Hemophilia A: Intrathymic Delivery for Tolerance and Longterm Expression of FVIII

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DESCRIPTION (provided by applicant): The current standard of care for hemophilia A is treatment with intravenous human factor VIII (hFVIII) protein infusions, either prophylactically or during bleeding episodes. A major disadvantage for both prophylactic and on- demand FVIII protein infusions is the formation of anti-human FVIII antibodies (inhibitors) in about 25% of treated individuals. Inhibitor formation is one of the most difficult complications of this bleeding disorder since inhibitors neutralize hFVIII and thus thwart hFVIII replacement therapy. The long-term goal of this project is to develop a therapy that can avoid the serious problem of inhibitor formation and provide constant amounts of FVIII protein. This proposal will exploit the unique properties of the thymus to induce tolerance to foreign proteins (hFVIII) by intrathymic injection of a lentiviral vector encoding human FVIII. Intrathymic injection of lentiviral vectors efficiently transduces the long-lived thymic stromal cells, which are the target cell types for sustained hFVIII protein production. Due to the immunological properties of the thymus, this approach should facilitate long-term expression of hFVIII without elimination by the immune system. The general feasibility of this approach is supported by several studies. For example, MHC-incompatible bone marrow stromal cells engineered to express a transgene and implanted into the thymus will not undergo immune rejection and can secrete the transgene product long-term. Work spanning forty years has shown that intrathymic exposure to foreign antigen can induce tolerance to a subsequent graft in the periphery. With respect to the delivery system, a non-primate lentiviral vector derived from Feline Immunodeficiency Virus (FIV) provides excellent transduction efficiency of a variety of cells and organs including stromal cells. Together, these results show that exploiting the immune privileged status of the thymus as a site for protein production has the potential to revolutionize treatment for hemophilia A. This proposal will explore intrathymic FIV-hFVIII vector injection to prevent inhibitor formation. FIV vectors will be used since they efficiently transduce non-proliferating cells including those that make up the thymic stroma, and stably integrate into the host genome potentially allowing lifelong expression of the transgene. Additional benefits are the viral particle's low toxicity, low immunogenicity and the lack of any viral genes after integration into the host genome. The hypothesis that the combination of an efficient and relatively non-immunogenic vector system with the intrathymic route of delivery will prevent inhibitor formation and allow secretion of hFVIII protein from transduced thymic stroma into peripheral blood will be tested. To demonstrate the feasibility of this approach for long-term hFVIII therapy, hFVIII protein and possible anti-hFVIII antibodies will be measured over six months in a mouse model, possible effects of FIV- hFVIII intrathymic injection on thymopoiesis will be assessed, and organs other than thymus will be analyzed for vector transduction and hFVIII mRNA expression. Data from these experiments will indicate whether intrathymic injection of FIV-hFVIII vectors will provide a valuable treatment alternative specifically aimed at reduced inhibitor formation. Exceeding the scope of this grant application, the proposed proof-of-concept studies will form the basis for future studies including a comparison of the intrathymic with the iv route of administration and the potential of intrathymic injection to reduce pre-existing inhibitors. The use of gene therapy to correct hemophilia A introduces the expression of the factor VIII protein. Factor VIII protein is low or missing in hemophilia patients and therefore a foreign protein for most patients. Exposure to any foreign protein runs the risk of the recipient patient mounting an immune response to the new protein. Such immune responses neutralize the therapeutic protein rendering it ineffective. Approximately 25% of all hemophilia A patients suffer from inhibitors, which leads to severe complications including uncontrolled bleeding, chronic joint disease and premature death. The proposed research will evaluate and optimize a means to "teach" the immune system that the new therapeutic protein is not foreign so that an immune response will not be generated and the function of the factor VIII protein to stop uncontrolled bleeding will be preserved.