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Neurotherapeutics : the Journal of the... Oct 2023Duchenne muscular dystrophy (DMD) is the most common childhood form of muscular dystrophy. It is caused by mutations in the DMD gene, leading to reduced or absent... (Review)
Review
Duchenne muscular dystrophy (DMD) is the most common childhood form of muscular dystrophy. It is caused by mutations in the DMD gene, leading to reduced or absent expression of the dystrophin protein. Clinically, this results in loss of ambulation, cardiomyopathy, respiratory failure, and eventually death. In the past decades, the use of corticosteroids has slowed down the disease progression. More recently, the development of genetically mediated therapies has emerged as the most promising treatment for DMD. These strategies include exon skipping with antisense oligonucleotides, gene replacement therapy with adeno-associated virus, and gene editing with CRISPR (clustered regularly interspaced short palindromic repeats) technology. In this review, we highlight the most up-to-date therapeutic progresses in the field, with emphasis on past and recent experiences, as well as the latest clinical results of DMD micro-dystrophin gene therapy. Additionally, we discuss the lessons learned along the way and the challenges encountered, all of which have helped advance the field, with the potential to finally alleviate such a devastating disease.
Topics: Humans; Child; Muscular Dystrophy, Duchenne; Dystrophin; Gene Editing; Exons; Genetic Therapy
PubMed: 37673849
DOI: 10.1007/s13311-023-01423-y -
Acta Neuropathologica Communications Oct 2023Duchenne muscular dystrophy (DMD) is a devastating X-linked muscular disease, caused by mutations in the DMD gene encoding Dystrophin and affecting 1:5000 boys...
Duchenne muscular dystrophy (DMD) is a devastating X-linked muscular disease, caused by mutations in the DMD gene encoding Dystrophin and affecting 1:5000 boys worldwide. Lack of Dystrophin leads to progressive muscle wasting and degeneration resulting in cardiorespiratory failure. Despite the absence of a definitive cure, innovative therapeutic avenues are emerging. Myopathologic studies are important to further understand the biological mechanisms of the disease and to identify histopathologic benchmarks for clinical evaluations. We conducted a myopathologic analysis on twenty-four muscle biopsies from DMD patients, with particular emphasis on regeneration, fibro-adipogenic progenitors and muscle stem cells behavior. We describe an increase in content of fibro-adipogenic progenitors, central orchestrators of fibrotic progression and lipid deposition, concurrently with a decline in muscle regenerative capacity. This regenerative impairment strongly correlates with compromised activation and expansion of muscle stem cells. Furthermore, our study uncovers an early acquisition of a senescence phenotype by DMD-afflicted muscle stem cells. Here we describe the myopathologic trajectory intrinsic to DMD and establish muscle stem cell senescence as a pivotal readout for future therapeutic interventions.
Topics: Humans; Male; Dystrophin; Fibrosis; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Regeneration; Satellite Cells, Skeletal Muscle; Cellular Senescence
PubMed: 37858263
DOI: 10.1186/s40478-023-01657-z -
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 -
Cell Reports Nov 2023Duchenne muscular dystrophy (DMD) is a severe genetic disease caused by the loss of the dystrophin protein. Exon skipping is a promising strategy to treat DMD by...
Duchenne muscular dystrophy (DMD) is a severe genetic disease caused by the loss of the dystrophin protein. Exon skipping is a promising strategy to treat DMD by restoring truncated dystrophin. Here, we demonstrate that base editors (e.g., targeted AID-mediated mutagenesis [TAM]) are able to efficiently induce exon skipping by disrupting functional redundant exonic splicing enhancers (ESEs). By developing an unbiased and high-throughput screening to interrogate exonic sequences, we successfully identify novel ESEs in DMD exons 51 and 53. TAM-CBE (cytidine base editor) induces near-complete skipping of the respective exons by targeting these ESEs in patients' induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Combined with strategies to disrupt splice sites, we identify suitable single guide RNAs (sgRNAs) with TAM-CBE to efficiently skip most DMD hotspot exons without substantial double-stranded breaks. Our study thus expands the repertoire of potential targets for CBE-mediated exon skipping in treating DMD and other RNA mis-splicing diseases.
Topics: Humans; Dystrophin; RNA, Guide, CRISPR-Cas Systems; Muscular Dystrophy, Duchenne; RNA Splicing; Exons
PubMed: 37906593
DOI: 10.1016/j.celrep.2023.113340 -
Biophysical Journal Aug 2018We have used pulsed electron paramagnetic resonance, calorimetry, and molecular dynamics simulations to examine the structural mechanism of binding for dystrophin's...
We have used pulsed electron paramagnetic resonance, calorimetry, and molecular dynamics simulations to examine the structural mechanism of binding for dystrophin's N-terminal actin-binding domain (ABD1) and compare it to utrophin's ABD1. Like other members of the spectrin superfamily, dystrophin's ABD1 consists of two calponin-homology (CH) domains, CH1 and CH2. Several mutations within dystrophin's ABD1 are associated with the development of severe degenerative muscle disorders Duchenne and Becker muscular dystrophies, highlighting the importance of understanding its structural biology. To investigate structural changes within dystrophin ABD1 upon binding to actin, we labeled the protein with spin probes and measured changes in inter-CH domain distance using double-electron electron resonance. Previous studies on the homologous protein utrophin showed that actin binding induces a complete structural opening of the CH domains, resulting in a highly ordered ABD1-actin complex. In this study, double-electron electron resonance shows that dystrophin ABD1 also undergoes a conformational opening upon binding F-actin, but this change is less complete and significantly more structurally disordered than observed for utrophin. Using molecular dynamics simulations, we identified a hinge in the linker region between the two CH domains that grants conformational flexibility to ABD1. The conformational dynamics of both dystrophin's and utrophin's ABD1 showed that compact conformations driven by hydrophobic interactions are preferred and that extended conformations are energetically accessible through a flat free-energy surface. Considering that the binding free energy of ABD1 to actin is on the order of 6-7 kcal/mole, our data are compatible with a mechanism in which binding to actin is largely dictated by specific interactions with CH1, but fine tuning of the binding affinity is achieved by the overlap between conformational ensembles of ABD1 free and bound to actin.
Topics: Actins; Dystrophin; Electron Spin Resonance Spectroscopy; Molecular Dynamics Simulation; Protein Binding; Protein Domains
PubMed: 30007583
DOI: 10.1016/j.bpj.2018.05.039 -
ILAR Journal 2015Duchenne muscular dystrophy is a progressive, fatal, X-linked disease caused by a failure to accumulate the cytoskeletal protein dystrophin. This disease has been... (Review)
Review
Duchenne muscular dystrophy is a progressive, fatal, X-linked disease caused by a failure to accumulate the cytoskeletal protein dystrophin. This disease has been studied using a variety of animal models including fish, mice, rats, and dogs. While these models have contributed substantially to our mechanistic understanding of the disease and disease progression, limitations inherent to each model have slowed the clinical advancement of therapies, which necessitates the development of novel large-animal models. Several porcine dystrophin-deficient models have been identified, although disease severity may be so severe as to limit their potential contributions to the field. We have recently identified and completed the initial characterization of a natural porcine model of dystrophin insufficiency. Muscles from these animals display characteristic focal necrosis concomitant with decreased abundance and localization of dystrophin-glycoprotein complex components. These pigs recapitulate many of the cardinal features of muscular dystrophy, have elevated serum creatine kinase activity, and preliminarily appear to display altered locomotion. They also suffer from sudden death preceded by EKG abnormalities. Pig dystrophinopathy models could allow refinement of dosing strategies in human-sized animals in preparation for clinical trials. From an animal handling perspective, these pigs can generally be treated normally, with the understanding that acute stress can lead to sudden death. In summary, the ability to create genetically modified pig models and the serendipitous discovery of genetic disease in the swine industry has resulted in the emergence of new animal tools to facilitate the critical objective of improving the quality and length of life for boys afflicted with such a devastating disease.
Topics: Animals; Disease Models, Animal; Dogs; Dystrophin; Mice; Muscular Dystrophy, Duchenne; Rats; Swine; Swine Diseases
PubMed: 25991703
DOI: 10.1093/ilar/ilv015 -
International Journal of Molecular... Aug 2022Duchenne muscular dystrophy (DMD) is the most common fatal muscle disease, with an estimated incidence of 1/3500-1/5000 male births, and it is associated with mutations...
Duchenne muscular dystrophy (DMD) is the most common fatal muscle disease, with an estimated incidence of 1/3500-1/5000 male births, and it is associated with mutations in the X-linked gene encoding dystrophin, the largest known human gene. There is currently no cure for DMD. The large size of the gene hampers exogenous gene addition and delivery. The genetic correction of DMD patient-derived induced pluripotent stem cells (DMD-iPSCs) and differentiation into suitable cells for transplantation is a promising autologous therapeutic strategy for DMD. In this study, using CRISPR/Cas9, the full-length dystrophin coding sequence was reconstructed in an exon-50-deleted DMD-iPSCs by the targeted addition of exon 50 at the junction of exon 49 and intron 49 via homologous-directed recombination (HDR), with a high targeting efficiency of 5/15, and the genetically corrected iPSCs were differentiated into cardiomyocytes (iCMs). Importantly, the full-length dystrophin expression and membrane localization were restored in genetically corrected iPSCs and iCMs. Thus, this is the first study demonstrating that full-length dystrophin can be restored in iPSCs and iCMs via targeted exon addition, indicating potential clinical prospects for DMD gene therapy.
Topics: Dystrophin; Exons; Humans; Induced Pluripotent Stem Cells; Male; Muscular Dystrophy, Duchenne; Myocytes, Cardiac
PubMed: 36012442
DOI: 10.3390/ijms23169176 -
BioDrugs : Clinical Immunotherapeutics,... Jan 2024Duchenne muscular dystrophy is a devastating disease that leads to progressive muscle loss and premature death. While medical management focuses mostly on symptomatic... (Comparative Study)
Comparative Study Review
Duchenne muscular dystrophy is a devastating disease that leads to progressive muscle loss and premature death. While medical management focuses mostly on symptomatic treatment, decades of research have resulted in first therapeutics able to restore the affected reading frame of dystrophin transcripts or induce synthesis of a truncated dystrophin protein from a vector, with other strategies based on gene therapy and cell signaling in preclinical or clinical development. Nevertheless, recent reports show that potentially therapeutic dystrophins can be immunogenic in patients. This raises the question of whether a dystrophin paralog, utrophin, could be a more suitable therapeutic protein. Here, we compare dystrophin and utrophin amino acid sequences and structures, combining published data with our extended in silico analyses. We then discuss these results in the context of therapeutic approaches for Duchenne muscular dystrophy. Specifically, we focus on strategies based on delivery of micro-dystrophin and micro-utrophin genes with recombinant adeno-associated viral vectors, exon skipping of the mutated dystrophin pre-mRNAs, reading through termination codons with small molecules that mask premature stop codons, dystrophin gene repair by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated genetic engineering, and increasing utrophin levels. Our analyses highlight the importance of various dystrophin and utrophin domains in Duchenne muscular dystrophy treatment, providing insights into designing novel therapeutic compounds with improved efficacy and decreased immunoreactivity. While the necessary actin and β-dystroglycan binding sites are present in both proteins, important functional distinctions can be identified in these domains and some other parts of truncated dystrophins might need redesigning due to their potentially immunogenic qualities. Alternatively, therapies based on utrophins might provide a safer and more effective approach.
Topics: Humans; Dystrophin; Genetic Therapy; Muscular Dystrophy, Duchenne; Utrophin
PubMed: 37917377
DOI: 10.1007/s40259-023-00632-3 -
Skeletal Muscle Nov 2023The lack of functional dystrophin protein in Duchenne muscular dystrophy (DMD) causes chronic skeletal muscle inflammation and degeneration. Therefore, the restoration...
BACKGROUND
The lack of functional dystrophin protein in Duchenne muscular dystrophy (DMD) causes chronic skeletal muscle inflammation and degeneration. Therefore, the restoration of functional dystrophin levels is a fundamental approach for DMD therapy. Electrical impedance myography (EIM) is an emerging tool that provides noninvasive monitoring of muscle conditions and has been suggested as a treatment response biomarker in diverse indications. Although magnetic resonance imaging (MRI) of skeletal muscles has become a standard measurement in clinical trials for DMD, EIM offers distinct advantages, such as portability, user-friendliness, and reduced cost, allowing for remote monitoring of disease progression or response to therapy. To investigate the potential of EIM as a biomarker for DMD, we compared longitudinal EIM data with MRI/histopathological data from an X-linked muscular dystrophy (mdx) mouse model of DMD. In addition, we investigated whether EIM could detect dystrophin-related changes in muscles using antisense-mediated exon skipping in mdx mice.
METHODS
The MRI data for muscle T2, the magnetic resonance spectroscopy (MRS) data for fat fraction, and three EIM parameters with histopathology were longitudinally obtained from the hindlimb muscles of wild-type (WT) and mdx mice. In the EIM study, a cell-penetrating peptide (Pip9b2) conjugated antisense phosphorodiamidate morpholino oligomer (PPMO), designed to induce exon-skipping and restore functional dystrophin production, was administered intravenously to mdx mice.
RESULTS
MRI imaging in mdx mice showed higher T2 intensity at 6 weeks of age in hindlimb muscles compared to WT mice, which decreased at ≥ 9 weeks of age. In contrast, EIM reactance began to decline at 12 weeks of age, with peak reduction at 18 weeks of age in mdx mice. This decline was associated with myofiber atrophy and connective tissue infiltration in the skeletal muscles. Repeated dosing of PPMO (10 mg/kg, 4 times every 2 weeks) in mdx mice led to an increase in muscular dystrophin protein and reversed the decrease in EIM reactance.
CONCLUSIONS
These findings suggest that muscle T2 MRI is sensitive to the early inflammatory response associated with dystrophin deficiency, whereas EIM provides a valuable biomarker for the noninvasive monitoring of subsequent changes in skeletal muscle composition. Furthermore, EIM reactance has the potential to monitor dystrophin-deficient muscle abnormalities and their recovery in response to antisense-mediated exon skipping.
Topics: Mice; Animals; Dystrophin; Mice, Inbred mdx; Electric Impedance; Mice, Inbred C57BL; Muscular Dystrophy, Duchenne; Muscle, Skeletal; Morpholinos; Myography; Biomarkers
PubMed: 37980539
DOI: 10.1186/s13395-023-00331-1 -
Cell Cycle (Georgetown, Tex.) 2015
Topics: Animals; Dystrophin; Female; Humans; Male; Muscular Dystrophies; Sarcoma
PubMed: 26101897
DOI: 10.1080/15384101.2015.1064676