Lentiviral-mediated phenotypic correction of type 1 and type 2 ABCA3 pathogenic variants

The ATP-binding cassette subfamily A member 3 (ABCA3) protein localizes to lamellar bodies in alveolar type 2 (AT2) cells and transports phospholipids required for pulmonary surfactant assembly. ABCA3 deficiency results from biallelic pathogenic variants in ABCA3 and causes progressive neonatal respiratory failure or childhood interstitial lung disease (chILD). Supportive care or lung transplantation are the only current treatments for ABCA3 deficiency. Complementing defective ABCA3 production by gene addition has therapeutic potential. Previous studies show that repairing or complementing ABCA3 in induced pluripotent stem cell (iPSC) derived AT2 cells restores defining phenotypes (e.g., lamellar body morphology and surfactant phospholipid composition). ABCA3 pathogenic missense variants are rare or private and disrupt ABCA3 function through altered protein trafficking (type 1) or impairing phospholipid transport (type 2) into the lamellar bodies. Little is known about cellular responses to complementing mistrafficking or phospholipid transport variants. Here we used CRISPR/Cas9 to knockout ABCA3 from the A549 human pulmonary epithelial cell line to generate A549/ABCA3-/- cells stably expressing ABCA3 missense variant cDNAs from a single genomic locus: L101P (type 1), E292V (type 2), or E690K (type 2), or wild-type cDNA. Lentiviral-mediated ABCA3 delivery to each cell line phenotypically corrected the mutant cells. We observed restoration of lamellar body morphology and cell proliferation in ABCA3 complemented cells. Bulk RNA sequencing suggested that ABCA3 delivery ameliorated aberrant NF-kB signaling in p.E292V or p.E690K mutant cell lines, but not in p.L101P or knockout lines. These studies highlight important differences between ABCA3 pathogenic variant types that may influence genetic therapy outcomes.