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Disease Models & Mechanisms Mar 2015Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder. It is caused by loss-of-function mutations in the dystrophin gene. Currently, there is no... (Review)
Review
Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder. It is caused by loss-of-function mutations in the dystrophin gene. Currently, there is no cure. A highly promising therapeutic strategy is to replace or repair the defective dystrophin gene by gene therapy. Numerous animal models of DMD have been developed over the last 30 years, ranging from invertebrate to large mammalian models. mdx mice are the most commonly employed models in DMD research and have been used to lay the groundwork for DMD gene therapy. After ~30 years of development, the field has reached the stage at which the results in mdx mice can be validated and scaled-up in symptomatic large animals. The canine DMD (cDMD) model will be excellent for these studies. In this article, we review the animal models for DMD, the pros and cons of each model system, and the history and progress of preclinical DMD gene therapy research in the animal models. We also discuss the current and emerging challenges in this field and ways to address these challenges using animal models, in particular cDMD dogs.
Topics: Animals; Cardiomyopathies; Disease Models, Animal; Dystrophin; Genetic Therapy; Muscular Dystrophy, Duchenne; Neurons
PubMed: 25740330
DOI: 10.1242/dmm.018424 -
Presse Medicale (Paris, France : 1983) May 1994The fantastic advances in fundamental research into muscular dystrophy over the past few years have led to the discovery of a cytoskeleton protein called dystrophin and... (Review)
Review
The fantastic advances in fundamental research into muscular dystrophy over the past few years have led to the discovery of a cytoskeleton protein called dystrophin and a group of associated and analogous molecules. The understanding of their molecular structure and the modes of genetic and functional regulation have forced us to take a new look at our concept of the pathogenesis of muscular dystrophy. Today, the group of diseases called X-linked hereditary muscular dystrophies must now be conceived in light of a deficiency in dystrophin. The exact physiopathology of the process involved is not yet fully understood but most undoubtedly, although the lack of dystrophin can trigger the disease, the deficiency cannot in itself impede muscle contraction. As a result of these discoveries, diagnostic methods have changed drastically, genetic counselling has become more precise, and most importantly, new treatment rationales can call upon pharmacological processes capable of stopping disease progression. Genetic therapy is another approach. The aim is to provide the deficient cells with the capacity of synthesizing normal dystrophin or an analogous molecule, thus halting the cascade of events leading to necrosis. The transfer of the technical means which have made these fundamental advances in basic research possible to successful clinical applications in the treatment of muscular dystrophy will be another of the lessons to be learned from dystrophin.
Topics: Dystrophin; Female; Genetic Therapy; Humans; Male; Muscular Dystrophies; Polymerase Chain Reaction; Pregnancy; Prenatal Diagnosis; X Chromosome
PubMed: 7937619
DOI: No ID Found -
Gene Therapy Nov 2022Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disorder caused by mutations in the DMD gene, leading to severe reduction or absence of the protein...
Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disorder caused by mutations in the DMD gene, leading to severe reduction or absence of the protein dystrophin. Gene therapy strategies that aim to increase expression of a functional dystrophin protein (mini-dystrophin) are under investigation. The ability to accurately quantify dystrophin/mini-dystrophin is essential in assessing the level of gene transduction. We demonstrated the validation and application of a novel peptide immunoaffinity liquid chromatography-tandem mass spectrometry (IA-LC-MS/MS) assay. Data showed that dystrophin expression in Becker muscular dystrophy and DMD tissues, normalized against the mean of non-dystrophic control tissues (n = 20), was 4-84.5% (mean 32%, n = 20) and 0.4-24.1% (mean 5%, n = 20), respectively. In a DMD rat model, biceps femoris tissue from dystrophin-deficient rats treated with AAV9.hCK.Hopti-Dys3978.spA, an adeno-associated virus vector containing a mini-dystrophin transgene, showed a dose-dependent increase in mini-dystrophin expression at 6 months post-dose, exceeding wildtype dystrophin levels at high doses. Validation data showed that inter- and intra-assay precision were ≤20% (≤25% at the lower limit of quantification [LLOQ]) and inter- and intra-run relative error was within ±20% (±25% at LLOQ). IA-LC-MS/MS accurately quantifies dystrophin/mini-dystrophin in human and preclinical species with sufficient sensitivity for immediate application in preclinical/clinical trials.
Topics: Humans; Rats; Animals; Dystrophin; Muscular Dystrophy, Duchenne; Chromatography, Liquid; Tandem Mass Spectrometry; Muscle, Skeletal; Genetic Therapy
PubMed: 34737451
DOI: 10.1038/s41434-021-00300-7 -
Human Molecular Genetics Sep 2016Dystrophin is a large sub-sarcolemmal protein. Its absence leads to Duchenne muscular dystrophy (DMD). Binding to the sarcolemma is essential for dystrophin to protect...
Dystrophin is a large sub-sarcolemmal protein. Its absence leads to Duchenne muscular dystrophy (DMD). Binding to the sarcolemma is essential for dystrophin to protect muscle from contraction-induced injury. It has long been thought that membrane binding of dystrophin depends on its cysteine-rich (CR) domain. Here, we provide in vivo evidence suggesting that dystrophin contains three additional membrane-binding domains including spectrin-like repeats (R)1-3, R10-12 and C-terminus (CT). To systematically study dystrophin membrane binding, we split full-length dystrophin into ten fragments and examined subcellular localizations of each fragment by adeno-associated virus-mediated gene transfer. In skeletal muscle, R1-3, CR domain and CT were exclusively localized at the sarcolemma. R10-12 showed both cytosolic and sarcolemmal localization. Importantly, the CR-independent membrane binding was conserved in murine and canine muscles. A critical function of the CR-mediated membrane interaction is the assembly of the dystrophin-associated glycoprotein complex (DGC). While R1-3 and R10-12 did not restore the DGC, surprisingly, CT alone was sufficient to establish the DGC at the sarcolemma. Additional studies suggest that R1-3 and CT also bind to the sarcolemma in the heart, though relatively weak. Taken together, our study provides the first conclusive in vivo evidence that dystrophin contains multiple independent membrane-binding domains. These structurally and functionally distinctive membrane-binding domains provide a molecular framework for dystrophin to function as a shock absorber and signaling hub. Our results not only shed critical light on dystrophin biology and DMD pathogenesis, but also provide a foundation for rationally engineering minimized dystrophins for DMD gene therapy.
Topics: Animals; Binding Sites; Conserved Sequence; Cytosol; Dogs; Dystrophin; Glycoproteins; Humans; Mice; Mice, Inbred mdx; Muscular Dystrophy, Animal; Myocardium; Protein Domains; Sarcolemma
PubMed: 27378693
DOI: 10.1093/hmg/ddw210 -
Critical Reviews in Eukaryotic Gene... 2009The dystrophin/dystrobrevin/dystrotelin superfamily is marked by a common constellation of domains whose juxtaposition is tightly constrained in all three subfamilies.... (Review)
Review
The dystrophin/dystrobrevin/dystrotelin superfamily is marked by a common constellation of domains whose juxtaposition is tightly constrained in all three subfamilies. These domains comprise a cluster of four closely packed EF hands, a ZZ domain, and two coiled-coil regions. In addition, the dystrophin and dystrobrevin branches share one or two binding sites for members of the syntrophin family of adaptor proteins, and the dystrophins and some dystrotelins share a WW domain. Much of the exon structure of the genes encoding these domains is also shared, confirming the monophyletic status of the superfamily. Almost all animals have at least one member of each branch, with multiple paralogous members of the dystrophin and dystrobrevin branches in the vertebrates. Thus, in humans, 6 genes, 19 promoters, and 10 alternatively spliced exons conspire to generate a staggering array of proteins, with at least one in almost every tissue of the body. This review aims to summarize what is known of this complexity of expression of members of the dystrophin/dystrobrevin/dystrotelin superfamily across the animal kingdom. The widespread expression in adult vertebrates, together with elaborate and dynamic patterns during development, paints a picture of a superfamily whose fundamental biological function is still poorly understood.
Topics: Alternative Splicing; Animals; Dystrophin; Dystrophin-Associated Proteins; Gene Expression Profiling; Invertebrates; Phylogeny; Protein Isoforms; Vertebrates
PubMed: 19392646
DOI: 10.1615/critreveukargeneexpr.v19.i2.10 -
Cell Cycle (Georgetown, Tex.) 2015
Topics: Animals; Dystrophin; Female; Humans; Male; Muscular Dystrophies; Sarcoma
PubMed: 26101897
DOI: 10.1080/15384101.2015.1064676 -
Biochimica Et Biophysica Acta.... Jan 2021The molecular and cellular basis for cataract development in mice lacking dystrophin, a scaffolding protein that links the cytoskeleton to the extracellular matrix, is...
The molecular and cellular basis for cataract development in mice lacking dystrophin, a scaffolding protein that links the cytoskeleton to the extracellular matrix, is poorly understood. In this study, we characterized lenses derived from the dystrophin-deficient mdx mouse model. Expression of Dp71, a predominant isoform of dystrophin in the lens, was induced during lens fiber cell differentiation. Dp71 was found to co-distribute with dystroglycan, connexin-50 and 46, aquaporin-0, and NrCAM as a large cluster at the center of long arms of the hexagonal fibers. Although mdx mouse lenses exhibited dramatically reduced levels of Dp71, only older lenses revealed punctate nuclear opacities compared to littermate wild type (WT) lenses. The levels of dystroglycan, syntrophin, and dystrobrevin which comprise the dystrophin-associated protein complex (DAPC), and NrCAM, connexin-50, and aquaporin-0, were significantly lower in the lens membrane fraction of adult mdx mice compared to WT mice. Additionally, decreases were observed in myosin light chain phosphorylation and lens stiffness together with a significant elevation in the levels of utrophin, a functional homolog of dystrophin in mdx mouse lenses compared to WT lenses. The levels of perlecan and laminin (ligands of α-dystroglycan) remained normal in dystrophin-deficient lens fibers. Taken together, although mdx mouse lenses exhibit only minor defects in lens clarity possibly due to a compensatory increase in utrophin, the noted disruptions of DAPC, stability, and organization of membrane integral proteins of fibers, and stiffness of mdx lenses reveal the importance of dystrophin and DAPC in maintaining lens clarity and function.
Topics: Animals; Dystrophin; Eye Proteins; Gene Expression Regulation; Lens, Crystalline; Mice; Mice, Inbred mdx
PubMed: 33127476
DOI: 10.1016/j.bbadis.2020.165998 -
Molecular Genetics and Metabolism 2001Duchenne muscular dystrophy was described in the medical literature in the early 1850s but the molecular basis of the disease was not determined until the late 1980s.... (Review)
Review
Duchenne muscular dystrophy was described in the medical literature in the early 1850s but the molecular basis of the disease was not determined until the late 1980s. The cloning of dystrophin led to the identification of a large complex of proteins that plays an important, although not yet well understood, role in muscle biology. Concomitant with the elucidation of the function of dystrophin and its associated proteins has been the pursuit of therapeutic options for muscular dystrophy. Although there is still no cure for this disorder, great advances are being made in the areas of gene introduction and cell transplant therapy.
Topics: Animals; Cell Transplantation; Cloning, Molecular; Disease Models, Animal; Dystrophin; Forecasting; Genetic Therapy; Humans; Muscle, Skeletal; Muscular Dystrophies
PubMed: 11592805
DOI: 10.1006/mgme.2001.3220 -
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 -
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