Thématiques

Study of the epithelial ion transport in physiology and human diseases (cystic fibrosis, Dry Eye Disease) caused by dysfunction of ion channels (channelopathies)

Responsable :  Frédéric BECQ

Recherche

Based on our expertise, know-how, experimental facilities and screening platform, in epithelial physiology and pathologies, we will develop our project in 2 directions, CF-Lab and DED-Lab.

CF-Lab project

Our aim is to better understand the molecular basis and pharmacology of epithelial transport in physiology and human pathology (Cystic Fibrosis). We will pursue the study of transepithelial ionic transport using a broad range of methodologies (Patch-clamp, Ussing chambers, imaging, biochemistry and molecular biology) and will develop pharmacological tools and mechanism of action thereof (11–13). Our cell models are human epithelial airway cells: cell lines (normal and CFTR mutated), primary culture of human bronchial epithelial cells (normal and F508del/F508del) in collaboration with Dr Coraux (INSERM, Reims) and Dr Sage (Foch Hospital, Paris). We are exploring the mechanism of action and effect of first and second generation therapeutics such as Trikafta, Symdeko, Orkambi, as well as novel compounds in pre-clinical development (collaboration with ManRos Therapeutics, Roscoff, France; collaboration with V Chappe, Halifax, Canada; collaboration with JL Decout, UJF, Grenoble; collaboration with I. Callebaut, Paris).

The epithelial CFTR channel is our main target, but we are also studying epithelial Ca2+ channels (such as TRPV6) and Ca2+-dependent Cl channels (such as TMEM16A). We and others showed the importance of calcium or pathways activated by Ca2+ in CFTR activation. Recently, we focused on phospholipase C (PLC) and studied its role in forskolin-stimulated CFTR-dependent short‑circuit current in normal airway epithelial 16HBE14o-, CFBE‑WT cells and in temperature corrected CFBE‑F508del (14). We are pursuing our investigation on Ca2+ homeostasis deregulation in CF by testing new therapeutic molecules on Ca2+ transport in CF versus corrected CF cells to better understand the link between Ca2+ homeostasis and CFTR traffic/activity. In addition, we are launching a study on the impact of hypoxia on bronchial epithelial cells to study the modulations of ion channel activities in this experimental condition that should better mimic the physiological and pathological environment of epithelial cells in the airways in pulmonary diseases. Cells will be incubated with various oxygen concentrations with our hypoxic chamber and transepithelial current will be measured and compared to cells in normoxia. The effect of therapeutics will be compared under hypoxia vs normoxia. One objective is to set experimental conditions to study airway epithelial cells in conditions that are close to pathology in vivo (collaboration with T Ducret & C Guibert, INSERM U1045, Bordeaux, France). Finally, we recently developed the quadriwave lateral shearing interferometry (QWLSI) method to record water fluxes in cultured living epithelial cells (15) and highlighted the role of CFTR in a cAMP‑dependent and mercury–sensitive water transport in various cells involving AQP3. We will use the QWLSI method to study in detail the water movement in CF cells following treatment with therapeutics in relation to AQP isoforms (collaboration with Pascal Jourdain and Pierre Marquet, Lausanne, Switzerland).

Funding: VLM grants and charities

DED-Lab Project

We initiated in 2016 a collaboration with H4 Orphan Pharma a Dijon-based pharmaceutic company to develop pharmacological compounds in CF. In 2021, we’ll start the common laboratory DED-Lab to develop a new R&D axe in ocular area and in particular in Dry Eye Diseases (DED). This project will be funded for the period 2021-2025 by the ANR (350 000€) with cofundings from Région Nouvelle Aquitaine and Feder funds.

DED is a multifactorial malady of the tears and ocular surface that results in symptoms of discomfort, visual disturbances and tears film instability with potential damage to the ocular surface.  It is accompanied by increased osmolarity  of  tear  film  and  inflammation  of  the  ocular surface. It affects millions of peoples worldwide with no efficient treatment so far. There is thus an unmet need for DED. The DED-Line project will put in synergy our respective complementary scientific and entrepreneurial expertise to develop human DED cellular models to study in vitro novel therapeutic pharmacological agents and concepts. With H4 Orphan Pharma, we identified the potential of 2 small compounds (554-001 and 554-002) as CFTR ion channel agonists (confidential data). We thus aimed to lift scientific and technical locks and barriers by creating ocular human DED cell cultures, mimicking DED, allowing their biobanking, dissemination and use to screen on-demand chemicals and to define the mechanism of action of our own lead compounds. The DED-Line project will strengthen the second axe of the company R&D and will help to positioned PRéTI laboratory at the frontline, in France and Europe, of research in DED and ocular physiopathology.

Funding: ANR Labcom 2021-2025