461 - Functional Characterization of Disease-Associated ABCA3 Variants in a Human Pulmonary Epithelial Cell Line

Poster Number: 461
Publication Number: 461.432

Jennifer Wambach, Washington University in St. Louis School of Medicine, St. Louis, MO, United States; Ping Yang, Washington University in St. Louis School of Medicine, St. Louis, MO, United States; Daniel J. Wegner, Washington University in St. Louis School of Medicine, St. Louis, MO, United States; Hillary Heins, Washington University in St. Louis School of Medicine, St. Louis, MO, United States; F. Sessions Cole, Washington University in St. Louis School of Medicine, St. Louis, MO, United States

Background: ATP-binding cassette transporter (ABCA3) transports phospholipids required for surfactant assembly and function from the cytoplasm into lamellar bodies in alveolar type II cells. Rare, usually private, biallelic pathogenic variants in the ABCA3 gene ( >300 identified to date) cause lethal neonatal respiratory failure in term infants and childhood interstitial lung disease by disrupting intracellular trafficking or phospholipid transport into the lysosomally-derived lamellar bodies. Current therapies (corticosteroids, azithromycin, hydroxychloroquine) are non-specific with limited efficacy. Lung transplantation remains the definitive treatment for infants and children with progressive respiratory failure.

Objective: To use site-directed, Cre recombinase-mediated integration of ABCA3 disease-associated variants for functional characterization in a human, pulmonary epithelial cell line (A549).

Design/Methods: After CRISPR-Cas9-mediated abrogation of endogenous ABCA3 expression in A549 cells, we transduced cells with lentivirus containing a ‘landing pad’ cassette tagged with GFP, expanded cells under hygromycin selection, performed fluorescence-activated cell sorting (FACS) for GFP-positive cells and expanded sorted cells. We used inverse PCR to map the genomic location of the landing pad to an intergenic locus on chromosome 12. We co-transfected lox-flanked ABCA3-mCherry constructs (wild-type ABCA3 and 11 disease-associated ABCA3 variants including previously characterized mistrafficking (L101P) and impaired phospholipid transport (E292V) mutants) with Cre recombinase, performed FACS for GFP negative/mCherry positive cells, expanded cells and sequenced to confirm location of integration. We performed functional characterization using protein immunoblotting and immunocolocalization with markers of the endoplasmic reticulum (SF_ER) and lysosomes (anti-CD63).

Results: We identified 3 mistrafficking ABCA3 variants that cause ER retention of the mutant protein (p.Q215K, p.L982P, p.L1553P), 4 variants with partial mistrafficking (p.W179C, p.S693L, p.K1388N, p.G1421R) and 4 variants with intracellular trafficking/processing similar to WT (p.R280C, p.N568D, p.E690K, p.V1399M).Conclusion(s): The landing pad system permits functional characterization of ABCA3 variants in A549 cells without confounding effects of viral transduction or random integration. A549 lines that stably express ABCA3 variants provide a genetically versatile, human pulmonary epithelial cell line-based system for discovery of variant-specific mechanisms that disrupt surfactant metabolism and development of personalized therapeutic strategies.