The development of inhibitory antibodies against FVIII is not only a severe and important complication of protein replacement therapy, but also a major concern in gene therapy of hemophilia A. Generation of such inhibitors might potentially preclude gene therapy for hemophilia A. In this project, we propose to investigate a novel gene therapy approach that will provide therapeutic FVIII protein and induce immune tolerance for hemophilia A and hemophilia A with inhibitors based on the hypothesis that targeting the production of FVIII to platelets that activate at the site where FVIII is needed could overcome the presence of inhibitory antibodies; in addition, platelets contain immunomodulatory molecule, transforming growth factor beta (TGF), would induce antigen-specific regulatory T cells, promoting immunologic tolerance. We have developed a clinically translatable gene therapy protocol for hemophilia A using lentiviral (LV) gene delivery of the FVIII expression cassette under control of the platelet-specific aIIb promoter (2bFVIII) to hematopoietic stem cells resulting in FVIII expression in platelets. Our studies have demonstrated that sustained therapeutic levels of platelet-FVIII expression are obtained while inhibitor titers decline with tie in 2bFVIIILV-transduced FVIII-primed hemophilia A mice (inhibitor model). Our most recent studies show that 2bFVIII gene therapy can not only restore hemostasis but also induce immune tolerance in the non-inhibitor model mice. In the current application, we propose to explore how immune tolerance is induced in hemophilia A mice after platelet gene therapy and the efficacy of this novel gene therapy approach in inducing immunologic tolerance in the clinical setting of pre-existing FVIII inhibitory antibodies. We will also investigate how the clinically relevant non-myeloablative conditioning regimens affect the immunologic consequences in platelet gene therapy of hemophilia A and hemophilia A with pre-existing anti-FVIII immunity. These studies should help us to further understand the biological characteristics of 2bFVIII gene therapy, with a potential developing a gene therapy approach that can not only provide therapeutic protein, but also induce the antigen-specific immune tolerance for the clinical treatment of HA patients and patients with inhibitors, as well as non-hereditary hemophilic patients with acquired inhibitory antibodies that can also have life-threatening clinica bleeding.

Public Health Relevance

Hemophilia A is bleeding disorders resulting from factor VII (FVIII) deficiency. The development of inhibitory antibodies against FVIII is not only a severe and important complication of protein replacement therapy; bit also a major concern in gene therapy of hemophilia A. In this project, we will develop a novel approach by targeting the synthesis of FVII to blood platelets to provide therapeutic protein and induce immune tolerance in hemophilia A even in the presence of inhibitory antibodies.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hemostasis and Thrombosis Study Section (HT)
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Sarkar, Rita
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Medical College of Wisconsin
Schools of Medicine
United States
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Shi, Qizhen (2018) Platelet-Targeted Gene Therapy for Hemophilia. Mol Ther Methods Clin Dev 9:100-108
Luo, Xiaofeng; Chen, Juan; Schroeder, Jocelyn A et al. (2018) Platelet Gene Therapy Promotes Targeted Peripheral Tolerance by Clonal Deletion and Induction of Antigen-Specific Regulatory T Cells. Front Immunol 9:1950
Baumgartner, C K; Mattson, J G; Weiler, H et al. (2017) Targeting factor VIII expression to platelets for hemophilia A gene therapy does not induce an apparent thrombotic risk in mice. J Thromb Haemost 15:98-109
Chen, Juan; Schroeder, Jocelyn A; Luo, Xiaofeng et al. (2017) The impact of von Willebrand factor on factor VIII memory immune responses. Blood Adv 1:1565-1574
Chen, Y; Luo, X; Schroeder, J A et al. (2017) Immune tolerance induced by platelet-targeted factor VIII gene therapy in hemophilia A mice is CD4 T cell mediated. J Thromb Haemost 15:1994-2004
Chen, Yingyu; Schroeder, Jocelyn A; Chen, Juan et al. (2016) The immunogenicity of platelet-derived FVIII in hemophilia A mice with or without preexisting anti-FVIII immunity. Blood 127:1346-54
Haribhai, Dipica; Luo, Xiaofeng; Chen, Juan et al. (2016) TGF-?1 along with other platelet contents augments Treg cells to suppress anti-FVIII immune responses in hemophilia A mice. Blood Adv 1:139-151
Baumgartner, C K; Zhang, G; Kuether, E L et al. (2015) Comparison of platelet-derived and plasma factor VIII efficacy using a novel native whole blood thrombin generation assay. J Thromb Haemost 13:2210-9
Shi, Q; Schroeder, J A; Kuether, E L et al. (2015) The important role of von Willebrand factor in platelet-derived FVIII gene therapy for murine hemophilia A in the presence of inhibitory antibodies. J Thromb Haemost 13:1301-9
Chen, Yingyu; Schroeder, Jocelyn A; Kuether, Erin L et al. (2014) Platelet gene therapy by lentiviral gene delivery to hematopoietic stem cells restores hemostasis and induces humoral immune tolerance in FIX(null) mice. Mol Ther 22:169-77

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