992. Induction of Immune Tolerance to Transgene Products by Mixed Chimerism with Low Dose Irradiation
Authors: Satoru Otsuru, Katsuto Tamai, Takehiko Yamazaki, Takenao Chino, Hideki Yoshikawa, Yasufumi Kaneda
Journal: Molecular Therapy (2006)

Gene therapy has great potential to treat various intractable diseases, especially being expected to provide novel approach for genetic disorders with no other therapeutic strategy. Varieties of gene delivery systems as well as the optimal gene therapy protocols have been established. To achieve successful outcome of gene therapy for genetic diseases, however, regulation of the immunological reaction against transgene products must be critical to ensure sustained effect of the therapy. To surmount the immune-mediated rejection, specific immune tolerance to the transgene product has to be induced before gene therapy. It has been demonstrated that hematopoietic chimerism with transplanted bone morrow cells leaded to immune tolerance against donor cells, and full hematopoietic chimerism could be induced by bone marrow transplantation (BMT) with the conventional lethal dose (10Gy) irradiation. Since lethal dose irradiation has many risks such as severe immunosuppression, it would be more acceptable if immune tolerance could be induced with lower dose irradiation which is applicable for gene therapy.In this study, first evaluated minimal irradiation dose (MID) required for enhanced green fluorescence protein (EGFP)-transgenic BMT in mouse model to induce immune tolerance against cells expressing EGFP. Repeated analysis of MID for EGFP-BMT revealed that 4Gy was minimal but sufficient irradiation dose to induce mixed chimerism in mouse bone morrow. FACS analysis showed that about 20% chimeras were maintained at least for 12 months after 4Gy irradiation, whereas no chimerism was obtained by irradiation with the lower doses. Furthermore, the mixed chimerism of EGFP positive cells was found not only in the bone marrow but also in the thymus, spleen and peripheral blood, suggesting successful induction of immune tolerance to EGFP. To confirm the induction of specific immune tolerance to the transgenic EGFP, the EGFP-transgenic skin was grafted on the mice with EGFP-transgenic BMT after various dose irradiations. Successful engraftment of EGFP-skin was observed on the EGFP-BMT mice with 4Gy or with higher dose irradiation even 15 weeks after skin transplantation. On the contrary, basically all EGFP-skin grafts were rejected by BMT mice irradiated with 3Gy or lower dose, further indicating that 4Gy was MID. Furthermore, we found the high levels of anti-EGFP antibody were induced in the mice with 3Gy or less dose irradiation during rejection process of the EGFP- skin transplantations, while no anti-EGFP antibody was raised in the mice with 4Gy or more irradiation. These data strongly suggest that 4Gy irradiation is established MID for inducing immune tolerance to transgene products in transgenic BMT of syngenic mouse. Since BMT with lethal dose irradiation is already established clinical protocols, non-lethal 4Gy irradiation before gene-corrected syngenic BMT to patients with intractable gene-defective disorders might have great promise in a future clinical application.