-
Journal of Cachexia, Sarcopenia and... Apr 2022Duchenne muscular dystrophy (DMD) is caused by DMD mutations leading to dystrophin loss. Full-length Dp427 is the primary dystrophin isoform expressed in muscle and is...
BACKGROUND
Duchenne muscular dystrophy (DMD) is caused by DMD mutations leading to dystrophin loss. Full-length Dp427 is the primary dystrophin isoform expressed in muscle and is also expressed in the central nervous system (CNS). Two shorter isoforms, Dp140 and Dp71, are highly expressed in the CNS. While a role for Dp140 and Dp71 on DMD CNS comorbidities is well known, relationships between mutations expected to disrupt Dp140 and Dp71 and motor outcomes are not.
METHODS
Functional outcome data from 387 DMD boys aged 4-15 years were subdivided by DMD mutation expected effects on dystrophin isoform expression; Group 1 (Dp427 absent, Dp140/Dp71 present, n = 201); Group 2 (Dp427/Dp140 absent, Dp71 present, n = 152); and Group 3 (Dp427/Dp140/Dp71 absent, n = 34). Relationships between isoform group and North Star ambulatory assessment (NSAA) scores, 10 m walk/run velocities and rise time velocities were explored using regression analysis. Western blot analysis was used to study Dp427, Dp140 and Dp71 production in myogenic cells (control and DMD human), control skeletal muscle, DMD skeletal muscle from the three isoform groups and cerebral cortex from mice (wild-type and DMD models). Grip strength and rotarod running test were studied in wild-type mice and DMD mouse models. DMD mouse models were mdx (Dp427 absent, Dp140/Dp71 present), mdx52 (Dp427/Dp140 absent, Dp71 present) and DMD-null (lacking all isoforms).
RESULTS
In DMD boys, mean NSAA scores at 5 years of age were 6.1 points lower in Group 3 than Group 1 (P < 0.01) and 4.9 points lower in Group 3 than Group 2 (P = 0.05). Mean peak NSAA scores were 4.0 points lower in Group 3 than Group 1 (P < 0.01) and 1.6 points lower in Group 2 than Group 1 (P = 0.04). Mean four-limb grip strength was 1.5 g/g lower in mdx52 than mdx mice (P = 0.003) and 1.5 g/g lower in DMD-null than mdx mice (P = 0.002). Dp71 was produced in myogenic cells (control and DMD human) and skeletal muscle from humans in Groups 1 and 2 and mdx mice, but not skeletal muscle from human controls, myogenic cells and skeletal muscle from humans in Group 3 or skeletal muscle from wild-type, mdx52 or DMD-null mice.
CONCLUSIONS
Our results highlight the importance of considering expected effects of DMD mutations on dystrophin isoform production when considering patterns of DMD motor impairment and the implications for clinical practice and clinical trials. Our results suggest a complex relationship between dystrophin isoforms expressed in the brain and DMD motor function.
Topics: Animals; Dystrophin; Humans; Mice; Mice, Inbred mdx; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Protein Isoforms
PubMed: 35083887
DOI: 10.1002/jcsm.12914 -
Disease Models & Mechanisms Mar 2022Duchenne muscular dystrophy (DMD), a fatal musculoskeletal disease, is associated with neurodevelopmental disorders and cognitive impairment caused by brain dystrophin...
Duchenne muscular dystrophy (DMD), a fatal musculoskeletal disease, is associated with neurodevelopmental disorders and cognitive impairment caused by brain dystrophin deficiency. Dog models of DMD represent key translational tools to study dystrophin biology and to develop novel therapeutics. However, characterisation of dystrophin expression and function in the canine brain is lacking. We studied the DE50-MD canine model of DMD that has a missense mutation in the donor splice site of exon 50. Using a battery of cognitive tests, we detected a neurocognitive phenotype in DE50-MD dogs, including reduced attention, problem solving and exploration of novel objects. Through a combination of capillary immunoelectrophoresis, immunolabelling, quantitative PCR and RNAScope in situ hybridisation, we show that regional dystrophin expression in the adult canine brain reflects that of humans, and that the DE50-MD dog lacks full-length dystrophin (Dp427) protein expression but retains expression of the two shorter brain-expressed isoforms, Dp140 and Dp71. Thus, the DE50-MD dog is a translationally relevant pre-clinical model to study the consequences of Dp427 deficiency in the brain and to develop therapeutic strategies for the neurological sequelae of DMD.
Topics: Animals; Brain; Dogs; Dystrophin; Exons; Muscular Dystrophy, Duchenne; Phenotype
PubMed: 35019137
DOI: 10.1242/dmm.049291 -
International Journal of Molecular... Jul 2023Duchenne muscular dystrophy (DMD) is a yet incurable rare genetic disease that affects the skeletal and cardiac muscles, leading to progressive muscle wasting and...
Duchenne muscular dystrophy (DMD) is a yet incurable rare genetic disease that affects the skeletal and cardiac muscles, leading to progressive muscle wasting and premature death. DMD is caused by the lack of dystrophin, a muscle protein essential for the biochemical support and integrity of muscle fibers. Gene replacement strategies for Duchenne muscular dystrophy (DMD) employing the adeno-associated virus (AAV) face the challenge imposed by the limited packaging capacity of AAV, only allowing the accommodation of a short version of dystrophin (µDys) that is still far removed from correcting human disease. The need to develop strategies leading to the expression of a best performing dystrophin variant led to only few studies reporting on the use of dual vectors, but none reported on a method to assess in vivo transgene reconstitution efficiency, the degree of which directly affects the use of safe AAV dosing. We report here on the generation of a dual AAV vector approach for the expression of a larger dystrophin version (quasidystrophin) based on homologous recombination, and the development of a methodology employing a strategic droplet digital PCR design, to determine the recombination efficiency as well as the occurrence of unwanted concatemerization events or aberrant expression from the single vectors. We demonstrated that, upon systemic delivery in the dystrophic D2.B10-Dmd/J (DBA2mdx) mice, our dual AAV approach led to high transgene reconstitution efficiency and negligible Inverted Terminal Repeats (ITR)-dependent concatemerization, with consequent remarkable protein restoration in muscles and improvement of muscle pathology. This evidence supports the suitability of our system for gene therapy application and the potential of this methodology to assess and improve the feasibility for therapeutic translation of multiple vector approaches.
Topics: Mice; Humans; Animals; Muscular Dystrophy, Duchenne; Dystrophin; Dependovirus; Muscle, Skeletal; Mice, Inbred mdx; Genetic Vectors
PubMed: 37511179
DOI: 10.3390/ijms241411421 -
International Journal of Molecular... May 2023Mutations that prevent the production of proteins in the gene cause Duchenne muscular dystrophy. Most frequently, these are deletions leading to reading-frame shift....
Mutations that prevent the production of proteins in the gene cause Duchenne muscular dystrophy. Most frequently, these are deletions leading to reading-frame shift. The "reading-frame rule" states that deletions that preserve ORF result in a milder Becker muscular dystrophy. By removing several exons, new genome editing tools enable reading-frame restoration in DMD with the production of BMD-like dystrophins. However, not every truncated dystrophin with a significant internal loss functions properly. To determine the effectiveness of potential genome editing, each variant should be carefully studied in vitro or in vivo. In this study, we focused on the deletion of exons 8-50 as a potential reading-frame restoration option. Using the CRISPR-Cas9 tool, we created the novel mouse model DMDdel8-50, which has an in-frame deletion in the gene. We compared DMDdel8-50 mice to C57Bl6/CBA background control mice and previously generated DMDdel8-34 KO mice. We discovered that the shortened protein was expressed and correctly localized on the sarcolemma. The truncated protein, on the other hand, was unable to function like a full-length dystrophin and prevent disease progression. On the basis of protein expression, histological examination, and physical assessment of the mice, we concluded that the deletion of exons 8-50 is an exception to the reading-frame rule.
Topics: Mice; Animals; Dystrophin; Mice, Inbred CBA; Muscular Dystrophy, Duchenne; Phenotype; Exons; Gene Deletion
PubMed: 37298068
DOI: 10.3390/ijms24119117 -
Annals of Clinical and Translational... Dec 2020The novel morpholino antisense oligonucleotide viltolarsen targets exon 53 of the dystrophin gene, and could be an effective treatment for patients with Duchenne...
OBJECTIVE
The novel morpholino antisense oligonucleotide viltolarsen targets exon 53 of the dystrophin gene, and could be an effective treatment for patients with Duchenne muscular dystrophy (DMD). We investigated viltolarsen's ability to induce dystrophin expression and examined its safety in DMD patients.
METHODS
In this open-label, multicenter, parallel-group, phase 1/2, exploratory study, 16 ambulant and nonambulant males aged 5-12 years with DMD received viltolarsen 40 or 80 mg/kg/week via intravenous infusion for 24 weeks. Primary endpoints were dystrophin expression and exon 53 skipping levels.
RESULTS
In western blot analysis, mean changes in dystrophin expression (% normal) from baseline to Weeks 12 and 24 were - 1.21 (P = 0.5136) and 1.46 (P = 0.1636), respectively, in the 40 mg/kg group, and 0.76 (P = 0.2367) and 4.81 (P = 0.0536), respectively, in the 80 mg/kg group. The increase in mean dystrophin level at Weeks 12 and 24 was significant in the 80 mg/kg group (2.78%; P = 0.0364). Patients receiving 80 mg/kg showed a higher mean exon 53 skipping level (42.4%) than those receiving 40 mg/kg (21.8%). All adverse events were judged to be mild or moderate in intensity and none led to study discontinuation.
INTERPRETATION
Treatment with viltolarsen 40 or 80 mg/kg elicited an increasing trend in dystrophin expression and exon 53 skipping levels, and was safe and well tolerated. The decline in motor function appeared less marked in patients with higher dystrophin levels; this may warrant further investigation. This study supports the potential clinical benefit of viltolarsen.
Topics: Child; Child, Preschool; Dystrophin; Humans; Japan; Male; Muscular Dystrophy, Duchenne; Oligonucleotides; Outcome Assessment, Health Care
PubMed: 33285037
DOI: 10.1002/acn3.51235 -
The AAPS Journal Dec 2022Duchenne muscular dystrophy (DMD) is a degenerative muscular disease affecting roughly one in 5000 males at birth. The disease is often caused by inherited X-linked...
Duchenne muscular dystrophy (DMD) is a degenerative muscular disease affecting roughly one in 5000 males at birth. The disease is often caused by inherited X-linked recessive pathogenic variants in the dystrophin gene, but may also arise from de novo mutations. Disease-causing variants include nonsense, out of frame deletions or duplications that result in loss of dystrophin protein expression. There is currently no cure for DMD and the few treatment options available aim at slowing muscle degradation. New advances in gene therapy and understanding of dystrophin (DYS) expression in other muscular dystrophies have opened new opportunities for treatment. Therefore, reliable methods are needed to monitor dystrophin expression and assess the efficacy of new therapies for muscular dystrophies such as DMD and Becker muscular dystrophy (BMD). Here, we describe the validation of a novel Western blot (WB) method for the quantitation of mini-dystrophin protein in human skeletal muscle tissues that is easy to adopt in most laboratory settings. This WB method was assessed through precision, accuracy, selectivity, dilution linearity, stability, and repeatability. Based on mini-DYS standard performance, the assay has a dynamic range of 0.5-15 ng protein (per 5 µg total protein per lane), precision of 3.3 to 25.5%, and accuracy of - 7.5 to 3.3%. Our stability assessment showed that the protein is stable after 4 F/T cycles, up to 2 h at RT and after 7 months at - 70°C. Furthermore, our WB method was compared to the results from our recently published LC-MS method. Workflow for our quantitative WB method to determine mini-dystrophin levels in muscle tissues (created in Biorender.com). Step 1 involves protein extraction from skeletal muscle tissue lysates from control, DMD, or BMD biospecimen. Step 2 measures total protein concentrations. Step 3 involves running gel electrophoresis with wild-type dystrophin (wt-DYS) from muscle tissue extracts alongside mini-dystrophin STD curve and mini-DYS and protein normalization with housekeeping GAPDH.
Topics: Male; Infant, Newborn; Humans; Dystrophin; Muscular Dystrophy, Duchenne; Muscle, Skeletal; Biopsy; Blotting, Western
PubMed: 36539515
DOI: 10.1208/s12248-022-00776-0 -
Skeletal Muscle Jan 2023The dystrophin-glycoprotein complex (DGC) is a critical adhesion complex of the muscle cell membrane, providing a mechanical link between the extracellular matrix (ECM)...
Multi-omics analysis of sarcospan overexpression in mdx skeletal muscle reveals compensatory remodeling of cytoskeleton-matrix interactions that promote mechanotransduction pathways.
BACKGROUND
The dystrophin-glycoprotein complex (DGC) is a critical adhesion complex of the muscle cell membrane, providing a mechanical link between the extracellular matrix (ECM) and the cortical cytoskeleton that stabilizes the sarcolemma during repeated muscle contractions. One integral component of the DGC is the transmembrane protein, sarcospan (SSPN). Overexpression of SSPN in the skeletal muscle of mdx mice (murine model of DMD) restores muscle fiber attachment to the ECM in part through an associated increase in utrophin and integrin adhesion complexes at the cell membrane, protecting the muscle from contraction-induced injury. In this study, we utilized transcriptomic and ECM protein-optimized proteomics data sets from wild-type, mdx, and mdx transgenic (mdx) skeletal muscle tissues to identify pathways and proteins driving the compensatory action of SSPN overexpression.
METHODS
The tibialis anterior and quadriceps muscles were isolated from wild-type, mdx, and mdx mice and subjected to bulk RNA-Seq and global proteomics analysis using methods to enhance capture of ECM proteins. Data sets were further analyzed through the ingenuity pathway analysis (QIAGEN) and integrative gene set enrichment to identify candidate networks, signaling pathways, and upstream regulators.
RESULTS
Through our multi-omics approach, we identified 3 classes of differentially expressed genes and proteins in mdx muscle, including those that were (1) unrestored (significantly different from wild type, but not from mdx), (2) restored (significantly different from mdx, but not from wild type), and (3) compensatory (significantly different from both wild type and mdx). We identified signaling pathways that may contribute to the rescue phenotype, most notably cytoskeleton and ECM organization pathways. ECM-optimized proteomics revealed an increased abundance of collagens II, V, and XI, along with β-spectrin in mdx samples. Using ingenuity pathway analysis, we identified upstream regulators that are computationally predicted to drive compensatory changes, revealing a possible mechanism of SSPN rescue through a rewiring of cell-ECM bidirectional communication. We found that SSPN overexpression results in upregulation of key signaling molecules associated with regulation of cytoskeleton organization and mechanotransduction, including Yap1, Sox9, Rho, RAC, and Wnt.
CONCLUSIONS
Our findings indicate that SSPN overexpression rescues dystrophin deficiency partially through mechanotransduction signaling cascades mediated through components of the ECM and the cortical cytoskeleton.
Topics: Mice; Animals; Dystrophin; Muscular Dystrophy, Duchenne; Mechanotransduction, Cellular; Multiomics; Mice, Inbred mdx; Muscle, Skeletal; Cytoskeleton; Membrane Proteins; Neoplasm Proteins
PubMed: 36609344
DOI: 10.1186/s13395-022-00311-x -
The Journal of Pathology Aug 2021Duchenne muscular dystrophy (DMD) is a muscle-wasting disease caused by dystrophin deficiency. Vascular dysfunction has been suggested as an underlying pathogenic...
Duchenne muscular dystrophy (DMD) is a muscle-wasting disease caused by dystrophin deficiency. Vascular dysfunction has been suggested as an underlying pathogenic mechanism in DMD. However, this has not been thoroughly studied in a large animal model. Here we investigated structural and functional changes in the vascular smooth muscle and endothelium of the canine DMD model. The expression of dystrophin and endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS), and the structure and function of the femoral artery from 15 normal and 16 affected adult dogs were evaluated. Full-length dystrophin was detected in the endothelium and smooth muscle in normal but not affected dog arteries. Normal arteries lacked nNOS but expressed eNOS in the endothelium. NOS activity and eNOS expression were reduced in the endothelium of dystrophic dogs. Dystrophin deficiency resulted in structural remodeling of the artery. In affected dogs, the maximum tension induced by vasoconstrictor phenylephrine and endothelin-1 was significantly reduced. In addition, acetylcholine-mediated vasorelaxation was significantly impaired, whereas exogenous nitric oxide-induced vasorelaxation was significantly enhanced. Our results suggest that dystrophin plays a crucial role in maintaining the structure and function of vascular endothelium and smooth muscle in large mammals. Vascular defects may contribute to DMD pathogenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Topics: Animals; Disease Models, Animal; Dogs; Dystrophin; Endothelium, Vascular; Muscle, Smooth, Vascular; Muscular Dystrophy, Animal; Muscular Dystrophy, Duchenne
PubMed: 33999411
DOI: 10.1002/path.5704 -
Cells Oct 2023Understanding and countering the well-established negative health consequences of spaceflight remains a primary challenge preventing safe deep space exploration....
Understanding and countering the well-established negative health consequences of spaceflight remains a primary challenge preventing safe deep space exploration. Targeted/personalized therapeutics are at the forefront of space medicine strategies, and cross-species molecular signatures now define the 'typical' spaceflight response. However, a lack of direct genotype-phenotype associations currently limits the robustness and, therefore, the therapeutic utility of putative mechanisms underpinning pathological changes in flight. We employed the worm as a validated model of space biology, combined with 'NemaFlex-S' microfluidic devices for assessing animal strength production as one of the most reproducible physiological responses to spaceflight. Wild-type and (BZ33) strains (a Duchenne muscular dystrophy (DMD) model for comparing predisposed muscle weak animals) were cultured on the International Space Station in chemically defined media before loading second-generation gravid adults into NemaFlex-S devices to assess individual animal strength. These same cultures were then frozen on orbit before returning to Earth for next-generation sequencing transcriptomic analysis. Neuromuscular strength was lower in flight ground controls (16.6% decline, < 0.05), with significantly more (23% less strength, < 0.01) affected than wild types. The transcriptional gene ontology signatures characterizing both strains of weaker animals in flight strongly corroborate previous results across species, enriched for upregulated stress response pathways and downregulated mitochondrial and cytoskeletal processes. Functional gene cluster analysis extended this to implicate decreased neuronal function, including abnormal calcium handling and acetylcholine signaling, in space-induced strength declines under the predicted control of UNC-89 and DAF-19 transcription factors. Finally, gene modules specifically altered in animals in flight again cluster to neuronal/neuromuscular pathways, suggesting strength loss in DMD comprises a strong neuronal component that predisposes these animals to exacerbated strength loss in space. Highly reproducible gene signatures are strongly associated with space-induced neuromuscular strength loss across species and neuronal changes in calcium/acetylcholine signaling require further study. These results promote targeted medical efforts towards and provide an in vivo model for safely sending animals and people into deep space in the near future.
Topics: Humans; Animals; Caenorhabditis elegans; Acetylcholine; Calcium; Space Flight; Caenorhabditis elegans Proteins; Dystrophin
PubMed: 37887314
DOI: 10.3390/cells12202470 -
Annals of Neurology Feb 2021This study was undertaken to determine whether a low residual quantity of dystrophin protein is associated with delayed clinical milestones in patients with DMD...
OBJECTIVE
This study was undertaken to determine whether a low residual quantity of dystrophin protein is associated with delayed clinical milestones in patients with DMD mutations.
METHODS
We performed a retrospective multicentric cohort study by using molecular and clinical data from patients with DMD mutations registered in the Universal Mutation Database-DMD France database. Patients with intronic, splice site, or nonsense DMD mutations, with available muscle biopsy Western blot data, were included irrespective of whether they presented with severe Duchenne muscular dystrophy (DMD) or milder Becker muscular dystrophy (BMD). Patients were separated into 3 groups based on dystrophin protein levels. Clinical outcomes were ages at appearance of first symptoms; loss of ambulation; fall in vital capacity and left ventricular ejection fraction; interventions such as spinal fusion, tracheostomy, and noninvasive ventilation; and death.
RESULTS
Of 3,880 patients with DMD mutations, 90 with mutations of interest were included. Forty-two patients expressed no dystrophin (group A), and 31 of 42 (74%) developed DMD. Thirty-four patients had dystrophin quantities < 5% (group B), and 21 of 34 (61%) developed BMD. Fourteen patients had dystrophin quantities ≥ 5% (group C), and all but 4 who lost ambulation beyond 24 years of age were ambulant. Dystrophin quantities of <5%, as low as <0.5%, were associated with milder phenotype for most of the evaluated clinical outcomes, including age at loss of ambulation (p < 0.001).
INTERPRETATION
Very low residual dystrophin protein quantity can cause a shift in disease phenotype from DMD toward BMD. ANN NEUROL 2021;89:280-292.
Topics: Adolescent; Adrenal Cortex Hormones; Adult; Age of Onset; Angiotensin-Converting Enzyme Inhibitors; Blotting, Western; Child; Cohort Studies; Disease Progression; Dystrophin; Humans; Male; Mobility Limitation; Mortality; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Noninvasive Ventilation; Oxadiazoles; Phenotype; Proportional Hazards Models; Retrospective Studies; Severity of Illness Index; Spinal Fusion; Stroke Volume; Tracheostomy; Vital Capacity; Young Adult
PubMed: 33159473
DOI: 10.1002/ana.25951