Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder associated with lethal degeneration of cardiac and skeletal muscle. DMD affects approximately 1 in 5000 males and is caused by more than 3000 different mutations in the X-linked dystrophin gene (DMD). These mutations are highly varied and include large deletions or duplications, point mutations, and other small mutations. Given that thousands of assortment of mutations cause DMD, genome editing approaches that traditionally target a single genetic location have faced a quandary with DMD.
Despite the diversity of DMD mutations, many of these mutations are clustered in specific “hotspot” areas of the gene. These “hotspots” (exons 45 to 55 and exons 2 to 10) are organized such that skipping a few exons within or nearby the hotspots can rescue dystrophin function in a majority (~60%) of DMD patients. This clever approach exploits CRISPR-Cas9 to target sites that induce skipping of the most problematic exons. CRISPR-Cas9 was directed to destroy conserved splice acceptor or donor sites preceding DMD mutations or to bypass mutant or out-of-frame exons. The resulting insertion/deletion mutations generated by nonhomologous end joining potentially allow skipping of the most common mutant or out-of-frame DMD exons within or nearby mutational hotspots, thereby allowing splicing between surrounding exons to recreate in-frame dystrophin proteins lacking the mutations. The correction of DMD mutations by exon skipping was termed “myoediting.”
Myoediting was tested in patient-derived induced pluripotent stem cells (iPSCs) with varied mutations within the DMD gene. By abolishing RNA splice sites and removing the most common mutations, myoediting was able to efficiently restore dystrophin protein expression in derivative cardiomyocytes. Furthermore, in three-dimensional engineered heart muscle, myoediting of DMD mutations restored dystrophin expression and the corresponding mechanical force of contraction. Altogether, this genome editing approach represents a promising means of addressing the complex genetic basis of DMD.
The article titled, “Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing,” was published in Science Advances.