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CRISPR/Cas9-based Exon Skipping Returns Dystrophin Expression to a Canine Model of DMD

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Review of “Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy” from Science by Stuart P. Atkinson 

Many of the mutations in the Dystrophin gene that lead to the development of Duchenne muscular dystrophy (DMD), a progressive muscle degeneration disorder [1], prevent the expression of a functional form of the dystrophin protein [2, 3]. However, CRISPR/Cas9-based exon “skipping”-based approaches permit the expression of a truncated, but functional, form of dystrophin and this strategy has the potential to benefit a significant proportion of DMD patients [4]. As an example, a previous Stem Cells Portal article described how CRISPR/Cas9-mediated exon skipping returned contractile activity to cardiomyocytes derived from DMD patient induced pluripotent stem cells (iPSCs) [5].

To test the efficacy and safety of this approach in a large animal model, researchers from the laboratory of Eric N. Olson (University of Texas Southwestern Medical Center, Dallas, TX, USA) assessed CRISPR/Cas9-mediated exon skipping in a canine model of DMD (deltaE50-MD) that exhibits many features of the human disease, such as muscle weakness, atrophy, and fibrosis [6].

Reporting in Science [7], Amoasii et al. employed adeno-associated viruses (AAV9) for the intramuscular or systemic delivery of the required CRISPR components (S. pyogenes Cas9 and a single guide RNA that targeted a region adjacent to the exon 51 splice acceptor site) in four one-month-old dogs and examined dystrophin protein expression after six and eight weeks, respectively. Of note, the AAV9 viruses employed display preferential tropism for skeletal muscle and heart tissue and the application of a muscle-specific creatine kinase regulatory cassette driving Cas9 expression further promoted muscle-specific exon-skipping.

Astonishingly, the authors report that both delivery routes permitted dystrophin restoration, and while restoration levels depended on muscle type, cardiac muscle dystrophin levels reached over 90% of normal dystrophin levels following systemic delivery. Additionally, each of the four treated dogs exhibited improved muscle histology and a lack of inflammatory cell infiltration, with deep sequencing at the top predicted off-target sites within protein-coding exons revealing no significant off-target effects.

While this study in a suitable large animal model provides evidence that CRISPR/Cas9 genome-editing approaches may prove clinically useful for the treatment of DMD, the authors acknowledge the need for longer-term studies in an expanded number of dogs to investigate off-target effects, the long-term genomic stability of gene-edited muscle tissues, and the possible immunogenicity of Cas9.

For more on novel treatment approaches for DMD and the ever-evolving field of CRISPR/Cas9, stay tuned to the Stem Cells Portal!

References

  1. Muntoni F, Torelli S, and Ferlini A, Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet Neurol 2003;2:731-40.
  2. Bladen CL, Salgado D, Monges S, et al., The TREAT-NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations. Hum Mutat 2015;36:395-402.
  3. Li Y, Liu Z, OuYang S, et al., Distribution of dystrophin gene deletions in a Chinese population. J Int Med Res 2016;44:99-108.
  4. Aartsma-Rus A, Fokkema I, Verschuuren J, et al., Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations. Hum Mutat 2009;30:293-9.
  5. Long C, Li H, Tiburcy M, et al., Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing. Science Advances 2018;4.
  6. Hildyard J, Rawson F, Harron R, et al., Characterising the skeletal muscle histological phenotype of the DeltaE50-MD dog, a preclinical model of Duchenne muscular dystrophy. Neuromuscular Disorders 2018;28:S18.
  7. Amoasii L, Hildyard JCW, Li H, et al., Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Science 2018;362:86-91.