Surfactant air liquid interface model of ABCA3 surfactant protein deficiency

To aid progression of gene therapies for lung diseases to the clinic, we developed a physiologically relevant model of the lung parenchyma based on human pulmonary epithelial H441 cells. When grown at the air-liquid interface, in medium supporting polarisation, this cell culture model recapitulates the expression profile of human Alveolar Type II (ATII) pneumocytes (PMID:34977272). Our surfactant air-liquid interface (SALI) model is also sustained in long-term culture unlike primary ATII cells. We used the SALI culture system to generate a model of ABCA3 surfactant protein deficiency, a rare recessive disorder causing severe respiratory distress in babies, to progress lentiviral gene therapy for this condition. To generate homozygous ABCA3 knockout (KO) H441 cells, three separate gRNAs targeting exon 5 of ABCA3 were designed. Submerged cells were transfected with the synthetic gRNAs and Cas9 ribonucleoprotein (RNP) complexes using lipofectamine. H441 cells were cultured for three passages and sorted for single cells using FACS. Individual clones (n=95) were genotyped using Sanger sequencing. One clone showing a frameshift mutation and premature stop codons in exon 5 also showed a complete absence of ABCA3 protein by Western blotting. The H441 ABCA3-KO cells have been grown as SALI cultures to assess the shift in expression of surfactant related genes after airlift. Functional studies include measurement of transepithelial electrical resistance (TEER), lipid dysregulation by DPPC-release assay, and the presence of surfactant organelles (lamellar bodies). In the absence of a robust murine model for ABCA3 deficiency this ABCA3-KO SALI model can be used to test genetic therapies.