GeneTAC™ small molecules reduce toxic nuclear foci and restore normal splicing in corneal endothelial cells derived from patients with Fuchs endothelial corneal dystrophy (FECD) harboring repeat expansions in transcription factor 4 (TCF4)

Abstract

Purpose : Most cases of FECD are caused by a CTG trinucleotide repeat expansion in intron 3 of the TCF4 gene leading to production of toxic RNA foci, global splicing dysregulation, and cellular dysfunction. The mainstay of FECD management is keratoplasty; no disease-modifying therapies are currently approved. GeneTAC™ molecules are small molecule gene targeted chimera compounds designed to target specific genomic sequences through a DNA-binding moiety and modulate transcription. In this study, we evaluated GeneTAC™ molecules designed to selectively target the expanded CTG repeats in TCF4 intron 3 for their effects on RNA foci formation and mis-splicing.

Methods : GeneTAC™ molecules were synthesized and selected for RNA foci-reducing activity in immortalized F35T corneal endothelial cells (CECs). Primary FECD patient-derived CEC lines (n=4) were treated continuously for 2 to 9 days with 7 unique GeneTAC™ molecules at concentrations up to 300 nM. Toxic nuclear RNA foci containing expanded CUG repeats were evaluated using fluorescence in situ hybridization, and disease-related splicing defects in MBNL1, MBNL2 and NUMA1 were evaluated using PCR.

Results : In FECD patient-derived primary CEC lines harboring mono- and biallelic TCF4 repeat expansions, treatment with GeneTAC™ molecules reduced the average number of foci by 40 to 99% in a time- and concentration-dependent manner. Changes in splicing of MBNL1, MBNL2 and NUMA1 mRNA demonstrated that the most active GeneTAC™ molecules corrected splicing to levels observed in unaffected CECs at concentrations > 33 nM.

Conclusions : Treatment with GeneTAC™ molecules corrected key molecular hallmarks in FECD patient-derived CECs. These findings support continuing development of GeneTAC™ molecules as a potential disease-modifying therapy for FECD.

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