Beyond Kaftrio: mechanistic insights to maximize N1303K-CFTR rescue

Abstract Introduction N1303K is the fourth most frequent Cystic Fibrosis (CF) causing mutation. People with CF (pwCF) clinical status can be improved by Elexacaftor(ELX)/Tezacaftor(TEZ)/Ivacaftor (ETI) combotherapy. We investigated the mechanism underlying N1303K-CFTR rescue. Methods N1303K-CFTR expression and maturation was evaluated by Western Blot in cell lines and Human Nasal Epithelial Primary Cells (HNECs). Cell surface expression was studied by nanoluciferase complementation assay and TurboID proximity labeling. Functional rescue was tested in vitro by YFP-Based Assay and Short Circuit Current. Results Correction by ELX/TEZ increases N1303K-CFTR amounts, but not its maturation in CFTR-expressing HEK and 16HBEge cell lines and in HNECs. In control conditions, N1303K-CFTR is more distributed at the cell surface and significantly more surface partners are identified in the N1303K-CFTR interactome as compared to F508del-CFTR in HEK cells. ELX/TEZ induces a global stabilization of N1303K-CFTR without favoring its plasma membrane relocation in contrast to F508del-CFTR which is redistributed to the membrane. ETI increases N1303K-CFTR activity in HNECs and can be increased by API co-potentiation with a predicted increase in Forced Expiratory Volume in 1 second (ppFEV1) by respectively 13(2)% and 18%(3). This is consistent with a gain in ppFEV1 reported in pwCF carrying the N1303K mutation and additional improvement by API in a patient. Conclusion These results support the expansion of ETI approval to N1303K mutation but highlight different mechanisms of action than for F508del. ### Competing Interest Statement Stefano Pantano declares Vertex pharmaceuticals support in sample collection without financial contribution. Stefano Costa declares payment or honoraria for speakers bureaus from Vertex pharmaceuticals. Sonia Volpi declares payment of honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Vertex pharmaceuticals and DMF Pharma FoodAR and support for attending meetings and/or travel from Chiesi. Stephanie Bui declares participation in the protocoles of Vertex pharmaceuticals studies as principal investigator. Clemence Martin reports payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Chiesi, Astra Zeneca, Boehringer Ingelheim, GSK. Support for attending meetings and/or travel from Chiesi, Boehringer Ingelheim. Nicoletta Pedemonte declares payment or honoraria from Vertex Pharmaceuticals for lectures, presentations, speakers bureaus, manuscript writing or educational events - Speaker for a lecture at the 45th European Cystic Fibrosis Society Conference, Rotterdam, June 2022. Pierre Regis Burgel reports grants from Vertex pharmaceuticals, GSK, outside the submitted work. Luis J.V. Galietta declares Patents planned, issued or pending. Compounds described are not present in the submitted paper. Isabelle Sermet-Gaudelus reports support for the present manuscript from Vaincre La Mucoviscidose and Mucoviscidose ABCF2. Isabelle Sermet-Gaudelus also reports, outside the submitted work, grants from Agence Nationale pour la Recherche, Assistance Publique Hopitaux de Paris, Vertex Innovation Award; consulting fees and travel support from Vertex therapeutics.