Higher throughput drug screening for rare respiratory diseases: Readthrough therapy in primary ciliary dyskinesia

Abstract

Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies, followed by ciliated differentiation at air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that it is possible to expand from such biopsies. Here, we describe an immunofluorescence screening method, enabled by extensive expansion of PCD patient basal cells and their culture into differentiated respiratory epithelium in miniaturised 96-well transwell format ALI cultures. Analyses of ciliary ultrastructure, beat pattern and beat frequency indicate that a range of different PCD defects can be retained in these cultures. As proof-of-principle, we performed a personalized investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia, RGMC), in this case caused by a homozygous nonsense mutation in the MCIDAS gene. The screening system allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. Restoration of basal body formation in the patient's nasal epithelial cells was seen in vitro, suggesting a novel avenue for drug evaluation and development in PCD.