Clathrin-mediated endocytosis is the best characterized process for the entry of proteins and lipids at the plasma membrane of eukaryotic cells. It is also the Achilles’ heel for human diseases such as neurodegenerative diseases and congenital myopathies. While neuronal axons form small clathrin-coated pits for fast endocytosis at synapses, muscle cells form large flat clathrin-coated plaques for cell adhesion at costameres, a cornerstone of mechanotransduction in muscle through their role as physical and functional links between the extracellular matrix and the cytoskeleton. We have demonstrated that the formation of actin networks capable of scaffolding intermediate filaments around clathrin plaques requires dynamin 2 (DNM2), another member of the endocytosis machinery crucial for the formation and release of clathrin-coated vesicles. Mutations in DNM2, amphiphysin 2 (BIN1) and myotubularin (MTM1) lead to centronuclear myopathy (CNM) and we showed that clathrin plaques are altered in CNM cells and mouse models. One common denominator in all three forms is a defective actin and cortical cytoskeleton leading to disorganized costameres, suggesting a defect of mechanotransduction as a common pathomechanism in CNMs. Our preliminary results strongly indicate that this alteration is associated with defective YAP/TAZ mechanosensitive pathway leading to altered transcription of their target genes. The first objective of CLASS is to decipher the function of clathrin plaques in YAP/TAZ mechanotransduction and to highlight involvement of this function in the pathomechanisms of centronuclear myopathy. We will (1) analyse clathrin plaque response to mechanical cues, (2) dissect mechanotransduction signaling in CNM and (3) reveal the benefit of releasing YAP/TAZ nuclear sequestration on CNM myotubes. While our results point towards a specialized function for clathrin plaques at muscle costameres, one fundamental question remains: Why do some cells exclusively form coated pits while others form plaques? We recently achieved the proof of concept that formation and plasticity of CCS are controlled by tissue-specific alternative splicing events through investigation of clathrin heavy chain's exon 31, which is aberrantly spliced in myotonic dystrophy (DM1), a "spliceopathy" affecting in particular muscle and central nervous system (CNS) functions. In DM1, functional loss of RNA binding proteins MBNL1 and CELF1 disturbs the developmentally regulated splicing program resulting in aberrant expression of embryonic isoforms in adult tissues. The second objective is to reveal the mechanisms by which muscle cells form plaques using alternative splicing isoforms of the endocytosis machinery and provide novel insight to DM1 pathophysiology. We will (4) reveal the effect of skipping key exons of developmentally regulated splicing variants of endocytosis proteins in neurons and myotubes, (5) decipher the effect of mis-regulated splicing of the endocytosis machinery in DM1 cells and (6) force exon skipping of developmentally regulated splicing variants in mice CLASS offers a genuinely innovative opportunity to push beyond the actual limitations concerning the fundamental biology of endocytosis: We will unravel how alternative splicing can modulate clathrin polymerization and induce formation of mechano-signaling platforms. This will be a major conceptual turning point in the endocytosis field by proving with unambiguous visual evidence that the clathrin machinery is genetically regulated to produce functional diversity. Our technical capabilities and leading expertise in the clathrin field, CNM and DM1 pathophysiology, our unique expertise in producing genetically modified hiPSC clones and differentiating them in neurons and muscle cells, and our powerful expertise on RNA splicing, put us in a unique position to undertake the dissection of genetic determinants in clathrin structural plasticity in healthy and diseased states.

The SEDUCE proposal aims at establishing an innovative approach to identify new therapeutic molecules for alternate-splicing related diseases and to develop new screening tools for drug discovery approaches driven by academia and industry. Pre-mRNA splicing is a fundamental process in mammalian gene expression and alternative RNA splicing plays a considerable role in generating protein diversity. During this process, particular exons of a gene will be included within or excluded from the final maturated mRNA, and the resulting transcripts generate diverse protein isoforms. Alternative splicing has been proposed as one of the major mechanisms contributing to protein diversity. Increasing evidence has shown that the disruption of alternative splicing negatively impacts health and contributes to human diseases, including cancer, diabetes, and neuromuscular diseases. The recent realization that up to 50% of genetic diseases involve splicing mutations has driven the development of several therapeutic approaches to correct aberrant splicing. Among these, the identification of small molecules capable to modulate splicing has been accelerated in the last decade with the technical development of large-scale cell-based screens. However, up to date, the success rate for identifying a splicing modulator that reaches the market is extremely low. Several parameters can explain this failure 1). the use of reporter constructs that contain only a restricted part of the gene of interest 2). the use of transformed cells instead of disease-relevant cell types. Therefore, one of the most important challenges for the future development of small molecule modulator will depend upon how well the specificity of the effects can be optimized. Altogether, these bottlenecks largely block the deployment of drug discovery campaigns and therefore abrogate the development of new medicines curing alternate splicing related diseases early on in the drug development process. In this context, SEDUCE consortium proposes to combine the use of disease-specific human stem cells differentiated into relevant cell types with high throughput RT-qPCR screening to identify new therapeutics for two distinct alternative splicing related neuromuscular diseases: spinal muscular atrophy and myotonic dystrophy type 1. Overall, our proposal has several objectives (1) identify and optimize new splice modulators for SMA and DM1, (2) decipher their mechanisms of action (3) validate their action in vivo and (4) convert these new molecules into marketable products. Ultimately, our aim is to deliver a meaningful technology that will accelerate the development of therapeutics for the growing list of diseases in which the process of pre-mRNA splicing is altered while ensuring its direct availability to academic lab or pharmaceuticals companies for screening campaigns. To achieve these goals, the consortium has secured all the necessary expertise : Partner 1 (Cécile Martinat, I-Stem) was one of the first laboratory developing new cellular models based on disease-specific human pluripotent stem cells such as DM1 and SMA. Partner 2 (Eric Perret, Evotec) brings an industrial expertise in the development of successful RT-qPCR HTS based, in the access and optimization of chemical librairies and the expertise of hit optimization. Partner 3 and Partner 4 (Denis Furling, Institute of Myology and Nicolas Charlet, IGBMC) are leading experts in deciphering molecular mechanisms involved in normal and pathological alternate splicing as well as in testing splicing and phenotype correction in vivo in mouse models. Our proposal offers a genuinely innovative opportunity to push beyond the limitations of current models and promises to open up major new “assay development space” by increasing our understanding of the regulation of alternate splicing, identifying disease specific splicing modulators and offering a platform that can be applied to a wide range of research areas.

In developed countries, retinal degenerative diseases affecting Retinal Pigmented Epithelium (RPE), including Aged-Macular Dystrophy (AMD) and inherited retinal diseases such Retinitis Pigmentosa (RP), are the predominant causes of human blindness worldwide and are responsible for more than 1.5 million cases in France (30 million worldwide). While AMD, the leading cause of blindness in western countries, may originate from complex interactions of genetic and environmental factors, RP is engendered from monogenic mutations and affect a younger population. The neuroretina is a complex structure whose health depends on blood vessels and RPE, each of which is affected differently in the spectrum of retinal disease. At present, despite the scientific advances achieved in the last years, there is no cure for such diseases. Gene therapy to restore functions of RPE is rendered difficult due to the heterogeneity of causal mutations. In this context, we are ambitioning to develop an Advanced Therapy Medicinal Product (ATMP) based on our expertise in tissue engineering and in the manipulation of pluripotent stem cells. This novel Tissue Engineered Product (TEP) consists in RPE cells derived from clinical grade human embryonic stem cells (hESC) disposed on a biocompatible substrate allowing the formation of 3D functional retinal pigmented epithelium, suitable for transplantation. To reach this aim, (i) we will first optimize a Good Manufacturing Process (GMP)-scalable process for industrial manufacturing of the TEP for clinical use. We already set up and validated a reproducible differentiation protocol to generate pure RPE cells from clinically compatible hESC lines. When cultured onto a biocompatible substrate, RPE cells are polarized and functional. The second aspect of our proposal (ii) will be to test the safety and efficacy of our TEP in animal models (rodents and non-human primates). We have already performed functional experiment in the RCS rat, a rodent model of RP, and demonstrated that the TEP treatment leads to better visual performance that an RPE suspension treatment into the subretinal space of RCS rats and that this recovery was maintained for a longer period of time. We will estimate the risk of hESC contamination leading to side effects like teratoma, as well as shedding and toxicity of our TEP in immunodeficient rodents. This is a prerequisite from regulatory agencies for TEP approval before its first-in-man administration. We will validate the surgical approach and the absence of side-effects in non-human primates. Finally (iii), all the manufacturing procedures will be transferred to Contract Manufacturing Organizations (CMO) for manufacturing of the RPE bank and the TEP clinical lots. The preclinical development program proposed here will lay the foundations for clinical studies early 2019.

Identification and characterization of novel genetic mutations in Amyotrophic Lateral Sclerosis. Amyotrophic Lateral Sclerosis (ALS) is an incurable neurodegenerative disease characterized by loss of upper and lower motor neurons causing skeletal muscle atrophy and paralysis. An expansion of GGGGCC (G4C2) repeats in the C9ORF72 gene is the most common cause of ALS. These repeats are pathogenic through translation into toxic DPR proteins and through impaired expression of the C9ORF72 protein. We previously found that decreased expression of C9ORF72 causes motor neuronal cell death in zebrafish (Ciura et al., 2013) and that C9ORF72 regulates autophagy (Sellier et al., 2016), a protein clearance mechanism essential for neurons. Our most recent data identify novel missense mutations in the C9ORF72 gene. These coding mutations are found in ALS patients but not in control cohorts or control exome database. Our preliminary results in transfected neuronal cells and injected zebrafish indicate that these mutants alter autophagy and cause neuronal cell death. These results are important as they identify novel genetic mutations that may be pathogenic in ALS. Furthermore, these data reinforce the importance of C9ORF72 in this devastating disease. Thus, it is now crucial to confirm these results and explore further the pathogenic importance of these mutations. Moreover, the relationship, if any exist, between these missense mutations and the G4C2 repeat expansion in C9ORF72 is unclear. Finally, there is no treatment for this devastating disease. Thus, we propose to: 1 - Develop state-of-the-art iPS cell and animal (zebrafish and mouse) models of C9ORF72 mutations. 2 - Explore by which mechanisms C9ORF72 mutations alter autophagy. 3 - Explore whether these mutations can help to clarify the pathogenic effect of the G4C2 expansion. 4 - Identify compounds correcting autophagy alterations due to C9ORF72 mutations. Overall, our proposal is highly innovative as it identify novel genetic mutations in ALS-FTD. Furthermore, our project will help to better understand the causes of ALS and open novel routes toward treatment for that devastating disease.