RNA toxicity induced by TCF4 CTG expansions is ameliorated by antisense therapeutics in a patient-derived cell model of Fuchs corneal endothelial dystrophy (FECD).

Abstract

Purpose : To identify the incidence of TCF4 CTG18.1 expansions within a large FECD patient cohort (n=450) and its association with disease. To develop a patient-derived corneal endothelial cell (CEC) model to investigate CTG18.1 expansion-related pathology and explore therapeutic approaches for this repeat expansion-mediated disease, within its native cellular and genomic context.

Methods : A total of 450 FECD patients were recruited to the study; either with clinical signs of FECD (numerous corneal guttae on slit-lamp biomicroscopy) or prior corneal transplantation surgery for FECD. CTG18.1 was genotyped using a short tandem repeat assay. CECs were cultured using a dual media approach and RNA foci were visualised by fluorescence in situ hybridisation, prior to quantitative image analysis. Immunocytochemistry was performed to determined MBNL1 and MBNL2 cellular localisation. Differential splicing events were analysed by reverse transcription (RT)-PCR and antisense oligonucleotides (ASOs) were transfected using standard methodologies.

Results : We determine that a non-coding trinucleotide repeat expansion in TCF4 confers significant (p=5.69 x10^-71) risk for FECD (>76-fold) in our large patient cohort. Investigation of 60 independent patient-derived CEC lines collectively demonstrated that, incidence of nuclear RNA foci, sequestration of splicing factor proteins to foci, and differential alternative splicing are all concordant with expanded genotype status. Importantly we show that an ASO treatment reduced RNA foci incidence and rescued MBNL1 sequestration and differential splicing events elicited by the expanded repeat in multiple, independent, patient-derived CEC lines.

Conclusions : We demonstrate that the TCF4 CTG18.1 expansion confers highly significant disease risk in our large FECD patient cohort and the trinucleotide repeat size is a fundamental driver of the incidence of RNA foci. Furthermore, we have defined downstream markers of RNA toxicity and demonstrate the translational potential of an ASO therapy.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.