ABCMdb

PMID: 25467948

Dekkers R, Vijftigschild LA, Vonk AM, Kruisselbrink E, de Winter-de Groot KM, Janssens HM, van der Ent CK, Beekman JM
A bioassay using intestinal organoids to measure CFTR modulators in human plasma.
J Cyst Fibros. 2015 Mar;14(2):178-81. doi: 10.1016/j.jcf.2014.10.007. Epub 2014 Nov 6., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Gly551Asp ABCC7 p.Ser1251Asn Introduction The CFTR potentiator VX-770 (ivacaftor) is registered for the CFTR gating mutation CFTR-G551D and recently also for eight other gating mutations including CFTR-S1251N (http://investors. Login to comment 26 ABCC7 p.Arg117His ABCC7 p.Ser1251Asn CFTR function measurements in organoids Intestinal crypts were isolated from cystic fibrosis patients (R117H/F508del, S1251N/F508del and F508del/F508del), and CFTR function was quantified by measurement of forskolin-induced swelling of intestinal organoid cultures as previously described [9]. Login to comment 31 ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn CFTR-S1251N potentiation by VX-770 in pooled plasma To determine assay conditions that allow us to discriminate between relevant concentrations of VX-770 in plasma, we stimulated organoids expressing the gating mutant CFTR-S1251N with various amounts of pooled plasma from ten healthy individuals in combination with VX-770 concentrations. Login to comment 33 ABCC7 p.Ser1251Asn We stimulated organoids with a suboptimal amount of forskolin (0.4 bc;M) to facilitate the optimal detection of VX-770-induced CFTR function since considerable residual function associated with S1251N at high forskolin levels limits the VX-770-dependent swelling (Fig. S2A and B). Login to comment 37 ABCC7 p.Ser1251Asn (A) Pooled plasma from ten healthy donors, VX-770 and forskolin (0.4 bc;M) were combined to stimulate CFTR-S1251N expressing organoids. Login to comment 43 ABCC7 p.Arg117His (D) Swelling of R117H organoids after stimulation with forskolin (0.05 bc;M) and serial dilution of plasma containing VX-770 (2 bc;M in 100% plasma). Login to comment 50 ABCC7 p.Arg117His Stimulating CFTR-R117H organoids with forkolin (0.05 bc;M) and increasing amounts of plasma containing VX-770 (2 bc;M in 100% plasma) resulted in a dose-dependent increase in organoid swelling (Fig. 1D). Login to comment 54 ABCC7 p.Ser1251Asn Variability of VX-770-dependent swelling between plasma samples We investigated the variability of VX-770-dependent organoid swelling in plasma samples derived from 9 different healthy donors in the presence of VX-770 on CFTR-S1251N expressing organoids. Login to comment 73 ABCC7 p.Ser1251Asn (A) CFTR-S1251N expressing organoids were stimulated with 6% plasma from 9 different donors in the absence or presence of VX-770 (60 nM, representing 1 bc;M in 100% plasma) and forskolin (0.5 bc;M). Login to comment