Exon skipping with morpholino oligomers: new treatment option for cardiomyopathy in Duchenne muscular dystrophy?

The dystrophin gene encodes an essential component of the transmembrane dystrophin–glycoprotein complex (DGC), which plays a critical role in maintaining membrane stability in cardiac and skeletal muscles. Defects in dystrophin destabilize the entire complex, which results in an abnormal susceptibility to sarcolemmal injury in response to contractile stress. Mutations that cause slight sequence alterations or loss of internal exons but conserve the reading frame result in Becker muscular dystrophy with a mild phenotype. Complete absence of dystrophin causes Duchenne muscular dystrophy (DMD), a X-chromosomal, fatal, and inherited muscle disease. Affected boys show progressive muscle weakness, leading to early immobility, respiratory failure, and markedly reduced life expectancy. Cardiomyopathy is an almost invariable complication and a frequent cause of death in these patients. As a consequence of improvements in overall management for DMD patients, survival has steadily improved. In older patients, however, there is an increasing proportion of patients experiencing premature death due to ventricular dysfunction. Therefore, appropriate treatment strategies to target the cardiomyopathy become more and more crucial to reduce mortality. Thus far, there is no established curative approach to DMD. However, there now seems to be hope since the identification of the molecular basis of this inherited disease has translated into recent therapeutic principles to causally treat dystrophin deficiency: cell replacement (myoblasts or stem-cells), pharmacological approaches to induce ribosomal readthrough of premature termination codons, viral gene transfer with micro/mini-dystrophin cDNA, and antisense oligonucleotide-mediated exon skipping are the most promising treatment strategies. 10 Although these therapies have been shown to restore dystrophin expression locally in dystrophic skeletal muscles, they may leave the cardiomyopathy essentially untreated. Currently, the approach of exon skipping is especially considered to have the potential for efficient treatment of boys with DMD. Antisense oligonucleotide (AON)-mediated exon skipping functions to restore the open-reading frame by removing specific exons from the altered dystrophin transcript, creating shortened, but functional, proteins that should be able to convert the Duchenne into a Becker phenotype. Recently, two different types of AONs have successfully been tested in a small number of patients with DMD: AONs of a 20-O-methyl phosphorothioate RNA chemistry or phosphorodiamidate morpholino oligomers (PMOs). In these first clinical proof-of-concept studies, local intramuscular injections of AONs appeared to be safe and to have induced local expression of a significant amount of dystrophin protein in defined ‘isolated’ muscles. However, an improvement of morbidity or mortality probably requires treatment of comprehensive muscle groups with higher doses of the respective agent. Thus far, systemic applications of AONs have only been tested in animal models of DMD. Weekly intravenous injections of PMOs into dystrophin-deficient mdx mice, a murine model of DMD with a nonsense-mutation in exon 23 of the dystrophin gene, resulted in body-wide expression of functional levels of dystrophin in skeletal muscles. However, these effects were disappointingly absent in cardiac muscle of these mice. This might be a crucial limitation of the treatment with AONs, especially if one bears in mind that an increase in physical activity by targeted repair of skeletal muscles in mdx mice worsens cardiac injury and dilated cardiomyopathy. Jearawiriyapaisarn et al. report on a new strategy for using PMOs. On the basis of the recent observation that conjugation of arginine-rich, cell-penetrating peptides to PMOs allows an efficient cardiac transfer, 15 the authors investigated the effect of a peptideconjugated PMO (PPMO AVI-5225) on morphological alterations and contractile function in mdx mouse hearts. Skipping of exon 23 induced by PPMO AVI-5225 achieved cardiac expression of a shortened but functional dystrophin protein sufficient to ameliorate sarcolemmal damage, hypertrophy, and diastolic dysfunction in the hearts of mdx mice. The authors highlight that the combination of positively charged peptides with neutral oligomers that sequence-specifically target pre-mRNA is responsible for the effective cardiac expression