Pharmacological and molecular features of DT-168, a topical GeneTAC^TM small molecule being developed as potential treatment for Fuchs Endothelial Corneal Dystrophy caused by CTG repeat expansions in the TCF4 gene

Abstract

Purpose : Over 70% of Fuchs endothelial corneal dystrophy (FECD) cases are caused by cytosine-thymine-guanine (CTG) nucleotide repeat expansions in the TCF4 gene (CTG18.1) transcribed into toxic TCF4 RNA. This pathogenic RNA sequesters splicing proteins and forms nuclear foci, elicits transcript mis-splicing (spliceopathy), and drives corneal endothelial cell (CEC) death. We evaluated the tolerability, corneal biodistribution, and pharmacologic activity of topical ophthalmic DT-168 – a novel small molecule inhibitor of CTG18.1 transcription under development as a potential treatment for FECD.

Methods : CECs were cultured from donors with a clinical diagnosis of FECD who underwent endothelial keratoplasty and from unaffected cadavers (control CECs). Repeat expansions in the TCF4 gene were measured using optical genome mapping. FECD and control CECs were treated in vitro with DMSO or DT-168 and evaluated for i) nuclear foci labeling via fluorescent in situ hybridization and ii) splicing analysis via RNA sequencing. Frequency of exon inclusion and splice junction use, termed percent spliced-in (PSI), was used to establish a panel of splicing events that differed significantly between DMSO treated control and FECD CECs. In addition, rabbits were treated twice-daily for 14 days with DT-168 eye drops to assess safety based on standard ophthalmologic assessments and biodistribution.

Results : FECD CECs had CTG repeat expansions of ~9 to 21 kilobases in the TCF4 gene; expansions were not detected in control CECs. Untreated FECD CECs averaged 2.9 ± 0.2 foci per nucleus, while levels in control CECs were negligible. DT-168 reduced foci in FECD CECs to control levels, with IC₅₀ values of 24 to 102 nM. DT-168 significantly improved splicing in FECD CECs for all panel genes, with correction that exceeded 50% of control values. DT-168 eye drops were well-tolerated and distributed throughout the cornea, including CECs. Plasma levels of DT-168 were negligible.

Conclusions : DT-168 markedly reduced nuclear foci and improved spliceopathy in FECD CEC cultures, was well-tolerated in rabbits, and had good biodistribution throughout the cornea. These findings show that DT-168 could address the most common genetic cause of FECD and support development of DT-168 as a potential disease-modifying therapy.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.