How much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse

Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly evaluate functional improvement, we used in situ protocols in the mdx mouse to measure muscle strength and resistance to eccentric contraction-induced damage. Here, we modelled the treatment of muscle with pre-existing dystrophic pathology using antisense oligonucleotides conjugated to a cell-penetrating peptide. We reveal that 15% homogeneous dystrophin expression is sufficient to protect against eccentric contraction-induced injury. In addition, we demonstrate a >40% increase in specific isometric force following repeated administrations. Strikingly, we show that changes in muscle strength are proportional to dystrophin expression levels. These data define the dystrophin restoration levels required to slow down or prevent disease progression and improve overall muscle function once a dystrophic environment has been established in the mdx mouse model

Cmah-dystrophin deficient mdx mice display an accelerated cardiac phenotype that is improved following peptide-PMO exon skipping treatment

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin protein, leading to progressive muscle weakness and premature death due to respiratory and/or cardiac complications. Cardiac involvement is characterized by progressive dilated cardiomyopathy, decreased fractional shortening and metabolic dysfunction involving reduced metabolism of fatty acids—the major cardiac metabolic substrate. Several mouse models have been developed to study molecular and pathological consequences of dystrophin deficiency, but do not recapitulate all aspects of human disease pathology and exhibit a mild cardiac phenotype. Here we demonstrate that Cmah (cytidine monophosphate-sialic acid hydroxylase)-deficient mdx mice (Cmah−/−;mdx) have an accelerated cardiac phenotype compared to the established mdx model. Cmah−/−;mdx mice display earlier functional deterioration, specifically a reduction in right ventricle (RV) ejection fraction and stroke volume (SV) at 12 weeks of age and decreased left ventricle diastolic volume with subsequent reduced SV compared to mdx mice by 24 weeks. They further show earlier elevation of cardiac damage markers for fibrosis (Ctgf), oxidative damage (Nox4) and haemodynamic load (Nppa). Cardiac metabolic substrate requirement was assessed using hyperpolarized magnetic resonance spectroscopy indicating increased in vivo glycolytic flux in Cmah−/−;mdx mice. Early upregulation of mitochondrial genes (Ucp3 and Cpt1) and downregulation of key glycolytic genes (Pdk1, Pdk4, Ppara), also denote disturbed cardiac metabolism and shift towards glucose utilization in Cmah−/−;mdx mice. Moreover, we show long-term treatment with peptide-conjugated exon skipping antisense oligonucleotides (20-week regimen), resulted in 20% cardiac dystrophin protein restoration and significantly improved RV cardiac function. Therefore, Cmah−/−;mdx mice represent an appropriate model for evaluating cardiac benefit of novel DMD therapeutics

THE PHYSIOLOGICAL CONSEQUENCES OF DIFFERENT LEVELS OF DYSTROPHIN FOLLOWING ANTISENSE BASED EXON-SKIPPING IN THE MDX MOUSE

We examined the effects on muscle physiology of restoring different levels of dystrophin in mdx mice with established dystrophic pathophysiology (12 weeks and older). Dystrophin expression was induced very efficiently using cell penetrating peptides linked to an antisense sequencing targeting exon 23 which contains a premature stop mutation. We assessed muscle physiology in the tibialis anterior (TA) muscle of the mouse using a terminally anaesthetised in situ protocol. To assess muscle physiology in the diaphragm we used strips of diaphragm in an in-vitro system. In both cases we examined the force–frequency relationship and established maximum specific tetanic force. We then subjected the muscles to a 10% stretch while stimulating them to contract. This eccentric exercise was highly damaging to dystrophic muscle. We present data showing that 15% of normal levels of dystrophin were sufficient to prevent eccentric exercise induced damage following a single dose of Pip6a-PMO. Chronic intravenous (IV) administration had a cumulative effect and we show that restoration of 50% of normal levels of dystrophin produced a 40% improvement in maximum specific force. Intraperitoneal administration of a single dose of B-PMO produced an 88% increase in maximum specific force as well as protecting against eccentric exercise induced damage in the diaphragm. Similar results were obtained in the diaphragm with chronic IV delivery of Pip6a-PMO at the same dose as the studies in the TA, even when treating older mice with extensive fibrosis in the diaphragm. While caution must be applied when extrapolating these results to DMD patients, the results suggest that moderate levels of dystrophin may be sufficient to slow-down or possibly prevent disease progression whereas higher levels of dystrophin will also improve muscle force production