Abstract
Purpose :
The zinc finger e-box binding homeobox 1 (ZEB1) gene encodes a transcription factor involved in regulating cell state transitions critical in development and disease. Hypothesized to lead to ZEB1 insufficiency, mutations in ZEB1 are associated with posterior polymorphous corneal dystrophy (PPCD). To characterize the impact of PPCD on the corneal endothelial cell (CEnC) state and gain a better understanding of the pathogenesis of PPCD, we generated a cell-based model of PPCD using CRISPR-Cas9 gene editing.
Methods :
Using RNA-seq data derived from corneal endothelium of individuals with PPCD, differentially expressed genes (DEGs) in PPCD were compared to genes highly-associated with ex vivo corneal epithelial cells (evCEpC) or with ex vivo corneal endothelial cells (evCEnC). CRISPR-Cas9 gene editing was performed in CEnC to produce a monoallelic knockout of ZEB1 (ZEB1+/-). To characterize the rescue of the ZEB1 insufficiency phenotype, ZEB1+/- and the reference cell line, ZEB1+/+, were transduced with lentivirus containing ZEB1 cDNA, subsequently generating transgenic cell lines ZEB1+/+ +LV and ZEB1+/- +LV. Biological triplicates of each of the four cell lines were sub-cloned and validated with off-target site screening, qPCR, and Western blot. RNA-seq and transcriptomic analyses were performed.
Results :
We identified 1715 DEGs in PPCD endothelium compared to age-matched controls, of which 920 were up-regulated and 795 were down-regulated. Comparison of the DEGs in PPCD with evCEpC or evCEnC genes demonstrated 26% (65/249) of evCEpC were upregulated and 37% (40/108) of evCEnC were downregulated. By comparing ZEB1+/- -LV, ZEB1+/+ +LV, and ZEB1+/- +LV to the reference cell line, ZEB1+/+ -LV, 2222 DEGs were identified. Reconstitution of the ZEB1+/- -LV cell line with ZEB1 (ZEB1+/- +LV) increased its Spearman correlation with the ZEB1+/+ -LV reference cell line from 0.89 to 0.95, the highest correlation between any two groups from our samples; PCA and hierarchical clustering results corroborated this high correlation.
Conclusions :
Gene expression changes observed in PPCD are consistent with an endothelial to epithelial-like transition. In addition, using a transcriptomic approach, we have demonstrated the CRISPR-Cas9 generated ZEB1+/- HCEnC as a viable cell-based model of PPCD to be used for performing relevant functional studies.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.