Induction of Tolerance to FVIII in Hemophilic Mice by Delivery of Apoptotic Syngeneic Fibroblasts Expressing a FVIII Transgene.

In response to treatment with factor concentrates approximately 30% of patients with severe hemophilia A develop high titer inhibitory antibodies to Factor VIII. Inhibitor formation represents a major obstacle in treating patients of hemophilia A. We are investigating ways to prevent and treat inhibitors, in the mouse model of hemophilia A, through tolerance induction by infusion of FVIII vector modified apoptotic syngeneic fibroblasts. We generated a fibroblast cell line from a tail snip of a 129Sv−FVIIIKO mouse. We then transduced these cells with a bi−cistronic foamy virus vector expressing both the full length FVIII coding sequence and the zeocin resistance gene. Production of FVIII by these modified cells was confirmed by ELISA on cell culture medium. A cell line transduced with a control vector expressing only the zeocin resistance gene was also generated. Both vector modified cell lines were maintained under selective pressure and induced to apoptose by UV irradiation just prior to infusion into 129Sv−FVIIIKO mice. Mice were treated with two infusions of UV−irradiated FVIII expressing or control fibroblasts at three different weekly cell doses: 1 x 10⁷, 2 x 10⁶, or 2 x 10⁵. Ten days later they were inoculated with 4 weekly intravenous doses of 0.2 μg albumin free recombinant human FVIII (ADVATE). An additional set of control mice were not given any apoptotic cells prior to inoculation with ADVATE. Blood was collected to determine anti−FVIII inhibitor titers one week after the final ADVATE dose. The Bethesda titers in mice treated with apoptotic FVIII expressing fibroblasts, at all 3 cell doses, were 3–5 fold lower than mice that received no cells prior to intravenous FVIII challenge (p < 0.02 for all 3 cohorts, Welch’s t−test). A pair wise comparison with mice that received equivalent numbers of control apoptotic cells demonstrated significantly lower Bethesda titers in the mice given two weekly doses of 2 x10⁶ FVIII expressing cells (436 ± 140BU versus 1230 ± 433BU, p = 0.04). In mice that received 1 x 10⁷ and 2 x 10⁵ apoptotic FVIII expressing cells there was a trend for the development of lower Bethesda titers when compared to mice that received an equivalent numbers of apoptotic cells modified by the control vector. Four months after treatment with ADVATE mice from the no cell group and both the 1 x 10⁷ control and FVIII vector expressing groups were re−challenged with 4 additional weekly doses of ADVATE. T cell proliferation assays showed the highest stimulation index in mice that received no cells and the lowest index in mice that were treated with apoptotic FVIII expressing fibroblasts. Bethesda titer results from these re−challenged mice are currently pending. Our data show that anti−Factor VIII inhibitory antibody titers can be reduced by the prior infusion of apoptotic syngeneic cells stably transduced with a FVIII expressing foamy virus vector. Furthermore, the higher titers seen in mice treated with cells containing a control vector demonstrate that the effect is dependent on delivery of FVIII within the apoptotic cells. Moreover, the T cell stimulation data seen in the mice re−challenged 4 months after initial inoculation with FVIII indicate that the decrease in immune priming is due to the induction of durable tolerance. Future work will focus on elucidating the mechanisms of tolerance induction in this model and on combining apoptotic cell delivery with other immune modulators to optimize results.