Abstract 3156

At present, the most serious complication in hemophilia therapy is the development of neutralizing antibodies (inhibitors) to intravenous administrated recombinant protein, which compromises therapy, creates immune-toxicity, and increases costs. Although inhibitor formation is less frequent in hemophilia B, it is more prevalent in severe hemophilia B patients, often with additional consequences - up to 50% patients with inhibitors to factor IX (F.IX) develop anaphylactic reactions. These further increase risks of morbidity and mortality. Available bypass therapy is expensive and at risk for thrombosis. Clinical immune tolerance induction (ITI) protocols are lengthy, expensive, and are often terminated in hemophilia B due to anaphylactic reactions or nephrotic syndrome. Therefore, effective protocols to induce immune tolerance to F.IX are urgently needed. B cells have been identified as antigen presenting cells with potentially immune suppressive/regulatory roles. Upon gene transfer, primary B cells were found to induce tolerance to the expressed transgene product. Hence, we use autologous gene-modified primary B cells expressing F.IX antigen fused with immunoglobulin-G heavy chain in a murine model of hemophilia B. Our murine hemophilia B model is unique in both developing high-titer inhibitors and fatal anaphylactic reactions to protein replacement therapy. Retroviral transduced B cells, expressing either full-length or shorter version of F.IX, markedly reduced inhibitor titers up to 30-fold and completely prevented fatal anaphylactic reactions. After 7 weeks of treatment with recombinant human F.IX (IV, 1 IU/mouse, once per week), mice receiving control B cells (n=6) had developed inhibitor titers of 23±8 BU, and 50% died after the last injection. Mice tolerized to F.IX by B cell transplant (n=7) had formed <1 BU, essentially undetectable by this assay, and all survived without anaphylactic reactions. We also tested the B cell-based therapy in already primed mice. Animals receiving B cells expressing the F.IX-IgG fusion successfully reversed the inhibitor and total anti-F.IX IgG titers markedly, whereas animals receiving B cells expressing IgG control had insignificant changes of inhibitor/antibody levels. Our data suggested that B cell-based gene therapy is a promising strategy in not only prevention but also treatment of inhibitors against F.IX. Besides retroviral gene transfer, we tested alternative methods such as DNA nucleofection. Interestingly, although achieving higher gene transfer efficiency, nucleofection of the plasmid encoding the retroviral expression cassette increased rather than decreased immune responses to F.IX. This was likely caused by activation of innate immune mediators and inflammatory cytokine expression as indicated by expression array analysis. Among the 29 genes tested, IL-6 and type I IFN were significantly upregulated in nucleofected B cells compared with retroviral infected B cells, which was further confirmed by ELISA. IL-6 and type I IFN are known to abrogate tolerance such as in transplant rejection and anti-tumor immunity. We suspected that the endosomal DNA sensor TLR9 may induce these cytokines in response to nucleofection. Consistent with this hypothesis, using a TLR9 inhibitory oligodeoxynucleotide (ODN 2088), we significantly reduced nucleofection-associated IL-6 and type I IFN production compared to passive ODN control. These data provide insights into the mechanisms that control the immune phenotype of gene-modified primary B cells, which become tolerogenic under conditions of limited innate responses and immunogenic upon activation of inflammatory and IFN I gene expression.


Herzog:Genzyme Corp.: Royalties, AAV-FIX technology, Royalties, AAV-FIX technology Patents & Royalties.

Author notes


Asterisk with author names denotes non-ASH members.