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.

Background/objectives: KRAS-mutant lung adenocarcinoma remains without effective targeted therapies for most patients, particularly those with non-G12C alleles or resistance to KRAS ^G12C inhibitors. RAF1 is essential for KRAS-driven tumor maintenance through kinase-independent survival functions, making it an attractive candidate for targeted protein degradation. However, the therapeutic impact and safety of co-targeting RAF1 with related kinases remain unclear. Methods: We used dual-recombinase genetically engineered mouse models of Kras ^+/G12V ;Trp53 ^-/- lung cancer to evaluate the effects of Raf1 ablation alone or in combination with Araf , Egfr , or Ddr1 . Lung tumors were initiated by intranasal Ad5-CMV-FLPo delivery and allowed to reach CT-detectable size before inducing systemic gene deletion via tamoxifen-activated CreERT2. Tumor burden was monitored by longitudinal CT imaging and classified using RECIST-like criteria. Toxicity was assessed by body weight monitoring, histopathology of major organs, and survival analysis. Results: Raf1 deletion induced robust tumor regression within two months, in more than 60% of lesions. Araf ablation alone or combined with Raf1 did not affect tumor initiation, progression, or regression rates. Similarly, neither genetic nor pharmacological EGFR inhibition (afatinib) improved responses to Raf1 ablation. Ddr1 co-deletion also failed to enhance therapeutic efficacy and slightly reduced response rates. None of the dual-targeting strategies increased systemic toxicity. Conclusions: RAF1 is a key, non-redundant vulnerability in KRAS-driven lung adenocarcinoma. Co-targeting ARAF, EGFR, or DDR1 provides no additional therapeutic benefit in established disease. The absence of adverse effects from ARAF co-deletion suggests that RAF1 degraders with partial cross-activity towards ARAF are likely to be safe. These findings provide a strong preclinical rationale for developing RAF1-targeted degradation as a monotherapy for these malignancies.

Gene edition methods are an attractive putative therapeutic option for Duchenne muscular dystrophy and they have an immediate application in the generation of research models. To generate two new edited myoblast cultures that could be useful 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: DMDΔ52-Model, 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 and, to show their use in the assessment of DMD treatments, we have performed exon skipping in the DMDΔ52-Model and have used the unedited 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 edition protocol, DMD-UTRN-Model offers a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.