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Molecular Therapy. Methods & Clinical... Sep 2023Duchenne muscular dystrophy (DMD) is a disease with a life-threatening trajectory resulting from mutations in the dystrophin gene, leading to degeneration of skeletal...
Duchenne muscular dystrophy (DMD) is a disease with a life-threatening trajectory resulting from mutations in the dystrophin gene, leading to degeneration of skeletal muscle and fibrosis of cardiac muscle. The overwhelming majority of mutations are multiexonic deletions. We previously established a dystrophic mouse model with deletion of exons 52-54 in that develops an early-onset cardiac phenotype similar to DMD patients. Here we employed CRISPR-Cas9 delivered intravenously by adeno-associated virus (AAV) vectors to restore functional dystrophin expression via excision or skipping of exon 55. Exon skipping with a solitary guide significantly improved editing outcomes and dystrophin recovery over dual guide excision. Some improvements to genomic and transcript editing levels were observed when the guide dose was enhanced, but dystrophin restoration did not improve considerably. Editing and dystrophin recovery were restricted primarily to cardiac tissue. Remarkably, our exon skipping approach completely prevented onset of the cardiac phenotype in treated mice up to 12 weeks. Thus, our results demonstrate that intravenous delivery of a single-cut CRISPR-Cas9-mediated exon skipping therapy can prevent heart dysfunction in DMD .
PubMed: 37545481
DOI: 10.1016/j.omtm.2023.07.004 -
Advances in Therapy Apr 2024Duchenne muscular dystrophy (DMD) is one of the most prevalent X-linked inherited neuromuscular disorders, with an estimated incidence between 1 in 3500 and 5000 live... (Review)
Review
Duchenne muscular dystrophy (DMD) is one of the most prevalent X-linked inherited neuromuscular disorders, with an estimated incidence between 1 in 3500 and 5000 live male births. The median life expectancy at birth is around 30 years due to a rapid and severe disease progression. Currently, there is no cure for DMD, and the standard of care is mainly palliative therapy and glucocorticoids to mitigate symptoms and improve quality of life. Recent advances in phosphorodiamidate morpholino antisense oligonucleotide (PMO) technology has proven optimistic in providing a disease-modifying therapy rather than a palliative treatment option through correcting the primary genetic defect of DMD by exon skipping. However, as a result of the high variance in mutations of the dystrophin gene causing DMD, it has been challenging to tailor an effective therapy in most patients. Viltolarsen is effective in 8% of patients and accurately skips exon 53, reestablishing the reading frame and producing a functional form of dystrophin and milder disease phenotype. Results of recently concluded preclinical and clinical trials show significantly increased dystrophin protein expression, no severe adverse effects, and stabilization of motor function. In summary, viltolarsen has provided hope for those working toward giving patients a safe and viable treatment option for managing DMD. This review summarizes an overview of the presentation, pathophysiology, genetics, and current treatment guidelines of DMD, pharmacological profile of viltolarsen, and a summary of the safety and efficacy with additional insights using recent clinical trial data.
Topics: Infant, Newborn; Humans; Male; Muscular Dystrophy, Duchenne; Dystrophin; Quality of Life; Oligonucleotides
PubMed: 38376743
DOI: 10.1007/s12325-024-02801-4 -
Progress in Retinal and Eye Research Jul 2023Duchenne muscular dystrophy (DMD) is caused by X-linked inherited or de novo DMD gene mutations predominantly affecting males who develop early-onset muscle... (Review)
Review
Duchenne muscular dystrophy (DMD) is caused by X-linked inherited or de novo DMD gene mutations predominantly affecting males who develop early-onset muscle degeneration, severely affecting their quality of life and leading to reduced life expectancy. DMD patients may also develop proliferative retinopathy, cataract, ERG abnormalities, altered contrast sensitivity, color vision losses, and elevated flash detection thresholds during dark adaptation. Depending on the position of the genetic alteration in the large DMD gene, it is associated with a lack of the full-length dystrophin protein possibly with an additional loss of one or several other dystrophins, which are normally transcribed from internal promoters in retina and crystalline lens. During the last decades, the properties of the dystrophins have been characterized in patients with different genetic alterations and in genetic mouse models of DMD. The complex expression pattern of the dystrophins in photoreceptors, Müller glial cells and astrocytes, likely influences synaptic transmission, ionic balance and vascular integrity of the retina. However, the specific function of each retinal dystrophin remains largely unknown. This review describes the current knowledge on dystrophin expression, the putative molecular, structural, and physiological properties of retinal dystrophins, and the main clinical implications associated with the loss of dystrophins in DMD patients and mouse models. Current data and working hypotheses warrant future research on retinal dystrophins to increase our understanding of dystrophin function in the central nervous system in general and to unveil new retinal mechanisms and therapeutic avenues for retinal diseases.
Topics: Male; Mice; Animals; Dystrophin; Muscular Dystrophy, Duchenne; Quality of Life; Retina; Retinal Diseases
PubMed: 36404230
DOI: 10.1016/j.preteyeres.2022.101137 -
Biomolecules Nov 2023mice with a spontaneous mutation in exon 23 of the gene represent the most common model to investigate the pathophysiology of Duchenne muscular dystrophy (DMD). The...
mice with a spontaneous mutation in exon 23 of the gene represent the most common model to investigate the pathophysiology of Duchenne muscular dystrophy (DMD). The disease, caused by the lack of functional dystrophin, is characterized by irreversible impairment of muscle functions, with the diaphragm affected earlier and more severely than other skeletal muscles. We applied a label-free (LF) method and the more thorough tandem mass tag (TMT)-based method to analyze differentially expressed proteins in the diaphragm of 6-week-old mice. The comparison of both methods revealed 88 commonly changed proteins. A more in-depth analysis of the TMT-based method showed 953 significantly changed proteins, with 867 increased and 86 decreased in dystrophic animals (-value < 0.05, fold-change threshold: 1.5). Consequently, several dysregulated processes were demonstrated, including the immune response, fibrosis, translation, and programmed cell death. Interestingly, in the dystrophic diaphragm, we found a significant decrease in the expression of enzymes generating hydrogen sulfide (HS), suggesting that alterations in the metabolism of this gaseous mediator could modulate DMD progression, which could be a potential target for pharmacological intervention.
Topics: Animals; Mice; Mice, Inbred mdx; Diaphragm; Proteome; Muscular Dystrophy, Duchenne; Muscle, Skeletal; Mice, Inbred C57BL
PubMed: 38002330
DOI: 10.3390/biom13111648 -
Biomolecules Jul 2023The progressive degeneration of the skeletal musculature in Duchenne muscular dystrophy is accompanied by reactive myofibrosis, fat substitution, and chronic... (Review)
Review
The progressive degeneration of the skeletal musculature in Duchenne muscular dystrophy is accompanied by reactive myofibrosis, fat substitution, and chronic inflammation. Fibrotic changes and reduced tissue elasticity correlate with the loss in motor function in this X-chromosomal disorder. Thus, although dystrophinopathies are due to primary abnormalities in the gene causing the almost-complete absence of the cytoskeletal Dp427-M isoform of dystrophin in voluntary muscles, the excessive accumulation of extracellular matrix proteins presents a key histopathological hallmark of muscular dystrophy. Animal model research has been instrumental in the characterization of dystrophic muscles and has contributed to a better understanding of the complex pathogenesis of dystrophinopathies, the discovery of new disease biomarkers, and the testing of novel therapeutic strategies. In this article, we review how mass-spectrometry-based proteomics can be used to study changes in key components of the endomysium, perimysium, and epimysium, such as collagens, proteoglycans, matricellular proteins, and adhesion receptors. The mouse diaphragm displays severe myofibrosis, making it an ideal model system for large-scale surveys of systematic alterations in the matrisome of dystrophic fibers. Novel biomarkers of myofibrosis can now be tested for their appropriateness in the preclinical and clinical setting as diagnostic, pharmacodynamic, prognostic, and/or therapeutic monitoring indicators.
Topics: Animals; Mice; Mice, Inbred mdx; Diaphragm; Proteomics; Muscular Dystrophy, Duchenne; Muscle, Skeletal; Extracellular Matrix; Extracellular Matrix Proteins; Biomarkers
PubMed: 37509144
DOI: 10.3390/biom13071108 -
Current Journal of Neurology Oct 2023Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are inherited X-linked disorders resulting from alterations in the dystrophin gene....
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are inherited X-linked disorders resulting from alterations in the dystrophin gene. Genotype-phenotype matching studies have revealed a link between disease severity, the amount of muscle dystrophin, and the extent of mutation/deletion on the dystrophin gene. This study aimed to assess the relationship between genetic alterations in the dystrophin gene and the clinical status of patients with dystrophinopathies among the Iranian population. This cross-sectional study examined 54 patients with muscle weakness caused by abnormalities in the dystrophin gene at a hospital affiliated to Isfahan University of Medical Sciences, Isfahan, Iran, in 2021. The participants' demographic information, including age, family history of muscle dystrophies, and family history of other medical diseases as well as the type of muscular dystrophy were recorded. Furthermore, the number and region of deleted exons based on dystrophy types were also evaluated using multiplex ligation-dependent probe amplification (MLPA). The patients' gaits were also assessed as using a wheelchair, the presence of waddling gaits, or toe gaits. The patients' clinical status and the coexistence of pulmonary, bulbar, and mental conditions were also examined and compared between the two groups of dystrophinopathies. In this study, 54 patients with dystrophinopathy with the mean age of 16.63 ± 12.10 years were evaluated, of whom 22 (40.7%) and 30 (55.6%) patients were classified as BMD and DMD, respectively. The most affected regions with deleted exons were exons 45-47 (n = 5) and 45-48 (n = 4) in patients with BMD, while exons 45, 48-52, 51-55, and 53 (2 cases per exon) were the most common affected exons in patients with DMD. Further analyses revealed that deletions in exons 45-47 and 51-55 were significantly associated with older and younger ages at the onset of becoming wheelchair-bound in patients with dystrophy, respectively. The hotspot range in both BMD and DMD was within exons 45-55 (n = 15 for each group); 63% of the patients had alterations on the dystrophin gene within this range [30 patients (68.18%) in the BMD group, 15 patients (53.57%) in the DMD group]. Exon deletion was the most common genetic alteration in patients with dystrophinopathies. No significant difference was observed between DMD and BMD regarding the number of deleted exons. Deletions in exons 45-47 and 51-55 were linked to later and earlier onset of becoming wheelchair-bound, respectively.
PubMed: 38425356
DOI: 10.18502/cjn.v22i4.14528 -
Molecular Therapy. Methods & Clinical... Sep 2023Duchenne muscular dystrophy (DMD) is an X-linked disease caused by loss-of-function mutations in the gene and is characterized by muscle wasting and early mortality....
Duchenne muscular dystrophy (DMD) is an X-linked disease caused by loss-of-function mutations in the gene and is characterized by muscle wasting and early mortality. Adeno-associated virus-mediated gene therapy is being investigated as a treatment for DMD. In the nonclinical study documented here, we determined the effective dose of fordadistrogene movaparvovec, a clinical candidate adeno-associated virus serotype 9 vector carrying a human mini-dystrophin transgene, after single intravenous injection in a dystrophin-deficient (DMD) rat model of DMD. Overall, we found that transduction efficiency, number of muscle fibers expressing the human mini-dystrophin polypeptide, improvement of the skeletal and cardiac muscle tissue architecture, correction of muscle strength and fatigability, and improvement of diastolic and systolic cardiac function were directly correlated with the amount of vector administered. The effective dose was then tested in older DMD rats with a more dystrophic phenotype similar to the pathology observed in older patients with DMD. Except for a less complete rescue of muscle function in the oldest cohort, fordadistrogene movaparvovec was also found to be therapeutically effective in older DMD rats, suggesting that this product may be appropriate for evaluation in patients with DMD at all stages of disease.
PubMed: 37746247
DOI: 10.1016/j.omtm.2023.05.017 -
Cells Mar 2024Duchenne muscular dystrophy (DMD) is a genetic progressive muscle-wasting disorder that leads to rapid loss of mobility and premature death. The absence of functional... (Review)
Review
Duchenne muscular dystrophy (DMD) is a genetic progressive muscle-wasting disorder that leads to rapid loss of mobility and premature death. The absence of functional dystrophin in DMD patients reduces sarcolemma stiffness and increases contraction damage, triggering a cascade of events leading to muscle cell degeneration, chronic inflammation, and deposition of fibrotic and adipose tissue. Efforts in the last decade have led to the clinical approval of novel drugs for DMD that aim to restore dystrophin function. However, combination therapies able to restore dystrophin expression and target the myriad of cellular events found impaired in dystrophic muscle are desirable. Muscles are higher energy consumers susceptible to mitochondrial defects. Mitochondria generate a significant source of reactive oxygen species (ROS), and they are, in turn, sensitive to proper redox balance. In both DMD patients and animal models there is compelling evidence that mitochondrial impairments have a key role in the failure of energy homeostasis. Here, we highlighted the main aspects of mitochondrial dysfunction and oxidative stress in DMD and discussed the recent findings linked to mitochondria/ROS-targeted molecules as a therapeutic approach. In this respect, dual targeting of both mitochondria and redox homeostasis emerges as a potential clinical option in DMD.
Topics: Animals; Humans; Muscular Dystrophy, Duchenne; Dystrophin; Reactive Oxygen Species; Muscle, Skeletal; Mitochondria
PubMed: 38607013
DOI: 10.3390/cells13070574 -
Particle and Fibre Toxicology Jul 2023Exposure to particulate matter (PM) with an aerodynamic diameter less than 2.5 μm (PM) is a risk factor for developing pulmonary diseases and the worsening of ongoing...
BACKGROUND
Exposure to particulate matter (PM) with an aerodynamic diameter less than 2.5 μm (PM) is a risk factor for developing pulmonary diseases and the worsening of ongoing disease. Mitochondrial fission and fusion are essential processes underlying mitochondrial homeostasis in health and disease. We examined the role of mitochondrial fission and fusion in PM-induced alveolar epithelial cell damage and lung injury. Key genes in these processes include dystrophin-related protein 1 (DRP1) and optic atrophy 1 (OPA1) respectively.
METHODS
Alveolar epithelial (A549) cells were treated with PM (32 µg/ml) in the presence and absence of Mdivi-1 (10µM, a DRP1 inhibitor) or BGP-15 (10µM, an OPA1 activator). Results were validated using DRP1-knockdown (KD) and OPA1-overexpression (OE). Mice were injected intraperitoneally with Mdivi-1 (20 mg/kg), BGP-15 (20 mg/kg) or distilled water (control) one hour before intranasal instillation of PM (7.8 mg/kg) or distilled water for two consecutive days.
RESULTS
PM exposure of A549 cells caused oxidative stress, enhanced inflammation, necroptosis, mitophagy and mitochondrial dysfunction indicated by abnormal mitochondrial morphology, decreased mitochondrial membrane potential (ΔΨm), reduced mitochondrial respiration and disrupted mitochondrial fission and fusion. Regulating mitochondrial fission and fusion pharmacologically using Mdivi-1 and BGP-15 and genetically using DRP1-KD and OPA1-OE prevented PM-induced celluar damage in A549 cells. Mdivi-1 and BGP-15 attenuated PM-induced acute lung injury in mice.
CONCLUSION
Increased mitochondrial fission and decreased mitochondrial fusion may underlie PM-induced alveolar epithelial cell damage in vitro and lung injury in vivo.
Topics: Mice; Animals; Particulate Matter; Mitochondrial Dynamics; Alveolar Epithelial Cells; Lung Injury; Water
PubMed: 37464447
DOI: 10.1186/s12989-023-00534-w -
Science Signaling Jul 2023Precise synapse formation is essential for normal functioning of the nervous system. Retinal photoreceptors establish selective contacts with bipolar cells, aligning the...
Precise synapse formation is essential for normal functioning of the nervous system. Retinal photoreceptors establish selective contacts with bipolar cells, aligning the neurotransmitter release apparatus with postsynaptic signaling cascades. This involves transsynaptic assembly between the dystroglycan-dystrophin complex on the photoreceptor and the orphan receptor GPR179 on the bipolar cell, which is mediated by the extracellular matrix protein pikachurin (also known as EGFLAM). This complex plays a critical role in the synaptic organization of photoreceptors and signal transmission, and mutations affecting its components cause blinding disorders in humans. Here, we investigated the structural organization and molecular mechanisms by which pikachurin orchestrates transsynaptic assembly and solved structures of the human pikachurin domains by x-ray crystallography and of the GPR179-pikachurin complex by single-particle, cryo-electron microscopy. The structures reveal molecular recognition principles of pikachurin by the Cache domains of GPR179 and show how the interaction is involved in the transsynaptic alignment of the signaling machinery. Together, these data provide a structural basis for understanding the synaptic organization of photoreceptors and ocular pathology.
Topics: Humans; Carrier Proteins; Cryoelectron Microscopy; Extracellular Matrix Proteins; Photoreceptor Cells; Synapses
PubMed: 37490546
DOI: 10.1126/scisignal.add9539