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Cells Sep 2022Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease arising from loss-of-function mutations in the gene and characterized by progressive muscle... (Review)
Review
Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease arising from loss-of-function mutations in the gene and characterized by progressive muscle degeneration, respiratory insufficiency, cardiac failure, and premature death by the age of thirty. Albeit DMD is one of the most common types of fatal genetic diseases, there is no curative treatment for this devastating disorder. In recent years, gene editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system has paved a new path toward correcting pathological mutations at the genetic source, thus enabling the permanent restoration of dystrophin expression and function throughout the musculature. To date, the therapeutic benefits of CRISPR genome-editing systems have been successfully demonstrated in human cells, rodents, canines, and piglets with diverse DMD mutations. Nevertheless, there remain some nonignorable challenges to be solved before the clinical application of CRISPR-based gene therapy. Herein, we provide an overview of therapeutic CRISPR genome-editing systems, summarize recent advancements in their applications in DMD contexts, and discuss several potential obstacles lying ahead of clinical translation.
Topics: Animals; CRISPR-Cas Systems; Dogs; Dystrophin; Gene Editing; Genetic Therapy; Humans; Muscular Dystrophy, Duchenne; Swine
PubMed: 36230926
DOI: 10.3390/cells11192964 -
Journal of Muscle Research and Cell... Dec 2013Heart disease is a leading cause of death in patients with Duchenne muscular dystrophy (DMD). Patients with DMD lack the protein dystrophin, which is widely expressed in...
Heart disease is a leading cause of death in patients with Duchenne muscular dystrophy (DMD). Patients with DMD lack the protein dystrophin, which is widely expressed in striated muscle. In skeletal muscle, the loss of dystrophin results in dramatically decreased expression of the dystrophin associated glycoprotein complex (DGC). Interestingly, in the heart the DGC is normally expressed without dystrophin; this has been attributed to presence of the dystrophin homologue utrophin. We demonstrate here that neither utrophin nor dystrophin are required for the expression of the cardiac DGC. However, alpha-dystroglycan (α-DG), a major component of the DGC, is differentially glycosylated in dystrophin-(mdx) and dystrophin-/utrophin-(dko) deficient mouse hearts. In both models the altered α-DG retains laminin binding activity, but has an altered localization at the sarcolemma. In hearts lacking both dystrophin and utrophin, the alterations in α-DG glycosylation are even more dramatic with changes in gel migration equivalent to 24 ± 3 kDa. These data show that the absence of dystrophin and utrophin alters the processing of α-DG; however it is not clear if these alterations are a consequence of the loss of a direct interaction with dystrophin/utrophin or results from an indirect response to the presence of severe pathology. Recently there have been great advances in our understanding of the glycosylation of α-DG regarding its role as a laminin receptor. Here we present data that alterations in glycosylation occur in the hearts of animal models of DMD, but these changes do not affect laminin binding. The physiological consequences of these alterations remain unknown, but may have significant implications for the development of therapies for DMD.
Topics: Animals; Cytoskeletal Proteins; Dystrophin; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Glycosylation; Immunohistochemistry; Mice; Muscular Dystrophy, Duchenne; Utrophin
PubMed: 24096570
DOI: 10.1007/s10974-013-9362-9 -
Cellular and Molecular Life Sciences :... Jun 2021Duchenne muscular dystrophy (DMD) is a devastating chromosome X-linked disease that manifests predominantly in progressive skeletal muscle wasting and dysfunctions in... (Review)
Review
Duchenne muscular dystrophy (DMD) is a devastating chromosome X-linked disease that manifests predominantly in progressive skeletal muscle wasting and dysfunctions in the heart and diaphragm. Approximately 1/5000 boys and 1/50,000,000 girls suffer from DMD, and to date, the disease is incurable and leads to premature death. This phenotypic severity is due to mutations in the DMD gene, which result in the absence of functional dystrophin protein. Initially, dystrophin was thought to be a force transducer; however, it is now considered an essential component of the dystrophin-associated protein complex (DAPC), viewed as a multicomponent mechanical scaffold and a signal transduction hub. Modulating signal pathway activation or gene expression through epigenetic modifications has emerged at the forefront of therapeutic approaches as either an adjunct or stand-alone strategy. In this review, we propose a broader perspective by considering DMD to be a disease that affects myofibers and muscle stem (satellite) cells, as well as a disorder in which abrogated communication between different cell types occurs. We believe that by taking this systemic view, we can achieve safe and holistic treatments that can restore correct signal transmission and gene expression in diseased DMD tissues.
Topics: Animals; Bone and Bones; Cell Communication; Dystrophin; Humans; Microvessels; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Neurons; Signal Transduction
PubMed: 33825942
DOI: 10.1007/s00018-021-03821-x -
Journal of Cachexia, Sarcopenia and... Aug 2023Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, leads to progressive and fatal muscle weakness through yet-to-be-fully deciphered molecular...
BACKGROUND
Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, leads to progressive and fatal muscle weakness through yet-to-be-fully deciphered molecular perturbations. Emerging evidence implicates RhoA/Rho-associated protein kinase (ROCK) signalling in DMD pathology, yet its direct role in DMD muscle function, and related mechanisms, are unknown.
METHODS
Three-dimensionally engineered dystrophin-deficient mdx skeletal muscles and mdx mice were used to test the role of ROCK in DMD muscle function in vitro and in situ, respectively. The role of ARHGEF3, one of the RhoA guanine nucleotide exchange factors (GEFs), in RhoA/ROCK signalling and DMD pathology was examined by generating Arhgef3 knockout mdx mice. The role of RhoA/ROCK signalling in mediating the function of ARHGEF3 was determined by evaluating the effects of wild-type or GEF-inactive ARHGEF3 overexpression with ROCK inhibitor treatment. To gain more mechanistic insights, autophagy flux and the role of autophagy were assessed in various conditions with chloroquine.
RESULTS
Inhibition of ROCK with Y-27632 improved muscle force production in 3D-engineered mdx muscles (+25% from three independent experiments, P < 0.05) and in mice (+25%, P < 0.001). Unlike suggested by previous studies, this improvement was independent of muscle differentiation or quantity and instead related to increased muscle quality. We found that ARHGEF3 was elevated and responsible for RhoA/ROCK activation in mdx muscles, and that depleting ARHGEF3 in mdx mice restored muscle quality (up to +36%, P < 0.01) and morphology without affecting regeneration. Conversely, overexpressing ARHGEF3 further compromised mdx muscle quality (-13% vs. empty vector control, P < 0.01) in GEF activity- and ROCK-dependent manner. Notably, ARHGEF3/ROCK inhibition exerted the effects by rescuing autophagy which is commonly impaired in dystrophic muscles.
CONCLUSIONS
Our findings uncover a new pathological mechanism of muscle weakness in DMD involving the ARHGEF3-ROCK-autophagy pathway and the therapeutic potential of targeting ARHGEF3 in DMD.
Topics: Animals; Mice; Dystrophin; Mice, Inbred mdx; Muscle Weakness; Muscle, Skeletal; Muscular Dystrophy, Duchenne
PubMed: 37311604
DOI: 10.1002/jcsm.13278 -
Disease Models & Mechanisms Dec 2019Louis Kunkel has devoted his career to understanding the causes, mechanisms and treatment of muscular dystrophies. Dr Kunkel is the past Director of the Genomics Program...
Louis Kunkel has devoted his career to understanding the causes, mechanisms and treatment of muscular dystrophies. Dr Kunkel is the past Director of the Genomics Program at Boston Children's Hospital and Professor of Genetics and Pediatrics at Harvard Medical School. In this interview, he talks about his discovery of dystrophin, including patients in preclinical research, and bearded irises.
Topics: Animals; Dystrophin; History, 20th Century; History, 21st Century; Humans; Muscular Dystrophies
PubMed: 31843755
DOI: 10.1242/dmm.043018 -
Biology Open Aug 2020Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutation of the gene. Pharmacological therapies that function independently of...
Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutation of the gene. Pharmacological therapies that function independently of dystrophin and complement strategies aimed at dystrophin restoration could significantly improve patient outcomes. Previous observations have suggested that serotonin pathway modulation ameliorates dystrophic pathology, and re-application of serotonin modulators already used clinically would potentially hasten availability to DMD patients. In our study, we used dystrophin-deficient and zebrafish models of DMD for rapid and easy screening of several classes of serotonin pathway modulators as potential therapeutics. None of the candidate drugs tested significantly decreased the percentage of zebrafish exhibiting the dystrophic muscle phenotype in the short-term birefringence assay or lengthened the lifespan in the long-term survival assay. Although we did not identify an effective drug, we believe our data is of value to the DMD research community for future studies, and there is evidence that suggests serotonin modulation may still be a viable treatment strategy with further investigation. Given the widespread clinical use of selective serotonin reuptake inhibitors, tricyclic antidepressants and reversible inhibitors of monoamine oxidase, their reapplication to DMD is an attractive strategy in the field's pursuit to identify pharmacological therapies to complement dystrophin restoration strategies.
Topics: Animals; Birefringence; Drug Evaluation, Preclinical; Dystrophin; Monoamine Oxidase Inhibitors; Receptors, Serotonin; Serotonin; Serotonin Receptor Agonists; Selective Serotonin Reuptake Inhibitors; Survival Analysis; Zebrafish
PubMed: 32718931
DOI: 10.1242/bio.053363 -
Neurology Nov 2014We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its...
OBJECTIVE
We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials.
METHODS
Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels.
RESULTS
Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable.
CONCLUSIONS
Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval.
Topics: Dystrophin; Humans; Medical Laboratory Science; Muscular Dystrophy, Duchenne; Observer Variation; Translational Research, Biomedical
PubMed: 25355828
DOI: 10.1212/WNL.0000000000001025 -
Genes Jul 2020Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive condition caused primarily by out-of-frame mutations in the dystrophin gene. In males, DMD presents with... (Review)
Review
Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive condition caused primarily by out-of-frame mutations in the dystrophin gene. In males, DMD presents with progressive body-wide muscle deterioration, culminating in death as a result of cardiac or respiratory failure. A milder form of DMD exists, called Becker muscular dystrophy (BMD), which is typically caused by in-frame dystrophin gene mutations. It should be emphasized that DMD and BMD are not exclusive to males, as some female dystrophin mutation carriers do present with similar symptoms, generally at reduced levels of severity. Cardiac involvement in particular is a pressing concern among manifesting females, as it may develop into serious heart failure or could predispose them to certain risks during pregnancy or daily life activities. It is known that about 8% of carriers present with dilated cardiomyopathy, though it may vary from 0% to 16.7%, depending on if the carrier is classified as having DMD or BMD. Understanding the genetic and molecular mechanisms underlying cardiac manifestations in dystrophin-deficient females is therefore of critical importance. In this article, we review available information from the literature on this subject, as well as discuss the implications of female carrier studies on the development of therapies aiming to increase dystrophin levels in the heart.
Topics: Animals; Dystrophin; Female; Heart; Heterozygote; Humans; Muscular Dystrophy, Duchenne; Phenotype
PubMed: 32650403
DOI: 10.3390/genes11070765 -
Cells, Tissues, Organs 2022Duchenne muscular dystrophy (DMD) is a devastating and debilitating muscle degenerative disease affecting 1 in every 3,500 male births worldwide. DMD is progressive and... (Review)
Review
Duchenne muscular dystrophy (DMD) is a devastating and debilitating muscle degenerative disease affecting 1 in every 3,500 male births worldwide. DMD is progressive and fatal; accumulated weakening of the muscle tissue leads to an inability to walk and eventual loss of life due to respiratory and cardiac failure. Importantly, there remains no effective cure for DMD. DMD is caused by defective expression of the DMD gene, which encodes for dystrophin, a component of the dystrophin glycoprotein complex. In muscle fibers, this protein complex plays a critical role in maintaining muscle membrane integrity. Emerging studies have shown that muscle stem cells, which are adult stem cells responsible for muscle repair, are also affected in DMD. DMD muscle stem cells do not function as healthy muscle stem cells, and their impairment contributes to disease progression. Deficiencies in muscle stem cell function include impaired establishment of cell polarity leading to defective asymmetric stem cell division, reduced myogenic commitment, impaired differentiation, altered metabolism, and enhanced entry into senescence. Altogether, these findings indicate that DMD muscle stem cells are dysfunctional and have impaired regenerative potential. Although recent advances in adeno-associated vector and antisense oligonucleotide-mediated mechanisms for gene therapy have shown clinical promise, the current therapeutic strategies for muscular dystrophy do not effectively target muscle stem cells and do not address the deficiencies in muscle stem cell function. Here, we discuss the merits of restoring endogenous muscle stem cell function in degenerating muscle as a viable regenerative medicine strategy to mitigate DMD.
Topics: Adult; Male; Humans; Muscular Dystrophy, Duchenne; Dystrophin; Satellite Cells, Skeletal Muscle; Muscle Fibers, Skeletal; Power, Psychological
PubMed: 33910206
DOI: 10.1159/000514305 -
Scientific Reports Jan 2021Emerging and promising therapeutic interventions for Duchenne muscular dystrophy (DMD) are confounded by the challenges of quantifying dystrophin. Current approaches...
Emerging and promising therapeutic interventions for Duchenne muscular dystrophy (DMD) are confounded by the challenges of quantifying dystrophin. Current approaches have poor precision, require large amounts of tissue, and are difficult to standardize. This paper presents an immuno-mass spectrometry imaging method using gadolinium (Gd)-labeled anti-dystrophin antibodies and laser ablation-inductively coupled plasma-mass spectrometry to simultaneously quantify and localize dystrophin in muscle sections. Gd is quantified as a proxy for the relative expression of dystrophin and was validated in murine and human skeletal muscle sections following k-means clustering segmentation, before application to DMD patients with different gene mutations where dystrophin expression was measured up to 100 µg kg Gd. These results demonstrate that immuno-mass spectrometry imaging is a viable approach for pre-clinical to clinical research in DMD. It rapidly quantified relative dystrophin in single tissue sections, efficiently used valuable patient resources, and may provide information on drug efficacy for clinical translation.
Topics: Adolescent; Aged, 80 and over; Animals; Child; Dystrophin; Female; Fluorescent Antibody Technique; Gadolinium; Humans; Immunohistochemistry; Male; Mass Spectrometry; Mice; Muscle Fibers, Skeletal; Muscular Dystrophy, Duchenne; Mutation; Quadriceps Muscle
PubMed: 33441839
DOI: 10.1038/s41598-020-80495-8