32. Tolerance after Neonatal Gene Transfer of a Human Factor IX-Expressing Retroviral Vector May Involve Clonal Deletion in C3H Mice and Is Effective in Hemophilia B Dogs
Authors: Lingfei Xu, Mark E. Haskins, Timothy C. Nichols, Manxue Mei, Patty O'Donnell, Dwight A. Bellinger, Stephanie McCorquodale, Katherine P. Ponder
Journal: Molecular Therapy (2005)

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Hemophilia B is a bleeding disorder due to deficiency of factor IX (FIX). Antibodies that inhibit the function of FIX can develop after protein or gene therapy and reduce the effectiveness of treatment. We previously showed that neonatal gene transfer with a high dose of an amphotropic gamma retroviral vector (RV) expressing human FIX (hFIX) from the human |[alpha]|1-antitrypsin promoter did not induce antibodies to hFIX in hemophilia A mice or in normal dogs. In this study, we attempted to identify the mechanism of tolerance after neonatal gene therapy in mice, and to further explore the expression level needed for tolerance in dogs. Normal C3H mice make antibodies to hFIX and do not bleed after injections. Neonatal C3H mice that were injected IV at 2 or 3 days after birth with RV at 1010, 109, or 108 transducing units (TU)/kg achieved 373|[plusmn]|47 (7.5%), 102|[plusmn]|22, and 1.9|[plusmn]|0.7 ng/ml of hFIX in plasma, respectively. Only 1 of 41 mice developed anti-hFIX antibodies after gene transfer. Challenge with 10 doses of 30U/kg/dose of hFIX protein as adults resulted in anti-hFIX antibodies in 0 of 8, 2 of 10 and 11 of 13 mice in the high, medium and low dose groups, respectively. Lymphocytes from mice that were tolerized with a high RV dose as newborns, and then challenged with hFIX protein did not secrete interferon |[atilde]|, interleukin 2 (IL-2), or IL-10 in response to hFIX in vitro. In contrast, lymphocytes from mice that were immunized without preceding neonatal gene transfer secreted these cytokines. Since CD25+ suppressor cells secrete IL-10, these data are consistent with clonal deletion as a major factor in tolerance induction. Adoptive transfer experiments showed that most recipients of lymphocytes from tolerant mice produced anti-hFIX antibodies, which is consistent with clonal deletion of hFIX-responsive cells being a major mechanism of neonatal tolerance. Since the newborn immune system is more mature in large animals, we further explored if neonatal gene therapy could avoid antibody formation in dogs. Normal dogs that were injected IV with 3|[times]|108 (N=4) or 6|[times]|107 (N=3) TU/kg of RV at 2 or 3 days after birth achieved 28|[plusmn]|14 and 1.8|[plusmn]|0.3 ng/ml of hFIX in plasma, respectively, and did not develop anti-hFIX antibodies after 10 doses of hFIX protein challenge starting at 2 months. In addition, hemophilia B dogs (N=3) treated with 3|[times]|109 TU/kg of RV at 2 or 3 days after birth achieved 520|[plusmn]|215 ng/ml of hFIX in plasma, and none developed anti-hFIX antibodies. We conclude that achieving >10 ng/ml of hFIX is critical for tolerance induction after neonatal gene transfer in C3H mice, and the mechanism may involve clonal deletion. The level of expression needed to induce neonatal tolerance to hFIX is lower in normal dogs (2 ng/ml) than in mice, and neonatal tolerance is effective in hemophilia B dogs.