De nombreuses maladies génétiques sont directement liées à la reconnaissance de protéines mal repliées par le contrôle qualité du réticulum endoplasmique (RE), conduisant à leur rétrotranslocation vers le cytosol puis leur dégradation. Dans le cas des glycoprotéines mutées, comme la protéine F508del-CFTR (Cystic Fibrosis Transmembrane conductance Regulator), la démannosylation extensive de leur résidus mannoses constitue une étape clé dans ce processus. L’objectif de ce travail a été d’identifier et de caractériser des molécules capables de rétablir l’expression membranaire de F508del-CFTR en ciblant cette activité enzymatique. Dans un premier temps, nous avons testé des dérivés multivalents basés sur le Deoxymannojirimycin (DMJ), un inhibiteur spécifique de la classe I des α-mannosidases et révélé leur effet correcteur puissant sur F508del-CFTR. Nous avons par la suite mis en évidence leur mécanisme d’action et tenté d’expliquer l’augmentation d’efficacité observée entre les monovalents et les multivalents. Nous nous sommes enfin focalisés sur le rôle des principales mannosidases dans le RE, l’α1,2-mannosidase I du RE (ERManI) et la famille des lectines EDEM (ER degradation-enhancing α-mannosidase-like protein). Nous avons montré par une stratégie de siRNA qu’ERManI, EDEM1 et EDEM2 sont impliquées dans la rétention réticulaire du F508del-CFTR.Notre étude ouvre ainsi de nouvelles perspectives quant à l’identification de nouveaux agents pharmacologiques ciblant ces protéines. / Numerous genetic diseases are directly associated to the recognition of misfolded proteins by the endoplasmic reticulum (ER) quality control, leading to their retention and subsequently their retrotranslocation to the cytosol for degradation. In the case of mutated glycoproteins such as F508del-CFTR (Cystic Fibrosis Transmembrane conductance Regulator), causing CF pathology, mannose trimming is a key step of this process. Our objective was to identify and characterize molecules targeting ER-mannosidases activity, with the goal to restore F508del-CFTR to the plasma membrane.First, we tested multivalent derivatives, based mainly on Deoxymannojirimycin (DMJ), a specific inhibitor of α-mannosidasesI and revealed their better corrective effect on F508del-CFTR and their mechanism of action. Then we explored the mechanism explaining the higher efficiency of the multivalents compared to the monovalent. Finally, we focused on the role of key players of mannose trimming, ER-α1,2-mannosidase I (ERManI) and EDEMs (ER degradation-enhancing α-mannosidase-like protein) proteins on F508del-CFTR trafficking defect. We showed the implication of ERManI, EDEM1 and EDEM2 in F508del-CFTR retention, using a siRNA strategy. In conclusion, our study highlights these proteins as potential pharmacological targets to develop correctors for the F508del-CFTR trafficking defect.

Cystic Fibrosis (CF) is a common life-shortening autosomal recessive disease that affects over 100.000 people worldwide people worldwide. It is caused by mutations in the CF trans-membrane conductance regulator (CFTR) gene, that encodes for a membrane channel localized at the apical surface of epithelial cells where it has a crucial role in the secretion of chloride and bicarbonate. Over 2100 different CFTR mutations have been reported and among the pathogenic once the most common is F508del, located in the nucleotide-binding domain 1 (NBD1). F508del is a three-nucleotide deletion that results in the loss of a phenylalanine at position 508 in the protein and in the consequent CFTR degradation by the ubiquitin-proteasome system. Different attempts to correct F508del-CFTR gene were made using genome editing approaches, however deletions like F508del remain difficult to be repaired. Several studies reported that additional mutations (revertant mutations) in the F508del-CFTR gene can rescue both CFTR folding and activity, suggesting a potential novel strategy to correct F508del. For this reason, the first aim of this work was the identification of novel F508del-CFTR revertants that can rescue CFTR localization and function. We generated a library of mutants introducing random substitutions into the F508del-CFTR gene. Revertant mutations were isolated based on their ability to rescue the presence of CFTR at the plasma membrane (PM) in HEK293T cells and identified by Sanger sequencing. Restoration of CFTR maturation, localization, and function of the identified revertants was evaluated by western blot, flow cytometry analysis and YFP assay, reaching levels similar to the wild type CFTR. Then we used CRISPR-Cas technology to introduce selected revertant mutations, such as I539T, R553Q, G550E, R555K and R1070W, in the endogenous F508del-CFTR gene. Adenine and cytosine base editors (ABE and CBE) allow the insertion of the desired base conversion without the formation of double strand breaks. Efficient editing was evaluated through Sanger sequencing, reaching up to 60% of base conversion. CFTR rescue at the PM in edited cells was analyzed by flow cytometry showing different degrees of recovery compared to the wild type CFTR. In this work, we confirmed that revertant mutations can rescue F508del CFTR localization and function. In addition, we demonstrated that CRISPR-base editors are valid tools to introduce these mutations in the endogenous F508del-CFTR gene, leading to a permanent correction. The proposed strategy could overcome the limits that genome editing strategies faced till now in the correction of F508del, providing a new potential therapeutic approach to treat CF.