Myotonic dystrophy type I (DM1) is the most common form of adult muscular dystrophy, caused by expansion of a CTG triplet repeat in the 3′ untranslated region (3′UTR) of the myotonic dystrophy protein kinase (DMPK) gene. The pathological CTG repeats result in protein trapping by expanded transcripts, a decreased DMPK translation and the disruption of the chromatin structure, affecting neighboring genes expression. The muscleblind-like (MBNL) and CUG-BP and ETR-3-like factors (CELF) are two families of tissue-specific regulators of developmentally programmed alternative splicing that act as antagonist regulators of several pre-mRNA targets, including troponin 2 (TNNT2), insulin receptor (INSR), chloride channel 1 (CLCN1) and MBNL2. Sequestration of MBNL proteins and up-regulation of CELF1 are key to DM1 pathology, inducing a spliceopathy that leads to a developmental remodelling of the transcriptome due to an adult-to-foetal splicing switch, which results in the loss of cell function and viability. Moreover, recent studies indicate that additional pathogenic mechanisms may also contribute to disease pathology, including a misregulation of cellular mRNA translation, localization and stability. This review focuses on the cause and effects of MBNL and CELF1 deregulation in DM1, describing the molecular mechanisms underlying alternative splicing misregulation for a deeper understanding of DM1 complexity. To contribute to this analysis, we have prepared a comprehensive list of transcript alterations involved in DM1 pathogenesis, as well as other deregulated mRNA processing pathways implications.

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.

Utrophin is an autosomal paralogue of dystrophin, a protein whose deficit causes Duchenne and Becker muscular dystrophies (DMD/BMD). Utrophin is naturally overexpressed at the sarcolemma of mature dystrophin‐deficient fibres in DMD and BMD patients as well as in the mdx Duchenne mouse model. Dystrophin and utrophin can co‐localise in human foetal muscle, in the dystrophin‐competent fibres from DMD/BMD carriers, and revertant fibre clusters in biopsies from DMD patients. These findings suggest that utrophin overexpression could act as a surrogate, compensating for the lack of dystrophin, and, as such, it could be used in combination with dystrophin restoration therapies. Different strategies to overexpress utrophin are currently under investigation. In recent years, many compounds have been reported to modulate utrophin expression efficiently in preclinical studies and ameliorate the dystrophic phenotype in animal models of the disease. In this manuscript, we discuss the current knowledge on utrophin protein and the different mechanisms that modulate its expression in skeletal muscle. We also include a comprehensive review of compounds proposed as utrophin regulators and, as such, potential therapeutic candidates for these muscular dystrophies.

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.

Several exon skipping antisense oligonucleotides (eteplirsen, golodirsen, viltolarsen, and casimersen) have been approved for the treatment of Duchenne muscular dystrophy, but many more are in development targeting an array of different DMD exons. Preclinical screening of the new oligonucleotide sequences is routinely performed using patient-derived cell cultures, and evaluation of their efficacy may be performed at RNA and/or protein level. While several methods to assess exon skipping and dystrophin expression in cell culture have been developed, the choice of methodology often depends on the availability of specific research equipment.In this chapter, we describe and indicate the relevant bibliography of all the methods that may be used in this evaluation and describe in detail the protocols routinely followed at our institution, one to evaluate the efficacy of skipping at RNA level (nested PCR) and the other the restoration of protein expression (myoblot), which provide good results using equipment largely available to most research laboratories.

Neuromuscular Disorders 31 (2021) S47-S162 ability to accurately quantify dystrophin/mini-dystrophin is essential in determining the level of gene transduction. We developed and validated a novel, sensitive and selective peptide immunoaffinity liquid chromatographytandem mass spectrometry (IA-LC-MS/MS) assay, which demonstrated accurate dystrophin/mini-dystrophin quantification in a DMD rat model and human muscle tissue. Biceps femoris tissue from dystrophin-deficient rats treated with AAV9.hCK.Hopti-Dys3978.spA, an investigational adeno-

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