Bioencapsulated Factor IX for Oral Tolerance in Hemophilia B

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DESCRIPTION (provided by applicant): Hemophilia is the X-linked bleeding disorder caused by mutations in the gene encoding blood coagulation factor VIII (hemophilia A) or factor IX (hemophilia B). Current treatment is based on intravenous infusion of plasma-derived or recombinant clotting factor concentrates. More recently, substantial progress toward gene therapy has been made. Formation of inhibitory antibodies ("inhibitors") to the therapeutic clotting factor represents a serious complication of treatment. Elimination of inhibitors by current immune tolerance induction protocols is expensive, not always effective, and less successful for antibodies against factor IX (F.IX). No prophylactic protocols to prevent inhibitor formation are available. Since inhibitor formation is dependent on T help, CD4+ T cells represent an alternative target for tolerance induction. Specific routes of antigen administration such as mucosal (oral or nasal) are known to induce regulatory T cells and T cell tolerance. This proposal is based on the hypothesis that mucosal antigen administration or a combination of mucosal and systemic antigen delivery can prevent inhibitor formation. In order to develop an effective, cost- efficient, and clinically applicable oral tolerance protocol, we will generate transgenic plants expressing high levels of human F.IX (hF.IX) in the chloroplast. Bio-encapsulated hF.IX will be fed to mice to test for prevention of antibody formation to hF.IX upon repeated intravenous injected of hF.IX protein. Because this approach is based on antigen presentation to CD4+ T cells specific for hF.IX-derived peptide epitopes, biological activity of orally delivered hF.IX is not required. An optimized protocol will be evaluated for tolerance induction in hemophilia B mice with a large F.IX gene deletion. A strain of mice known to frequently form inhibitors to hF.IX is chosen for these studies. We hypothesize that optimal activation of regulatory T cells following presentation of plant-derived hF.IX antigen will result in the ability to provide protein- or gene-based therapy for hemophilia B without inhibitor formation. The protocol will also be applied to animals with pre-existing inhibitors due to prior protein therapy in order to investigate the ability to eliminate inhibitors. In future studies, the oral tolerance protocol will be further optimized and adapted to prevention of inhibitors to factor VIII in treatment of hemophilia A.