July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
RNA toxicity induced by TCF4 CTG expansions is ameliorated by antisense therapeutics in a patient-derived cell model of Fuchs corneal endothelial dystrophy (FECD).
Author Affiliations & Notes
  • Alice E Davidson
    Institute of Ophthalmology, UCL, London , England, United Kingdom
  • Christina Zarouchlioti
    Institute of Ophthalmology, UCL, London , England, United Kingdom
  • Beatriz Sanchez-Pintado
    Institute of Ophthalmology, UCL, London , England, United Kingdom
  • Nathaniel Hafford Tear
    Institute of Ophthalmology, UCL, London , England, United Kingdom
  • Pontus Klein
    ProQR therapeutics, Leiden, Netherlands
  • Petra Liskova
    Charles University , Prague, Czechia
  • Kalyan Dulla
    ProQR therapeutics, Leiden, Netherlands
  • Kirithika Muthusamy
    Moorfeilds Eye Hospital , London , United Kingdom
  • Lubica Dudakova
    Charles University , Prague, Czechia
  • Pavlina Skalicka
    Charles University , Prague, Czechia
  • Pirro G Hysi
    King's College London, London , United Kingdom
  • Michael E. Cheetham
    Institute of Ophthalmology, UCL, London , England, United Kingdom
  • Stephen J. Tuft
    Moorfeilds Eye Hospital , London , United Kingdom
  • Peter S Adamson
    ProQR therapeutics, Leiden, Netherlands
  • Alison J Hardcastle
    Institute of Ophthalmology, UCL, London , England, United Kingdom
  • Footnotes
    Commercial Relationships   Alice Davidson, None; Christina Zarouchlioti, None; Beatriz Sanchez-Pintado, None; Nathaniel Hafford Tear, None; Pontus Klein, ProQR Therapeutics (E); Petra Liskova, None; Kalyan Dulla, ProQR Therapeutics (E); Kirithika Muthusamy, None; Lubica Dudakova, None; Pavlina Skalicka, None; Pirro Hysi, None; Michael Cheetham, None; Stephen Tuft, None; Peter Adamson, ProQR Therapeutics (E); Alison Hardcastle, None
  • Footnotes
    Support  We were supported by funding from Fight for Sight Early Career Investigator Award (A.E.D), Academy of Medical Sciences Springboard (A.E.D), The National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital National Health ServiceFoundation Trust and UCL Institute of Ophthalmology, Rosetrees Trust (A.E.D and A.J.H), Wellcome Trust (M.E.C) and ProQR NV via a Research Collaboration Award. LD, PS and PL were supported by PROGRES-Q26/LF1 andGACR 17-12355S. PS was supported by GAUK227015/2017.
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3022. doi:
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      Alice E Davidson, Christina Zarouchlioti, Beatriz Sanchez-Pintado, Nathaniel Hafford Tear, Pontus Klein, Petra Liskova, Kalyan Dulla, Kirithika Muthusamy, Lubica Dudakova, Pavlina Skalicka, Pirro G Hysi, Michael E. Cheetham, Stephen J. Tuft, Peter S Adamson, Alison J Hardcastle; RNA toxicity induced by TCF4 CTG expansions is ameliorated by antisense therapeutics in a patient-derived cell model of Fuchs corneal endothelial dystrophy (FECD).. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3022.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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