BackgroundSystemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle (exon skipping) is a promising therapy. Treatment with Phosphorodiamidate morpholino oligomers (PMO) lead to shorter de novo dystrophin protein in both animal models and DMD boys who otherwise lack dystrophin; however, restoration of dystrophin has been observed to be highly variable. Understanding the factors causing highly variable induction of dystrophin expression in pre-clinical models would likely lead to more effective means of exon skipping in both pre-clinical studies and human clinical trials.MethodsIn the present study, we investigated possible factors that might lead to the variable success of exon skipping using morpholino drugs in the mdx mouse model. We tested whether specific muscle groups or fiber types showed better success than others and also correlated residual PMO concentration in muscle with the amount of de novo dystrophin protein 1 month after a single high-dose morpholino injection (800 mg/kg). We compared the results from six muscle groups using three different methods of dystrophin quantification: immunostaining, immunoblotting, and mass spectrometry assays.ResultsThe triceps muscle showed the greatest degree of rescue (average 38±28 % by immunostaining). All three dystrophin detection methods were generally concordant for all muscles. We show that dystrophin rescue occurs in a sporadic patchy pattern with high geographic variability across muscle sections. We did not find a correlation between residual morpholino drug in muscle tissue and the degree of dystrophin expression.ConclusionsWhile we found some evidence of muscle group enhancement and successful rescue, our data also suggest that other yet-undefined factors may underlie the observed variability in the success of exon skipping. Our study highlights the challenges associated with quantifying dystrophin in clinical trials where a single small muscle biopsy is taken from a DMD patient.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0070-6) contains supplementary material, which is available to authorized users.

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 evaluated the effect of chronic phosphorodiamidate morpholino oligomer (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 chronically with either 800 mg/kg/month PMO for 6 months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum‐circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin‐specific antibodies were observed in the control saline‐treated mdx cohorts (n = 8) or in aged (12‐month‐old) mdx mice with expanded ‘revertant’ dystrophin‐expressing fibers. Reactive antibodies recognized both full‐length and truncated exon‐skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen‐specific T‐cell cytokine responses (e.g. IFN‐g, IL‐2) in dystrophin antibody‐positive mice than in dystrophin antibody‐negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin‐expressing fibers along with CD8+ and perforin‐positive T cells in the vicinity, suggesting an activation of cell‐mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody‐negative mice than on those of dystrophin antibody‐positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell‐mediated and humoral immune responses in mdx mice. Our data highlights the need to further investigate the autoimmune response and its long‐term consequences after exon‐skipping therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.