Drug screening in a zebrafish model of Duchenne muscular dystrophy.
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ABSTRACT:
SUBMITTER: Kawahara G
PROVIDER: S-EPMC3069215 | biostudies-literature | 2011 Mar
REPOSITORIES: biostudies-literature
Publications
Drug screening in a zebrafish model of Duchenne muscular dystrophy.
Kawahara Genri G Karpf Jeremy A JA Myers Jennifer A JA Alexander Matthew S MS Guyon Jeffrey R JR Kunkel Louis M LM
Proceedings of the National Academy of Sciences of the United States of America 20110314 13
Two known zebrafish dystrophin mutants, sapje and sapje-like (sap(c/100)), represent excellent small-animal models of human muscular dystrophy. Using these dystrophin-null zebrafish, we have screened the Prestwick chemical library for small molecules that modulate the muscle phenotype in these fish. With a quick and easy birefringence assay, we have identified seven small molecules that influence muscle pathology in dystrophin-null zebrafish without restoration of dystrophin expression. Three of ...[more]
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Project description:BackgroundDuchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin, a muscle cytoskeletal protein. Utrophin is a homologue of dystrophin that can functionally compensate for its absence when expressed at increased levels in the myofibre, as shown by studies in dystrophin-deficient mice. Utrophin upregulation is therefore a promising therapeutic approach for DMD. The use of a small, drug-like molecule to achieve utrophin upregulation offers obvious advantages in terms of delivery and bioavailability. Furthermore, much of the time and expense involved in the development of a new drug can be eliminated by screening molecules that are already approved for clinical use.Methodology/principal findingsWe developed and validated a cell-based, high-throughput screening assay for utrophin promoter activation, and used it to screen the Prestwick Chemical Library of marketed drugs and natural compounds. Initial screening produced 20 hit molecules, 14 of which exhibited dose-dependent activation of the utrophin promoter and were confirmed as hits. Independent validation demonstrated that one of these compounds, nabumetone, is able to upregulate endogenous utrophin mRNA and protein, in C2C12 muscle cells.Conclusions/significanceWe have developed a cell-based, high-throughput screening utrophin promoter assay. Using this assay, we identified and validated a utrophin promoter-activating drug, nabumetone, for which pharmacokinetics and safety in humans are already well described, and which represents a lead compound for utrophin upregulation as a therapy for DMD.
| S-EPMC3197614 | biostudies-literature
Project description:Duchenne muscular dystrophy (DMD) is a common and relentlessly progressive muscle disease. Some interventions have been identified that modestly slow progression and prolong survival, but more meaningful therapies are lacking. The goal of this study is to identify new therapeutic pathways for DMD using a zebrafish model of the disease. To accomplish this, we performed a non-biased drug screen in sapje, a zebrafish line with a recessive nonsense mutation in dystrophin. We identified 6 positive hits (out of 640 total drugs tested) by their ability to prevent abnormal birefringence in sapje. Follow-up analyses demonstrated that fluoxetine, a selective serotonin reuptake inhibitor (SSRI), provided the most substantial benefit. Morpholino-based experimentation confirmed that modulation of the serotonin pathway alone can prevent the dystrophic phenotype, and transcriptomic analysis revealed changes in calcium homeostasis as a potential mechanism. In all, we demonstrate that monoamine agonists can prevent disease in a vertebrate model of DMD. Given the safe and widespread use of SSRIs in clinical practice, our study identifies an attractive target pathway for therapy development.
| S-EPMC4119416 | biostudies-literature
Project description:We developed a novel somite-based step-wise strategy for the efficient derivation of functional human myocytes, suggesting that past failures were due to incomplete specification. Treatment with two small molecules inhibiting glycogen synthase kinase 3β (GSK-3β) and the Notch signaling pathway in undifferentiated hPSCs results in the formation of somite-like cells by Day 4 and, subsequently contractile myotubes in vitro around Day 25 with the ability to engraft and actively participate in muscle repair in vivo. Antibody-based purification can enrich homogenous myocyte populations exhibiting genuine myogenic molecular and cellular characteristics, including extraocular muscle-like features. Furthermore, hPSCs derived from patients with multiple neuromuscular diseases successfully give rise to patient-specific skeletal muscle cells bearing signature phenotypes. Human embryonic stem cell were differentiated to skeletal muscle and compared to non-differentiated cells in triplicate biological replicates. For the confirmation of differentiated cell's characteristics and the global mRNA profiles, We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated genes during this process. Further more, we used microarrys for the comparing control and disease gene expression profiles, again in triplicate.
2016-06-16 | E-GEOD-70955 | biostudies-arrayexpress
Project description:Duchenne muscular dystrophy (DMD) is a severe, progressive, and incurable X-linked disorder caused by mutations in the dystrophin gene. Patients with DMD have an absence of functional dystrophin protein, which results in chronic damage of muscle fibers during contraction, thus leading to deterioration of muscle quality and loss of muscle mass over time. Although there is currently no cure for DMD, improvements in treatment care and management could delay disease progression and improve quality of life, thereby prolonging life expectancy for these patients. Furthermore, active research efforts are ongoing to develop therapeutic strategies that target dystrophin deficiency, such as gene replacement therapies, exon skipping, and readthrough therapy, as well as strategies that target secondary pathology of DMD, such as novel anti-inflammatory compounds, myostatin inhibitors, and cardioprotective compounds. Furthermore, longitudinal modeling approaches have been used to characterize the progression of MRI and functional endpoints for predictive purposes to inform Go/No Go decisions in drug development. This review showcases approved drugs or drug candidates along their development paths and also provides information on primary endpoints and enrollment size of Ph2/3 and Ph3 trials in the DMD space.
| S-EPMC9353054 | biostudies-literature
Project description:Dystrophin deficiency results in lethal Duchenne muscular dystrophy (DMD). Substituting missing dystrophin with abbreviated microdystrophin has dramatically alleviated disease in mouse DMD models. Unfortunately, translation of microdystrophin therapy has been unsuccessful in dystrophic dogs, the only large mammalian model. Approximately 70% of the dystrophin-coding sequence is removed in microdystrophin. Intriguingly, loss of ?50% dystrophin frequently results in severe disease in patients. To test whether the small gene size constitutes a fundamental design error for large mammalian muscle, we performed a comprehensive study using 22 dogs (8 normal and 14 dystrophic). We delivered the ?R2-15/?R18-19/?R20-23/?C microdystrophin gene to eight extensor carpi ulnaris (ECU) muscles in six dystrophic dogs using Y713F tyrosine mutant adeno-associated virus (AAV)-9 (2.6 × 10(13) viral genome (vg) particles/muscle). Robust expression was observed 2 months later despite T-cell infiltration. Major components of the dystrophin-associated glycoprotein complex (DGC) were restored by microdystrophin. Treated muscle showed less inflammation, fibrosis, and calcification. Importantly, therapy significantly preserved muscle force under the stress of repeated cycles of eccentric contraction. Our results have established the proof-of-concept for microdystrophin therapy in dystrophic muscles of large mammals and set the stage for clinical trial in human patients.
| S-EPMC3616540 | biostudies-other
Project description:We developed a novel somite-based step-wise strategy for the efficient derivation of functional human myocytes, suggesting that past failures were due to incomplete specification. Treatment with two small molecules inhibiting glycogen synthase kinase 3β (GSK-3β) and the Notch signaling pathway in undifferentiated hPSCs results in the formation of somite-like cells by Day 4 and, subsequently contractile myotubes in vitro around Day 25 with the ability to engraft and actively participate in muscle repair in vivo. Antibody-based purification can enrich homogenous myocyte populations exhibiting genuine myogenic molecular and cellular characteristics, including extraocular muscle-like features. Furthermore, hPSCs derived from patients with multiple neuromuscular diseases successfully give rise to patient-specific skeletal muscle cells bearing signature phenotypes.
2016-06-16 | GSE70955 | GEO
Project description:Duchenne muscular dystrophy is a severe, progressive, muscle-wasting disease that leads to difficulties with movement and, eventually, to the need for assisted ventilation and premature death. The disease is caused by mutations in DMD (encoding dystrophin) that abolish the production of dystrophin in muscle. Muscles without dystrophin are more sensitive to damage, resulting in progressive loss of muscle tissue and function, in addition to cardiomyopathy. Recent studies have greatly deepened our understanding of the primary and secondary pathogenetic mechanisms. Guidelines for the multidisciplinary care for Duchenne muscular dystrophy that address obtaining a genetic diagnosis and managing the various aspects of the disease have been established. In addition, a number of therapies that aim to restore the missing dystrophin protein or address secondary pathology have received regulatory approval and many others are in clinical development.
| S-EPMC10557455 | biostudies-literature
Project description:Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in the DMD gene and loss of the protein dystrophin. The absence of dystrophin leads to myofiber membrane fragility and necrosis, with eventual muscle atrophy and contractures. Affected boys typically die in their second or third decade due to either respiratory failure or cardiomyopathy. Despite extensive attempts to develop definitive therapies for DMD, the standard of care remains prednisone, which has only palliative benefits. Animal models, mainly the mdx mouse and golden retriever muscular dystrophy (GRMD) dog, have played a key role in studies of DMD pathogenesis and treatment development. Because the GRMD clinical syndrome is more severe than in mice, better aligning with the progressive course of DMD, canine studies may translate better to humans. The original founder dog for all GRMD colonies worldwide was identified in the early 1980s before the discovery of the DMD gene and dystrophin. Accordingly, analogies to DMD were initially drawn based on similar clinical features, ranging from the X-linked pattern of inheritance to overlapping histopathologic lesions. Confirmation of genetic homology between DMD and GRMD came with identification of the underlying GRMD mutation, a single nucleotide change that leads to exon skipping and an out-of-frame DMD transcript. GRMD colonies have subsequently been established to conduct pathogenetic and preclinical treatment studies. Simultaneous with the onset of GRMD treatment trials, phenotypic biomarkers were developed, allowing definitive characterization of treatment effect. Importantly, GRMD studies have not always substantiated findings from mdx mice and have sometimes identified serious treatment side effects. While the GRMD model may be more clinically relevant than the mdx mouse, usage has been limited by practical considerations related to expense and the number of dogs available. This further complicates ongoing broader concerns about the poor rate of translation of animal model preclinical studies to humans with analogous diseases. Accordingly, in performing GRMD trials, special attention must be paid to experimental design to align with the approach used in DMD clinical trials. This review provides context for the GRMD model, beginning with its original description and extending to its use in preclinical trials.
| S-EPMC5438519 | biostudies-literature
Project description:BackgroundThe aim of this study was to pool multiple data sets to build a patient-centric, data-informed, natural history model (NHM) for Duchenne muscular dystrophy (DMD) to estimate disease trajectory across patient lifetime under current standard of care in future economic evaluations. The study was conducted as part of Project HERCULES, a multi-stakeholder collaboration to develop tools to support health technology assessments of new treatments for DMD.MethodsHealth states were informed by a review of NHMs for DMD and input from clinicians, patients and caregivers, and defined using common outcomes in clinical trials and real-world practice. The primary source informing the NHM was the Critical Path Institute Duchenne Regulatory Science Consortium (D-RSC) database. This was supplemented with expert input obtained via an elicitation exercise, and a systematic literature review and meta-analysis of mortality data.ResultsThe NHM includes ambulatory, transfer and non-ambulatory phases, which capture loss of ambulation, ability to weight bear and upper body and respiratory function, respectively. The NHM estimates patients spend approximately 9.5 years in ambulatory states, 1.5 years in the transfer state and the remainder of their lives in non-ambulatory states. Median predicted survival is 34.8 years (95% CI 34.1-35.8).ConclusionThe model includes a detailed disease pathway for DMD, including the clinically and economically important transfer state. The NHM may be used to estimate the current trajectory of DMD in economic evaluations of new treatments, facilitating inclusion of a lifetime time horizon, and will help identify areas for further research.
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Project description:In order to understand the chronic hypoxia (CH) effect upon the absence of dystrophin, Drosophila melanogaster wild type and the model for DMD (dmDys), in which all dystrophins expression was knocked out by iRNA, were exposed to high altitude hypoxia (hypobaric hypoxia) during a 16-day climbing period reaching the summit of Mount McKinley (6194 meters above sea level). Furthermore, dmDys and Drosophila wild type were exposed to normobaric hypoxia (hypoxic chamber) following the same oxygen levels observed during the climbing expedition and to normoxic conditions for comparison. Affymetrix GeneChip® profiling was performed for individual flies from each experimental group. CH-dmDys differentially expressed 1281 genes, whereas control group differentially expressed 57 genes. Eight heat shock protein genes detected in the CH-dmDys microarray study were down-regulated, instead of up-regulated as seen in wild type hypoxic flies. This result suggests a differential gene expression response to CH, which could affect muscle performance.These results suggest that dmDys is more sensitive to CH due to reduced muscle function and hypoxic stress response. In order to understand the chronic hypoxia (CH) effect upon the absence of dystrophin, Drosophila melanogaster wild type and the model for DMD (dmDys), in which all dystrophins expression was knocked out by iRNA, were exposed to high altitude hypoxia (hypobaric hypoxia) during a 16-day climbing period reaching the summit of Mount McKinley (6194 meters above sea level). Furthermore, dmDys and Drosophila wild type were exposed to normobaric hypoxia (hypoxic chamber) following the same oxygen levels observed during the climbing expedition and to normoxic conditions for comparison. Affymetrix GeneChip® profiling was performed for individual flies from each experimental group. CH-dmDys differentially expressed 1281 genes, whereas control group differentially expressed 57 genes. Eight heat shock protein genes detected in the CH-dmDys microarray study were down-regulated, instead of up-regulated as seen in wild type hypoxic flies. This result suggests a differential gene expression response to CH, which could affect muscle performance.These results suggest that dmDys is more sensitive to CH due to reduced muscle function and hypoxic stress response. Overall design: Adults wild type and dystrophic flies (3-5 days old) were exposed to hypobaric hypoxia for two weeks during the summer expedition to Mount McKinley, Alaska (6194 MASL). Another set of wild types and dystrophic flies were exposed to normobaric hypoxia according to the table I obtained during the climbing expedition. During the expedition, the flies were maintained in vials with regular molasses and covered by thermo isolation to avoid low temperature, keeping the temperature at 25C. The experiment performed in the laboratory also used vials with regular molasses and at 25C. Table I. Expedition log book for mount McKinley ascent. Information obtained during the ascent and summit of Mount McKinley, June 1st to June 16th of 2007. The oxygen pressure (PO2) was calculated from the barometric pressure. GNB means go and back from the mentioned point. DAY In order to understand the chronic hypoxia (CH) effect upon the absence of dystrophin, Drosophila melanogaster wild type and the model for DMD (dmDys), in which all dystrophins expression was knocked out by iRNA, were exposed to high altitude hypoxia (hypobaric hypoxia) during a 16-day climbing period reaching the summit of Mount McKinley (6194 meters above sea level). Furthermore, dmDys and Drosophila wild type were exposed to normobaric hypoxia (hypoxic chamber) following the same oxygen levels observed during the climbing expedition and to normoxic conditions for comparison. Affymetrix GeneChip® profiling was performed for individual flies from each experimental group. CH-dmDys differentially expressed 1281 genes, whereas control group differentially expressed 57 genes. Eight heat shock protein genes detected in the CH-dmDys microarray study were down-regulated, instead of up-regulated as seen in wild type hypoxic flies. This result suggests a differential gene expression response to CH, which could affect muscle performance.These results suggest that dmDys is more sensitive to CH due to reduced muscle function and hypoxic stress response. In order to understand the chronic hypoxia (CH) effect upon the absence of dystrophin, Drosophila melanogaster wild type and the model for DMD (dmDys), in which all dystrophins expression was knocked out by iRNA, were exposed to high altitude hypoxia (hypobaric hypoxia) during a 16-day climbing period reaching the summit of Mount McKinley (6194 meters above sea level). Furthermore, dmDys and Drosophila wild type were exposed to normobaric hypoxia (hypoxic chamber) following the same oxygen levels observed during the climbing expedition and to normoxic conditions for comparison. Affymetrix GeneChip® profiling was performed for individual flies from each experimental group. CH-dmDys differentially expressed 1281 genes, whereas control group differentially expressed 57 genes. Eight heat shock protein genes detected in the CH-dmDys microarray study were down-regulated, instead of up-regulated as seen in wild type hypoxic flies. This result suggests a differential gene expression response to CH, which could affect muscle performance.These results suggest that dmDys is more sensitive to CH due to reduced muscle function and hypoxic stress response. Overall design: Adults wild type and dystrophic flies (3-5 days old) were exposed to hypobaric hypoxia for two weeks during the summer expedition to Mount McKinley, Alaska (6194 MASL). Another set of wild types and dystrophic flies were exposed to normobaric hypoxia according to the table I obtained during the climbing expedition. During the expedition, the flies were maintained in vials with regular molasses and covered by thermo isolation to avoid low temperature, keeping the temperature at 25C. The experiment performed in the laboratory also used vials with regular molasses and at 25C. Table I. Expedition log book for mount McKinley ascent. Information obtained during the ascent and summit of Mount McKinley, June 1st to June 16th of 2007. The oxygen pressure (PO2) was calculated from the barometric pressure. GNB means go and back from the mentioned point. DAY LOCATION ALTITUDE m PO2 mmHg (%) 1 Base Camp 2200 123.6 (16.3%) 2 Base Camp 2200 123.6 (16.3%) 3 Base Camp 2200 123.6 (16.3%) 4 Ski Hill 2400 120.7 (15.9%) 5 Kahiltna Pass 2950 113.0 (14.9%) 6 Motorcycle Hill 3350 107.7 (14.2%) 7 Motorcycle Hill 3350 107.7 (14.2%) 8 GNB from Motorcycle 4150 (5 hours) 97.7 (12.9%) 9 Medical Camp 4350 95.3 (12.5%) 10 GNB from Medical Camp 4150 (5 hours) 97.7 (12.9%) 11 Medical Camp 4350 95.3 (12.5%) 12 GNB from Medical Camp 4900 89.0 (11.7%) 13 Medical Camp 4350 95.3 (12.5%) 14 High Camp 5250 85.1 (11.2%) 15 Summit 6194 (0.3 hours) 75.4 (9.9%) 16 High Camp 5250 85.1 (11.2%)
2010-07-31 | E-GEOD-15879 | biostudies-arrayexpress