Mutations in the gene encoding the CFTR protein affect mucus-producing epithelia, including the lungs and gastrointestinal tract. In the lungs, viscous mucus obstructs the bronchi, creating an environment conducive to chronic bacterial infection, which leads to the destruction of the lung parenchyma and the death of patients with Cystic Fibrosis. To date, ~65% of patients can be treated with drugs based on Ivacaftor (ex Kaftrio). Despite significant clinical benefits, in particular respiratory, have been reported, side effects may lead to the reduction and/or discontinuation of treatment due to the hepatic and pancreatic insufficiency inherent in the disease. Moreover, ~35% of patients with very rare mutated alleles are currently without a therapeutic solution. This may explain why gene therapy is a credible option to treat all patients regardless of their mutations. Since lung failure is responsible for mortality, gene therapy must be aerosolized to deliver the transgene. However, the high viscosity of CF mucus traps both viral and non-viral vectors and limits their clinical impact. In this context, extracellular vesicles (EVs) exhibiting a natural ability to diffuse into dense extracellular matrices, are relevant candidates to diffuse into mucus. The interest of ES derived from MSCs has been established in several pulmonary pathologies, mainly due to their natural antibacterial, anti-inflammatory and anti-fibrotic effects. Few studies have reported delivery of MSC-EV to the lungs after nebulization, demonstrating their safety but also showing heterogeneous results likely due to lack of stability during nebulization. Also, our project aims to nebulize the hMSC-EVs optimized to deliver an mRNA encoding CFTR and to evaluate them in the specific CF context.

Rational: Venous thromboembolism (VTE), clinically presenting as deep vein thrombosis (DVT) or pulmonary embolism (PE), is the third most frequent acute cardiovascular disease after myocardial infarction and stroke, and is associated with high mortality (around 10%). The frequency and the severity of VTE have not changed over the past 20 years despite major advances in diagnostic tests and therapeutic improvement. Particularly, distinguishing recurrent VTE from residual clot is of foremost importance. Indeed, therapeutic implications differ significantly whether the patient is facing an acute VTE versus a relatively passive residual thrombus whereas it remains challenging with the current imaging modalities to have a definite diagnosis. In deed, current imaging techniques although well validated for the exclusion of VTE at the acute phase, do not provide any information about the composition of the thrombus and can not distinguish between residual and newly formed clots. Molecular imaging has emerged at the end of the last century as a new in vivo imaging method allowing the visualization, characterization, and measurement of biological processes at a cellular or molecular level. In the setting of VTE, molecular imaging allows noninvasive direct targeting of the thrombus with high specificity and sensitivity with a potential of whole-body imaging. Targeting directly the active venous clot may allow a direct visualization of a specific part of the thrombus. Many studies since the end of the 1970s have already investigated the role of molecular imaging for the diagnosis of VTE targeting various contributors of thrombosis, as well as the different components of the venous clot. However, studies were performed using conventional planar or single photon emission computed tomography (SPECT) imaging, resulting in insufficient spatial resolution and detectability. Nuclear medicine and molecular imaging have undergone a technologic revolution with the development of an increasing array of new positron emission tomography (PET) tracers. Technical advantages of PET compared to SPECT include higher sensitivity, higher spatial resolution (4 mm for PET vs. 12 mm for SPECT), and superior quantitative capability. Objectives: The objective of this project is to develop new radiotracers for VTE diagnosis using PET technology, derived from tracers already assessed in humans with SPECT technology and to test them in pre-clinical models of thrombosis. Research hypotheses and methodology: We have selected three biomolecules corresponding to different constituents and steps of thrombus formation, that may potentially distinguish fresh (i.e recurrent VTE) from old thrombi (i.e residual clot), which is of foremost importance for clinicians. The challenge of this project is to find the appropriate combination of [targeting biomolecule/radionuclide with the suitable half-life/corresponding radioisotope-chelating agent] for a safe use and to propose the relevant biological and radiopharmaceutical approach from in vitro to in vivo studies.The three radiotracers will be produced by combining selected biomolecules targeting early and late thrombotic processes, and labeled with 64-copper (WP1). The binding capacity of synthetized radiopharmaceuticals to venous clots will be evaluated using ex vivo models of thrombus formation (WP2). Biodistribution, pharmacokinetic and binding capacity of synthetized radiopharmaceuticals to a clot will be evaluated in a mouse model of venous thrombosis (WP3). Expected results: We expect to improve the sensitivity of existing tracers using PET technology that will greatly facilitate their translation into humans, leading to the development of the world’s first PET radiopharmaceutical dedicated to recurrent VTE diagnosis with high specificity and sensitivity that could strongly modify patient care.

Dormancy is an adaptive trait that is established during seed maturation and prevents seed germination on the parent plant or out of proper season after seed dispersal. It is also an important agronomic trait as germination before harvest (vivipary) is a major cause of crop yield losses. Abscisic acid (ABA) is the key phytohormone promoting dormancy whereas nitrate (NO3-) stimulates germination by triggering ABA catabolism. Partners 1 & 2 have previously identified a new whole MAPK (Mitogen-activated protein kinase) module which is activated by both ABA and NO3- in Arabidopsis plantlets. They have also recently shown that mutants impaired in this module produce seeds which are more dormant. Strikingly and coherently, mutations in homologous MAPK genes in wheat and barley were reported to reduce vivipary. Taken together, these preliminary results suggest that this MAPK module is a new player controlling seed dormancy conserved throughout angiosperms. The fact that this module is activated by both ABA and NO3- also suggests that it may have a pivotal role as integrator of signaling pathways controlling dormancy. This project aims to better characterize this module in the frame of seed germination using Arabidopsis as a model plant and to exploit the results to develop new strategies to manipulate crop germination in the field. To achieve these goals, the first WP will aim to functionally validate the module by unveiling where and when it is required to modulate seed dormancy and which are the kinases involved in this function, a MAPK module being composed of at least 3 kinases. Importantly, we will test how the MAPK signaling module is modulated by and/or modulates ABA and NO3- signaling by using a combination of biochemical and genetic approaches to study mutants impaired in these signaling cascades. Furthermore, the MAPK module presents unique features when compared to other plant and animal MAPK modules described so far. The second WP will thus be devoted to the characterization of these specificities and will particularly study the translational and post-translational regulations of the module as well as decipher the unknown function of protein domains in the central MAP2K. The third WP will focus on the downstream events that are regulated by the module. Firstly, we will identify substrates which are phosphorylated by MAPKs and are important to control seed dormancy. Secondly, we will unveil the cellular processes which are regulated by the module by performing transcriptomic and metabolomic studies of mutants impaired in the module. Finally, a fourth WP will aim to identify molecules targeting this MAPK module and use them as chemical probes to investigate to which extent, across the plant kingdom, this module is important for seed physiology and to modulate seed dormancy in crops. This project relies on the collaboration of 4 groups recognized as leaders in their respective fields, who will bring their expertise and skills to challenge the novel hypothesis that the recently discovered MAPK module integrates distinct environmental signaling pathways to trigger the downstream processes that determine whether seeds germinate or not. Their joint work will lead to a better understanding of the factors that control seed physiological traits and new essential knowledge to enhance resilience through advanced breeding programs and to provide guidelines for optimal seed production, treatment and storage. It will also use an original strategy based on chemical genetics in yeast aiming at the identification of small molecules that modify the activity of the MAPK module and modulate dormancy in model species and crops. Thus, the MAPKSEED project brings together multidisciplinary expertises to tackle an important issue for optimizing sustainable agriculture in a changing environment by novel and original basic research.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, with an estimated prevalence of ~1 in 1000 to 1 in 2500 individuals. Its course is characterized by the development of multiple kidney cysts, causing progressive loss of kidney function and frequently leading to end-stage kidney disease during or after the sixth decade. The most severe extra renal manifestation in ADPKD is the development of intracranial aneurysm (IA). IA prevalence is fivefold higher in ADPKD than in the general population, with estimates ranging from 9 to 12%. The main complication of IAs is subarachnoid hemorrhage following rupture, with a mean age of 41 years in ADPKD patients versus 51 years in the general population, and a high rate of mortality and morbidity. The only identified risk factor for IA in ADPKD is a familial history of unruptured or ruptured IA, with a prevalence of 22% in patients with a familial risk of IA, versus 6-8% in the absence of family history. Until now, despite strong familial clustering of IA cases in some ADPKD pedigrees, no genetic factor associated with the development of IA has been identified. Indication, timing, and frequency of IA screening in ADPKD patients are not well defined. In this translational research project, we aim to identify the genetic determinants of IA formation in ADPKD patients. The GENOVAS-PKD study will take advantage of international and local ongoing collaborations, allowing us to gather the largest cohort of ADPKD patients affected by IA worldwide. We will evaluate the respective influences of genetic and non-genetic risk factors, and revisit the genic and allelic influence of the ADPKD genes on IA risk. We will perform whole exome-sequencing in informative pedigrees and in a large cohort of unrelated ADPKD individuals with a past history of IA to identify candidate genes, and we will employ targeted next-generation sequencing to analyze these candidate genes in a large replication cohort. We will develop first-line functional assays to understand the pathogenic mechanisms resulting from the variants identified. Through the identification of genes influencing the development of IA in ADPKD patients, our hope is to improve the pre-symptomatic screening strategy, which will alleviate the mortality and morbidity burdens associated with this severe complication. Understanding the genetic determinants of IA formation will provide a better understanding of IA pathogenesis, hopefully translating into the discovery of pathways amenable to disease-modifying therapies.