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

Dani Do Hyang Lee,Ersilia Nigro,Satyanarayana Somavarapu,Daniela Cardinale,Andrew Rutman,Hannah M Mitchison,Robin Ketteler,Elisabeth Forsythe,Daniel Peckham,Stephen L Hart,Robert A Hirst,Claire M Smith,Robert E Hynds,Sam M Janes,Evie Robson,Alexander Agrotis,Philip L Beales,Dale Moulding,Mahmoud R Fassad ,Colin Butler ,Christopher O’callaghan

doi:10.1183/13993003.00455-2020

Abstract

BackgroundDevelopment 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.MethodsWe 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 MCIDAS gene.ResultsInitial 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 in vitro.ConclusionOur study provides a platform for higher throughput analyses of airway epithelia that is applicable in a range of settings and suggests novel avenues for drug evaluation and development in PCD caused by nonsense mutations.

Highlights

Primary ciliary dyskinesia (PCD) is a rare autosomal recessive genetic disorder arising from abnormalities in the structure and function of motile cilia
As proof-of-principle, we performed a personalized investigation in a patient with a rare and severe form of primary ciliary dyskinesia (PCD), in this case caused by a homozygous nonsense mutation in the MCIDAS gene
Brush biopsy-derived nasal epithelial cells were cultured from 12 PCD patients with a range of genetic defects, one patient with a reduced generation of motile cilia (RGMC) ciliopathy and 11 healthy donors with no known ciliary defects (Table 1)

Summary

Introduction

Primary ciliary dyskinesia (PCD) is a rare autosomal recessive genetic disorder arising from abnormalities in the structure and function of motile cilia. As a result of disease diversity, there is no single PCD diagnostic test: clinical history, genetic analyses, low nasal nitric oxide levels and the direct observation by light microscopy of abnormal ciliated epithelial cells in nasal brush biopsies using high-speed video analysis as well as analyses of ciliary fine structure by electron microscopy are used, often in combination, to reach a PCD diagnosis [5]. Examining cultured cells can reduce the requirement for re-biopsy when biopsy samples do not contain ciliated cells [6] and is advantageous when characterizing atypical PCD phenotypes such as inner dynein arm defects, ciliary disorientation or reduced generation of multiple motile cilia

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Conclusion