Morpholino‐induced exon skipping stimulates cell‐mediated and humoral responses to dystrophin in mdx mice

Abstract

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.

Muscle and cardiac therapeutic strategies for Duchenne muscular dystrophy: past, present, and future

“…One concern of dystrophin replacement therapy is related to the immune reaction in response to the newly generated dystrophin protein [ 34 , 54 ]. Immunosuppressant drugs may, therefore, be necessary.…”

Emerging proteomic biomarkers of X-linked muscular dystrophy

“…These new methodologies include stem cell therapy, myoblast transfer, utrophin substitution therapy, enhancement of the cellular stress response, transcutaneous electrical nerve stimulation and improved steroid therapy, as well as a variety of direct or indirect genetic approaches including plasmid transfer, viral transfer, naked DNA transfer, mini-dystrophin delivery, stop codon read-through therapy, exon-skipping therapy and CRISPR/Cas9-based partial genome editing [22][23][24]. Potentially dangerous side effects due to de novo dystrophin expression following gene therapy were recently highlighted by the immunological evaluation of exon skipping in the mdx mouse [25]. Newly synthesised dystrophin molecules were shown to trigger both cell-mediated and humoral immune responses, which suggests that exon skipping therapy might be complicated by a long-term autoimmune response and associated cellular damage [25].…”

“…Potentially dangerous side effects due to de novo dystrophin expression following gene therapy were recently highlighted by the immunological evaluation of exon skipping in the mdx mouse [25]. Newly synthesised dystrophin molecules were shown to trigger both cell-mediated and humoral immune responses, which suggests that exon skipping therapy might be complicated by a long-term autoimmune response and associated cellular damage [25].…”

Autoimmune response and its long‐term consequences after exon‐skipping therapy in a Duchenne muscular dystrophy mouse model

“…In a recent issue of The Journal of Pathology , Vila et al highlighted the need to further investigate the autoimmune response against de novo synthesised dystrophin protein and its long‐term consequences after exon‐skipping therapy for Duchenne muscular dystrophy (DMD), a fatal, inherited progressive degenerative muscle disorder. Typically, DMD occurs as a result of truncating mutations in the DMD gene that result in an almost total lack of dystrophin protein in muscle fibres.…”

“…In the present study , a U‐PLEX cytokine panel was performed to assay T‐cell reactivity against dystrophin. However, in future studies, we think it would be important to perform further profiling of the T‐cell response.…”