BACKGROUND: For most of the >2000 CFTR gene variants reported, neither the associated disease liability nor the underlying basic defect are known, and yet these are essential for disease prognosis and CFTR-based therapeutics. Here we aimed to characterize two ultra-rare mutations - 1717-2A > G (c.1585-2A > G) and S955P (p.Ser955Pro) - as case studies for personalized medicine. METHODS: Patient-derived rectal biopsies and intestinal organoids from two individuals with each of these mutations and F508del (p.Phe508del) in the other allele were used to assess CFTR function, response to modulators and RNA splicing pattern. In parallel, we used cellular models to further characterize S955P independently of F508del and to assess its response to CFTR modulators. RESULTS: Results in both rectal biopsies and intestinal organoids from both patients evidence residual CFTR function. Further characterization shows that 1717-2A > G leads to alternative splicing generating <1% normal CFTR mRNA and that S955P affects CFTR gating. Finally, studies in organoids predict that both patients are responders to VX-770 alone and even more to VX-770 combined with VX-809 or VX-661, although to different levels. CONCLUSION: This study demonstrates the high potential of personalized medicine through theranostics to extend the label of approved drugs to patients with rare mutations.

Dalton Cardiovascular Research Center University of Missouri Columbia MO United States of America

Department of Biology and Medical Genetics 2nd Faculty of Medicine Charles University and University Hospital Motol 5 Uvalu 84 Prague 5 150 06 Prague Czech Republic

Department of Medical Genetics Masaryk University Brno and University Hospital Brno Jihlavská 20 Brno 625 00 Czech Republic

Department of Pediatrics 2nd Faculty of Medicine Charles University and University Hospital Motol 5 Uvalu 84 Prague 5 150 06 Prague Czech Republic

Institut für Physiologie Universität Regensburg Universitätsstraße 31 D 93053 Regensburg Germany

University of Lisboa Faculty of Sciences BioISI Biosystems and Integrative Sciences Institute Campo Grande C8 bdg 1749 016 Lisboa Portugal

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$a Organoids as a personalized medicine tool for ultra-rare mutations in cystic fibrosis: The case of S955P and 1717-2A>G / $c IAL. Silva, T. Doušová, S. Ramalho, R. Centeio, LA. Clarke, V. Railean, HM. Botelho, A. Holubová, I. Valášková, JT. Yeh, TC. Hwang, CM. Farinha, K. Kunzelmann, MD. Amaral,

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$a BACKGROUND: For most of the >2000 CFTR gene variants reported, neither the associated disease liability nor the underlying basic defect are known, and yet these are essential for disease prognosis and CFTR-based therapeutics. Here we aimed to characterize two ultra-rare mutations - 1717-2A > G (c.1585-2A > G) and S955P (p.Ser955Pro) - as case studies for personalized medicine. METHODS: Patient-derived rectal biopsies and intestinal organoids from two individuals with each of these mutations and F508del (p.Phe508del) in the other allele were used to assess CFTR function, response to modulators and RNA splicing pattern. In parallel, we used cellular models to further characterize S955P independently of F508del and to assess its response to CFTR modulators. RESULTS: Results in both rectal biopsies and intestinal organoids from both patients evidence residual CFTR function. Further characterization shows that 1717-2A > G leads to alternative splicing generating <1% normal CFTR mRNA and that S955P affects CFTR gating. Finally, studies in organoids predict that both patients are responders to VX-770 alone and even more to VX-770 combined with VX-809 or VX-661, although to different levels. CONCLUSION: This study demonstrates the high potential of personalized medicine through theranostics to extend the label of approved drugs to patients with rare mutations.

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$a Doušová, Tereza $u Department of Pediatrics, 2nd Faculty of Medicine, Charles University and University Hospital Motol, V Uvalu 84,Prague 5, 150 06 Prague, Czech Republic.

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$a Ramalho, Sofia $u University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, C8 bdg, 1749-016 Lisboa, Portugal.

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$a Centeio, Raquel $u University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, C8 bdg, 1749-016 Lisboa, Portugal.

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$a Holubová, Andrea $u Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Charles University and University Hospital Motol, V Uvalu 84,Prague 5, 150 06 Prague, Czech Republic.

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$a Valášková, Iveta $u Department of Medical Genetics, Masaryk University Brno and University Hospital Brno, Jihlavská 20, Brno 625 00, Czech Republic.

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$a Yeh, Jiunn-Tyng $u Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO, United States of America.

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$a Hwang, Tzyh-Chang $u Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO, United States of America.

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$a Farinha, Carlos M $u University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, C8 bdg, 1749-016 Lisboa, Portugal.

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$a Kunzelmann, Karl $u Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany.

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$a Amaral, Margarida D $u University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, C8 bdg, 1749-016 Lisboa, Portugal. Electronic address: mdamaral@fc.ul.pt.

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