Beyond Trikafta: new models to boost tissue dependent rescue of N1303K-CFTR

Iwona Pranke^1,2Valeria Capurro³Benoit Chevalier^1,2Emanuela Pesce³Valeria Tomati³Cristina Pastorino³Mairead Aubert^1,2Aurelie Hatton^1,2Elise Dreano^1,2Mariateresa Lena^3,4Renata Bocciardi^3,4Federico Zara^3,4Stefano Pantano⁵Vito Terlizzi⁶Cristina Lucanto⁷Stefano Costa⁸Laura Claut⁹Valeria Daccò⁹Piercarlo Poli¹⁰Massimo Maschio¹¹Benedetta Fabrizzi¹²Nicole Caporelli¹²Marco Cipolli¹³Sonia Volpi¹³Frederique Chedevergne¹⁴Laure Cosson¹⁵Julie Macey¹⁶Sophie Ramel¹⁷Laurence Weiss¹⁸Dominique Grenet¹⁹Laurence LE CLAINCHE²⁰Benoit Douvry²¹Bruno Ravoninjatovo²²Camille Audousset²³Aurélie Tatopoulos²⁴Bénédicte Richaud-Thiriez²⁵Melissa Baravalle²⁶Guillaume Thouvenin²⁷Guillaume Labbé²⁸Marie Mittaine²⁹Philippe Reix³⁰Isabelle Durieu³¹Julie Mankikian³²Stephanie Bui³³Thao Nguyen-Khoa^1,2,34Karim Khoukh³⁵Clémence Martin^36,37,38Jennifer Da Silva^37,38Paola De Carli³⁹Carlo Castellani⁴⁰Federico Cresta⁴⁰Luis Galietta⁴¹Anne Guillemaut⁴²Emmanuelle Girodon^1,2,43Natacha Remus²¹Mathis Bulcaen⁴⁴Marjolein Ensinck⁴⁴Miroslaw Zajac⁴⁵Marianne Carlon⁴⁴Jean Lebihan¹⁷Pierre Régis Burgel^2,36,37,46Isabelle Sermet-Gaudelus^1,14,2,38*Alexandre Hinzpeter^1,2Nicoletta Pedemonte^3*

• ¹INSERM, CNRS, Institut Necker Enfants Malades, Paris, France
• ²Universite Paris Cite, Paris, France
• ³UOC Genetica Medica, Giannina Gaslini Institute (IRCCS), Genoa, Italy
• ⁴Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DINOGMI), University of Genoa, Genoa, Italy
• ⁵UOSD CRR Fibrosi Cistica, P.O. San Liberatore, Atri, Italy
• ⁶Department of Pediatric Medicine, Meyer Children's Hospital IRCCS, Cystic Fibrosis Regional Reference Center, Florence, Italy
• ⁷Centro Hub Fibrosi Cistica, Azienda Ospedaliera Universitaria Policlinico G. Martino, Messina, Italy
• ⁸University Hospital "G Martino", Department of Pediatrics, Messina, Italy
• ⁹Department of Pediatrics, Cystic Fibrosis Center, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milano, Italy
• ¹⁰Department of Pediatrics, Cystic Fibrosis Regional Support Center, University of Brescia, ASST Spedali Civili Brescia, Brescia, Italy
• ¹¹IRCCS Materno Infantile Burlo Garofolo, Trieste, Italy
• ¹²Cystic Fibrosis Regional Centre, Unit of Emerging and Immunosuppressed Infectious Diseases, Department of Gastroenterology and Transplantation, Azienda Ospedaliero-Universitaria ‘Ospedali Riuniti’, Ancona, Italy
• ¹³Cystic Fibrosis Center of Verona, Azienda Ospedaliera Universitaria Integrata, Verona, Italy
• ¹⁴Cystic Fibrosis National Pediatric Reference Center, Pneumo-Allergologie Pédiatrique, Hôpital Necker Enfants Malades, AP-HP, Paris, France
• ¹⁵Centre de Ressources et de Compétence de la Mucoviscidose Enfants, Hôpital de Clocheville, Tours, France
• ¹⁶Centre de Ressources et de Compétence de la Mucoviscidose, CHU Pellegrin, Bordeaux, France
• ¹⁷Centre de Ressources et de Compétence de la Mucoviscidose Adulte, Centre de Perharidy, Roscoff, France
• ¹⁸Centre de Ressources et de Compétence de la Mucoviscidose Pédiatrique, CHU, Strasbourg, France
• ¹⁹Centre de Ressources et de Compétence de la Mucoviscidose, Hôpital Foch, Suresnes, France
• ²⁰Centre de Ressources et de Compétence de la Mucoviscidose Pédiatrique, Hôpital Robert Debré, Paris, France
• ²¹Centre de Ressources et de Compétence de la Mucoviscidose Mixte, CHIC, Creteil, France
• ²²Centre de Ressources et de Compétence de la Mucoviscidose, American Memoral Hospital, Reims, France
• ²³Centre de Ressources et de Competences de la Mucoviscidose Adulte, Lille, France
• ²⁴Centre Hospitalier Regional Universitaire de Nancy Hopitaux de Brabois, Vandœuvre-lès-Nancy, France
• ²⁵Centre de Ressources et de Compétence de la Mucoviscidose Adulte, Centre Hospitalier Jean Minjoz, Besancon, France
• ²⁶Centre de Ressources et de Compétence de la Mucoviscidose Enfants, Hôpital d'Enfants de la Timone, Marseille, France
• ²⁷Centre de Ressources et de Compétence de la Mucoviscidose Enfants, Hôpital Trousseau, Paris, France
• ²⁸Centre de Ressources et de Compétence de la Mucoviscidose, CHU Estaing, Clermont-Ferrand, France
• ²⁹Centre de ressources et de compétences pour la mucoviscidose, Hôpital des enfants, CHU Toulouse, Toulouse, France
• ³⁰Centre de Ressources et de Compétence de la Mucoviscidose Pédiatrique, Hospices Civils de Lyon, Bron, France
• ³¹Centre de Référence Adulte de la Mucoviscidose, Hospices Civils de Lyon, Université de Lyon, Lyon, France
• ³²Centre hospitalier régional universitaire Bretonneau, Tours, France
• ³³Université de Bordeaux, CRCM pédiatrique, center de Recherche Cardio-thoracique de Bordeaux, INSERM U1045, Bordeaux Imaging Center, Bordeaux, France
• ³⁴Laboratory of Biochemistry, Hôpital Universitaire Necker Enfants Malades AP-HP Centre, Paris, France
• ³⁵Pharmacie Delpech, Paris, France
• ³⁶Institut Cochin, Inserm U1016, Paris, France
• ³⁷Respiratory Medicine and Cystic Fibrosis National Reference Center, Hôpital Cochin, AP-HP. Centre Université Paris Cité, Paris, France
• ³⁸ERN-Lung CF Network, Frankfurt, Germany
• ³⁹Association Vaincre la Mucoviscidose, Paris, France
• ⁴⁰UOSD Fibrosi Cistica, Giannina Gaslini Institute (IRCCS), Genoa, Italy
• ⁴¹Telethon Institute of Genetics and Medicine, Pozzuoli, Italy
• ⁴²Centre de ressources et de compétences pour la mucoviscidose Adultes, Centre hospitalier régional universitaire de Nancy, Vandœuvre-Lès-Nancy, France
• ⁴³Service de Médecine Génomique des Maladies de Système et d'Organe, Hôpital Cochin, Paris, France
• ⁴⁴Laboratory of Respiratory Diseases and Thoracic Surgery, KU Leuven, Leuven, Belgium
• ⁴⁵Department of Physics and Biophysics, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland
• ⁴⁶ERN-Lung CF network, Frankfurt, France

Rationale Respiratory status of people with Cystic Fibrosis (pwCF) carrying N1303K is improved by Elexacaftor/Tezacaftor/Ivacaftor (ETI) but, contrary to other mutations, the impact on sweat test results is limited. Methods To explore this discrepancy, we implemented new sweat gland and respiratory cell lines stably expressing Wild type (WT)-, F508del- and N1303K-CFTR. CFTR dependent chloride (Cl-) and bicarbonate (HCO3-) transport was measured by short circuit current in these new models and in primary Human Nasal Epithelial Cells (HNECs). CFTR expression was evaluated by Western blot. Results In the airway and the sweat gland cells expressing F508del-CFTR, ETI induced maturation of CFTR and increased Cl- transport. In the respiratory cell lines and HNECs, N1303K-CFTR generated both immature and mature forms of CFTR. Correction by ETI increased CFTR amounts without promoting its maturation and improved Cl- secretion. N1303K-CFTR channel activity was markedly increased by co-potentiation of IVA with Apigenin. In the sweat gland, N1303K-CFTR was expressed as a globally misfolded protein, non-rescuable by ETI. API treatment to 2 patients improved FEV1 without lowering sweat Cl- content. Conclusion N1303K-CFTR shows tissue specific correction and suboptimal response to ETI which can be improved by API.