MRSU 974 – Research Center in Myology

Muscle Organization & Therapy of Dominant Centronuclear Myopathy

Team 2 : Marc Bitoun

Strengthening knowledge on fundamental aspects of muscle biology is one central challenge in order to decipher pathomechanisms and identify targets for therapeutic intervention for neuromuscular disorders. This is particularly true for diseases due to mutations in genes encoding proteins with pleiotropic roles.

One archetypal example is illustrated by the Dynamin 2-related centronuclear myopathy (CNM). There is no available treatment and the pathomechanisms are still not fully understood for this disease characterized by abnormal nuclear centralization. The team has a long-lasting interest in the dominant CNM exemplified by identification of the causing gene, characterization of clinical and histopathological phenotypes, development of animal models, characterization of the role of the endocytosis machinery in muscle, identification of several pathomechanisms and development of gene-based therapies. The objectives of the team are: i) to dissect fundamental mechanisms of muscle cells, relevant to primarily understand the dominant CNM, and beyond, numerous other neuromuscular disorders, and ii) to develop experimental therapies for the dominant CNM.

In this context, the following projects are developed:

• Defining the role of the endocytosis machinery in muscle (S. Vassilopoulos).
• Investigating the connections between cytoskeletons and nuclear envelope (B. Cadot, more information in https://cadotbruno.com/).
• Understanding the Mechanobiology in healthy and pathological muscle (C. Coirault).
• Studying the pathomechanisms and developing therapies for dominant CNM (D. Trochet)
• Optimizing the AAV-mediated transduction efficacy in pathological muscle (S. Benkhelifa-Ziyyat).
• Characterizing the histopathological features of congenital myopathies (N. Romero, see also the page dedicated to the morphological unit of the Institute).

Contact

Marc Bitoun : m.bitoun@institut-myologie.org
Phone : +33 1 42 16 57 18

Cell and Molecular Orchestration in Muscle Regeneration, Ageing and Diseases

Team 3 : Capucine Trollet

Team 3, headed by Capucine Trollet and Vincent Mouly, is working on the molecular and cellular actors involved in human muscle regeneration, in muscle ageing and in muscular dystrophies including oculopharyngeal muscular dystrophy (OPMD) and Duchenne muscular dystrophy (DMD).

More precisely we are working on RNA metabolism, muscle regeneration, muscle stem cells, and fibrosis, with the final aim of developing innovative therapeutic approaches.

We have a strong expertise on human cellular models (this includes the immortalization facility that we have initiated) – and xenotransplantation (this includes several immunodeficient mouse models and grafting procedures).

Themes currently developed in parallel and in synergy

• Dissection of the molecular mechanisms relevant to OPMD and muscle ageing
• Dissection of cellular communication during muscle ageing, fibrosis and regeneration
• Development of therapeutic approaches.

Contact

Vincent Mouly : vincent.mouly@sorbonne-universite.fr
Capucine Trollet : capucine.trollet@sorbonne-universite.fr

Gene therapy for DMD and pathophysiology of skeletal muscle group

Team 4 : France Piétri-Rouxel

Optimizing therapeutic approach to cure Duchenne Muscular Dystrophy

The dystrophinopathies are pathologies caused by anomalies in the DMD gene encoding for a protein called dystrophin. This protein is absent in Duchenne muscular dystrophy (DMD) while it is present but qualitatively and/or quantitatively altered in the Becker muscular dystrophy (BMD). It is known that the modular structure of dystrophin tolerates large internal deletions. This observation led to the development of two main therapeutic strategies: classical gene therapy with transfer of functional mini- or micro-dystrophin cDNAs in muscles, and targeted exon skipping. Exon skipping strategy, using antisense molecules or gene therapy with AAV-U7, converts an out-of-frame mutation into an in-frame mutation leading to an internally deleted dystrophin. However, in preclinical DMD models, dystrophin restoration by AAV-U7-mediated exon-skipping therapy was shown to drastically decrease after one year in treated animals. We recently showed that pre-treating dystrophic mice muscle with a single dose of peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotides led to transitory dystrophin expression at the sarcolemma and allowed efficient maintenance of AAV genomes enhancing significantly the long-term effect of AAV-U7 therapy. Currently, we evaluate this combined treatment by addressing the benefit of systemic injection of therapeutic PPMO and AAV-U7 vector to a severe DMD model (dystrophin/utrophin double-knockout mouse (dKO)). These mice suffer from a much more severe and progressive muscle wasting, heart and diaphragm functions, impaired mobility and premature death, mimicking pathophysiology of DMD patients.

Phenotypic and genomic characterization of Becker dystrophy patients with 45 to 55 exons deletion
BMD displays 1/30000 live births incidence and is characterized by a progressive muscular dystrophy with or without cardiomyopathy. We present a population of 49 BMD patients with a DMD gene in-phase deletion of exons 45 to 55 (BMDdel45-55). As described, 63% of Duchenne patients are eligible to a multiexon skipping therapy by skipping exons 45 to 55 transforming DMD to BMDdel45-55 patients, it is thus crucial to study the genomic/phenotype link in this BMD cohort. Interestingly, emerging regulatory actors as lncRNA are localized in introns 44 and 55. Thus, the specific neo-introns of each patient could create or modify the lncRNA and/or RNA non-coding sequences. The objective of this study is to identify modifier factors involved in phenotypic variability in BMDdel45-55 patients We performed (i) a phenotypic characterization of 49 patients, (ii) a lncRNA profile in 40/49patients and (iii) a WGS in 19/49patients.

Proteins connecting voltage sensing with muscle mass homeostasis
Deciphering the mechanisms governing skeletal muscle plasticity is essential for understanding pathophysiological processes, including muscle dystrophy and age-related sarcopenia. Muscle activity reverses atrophy, but the connection between these processes is unknown. The voltage sensor CaV1.1 has a central role in excitation–contraction coupling, raising the possibility that it may also initiate the adaptive response to changes in muscle activity. We revealed the existence of a transcription switch for the beta subunit of CaV1.1 (CaVβ1) that depends on the innervation state of the muscle. We showed that denervation increases the expression of a novel embryonic isoform, CaVβ1E. CaVβ1E boosts downstream GDF5 signaling to counteract muscle loss after denervation. We reported that aged muscle expresses significantly reduced levels of CaVβ1E and that CaVβ1E overexpression in aging muscle reduces mass waste by rescuing GDF5 expression. Crucially, we also identified human CaVβ1E and showed a tight negative correlation between hCaVβ1E expression and age-related muscle decline in people, suggesting that the mechanisms underlying muscle mass homeostasis are conserved across species. Actually, we have preliminary data indicating a promising therapeutic approach to improve age-related muscle waste due to the implementation of the recombinant protein (rGdf5).

Contact

France Piétri-Rouxel : france.pietri-rouxel@sorbonne-universite.fr

SLA &SMA group

Biotherapy of Motor Neuron Diseases (SLA & SMA)

Team 6 : Maria Grazia BIFERI

The main goal of our team is to identify efficient strategies to target the central nervous system (CNS) and to develop novel therapies for motor neuron disorders. In particular, we focus on Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS), two yet incurable diseases.

Our studies opened new perspectives and applications for the treatment of CNS diseases and other diseases, based on the use of Adeno-Associated Virus (AAV) vectors. Indeed, our team discovered the unique therapeutic potential of self-complementary AAV serotype 9 vectors (AAV9) in the setting of global gene transfer to the CNS following systemic (intravenous and intramuscular) delivery (Barkats, patent PCT/EP2008/063297, 2007; Duque et al., 2009; Benkhelifa-Ziyyat et al., 2013). We also recently demonstrated that AAV serotype 10 (AAV10), delivered systemically, outperforms AAV9 for gene transfer to CNS and peripheral organs (Tanguy et al., 2015).

Taking advantage of AAV vectors potential to target CNS, we developed an efficient gene therapy for SMA able to extend survival and improve the phenotype of a severe SMA mouse model (Dominguez et al., 2011). Interestingly, a phase I/II clinical trial, using a similar approach, is currently undergoing in USA for SMA type 1 patients (NCT02122952).
More recently, we have developed an innovative technique for the treatment of ALS linked to Superoxide dismutase 1 (SOD1) mutations. Using an exon-skipping approach and AAV vectors, we induced a strong, body-wide reduction of mutant hSOD1 in the well-characterized SOD1G93A mouse model (Biferi et al., 2017). This therapeutic strategy has been awarded by the “THE $1M AVI KREMER ALS TREATMENT PRIZE4LIFE”.

Projects

Our projects are aimed to:

1. Optimize the gene therapies for SMA and ALS
2. Develop new therapeutic approaches for motor neuron disorders
3. Understand the pathophysiology of these complex diseases

Contact

Maria Grazia BIFERI :  mg.biferi@institut-myologie.org

Myasthenia Gravis, Etiology, Pathophysiology & Therapeutic Approach

Team 7 : Rozen Le Panse

Myasthenia gravis (MG) is an autoimmune disease caused by autoantibodies directed against components of the neuromuscular junction and leading to abnormal muscle fatigability. In most cases, the autoantibodies are directed against the acetylcholine receptor (AChR). However, in a minority of cases, patients have antibodies directed against other molecules of the muscle endplate, including the muscle specific tyrosine kinase (MuSK) or the lipoprotein-related protein 4 (LRP4).

The thymus is most likely the site of initiation of the disease in patients with anti- AChR antibodies. Indeed thymic histological abnormalities are very common in this subgroup of patients: 50-60 % of patients have a thymic follicular hyperplasia with germinal centers, 10 to 15% have a thymic tumor, and thymectomy is a beneficial treatment for many patients.

MG as all autoimmune diseases, is a multifactorial disease involving genetic and hormonal predisposing background, abnormalities of the immune system, and is triggered by unidentified factors. Among these, environmental factors such as viral infections or endocrine disrupters are highly suspected.

The team uses many immunological, cellular and molecular techniques, including omics approaches to identify factors that are responsible for the initiation of the disease, its development and its chronicity. The team is also developing new therapeutic approaches based on immune modulation.

Main projects

Our research aims to better understand the pathophysiological mechanisms in myasthenia gravis and to elucidate the events involved in the initiation and chronicity of the disease, with the long-term objective to propose new therapies.

Our specific objectives are to:

• Identify the triggering mechanisms that cause myasthenia. We study the role of genetic factors and sex hormones, and analyze how the activation of signaling pathways of innate immunity may lead to remodeling of the thymus. We also analyze the relative contribution of the environment and the genetics, namely by exploring epigenetic data in monozygotic twins that are discordant for MG.
• Analyze the pathophysiological mechanisms in the thymus and in the muscle. In the thymus, we study how the cytokines related to IL-17 and T follicular cells affect immune regulation processes. In the muscle, we study the effects of pathogenic antibodies on muscle function, and the ability of satellite cells to regenerate muscle after the autoimmune attack.
• Provide a proof of concept of new immunotherapeutic approaches. To overcome the immune dysregulation defects observed in patients, we test the therapeutic potential of mesenchymal stem cells and molecules that interfere with the IL-17 inflammatory pathway. To this end, we developed a new pre-clinical experimental model based on an immune-deficient mice grafted with thymic biopsies from MG patients. This model recapitulates very well the clinical observations of MG patients, including clinical symptoms and anti-acetylcholine receptor antibody production.
• Search for biomarkers in the serum of MG patients. The finding of such molecules is very important for the follow up of the patients and the analysis of their response to various treatments. To do so, in collaboration with clinicians, we investigate circulating micro-RNAs and various cytokines.

Contact

Rozen Le Panse : rozen.lepanse@sorbonne-universite.fr

Inflammatory Myopathies & Innovative Therapies

Team 8 : Olivier Benveniste

The team lead by Prof. Olivier Benveniste is committed to perform translational medicine studies focused on muscle immunology (including primary inflammatory myopathies (myositis), and immune responses elicited by enzyme replacement therapy procedures targeting muscle lysosomial diseases).

Myositis projects

Auto-immune myopathies are a rare and heterogeneous acquired muscle diseases since both muscular and extra-muscular manifestations may differ, leading to identify subgroups of patients based on clinical phenotype and muscle pathology. Life-threatening complications still exist. The pathophysiology of these disabling diseases remains largely unknown. To date, most of these auto-immune myopathies are associated with newly described myositis specific auto-antibodies (MSA). Whereas each MSA is associated with a relatively homogenous clinical phenotype, nothing is known concerning the specificity of muscle pathology associated with a given MSA. In addition, the specificity of muscular immune response associated with a given MSA is not described as well.

Our goal is to revisit the classification, physiopathology and then treatment of the different inflammatory myopathies based on their MSA.

To achieve this goal and since the opening of the team, we have setting-up an e-CRF for myositis. We established the ethical management rules and obtained approval from ethical committee and CCTIRS. We launched this e-CRF in our center but also in 8 other centers in France. We started to perform patient characterization and phenotyping (998 patients are in the database in April 2015, and for 230 of them we already completed clinical/biological notably serological/imaging/pathology parameters). We hired a PhD student with a project in epidemiology to feed and analyse this database. In parallel, we established sample banking in “Myosites ADN-ARNthèque Serothèque Cellulothèque” (MASC study) (reference Codecoh: DC-2011-1445). Using these tools, we sharply characterize inflammatory responses of patients, effects of MSA on myoblasts primary cultures / tubes, or in vivo on mouse.

Lysosomial diseases and immune reactions elicited by enzyme replacement therapies

Pompe disease (PD) is an inherited metabolic myopathy caused by a deficiency of the lysosomal enzyme acid α-glucosidase (GAA). Enzyme replacement therapy (ERT) has improved the outcome of PD, however immune responses directed against infused recombinant GAA are not uncommon and in some cases prevent therapeutic efficacy. While it is clear that the development neutralizing antibodies to GAA is associated with a poor prognosis in infantile PD patients, in subjects with the milder form of the disease, the late-onset Pompe diseases (LOPD), immunity against GAA in ERT is poorly characterized both from an immunological and a clinical perspective.

The aim of the current study is to better characterize T cell responses to recombinant GAA in LOPD subjects with high vs. low antibody titers to the protein and to compare results in untreated LOPD subjects and healthy donors.

A similar approach is applied in Fabry disease, another lysosomal disorder eligible for ERT.

Contact

Olivier Benveniste : olivier.benveniste@aphp.fr

Signaling Pathways & Striated Muscles

Team 9 : Antoine Muchir and Fabien Le Grand

Triated muscles comprise approximately 40% of total body weight, contain 50–75% of all body proteins and contribute significantly to multiple bodily functions. Two types of striated muscles exist: skeletal and cardiac muscles. They share a common architecture characterized by a very particular and well-described arrangement of muscle cells and associated connective tissue.

The team SPaSM is composed of the Le Grand and Muchir’s research groups that assemble complementary expertise in both heart and skeletal muscle basic cell biology and translational research. The focal point of the team will be the dissection of mechanisms leading to striated muscle dysfunction using an integrated multi-scale approach.Our goal is to identify specific targets for therapeutic development, which will be the basis for future clinical trials to treat muscular dystrophies and cardiomyopathies and develop personalized medicine.

Our research will revolve around three aims:

• Using single-cell technologies, we will dissect the cell type composition (RNA-sequencing; Mass Cytometry) of striated muscles during development, regeneration, ageing and in disease.
• We will study intracellular cytoskeleton dynamics as an endpoint of extracellular signaling pathways and decipher the cues that regulate skeletal myoblasts fusion and cardiac myocyte contractility.
• We found aberrant Wnt/Erk target gene expression in both cardiomyopathy and muscle dystrophies. We aim to understand the functional consequences of these mis-regulations in muscle cells.

Major projects

While most myopathies cannot be cured, symptoms can be alleviated. Development of therapeutic advances to ameliorate patient symptoms and life is a priority in our society. Knowledge of the molecular mechanisms that control the muscle stem cell pool or the homeostasis of differentiated cardiomyocytes will help to identify molecules able to modulate muscle cell fate in vivo, with an outcome for regenerative medicine. We study animal models of both muscular dystrophy and cardiomyopathy and develop novel pharmacological therapies based on our discoveries.

Our research revolve around 3 axes:

• Tissue organization of striated muscles in health and disease
• Signaling pathways regulating structure-function relationships in striated muscles
• Control of striated muscles gene expression by signaling pathways

Contact

Antoine Muchir : antoine.muchir@sorbonne-universite.fr

Fabien Le Grand : fabien.le-grand@inserm.fr

Neuromuscular Connectivity in Health & Diseases

Team 10 : Bertrand Fontaine

Our laboratory aims to understand the cellular and molecular mechanisms underlying neuromuscular synapse assembly and maintenance in physiological and pathological conditions.

The Neuromuscular junction (NMJ) is the contact zone between motor neurons and skeletal muscles. This synapse drives the precise initiation and control of motion. Therefore, much of our behavior and wellness relies on the appropriate functioning of this specialised structure. Neuromuscular transmission deficiency occurs in a large array of rare human diseases including channelopathies, congenital or acquired myasthenia and amyotrophic lateral sclerosis. Most of these pathologies are untreatable and life threatening with devastating economic and societal consequences in terms of loss of quality of life and of the burden of disability. The patients display complex clinical phenotypes mainly characterized by a profound muscle weakness and loss of mobility.

We combine unbiased screens and a large array of functional assays including quantitative morphological imaging, behavioral analysis and electrophysiology using mouse models and/or human-derived specimens to explore the complexity of the trans-synaptic mechanisms controlling neuromuscular connectivity. Our overarching goal is to improve physiopathological knowledge that can be used not only for molecular diagnosis and genetic counselling of families affected with the diseases of interest, but also to design new targets of therapeutic interest.

To achieve this goal, our team is built on an organization that favors tight interactions between practicing neurologists from the Paris Est French reference center for neuromuscular diseases and researchers/research assistants together with a large network of national/international collaborations to share knowledge and expertise.

We defined three main aims of research

1. Characterize the molecular determinants underlying NMJ assembly and maintenance (PI: Laure Strochlic/Julien Messéant)
   We have recently identified a new trans-synaptic pathway at the NMJ and developed innovative tools using mouse genetics to dissect its molecular characteristics.
2. Understand how disruption of nerve/muscle communication leads to neuromuscular diseases such as myasthenia, amyotrophic lateral sclerosis and aged-related muscle wasting (PI: Stéphanie Godard-Bauché/Gaelle Bruneteau).
   Thanks to our clinical expertise and our national networks, we analyze the physiopathological mechanisms underlying the studied diseases in patients.
3. Modulate trans-synaptic function to restore appropriate synaptic connectivity in a pathological context as a basis for therapeutic interventions (PI: Bertrand Fontaine/Laure Strochlic).
   We develop innovative pharmacological or genetic strategies that promote trans-synaptic communication and nerve/muscle attachment to mitigate NMJ disease symptoms to ultimately prevent or compensate the progression of the loss of motor function in neuromuscular diseases.

Key words: Neuromuscular Junction, Neuromuscular disorders, disease modeling mouse models, human derived specimens, quantitative imaging, therapeutic strategies.

Contact

Bertrand Fontaine : bertrand.fontaine@aphp.fr