Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by ptosis, swallowing difficulties, proximal limb weakness and nuclear aggregates in ...skeletal muscles. OPMD is caused by a trinucleotide repeat expansion in the PABPN1 gene that results in an N-terminal expanded polyalanine tract in polyA-binding protein nuclear 1 (PABPN1). Here we show that the treatment of a mouse model of OPMD with an adeno-associated virus-based gene therapy combining complete knockdown of endogenous PABPN1 and its replacement by a wild-type PABPN1 substantially reduces the amount of insoluble aggregates, decreases muscle fibrosis, reverts muscle strength to the level of healthy muscles and normalizes the muscle transcriptome. The efficacy of the combined treatment is further confirmed in cells derived from OPMD patients. These results pave the way towards a gene replacement approach for OPMD treatment.
Aims
New therapies for neuromuscular disorders are often mutation specific and require to be studied in patient's cell cultures. In Duchenne muscular dystrophy (DMD) dystrophin restoration drugs are ...being developed but as muscle cell cultures from DMD patients are scarce and do not grow or differentiate well, only a limited number of candidate drugs are tested. Moreover, dystrophin quantification by western blotting requires a large number of cultured cells; so fewer compounds are as thoroughly screened as is desirable. We aimed to develop a quantitative assessment tool using fewer cells to contribute in the study of dystrophin and to identify better drug candidates.
Methods
An ‘in‐cell western’ assay is a quantitative immunofluorescence assay performed in cell culture microplates that allows protein quantification directly in culture, allowing a higher number of experimental repeats and throughput. We have optimized the assay (‘myoblot’) to be applied to the study of differentiated myoblast cultures.
Results
After an exhaustive optimization of the technique to adapt it to the growth and differentiation rates of our cultures and the low intrinsic expression of our proteins of interests, our myoblot protocol allows the quantification of dystrophin and other muscle‐associated proteins in muscle cell cultures. We are able to distinguish accurately between the different sets of patients based on their dystrophin expression and detect dystrophin restoration after treatment.
Conclusions
We expect that this new tool to quantify muscle proteins in DMD and other muscle disorders will aid in their diagnosis and in the development of new therapies.
Adeno Associated virus serotype 8 (AAV8) is of particular interest as a vector for pre-clinical and clinical trial for Duchenne Muscular Dystrophy (DMD). In several cell lines, this vector has been ...shown to enter cells through clathrin-mediated endocytosis followed by a trafficking through the microtubule network in various endosomal compartments toward the nucleus. To efficiently transduce cells, AAV must undergo multiple levels of regulation in these cellular compartments. In DMD, dystrophin deficiency results in disturbed balance of cellular events i.e., fiber centronucleation, disorganized cytoskeleton, presence of fibrosis. We have recently described a loss of virion genomes from both dogs and mice models of DMD treated with therapeutic molecules vectorized in AAV. Indeed, the pathophysiological state of DMD muscle should impact on virions fate and subsequently affect crucial steps for AAV effectiveness as viral uncoating, viral genome maintenance and consequently, the transduction efficiency of AAV. Our project aims to characterize cellular uptake and intracellular transport of AAV8 in DMD muscular cells, with the goal of optimizing AAV vector use to get the best transduction efficiency with the lowest AAV dose. Our first data showed that AAV8-GFP was less efficient to transduce DMD and control primary muscular cells compared to HeLa cells. Moreover, AAV8 traffics through same endosomal compartment in DMD and control myoblasts, but at different rates during early time points of the transduction. These results suggest that in muscle cells, AAV8 uses different entry and trafficking pathways from those previously described in HeLa cells and that dystrophic cellular status could affect subcellular processing of the vector particles. We will specify the relationship between AAV8 vector entry, trafficking, uncoating, and transduction efficiency in vitro in primary myoblasts/myotubes of DMD patients and controls.
Abstract The mechanisms underlying myoblast response to mechanical forces are critical for muscle development and functionality. To initiate adequate mechanotransduction events, myoblasts must be ...able to sense strains in their extracellular cellular matrix (ECM) substrate, transmit forces to the cell interior and activate intracellular signaling. The presence of lamin A/C is critical for intact nucleoskeleton and mechanical transmission between the nucleus and the ECM. Whether mutation in the LMNA gene (encoding for lamin A/C) causes mechanotransduction defects in human myoblasts has never been directly tested. Myoblasts with LMNA p.Lys32del mutation (D32) were obtained from a 12 year-old boy suffering from LMNA-related congenital muscular dystrophy (L-CMD). Normal sex- and age-matched human myoblasts (WT) were used as controls. Myoblasts were immortalized, cultured in a 3D fibrin matrix. 3D constructs were subjected to axial cyclic stretch (10% amplitude, 0.5 Hz, 4 h). Confocal microscopy imaging and expression of mechanosensitive genes (Iex-1 and Ccnd-1) were analyzed. Unstrained D32 myoblasts had larger and more abundant actin bundles and longer and thinner nuclei ( p < 0.01) compared with WT. Repetitive strains induced a thickening of actin stress fibers, and an alignment of cells along the longitudinal axis of the 3D matrix in WT absent in D32 cells, while a significant nuclear elongation was observed in D32 but not in WT. Addition of blebbistatin, a myosin inhibitor or Y-27632, a Rho kinase inhibitor, abolished the strain-induced WT cytoskeleton reorganization and myoblasts alignment without changing WT nuclear shape. Milder effects on actin bundles were observed in D32. Strain-induced changes in the expression of Iex-1 and Ccnd-1 were higher in WT than in D32 ( p < 0.001). We provide first evidence that lamin A/C mutation causes mechanotransduction and cytoskeleton organization defects in human myoblasts and may contribute to mechanical damage in striated muscles from L-CMD.
Oculopharyngeal muscular dystrophy (OPMD) is a late onset autosomal dominant inherited dystrophy due to an expansion of GCG repeats in the coding region of the ubiquitously expressed PABPN1 gene. The ...main muscular targets of OPMD are cricopharyngeal muscle (CPM) and elevator eyelid muscle whose progressive involvement leads to dysphagia and ptosis. We have recently demonstrated that OPMD CPM is characterized by atrophy, fibrosis and increased PAX7-positive cells compared to control CPM and to OPMD non-affected muscles such as quadriceps (QM). These results suggest that the specific involvement of affected muscles in OPMD correlates with an exacerbated fibrosis and a failure of the regenerative response. This led us to further study the skeletal muscle progenitor cells from CPM and their possible deregulation in OPMD. In vitro, primary cultures isolated from CPM biopsies of both OPMD ( n = 4) and control ( n = 4) subjects were characterized by a rapid fall of their myogenicity, whereas in primary cultures from control QM ( n = 3) the myogenicity was maintained throughout the course of the lifespan. Several hypotheses for this drastic loss of myogenicity have been investigated, e.g. proliferative deregulation between resident fibroblasts and myoblasts. In addition, CPM cells regeneration capacity was explored in vivo. Each primary culture was injected into cryodamaged tibialis anterior (TA) muscle of an immunodeficient mouse model. One month after injection, a high number of human PAX7-positive satellite cells were found in TA muscles injected with CPM myoblasts compared to control injected QM myoblasts. Moreover, an elevated number of human cells were revealed in the interstitial space of TA muscle injected by CPM myoblasts. Altogether, these results show that muscle primary cultures represent a good model to further study the deregulations observed in OPMD CPM. Cellular and molecular mechanisms deregulated in both fibroblasts and myoblasts fractions are now being investigated.
In recent years, therapeutic approaches for neuromuscular diseases have been developed and new strategies are currently emerging. Human cellular models adapted to each disease and each mutation, are ...needed to test these therapeutic strategies. Patient-derived muscle stem cells, also known as myoblasts can be isolated from biopsies, but their use is restricted by 1) the availability of muscle biopsies for research, 2) the limited proliferative capacity of the myoblasts and 3) their potential exhaustion in degenerative diseases. The Myoline platform of the Center of Research in Myology develops easy-to-use immortalized human myoblast models isolated from muscle tissue of patients suffering from various neuromuscular diseases. When access to muscle biopsies is not available, fibroblasts from skin biopsies can be immortalized and converted into muscle cells. Recently, we are also developing models of Fibro-Adipogenic Progenitors (FAPS) from muscles of patients with neuromuscular diseases. Human muscle cells are immortalized by transduction of the telomerase (hTERT) and cyclin-dependent kinase 4 (cdk4) genes. These immortalized muscle cells present an extended proliferation, and retain their ability to differentiate and fuse into myotubes. We have generated more than 168 human immortalized myoblast lines including 36 neuromuscular diseases such as DMD, DM1, LGMD2B, OPMD, FSHD and control subjects. Whenever access to muscle biopsies is not possible, an alternative cellular model has been developed using human skin fibroblasts: the transduction with hTERT provides an extended proliferation, while the conditional expression of the myogenic factor MyoD drives the muscle conversion and differentiation into multinucleated myotubes. Several human fibroblast lines have been produced, e.g. from DMD, DM1 or controls subjects. These cellular models have the great advantage of being easy to use and allow rapid testing of therapeutic strategies. They are made available to the academic international scientific community on a collaborative basis. For some of these cell lines, the patient consent allows their use by private companies. The Myoline platform can also be solicited to set up new models on request.
Abstract Skin fibroblasts are essential tools for biochemical, genetic and physiopathological investigations of mitochondrial diseases. Their immortalization has been previously performed to overcome ...the limited number of divisions of these primary cells but it has never been systematically evaluated with respect to efficacy and impact on the oxidative phosphorylation (OXPHOS) characteristics of the cells. We successfully immortalized with the human telomerase gene 15 human fibroblasts populations, 4 derived from controls and 11 from patients with diverse respiratory chain defects. Immortalization induced significant but mild modification of the OXPHOS characteristics of the cells with lower rates of oxygen consumption and ATP synthesis associated with their loose coupling. However, it never significantly altered the type and severity of any genetic OXPHOS defect present prior to immortalization. Furthermore, it did not significantly modify the cells’ dependence on glucose and sensitivity to galactose thus showing that immortalized cells could be screened by their nutritional requirement. Immortalized skin fibroblasts with significant OXPHOS defect provide reliable tools for the diagnosis and research of the genetic cause of mitochondrial defects. They also represent precious material to investigate the cellular responses to these defects, even though these should afterwards be verified in unmodified primary cells.
Lamins are the main constituents of the nuclear lamina, a protein meshwork underlining the inner face of the nuclear envelope (NE) and facing chromatin and nucleoplasm. A-type lamins (lamin A and C), ...encoded by the LMNA gene, have roles in the chromatin organization, through domains called Lamin-Associated Domains (LAD). Proper maintenance of chromatin organization is essential for normal cell function, and depends on nuclear envelope stability and tissue-specific nuclear envelope proteins. LMNA gene mutations are responsible for a wide spectrum of disorders called laminopathies, most of them affecting striated muscles. LMNA mutations have been associated with defects in LAD organization and gene expression at the nuclear periphery, contributing in the pathophysiological mechanisms of laminopathies. Laminopathies are characterized by a strong clinical variability and genetic or epigenetic factors, such as modifier genes or specific chromatin organization defects could explain such variability. We collected biological materials, through national and international collaborations and we received a majority of fibroblasts from LMNA-mutated patients. Genome organization being highly different between cell types, we myo-converted immortalized skin fibroblasts into myogenic cells, via overexpression of murine MyoD. We performed a study to validate this model as relevant to investigate LAD organization, by combining RNA-seq and ChIP-seq targeting Lamin A/C and histone marks. We showed that myotubes derived from these myo-converted fibroblasts underwent a clear phenotypic switch to myogenic cell type, at a transcriptomic and at a chromatin level, hence validating this cell system to study LAD organization in muscle cells.
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