Gene expression profile of intestinal organoids from people with cystic fibrosis upon exposure to elexacaftor/tezacaftor/ivacaftor

Background: The forskolin-induced swelling assay (FIS) in intestinal organoids (IOs), used to determine in vitro responsiveness to elexacaftor/tezacaftor/ivacaftor (ETI), showed variability in swelling among IOs obtained from people with CF (pwCF) carrying the same genotype. We aimed to characterise the effect of ETI on the transcriptional activity of IOs-derived cells to understand the intracellular processes triggered by ETI and the differences in treatment response. Methods: Six high- and six low-responding IOs to ETI, derived from F508del/F508del pwCF, were incubated with or without ETI for 2 to 6 hours. Gene expression was modelled using negative binomial models adjusted for subjects, having time and incubation with ETI as predictors. Results: Incubation with ETI resulted in a significant upregulation of several biological processes: mostly related to chemokines and signalling, chemotaxis, and tissue development processes. No changes were observed in abundance of the CFTR transcripts or in CFTR-related gene sets and pathways. The genes and pathways associated with ETI did not overlap with those whose expression changed with time only. IOs with a high FIS response did not significantly differ in any interpretable gene from the FIS-low organoids. Conclusions: The changes in the IOs gene expression upon the exposure to ETI cannot explain differences in the magnitude of IOs FIS-measured response to ETI. Genes of the CFTR-related pathways do not change their transcriptional activity; instead, overexpression was observed in genes of inflammatory-like cytokine response and receptor activation pathways, seemingly unrelated to the mechanisms of FIS, but possibly related to the effect of ETI itself. Overall design: The present study aimed to investigate the effect of ETI on the overall gene expression profile, in particular, on the transcriptional activity of CFTR and CF-modifying genes. The inclusion of organoids at opposite ends of the FIS response spectrum also allowed us to assess whether gene expression changes are related to the magnitude of the organoid response to ETI. Briefly, each IOs culture was split into seven culture wells, then three wells were incubated with ETI medium, while 4 control wells were incubated in ETI-free medium. One of the control wells was immediately harvested at 0 hours (baseline). Three pairs of ETI-free and ETI-containing cultures were harvested at 2, 4 and 6 hours. In total, 12 IOs from 12 pwCF (F508del homozygotes) were included, making 12 biological replicates. Of note, six IOs had the best and six IOs had the worst response to ETI when tested by the FIS.