{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T14:39:28Z","timestamp":1787582368899,"version":"build-2736575974"},"reference-count":31,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,2,23]],"date-time":"2026-02-23T00:00:00Z","timestamp":1771804800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Deutsche F\u00f6rderungsgesellschaft zur Mukoviszidoseforschung e.V."},{"name":"Ministry of Culture and Science of the State of North Rhine-Westphalia","award":["PB22-014"],"award-info":[{"award-number":["PB22-014"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the atypical ATP-binding cassette (ABC) family that functions as a phosphorylation-regulated epithelial anion channel. Cystic fibrosis (CF) is characterised by variants in the CFTR gene that lead to impaired epithelial chloride\u2013ion transport and increased mucus viscosity. Although CFTR modulators such as Trikafta\u00ae have transformed the care of many CF patients, individuals harbouring rare CFTR variants still have no effective treatment options. In this study, we used primary air\u2013liquid interface (ALI) airway cultures obtained from 21 CF patients (pwCF) and 21 healthy controls (HC) to evaluate the therapeutic efficacy of CFTR restoration based on chitosan-mediated CFTR mRNA and modulators. While modulators restored CFTR channel function in most cultures derived from CF patients, those with class I or other rare variants showed no improvement. Chitosan-mediated CFTR mRNA delivery successfully restored CFTR function in ALI cultures of patients carrying rare CFTR variants with limited or no observed clinical response to modulator therapy, assessed by electrophysiology using our newly developed Multi Transepithelial Current Clamp (MTECC) Ussing chamber. This was then confirmed by morphological visualisation of CFTR protein expression in modulator-responsive patient samples using immunofluorescence (IF) staining. IF revealed an increase in CFTR signal and the restoration of epithelial barrier integrity following chitosan-mRNA and modulator treatment as a secondary outcome alongside CFTR functional measurements. Notably, MUC5AC expression, a major gel-forming mucin expressed by airway goblet cells and mucus viscosity were elevated in CF cultures, but were markedly reduced following successful intervention, approaching the levels seen in HCs. These findings establish the potential of chitosan-mRNA delivery as a therapeutic approach for CF patients, particularly those who do not respond to modulators. They also provide a practical, comparative evaluation of advanced mRNA-based treatments in patient-derived airway models.<\/jats:p>","DOI":"10.3390\/ijms27042063","type":"journal-article","created":{"date-parts":[[2026,2,23]],"date-time":"2026-02-23T08:58:21Z","timestamp":1771837101000},"page":"2063","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Restoration of Defective CFTR in Human Nasal Respiratory Epithelial Cells by CFTR Modulators and mRNA Transfection"],"prefix":"10.3390","volume":"27","author":[{"given":"Roshani Narayan","family":"Singh","sequence":"first","affiliation":[{"name":"Department of General Paediatrics, University Hospital Muenster, Albert-Schweitzer-Campus 1, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marilia Marta","family":"Horn","sequence":"additional","affiliation":[{"name":"Institute of Molecular Microbiology and Biotechnology, University of Muenster, Corrensstr. 3, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marin","family":"Juko","sequence":"additional","affiliation":[{"name":"Department of General Paediatrics, University Hospital Muenster, Albert-Schweitzer-Campus 1, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ami","family":"Kampshoff","sequence":"additional","affiliation":[{"name":"Department of General Paediatrics, University Hospital Muenster, Albert-Schweitzer-Campus 1, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2557-5532","authenticated-orcid":false,"given":"Jochen","family":"Schmid","sequence":"additional","affiliation":[{"name":"Institute of Molecular Microbiology and Biotechnology, University of Muenster, Corrensstr. 3, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0282-6765","authenticated-orcid":false,"given":"Heymut","family":"Omran","sequence":"additional","affiliation":[{"name":"Department of General Paediatrics, University Hospital Muenster, Albert-Schweitzer-Campus 1, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dandan","family":"Zhang","sequence":"additional","affiliation":[{"name":"CCRC Hauner\u2014Comprehensive Childhood Research Centre, Ludwig-Maximilians University Munich, Lindwurmstr. 4, 80337 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9613-5393","authenticated-orcid":false,"given":"Joseph","family":"Rosenecker","sequence":"additional","affiliation":[{"name":"CCRC Hauner\u2014Comprehensive Childhood Research Centre, Ludwig-Maximilians University Munich, Lindwurmstr. 4, 80337 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-4044-1695","authenticated-orcid":false,"given":"Wolf-Michael","family":"Weber","sequence":"additional","affiliation":[{"name":"Department of General Paediatrics, University Hospital Muenster, Albert-Schweitzer-Campus 1, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J\u00f6rg","family":"Gro\u00dfe-Onnebrink","sequence":"additional","affiliation":[{"name":"Department of General Paediatrics, University Hospital Muenster, Albert-Schweitzer-Campus 1, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"456","DOI":"10.1016\/j.jcf.2022.01.009","article-title":"Worldwide rates of diagnosis and effective treatment for cystic fibrosis","volume":"21","author":"Guo","year":"2022","journal-title":"J. Cyst. Fibros."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1038\/s41572-024-00538-6","article-title":"Cystic Fibrosis","volume":"10","author":"Mall","year":"2024","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1038\/nature04712","article-title":"The ABC protein turned chloride channel whose failure causes cystic fibrosis","volume":"440","author":"Gadsby","year":"2006","journal-title":"Nature"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Ramananda, Y., Naren, A.P., and Arora, K. (2024). Functional consequences of CFTR interactions in cystic fibrosis. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25063384"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1016\/j.jcf.2018.05.004","article-title":"CFTR modulator theratyping: Current status, gaps and future directions","volume":"18","author":"Clancy","year":"2019","journal-title":"J. Cyst. Fibros."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Baroni, D. (2025). Unraveling the mechanism of action, binding sites, and therapeutic advances of CFTR modulators: A narrative review. Curr. Issues Mol. Biol., 47.","DOI":"10.3390\/cimb47020119"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1164\/rccm.202110-2249OC","article-title":"Effects of Elexacaftor\/Tezacaftor\/Ivacaftor Therapy on CFTR Function in Patients with Cystic Fibrosis and One or Two F508del Alleles","volume":"105","author":"Graeber","year":"2022","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Regard, L., Martin, C., Burnet, E., Da Silva, J., and Burgel, P.-R. (2022). CFTR modulators in people with cystic fibrosis: Real-world evidence in France. Cells, 11.","DOI":"10.3390\/cells11111769"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"102247","DOI":"10.1016\/j.coph.2022.102247","article-title":"CFTR RNA- and DNA-based therapies","volume":"65","author":"Harrison","year":"2022","journal-title":"Curr. Opin. Pharmacol."},{"key":"ref_10","first-page":"1","article-title":"Chitosan as a non-viral co-transfection system in a cystic fibrosis cell line","volume":"502","author":"Weber","year":"2016","journal-title":"Int. J. Pharm."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Kolonko, A.K., Effing, J., Gonz\u00e1lez-Espinosa, Y., Bangel-Ruland, N., van Driessche, W., Goycoolea, F.M., and Weber, W.-M. (2020). Capsaicin-loaded chitosan nanoparticles for wtCFTR-mRNA delivery to a cystic fibrosis cell line. Biomedicines, 8.","DOI":"10.3390\/biomedicines8090364"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"126104","DOI":"10.1016\/j.ijpharm.2025.126104","article-title":"Development of aerosolizable mRNA incorporated chitosan nanoparticles for efficient lung delivery","volume":"10","author":"Kota","year":"2025","journal-title":"Int. J. Pharmaceut."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2363","DOI":"10.1016\/j.bpj.2024.06.010","article-title":"Biophysics of claudin proteins in tight junction architecture: Three decades of progress","volume":"123","author":"Marsch","year":"2024","journal-title":"Biophys. J."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Singh, R.N., Mete, V., van Driessche, W., Omran, H., Weber, W.-M., and Grosse-Onnebrink, J. (2025). Assessing CFTR Function and Epithelial Morphology in Human Nasal Respiratory Cell Cultures: A Combined Immunofluorescence and Electrophysiological Study. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26157618"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"386","DOI":"10.1002\/jbm.a.36251","article-title":"Rheolgical properties of synthetic mucus for airway clearance","volume":"106","author":"Lafforgue","year":"2017","journal-title":"J. Biomed. Mater. Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"614","DOI":"10.1016\/j.colsurfb.2018.07.075","article-title":"Shear and extensional rheological characterisation of mucin solutions","volume":"171","author":"Ahmad","year":"2018","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Hanssens, L.S., Duchateau, J., and Casimir, G.J. (2021). CFTR protein: Not just a chloride channel?. Cells, 10.","DOI":"10.3390\/cells10112844"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Pankonien, I., Quaresma, M.C., Rodrigues, C.S., and Amaral, M.D. (2022). CFTR, cell junctions and the cytoskeleton. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23052688"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Abrami, M., Biasin, A., Tescione, F., Tierno, D., Dapas, B., Carbone, A., Grassi, G., Conese, M., Di Gioia, S., and Larobina, D. (2024). Mucus structure, viscoelastic properties, and composition in chronic respiratory diseases. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25031933"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2619","DOI":"10.1113\/JP287891","article-title":"The apical mucus layer alters the pharmacological properties of the airway epithelium","volume":"603","author":"Guidone","year":"2025","journal-title":"J. Physiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1745","DOI":"10.1261\/rna.701307","article-title":"Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells","volume":"13","author":"Jemielity","year":"2007","journal-title":"RNA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"7125","DOI":"10.1038\/s41467-022-34483-3","article-title":"Biotechnologically produced chitosans with nonrandom acetylation patterns differ from conventional chitosans in properties and activities","volume":"13","author":"Sreekumar","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_23","first-page":"1016","article-title":"Functional restoring of defect CFTR by transfection of CFTR-mRNA using chitosan","volume":"3","author":"Kolonko","year":"2016","journal-title":"JSM Genet. Genom."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1586","DOI":"10.1038\/s41587-022-01491-z","article-title":"Unlocking the promise of mRNA therapies","volume":"40","author":"Rohner","year":"2022","journal-title":"Nat. Biotechnol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"7511","DOI":"10.1093\/nar\/gkw551","article-title":"mRNA capping: Biological functions and applications","volume":"44","author":"Ramanathan","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1038\/s41586-018-0393-7","article-title":"A revised airway epithelial hierarchy includes CFTR-expressing ionocytes","volume":"560","author":"Montoro","year":"2018","journal-title":"Nature"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"656","DOI":"10.1016\/j.jcf.2023.04.008","article-title":"Inhaled mRNA therapy for treatment of cystic fibrosis: Interim results of a randomized, double-blind, placebo-controlled phase 1\/2 clinical study","volume":"22","author":"Rowe","year":"2023","journal-title":"J. Cyst. Fibros."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Ways, T.M.M., Lau, W.M., and Khutoryanskiy, V.V. (2018). Chitosan and its derivatives for application in mucoadhesive drug delivery systems. Polymers, 10.","DOI":"10.3390\/polym10030267"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1038\/nrd.2017.243","article-title":"mRNA vaccines\u2014A new era in vaccinology","volume":"17","author":"Pardi","year":"2018","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1539","DOI":"10.1083\/jcb.141.7.1539","article-title":"Claudin-1 and -2: Novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin","volume":"141","author":"Furuse","year":"1998","journal-title":"J. Cell Biol."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Saint-Criq, V., Delpiano, L., Casement, J., Onuora, J.C., Lin, J., and Gray, M.A. (2020). Choice of differentiation media significantly impacts cell lineage and response to CFTR modulators in fully differentiated primary cultures of cystic fibrosis human airway epithelial cells. 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