This project focuses on the most common inherited peripheral neuropathy in humans: Charcot-Marie-Tooth disease (CMT). More than 90 altered genes may be responsible for CMT. Around 10% of the CMT mutations are Premature STOP Codon (PTC). Our main objective is to develop effective therapeutic approaches to treat CMT patients harboring PTC mutations, thanks to the work of three complementary teams. The Limoges coordinator team managed the creation of human induced pluripotent stem cells (iPSc) and their differentiation into motoneurons (affected cells in CMT patients), providing then an excellent in vitro model to test drugs. The Bordeaux team is an expert in CRISPR-Cas9 technology and will be able to generate PTC mutations in CMT genes on Limoges’ iPSc. We will then investigate if the new readthrough molecules, identified by Lille team, are efficient on these generated motoneurons. This project could also be the proof of concept to treat other inherited diseases due to PTC mutations.

Survivors of sepsis – a severe infection leading to intensive care unit (ICU), are more and more numerous because of greater hospital care and an increasing aged population. However, their stay in Intensive Care Unit (ICU) is responsible for sustained consequences, known as the post-intensive care syndrome, that accelerate physiological aging and alter long-term prognosis. As such, survivors develop muscle weakness during their hospital stay and it persists months after ICU discharge. As in physiological aging, mitochondrial dysfunction and inflammation are key mechanisms in long-lasting ICU-acquired weakness. Importantly, data from our team and others suggest that inhibition of the Receptor for Advanced Glycation End-products (RAGE) – a multiligand pattern recognition receptor – is of potential interest to reduce those mechanisms. Therefore, we aim to study the effects of RAGE inhibition on sepsis-induced muscle weakness and to understand the underlying mechanisms. However, several obstacles must be overcome: (i) develop a clinically relevant model of sepsis-inducing muscle weakness, (ii) develop specific inhibitors against RAGE, and (iii) set up a device to test these molecules on human muscle. To do so, we will use cellular (differentiated murine and human myoblasts), animal (sepsis induced in mice by injection of heterologous stool), and human (human muscle development by tissue engineering from patient biopsies) models of sepsis-induced muscle weakness. Modulation of RAGE will be achieved by genetic invalidation or synthesis of pharmacological inhibitors of RAGE developed within the consortium. We expect to show that RAGE inhibition improves mitochondrial parameters, reduces inflammation and senescence, attenuates RAGE-related signaling pathways, and restores overall skeletal muscle function. By developing the first tissue bioengineered model of post-septic muscle weakness derived from critical care patients, we will advance our novel patentable inhibitors into the clinic. We hope to make a step towards personalized medicine for weakness acquired after ICU hospitalization. This study is critical to understanding the pathophysiology of sepsis-accelerated muscle aging, and to proposing a new individualized treatment for a key feature of the post-resuscitation syndrome. In the long run, we hope to improve the patient's quality of life and long-term survival.

Schistosomes are blood-dwelling parasites causing Schistosomiasis, or Bilharzia, the second most important parasitic disease after malaria. It affects 230 million people and is responsible for about 200 000 deaths per year. The pathology of schistosomiasis is mainly due to eggs in host tissues. These eggs cause the formation of granulomas and elicit inflammatory processes, which affect organ functions and increase the risk of cancer. Praziquantel (PZQ) is the unique drug recommended for the treatment of Schistosomiasis. There is an urgent need to discover new antischistosomal molecules. Schistosome studies are extremely tedious since experiment on adult worms are difficult outside of their mammal host. In particular, in vitro studies in Petri dish do not allow long-term survival and retention of basic biological functions like mobility and production of mature eggs that can hatch into larvae. This project is the result of a collaboration between V. Senez (LIMMS, IRL 2820) and J. Vicogne (CIIL, UMR 9017 CNRS, U1019 INSERM) and is also supported by a continuing collaboration between V. Senez and Pr. Y. Sakai (University of Tokyo) in the development of Organ-On-a-Chip (OOC) and their instrumentation in BioMEMS. We have already conducted several studies on the development of the 3D model of the target organs, namely the liver, and on that of a microfluidic environment imitating mesenteric veins. We have established the proof of concept that adult worms are able to settle and survive in a microfluidic system with significant egg production. We have finally shown a major effect of PZQ on worm motility and surface adhesion at doses as low as 50 nM, which were not considered as significant or lethal in regular in vitro assays. Our goal is to design two complementary microfluidic devices that will i) sustainably cultivate in vitro couples of adult worms by preserving their fertility and ii) produce a 3D model of liver in which eggs produced by the adult worms will mature thanks to its immunotolerant property. We will therefore develop a miniaturized reproduction of mesenteric veins to simulate the mass transport (nutrient, oxygen and drugs) between the intestinal capillary system and liver. We will also produce a perfused 3D model of liver tissue in which we will study the influence of different cellular and acellular components on egg maturation and, conversely, the influence of eggs on the response of liver tissue. Within these two microfluidic devices, arrayed in 96-wells format, we will show that we can perform efficient screening for therapeutic molecules both on the adult worm (mesenteric chip) and on the eggs (liver chip). In addition, this innovative and instrumented (electrical biosensor) in vitro model mimicking the environment of the host, we will also generate GFP expressing strains by in situ egg electroporation. In summary, our goal is to offer the community a highly predictive functional screening tool to identify new molecules against Schistosomiasis. This tool will be also able to generate viable in vitro multicellular larvae from adult worms making possible the generation of transgenic strains. Transgenic worms would be an unprecedented tool for the propagation of strains of worms with compromised phenotypes in infected geographical areas.

Given the paucity of effective treatments for idiopathic pulmonary fibrosis (IPF), efforts should be made to test and to better characterize new anti-fibrotic drug candidates. The recent development of RNA-targeted therapies using chemically modified oligonucleotide (ASOs) therapeutics show promising clinical outcomes in the treatment of several diseases. This approach is relevant for undruggable targets including non-coding RNAs. Our recent data have highlighted the importance of a family of ncRNAs as key specific regulators of the TGF-? pathway and myofibroblast (MYFs) activation during the pathogenesis of fibroproliferative disorders including IPF. Targeting these ncRNAs using ASO-based therapeutics appears as an attractive therapeutic option and preclinical studies have shown encouraging data. However, these approaches need further developments for translation to the clinic. Our main objective is to optimize the delivery of therapeutic ASOs and address their specificity in mice models of lung fibrosis. Non-invasive imaging will provide unique data on the biodistribution of ASO, as well as on the peak of therapeutic efficacy. Single-cell pharmacogenomics approaches will then be used to evaluate the efficacy and specificity of our drug candidates, providing more specific insights into their mechanism of action and off-target effects in the different cell populations of the fibrotic lung. Finally, as a first proof-of-concept to validate this ASO-based drug in a human context, the best ASO formulation will be evaluated in IPF-derived primary lung fibroblasts using various approaches including single-cell transcriptomics to capture the effect of the drug on the plasticity of these mesenchymal cells. Our project will improve the current knowledge on ASO delivery and safety but should also decipher ncRNA-associated regulatory circuits during MYF differentiation / de-differentiation and their potential interactions with developmental pathways (?-catenin, TGF-? / SMAD, FGFs) reactivated in IPF.

Cancer immunotherapies are here to stay. However, only a minority of patients experience durable survival from these therapies, highlighting the urgent need to develop immunotherapies with improved effectiveness and patient response rates. One mechanism used by tumors to evade the immune response is the generation of an acidic tumor microenvironment (TME). To create a more favorable niche for CAR T-cell function, T cell stimulatory cytokines (IL-2, IL-12) based combinatorial approaches are being explored for potentially improved outcomes compared to conventional CAR T-cell products. While these strategies may potentiate CAR T-cell function and efficacy, they may paradoxically increase the risk of adverse events due to increased pro-inflammatory signaling. Cytokines are pleiotropic that limits their therapeutic use. In previous work, we have demonstrated the use of yeast surface display for engineering novel cytokines with improve specificity. Unpublished work from our laboratory shows besides cell-intrinsic parameters, extracellular acidic pH environment (pHe) such that found in tumor and lymph nodes, inactivates T-cell stimulatory cytokines (IL-2, IL15), by preventing binding to their receptors. Here, we propose to use a yeast-display-based engineering strategy to generate novel pHe switchable IL-2 and IL-15 cytokines, termed as "SwitchKines" that potently activate T cells in acidic niches while exhibiting reduce activity in physiological pH- increasing their therapeutic utility. We will not only gain insights into how acidic pHe alters cytokine-cytokine receptor interactions but will help us to design potent therapies to overcome TME resistance to immunotherapies. We will test the therapeutic efficacy of "SwitchKines" to expand the scope of checkpoint inhibitors and chimeric antigen receptor (CAR)-T-based immunotherapies in therapy-resistant cancer models. This work will revitalize cytokine immunotherapies for treating cancer and autoimmune diseases.