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Duchenne Muscular Dystrophy (DMD)'s complex multi-system pathophysiology, coupled with the cost-prohibitive logistics of multi-year drug screening and follow-up, has hampered the pursuit of new therapeutic approaches. Here we conducted a systematic historical and text mining-based pilot feasibility study to explore the potential of established or previously tested drugs as prospective DMD therapeutic agents. Our approach utilized a Swanson linking-inspired method to uncover meaningful yet largely hidden deep semantic connections between pharmacologically significant DMD targets and drugs developed for unrelated diseases. Specifically, we focused on molecular target-based MeSH terms and categories as high-yield bioinformatic proxies, effectively tagging relevant literature with categorical metadata. To identify promising leads, we comprehensively assembled published reports from 2011 and sampling from subsequent years. We then determined the earliest year when distinct MeSH terms or category labels of the relevant cellular target were referenced in conjunction with the drug, as well as when the pertinent target itself was first conclusively identified as holding therapeutic value for DMD. By comparing the earliest year when the drug was identifiable as a DMD treatment candidate with that of the first actual report confirming this, we computed an Index of Delayed Discovery (IDD), which serves as a metric of Swanson-linked latent knowledge. Using these findings, we identified data from previously unlinked articles subsetted via MeSH-derived Swanson linking or from target classes within the DrugBank repository. This enabled us to identify new but untested high-prospect small-molecule candidates that are of particular interest in repurposing for DMD and warrant further investigations.

Muscle damage and fibro-fatty replacement of skeletal muscles is a main pathologic feature of Duchenne muscular dystrophy (DMD) with more proximal muscles affected earlier and more distal affected later in the disease course, suggesting that different skeletal muscle groups possess distinctive characteristics that influence their susceptibility to disease. To explore transcriptomic factors driving differential gene expression and modulating DMD skeletal muscle severity, we characterized the transcriptome of vastus lateralis (VL), a more proximal and susceptible muscle, relative to tibialis anterior (TA), a more distal and protected muscle, in 15 healthy individuals using bulk RNA sequencing to identify gene expression differences that may mediate their relative susceptibility to damage with loss of dystrophin. Matching single nuclei RNA sequencing data was generated for 3 of the healthy individuals, to infer cell composition in the bulk RNA sequencing dataset and to improve mapping of differentially expressed genes to their cell source of expression. A total of 3,410 differentially expressed genes were identified and mapped to cell type using single nuclei RNA sequencing of muscle, including long non-coding RNAs and protein coding genes. There was an enrichment of genes involved in calcium release from the sarcoplasmic reticulum, particularly in the myofibers and these myofiber genes were higher in the VL. There was an enrichment of genes in "Collagen-Containing Extracellular Matrix" expressed by fibroblasts, endothelial, smooth muscle and pericytes, with most genes higher in the TA, as well as genes in "Regulation Of Apoptotic Process" expressed across all cell types. Previously reported genetic modifiers were also enriched within the differentially expressed genes. We also identify 6 genes with differential isoform usage between the VL and TA. Lastly, we integrate our findings with DMD RNA sequencing data from the TA, and identify "Collagen-Containing Extracellular Matrix" and "Negative Regulation Of Apoptotic Process" as differentially expressed between DMD compared to healthy. Collectively, these findings propose novel candidate mechanisms that may mediate differential muscle susceptibility in muscular dystrophies and provide new insight into potential therapeutic targets.

In Duchenne muscular dystrophy, dystrophin loss leads to chronic muscle damage, dysregulation of repair, fibro-fatty replacement, and weakness. We develop methodology to efficiently isolate individual nuclei from minute quantities of frozen skeletal muscle, allowing single nuclei sequencing of irreplaceable archival samples and from very small samples. We apply this method to identify cell and gene expression dynamics within human DMD and mdx mouse muscle, characterizing effects of dystrophin rescue by exon skipping therapy at single nuclei resolution. DMD exon 23 skipping events are directly observed and increased in myonuclei from treated mice. We describe partial rescue of type IIa and IIx myofibers, expansion of an MDSC-like myeloid population, recovery of repair/remodeling M2-macrophage, and repression of inflammatory POSTN1 + fibroblasts in response to exon skipping and partial dystrophin restoration. Use of this method enables exploration of cellular and transcriptomic mechanisms of dystrophin loss and repair within an intact muscle environment. Our initial findings will scaffold our future work to more directly examine muscular dystrophies and putative recovery pathways.

In vitro models of patient-derived muscle allow for more efficient development of genetic medicines for the muscular dystrophies, which often present mutation-specific pathologies. One popular strategy to generate patient-specific myotubes involves reprogramming dermal fibroblasts to a muscle lineage through MyoD induction. However, creating physiologically relevant, reproducible tissues exhibiting multinucleated, aligned myotubes with organized striations is dependent on the introduction of physicochemical cues that mimic the native muscle microenvironment. Here, we engineered patient-specific control and dystrophic muscle tissues in vitro by culturing and differentiating MyoD-directly reprogrammed fibroblasts isolated from one healthy control subject, three patients with Duchenne muscular dystrophy (DMD), and two Limb Girdle 2A/R1 (LGMD2A/R1) patients on micromolded gelatin hydrogels. Engineered DMD and LGMD2A/R1 tissues demonstrated varying levels of defects in α-actinin expression and organization relative to control, depending on the mutation. In genetically relevant DMD tissues amenable to mRNA reframing by targeting exon 44 or 45 exclusion, exposure to exon skipping antisense oligonucleotides modestly increased myotube coverage and alignment and rescued dystrophin protein expression. These findings highlight the value of engineered culture substrates in guiding the organization of reprogrammed patient fibroblasts into aligned muscle tissues, thereby extending their value as tools for exploration and dissection of the cellular and molecular basis of genetic muscle defects, rescue, and repair.

Systemic delivery of antisense oligonucleotides (AO) for DMD exon skipping has proven effective for reframing DMD mRNA, rescuing dystrophin expression, and slowing disease progression in animal models. In humans with Duchenne muscular dystrophy treated with AOs, low levels of dystrophin have been induced, and modest slowing of disease progression has been observed, highlighting the need for improved efficiency of human skipping drugs. Here, we demonstrate that dantrolene and Rycals S107 and ARM210 potentiate AO-mediated exon skipping of exon 44 or exon 45 in patient-derived myotube cultures with appropriate mutations. Further, dantrolene is shown to boost AO-mediated exon skipping in patient-derived, induced cardiomyocyte cultures. Our findings further validate the ryanodine receptors (RyR) as the likely target responsible for exon skip boosting and demonstrate potential applicability beyond exon 51 skipping. These data provide preclinical support of dantrolene trial as an adjuvant to AO-mediated exon-skipping therapy in humans and identify a novel Rycal, ARM210, for development as a potential exon-skipping booster. Further, they highlight the value of mutation-specific DMD culture models for basic discovery, preclinical drug screening and translation of personalized genetic medicines.

Duchenne muscular dystrophy is caused by mutations in the dystrophin-encoding DMD gene. While Duchenne is most commonly caused by large intragenic deletions that cause frameshift and complete loss of dystrophin expression, in-frame deletions in DMD can result in the expression of internally truncated dystrophin proteins and may be associated with a milder phenotype. In this study, we describe two individuals with large in-frame 5' deletions (exon 3-23 and exon 3-28) that remove the majority of the N-terminal region, including part of the actin binding and central rod domains. Both patients had progressive muscle weakness during childhood but are observed to have a relatively mild disease course compared to typical Duchenne. We show that in muscle biopsies from both patients, truncated dystrophin is expressed at the sarcolemma. We have additionally developed a patient-specific fibroblast-derived cell model, which can be inducibly reprogrammed to form myotubes that largely recapitulate biopsy findings for the patient with the exon 3-23 deletion, providing a culture model for future investigation of this unusual case. We discuss these mutations in the context of previously reported 5' in-frame DMD deletions and relevant animal models, and review the spectrum of phenotypes associated with these deletions.

Dystrophinopathies are diseases caused by mutations in the Duchenne Muscular Dystrophy gene (DMD) encoding the dystrophin protein. Depending on the type of mutation, patients develop either the severe DMD or the milder Becker Muscular Dystrophy. Although substantial effort was made, the pathophysiology and variation in disease severity are still poorly understood. During the last two decades, relentless efforts were made to develop therapeutic strategies. Among these, gene therapy and cell replacement therapy appear very promising. These approaches are based on the replacement and/or repair of the mutated DMD gene or transcript at the molecular level, or at the cellular level via replacement of the damaged muscle cells. While highly successful in animal models, these therapies showed only modest efficacy in human clinical trials. More importantly, variable effects were observed in patients carrying the same mutation, suggesting that several factors (e.g., genetic modifiers, environmental factors) can affect treatment outcomes. In this review, we will describe recent advancements and new approaches of gene and cell therapies for dystrophinopathies that pave the way for a medicine "à la carte".

Skeletal muscle undergoes many micro-membrane lesions at physiological state. Based on their sizes and magnitude these lesions are repaired via different complexes on a specific spatio-temporal manner. One of the major repair complex is a dysferlin-dependent mechanism. Accordingly, mutations in the DYSF gene encoding dysferlin results in the development of several muscle pathologies called dysferlinopathies, where abnormalities of the membrane repair process have been characterized in patients and animal models. Recent efforts have been deployed to decipher the function of dysferlin, they shed light on its direct implication in sarcolemma resealing after injuries. These discoveries served as a strong ground to design therapeutic approaches for dysferlin-deficient patients. This review detailed the different partners and function of dysferlin and positions the sarcolemma repair in normal and pathological conditions.

Systemic delivery of antisense oligonucleotides (AO) for DMD exon skipping has proven effective for reframing DMD mRNA, rescuing dystrophin expression, and slowing disease progression in animal models. In humans with Duchenne muscular dystrophy treated with AOs, low levels of dystrophin have been induced, and modest slowing of disease progression has been observed, highlighting the need for improved efficiency of human skipping drugs. Here, we demonstrate that dantrolene and Rycals S107 and ARM210 potentiate AO-mediated exon skipping of exon 44 or exon 45 in patient-derived myotube cultures with appropriate mutations. Further, dantrolene is shown to boost AO-mediated exon skipping in patient-derived, induced cardiomyocyte cultures. Our findings further validate the ryanodine receptors (RyR) as the likely target responsible for exon skip boosting and demonstrate potential applicability beyond exon 51 skipping. These data provide preclinical support of dantrolene trial as an adjuvant to AO-mediated exon-skipping therapy in humans and identify a novel Rycal, ARM210, for development as a potential exon-skipping booster. Further, they highlight the value of mutation-specific DMD culture models for basic discovery, preclinical drug screening and translation of personalized genetic medicines., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dysferlinopathies are rare genetic diseases affecting muscles due to mutations in DYSF. Exon 32 of DYSF has been shown to be dispensable for dysferlin functions. Here we present a method to visualize the skipping of exon 32 at the RNA and protein levels using an antisense oligonucleotide on cells derived from a dysferlinopathy-affected patient.