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Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice

Nature Medicine volume 20, pages 992–1000 (2014)Cite this article

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A Corrigendum to this article was published on 07 May 2015

A Corrigendum to this article was published on 07 April 2015

This article has been updated

Abstract

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject–derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5′ exons of DMD.

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Figure 1: Protein and RNA studies from human biopsy samples.
Figure 2: Mapping of a dystrophin IRES in exon 5.
Figure 3: Stimulation of IRES activity by out-of-frame exon skipping in human subject–derived cell lines.
Figure 4: Intramuscular delivery of AAV1.
Figure 5: Glucorticoid activation of the dystrophin IRES.
Figure 6: Muscle pathology, membrane integrity and contraction-induced damage following expression of the IRES-driven isoform.

Accession codes

Primary accessions

Gene Expression Omnibus

Referenced accessions

NCBI Reference Sequence

Change history

  • 25 August 2014

     In the version of this article initially published online, the nucleotide numbering of the AS (–3 to + 16), B (+17 to +40) and C (+49 to –10) targeted sequences in Figure 3b was incorrect, and thus the schematic of the sequences was incorrectly drawn. The correct numbering is as follows: AS, –3 to + 18; B, +17 to +44; and C, +49 to –8. The correction has no impact on the results of the study or its conclusions. The error has been corrected for all versions of this article.

  • 13 March 2015

     In the version of this article initially published, three participants of the study were not included as co-authors. Also, one of the individuals mentioned in the Acknowledgments section of the report was incorrectly included and thus has been removed at their request, and the name of another individual mentioned in the Acknowledgments was originally misspelled (“Fabbri” should have been "Fabri"). The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This work has been supported by the US National Institutes of Health National Institute of Neurological Disorders and Stroke (R01 NS043264 (K.M.F., M.T.H. and R.B.W.)), the National Institute of General Medical Sciences (R01 GM038277 and R01 GM084177 (D.R.S.)), and CureDuchenne (K.M.F.). N.W. has received fellowship support from the Ohio State University/Nationwide Children's Hospital Muscle Group and the Philippe Foundation. We wish to acknowledge the technical assistance of A. Rutherford, Y. Kaminoh and L. Taylor and are grateful for the reagents provided by P. Sarnow (pRDEF; Stanford University) and G. Morris (hybridoma cell lines for antibodies MANEX1A, MANCHO3 and MANDAG2; MDA Monoclonal Antibody Resource at the Wolfson Centre for Inherited Neuromuscular Disease). The EU BIO-NMD project (no. 241665) provided support to A. Ferlini and C.A.-K.S., and the Duchenne Parent Project Italy (DMD Diagnostics Project) provided support to A. Ferlini. Thanks are also due to C. Rodolico and M. Fabris for their technical support. The Clinical Proteomics Mass Spectrometry core facility at Karolinska University Hospital and Science for Life Laboratory provided assistance in mass spectrometry and data analysis.

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Author notes

  1. Nicolas Wein and Adeline Vulin: These authors contributed equally to this work.

Authors and Affiliations

  1. The Center for Gene Therapy, Nationwide Children's Hospital, The Ohio State University, Columbus, Ohio, USA

    Nicolas Wein, Adeline Vulin, Andrew Findlay, Kristin N Heller, Louise R Rodino-Klapac & Kevin M Flanigan

  2. Department of Medical Sciences, Section of Microbiology and Medical Genetics, University of Ferrara, Ferrara, Italy

    Maria S Falzarano, Simona Passarelli, Matteo Bovolenta, Marcella Neri, Francesca Gualandi & Alessandra Ferlini

  3. Department of Proteomics and Nanobiotechnology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden

    Christina Al-Khalili Szigyarto & Mathias Uhlén

  4. Department of Neurology, Washington University School of Medicine, St. Louis, Missouri, USA

    Baijayanta Maiti

  5. Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA

    Baskar Bakthavachalu & Daniel R Schoenberg

  6. Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio, USA

    Baskar Bakthavachalu & Daniel R Schoenberg

  7. Department of Neuroscience, University of Messina and Centro Clinico Nemo Sud, Messina, Italy

    Sonia Messina & Giuseppe Vita

  8. Bambino Gesù Children's Hospital, Rome, Italy

    Chiara Passarelli

  9. Centre for Comparative Genomics, Murdoch University, Perth, Western Australia, Australia

    Steve D Wilton

  10. Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA

    Louise R Rodino-Klapac & Kevin M Flanigan

  11. Division of Biomedical Informatics, Department of Computer Science, University of Kentucky Lexington, Kentucky, USA

    Lin Yang

  12. Department of Human Genetics, The University of Utah School of Medicine, Salt Lake City, Utah, USA

    Diane M Dunn, Robert B Weiss & Michael T Howard

  13. Department of Neurology, The Ohio State University, Columbus, Ohio, USA

    Kevin M Flanigan

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  1. Nicolas Wein
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Contributions

N.W. performed the immunostaining staining on human and mouse muscle, generated reagents for and performed all luciferase experiments, immortalized and transdifferentiated human cell lines, performed immunoblot and RT-PCR experiments on C2C12 and FibroMyoD cells, and performed RT-PCR on mouse tissues. A.V. generated the Dup2 mouse model, injected all mice and performed all immublotting on mouse tissue and on muscle biopsies from the subjects with Dup2 and frameshift mutations. B.M. generated the master exon 1–5 construct and performed the initial dual luciferase assays. K.N.H. and L.R.R.-K. performed or analyzed the specific force and eccentric contraction–induced damage tests. L.Y. developed and performed the H&E and EBD quantification. A. Ferlini, C.A.-K.S. and M.U. designed and performed analysis of the exon 2–deleted muscle. S.M., G.V. and C.P. clinically characterized the subject with exon 2 deletion, and S.B., M.B., and M.N. performed the genetic characterization of the patient with exon 2 deletion. M.S.F. performed cell and AON studies on a subject with exon 2 deletion. S.D.W. provided critical reagents (AONs) for cell-based experiments. B.B. and D.R.S. designed and performed the formaldehyde RNA electrophoresis and northern blotting. D.M.D. and R.B.W. designed and performed the ribosome profiling experiments. A. Findlay generated all the U7 constructs. N.W., M.T.H. and K.M.F. designed the experiments and analyzed and interpreted the data. N.W., R.B.W. and K.M.F. wrote the manuscript and the compiled the figures, with contributions from A.V., F.G., R.B.W., A. Ferlini and D.R.S.

Corresponding author

Correspondence to Kevin M Flanigan.

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Wein, N., Vulin, A., Falzarano, M. et al. Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice. Nat Med 20, 992–1000 (2014). https://doi.org/10.1038/nm.3628

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