36. Permanent Phenotypic Correction of Haemophilia B in Immunocompetent Mice by Prenatal Gene Therapy

Abstract

Protein replacement treatments often trigger unwanted immune responses in the host. Induction of immune tolerance would be a desirable goal to mitigate such events, and would eliminate the need for life-long immunosuppressive therapy in affected individuals. Hemophilia B is an X-linked bleeding disorder caused by blood coagulation factor IX (FIX) deficiency. Some hemophiliacs develop neutralizing antibodies to FIX (inhibitors) that render the infused FIX ineffective. In this study we evaluated whether oral delivery of hFIX-DNA nanoparticles can downregulate the immune response against hFIX in immunized mice. A group of C57BL/6 mice was immunized with recombinant hFIX in Freund's adjuvant followed by three boost at day 7, 14, and 21. As expected, all mice developed high titer of anti-hFIX antibodies as measured by day 28. At this time, half the mice were treated with a single oral dose of plasmid DNA nanoparticles containing hFIX cDNA under the control of the b -actin promoter. The other half was left untreated, and served as control. By ELISA, we detected circulating levels of hFIX in the treated mice by day 3 after the oral treatment. In contrast, control mice had undetectable levels of circulating hFIX. Importantly, the titer of inhibitors against hFIX was measured by a Bethesda assay. Control mice had inhibitors, on average, of 40 BU/ml on day 21 after the oral administration of DNA. In contrast, the level of inhibitors became undetectable in the treated group by day 21. The cellular immune response was also assessed. Stimulation of splenocytes from orally treated mice with hFIX resulted in increased secretion of transforming growth factor-b, but no detectable interferon-g production. In contrast, stimulation of splenocytes from immunized control mice with hFIX showed an increase in the production of interferon-g, and no secretion of transforming growth factor-b. Additionally, a hFIX-specific cytotoxic T-lymphocyte (CTL) response was detected in the control, but not in the orally treated mice. Thus, our results suggest that the oral delivery of DNA nanoparticles downregulated the pre-existing immune responses against human FIX. This downregulation may result from the induction of a shift from a proinflammatory T helper cell type 1 to an anti-inflammatory T helper cell type 2 immune response. In conclusion, we have shown that pre-existing immune responses against hFIX can be overcome. This strategy may facilitate the delivery of proteins in a pre-sensitized host. Similar approaches may have potential applications for the induction of immune tolerance in autoimmune diseases. This strategy may also have implications for the prevention of unwanted immune responses elicited by the expression of novel transgenes.