Duchenne muscular dystrophy (DMD) represents the most frequent hereditary childhood myopathy, leading to progressive muscle degeneration and weakness, and to premature death due to respiratory and cardiac failure. The X-chromosomal location of DMD renders 1 in 3,500 to 5,000 male newborns affected.
The vast majority of patients carry frameshift mutations in the DMD gene encoding dystrophin (DMD), which are mainly exon deletions. Recently, Crispr/Cas9-based gene editing strategies have demonstrated an efficient and permanent genomic correction in murine mdx models. Moreover, intravenous (i.v.) application of AAV9 delivering CRISPR/Cas9 components in a beagle model of DMD (exon 50 deficiency) proved successful in restoring expression of a shortened dystrophin in various muscles, including the heart. However, functional data have not been reported as of yet.
In a consortium of DZHK-scientists from Internal Medicine at TUM as well as LMU and Helmholtz-Munich, pigs of a transgenic strain lacking dystrophin exon 52 were investigated with regard to skeletal muscle function and electrophysiological stability of the heart. The researchers were able to demonstrate that a systemic infusion of AAV9-Crispr-Cas9 combined with appropriate guide RNAs sufficed to express a shortened, but stable dystrophin in many muscles, including the diaphragm and heart.
Moreover, high-resolution electrophysiological mapping in the diseased pigs, which died of sudden cardiac death no later than 105 days after birth, revealed a large area of vulnerable myocardium. This area was found reduced by the AAV9-Cas9-gRNA treatment, extending life span of the treatment group significantly. Consistently, human iPS-derived cardiomyocytes subjected to analogous AAV6-Cas9-gRNA transduction demonstrated dystrophin expression combined with a normalization of calcium handling. These findings demonstrate in preclinical large animal experiments, that gene editing by Crispr-Cas9 in DMD is capable of inducing dystrophin expression, and improving heart and skeletal muscle functions.