W112 - Pharmacological and Genetic Perturbation of Large Airway Epithelial Cells in Air-liquid-interface Culture

Abstract Text: Airway epithelial barrier function is crucial for the development of Asthma and COPD pathogenesis. To identify mechanisms that drive epithelial dysfunction in Asthma/COPD, we aimed to establish a 3D in vitro culture system that recapitulates all airway epithelial cells types and enables functional studies of large airway epithelium. Protocols have emerged enabling airway-mimetic models by culturing primary lung basal cells in air-liquid interface (ALI) culture. We established a platform to study the impact of pharmacological and genetic perturbation of airway epithelial cells in ALIs and to quantify the effect on cellular phenotype as well as cytokine/chemokine release. These ALIs formed a pseudostratified layer exhibiting key morphological characteristics of lung epithelium, including mucous secreting cells, basal cells and club cells. As previously reported, treatment with IL-13 induced goblet cell metaplasia, as evidenced by induction of MUC5AC+ goblet cells.

To allow for the interrogation of key pathways associated with epithelial function, we have optimized conditions to transduce basal cells with lentivirus without compromising ALI cellular diversity or ciliation. Using lentiviral vectors coexpressing Cas9 and CRISPR sgRNAs, we confirmed robust knockout of CD81, a control target gene in large airway basal epithelial cells and are currently testing the effects of gene knockout on IL-13, EGF, RAR agonism induced goblet and/or squamous metaplasia, key pathologies seen in COPD and asthmatic patients.

This end-to-end platform of primary cell culture, organoid formation in ALIs, pharmacological and genetic perturbation, and multi-modal characterization forms a basis to identify and characterize new targets and mechanisms in Asthma and COPD.