(631) Comparing F508del CFTR modulator responses in human primary enteric monolayer (hPEM) and human bronchial epithelial (hBE) cultures

Background: Primary hBE ALI cultures have been pivotal in developing CFTR modulators, but scarcity of non-F508del CF hBE cells, limited expansion capability and lack of sensitivity hamper the discovery of novel therapeutics for nonsense and other rare CFTR mutations. Enteric cells can be obtained from rectal biopsies, extensively expanded in culture and grown into intestinal organoids (IOs). IOs have been utilized in the highly sensitive forskolin-induced swelling assay to assess CFTR function and rescue by modulators. Furthermore, IO-derived hPEMs have demonstrated substantial functional CFTR anion transport activity. To utilize these high CFTR expressing hPEM cultures in nonsense-CFTR screening assays, we first evaluated the hPEM model, comparing the effects of clinical CFTR modulators in F508del^+/+ hBE and hPEM cultures on CFTR function.

Methods: Primary hBEs were cultured using the ALI protocol published by Vertex [1]. Intestinal cells were expanded in Matrigel (Corning, USA) droplets and IntestiCult-SF culture media (StemCell Tech., USA) and passaged every 7-10 days. For hPEM preparation, IOs were dissociated using mechanical and enzymatic dissociation, seeded on Transwell filter plates (Corning, USA) and maintained in 1:1 Intesticult OGM (StemCell Technologies) and LWRN conditioned media (TTML, Univ. Michigan). hPEMs and hBEs were cultured on filters for 2 and 4 weeks respectively. Equivalent current (I_eq) measurements were performed using a TECC-24 (EP Design, Belgium). Correctors were added for 24hrs. For corrector combination experiments tezacaftor was held constant at 3 mM. CFTR currents were stimulated with in-assay additions of 10 mM Forskolin and 1 mM Ivacaftor.

Results: The potency order in both in vitro models was identical (Table 1). Both 1^st generation (lumacaftor, tezacaftor) CFTR modulators notably had lower potency in hPEMs compared to hBEs. Higher potency was observed in hPEMs for both VX-121 and the corrector combinations, while VX-121 and elexacaftor, when tested alone in hPEMs generated an E_max close to that achieved with the corrector combinations. However, in hBEs corrector combinations were more efficacious than any single modulator. Overall, hPEMs generated larger currents compared to hBEs, further supporting the notion that intestinal epithelia have higher CFTR expression than airway epithelia.

Conclusions: Primary hBE cells remain the gold standard for in vitro CFTR functional measurements. However, an alternative primary cell model would be highly valuable, particularly for testing future potential nonsense CFTR therapeutic approaches. Data presented here illustrate the sensitivity and assay window of the hPEM model compared to the hBE model. This, along with more readily accessible material of a wide genotype range and greater expansion capability, makes the hPEM model an attractive platform for assessing CFTR functional rescue in response to novel therapeutic agents.

Acknowledgements:

References: [1] Neuberger T, Burton B, Clark H, Van Goor F. Use of primary cultures of human bronchial epithelial cells isolated from cystic fibrosis patients for the pre-clinical testing of CFTR modulators. Methods Mol Biol. 2011;741:39-54. doi: 10.1007/978-1-61779-117-8_4. PMID: 21594777.