A breakthrough study from Duke University may offer promise for future gene therapies for Duchenne muscular dystrophy (DMD). With an incidence of approximately 1 in 3500 male births, DMD is the most commonly inherited muscular disorder in children. Symptoms present before age 5 and include progressive muscle weakness in the upper limbs, respiratory issues, and cardiomyopathy.1-3 A 2007 study by the Centers for Disease Control found that 90% of affected males between 15 and 24 years old were confined to a wheelchair and just 58% survived in the 20 to 24 age group.4
DMD is caused by mutations in the X-linked dystrophin gene, which encodes a structural protein required for muscle membrane stability.5 Dystrophin deficiency triggers cardiac and skeletal muscle wasting as well as replacement of muscle tissue by adipose tissue.6 Loss of function typically results from frameshift mutations (mainly deletions) that truncate the protein prematurely. Multiple studies have highlighted the use of exon skipping, the practice of removing exons adjacent to deletions that restores the reading frame and produces a functional protein.7-9 Skipping exon 51 alone could resolve up to 13% of all dystrophin deletions.
In a 2015 article published in Molecular Therapy, Ousterout et al. describe a genome editing method that utilizes zinc finger endonucleases (ZFNs) to target splice sequences of exon 51 and permanently exclude it from the dystrophin transcript. ZFNs include domains for DNA binding and cleavage, which is mediated through FokI activity. When ZFNs bind recognition sequences adjacent to one another, Fok1 moieties dimerize and initiate targeted double stranded breaks in the DNA. By localizing ZFNs to critical splice site sequences flanking dystrophin exon 51, Fok1-mediated double stranded DNA breaks trigger cellular DNA repair mechanisms that yield a dystrophin transcript lacking exon 51. 1,10
ZFNs targeting exon 51 were transfected into myoblasts of a DMD patient with a known deletion of dystrophin exons 48-50. After differentiation to myofibers, RNA was isolated and RT-PCR confirmed the loss of exon 51. Restored dystrophin function was confirmed by western blot analysis that revealed wild type antibody activity at the C-terminus and rod domain, critical regions to the structural activity of dystrophin, in the ZFN-transfected clone. Finally, corrected myoblasts were transplanted into immunodeficient mice and human dystrophin was measured at the sarcolemma membrane to confirm correct localization of the transplanted cells.1
To date, several approaches to gene editing have been described. CRISPR-Cas is a bacterial immune system harnessed for genome editing in which segments of invading DNA are inserted into the palindromic repeats of the CRISPR genome and silenced by the Cas9 protein.11 Alternatively, transcription activator-like effector nucleases (TALENs) are analogous to ZFNs for their incorporation of Fok1 endonuclease activity with a highly-customizable TALE repeat domain for sequence-specific binding.12 Ousterout et al. suggest that ZFNs may be a preferred method for DMD therapies since ZFN-based genome modifications have proceeded to clinical trials for other applications. Gene repair research is currently supported by the Muscular Dystrophy Association with the hope that emerging therapies can cease or prevent muscle damage and improve the quality of life for those affected by this aggressive disorder.
Medical Director Comments
Gene editing is a potentially transformative technology that is in very early stages of development. In broad terms, gene editing refers to a host of methods that repair genes by disrupting it or rewriting its sequence. Other technologies such as “read-through” therapies are in more advanced stages of development and are pending the results of human clinical trials. For example, the investigational oral drug ataluren is in development to treat Duchenne muscular dystrophy (DMD) resulting from nonsense mutations. The era of precision medicine is being heralded by treatments that are not necessarily disease specific but are targeted to the molecular mechanisms caused by pathogenic variants.
1) Ousterout DG, Kabadi AM, Thakore PI, et al. Correction of Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients Through Genomic Excision of Exon 51 by Zinc Finger Nucleases. Mol Ther. 2015; ePub ahead of print.
2) Bushby K, Finkel R, Birnkrant DJ, et al. Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management. Lancet Neurol. 2010; 9 (1): 77-93.
4) Centers for Disease Control. Prevalence of Duchenne/Becker Muscular Dystrophy Among Males Aged 5–24 Years — Four States, 2007. MMWR. 2009; 58 (40): 1119-1122.
6) Wang Z, Storb R, Halbert CL, et al. Successful regional delivery and long-term expression of a dystrophin gene in canine muscular dystrophy: a preclinical model for human therapies. Mol Ther. 2012; 20 (8): 1501-1507.
8) Cirak S, Arechavala-Gomeza V, Guglieri M, et al. Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet. 2011; 378 (9791): 595-605.
10) Carroll D. Genome engineering with targetable nucleases. Annu Rev Biochem. 2014; 83: 409-439.