Treating Muscular Dystrophy with CRISPR Gene Editing

By Mary Gearing

Having seen CRISPR’s success in basic research, researchers are eager to apply it in a clinical setting. CRISPR is often used for animal germline modification, to repair or add in disease-causing mutations, but, until recently it hadn’t been used to treat disease postnatally. Now, three papers published concurrently in Science have shown CRISPR can treat a genetic disease in a postnatal mouse model, an important proof of concept for future preclinical and clinical work.

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Duchenne muscular dystrophy: The most common inherited disease

The genetic disease targeted is Duchenne muscular dystrophy (DMD), an X-linked recessive disorder affecting approximately 1 in 5000 males. DMD is caused primarily by frameshift mutations in dystrophin, a protein essential for proper muscle function. Without functioning dystrophin, an individual experiences progressive muscle wasting leading to death at around 30 years of age. Despite the amount of research conducted on DMD, there is still no good treatment.

The dystrophin gene is very large (79 exons), but much of the sequence appears to be nonessential. Within the mutational hotspot for dystrophin, exons 45-55, there are multiple common deletions that maintain the protein’s reading frame, leading to the production of a smaller, but at least partially functional protein. Individuals with these mutations are often asymptomatic, or have only mild symptoms, a condition known as Becker muscular dystrophy (BMD).

Dystrophin’s size makes it difficult to deliver via gene therapy, so researchers have set their sights on another approach. Since shorter forms of dystrophin can still be functional, exon skipping is a good option for DMD treatment. Clinical trials have used oligonucleotide exon skipping (OEN) to remove mutated exons from the dystrophin transcript. Unfortunately, the oligonucleotides only modestly improve muscle function, and they must be injected regularly.

Dystrophin and genome editing

Since complex oligonucleotide treatment comes with many challenges, researchers have begun to explore genome editing approaches for exon skipping. Addgene depositor Charles Gersbach used paired zinc finger nucleases to remove exon 51 in DMD patient myoblasts. They observed a 13% removal rate of exon 51, which resulted in appropriately localized dystrophin. In a subsequent study, they used CRISPR with two gRNAs to delete exon 51 or exons 45-55 in patient myoblasts; when injected into DMD mice, these cells expressed functional dystrophin.

Editing genes in vivo is of course much more difficult than in cell culture, but Ran et al. have shown that CRISPR and AAV can be used together for postnatal genome editing in mice. Could this approach work with dystrophin exon skipping? For such a therapy to be successful, multiple requirements must be met. First, CRISPR would need to be delivered to both cardiac and skeletal muscle cells, where precision editing of the dystrophin gene would take place, with minimal risk of off-target editing. In order for the therapy to persist over time, stem cell editing would be highly desirable. Should stem cell editing occur, the CRISPR components would only need to be expressed for a short period of time, which would prevent the accumulation of unwanted mutations over time.

DMD is a good choice for a CRISPR proof of concept treatment, as the disease is especially well suited for genome editing. The homology-directed repair (HDR) pathway is downregulated in mature tissues - no problem, as exon skipping proceeds through the non-homologous end joining (NHEJ) pathway. It’s estimated that very little dystrophin correction (about 4%) is needed to see muscle improvement, with only 30% correction needed for normal function. Even low-frequency editing could make a huge difference in DMD, and it’s estimated that exon skipping therapies would be applicable to 80% of DMD patients.

exon skipping strategies for Duchenne muscular dystrophy

Long et al., Nelson et al., and Tabebordbar et al. each chose to try exon skipping in DMD mice, which have a mutation in dystrophin exon 23. Long et al. used SpCas9 in conjunction with AAV9, whereas Nelson et al. and Tabebordbar et al. used the shorter SaCas9 with AAV8 and AAV9, respectively. Although each study used slightly different methods, they each observed the same positive phenotypes in cardiac and skeletal muscle. Even when the genome editing frequency is low, the relative abundance of exon-skipped mRNA is high, likely because this mRNA is not subject to nonsense-mediated decay. Although Nelson et al. observed only 2% genome editing in one experiment, they found the exon-skipped transcript constituted 59% of total dystrophin mRNA, similar to the 39% observed by Tabebordbar et al. Long et al. show that the percentage of dystrophin-expressing muscle cells increases over time, and all three groups confirm dystrophin expression via Western blot, at <10% of the wild-type level. Muscle histology is improved, with vastly reduced inflammation and necrosis compared to unedited animals. In grip strength, specific force, and other muscle tests, muscle function is clearly improved, although not to wild-type levels. With regard to off-target effects, each group finds very low to no off-target activity at the ten highest-ranking predicted off-target sites.

Each paper characterizes additional, unique facets of CRISPR DMD therapy. Long et al. show that AAV-CRISPR does not cause obvious germline modification, an important finding given the controversy associated with such editing. Tabebordbar et al. show that muscle stem cells are modified by this approach, increasing the possibility that edits will persist long-term. In accordance with this result, Nelson et al. find that dystrophin restoration is maintained for at least six months.

Future directions and obstacles to clinical CRISPR editing

Given the success of the CRISPR-mediated exon skipping approach in mice, DMD researchers are very excited. This approach may also be applicable to a number of rare genetic diseases caused by splicing defects, including ataxia telangiectasia, congenital disorder of glycosylation, and Niemann-Pick disease type C. Although the three DMD studies referenced here represent a great step forward for CRISPR gene therapy, it’s important to realize that DMD is a simpler case than other genetic diseases we’d like to treat with CRISPR. As seen above, DMD can be treated with “one-size-fits-most” NHEJ-mediated editing, but most other diseases will require HDR-mediated precision editing tailored to smaller patient populations.

To bring DMD CRISPR therapy closer to the clinic, much work is still needed. First, CRISPR delivery must be optimized to:

1. Reach a high percentage of muscle cells throughout the body, especially stem cells

2. Remove any immunogenicity of the AAV vector

Once delivery has been optimized, it’s important to ascertain how long the rescue phenotype will last, and more importantly, if it does extend lifespan. This work should be done in mouse and large animal models with mutations in human-relevant exons 45-55, rather than in the traditional exon 23-mutated mouse model. Potential off-target effects in muscle, as well as unwanted germline editing, must be rigorously examined over a long period of time. High fidelity Cas9s such as eSpCas9 and SpCas9-HF should be explored to reduce off-target editing. Short-term CRISPR expression would be especially desirable, as it would reduce the potential of off-target editing over time, but this approach would require robust stem cell editing to maintain the desired phenotype.

Precision genome editing faces the challenges above and more. One chief challenge is upregulating HDR in mature tissues, as this process is necessary to precisely edit point mutations. In addition to upregulating HDR, NHEJ must be downregulated to prevent the introduction of new mutations by CRISPR; editing exonic regions comes with much more risk than the intronic editing used in the exon skipping approaches here. In most genetic diseases, the mutational landscape is broad and varied, necessitating the development of many distinct CRISPR therapies to match these different mutations. For each therapy, effectiveness and off-target risk must be evaluated separately, increasing the time to clinical approval.

Even with the challenges precision editing faces, it’s encouraging to see such progress in the more feasible case of DMD. If the safety and efficacy of this approach can be optimized, DMD could become one of the first diseases to be treated clinically with CRISPR. Many obstacles remain for CRISPR gene editing, but given the speed at which the technology is advancing, precision editing treatments may also be closer than we might expect.Download Addgene's CRISPR 101 eBook!



1. Kaiser J. CRISPR helps heal mice with muscular dystrophy. Science. 2015 Dec 31. doi:10.1126/science.aae0169

2. Long C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz E, Bhattacharyya S, Shelton JM, Bassel-Duby R, Olson EN. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 2015 Dec 31. pii: aad5725. PubMed PMID: 26721683.

3. Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Rivera RM, Madhavan S, Pan X, Ran FA, Yan WX, Asokan A, Zhang F, Duan D, Gersbach CA. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2015 Dec 31. pii: aad5143. PubMed PMID: 26721684.

4. Reardon S. Gene-editing summit supports some research in human embryos. Nature. 2015 Dec 3. doi:10.1038/nature.2015.18947

5. Siva K, Covello G, Denti MA. Exon-skipping antisense oligonucleotides to correct missplicing in neurogenetic diseases. Nucleic Acid Ther. 2014 Feb;24(1):69-86. PubMed PMID: 24506781. PubMed Central PMCID: PMC3922311.

6. Tabebordbar M, Zhu K, Cheng JK, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, Wagers AJ. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 2015 Dec 31. pii: aad5177. PubMed PMID: 26721686.

7. Ousterout DG, Kabadi AM, Thakore PI, Perez-Pinera P, Brown MT, Majoros WH, Reddy TE, Gersbach CA. Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases. Mol Ther. 2015 Mar;23(3):523-32. PubMed PMID: 25492562. PubMed Central PMCID: PMC4351462.

8. Ousterout DG, Kabadi AM, Thakore PI, Majoros WH, Reddy TE, Gersbach CA. Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat Commun. 2015 Feb 18;6:6244. PubMed PMID: 25692716. PubMed Central PMCID: PMC4335351.

9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015 Apr 9;520(7546);186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. PubMed PMID: 25830891. PubMed Central PMCID: PMC4393360.

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