Key Points
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Genetic neuromuscular conditions are debilitating and often prematurely fatal, and few standard treatment options are available
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Genome engineering provides a viable method of correcting the underlying genetic mutations in neuromuscular diseases
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A recent discovery, the highly adaptable CRISPR–Cas9 system, is already finding widespread use for cell therapy and in vivo gene editing
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Ongoing challenges include efficient gene delivery, identification and reduction of off-target interactions, and immunogenicity of genome engineering tools, delivery vectors and other neoantigens
Abstract
For many neuromuscular disorders, including Duchenne muscular dystrophy, spinal muscular atrophy and myotonic dystrophy, the genetic causes are well known. Gene therapy holds promise for the treatment of these monogenic neuromuscular diseases, and many such therapies have made substantial strides toward clinical translation. Recently, genome engineering tools, including targeted gene editing and gene regulation, have become available to correct the underlying genetic mutations that cause these diseases. In particular, meganucleases, zinc finger nucleases, TALENs, and the CRISPR–Cas9 system have been harnessed to make targeted and specific modifications to the genome. However, for most gene therapy applications, including genome engineering, gene delivery remains the primary hurdle to clinical translation. In preclinical models, genome engineering tools have been delivered via gene-modified cells or by non-viral or viral vectors to correct a diverse array of genetic diseases. In light of the positive results of these studies, genome engineering therapies are being enthusiastically explored for several genetic neuromuscular disorders. This Review summarizes the genome engineering strategies that are currently under preclinical evaluation for the treatment of degenerative neuromuscular disorders, with a focus on the molecular tools that show the greatest potential for clinical translation of these therapies.
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Acknowledgements
The authors' work has been supported by the Muscular Dystrophy Association (grant MDA277360), a Duke–Coulter Translational Partnership Grant, a Duke/UNC-Chapel Hill CTSA Consortium Collaborative Translational Research Award, a Hartwell Foundation Individual Biomedical Research Award, a March of Dimes Foundation Basil O'Connor Starter Scholar Award, NIH grants R01AR069085 and UH3TR000505, an NIH Director's New Innovator Award (DP2-OD008586), the Office of the Assistant Secretary of Defense for Health Affairs, through the Duchenne Muscular Dystrophy Research Program under awards W81XWH-15-1-0469 and W81XWH-16-1-0221, the Thorek Memorial Foundation, and the Allen Distinguished Investigator Program, through The Paul G. Allen Frontiers Group. Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the NIH or Department of Defense. C.E.N. is supported by a Hartwell Foundation Postdoctoral Fellowship and J.R.H. is supported by a National Science Foundation Graduate Research Fellowship and American Heart Association Predoctoral Fellowship.
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C.A.G., C.E.N. and J.R.H. are inventors on patent applications related to genome engineering for neuromuscular diseases.
Glossary
- Exon skipping
-
A technique in which antisense oligonucleotides are introduced that bind motifs within an mRNA, promoting alternative splicing and excluding the targeted exon from the final transcript.
- Nonsense readthrough compounds
-
Small molecules that suppress stop codon recognition and allow ribosomes to translate an mRNA, despite nonsense mutations.
- Morpholino
-
A non-ionic synthetic nucleic acid with DNA bases covalently attached to morpholine rings with a phosphorodiamidate backbone.
- Homology-directed repair
-
(HDR). A precise method of DNA repair that uses the sister chromosome or a provided DNA template to restore the damaged genome or introduce new DNA sequences.
- Safe-harbour locus
-
A genomic region where targeted integrations can be performed without disrupting endogenous gene activity or negatively affecting cell function.
- Non-homologous end joining
-
(NHEJ). A DNA repair pathway that religates double-strand breaks, with the possibility of producing small insertions or deletions that might lead to gene knockout.
- Directed evolution
-
A process that uses multiple rounds of mutagenesis and user-defined selection to alter the function of a protein or nucleic acid.
- Cys2–His2 zinc finger domain
-
A finger-like structure of ∼30 amino acids that recognizes three or four base pairs. Proteins containing multiple Cys2–His2 zinc finger domains can be used for sequence-specific DNA targeting.
- Golden Gate assembly
-
A cloning method that allows scarless ligation of multiple DNA fragments and is well-suited for cloning repetitive sequences, including TALE arrays.
- Protospacer adjacent motif
-
(PAM). A sequence motif that is recognized by the Cas9 enzyme. Several CRISPR systems have been described with varying PAM requirements.
- Small interfering RNA
-
Short, double-stranded RNA that mediates sequence-specific silencing of targeted mRNAs.
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Nelson, C., Robinson-Hamm, J. & Gersbach, C. Genome engineering: a new approach to gene therapy for neuromuscular disorders. Nat Rev Neurol 13, 647–661 (2017). https://doi.org/10.1038/nrneurol.2017.126
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DOI: https://doi.org/10.1038/nrneurol.2017.126
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