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.

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 slowdown or prevent disease progression and improve overall muscle function once a dystrophic environment has been established in the mdx mouse model.

Duchenne muscular dystrophy (DMD) is caused by absence of the integral structural protein, dystrophin, which renders muscle fibres susceptible to injury and degeneration. This ultimately results in cardiorespiratory dysfunction, which is the predominant cause of death in DMD patients, and highlights the importance of therapeutic targeting of the cardiorespiratory system. While there is some evidence to suggest that restoring dystrophin in the diaphragm improves both respiratory and cardiac function, the role of the diaphragm is not well understood. Here using exon skipping oligonucleotides we predominantly restored dystrophin in the diaphragm and assessed cardiac function by MRI. This approach reduced diaphragmatic pathophysiology and markedly improved diaphragm function but did not improve cardiac function or pathophysiology, with or without exercise. Interestingly, exercise resulted in a reduction of dystrophin protein and exon skipping in the diaphragm. This suggests that treatment regimens may require modification in more active patients. In conclusion, whilst the diaphragm is an important respiratory muscle, it is likely that dystrophin needs to be restored in other tissues, including multiple accessory respiratory muscles, and of course the heart itself for appropriate therapeutic outcomes. This supports the requirement of a body-wide therapy to treat DMD.

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.

Antisense directed exon-skipping has been shown to be a powerful method to induce expression of dystrophin in dystrophic muscle for a range of species including man. Dystrophin expression can be induced in the mdx mouse by oligonucleotides targeting exon 23 which removes the premature stop mutation in the murine dystrophin gene. We had previously shown improved resistance to eccentric exercise (10% length change of the active muscle) following intramuscular delivery of a phosphorodiamidate morpholino oligomer (PMO) targeting exon 23 in the mdx mouse (Sharp et al., 2011). Little improvement was seen in specific force which is commonly only 60% of wild-type in the mdx mouse. We now report similar physiology results following a single intravenous treatment with a peptide-PMO (PPMO) using the Pip6a peptide. Importantly, repeated intravenous PPMO treatment every two weeks starting at 12 weeks old and continuing for 10 doses, completely prevented the force drop associated with eccentric exercise and significantly improved the specific force, albeit not quite to wild-type levels. Both the acute and chronic dosing results correlated with the increased expression of dystrophin in muscle fibres and decreased levels of TIMP-1 in the serum. The chronic dosing study also showed a reduction in inflammation, restoration of a more normal fibre type pattern and reduction in fibre size variability. These studies show the potential of the PPMO formulation to significantly improve the results of exon-skipping in clinical trials. Antisense directed exon-skipping has been shown to be a powerful method to induce expression of dystrophin in dystrophic muscle for a range of species including man. Dystrophin expression can be induced in the mdx mouse by oligonucleotides targeting exon 23 which removes the premature stop mutation in the murine dystrophin gene. We had previously shown improved resistance to eccentric exercise (10% length change of the active muscle) following intramuscular delivery of a phosphorodiamidate morpholino oligomer (PMO) targeting exon 23 in the mdx mouse (Sharp et al., 2011). Little improvement was seen in specific force which is commonly only 60% of wild-type in the mdx mouse. We now report similar physiology results following a single intravenous treatment with a peptide-PMO (PPMO) using the Pip6a peptide. Importantly, repeated intravenous PPMO treatment every two weeks starting at 12 weeks old and continuing for 10 doses, completely prevented the force drop associated with eccentric exercise and significantly improved the specific force, albeit not quite to wild-type levels. Both the acute and chronic dosing results correlated with the increased expression of dystrophin in muscle fibres and decreased levels of TIMP-1 in the serum. The chronic dosing study also showed a reduction in inflammation, restoration of a more normal fibre type pattern and reduction in fibre size variability. These studies show the potential of the PPMO formulation to significantly improve the results of exon-skipping in clinical trials.

Duchenne muscle dystrophy is an X-linked muscle wasting disorder caused by mutations in the DMD gene preventing the production of a functional dystrophin protein. The lack of dystrophin de-stabilises the dystrophin associated protein complex (DAPC) and consequently DMD muscles are more susceptible to exercised induced muscle damage. We hypothesised stabilising the DAPC at the sarcolemma in mdx mice may reduce muscle susceptibility to exercise induced damage and consequently ameliorate muscle pathology. To evaluate this we crossed mice over-expressing the putative glycotransferase, LARGE, which facilitates the binding of alpha dystroglycan to laminin in the extracellular matrix, with mdx mice. The presence of the LARGE transgene in the LV5/mdx mice was established through genotyping and expression was confirmed through immunostaining. Pathological findings characterised by myofibre size variation, increased central nucleation, presence of inflammatory cells and calcium deposits was more serve in 8-week old LV5/mdx mice quadriceps and diaphragm muscles compared to mdx littermate controls. To determine if the over-expression of the LARGE transgene initiated muscle pathology early we analysed 3-week old quadriceps muscles. Stark degeneration was observed in LV5/mdx vastus muscles, in contrast to the rectus femoris which was well preserved. Few pathological findings were observed in the age matched mdx littermate controls. We also assessed the effect of over-expression of LARGE on muscle function using a standardised in vivo exercise test on the tibialis anterior muscle in situ. A significant drop in maximum force produced following eccentric contractions was observed in 22-week old LV5/mdx mice compared to littermate controls. Overall we show the over-expression of LARGE exacerbates DMD pathology in the mdx mice. Further work to establish an underlining mechanism for the deleterious effect of over-expressing LARGE in mdx mice is currently ongoing.

Antisense oligonucleotide (AON) therapy has been shown to be an effective treatment for restoring dystrophin expression in mdx mice and Duchenne muscular dystrophy (DMD) patients with out-of-frame mutations. However, to date, limited exon skipping studies have been conducted in DMD patients with large out-of-frame duplication mutations. Whilst exon-skipping one or two exons is a highly efficient process in cell culture, efficiency falls when targeting multiple exons, thus producing little of the desired end product. Therefore, we sought to understand the expression and function of large DMD duplication proteins when only two exons are removed in order to restore the reading frame. We constructed large “in-frame” DMD duplication mutation plasmids and evaluated their expression in vivo. Two out-of-frame DMD duplication mutations (dup ex22–29 and dup ex18–30) were modified to express in-frame human dystrophin transcripts (del ex21–22 dup ex23–29 and del ex17–18 dup ex19–30). In addition, an original “in-frame” DMD duplication mutation (dup ex3–25) was cloned to determine if over expression of the duplicated transcript would result in protein expression. Plasmids were electroporated into TA muscles of mdx mice, and human dystrophin expression was evaluated 7days later. Human dystrophin expression was detected at the sarcolemma for all the constructs, confirming the duplicated proteins were stable. In addition, all three proteins recruited the nNOs protein. Whilst this study indicates DMD patients with out-of-frame duplication mutations may be suitable candidates for AON therapy, further work to analyse the protective potential of restored in-frame duplication mutations is necessary. We are currently investigating DMD duplication protein function through the exclusion of Evans blue dye post treadmill exercises. In addition, plasmids will be administrated into neonatal mdx mice to assess the ability of the constructs to prevent muscle fibre degeneration.

Mutations in fukutin related protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms. We have now generated a mouse with a knock-down in Fkrp expression levels in the skeletal muscle but not the central nervous system (Sox1 Cre FKRPKD). The skeletal muscle of this mouse shows a marked reduction in glycosylated αdystroglycan and develops a clear muscle phenotype by 12 weeks of age. Previous work has shown that the over-expression of LARGE induces the hyperglycosylation of α-dystroglycan in both wild type and in cells from dystroglycanopathy patients, irrespective of their primary gene defect, strongly suggesting that LARGE could be an important therapeutic approach in these disorders. As a first step to confirming this on a disease background in vivo, we have now crossed the Sox1Cre FKRPKD line with one over-expressing LARGE. We present here our histological and physiological evaluation of the resulting phenotype.

The Sleeping beauty (SB) system is a non-viral DNA based vector that has been used to stably integrate therapeutic genes into disease models. Here we report the SB system is capable of stably integrating the ΔR4-R23/CTΔ micro-dystrophin gene into a conditionally immortal dystrophin deficient muscle cell-line, H2K SF1, a murine cell model for Duchenne muscular dystrophy. Genetically corrected H2K SF1 cells retained their myogenic properties in vitro. Moreover, upon transplantation ΔR4-R23/CTΔ micro-dystrophin expression was detected within mdx nu/nu mice. Our data suggests the SB system is an effective way of stably integrating therapeutic genes into myogenic cells.

A new conditionally immortal satellite cell-derived cell-line, H2K 2B4, was generated from the H2Kb-tsA58 immortomouse. Under permissive conditions H2K 2B4 cells terminally differentiate in vitro to form uniform myotubes with a myogenic protein profile comparable with freshly isolated satellite cells. Following engraftment into immunodeficient dystrophin-deficient mice, H2K 2B4 cells regenerated host muscle with donor derived myofibres that persisted for at least 24 weeks, without forming tumours. These cells were readily transfectable using both retrovirus and the non-viral transfection methods and importantly upon transplantation, were able to reconstitute the satellite cell niche with functional donor derived satellite cells. Finally using the Class II DNA transposon, Sleeping Beauty, we successfully integrated a reporter plasmid into the genome of H2K 2B4 cells without hindering the myogenic differentiation. Overall, these data suggest that H2K 2B4 cells represent a readily transfectable stable cell-line in which to investigate future stem cell based therapies for muscle disease.

Antisense mediated exon skipping to restore the open reading frame in dystrophin transcripts is a promising approach to therapy for Duchenne muscular dystrophy based on the results to date from cell culture, animal models and human clinical trials. It is also clear that whilst it is possible to skip one or two exons at relatively high efficiency, as is required to restore the open reading frame in patients with deletions and point mutations, it is considerably harder to efficiently skip multiple exons. Thus it is unlikely that exon skipping can be used to remove all of the excess exons in the larger DMD duplications. To assess the potential of limited exon skipping for the treatment of DMD duplications we have generated plasmid constructs containing in frame duplications and have tested these by gene transfer into mdx muscle and analysis by immunostaining for members of the dystrophin associated complex. We demonstrate that at least some of these extra-large dystrophins are functional.