Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by reading frame disrupting mutations in the DMD gene leading to absence of functional dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach aimed at restoring the reading frame at the pre-mRNA level, allowing the production of internally truncated partly functional dystrophin proteins. AONs work in a sequence specific manner, which warrants generating humanized mouse models for preclinical tests. To address this, we previously generated the hDMDdel52/mdx mouse model using transcription activator like effector nuclease (TALEN) technology. This model contains mutated murine and human DMD genes, and therefore lacks mouse and human dystrophin resulting in a dystrophic phenotype. It allows preclinical evaluation of AONs inducing the skipping of human DMD exons 51 and 53 and resulting in restoration of dystrophin synthesis. Here, we have further characterized this model genetically and functionally. We discovered that the hDMD and hDMDdel52 transgene is present twice per locus, in a tail-to-tail-orientation. Long-read sequencing revealed a partial deletion of exon 52 (first 25 bp), and a 2.3 kb inversion in intron 51 in both copies. These new findings on the genomic make-up of the hDMD and hDMDdel52 transgene do not affect exon 51 and/or 53 skipping, but do underline the need for extensive genetic analysis of mice generated with genome editing techniques to elucidate additional genetic changes that might have occurred. The hDMDdel52/mdx mice were also evaluated functionally using kinematic gait analysis. This revealed a clear and highly significant difference in overall gait between hDMDdel52/mdx mice and C57BL6/J controls. The motor deficit detected in the model confirms its suitability for preclinical testing of exon skipping AONs for human DMD at both the functional and molecular level.

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Duchenne muscular dystrophy (DMD) is a lethal muscle disease, caused by mutations in the DMD gene, encoding a protein that links the cytoskeleton and the extracellular matrix in skeletal and cardiac muscles. The primary consequences of disruption of the link due to lack of functional dystrophin involved sarcolemma destabilization, perturbation of Ca 2 + homeostasis, activation of proteases, mitochondrial damage, leading to tissue degeneration. A recently emphasized secondary aspect of the dystrophic process is a progressive metabolic change of the dystrophic tissue. However, the mechanism and nature of the metabolic dysregulation are yet poorly understood. In the present study, we characterized a molecular mechanism of metabolic perturbation in DMD. We sequenced plasma miRNA in a DMD cohort, comprising of 54 DMD patients treated or not by glucocorticoid, compared to 27 healthy controls, in three age groups. We identified 96 dysregulated miRNAs, 74 up and 22 down in DMD. We confirmed the dysregulation in DMD of the Dystro-miRs, Cardio-miRs and a large number of the DLK1-DIO3 miRNAs. We also identified numerous dysregulated miRNAs, yet unreported in DMD. We developed an original dual bioinformatics approach, based on both miRNAs' target and host genes, for the biological interpretation of miRNA dysregulation. This analysis predicted that dysregulation of lipid metabolism has a central role in DMD. Investigation of skeletal muscles of the mdx mouse, a model of DMD, revealed dysregulation of SREBP1 and SREBP2, two key transcription factors of cholesterol and fatty acid metabolism, perturbation of the mevalonate pathway, and accumulation of cholesterol. Elevated cholesterol was also found in biopsies of DMD patients. Treatment of mdx mice with Simvastatin, a hypolipidemic statin, normalized these perturbations and partially restored dystrophic parameters. This investigation supports that cholesterol metabolism and the mevalonate pathway are therapeutic targets in DMD.