Childhood interstitial lung disease (chILD) is a group of genetic diseases affecting infants and children. A subset of chILD can be caused by autosomal recessive mutations in the gene encoding ATP Binding Cassette A3 (ABCA3) protein, a lamellar body associated lipid transporter expressed in alveolar epithelial type II cells (AEC2s). ABCA3 mutations leading to disruption of surfactant homeostasis in AEC2s are thought to contribute to chILD pathogenesis. However, inaccessibility to perinatal tissue samples and the inherent instability of primary AEC2s in culture has limited studies on ABCA3 mutations in the cell type it affects. An in vitro disease model derived from patient-specific induced pluripotent stem cells (iPSC) would provide an inexhaustible supply of primary-like cells and present the first opportunity to study ABCA3 mutations and capture the inception of disease pathogenesis in its native cell type. Individuals with homozygous ABCA3 mutations are believed to develop lung disease due to two distinct pathological processes initiated in AEC2s, both of which can be mechanistically interrogated in patient specific iPSC-derived AEC2s. We hypothesize that mutation in the hydrolysis domain of the ABCA3 protein will impair surfactant lipid transport in AEC2s, resulting in surfactant deficiency, whereas mutation in the transmembrane domain of the ABCA3 protein will induce ABCA3 protein misfolding and accumulation of the mutated protein in the ER, leading to ER stress and AEC2 apoptosis. This hypothesis will be tested in two specific aims.
In aim 1, we will first characterize normal ABCA3 biology in our pluripotent stem cell (PSC) derived AEC2 model by using gene editing tools to target: a) a Tomato reporter construct to the AEC2 lineage-specific surfactant protein C (SFTPC) locus, and b) a GFP fluorescent reporter fused to the endogenous ABCA3 locus in a human PSC line. Combination of this bi-fluorescent model enables identification, isolation, and characterization of ABCA3 protein expression, function and trafficking in our PSC-derived AEC2.
In aim 2, we will generate ABCA3 mutant patient-derived iPSC lines corresponding to predicted hydrolysis and trafficking ABCA3 mutations and use CRISPR-Cas9 gene editing tools to correct each mutation. Parallel derivation of AEC2s from pre- and post-ABCA3 gene corrected patient iPSC and their characterization will enable identification of ABCA3 mutant-specific disease mechanisms which can potentially be used for future in vitro screens for the first mechanism-specific therapeutics for chILD patients.
The goal of this study is to create a novel in vitro model for studying diseases involving lung alveoli and use this model to examine children?s interstitial lung diseases (chILD) that are caused by mutations in the gene encoding ATP binding cassette A3 (ABCA3), a poorly understood condition for which there are currently no mechanism- specific therapies. We plan to generate induced pluripotent stem cells from chILD patients with ABCA3 mutations and use a directed differentiation approach following developmental pathways to differentiate them into alveolar epithelial-like cells. We will then apply this in vitro model in order to elucidate the mechanisms that initiate disease in these patients to set the foundation for targeted drug development for this disease.
