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High-speed Video Microscopy Analysis for First-line Diagnosis of Primary Ciliary Dyskinesia
Published on: January 19, 2022
Higher throughput drug screening for rare respiratory diseases: readthrough therapy in primary ciliary dyskinesia
Dani Do Hyang Lee1,2, Daniela Cardinale1,2, Ersilia Nigro3
1UCL Great Ormond Street Institute of Child Health, London, UK.
Background:
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.
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 MCIDAS gene.
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 in vitro. CONCLUSION: Our 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.
Insights
Researchers developed a new method for screening treatments for primary ciliary dyskinesia (PCD), a rare respiratory disease. This high-throughput system uses expanded patient cells to test drugs for rare genetic disorders like PCD.
Area of Science:
- Respiratory Medicine
- Genetics
- Cell Biology
Background:
- Therapeutic development for rare respiratory diseases is limited by the absence of high-throughput screening systems for patient-derived differentiated respiratory epithelium.
- Primary ciliary dyskinesia (PCD) is a rare genetic disorder affecting ciliary function and mucociliary transport, posing challenges for research and diagnostics.
- Current methods using primary cell cultures are restricted by the limited expansion of basal epithelial cells from patient biopsies.
Purpose of the Study:
- To establish a miniaturized, high-throughput screening platform for evaluating therapeutic approaches in primary ciliary dyskinesia (PCD).
- To enable the expansion and directed differentiation of basal epithelial cells from PCD patients into ciliated epithelium for pre-clinical research.
- To demonstrate the utility of this platform through a personalized investigation of a patient with a severe form of PCD caused by an MCIDAS gene mutation.
Main Methods:
- Developed an immunofluorescence screening method utilizing extensively expanded basal cells from PCD patients.
- Established miniaturized 96-well transwell air-liquid interface (ALI) cultures for directed differentiation into ciliated epithelium.
- Performed a proof-of-principle investigation on a patient with a homozygous nonsense mutation in the MCIDAS gene.
Main Results:
- The 96-well ALI cultures retained various PCD defects, including ciliary ultrastructure and beat patterns.
- The screening system facilitated the evaluation of drugs inducing translational readthrough, alone or with nonsense-mediated decay inhibitors.
- Observed restoration of basal body formation but not de novo cilia generation in patient-derived nasal epithelial cells in vitro.
Conclusions:
- The study presents a novel platform for high-throughput analysis of airway epithelia applicable to diverse rare respiratory diseases.
- This platform offers new avenues for drug evaluation and development, particularly for PCD caused by nonsense mutations.
- The findings support the potential for personalized therapeutic strategies in rare genetic respiratory disorders.
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