Adeno-Associated Virus Mediated Delivery of a TFPI Antibody Restores Hemostasis in Mouse Model of Hemophilia

Hemophilia is a family of rare bleeding disorders characterized by inadequate levels of intrinsic coagulation factors, Factor VIII (FVIII) in hemophilia A and Factor IX (FIX) in hemophilia B. This leads to insufficient thrombin generation for the conversion of fibrinogen to fibrin for development of a stable clot. Replacement factor therapies are provided as a prophylactic treatment to prevent bleeds or for on-demand treatment of an active bleed. Some patients develop inhibitory antibodies making them refractory to replacement therapies. Although hemophilic patients have defects in the intrinsic pathway, the extrinsic pathway remains intact. Augmenting the extrinsic tissue factor pathway is an attractive alternate approach to maintain hemostasis in hemophilic patients. Tissue factor pathway inhibitor (TFPI) is a Kunitz domain type inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation by rapidly inactivating protease functions of Factor Xa and Factor VIIa/Tissue Factor complex and as such, TFPI inhibition has been explored as an innovative option to restore thrombin generation. Previously, we demonstrated that antibody mediated TFPI inhibition restores hemostasis in mouse models of hemophilia (Jasuja et al, 2016). Here we investigated the impact of an adeno-associated virus (AAV) vector expressed anti-TFPI antibody in correcting bleeding disorders in hemophilic mice as a novel approach to restore hemostasis. An AAV vector expressing heavy and light chains of a neutralizing anti-TFPI monoclonal antibody was designed and constructed. In vitro evaluations confirmed that vector expressed antibody was functional using a dilute Prothrombin Time (dPT) assay. Next, male hemophilic A mice were systemically administered (intravenous) 1x1011 AAV vector genomes (vg) encoding anti-TFPI or control and had hemostasis evaluated 16 days post-treatment. Following recalcification of whole blood, thromboelastography (TEG) was used to measure clot formation times (R value), speed of clot formation (K value), as well as maximum amplitude. In hemophilia A mice treated with AAV-anti-TFPI, clot formation time (R value) was normalized to wild type levels, with a 10-fold reduction compared to hemophilia A mice administered a control Green fluorescent protein (GFP) encoding AAV vector. Mice administered the control AAV-GFP vector failed to correct additional parameters, including speed of clot formation (K value), angle and maximum amplitu