Correction of Murine Hemophilia A by Hematopoietic Stem Cell Gene Therapy.

A serious complication of current protein replacement therapy for hemophilia A patients with coagulation factor VIII (FVIII) deficiency is the frequent development of anti-FVIII inhibitor antibodies that preclude therapeutic benefit from further treatment. Induction of tolerance by persistent high-level FVIII synthesis following transplantation with hematopoietic stem cells expressing a retrovirally-delivered FVIII transgene offers the possibility to permanently correct the disease. Here, we transplanted bone marrow cells transduced with an optimized MSCV-based oncoretroviral vector encoding a secretion-enhanced B domain-deleted human FVIII transgene linked to a downstream EGFP reporter gene into immunocompetent hemophilia A mice (FVIII exon 16 knockout mice on a C57BL/6 background) that had been conditioned with a potentially lethal dose of irradiation (800 cGy), a sublethal dose of irradiation (550 cGy) or a nonmyelablative preparative regimen involving busulfan (two intraperitoneal doses of 10 mg/kg). Both groups of irradiated mice were transplanted with 2 × 10⁶ sorted EGFP⁺ bone marrow cells. At 26 weeks, 48 ± 24% (n = 10) and 18 ± 11% (n = 12) EGFP⁺ nucleated peripheral blood cells were detected in mice conditioned with 800 and 550 cGy irradiation, respectively. The busulfan-conditioned mice (n = 4) were transplanted with 15–20 × 10⁶ transduced unsorted bone marrow cells. One mouse died at 4 weeks posttransplant due to an unknown cause. The reconstitution kinetics of the remaining three mice was very similar to the group of mice conditioned with 550 cGy irradiation (18 ± 7% of their nucleated peripheral blood cells were EGFP⁺ at 26 weeks posttransplant). Broad transcriptional activity of the vector was observed in cells belonging to both the myeloid and lymphoid lineages in peripheral blood, and in donor-derived cells residing within the bone marrow, spleen and thymus. Importantly, therapeutic levels of FVIII (42%, 18% and 11% of normal, respectively, by COATEST assay) were detected in the plasma of all recipients 22–26 weeks posttransplant. When the mice were subsequently challenged with high doses of recombinant human FVIII (up to eight intravenous injections of 5–10 IU of recombinant full-length human FVIII at weekly intervals) to investigate the durability of tolerance induction, only minimal levels of inhibitor antibodies were detected in a subset of the corrected animals (0.8 ± 0.6, 0.7 ± 0.5 and 3 ± 4 Bethesda units per ml) in contrast to the robust anti-FVIII inhibitor response seen following immunization of naive hemophilia A mice (98 ± 48 Bethesda units per ml; n = 11). Suppression of the immune response to human FVIII was specific, as gene-treated mice mounted a normal humoral immune reaction to an unrelated antigen, tetanus toxoid. While we make no claims as to the nature of the hyporesponsive states elicited in each case, the results obtained in the small cohort of busulfan-conditioned animals are particularly exciting since the experimental protocol more closely approximates a clinically-acceptable situation, both in terms of a mild conditioning regimen as well as the lack of a preselection step for transduced bone marrow cells. These findings represent an encouraging advance toward potential clinical application and long-term amelioration or cure of this progressively debilitating, life-threatening bleeding disorder.