Capucine Trollet

Poly(A) binding protein nuclear 1 levels affect alternative polyadenylation

The choice for a polyadenylation site determines the length of the 3′-untranslated region (3′-UTRs) of an mRNA. Inclusion or exclusion of regulatory sequences in the 3′-UTR may ultimately affect gene expression levels. Poly(A) binding protein nuclear 1 (PABPN1) is involved in polyadenylation of pre-mRNAs. An alanine repeat expansion in PABPN1 (exp-PABPN1) causes oculopharyngeal muscular dystrophy (OPMD). We hypothesized that previously observed disturbed gene expression patterns in OPMD muscles may have been the result of an effect of PABPN1 on alternative polyadenylation, influencing mRNA stability, localization and translation. A single molecule polyadenylation site sequencing method was developed to explore polyadenylation site usage on a genome-wide level in mice overexpressing exp-PABPN1. We identified 2012 transcripts with altered polyadenylation site usage. In the far majority, more proximal alternative polyadenylation sites were used, resulting in shorter 3′-UTRs. 3′-UTR shortening was generally associated with increased expression. Similar changes in polyadenylation site usage were observed after knockdown or overexpression of expanded but not wild-type PABPN1 in cultured myogenic cells. Our data indicate that PABPN1 is important for polyadenylation site selection and that reduced availability of functional PABPN1 in OPMD muscles results in use of alternative polyadenylation sites, leading to large-scale deregulation of gene expression.

Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders

BackgroundInvestigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies.MethodsUsing transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders.ResultsThe immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice.ConclusionsDystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.

Codon and mRNA sequence optimization of microdystrophin transgenes improves expression and physiological outcome in dystrophic mdx mice following AAV2/8 gene transfer

Duchenne muscular dystrophy is a fatal muscle-wasting disorder. Lack of dystrophin compromises the integrity of the sarcolemma and results in myofibers that are highly prone to contraction-induced injury. Recombinant adeno-associated virus (rAAV)-mediated dystrophin gene transfer strategies to muscle for the treatment of Duchenne muscular dystrophy (DMD) have been limited by the small cloning capacity of rAAV vectors and high titers necessary to achieve efficient systemic gene transfer. In this study, we assess the impact of codon optimization on microdystrophin (DeltaAB/R3-R18/DeltaCT) expression and function in the mdx mouse and compare the function of two different configurations of codon-optimized microdystrophin genes (DeltaAB/R3-R18/DeltaCT and DeltaR4-R23/DeltaCT) under the control of a muscle-restrictive promoter (Spc5-12). Codon optimization of microdystrophin significantly increases levels of microdystrophin mRNA and protein after intramuscular and systemic administration of plasmid DNA or rAAV2/8. Physiological assessment demonstrates that codon optimization of DeltaAB/R3-R18/DeltaCT results in significant improvement in specific force, but does not improve resistance to eccentric contractions compared with noncodon-optimized DeltaAB/R3-R18/DeltaCT. However, codon-optimized microdystrophin DeltaR4-R23/DeltaCT completely restored specific force generation and provided substantial protection from contraction-induced injury. These results demonstrate that codon optimization of microdystrophin under the control of a muscle-specific promoter can significantly improve expression levels such that reduced titers of rAAV vectors will be required for efficient systemic administration.

Design of Phosphorodiamidate Morpholino Oligomers (PMOs) for the Induction of Exon Skipping of the Human DMD Gene

Duchenne muscular dystrophy (DMD) is caused by out-of-frame mutations of the human DMD gene. Antisense oligonucleotides (AOs) have previously been used to skip additional exons that border the deletions such that the reading frame is restored and internally truncated, but functional, dystrophin expressed. We have designed phosphorodiamidate morpholino oligomer (PMO) AOs to various exons of the human dystrophin gene. PMOs were designed to have their target sites overlapping areas of open RNA structure, as defined by hybridization-array analysis, and likely exonic splicing enhancer (ESE)/silencer sites on the target RNA. The ability of each PMO to produce exon skipping was tested in vitro in normal human skeletal muscle cells. Retrospective analysis of design parameters used and PMO variables revealed that active PMOs were longer, bound to their targets more strongly, had their target sites closer to the acceptor splice site of the exon, overlapped areas of open conformation (as defined by the hybridization or the RNA secondary structure prediction software), and could interfere with the binding of certain SR proteins. No other parameter appeared to show significant association to PMO-skipping efficacy. No design tool is strong enough in isolation; however, if used in conjunction with other significant parameters it can aid AO design.

Autologous Myoblast Transplantation for Oculopharyngeal Muscular Dystrophy: a Phase I/Iia Clinical Study

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominant genetic disease mainly characterized by ptosis and dysphagia. We conducted a phase I/IIa clinical study (ClinicalTrials.gov NCT00773227) using autologous myoblast transplantation following myotomy in adult OPMD patients. This study included 12 patients with clinical diagnosis of OPMD, indication for cricopharyngeal myotomy, and confirmed genetic diagnosis. The feasibility and safety end points of both autologous myoblast transplantation and the surgical procedure were assessed by videoendoscopy in addition to physical examinations. Potential therapeutic benefit was also assessed through videoendoscopy and videofluoroscopy of swallowing, quality of life score, dysphagia grade, and a drink test. Patients were injected with a median of 178 million myoblasts following myotomy. Short and long-term (2 years) safety and tolerability were observed in all the patients, with no adverse effects. There was an improvement in the quality of life score for all 12 patients, and no functional degradation in swallowing was observed for 10 patients. A cell dose-dependant improvement in swallowing was even observed in this study. This trial supports the hypothesis that a local injection of autologous myoblasts in the pharyngeal muscles is a safe and efficient procedure for OPMD patients.

Molecular and phenotypic characterization of a mouse model of oculopharyngeal muscular dystrophy reveals severe muscular atrophy restricted to fast glycolytic fibres

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by ptosis, dysphagia and proximal limb weakness. Autosomal-dominant OPMD is caused by a short (GCG)(8-13) expansions within the first exon of the poly(A)-binding protein nuclear 1 gene (PABPN1), leading to an expanded polyalanine tract in the mutated protein. Expanded PABPN1 forms insoluble aggregates in the nuclei of skeletal muscle fibres. In order to gain insight into the different physiological processes affected in OPMD muscles, we have used a transgenic mouse model of OPMD (A17.1) and performed transcriptomic studies combined with a detailed phenotypic characterization of this model at three time points. The transcriptomic analysis revealed a massive gene deregulation in the A17.1 mice, among which we identified a significant deregulation of pathways associated with muscle atrophy. Using a mathematical model for progression, we have identified that one-third of the progressive genes were also associated with muscle atrophy. Functional and histological analysis of the skeletal muscle of this mouse model confirmed a severe and progressive muscular atrophy associated with a reduction in muscle strength. Moreover, muscle atrophy in the A17.1 mice was restricted to fast glycolytic fibres, containing a large number of intranuclear inclusions (INIs). The soleus muscle and, in particular, oxidative fibres were spared, even though they contained INIs albeit to a lesser degree. These results demonstrate a fibre-type specificity of muscle atrophy in this OPMD model. This study improves our understanding of the biological pathways modified in OPMD to identify potential biomarkers and new therapeutic targets.

Gene therapy for muscular dystrophy: current progress and future prospects

Muscular dystrophies refer to a group of inherited disorders characterized by progressive muscle weakness, wasting and degeneration. So far, there is no effective treatment but new gene-based therapies are currently being developed with particular noted advances in using conventional gene replacement strategies, RNA-based approaches, or cell-based gene therapy with a main focus on Duchenne muscular dystrophy (DMD). DMD is the most common and severe form of muscular dystrophy and current treatments are far from adequate. However, genetic and cell-based therapies, in particular exon skipping induced by antisense strategies, and corrective gene therapy via functionally engineered dystrophin genes hold great promise, with several clinical trials ongoing. Proof-of-concept of exon skipping has been obtained in animal models, and most recently in clinical trials; this approach represents a promising therapy for a subset of patients. In addition, gene-delivery-based strategies exist both for antisense-induced reading frame restoration, and for highly efficient delivery of functional dystrophin mini- and micro-genes to muscle fibres in vivo and muscle stem cells ex-vivo. In particular, AAV-based vectors show efficient systemic gene delivery to skeletal muscle directly in vivo, and lentivirus-based vectors show promise of combining ex vivo gene modification strategies with cell-mediated therapies.

Delivery of AAV2/9-Microdystrophin Genes Incorporating Helix 1 of the Coiled-Coil Motif in the C-Terminal Domain of Dystrophin Improves Muscle Pathology and Restores the Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence-optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain-extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction-induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

Current advances in cell therapy strategies for muscular dystrophies

Introduction: Muscular dystrophies are a heterogeneous group of genetic diseases characterized by muscle weakness, wasting and degeneration. Cell therapy consists of delivering myogenic precursor cells to damaged tissue for the complementation of missing proteins and/or the regeneration of new muscle fibres. Areas covered: We focus on human candidate cells described so far (myoblasts, mesoangioblasts, pericytes, myoendothelial cells, CD133+ cells, aldehyde-dehydrogenase-positive cells, mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells), gene-based strategies developed to modify cells prior to injection, animal models (dystrophic and/or immunodeficient) used for pre-clinical studies, and clinical trials that have been performed using cell therapy strategies. The approaches are reviewed in terms of feasibility, hurdles, potential solutions and/or research areas from where the solution may come and potential application in terms of types of dystrophies and targets. Expert opinion: Cell therapy for muscular dystrophies should be put in the context of which dystrophy or muscle group is targeted, what tools are available at hand, but even more importantly what can cell therapy bring as compared with and/or in combination with other therapeutic strategies. The solution will probably be the right dosage of these combinations adapted to each dystrophy, or even to each type of mutation within a dystrophy.

RNAi-mediated knockdown of dystrophin expression in adult mice does not lead to overt muscular dystrophy pathology

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology. The peak of the pathology attributed to dystrophin deficiency happens between 3 and 8 weeks of age in mdx mice, the animal model of DMD. Accordingly, we hypothesized that the pathology observed with dystrophin deficiency may be developmentally regulated. Initially, we demonstrated that profound small interfering RNA-mediated dystrophin knockdown could be achieved in mouse primary muscle cultures. The use of adeno-associated virus vectors to express short-hairpin RNAs targeting dystrophin in skeletal muscle in vivo yielded a potent and specific dystrophin knockdown, but only after approximately 5 months, indicating the very long half-life of dystrophin. Interestingly, and in contrast to what is observed in congenital dystrophin deficiency, long-term ( approximately 1 year) dystrophin knockdown in adult mice did not result, per se, in overt dystrophic pathology or upregulation of utrophin. This supports our hypothesis and suggests new pathophysiology of the disease. Furthermore, taking into account the rather long half-life of dystrophin, and the notion that the development of pathology is age-dependent, it indicates that a single gene therapy approach before the onset of pathology might convey a long-term cure for DMD.