The development of inhibitory antibodies directed against human factor Vlll (h-fVIII) remains the most signiflcant clinical complication associated with the treatment of hemophilia A and is a critical barrier to gene therapy approaches. Not dissimilar to what occurs in the majority of monogenic diseases treated using protein replacement therapy, 20 - 30% of hemophilia A patients develop an immune response to the infused fVIII product that renders treatment ineffective. Therefore, the discovery and development of new methods to induce immune tolerance or hypo-responsiveness are needed. Hematopoietic stem cell transplantation (HSCT) protocols are showing promise in clinical trials for the treatment of a variety of human autoimmune disorders. Furthermore, HSCT combined with gene therapy has been shown, at least in animal models, to facilitate the introduction of and tolerance to neo or allo-antigens, such as those proteins deficient in monogenic diseases like hemophilia A. Our laboratories recently reported that transplantation of geneticallymodified HSCs containing a high-expression porcine (p)-fVIII transgene into hemophilia A mice results in the production of high levels of circulating fVlll activity. None of the recipient mice developed anti-fVlll antibodies following HSCT gene therapy, even in the context of non-myeloablative and non-radiation-based conditioning. Subsequently, we studied HSCT gene therapy in hemophilia A mice harboring anfi-hfVlll inhibitory antibody titers. Even though the majority of mice contained circulating antibodies that cross-reacted with and inhibited p-fVIII activity in vitro, transplantation of genetically-modified HSCs expressing p-fVlll into myeloablated hemophilia A mice restored circulating fVIII activity. Curative p-fVlll activity levels persisted and anti-h-fVIII antibody titers steadily declined throughout the course of the study in all mice. In contrast, the use of non-myeloablative total body irradiation (TBI) was only partially successful in promoting the engraftment of genetically-modified cells and resulted in a delayed reduction of anti-fVIII antibody titers. Therefore, pre-existing immunity presents an additional barrier to HSCT gene therapy. These data suggest that, while HSCT-based gene therapy incorporating a p-fVIII can be utilized successfully for the treatment of patients with refractory anti-hfVIII inhibitors, 1) more aggressive transplant conditioning is necessary to achieve disease correction, 2) the use of an orthologous (or other non-idenfical antigen) transgene product is critical, and 3) enduring tolerance/hyporesponsiveness can be achieved. The focus of this proposal is to develop an HSCT gene therapy strategy for the treatment of hemophilia A when complicated by inhibitors.

Public Health Relevance

Gene therapy offers the potential for long-term impact on the health of patients with hemophilia A. However, the immune response to fVIII remains a significant obstacle, and existing immunity represents a formidable challenge to the development of gene therapy treatments for hemophilia. The goal of this proposal is to develop a gene therapy treatment that can be applied to patients with fVlll inhibitors.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Specialized Center--Cooperative Agreements (U54)
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Special Emphasis Panel (ZHL1-CSR-C)
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Emory University
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