602. Induction of Tolerance to Human Factor VIII after Neonatal Gene Transfer of a Retroviral Vector Is Dose-Dependent in Mice and Is Not Effective in Cats
Authors: Lingfei Xu, Manxue Mei, Mark E. Haskins, Karyn Cullen, Katherine P. Ponder
Journal: Molecular Therapy (2005)

Hemophilia is a bleeding disorder due to deficiency of Factor VIII (FVIII) or Factor IX (FIX). Approximately 30% and 5% of patients with hemophilia A and B develop antibodies that inhibit the coagulation function of the factor (inhibitors). Neonatal gene transfer induces tolerance to human FIX in mice, dogs, and cats. However, it was unclear if this approach induces tolerance for the more immunogenic human FVIII (hFVIII) protein. Indeed, some mice that received a VSV-G pseudotyped retroviral vector (RV) expressing hFVIII as newborns developed inhibitors. In this study, an amphotropic gamma RV expressing human B domain-deleted FVIII (hFVIII) from the human |[alpha]|1-antitrypsin promoter was used to define the level of hFVIII necessary to achieve tolerance after neonatal gene transfer in mice. Hemophilia A mice were injected IV with 1010 transducing units (TU)/kg of RV at 2 to 3 days after birth. Plasma hFVIII antigen levels were 204|[plusmn]|33% of normal, and activity by COATEST assay was 326% of normal. None of 8 mice developed anti-hFVIII antibodies. To evaluate the level of hFVIII necessary to induce tolerance, a dose response study was done in normal C3H mice, which produced anti-hFVIII antibodies of 13|[plusmn]|4 mg/ml and inhibitor titers of 170 Bethesda units (BU)/ml after gene transfer into adults. Neonatal C3H mice were injected IV with 1010 (high), 109 (medium) or 108 (low) TU/kg of RV. The high dose group achieved 278|[plusmn]|45% of normal hFVIII, and 0 of 8 mice developed anti-hFVIII antibodies. The medium dose group achieved 19|[plusmn]|6% of normal hFVIII, and 8 of 11 mice developed anti-hFVIII antibodies. Average antibody levels for the group were 0.09|[plusmn]|0.03 mg/ml and 10|[plusmn]|4 BU/ml. These data suggest that 2|[times]|10|[ndash]|9 M of hFVIII in plasma induces tolerance, but 2|[times]|10|[ndash]|10 M is not sufficient. Nevertheless, neonatal transfer of the medium dose of RV resulted in antibody levels that were only 0.7% and Bethesda titers that were only 6% of those achieved after transfer into adults. Animals will be followed long-term to determine if antibody levels decline over time, which can occur after immune tolerance induction. Mice treated with low dose RV had undetectable levels of hFVIII (<3% of normal), and 0 of 13 developed anti-hFVIII antibodies, which was probably due to insufficient levels of hFVIII to stimulate an immune response. Mice will be challenged with hFVIII protein to determine if they are truly tolerant. The immune response to hFVIII after neonatal gene transfer was also evaluated in normal cats, which appear to have a more mature immune system. Four neonatal cats were injected IV with 9|[times]|109 TU/kg of the RV expressing hFVIII at day 5 after birth. Plasma hFVIII levels were undetectable, but 2 out of 4 cats developed anti-hFVIII IgG antibodies at 0.2 mg/ml within 4 weeks after gene transfer. We conclude that a high level of hFVIII is necessary to achieve tolerance after neonatal gene transfer in mice, and neonatal gene transfer is not effective at inducing tolerance in cats.