Clinical gene therapy trials for hemophilia A have failed due to insufficient target cell transduction or expression of factor VIII (fVIII). Animal studies have documented the extreme difficulty of expressing therapeutic levels of human fVIII from recombinant viral vectors without eliciting an anti-fVIII humoral response. We previously demonstrated curative fVIII activity levels in hemophilia A mice following a nonmyeloablative, genetically-modified bone marrow transplantation (Ide et al.,

). In these studies recipient hemophilia A mice received 3×105 bone marrow-derived sca-1+ cells transduced with a retrovirus encoding B-domain-deleted high-expression porcine fVIII (BDDpfVIII) after pre-transplantation conditioning with reduced-intensity TBI or busulfan followed by minimal immunosuppression with either costimulation blockade or antithymocyte serum (ATS). Using our high-expression fVIII transgene and busulfan or 3Gy TBI coupled with ATS resulted in sustained fVIII activity of >100% normal human levels. The main hematopoietic toxicity observed was transient T-cell suppression. High-level fVIII activity (>2 units/ml) was achieved without induction of anti-BDDpfVIII humoral immune responses. Furthermore because the development of inhibitory antibodies directed against fVIII remains a significant clinical complication associated with the treatment of hemophilia A, we tested the effectiveness of genetically-modified cells encoding BDDpfVIII in the setting of pre-existing fVIII immunity. Transplantation of genetically-modified HSCs into 5.5 Gy TBI/ATS or busulfan/ATS-conditioned hemophilia A mice that had significant anti-human fVIII inhibitory titers induced sustained, high-level fVIII activity. Following transplantation and stable engraftment, both naïve and pre-immunized mice were challenged with six weekly injections of 10 units human fVIII, an immunization regimen that results in antibody formation in 100% of naïve hemophilia A mice. All mice sustained their pre-challenge BDDpfVIII activity levels, and anti-fVIII antibodies were not detected. Surface markers of T-cell activation were the same between naïve hemophilia A mice and transplanted mice, for both naïve and preimmunized animals. In a mixed lymphocyte reaction, T-cells were found to be equally capable of reacting to allogenic antigens (i.e. irradiated Balb/C splenocytes) when isolated from mice with stable fVIII expression compared to nontransduced controls, demonstrating that the lack of anti-fVIII inhibitors is not due to a lack of immunocompetent T-cells. Further studies are underway assessing T-cell nonresponsiveness to BDDpfVIII in these transplanted mice. By demonstrating that transplanted mice are tolerant to BDDpfVIII, even in the context of pre-existing inhibitory antibodies and nonmyeloablative conditioning, this data further supports our previous studies showing that retroviral gene transfer using high-expression porcine fVIII elements is a compelling treatment for hemophilia A.

Author notes

Disclosure: No relevant conflicts of interest to declare.