Optimization of AAV vector to deliver FVa for hemophilia with inhibitors

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ABSTRACT Gene therapy with adeno-associated virus (AAV) vectors have been successfully applied in hemophilia patients. However, the patients with inhibitors (antibodies against coagulation factors) are excluded from these trials. AAV vectors have also been explored for delivery of a bypass product, FVIIa transgene, in preclinical animal models. Although long-term improvement of hemostasis was achieved, the complete phenotypic correction was not observed. During the coagulation cascade, FV (FVa) functions as a co-factor of FXa to amplify thrombin generation. We pioneered a study in which FVa driven by a liver specific promoter was constructed and packaged into AAV virions. After injection of AAV/FVa vectors into hemophilic mice, completely phenotypic correction was achieved over 28 weeks without obvious complications. In this proposal, we will explore an effective strategy using AAV vectors to deliver bypass products in the treatment of hemophilia with inhibitors. First, we will optimize FVa constructs by utilization of different hepatocyte promoters and modification of linker sequences between the FV heavy chain and light chain (Aim 1). Next, we will explore whether the combination of AAV/FVa and AAV/FVIIa has a synergistic effect on the improvement of hemostasis in hemophilic settings (Aim 2). Since the results obtained in mice experiments often do not translate to large animal models, we propose to examine the long term phenotypic correction effect in hemophilic dogs using AAV/FVa vector alone or in combination with AAV/FVIIa (Aim 3). Overall, the studies proposed in the project will establish the basis for AAV vector-mediated bypass product gene transfer in future clinical trials in hemophilia patients with inhibitors. !