June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
In vitro modelling of autosomal dominant TGFBI corneal dystrophies and development of a targeted therapeutic approach
Author Affiliations & Notes
  • Olivia Farah Rezek
    Institute of Ophthalmology, University College London, London, London, United Kingdom
  • Beatriz Sanchez-Pintado
    Institute of Ophthalmology, University College London, London, London, United Kingdom
  • Michael E Cheetham
    Institute of Ophthalmology, University College London, London, London, United Kingdom
  • Alice E Davidson
    Institute of Ophthalmology, University College London, London, London, United Kingdom
  • Stephen Tuft
    Moorfields Eye Hospital NHS Foundation Trust, London, London, United Kingdom
  • Alison J Hardcastle
    Institute of Ophthalmology, University College London, London, London, United Kingdom
  • Footnotes
    Commercial Relationships   Olivia Farah Rezek None; Beatriz Sanchez-Pintado None; Michael Cheetham ProQR, Code C (Consultant/Contractor), Alia Therapeutics, Code C (Consultant/Contractor), PYC, Code C (Consultant/Contractor); Alice Davidson None; Stephen Tuft None; Alison Hardcastle None
  • Footnotes
    Support  Moorfields Eye Charity 539960
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3844. doi:
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      Olivia Farah Rezek, Beatriz Sanchez-Pintado, Michael E Cheetham, Alice E Davidson, Stephen Tuft, Alison J Hardcastle; In vitro modelling of autosomal dominant TGFBI corneal dystrophies and development of a targeted therapeutic approach. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3844.

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

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Abstract

Purpose : TGFBI corneal dystrophies are autosomal dominant inherited diseases with distinctive phenotype-genotype correlations, characterised by the accumulation of TGFBI protein (TGFBIp) positive deposits in the sub-epithelial, Bowman and/or stromal layers of the cornea. Current surgical treatments do not target the underlying genetic cause of the disease, with disease symptoms frequently reoccurring. In order to generate disease models, patient derived induced pluripotent stem cells (iPSCs) were differentiated towards corneal epithelial-like cells to study the pathophysiology of TGFBI corneal dystrophies, and explore the use of targeted antisense oligonucleotides (ASOs) as a potential therapy.

Methods : Fibroblasts obtained from patients harbouring heterozygous hotspot mutations c.370C>T, p.R124C or c.371G>A, p.R124H were reprogrammed to iPSCs by nucleofection with the pluripotency factors Oct3/4, Kfl4, Sox2, L-Myc, Lin28 and mir302/367. iPSCs were differentiated towards a corneal epithelial-like cell lineage, in parallel with control iPSC, and characterised for expression of epithelial markers using quantitative reverse transcriptase PCR and immunocytochemistry. ASOs were designed to reduce expression of TGFBI mutant alleles and treatment effects were explored by reverse transcriptase (RT)-PCR, Sanger sequencing and quantitative RT-PCR.

Results : Immunofluorescence confirmed the expression of the stem cell markers Oct4, Nanog, SSEA4 and Tra-1-81 in all iPSC lines. Characterisation of the iPSC derived model confirmed a corneal epithelial-like phenotype with endogenous expression of markers KRT3, KRT14, P63, ABCG2 and E-cadherin at transcript and protein level. Following ASO treatment, a reduction of TGFBI transcript levels was observed by RT-PCR. Sanger sequencing demonstrated a specific reduction of the mutant allele in comparison to the wild type allele.

Conclusions : We have established a patient iPSC derived in vitro model to investigate the molecular mechanisms of TGFBI corneal dystrophies and test therapeutic approaches in an appropriate genomic and cellular context. Preliminary investigations indicate that ASO treatment targeting mutations at position p.R124 may be effective in reducing the expression of the mutant allele. Further investigations are required to further characterise the molecular mechanisms of disease and the effect of ASOs on TGFBIp.

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

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