Multiple endocrine neoplasia type 1 (MEN1) is a rare disease caused by mutations in the tumor suppressive gene MEN1. The 3 cardinal lesions are primary hyperparathyroidism, pituitary tumors, and neuroendocrine duodeno-pancreatic tumors. 27 to 70% of MEN1 patients die due to the disease. Despite a very specific phenotype, a large number of sporadic index cases receive a negative or uninformative genetic testing. The identification of mutation is essential to do the diagnosis in patients with an incomplete phenotype, but also to allow genetic counseling in families. Moreover, MEN1 is a long-life treating disease in which the lesions can occurred at any age, with no genotype-phenotype correlation. The disease penetrance is complete, but the phenotypical expression is variable, even within the same family. We reported that patients with MEN1 mosaic mutation have a phenotype as severe as patients with heterozygous mutation Our aim is to improve the diagnosis of MEN1 by developing i) tools to search for unconventional anomalies such as MEN1 mosaicism, promotor and deep-intronic mutation, and ii) functional analysis of sequence variants of uncertain significance. For that, we will use DNA and RNA sequencing of blood samples by next generation sequencing, and we will use a human organoid model of pituitary tumor that we developed in which the VSI will be introduced using Crispr-Cas9. Our second aim is to develop, characterize and compare by using a mosaic disease model, the tumor phenotypes of mouse models with mono- or bi-allelic inactivation of the Men1 gene in neural-crest derived cells at different time points of their pre- and post-natal differentiation. We will study the consequences of MEN1 deficiencies in tissues and the non-cell-autonomous factors that can lead to the development of tumors, in order to better understand the expression variability, and to identify, in very original way, new translational and actionable therapeutic leads.

The KCNQ2 gene encodes the Kv7.2 subunit of the potassium Kv7/M channel, known to control neuronal excitability in the brain and spinal chord via the M current (IM). Pathogenic variants in the KCNQ2 gene represent the major cause of early onset epileptic encephalopathy, leading to the concept of KCNQ2-related epileptic encephalopathies (KCNQ2-REE). The patients have a remarkably homogeneous phenotype at the beginning with frequent tonic seizures resulting in abnormal muscle contractions and apnoea in the first days after birth. This stormy phase of tonic seizures and abnormal EEG pattern lasts 2 to 15 weeks and usually gives place to a calmer period of rare seizures and significant amelioration of the EEG. Despite this apparent positive evolution in terms of seizures, the developmental process is definitively altered and leads to a severe and global neurological impairment. The vast majority of patients have no informative language, autistic behaviour, and significant motor impairment such as tetraplegia, spasticity, ataxia, global hypotonia or dystonia. The management of KCNQ2-REE patients is complex because it must take into account multiple disabilities: motor, cognitive and epileptic. Their vulnerability is indeed major and multifactorial: neurological (via tonic seizures that can lead to status epilepticus and death), respiratory, digestive and orthopaedic, due to permanent motor disability. To date none of the available anti-epileptic drugs has been demonstrated to be effective in KCNQ2-REE. The IMprove project is novel because it builds on the results on a previous ANR project that generated the concepts and tools that were necessary to build the current translational project. We were able to produce unique models (a knock-in mouse and human neuronal cultures derived from patient's iPS) that are still largely missing in the field and provide us with a strong competitive advantage. We are now able to study pathogenic mechanisms occurring in the whole mouse brain and directly in human Kcnq2-deficient neuronal cultures. Our previous results revealed that neuronal hyperexcitability is transient, suggesting the gradual establishment of a compensation phenomenon whose origin will be studied in the current project. Our models also allow to assess treatment response and to translate our findings to the bedside. The combination of expertise provided by our consortium will allow us to tackle the multiple dimensions of the subject (from natural history in patients to the study of human neuronal cultures, from omics studies to integrated cognitive and motor phenotyping, from electrophysiological characterization to drug testing). We believe that the multidisciplinary nature of our proposal will scale up its translational potential and be a key success factor, each partner having demonstrated its ability to collaborate successfully and to produce high-level publications. The strongest impact of such a project will be to propose new therapeutic approaches for KCNQ2-REE. Early onset epileptic encephalopathies are very severe diseases, without satisfactory therapeutic options. We believe that one way to move forward is such a pre-clinical project for diseases where clinical research is difficult (small number of patients, unethical placebo, late developmental endpoint). Our project has integrated an innovative dissemination activity with a specific action to design and organize practical trainings related to genetic epilepsies for relatives of children concerned by this pathology, through a three-day experimental course. IMprove is strongly supported by KCNQ2-France association, which brings together a large proportion of families affected by KCNQ2-REE.

The aim of the interdisciplinary project ICELARE is to combine cell biology with advanced laser-based techniques (laser-assisted printing, laser surface structuring, laser nanoparticle fabrication) for tissue engineering and regenerative medicine. The objective is to create and study 2D/3D microenvironments that best mimic the complexity and in vivo architecture of tissues in order to understand the interaction of cells with the environment in which they develop. In particular, we target the optimization of muscle cell differentiation and the formation of active neuromuscular junctions. The success of the project is based on the complementary expertise of the two laboratories involved, the Lasers, Plasmas and Phonic Processes Laboratory in the field of laser processes and the Marseille Medical Genetics Centre in the field of stem cells applied to muscle differentiation. The contractile function of the muscle can be modified in different situations such as traumas or certain diseases. If the muscle has good regenerative capacities, this faculty is limited when the trauma is too great. There is a great demand for the development of effective protocols to produce reproducible and standardized cellular models for pathologies affecting the muscles as well as the production of therapeutic grafts for the repair of functional tissues. One solution is the construction of muscle tissue in vitro and ex vivo but this is highly dependent on the ability to recreate the cellular complexity of the tissue to ensure the survival, vascularization and functional maturation of the grafted cells. The emergence of induced pluripotent stem cell technology, and their capacity for unlimited proliferation in culture as well as their potential for differentiation into all cell types, has led to the development of new therapies. But in the case of muscle cells the level of differentiation and tissue maturation remains limited and experimental development is still needed to obtain sufficiently differentiated cells. Until now, advances in muscle repair medicine and the modelling of acquired or congenital muscle pathologies using these approaches have been hampered by the lack of a satisfactory method for generating muscle cells from human stem cells. One way to improve these processes is to take advantage of tissue engineering. Thus, the ability to accurately position cells in complex 2D/3D models is proving to be of critical importance for the optimization and validation of ex vivo differentiation approaches and the development of new controlled and reproducible models. In addition, contact topography guidance in terms of micro/nano scale as well as the influence of nanoparticles in the culture medium are emerging parameters for successful differentiation and regeneration. This is a major challenge for future advances in tissue engineering, especially for effective on-site regenerative medicine. Within the framework of ICELARE, 3 processes will be implemented (bioprinting, surface structuration and addition of nanoparticules) with the assistance of pulsed lasers, which gives each of them clear advantages compared to traditional methods: clean method (no solvents or chemical additives), photonic process (no disturbances due to moving mechanical parts, no clogging of nozzles, very important precision and control), digital control... The expected results would represent an important breakthrough for biomedical research and tissue engineering with numerous applications: basic research, regenerative medicine, industrial developments in pharmacology, etc.

Cardiac valves control unidirectional blood flow. The valve leaflet is formed by a monolayer of valve endothelial cells on its outer surface while inside it is populated by valve interstitial cells that help to organize a complex extracellular matrix structure. The valve stenosis and calcification are frequent forms of valve disease that can be the result of congenital defects as well as ageing. Valve calcification can result from abnormal paracrine communication between the two valve cell types. Severe valve dysfunction lacks therapeutic approaches, and the only solution is to perform valve replacement with biologic or synthetic implants. Despite the importance of these diseases, much remains unknown on key signalling pathways regulating cardiac valve development, disease and even regeneration. New evidence reveals that multiple retinoic acid (RA)-related genes either strongly expressed in the adult valves or strongly regulated during valve development and/or regeneration. The overall objective of our project is to decipher the role of RA signalling in the development and regeneration of the cardiac valves. Using unique access to human samples in combination with in vivo mouse genetic lineage, functional and transcriptomic analyses together with recently established model of cardiac valve regeneration in zebrafish we will characterize the role of RA in valve development as well as in new valve formation. Our results will provide an in-depth characterization of the biological signal involved in valve development and thus gain key insights during valve remodeling. RAVAGE project will also help to understand the mechanisms of the alteration of RA signalling in pathological processes and will demonstrate its role in valve regeneration.

This project aims at understanding the role of the interaction between PIEZO1 a mechanically activated ion channel and KCNN4 a Ca2+ activated K+ channel in the pathophysiology of a rare red blood cell (RBC) disorder, the dehydrated Hereditary Stomatocytosis (DHSt). Patients with DHSt suffer from haemolytic anaemia and their RBCs are dehydrated due to an excess loss of KCl. The phenotype is highly heterogeneous from asymptomatic to severe form. While mutations in either PIEZO1 or KCNN4 have been associated to DHSt, the consequences of the functional interaction between both ion channels are poorly understood. We have previously shown that RBC dehydration in DHSt is due to the hyperactivity of KCNN4, whatever, the mutated channel KCNN4 or PIEZO1. Thus, there is a functional coupling between both channels and our preliminary results show also a molecular interaction between these two channels. We hypothesize that these interactions play a key role in RBC ion and water homeostasis, thus controlling RBC deformability. We propose that the different single point mutations identified in DHSt impairs interactions between the two channels, which contributes to DHSt phenotype variability. This proposal is an interdisciplinary approach combining genetic characterization of new mutations from a collection of patient blood samples, biochemical and functional characterization of the different mutants and bioinformatics analysis of the interaction to identify: 1/the interacting parts of the two channels, 2/the other proteins likely involved in the interaction, 3/ways to modify the interaction, 4/relevant targets to treat DHSt.