Functional Characterization of ABCA3 Genomic Variants

Washington University, Saint Louis MO

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Abstract

PROJECT SUMMARY Rare, usually private, biallelic pathogenic variants in the ATP binding cassette transporter A3 gene (ABCA3) are the most common monogenic cause of neonatal respiratory failure in term infants and childhood interstitial lung disease (chILD). ABCA3 transports phospholipids required for surfactant assembly and function across the lamellar body membrane in alveolar type 2 cells (AT2s). Broadly, ABCA3 pathogenic missense variants encode mutant proteins that (1) disrupt intracellular trafficking or (2) impair ATP-mediated, phospholipid transport into the lamellar body. However, the mechanism of ABCA3 variant-encoded disruption is not reliably predicted by location in the gene or protein and does not reliably correlate with associated heterogeneous lung disease phenotypes. Lack of neonatal symptoms among many children with chILD due to biallelic ABCA3 variants suggests that variant-specific mechanisms chronically disrupt AT2 cell metabolism and trigger differing lung disease phenotypes. Pathogenic variants in SFTPC, which encodes surfactant protein-C, activate intracellular stress and degradation pathways. However, there are limited data regarding activation of cell stress and proteostasis pathways by ABCA3 variants. Pharmacologic therapies for ABCA3 deficiency remain limited, non-specific, and unpredictably effective, and the 5-year survival for lung transplantation remains stagnant at ~50%. Variant-specific modulator therapies like those developed for individuals with cystic fibrosis due to biallelic variants in CFTR, which encodes another ATP binding cassette transport protein (also known as ABCC7), are needed. Recently, we observed similar cell-based features when ABCA3 pathogenic variants were expressed in either human induced pluripotent stem cell derived AT2 cells (iAT2s) or human pulmonary epithelial cell lines (A549). Further, iAT2 cells that express ABCA3 pathogenic variants demonstrate upregulation of pro-inflammatory pathways and reduced progenitor potential, consistent with an epithelial-intrinsic aberrant phenotype. For efficient characterization of cell stress and proteostasis pathways and for efficient high throughput screening of FDA-approved compounds that rescue ABCA3-encoded disruption, we will use A549 cell lines that stably express individual ABCA3 pathogenic variants. For confirmation of pharmacologic rescue of ABCA3 mutant AT2 cell phenotype, surfactant phospholipid composition, and lamellar body phenotype by screen-identified, FDA-approved compounds, we will use isogenic iAT2 cells edited to express the same ABCA3 variants. These specific aims will test the hypothesis that variant-encoded disruption of ABCA3 trafficking or phospholipid transport activates pathogenic cell stress and proteostasis pathways and can be corrected with FDA-approved compounds.