hemophilia B develop inhibitory antibodies to factors VIII and IX. Bypassing agents, such as prothrombinase complex and rhFVIIa, have been used to treat bleeding in hemophiliacs with inhibitors. FVIIa has also been used for secondary prophylaxis in hemophiliacs with inhibitors.¹ Continuous expression of FVIIa via gene transfer could address the problems of short half-life and expense that limit prophylactic FVIIa use. Successful FVIIa gene transfer in dogs with hemophilia is a critical step in the development of this approach for human trials. Problems complicating human hemophilia gene therapy trials with other vectors — low transduction efficiency, hepatoxicity, impaired longevity of expression, and immunogenicity — were not observed. Success in dog models has not consistently translated into success in humans, and the issue of thrombosis as a potential complication of FVIIa gene therapy remains a concern. Nonetheless, this study provides the hope of a compelling solution to a dreaded complication of hemophilia therapy.

1. Konkle BA, Ebbesen LS, Erhardtsen E, et al. Randomized, prospective clinical trial of recombinant factor VIIa for secondary prophylaxis in hemophilia patients with inhibitors. J Thromb Haemost. 2007;5:1904-13.
2. Margaritis P, Arruda VR, Aljamali M, et al. Novel therapeutic approach for hemophilia using gene delivery of an engineered secreted activated Factor VII. J Clin Invest. 2004;113:1025-31.
3. Aljamali MN, Margaritis P, Schlachterman A, et al. Long-term expression of murine activated factor VII is safe, but elevated levels cause premature mortality. J Clin Invest. 2008;118:1825-34.

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