Drug Repurposing for Cystic Fibrosis: Identification of Drugs That Induce CFTR-Independent Fluid Secretion in Nasal Organoids

Abstract

Individuals with cystic fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ~19% of pwCF cannot benefit from CFTR modulators Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here, we developed a medium-throughput 384-well screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR-dependent epithelial fluid secretion. From a ~1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including β2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in the Ussing chamber, and fluid secretion was not affected by TMEM16A, as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for medium-scale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.

Exploring intrinsic variability between cultured nasal and bronchial epithelia in cystic fibrosis

“…Our results further revealed clear differences in nasal and bronchial organoid fluid secretion. In earlier studies we showed that nasal organoids from subjects with CF displayed high intrinsic and cAMP-induced CFTR-independent fluid secretion 13 , 18 . In the current study we observed that bronchial organoids of subjects with CF lacked CFTR-independent fluid secretion.…”

“…This suggests differences in intrinsic fluid secretion between nasal and bronchial organoids, presumably independent of CFTR under basal culture conditions. To further investigate fluid secretion properties, we measured organoid swelling in response to the cAMP agonist forskolin, which we previously identified as an inducer of CFTR-independent fluid secretion in CF nasal organoids 13 , 18 . Nasal and bronchial organoids from HC subjects (unpaired samples) were included in this FIS assay, to discriminate between CFTR-dependent and CFTR-independent swelling responses.…”

“…Organoid size was quantified by use of the OrgaQuant convolutional neural network which automatically recognizes organoids in brightfield images 13 , 56 . The organoid surface area was estimated using OrgaQuant bounding boxes, assuming organoids had a disk shape.…”

“…These models commonly use undifferentiated airway basal progenitor cells from nasal or bronchial tissues 11 . Air–liquid interface (ALI) differentiated airway epithelial cell cultures are regarded as the golden standard 11 , although airway organoids are emerging as a novel advanced 3D model system, uniquely suited for investigating fluid secretion 12 , 13 .…”

Open questions in human lung organoid research

“…Organoid cultures are in vitro models derived from stem/progenitor cells, and involve the generation of a heterocellular structure that is reminiscent of the tissue of origin in a threedimensional cell culture environment (Barkauskas et al, 2017;Liberti and Morrisey, 2021;Sen et al, 2022). In the human respiratory system, nasal (Liu et al, 2020;Rodenburg et al, 2022), tracheobronchial (Rock et al, 2009;Sachs et al, 2019), small airway (Basil et al, 2022) and alveolar (Katsura et al, 2020;Salahudeen et al, 2020;Youk et al, 2020) epithelial organoid models have all been generated from post-natal tissue-resident stem cells. Additionally, organoid cultures derived from developing lung epithelia have been described (Nikolić et al, 2017;Miller et al, 2018), and the stepwise differentiation of pluripotent stem cells has been used to derive mature lung organoids and those resembling developmental intermediates (Jacob et al, 2017;Hawkins et al, 2021;Hein et al, 2022).…”