Duchenne muscular dystrophy (DMD) is a severe progressive muscular disorder caused by reading frame disrupting mutations in the DMD gene, preventing the synthesis of functional dystrophin. As dystrophin provides muscle fiber stability during contractions, dystrophin negative fibers are prone to exercise-induced damage. Upon exhaustion of the regenerative capacity, fibers will be replaced by fibrotic and fat tissue resulting in a progressive loss of function eventually leading to death in the early thirties. With several promising approaches for the treatment of DMD aiming at dystrophin restoration in clinical trials, there is an increasing need to determine more precisely which dystrophin levels are sufficient to restore muscle fiber integrity, protect against muscle damage and improve muscle function.To address this we generated a new mouse model (mdx-Xist Δhs) with varying, low dystrophin levels (3–47%, mean 22.7%, stdev 12.1, n = 24) due to skewed X-inactivation. Longitudinal sections revealed that within individual fibers, some nuclei did and some did not express dystrophin, resulting in a random, mosaic pattern of dystrophin expression within fibers. Mdx-Xist Δhs, mdx and wild type females underwent a 12 week functional test regime consisting of different tests to assess muscle function at base line, or after chronic treadmill running exercise. Overall, mdx-Xist Δhs mice with 3–14% dystrophin outperformed mdx mice in the functional tests. Improved histopathology was observed in mice with 15–29% dystrophin and these levels also resulted in normalized expression of pro-inflammatory biomarker genes, while for other parameters >30% of dystrophin was needed. Chronic exercise clearly worsened pathology, which needed dystrophin levels >20% for protection. Based on these findings, we conclude that while even dystrophin levels below 15% can improve pathology and performance, levels of >20% are needed to fully protect muscle fibers from exercise-induced damage.

Our study shows that dystrophin levels appear not to be a major determinant of disease severity in BMD, as long as it is above approximately 10%. A significant relation between age and disease course was only found in the exon 45-47 deletion subgroup. This suggests that at higher dystrophin levels, the disease course depends more on the mutation site than on the amount of the dystrophin protein produced.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by the lack of functional dystrophin. There is no cure, but several clinical trials aimed to restore the synthesis of functional dystrophin are underway. The dystrophin levels needed for improvement of muscle pathology, function, and overall vitality are not known. Here, we describe the mdx/utrn(-/-)/Xist(Δhs) mouse model, which expresses a range of low dystrophin levels, depending on the degree of skewing of X inactivation in a utrophin-negative background. Mdx/utrn(-/-) mice develop severe muscle weakness, kyphosis, respiratory and heart failure, and premature death closely resembling DMD pathology. We show that at dystrophin levels < 4%, survival and motor function in these animals are greatly improved. In mice expressing >4% dystrophin, histopathology is ameliorated, as well. These findings suggest that the dystrophin levels needed to benefit vitality and functioning of patients with DMD might be lower than those needed for full protection against muscle damage.

The genetic defect of mdx mice resembles that of Duchenne muscular dystrophy, although their functional performance and life expectancy is nearly normal. By contrast, mice lacking utrophin and dystrophin (mdx/utrn -/-) are severely affected and die prematurely. Mice with one utrophin allele (mdx/utrn +/-) are more severely affected than mdx mice, but outlive mdx/utrn -/- mice. We subjected mdx/utrn +/+, +/-, -/- and wild type males to a 12week functional test regime of four different functional tests. Mdx/utrn +/+ and +/- mice completed the regime, while mdx/utrn -/- mice died prematurely. Mdx/utrn +/- mice performed significantly worse compared to mdx/utrn +/+ mice in functional tests. Creatine kinase levels, percentage of fibrotic/necrotic tissue, morphology of neuromuscular synapses and expression of biomarker genes were comparable, whereas mdx/utrn +/- and -/- mice had increased levels of regenerating fibers. This makes mdx/utrn +/- mice valuable for testing the benefit of potential therapies on muscle function parameters.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON)-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.

Background Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by genetic loss of dystrophin protein. Extracellular microRNAs (ex-miRNAs) are putative, minimally invasive biomarkers of DMD. Specific ex-miRNAs (e.g. miR-1, miR-133a, miR-206, and miR-483) are highly up-regulated in the serum of DMD patients and dystrophic animal models and are restored to wild-type levels following exon skipping-mediated dystrophin rescue in mdx mice. As such, ex-miRNAs are promising pharmacodynamic biomarkers of exon skipping efficacy. Here, we aimed to determine the degree to which ex-miRNA levels reflect the underlying level of dystrophin protein expression in dystrophic muscle. Methods Candidate ex-miRNA biomarker levels were investigated in mdx mice in which dystrophin was restored with peptide-PMO (PPMO) exon skipping conjugates and in mdx-Xist Δhs mice that express variable amounts of dystrophin from birth as a consequence of skewed X-chromosome inactivation. miRNA profiling was performed in mdx-Xist Δhs mice using the FirePlex methodology and key results validated by small RNA TaqMan RT-qPCR. The muscles from each animal model were further characterized by dystrophin western blot and immunofluorescence staining. Results The restoration of ex-myomiR abundance observed following PPMO treatment was not recapitulated in the high dystrophin-expressing mdx-Xist Δhs group, despite these animals expressing similar amounts of total dystrophin protein (~37% of wild-type levels). Instead, ex-miRNAs were present at high levels in mdx-Xist Δhs mice regardless of dystrophin expression. PPMO-treated muscles exhibited a uniform pattern of dystrophin localization and were devoid of regenerating fibres, whereas mdx-Xist Δhs muscles showed non-homogeneous dystrophin staining and sporadic regenerating foci. Conclusions Uniform dystrophin expression is required to prevent ex-miRNA release, stabilize myofiber turnover, and attenuate pathology in dystrophic muscle.

Duchenne muscular dystrophy is caused by mutations that prevent synthesis of functional dystrophin. All patients develop dilated cardiomyopathy. Promising therapeutic approaches are underway that successfully restore dystrophin expression in skeletal muscle. However, their efficiency in the heart is limited. Improved quality and function of only skeletal muscle potentially accelerate the development of cardiomyopathy. Our study aimed to elucidate which dystrophin levels in the heart are required to prevent or delay cardiomyopathy in mice. Heart function and pathology assessed with magnetic resonance imaging and histopathological analysis were compared between 2, 6 and 10-month-old female mdx-Xist(Δhs) mice, expressing low dystrophin levels (3-15%) in a mosaic manner based on skewed X-inactivation, dystrophin-negative mdx mice, and wild type mice of corresponding genetic backgrounds and gender. With age mdx mice developed dilated cardiomyopathy and hypertrophy, whereas the onset of heart pathology was delayed and function improved in mdx-Xist(Δhs) mice. The ejection fraction, the most severely affected parameter for both ventricles, correlated to dystrophin expression and the percentage of fibrosis. Fibrosis was partly reduced from 9.8% in mdx to 5.4% in 10 month old mdx-Xist(Δhs) mice. These data suggest that mosaic expression of 4-15% dystrophin in the heart is sufficient to delay the onset and ameliorate cardiomyopathy in mice.

This study provides mutation specific data on the course of disease in patients with BMD. It shows that the disease course of patients with BMD, with a mutation equalling a 'skipped' DMD mutation is relatively mild. This finding strongly supports the potential benefit of exon skipping in patients with DMD.