Humoral and cell mediated immune response to new dystrophin after morpholino-induced exon skipping therapy in dystrophin-deficient mdx mice

Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have now evaluated the effect of chronic morpholino (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated with either 800mg/kg/month PMO for 6 months (n=8) or 100mg/kg/week PMO for 12 weeks (n=11). We found circulating antibodies directed against de novo dystrophin in 38% of the high-dose monthly PMO-treated cohort and 55% of the low-dose weekly PMO-treated cohort, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts or in aged mdx mice with expanded "revertant" dysttophin expressing fibers. Further, anti-dystrophin IgG antibodies recognized both full-length mouse dystrophin (normal) as well as Becker-like truncated dystrophin protein (exon-skipped) in muscle. Finally, we show that antigen-specific T cell interferon gamma, TNF-alpha and IL-6 production results from de novo dystrophin expression. More importantly, we found CD8+ Cytotoxic T-cells inthe vicintity of Major Histocompatibility Complex (MHC) class -1 and dystrophin-positive muscle fibers. Our results show that de novo dystrophin expression in response to exon skipping triggers both humoral aned cell-mediated immune responses in mdx mice. The implications of these findings for the long-term success of exon-skipping therapy withou immunosuppression are still unknown. Our data highlight the need to further investigate the nature and magnitute of the autoimmune response to newly synthesized dystrophin after exon-skipping therapy.