Hepatic Gene Transfer of Factor IX Reverses Inhibitors and Protects From Anaphylaxis in a Murine Hemophilia B Model

Abstract 669

Hemophilia B is a X-linked bleeding disorder caused by loss of coagulation factor IX (F.IX) function. Under current treatment protocols, approximately 2–4% of hemophilia B patients develop inhibitory antibodies to F.IX protein, and those with F.IX gene deletions are at risk for anaphylaxis. We work with a murine model for this pathogenic antibody response, namely C3H/HeJ hemophilia B (HB) mice with a F9 gene deletion. Anaphylactic reactions to intravenously administered F.IX protein are the result of a strong Th2-driven antibody response, comprised of IgG1 and IgE. Interestingly, we find that IgE levels correlate with the dose of recombinant F.IX protein. HB mice treated with weekly intravenous doses of 0.1 or 0.3 IU F.IX were free from allergic/anaphylactic reactions during the course of treatment (1 IP and 5 weekly IV injections of F.IX protein) and had very low to undetectable IgE against F.IX. In contrast, a dose of 1 IU induced IgE formation and caused fatal anaphylactic reactions after repeated administration. These results suggest that prophylaxis with lower F.IX doses may be recommended in individuals with F.IX gene deletion (Mice with missense or nonsense F.IX mutations did not show inhibitor formation or anaphylaxis at the high dose). In an attempt to reverse the response and desensitize the gene deletion HB animals, F.IX-treated mice with inhibitors, including those with detectable circulating IgE, received hepatic AAV8-F.IX gene transfer (1e11 vg/mouse). Within one month, these mice lost detectable IgG and IgE against F.IX and showed a level of correction of coagulation comparable to gene transfer in naive mice. Subsequent intravenous injections of 1 IU F.IX (weekly for 1 month) did not cause anaphylaxis, demonstrating successful desensitization. Control mice (no gene transfer) maintained their IgE levels during the course of the experiment. We are currently investigating if a lower AAV8 F.IX vector dose (1e10 vg) is similarly capable of reversing existing F.IX inhibitors/anaphylaxis. In order to determine the fate of F.IX antibody-secreting cells (ASC), we performed a B cell ELISPOT assay on bone marrow cells and splenocytes in F.IX immunized HB mice left untreated or treated with AAV8 F.IX vector. Control immunized mice showed predominantly ASC in the spleen with a few detected cells from bone marrow. ASC were nearly undetectable in vector-treated mice. These results suggest that AAV8 liver gene transfer not only suppresses F.IX-specific ASC, but may also prevent the re-activation of memory B cells. Experiments are ongoing to explore the role of induced regulatory T cells in modulating ASC. To characterize T cell responses against F.IX, we isolated splenocytes isolated from control immunized and vector treated mice, stimulated these cells in vitro with F.IX protein, and collected RNA for analysis with RT-PCR array. Control mice showed an upregulation of Th2 cytokines IL-4 and IL-13, which are known to induce B cell class switching to IgG1 and to IgE, corresponding to the observed antibody formation. Vector treated immunized mice did not show up regulation of any cytokines representative of Th1 or Th2 responses, indicating down-regulation of T help required for the antibody response. In summary, our data show that liver directed AAV8 F.IX gene transfer may not only induce tolerance to those at risk of developing inhibitors, but may offer an alternative treatment approach to expensive and long-term immune tolerance induction (ITI) protocols in those with existing pathogenic antibody responses. This concept may also apply to other genetic diseases, in which antibodies complicate protein replacement therapy.