Functional genomics of ABCA3 variants

Rare or private, biallelic variants in the ABCA3 (ATP-binding cassette transporter A3) gene are themost common monogenic cause of lethal neonatal respiratory failure and childhood interstitial lung disease. Functional characterization of fewer than 10% of over 200 diseaseassociated ABCA3 variants (majority missense) suggests either disruption of ABCA3 protein trafficking (type I) or of ATPasemediated phospholipid transport (type II). Therapies remain limited and nonspecific. A scalable platform is required for functional characterization of ABCA3 variants and discovery of pharmacologic correctors. To address this need, we first silenced the endogenous ABCA3 locus inA549 cellswithCRISPR/Cas9 genome editing.Next, to generate a parent cell line (A549/ABCA3-/-) with a single recombination target site for genomic integration and stable expression of individual ABCA3 missense variant cDNAs, we used lentiviral-mediated integration of a LoxFAS cassette, FACS, and dilutional cloning. To assess the fidelity of this cell-based model, we compared functional characterization (ABCA3 protein processing, ABCA3 immunofluorescence colocalization with intracellular markers, ultrastructural vesicle phenotype) of two individual ABCA3 mutants (type I mutant, p.L101P; type II mutant, p.E292V) in A549/ABCA3-/- cells and in both A549 cells and primary, human alveolar type II cells that transiently express each cDNA after adenoviral-mediated transduction. We also confirmed pharmacologic rescue of ABCA3 variant-encodedmistrafficking and vesicle diameter in A549/ABCA3-/- cells that express p.G1421R (type I mutant). A549/ABCA3-/- cells provide a scalable, genetically versatile, physiologically relevant functional genomics platformfor discovery of variant-specific mechanisms that disrupt ABCA3 function and for screening of potential ABCA3 pharmacologic correctors.