BACKGROUND: Despite the approval of CFTR modulator (CFTRm) drugs for specific variants, many people with cystic fibrosis with non-eligible genotypes may still benefit from these medications. Indeed, recent studies show that some rare CFTR variants can be rescued by approved CFTRm drugs. OBJECTIVE: we assessed the efficacy of CFTRm drugs on eight rare CFTR variants: p.Pro5Leu, p.Pro205Ser, p.Leu206Trp, p.Arg347Pro, p.Ile507del, p.Ser945Leu, p.Met1137Arg, and p.Asp1152His. METHODS: Patient-derived intestinal organoids with these variants in heterozygosity with other cystic fibrosis-causing ones were analyzed by the forskolin-induced swelling assay. Clinical data from individuals undergoing CFTRm therapy were collected both before and after treatment to evaluate clinical benefit. Furthermore, we characterized the molecular defect of those eight variants individually in cystic fibrosis bronchial epithelial cells. RESULTS: CFTR function in intestinal organoids with genotypes p.Asp1152His/p.Phe508del, p.Asp1152His/p.Asn1303Lys, p.Pro5Leu/p.Phe508del, p.Leu206Trp/p.Phe508del, p.Ser945Leu/p.Phe508del, p.Pro205Ser/p.Tyr1092Ter, and p.Met1137Arg/c.2657+5G>A was rescued by currently available CFTRm drugs, this was not observed for organoids with genotypes p.Arg347Pro/p.Phe508del (elexacaftor/tezacaftor/ivacaftor was not tested) and p.Ile507del/p.Gln890Ter. People with cystic fibrosis who, based on our data, started CFTRm therapy showed a clinical improvement, including an increase in lung function, and a reduction in sweat chloride levels. A positive correlation was observed between forskolin-induced swelling values and change in forced expiratory volume in 1 second. This study provides evidence that the forskolin-induced swelling assay using patient-derived intestinal organoids can effectively predict the clinical outcome of CFTRm treatment. In CFBE cells, all eight variants were found to have a processing defect and variants p.Pro5Leu, p.Pro205Ser, p.Leu206Trp, p.Arg347Pro, p.Ser945Leu, and p.Met1137Arg were functionally rescued by the current available CFTRm drug. The CFTRm drug did not elicit the appearance of mature p.Ile507del-CFTR and increased, albeit not significantly, the processing efficiency of p.Asp1152His-CFTR. CONCLUSIONS: This work highlights the importance of using patient-derived intestinal organoids as a theranostic tool to predict the clinical benefit and thus increase the number of people with cystic fibrosis with access to the currently approved CFTRm therapies. While theratyping in cell lines is a valuable approach, additional testing in cystic fibrosis-derived organoids provides more reliable predictions for the individuals carrying rare CFTR variants.

BioISI Biosystems and Integrative Sciences Institute Faculty of Sciences University of Lisboa Campo Grande Lisbon Portugal

Cystic Fibrosis Reference Centre Santa Maria Hospital Lisboa North Hospital Centre EPE Lisbon Portugal

Department of Paediatrics 2nd Faculty of Medicine Charles University and Motol University Hospital Prague Czech Republic

Paediatric Gastroenterology and Nutrition Unit Paediatric Hospital Coimbra Hospital and University Centre Coimbra Portugal

Paediatric Pulmonology Unit Department of Paediatrics Dona Estefânia Hospital Central Lisboa University Hospital Centre Lisbon Portugal

S2AQUAcoLAB EPPO Olhão Portugal

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