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.

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.

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.

Inherited muscular dystrophies are a heterogeneous group of diseases, caused by different types of genetic mutations. RNA therapies, and particularly antisense oligonucleotides, offer a palette of therapeutic strategies to either reduce the production of harmful proteins or to restore or increase protein expression. Consequently, they offer therapeutic promise for multiple forms of muscular dystrophies. This review outlines the different RNA therapy types considered for the treatment of Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy and myotonic dystrophy, emphasizing the strategies used to deliver these therapies to skeletal muscle with a focus on approaches that have reached the clinical trial stage.

Myotonic dystrophy type I (DM1) is caused by CTG repeat expansions in the DMPK gene leading to mRNA toxicity and sequestration of the splicing regulator MBNL1, affecting many tissues. We have developed an in vitro screening platform based on ddPCR and in-cell western to quantify these mRNAs and proteins and characterized >20 cell models to define DM1 biomarkers that could be useful for drug screening. DMPK protein levels were reduced in DM1-immortalized myoblasts and myotubes, but not in fibroblasts, while MBNL1 protein was consistently lower in all DM1 myogenic cultures, whether primary or immortalized. Myogenic differentiation of cultures led to an increase in DMPK mRNA expression, which was translated into increased MBNL1 sequestration in foci. We further corroborated the platform’s ability to assess therapeutic outcomes, evaluating the effect of a DMPK gapmer ASO and one siRNA: while the gapmer increased MBNL1 protein levels, the siRNA had no significant effect on MBNL1 release. Our platform and the in-depth characterization of some of the most used models would be of use to the DM1 research community.

Myotonic dystrophy type I (DM1) is caused by CTG repeat expansions in the DMPK gene leading to mRNA toxicity and sequestration of the splicing regulator MBNL1, affecting many tissues. We have developed an in vitro screening platform based on ddPCR and in-cell western to quantify these mRNAs and proteins and characterised more than 20 cell models to define DM1 biomarkers that could be useful for drug screening. DMPK protein levels were reduced in DM1-immortalised myoblasts and myotubes, but not in fibroblasts, while MBNL1 protein was consistently lower in all DM1 myogenic cultures, whether primary or immortalised. Myogenic differentiation of cultures led to an increase in DMPK mRNA expression, which was translated into increased MBNL1 sequestration in foci. We further corroborated the platform’s ability to assess therapeutic outcomes, evaluating the effect of a DMPK gapmer ASO and one siRNA: while the gapmer increased MBNL1 protein levels, the siRNA had no significant effect on MBNL1 release. Our platform and the in-depth characterisation of some of the most used models would be of use to the DM1 research community. Significance statement: Myotonic dystrophy type I (DM1) is a multisystemic disease with a complex pathogenesis and multiple outcome measures for drug assessment in vitro . In the last years, the increasing number of new potential therapies targeting DM1 in clinical trials has increased the need for robust and rapid evaluation of preclinical candidates, as well as in-depth knowledge of the cell models used. Here, we present a new cell-based platform that enables robust quantification of DMPK and MBNL1 in cell culture for cell model characterisation and drug screening. Indeed, we highlight the differences observed in DMPK and MBNL1 protein quantification in primary fibroblasts and myotubes, immortalised fibroblasts, myoblasts and myotubes. We then, we performed a proof-of-concept drug evaluation of potential therapeutic strategies targeting DMPK, showing the most suitable for targeting the DMPK expanded transcript.