Understanding Congenital Hereditary Endothelial Dystrophy pathogenesis using SLC4A11 knockout cellular model generated via CRISPR/Cas9 gene editing

Abstract

Purpose : Congenital Hereditary Endothelial Dystrophy (CHED) is a rare bilateral inherited disorder characterized by progressive corneal opacification and visual impairment due to SLC4A11 gene variations. This study aims to analyze SLC4A11 mutational spectrum in CHED cases and targeting the identified SLC4A11 variation for the generation of SLC4A11 knockout cellular models using CRISPR/Cas9 gene editing.

Methods : Clinically confirmed CHED cases(n=7) underwent genetic screening by direct sequencing of SLC4A11 gene and whole exome sequencing (WES) for SLC4A11-negative cases(n=2). The guideRNA targeting the identified SLC4A11 variation was designed and cloned into a PX458-CRISPR vector, followed by transfection into HEK293 cells. The transfection efficiency and indels were assessed by flow cytometry and restriction digestion, respectively. Pure population of CRISPR-edited cells were obtained by single-cell expansion followed by Sanger sequencing, qPCR, and functional characterization of the clones.

Results : Genetic screening of SLC4A11 in CHED familial(n=2) and sporadic(n=2) cases showed homozygous p.Ser516Trp variation in family1(n=3 affected cases), homozygous p.Arg209Trp, p.Thr463Thr, and p.Asn553Asp variations in family2(n=2 affected cases) and no SLC4A11 variation in sporadic cases. WES showed double heterozygous p.Gln253Leu in GDF6 and p.Gly3753fs*13 variation in ADGRV1 in sporadic case1 and heterozygous p.Gly1011_Glu1014del variation in RPGR (sporadic case2). In-silico analysis of identified variants revealed varying degrees of pathogenic effects. A transfection efficiency of ~35-40% was observed with the designed sgRNA-PX458 vector in HEK293 cells. HinCII-mediated RE digestion confirmed the indels at the targeted region of SLC4A11 gene. Immunofluorescence showed reduced expression of SLC4A11 and increased oxidative stress in transfected HEK293 cells. SLC4A11 knockouts p.Lys514fs*657 and p.Phe516fs*538 were obtained with 102bp intron-exon deletion and exonic T insertion, respectively. The CRISPR clones showed altered cell morphology, cell proliferation, ROS generation and absence of SLC4A11 expression.

Conclusions : The developed SLC4A11 knockout cellular models may enable a better understanding of the CHED and aid in development of potential therapeutics.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.