A Prophylactic Protocol for the Prevention of Inhibitor Formation in Gene Therapy for Hemophilia B by Shifting the Balance from An Effector to a Regulatory T Cell Response

Gene and protein treatments for severe hemophilia have been limited by immune responses to the therapeutic protein. Prophylactic protocols for the prevention of immune responses may be key to successful therapy. Our previous work has demonstrated the importance of regulatory T cells (Treg) in immune tolerance to therapeutic gene products. We attempted to induce antigen-specific tolerance to F.IX in AAV1-mediated muscle gene transfer with a novel protocol utilizing a combination of the immune suppressive drug Rapamycin (rapa), IL-10 cytokine, and an antigen specific CD4+ T cell epitope. Treg deficient (DO.11.10-tg Rag2−/−) BALB/c mice transgenic for ovalbumin -specific T cell receptor were used to determine the mechanism of tolerance. Groups of mice (n=4) were injected (IP) with rapa/IL-10/ova or Rapa/IL-10/irrelavent peptide (control) 3 times per week for 1, 3, or 4 weeks. Rapa/IL-10/ova treatment resulted in significant deletion of CD4+ cells from >30% of total splenic lymphocytes to 6% by 4 weeks. Apoptosis increased from 6% to 18% of CD4+ T cells over the duration of treatment. Apoptotic cells expressed FasL, showed an increase in activation marker CD69 and a decrease in CD62L, indicating Activation Induced Cell Death. Concomitant with the deletion of effector T cells, an increase in CD4+CD25+FoxP3+ ova-specific T cells was observed. Comparative studies after 3 weeks of treatment showed that the percentage of CD4+CD25+FoxP3+ ova-specific T cells in the peripheral blood, spleen, lymph nodes, and thymus averaged 28%, 19%, 22% and 2.4% in the rapa/IL-10/ova treated animals as opposed to 0.9%, 0.4%, 0% and 0.02% in the Rapa/IL-10/irrelavent peptide controls, respectively. In vitro suppression assay depicted absence of proliferation of effector T cells in presence of CD4+CD25+FoxP3+ isolated after 3 weeks of treatment. Flow cytometric analyses showed that the rate of apoptosis was higher in Teff compared to Treg, indicating a greater resistance of Treg to inhibition by rapamycin. Next, we tested this protocol for prevention of inhibitor formation in hemophilia B. Factor IX knockout mice (C3H/HeJ F9−/−) were injected IM with a dose of 1×10^11 vg AAV1-CMV-hFIX during the third week of treatment with rap/IL-10/hF.IX-specific peptide or Rap/IL-10/irrelavent peptide (control) in a 4-week treatment protocol. Substantial reduction in the inhibitor titers was observed after 2 months (<2 BU, representing the background of our assay) in the specific peptide protocol as compared to the irrelevant peptide controls (4–5 BU) and gene transfer only controls (4–19 BU). Clotting times (aPTT) were reduced to 45–53 seconds in the rap/IL-10/specific-peptide treated animals compared to 61– 86 seconds in the rap/IL-10/irrelavent peptide controls and 64–89 seconds in the non-immune suppressed controls. Animals treated with the hF.IX-specific tolerance protocol remained inhibitor-free for >5 months. In these immune competent animals, rapamycin administration caused a transient decline in T and B lymphocyte numbers and neutrophilia, which resolved between 5 and 7 months. In summary, our results demonstrate that the proposed prophylactic protocol can be successful in preventing immune responses to F.IX and may also be applicable to treatment of other inherited protein deficiencies.