High-content screening for rare respiratory diseases: readthrough therapy in primary ciliary dyskinesia
| Autor: | Mahmoud R. Fassad, Colin R. Butler, Satyanarayana Somavarapu, Sam M. Janes, Stephen L. Hart, Daniela Cardinale, Philip L. Beales, Dani Do Hyang Lee, Elisabeth Forsythe, Ersilia Nigro, Daniel Peckham, Hannah M. Mitchison, Andrew Rutman, Alexander Agrotis, Claire Smith, Christopher O'Callaghan, Robert E. Hynds, Robin Ketteler, Evie Robson, Dale Moulding, Robert A. Hirst |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0303 health sciences
Pathology medicine.medical_specialty business.industry Translational readthrough medicine.disease 3. Good health 03 medical and health sciences Basal (phylogenetics) 0302 clinical medicine 030228 respiratory system Cell culture High-content screening medicine Motile cilium otorhinolaryngologic diseases Respiratory epithelium Basal body business 030304 developmental biology Primary ciliary dyskinesia |
| DOI: | 10.1101/2020.02.28.959189 |
| Popis: | 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 limited by the number of basal cells that it is possible to expand from such biopsies. Here, we describe a high-content, imaging-based screening method, enabled by extensive expansion of PCD patient basal cells and their culture into differentiated human respiratory epithelium in miniaturised 96-well transwell format ALI cultures. Analyses of ciliary beat pattern, beat frequency and ultrastructure indicate that a range of different PCD defects are retained in these cultures. We perform a proof-of-principle personalized investigation in reduced generation of motile cilia (RGMC), a rare and very severe form of PCD, in this case caused by a homozygous nonsense mutation (c.441C>A; p.Cys147*) in theMCIDASgene. The screening system allowed multiple drugs inducing translational readthrough to be evaluated alone or in combination with inhibitors of nonsense-mediated decay. Restoration of basal body formation in the patient’s nasal epithelial cells was seenin vitro, suggesting a novel avenue for drug evaluation and development in PCD.SummaryWe describe primary cell culture of nasal epithelial cells from patients with primary ciliary dyskinesia including differentiatiation of these to a ciliary phenotype and high-content screening in miniaturised air-liquid interface cultures. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|