Skeletal muscle symptoms strongly contribute to mortality of myotonic dystrophy type 1 (DM1) patients. DM1 is a neuromuscular genetic disease caused by CTG repeat expansions that, upon transcription, sequester the Muscleblind-like family of proteins and dysregulate alternative splicing of hundreds of genes. However, mis-splicing does not satisfactorily explain muscle atrophy and wasting, and several other contributing factors have been suggested, including hyperactivated autophagy leading to excessive catabolism. MicroRNA ( miR ) -7 has been demonstrated to be necessary and sufficient to repress the autophagy pathway in cell models of the disease, but the origin of its low levels in DM1 was unknown. We have found that the RNA-binding protein Musashi-2 (MSI2) is upregulated in patient-derived myoblasts and biopsy samples. Because it has been previously reported that MSI2 controls miR-7 biogenesis, we tested the hypothesis that excessive MSI2 was repressing miR-7 maturation. Using gene-silencing strategies (small interfering RNAs [siRNAs] and gapmers) and the small molecule MSI2-inhibitor Ro 08-2750, we demonstrate that reducing MSI2 levels or activity boosts miR-7 expression, represses excessive autophagy, and downregulates atrophy-related genes of the UPS system. We also detect a significant upregulation of MBNL1 upon MSI2 silencing. Taken together, we propose MSI2 as a new therapeutic target to treat muscle dysfunction in DM1.

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient’s immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and β-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

Duchenne muscular dystrophy (DMD) exon 45-55 deletion (del45-55) has been postulated as a model that could treat up to 60% of DMD patients, but the associated clinical variability and complications require clarification. We aimed to understand the phenotypes and potential modifying factors of this dystrophinopathy subset. Methods: This cross-sectional, multicenter cohort study applied clinical and functional evaluation. Next generation sequencing was employed to identify intronic breakpoints and their impact on the Dp140 promotor, intronic long noncoding RNA, and regulatory splicing sequences. DMD modifiers (SPP1, LTBP4, ACTN3) and concomitant mutations were also assessed. Haplotypes were built using DMD single nucleotide polymorphisms. Dystrophin expression was

Background Laing myopathy is characterized by broad clinical and pathological variability. They are limited in number and protocol of study. We aimed to delineate muscle imaging profiles and validate imaging analysis as an outcome measure. Methods This was a cross‐sectional and longitudinal cohort study. Data from clinical, functional and semi‐quantitative muscle imaging (60 magnetic resonance imaging [MRI] and six computed tomography scans) were studied. Hierarchical analysis, graphic heatmap representation and correlation between imaging and clinical data using Bayesian statistics were carried out. Results The study cohort comprised 42 patients from 13 families harbouring five MYH7 mutations. The cohort had a wide range of ages, age at onset, disease duration, and myopathy extension and Gardner‐Medwin and Walton (GMW) functional scores. Intramuscular fat was evident in all but two asymptomatic/pauci‐symptomatic patients. Anterior leg compartment muscles were the only affected muscles in 12% of the patients. Widespread extension to the thigh, hip, paravertebral and calf muscles and, less frequently, the scapulohumeral muscles was commonly observed, depicting distinct patterns and rates of progression. Foot muscles were involved in 40% of patients, evolving in parallel to other regions with absence of a disto‐proximal gradient. Whole cumulative imaging score, ranging from 0 to 2.9 out of 4, was associated with disease duration and with myopathy extension and GMW scales. Follow‐up MRI studies in 24 patients showed significant score progression at a variable rate. Conclusions We confirmed that the anterior leg compartment is systematically affected in Laing myopathy and may represent the only manifestation of this disorder. However, widespread muscle involvement in preferential but variable and not distance‐dependent patterns was frequently observed. Imaging score analysis is useful to categorize patients and to follow disease progression over time.

CRISPR/Cas9-mediated gene editing may allow treating and studying rare genetic disorders by respectively, correcting disease mutations in patients, or introducing them in cell cultures. Both applications are highly dependent on Cas9 and sgRNA delivery efficiency. While gene editing methods are usually efficiently applied to cell lines such as HEK293 or hiPSCs, CRISPR/Cas9 editing in vivo or in cultured myoblasts prove to be much less efficient, limiting its use. After a careful optimisation of different steps of the editing protocol, we established a consistent approach to generate human immortalised myoblasts disease models through CRISPR/Cas9 editing. Using this protocol we successfully created a coding deletion of exon 52 of the DYSTROPHIN (DMD) gene in wild type immortalised myoblasts modelling Duchenne muscular dystrophy (DMD), and a microRNA binding sites deletion in the regulatory region of the UTROPHIN (UTRN) gene leading to utrophin upregulation in in Duchenne muscular dystrophy patient immortalised cultures. Sanger sequencing confirmed the presence of the corresponding genomic alterations and protein expression was characterised using myoblots. To show the utility of these cultures as platforms for assessing the efficiency of DMD treatments, we used them to evaluate the impact of exon skipping therapy and ezutromid treatment. Our editing protocol may be useful to others interested in genetically manipulating myoblasts and the resulting edited cultures for studying DMD disease mechanisms and assessing therapeutic approaches.SummaryWe report two novel immortalised myoblast culture models for studying Duchenne muscular dystrophy (DMD), generated through CRISPR/Cas9 gene editing: one recapitulates a common DYSTROPHIN (DMD) deletion and the other a regulatory mutation leading to UTROPHIN (UTRN) ectopic upregulation.

Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by expansion of unstable CTG repeats in a non-coding region of the DMPK gene. CUG expansions in mutant DMPK transcripts sequester MBNL1 proteins in ribonuclear foci. Depletion of this protein is a primary contributor to disease symptoms such as muscle weakness and atrophy and myotonia, yet upregulation of endogenous MBNL1 levels may compensate for this sequestration. Having previously demonstrated that antisense oligonucleotides against miR-218 boost MBNL1 expression and rescue phenotypes in disease models, here we provide preclinical characterization of an anta-gomiR-218 molecule using the HSA LR mouse model and patient-derived myotubes. In HSA LR , antagomiR-218 reached 40-60 pM 2 weeks after injection, rescued molecular and functional phenotypes in a dose-and time-dependent manner, and showed a good toxicity profile after a single subcutaneous administration. In muscle tissue, antagomiR rescued the normal subcellular distribution of Mbnl1 and did not alter the proportion of myonuclei containing CUG foci. In patient-derived cells, antagomiR-218 improved defective fusion and differentiation and rescued up to 34% of the gene expression alterations found in the transcriptome of patient cells. Importantly, miR-218 was found to be upregulated in DM1 muscle biopsies, pinpointing this microRNA (miRNA) as a relevant therapeutic target.

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