of Project 2 Hemophilia, an inherited bleeding disorder, is currently treated by protein replacement therapy that provides only transient correction and is a significant financial burden due to the need for frequent infusions. For those with access to concentrate, the most serious complication in the management of the disorder is the development of neutralizing antibodies to the infused factor that occur in up to 30% of those with hemophilia A. For such patients, control of bleeds by infusion of high-dose recombinant activated Factor Vll (rFVlla) results in effective liemostasis. However, this treatment carries a substantial cost due to the short half-life of the protein and the need for repeated dosing. To address these shortcomings we previously developed a novel gene transfer approach whereby we can effect hemostasis using a FVII transgene, engineered to be secreted in the active, two chain form (FVIIa). Using the liver as the target tissue, we demonstrated that continuous expression of FVlla following adeno-associated virus (AAV) gene transfer can correct the phenotypic defect in hemophilic mice and dogs. This approach has immunological advantages over Factor IX (FIX) or Factor Vlll (FVlll) gene strategies since the recipient is tolerant to the transgene product. Moreover it can effect hemostasis even in the face of neutralizing antibodies to FVIII or FIX. Additionally, since rFVlla is used for hemostasis management for other coagulation defects (such a platelet disorders), in contrast to FVIII/FIX gene-based approaches, FVIIa gene transfer may unify the treatment of several coagulation defects to a single product. However, a substantial proportion of hemophilia patients have advanced liver disease due to viral hepatitis, thus rendering the liver an unsuitable tissue for viral vector-mediated gene transfer. We developed a method for transducing extensive areas of muscle by an AAV vector expressing biologically active canine FIX and demonstrated multi- year hemostatic efficacy in hemophilia B dogs. Therefore, as a theme for this project, we propose to use skeletal muscle as the target tissue for expression and secretion of FVlla, using the canine model(s) of hemophilia that closely mimic the human condition. Initially, we wish to perform a systematic study of promoters and AAV serotypes for muscle expression of FVlla, and study the hemostatic efficacy and safety of canine FVlla (cFVlla) expression under optimal conditions, including dogs with inhibitors (Aim 1). In subsequent experiments, we will determine the therapeutic index of a variant of cFVlla with increased activity, as a means to reduce the effective vector dose/viral load per cell and thus address any potential limitations of muscle to perform post-translational modifications necessary for cFVlla biological activity, due to increased antigen expression (Aim 2). Lastly, we will develop a regulated system for cFVlla expression from muscle via a small, orally bioavailable drug (doxycycline) that will enhance the safety of FVIIa gene transfer (Aim 3). Overall, the experiments proposed in the project will establish in a comprehensive manner the pre-clinical basis for muscle-derived FVlla gene transfer.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
Project #
Application #
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Children's Hospital of Philadelphia
United States
Zip Code
French, Robert A; Samelson-Jones, Benjamin J; Niemeyer, Glenn P et al. (2018) Complete correction of hemophilia B phenotype by FIX-Padua skeletal muscle gene therapy in an inhibitor-prone dog model. Blood Adv 2:505-508
George, Lindsey A; Sullivan, Spencer K; Giermasz, Adam et al. (2017) Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant. N Engl J Med 377:2215-2227
Gollomp, Kandace; Lambert, Michele P; Poncz, Mortimer (2017) Current status of blood 'pharming': megakaryoctye transfusions as a source of platelets. Curr Opin Hematol 24:565-571
Sim, Xiuli; Poncz, Mortimer; Gadue, Paul et al. (2016) Understanding platelet generation from megakaryocytes: implications for in vitro-derived platelets. Blood 127:1227-33
Marcos-Contreras, Oscar A; Smith, Shannon M; Bellinger, Dwight A et al. (2016) Sustained correction of FVII deficiency in dogs using AAV-mediated expression of zymogen FVII. Blood 127:565-71
Arruda, V R; Samelson-Jones, B J (2016) Gene therapy for immune tolerance induction in hemophilia with inhibitors. J Thromb Haemost 14:1121-34
High, Katherine A; Anguela, Xavier M (2016) Adeno-associated viral vectors for the treatment of hemophilia. Hum Mol Genet 25:R36-41
Siner, Joshua I; Samelson-Jones, Benjamin J; Crudele, Julie M et al. (2016) Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models. JCI Insight 1:e89371
Zhang, Nanyan; Zhi, Huiying; Curtis, Brian R et al. (2016) CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes. Blood 127:675-80
Lyde, R; Sabatino, D; Sullivan, S K et al. (2015) Platelet-delivered therapeutics. J Thromb Haemost 13 Suppl 1:S143-50

Showing the most recent 10 out of 90 publications