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

Cilia Ciliary Motility Disorders / diagnosis Drug Evaluation, Preclinical High-Throughput Screening Assays Humans Kartagener Syndrome / diagnosis Mucociliary Clearance

Journal

The European respiratory journal

ISSN: 1399-3003

Titre abrégé: Eur Respir J

Pays: England

ID NLM: 8803460

Informations de publication

Date de publication:
10 2021

Historique:

received: 27 02 2020

accepted: 01 03 2021

pubmed: 3 4 2021

medline: 28 10 2021

entrez: 2 4 2021

Statut: epublish

Résumé

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 the 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 can be expanded from such biopsies. We describe an immunofluorescence screening method, enabled by extensive expansion of basal cells from PCD patients and the directed differentiation of these cells into ciliated epithelium in miniaturised 96-well transwell format ALI cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia), in this case caused by a homozygous nonsense mutation in the Initial analyses of ciliary ultrastructure, beat pattern and beat frequency in the 96-well transwell format ALI cultures indicate that a range of different PCD defects can be retained in these cultures. The screening system in our proof-of-principal investigation allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. We observed restoration of basal body formation but not the generation of cilia in the patient's nasal epithelial cells

Sections du résumé

BACKGROUND

METHODS

We describe an immunofluorescence screening method, enabled by extensive expansion of basal cells from PCD patients and the directed differentiation of these cells into ciliated epithelium in miniaturised 96-well transwell format ALI cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia), in this case caused by a homozygous nonsense mutation in the

RESULTS

Initial analyses of ciliary ultrastructure, beat pattern and beat frequency in the 96-well transwell format ALI cultures indicate that a range of different PCD defects can be retained in these cultures. The screening system in our proof-of-principal investigation allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. We observed restoration of basal body formation but not the generation of cilia in the patient's nasal epithelial cells

Identifiants

DOI: 10.1183/13993003.00455-2020 PMID: 33795320 PMC: PMC8514977

pubmed: 33795320

pii: 13993003.00455-2020

doi: 10.1183/13993003.00455-2020

pmc: PMC8514977

pii:

doi:

Types de publication

Journal Article Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

Subventions

Organisme : Wellcome Trust

ID : WT107963AIA

Pays : United Kingdom

Organisme : Department of Health

ID : 17DD08

Pays : United Kingdom

Organisme : Wellcome Trust

ID : WT209199/Z/17/Z

Pays : United Kingdom