View Abstract

A2460 - Correction of Type 2 Alveolar Epithelial Cell Dysfunction by Gene Editing Patient-Specific Induced Pluripotent Stem Cells
Author Block: K. Alysandratos1, A. Jacob1, S. H. Guttentag2, M. F. Beers3, D. N. Kotton1; 1The Pulmonary Center and the Center for Regenerative Medicine, Boston University School of Medicine, Boston, MA, United States, 2Pediatrics/Neonatology, Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, TN, United States, 3Medicine-Pulmonary, University of Pennsylvania, Philadelphia, PA, United States.
Rationale:
Alveolar epithelial type 2 (AT2) cell dysfunction has been implicated in the pathogenesis of many poorly understood lung diseases. Specifically, mutations affecting surfactant protein B and C (SFTPB and SFTPC) genes, which are highly expressed in AT2 cells, have been associated with neonatal respiratory distress syndrome and early-onset interstitial lung disease. Elucidating the molecular pathogenesis of AT2 cell dysfunction caused by these mutations is likely to inform the broader mechanisms by which AT2 cell dysfunction leads to chronic lung disease. As access to primary AT2 cells from patients with these mutations is limited and these cells are difficult to maintain in culture, we sought to engineer an in vitro disease model utilizing patient-specific induced pluripotent stem cells (iPSCs).
Methods:
We generated iPSC lines from patients carrying a homozygous SFTPB mutation (SFTPB121ins2/121ins2) and a heterozygous SFTPC (SFTPCI73T/WT) mutation. Using the CRISPR/Cas9 gene-editing technology we generated a syngeneic line corrected for the SFTPB121ins2 mutation (SFTPBWT/WT). We also targeted the endogenous SFTPC locus of the SFTPCI73T/WT line with a tdTomato reporter resulting in the generation of two syngeneic lines (SFTPtdTomato/WT and SFTPCI73T/tdTomato). Using a directed differentiation protocol developed by our lab, we derived alveolar epithelium from each line to perform head-to-head in vitro comparisons between mutant and corrected alveolar cells.
Results:
SFTPB121ins2/121ins2 alveolar epithelium expressed lower levels of SFTPB transcript compared to SFTPBWT/WT alveolar epithelium, no detectable SFTPB protein, no detectable lamellar bodies by electron microscopy, and misprocessed SFTPC protein to its 6 to 10-kD pro-protein form. Correction of the SFTPB121ins2/121ins2 mutation resulted in the appearance of lamellar bodies, reconstitution of the mature 8-kD SFTPB protein, and disappearance of the misprocessed pro-
SFTPC protein form. In contrast alveolospheres made from SFTPC mutant cells displayed intact SFTPB protein processing but misprocessed SFTPC protein.
Conclusions:
We show here that patient-specific PSC-derived AT2 cells provide a potential platform for disease modeling, and we plan to employ this novel in vitro human model system to provide further insights into the molecular mechanisms of AT2 cell dysfunction.