Prevention of Gene Transfer-Induced Inhibitor Formation by Nasal Administration of Human F.IX T Cell Epitope in a Murine Model of Hemophilia B.
Authors: Cao O. , Armstrong E. , Conti-Fine B. M. , High K. A. , Herzog R. W. 
Published: April 2020

Abstract
Hemophilia B is the X-linked bleeding disorder caused by absence of functional coagulation factor IX (F. IX). Gene therapy is a promising novel treatment strategy that can provide a continuous supply of F.IX through stable gene transfer. However, immune responses to the F.IX transgene product pose a concern. In particular, formation of inhibitory antibodies against F.IX represents a serious complication of treatment. In published work, we demonstrated induction of immune tolerance to human F.IX (hFIX) by hepatic, adeno-associated (AAV) vector-mediated gene transfer. However, not all strains of mice can be tolerized by this protocol (JCI 111:1347). Hemophilia B mice (with a large F.IX gene deletion) on a C3H genetic background form inhibitors to hF.IX after hepatic gene transfer. Antibody formation is dependent on activation of hF.IX-specific CD4+ T cells. Using a peptide library spanning the entire hF.IX protein, we identified peptide TEQKRNVIRIIPHHNYNAAI to contain an immunodominant CD4+ T cell epitope for hemophilia B C3H/HeJ mice. T cells can be specifically tolerized by giving antigen through specific routes, such as oral or nasal administration. Such procedures have been shown to prevent or treat T cell-mediated autoimmune diseases, and can induce T cell anergy, deletion, and activation of regulatory Th3 cells, which suppress T cell responses through secretion of the TGF- β cytokine. Short T epitope sequence may be safer for T cell tolerance procedures, since peptide-specific antibodies rarely cross-react with the cognate native Ag. Nasal administration does not need to overcome the proteolytic barriers present in the digestive system, and peptides cross epithelia more easily than large proteins. The hF.IX peptide described above (25 μl of PBS containing 100 μg peptide, n=10) or saline control (n=16) was administered into both nostrils of hemophilia B C3H/HeJ mice twice a week, starting 2 weeks before hepatic gene delivery of AAV-hFIX vector (1x1011 vg/mouse) in C3H hemophilia B mice. Mouse plasma samples were collected 2 and 4 weeks after vector administration. All control mice formed inhibitors (40% had Bethesda titers of 1–2 BU, 60% showed 2–6 BU). Antibodies were mostly IgG1, while IgG2a and IgG2b responses were also measured. In contrast, peptide-treated mice did not develop anti-hF.IX (50%) or had only low-titer antibodies (1–2 BU, 50%). Interestingly, 70–75% of both experimental groups had circulating hF.IX antigen following gene transfer. However, aPTT coagulation times of control mice were either uncorrected, 87%(&gt;50 sec) or mildly corrected, 13%(48–50 sec), while 60% of peptide-treated mice clearly had partial correction of coagulation (40–46 sec compared to 20–30 sec in normal mice). These data indicate that anti-hF.IX, while often not eliminating systemic expression, inhibited hF.IX activity in mice that did not receive peptide therapy. Successfully treated animals expressed 27–333 ng hF.IX/ml plasma. In summary, nasal administration of hF.IX-derived peptide antigen substantially reduced the risk of an inhibitor response in the context of gene transfer-based treatment of hemophilia B. Therefore, mucosal antigen presentation could be a promising new tool for improved safety of gene therapy or other types of treatments for hemophilia in the context of gene deletions or other settings of increased risk for immune responses to coagulation factors.