Development of a research pipeline to identify novel treatments for Fuchs Endothelial Corneal Dystrophy

Abstract

Purpose : Mutations in the SLC4A11 gene are associated with Fuchs endothelial corneal dystrophy (FECD). SLC4A11 is expressed by corneal endothelial cells (CECs) and regulates corneal fluid balance and ion homeostasis. SLC4A11 mutations can cause disease through protein misfolding and an unfolded protein response. Prior studies showed that correcting mutant SLC4A11 misfolding can restore CEC functional activity. Thus, a safe and effective chemical corrector for SLC4A11 mutations related to FECD may be identified through an advanced research pipeline consisting of cell-based assays, CRISPR screens, and high content imaging (HCI) technologies. The purpose of this study is to develop a cell-based system for our research pipeline.

Methods : Plasmids suitable for HCI were generated to express doxycycline-inducible (Tet-on) GFP-SLC4A11 wild-type or mutant (G709E) fusion proteins. The plasmids were stably transfected into a HEK293T dCas9-KRAB cell line to allow for CRISPR genetic screens. Confocal microscopy was used to assess the cellular localization of GFP-tagged SLC4A11^WT and SLC4A11^G709E in the absence or presence of doxycycline and glafenine (positive control compound). The cell culture assay was then miniaturized to 384-well plate format and optimized for high throughput phenotypic screens (HTS). A screening Z’ score was determined as a statistical parameter for predicting the success of HTS campaigns.

Results : Confocal microscopy showed that expression of both proteins was doxycycline inducible, but the WT protein was properly trafficked to the plasma membrane while the G709E mutant was retained in puncta. Addition of glafenine partially restored normal trafficking of SLC4A11^G709E. The doxycycline concentration (2 μg/mL), assay volume (20 μL), treatment time (48 hrs) and number of cells/well (3,000 cells) were successfully optimized. Dose-response curves identified the concentration required for maximal activity (40 μM) and confirmed that the negative control (DMSO) had no measurable effect on fluorescence or cell viability. The calculated Z’ score for 40 μM glafenine was 0.32.

Conclusions : We have developed a cell-based assay that is ready for HCI and CRISPR genetic screens for discovering correctors for FECD-associated mutations. More broadly, this assay and workflow can be readily adapted for use in other genetic causes of FECD and other ocular disorders.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.