Gene Therapy for Duchenne Muscular Dystrophy


Duchenne muscular dystrophy is a severe muscle wasting disorder caused by lack of functional dystrophin, due to reading‐frame disrupting mutations in the DMD gene. There is no cure, but several genetic therapeutic approaches aiming to delay disease progression by restoring, manipulating or replacing the DMD gene are under investigation in (pre)clinical settings. The antisense‐mediated exon skipping approach, which restores the disrupted reading frame allowing synthesis of partly functional dystrophin proteins is closest to clinical application. The first trials with viral vectors to deliver minidystrophin genes and compounds to force readthrough of premature stop codons by the translation machinery have also been undertaken, whereas other approaches, like genome editing, are still in the early development phase. Despite some remaining hurdles that have to be overcome, lessons learned can be used to catalyse the development of potential therapies.

Key Concepts:

  • Duchenne muscular dystrophy (DMD) is caused by reading‐frame disrupting mutations in the DMD gene, resulting in premature termination of dystrophin synthesis.

  • Inframe mutations allow synthesis of shorter, but partly functional dystrophin proteins, resulting in the milder Becker muscular dystrophy (BMD) phenotype.

  • Antisense oligonucleotide‐mediated exon skipping aims to restore the reading frame enabling synthesis of BMD‐like dystrophins, thereby converting the severe DMD into the milder BMD disease.

  • Gene replacement therapy (replacing the dysfunctional dystrophin cDNA) is hampered by the size and complexity of the DMD gene and dystrophin cDNA.

  • AAV vectors have a limited loading capacity of ∼4.5 kb, requiring the use of mini‐, or microdystrophin genes that encode only the most crucial functional domains.

  • Ataluren can be used to force the translational machinery to ignore premature stop codons enabling synthesis of intact dystrophin.

  • Endonuclease‐mediated generation of double‐strand breaks can be used to correct the genetic mutation or restore the reading frame.

  • Many therapeutic approaches have progressed to clinical trials.

Keywords: duchenne muscular dystrophy; gene therapy; dystrophin; exon skipping; antisense oligonucleotide; adeno‐associated viruses; readthrough of premature stop codons; genome editing

Figure 1.

(a) In healthy individuals, pre‐mRNA is converted into mRNA by the splicing machinery, which is translated into full‐length dystrophin. (b) Example of a DMD patient with a deletion of exons 52–58, which disrupts the reading frame and prevents translation of the complete dystrophin protein. (c) AONs targeting exon 51 hide this exon from the splicing machinery so that it is spliced out together with the flanking introns. This restores the reading frame so that a BMD‐like dystrophin protein can be generated.

Figure 2.

(a) Schematic overview of the full‐length dystrophin protein, which consists of four domains. At the N‐terminus, it has an actin‐binding domain 1 (A‐BD1), followed by the central rod domain that contains four proline‐rich hinge regions (H1–4) and 24 spectrin‐like repeats. Within the rod domain, a second actin‐binding domain (A‐BD2) and an nNOS‐binding domain (nNOS BD) are present located between repeats 11–17 and 16–17, respectively. The C‐terminal part consists of three binding domains for β‐dystroglycan (Dg‐BD), α‐sarcoglycan (S‐BD) and α‐dystrobrevin (Db‐BD). (b) The rAAV2.5‐CMV‐mini‐dystrophin protein, used for the clinical trial by Bowles et al., contains the first actin‐binding domain, three hinge regions, five spectrin‐like repeats and the β‐dystroglycan‐binding domain.

Figure 3.

(a) Example of a DMD patient with a stop codon (UAG) mutation in exon 9 resulting in premature termination of protein translation. (b) Administration of ataluren/PTC124 ensures readthrough of the translational machinery ignoring the premature stop codon enabling synthesis of full‐length dystrophin.

Figure 4.

(a) Genome editing can be used to restore a point mutation (indicated by star) in the DMD gene using a nuclease to induce a specific double‐stranded DNA break and a wild‐type template (pink). Once the mutation has been corrected, synthesis of full‐length dystrophin is feasible. (b) Deletions (or duplications) that disrupt the reading frame (indicated by star) can also be repaired by genome editing. Either the exon splice site or the mutation can be targeted. When the splice site (of the exon containing the mutation) is targeted by double‐strand breaks one or multiple base pairs are inserted or deleted, thereby abolishing the splice site, leading to skipping of the exon. Alternatively, when the mutation is targeted directly a random number of nucleotides is included or deleted, which restore the reading frame in ∼1:3 of cases. Exon skipping and reading frame restoration will both lead to internally deleted, BMD‐like dystrophin proteins.



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Further Reading

Aartsma‐Rus A (2010) Antisense‐mediated modulation of splicing: therapeutic implications for Duchenne muscular dystrophy. RNA Biology 7: 453–461.

Goyenvalle A, Seto JT, Davies KE and Chamberlain J (2011) Therapeutic approaches to muscular dystrophy. Human Molecular Genetics 20: R69–R78.

Lai Y and Duan D (2012) Progress in gene therapy of dystrophic heart disease. Gene Therapy 19: 678–685.

Lu QL, Yokota T, Takeda S et al. (2011) The status of exon skipping as a therapeutic approach to duchenne muscular dystrophy. Molecular Therapeutics 19: 9–15.

Mendell JR, Rodino‐Klapac L, Sahenk Z et al. (2012) Gene therapy for muscular dystrophy: lessons learned and path forward. Neuroscience Letters 527: 90–99.

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van Putten, Maaike, and Aartsma‐Rus, Annemieke(Sep 2013) Gene Therapy for Duchenne Muscular Dystrophy. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0025029]