Introduction: In vivo and ex vivo measurements of CFTR function in human cells and tissues can be used for screening and monitoring new ther- apies and phenotyping of controversial CFTR genotypes. Among the tools currently available, a technique enabling intestinal stem cells to expand into closed organoids containing crypt-like structures and an internal lumen lined by differentiated cells, recapitulating the in vivo tissue architecture (Sato T, et al. Gastroenterology. 2011;141:1762-72) was set up in our lab and used for measuring CFTR function by a simple and quantitative assay. Material and Methods: We developed intestinal organoids from 14 non-CF and 13 CF subjects and measured forskolin-induced swelling (FIS) (Dekkers JF, et al. Nat Med 2013;19:939-45). Results: In non-CF organoids swelling was completely blocked by the CFTR (inh)-172 and significantly enhanced following treatment with the potentiator ivacaftor (VX-770). Swelling rates in CF organoids were variable, dependent on the CFTR mutation. Remarkably, in organ- oids from a CF patient carrying the W1282X/R117H CFTR genotype we observed swelling following 24h exposure to the premature termination corrector ataluren (PTC124), but not in its absence. We also performed the membrane depolarization assay in monocytes (Sorio C, et al. PLoS One. 2011;6:e22212) carrying different CFTR mutations and detected resto- ration of CFTR function following 24h exposure to the corrector VRT-325 or the combination of lumacaftor (VX-809)+VX-770 (F508del mutation) and to PTC124 (nonsense mutations). Nasal potential difference (NPD) was abnormal but intestinal current measurement (ICM) was normal in the patient with the W1282X/R117H CFTR mutations and in a CF patient with F508del/T5TG13 CFTR genotype while sweat Cl - was 70 and 79mEq/L, respectively. ICM was abnormal in all other CF patients tested. Conclusions: This study explored and compared several robust assays for individualized medicine approaches aiming at supporting diagnosis, CFTR functional studies and new drug development and monitoring. We have been able to grow intestinal organoids from CF and non-CF subjects and to evaluate CFTR function by FIS assays showing partial restoration of CFTR activity in response to a CFTR potentiator, a CFTR corrector and a premature termination corrector. We can use a combination of CFTR func- tional tests covering multiple tissues and cell types including leukocytes (membrane potential), native intestinal epithelium (ICM), intestinal organ- oids (FIS), nasal epithelium (NPD), sweat gland duct (Gibson-Cooke sweat test) and sweat gland coil (ratiometric sweat rate measurements: Wine JJ, et al. PLoS One. 2013;8:e77114) for individualized approaches for CF diag- nosis, drug development and monitoring of new therapies targeting the basic defect.
Intestinal epithelial organoids contribute to combination of CFTR functional tests supporting drug development and diagnosis
Caldrer, Sara;Vercellone, Silvia;Sandri, Angela;SORIO, Claudio;BUFFELLI, Mario Rosario;MELOTTI, Paola Maria
2015-01-01
Abstract
Introduction: In vivo and ex vivo measurements of CFTR function in human cells and tissues can be used for screening and monitoring new ther- apies and phenotyping of controversial CFTR genotypes. Among the tools currently available, a technique enabling intestinal stem cells to expand into closed organoids containing crypt-like structures and an internal lumen lined by differentiated cells, recapitulating the in vivo tissue architecture (Sato T, et al. Gastroenterology. 2011;141:1762-72) was set up in our lab and used for measuring CFTR function by a simple and quantitative assay. Material and Methods: We developed intestinal organoids from 14 non-CF and 13 CF subjects and measured forskolin-induced swelling (FIS) (Dekkers JF, et al. Nat Med 2013;19:939-45). Results: In non-CF organoids swelling was completely blocked by the CFTR (inh)-172 and significantly enhanced following treatment with the potentiator ivacaftor (VX-770). Swelling rates in CF organoids were variable, dependent on the CFTR mutation. Remarkably, in organ- oids from a CF patient carrying the W1282X/R117H CFTR genotype we observed swelling following 24h exposure to the premature termination corrector ataluren (PTC124), but not in its absence. We also performed the membrane depolarization assay in monocytes (Sorio C, et al. PLoS One. 2011;6:e22212) carrying different CFTR mutations and detected resto- ration of CFTR function following 24h exposure to the corrector VRT-325 or the combination of lumacaftor (VX-809)+VX-770 (F508del mutation) and to PTC124 (nonsense mutations). Nasal potential difference (NPD) was abnormal but intestinal current measurement (ICM) was normal in the patient with the W1282X/R117H CFTR mutations and in a CF patient with F508del/T5TG13 CFTR genotype while sweat Cl - was 70 and 79mEq/L, respectively. ICM was abnormal in all other CF patients tested. Conclusions: This study explored and compared several robust assays for individualized medicine approaches aiming at supporting diagnosis, CFTR functional studies and new drug development and monitoring. We have been able to grow intestinal organoids from CF and non-CF subjects and to evaluate CFTR function by FIS assays showing partial restoration of CFTR activity in response to a CFTR potentiator, a CFTR corrector and a premature termination corrector. We can use a combination of CFTR func- tional tests covering multiple tissues and cell types including leukocytes (membrane potential), native intestinal epithelium (ICM), intestinal organ- oids (FIS), nasal epithelium (NPD), sweat gland duct (Gibson-Cooke sweat test) and sweat gland coil (ratiometric sweat rate measurements: Wine JJ, et al. PLoS One. 2013;8:e77114) for individualized approaches for CF diag- nosis, drug development and monitoring of new therapies targeting the basic defect.
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
