Gene editing of alveolar organoids reveals AT2 dysfunction due to endosomal SFTPC accumulation.
TL;DR
Endosomal dysfunction and polarity loss are established as drivers of SFTPC-I73T-mediated epithelial injury and mechanisms that may underlie AT2 dysfunction in disease more broadly are highlighted.
Abstract
Alveolar type 2 (AT2) cell dysfunction is key to the development of many lung diseases including pulmonary fibrosis (PF). The underlying mechanisms often remain poorly understood due to a paucity of manipulable primary human models and lack of defined triggers. Monogenic forms of familial pulmonary fibrosis combined with recent advances in in vitro culture techniques offer a unique opportunity to interrogate early pathogenic events in PF. To model toxic gain-of-function disease caused by the SFTPC variant I73T, we employed a base editing strategy in human lung-derived AT2 (fdAT2) organoids to edit the endogenous SFTPC locus and generate a heterozygous SFTPC-I73T-expressing disease model which we interrogated when grown in standard 3D culture, monolayer culture, and at air-liquid interface. SFTPC-I73T expressing organoids failed to form lumens and displayed disrupted epithelial polarity. This was due to SFTPC accumulation in enlarged early endosomes resulting in impaired apico-basal trafficking of polarity and adhesion proteins. Air exposure exacerbated these defects, causing epithelial barrier breakdown and impaired wound healing. Together, we demonstrate the ability to edit endogenous loci in differentiated alveolar organoids to generate disease models that provide mechanistic insights into disease. We establish endosomal dysfunction and polarity loss as drivers of SFTPC-I73T-mediated epithelial injury and highlight mechanisms that may underlie AT2 dysfunction in disease more broadly.
