Rezek, Farah Olivia; (2024) An in vitro iPSC-corneal epithelial cell model for the study of TGFBI corneal dystrophies and development of an antisense oligonucleotide treatment. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

TGFBI corneal dystrophies (CDs) are autosomal dominant inherited diseases with distinctive phenotype-genotype correlations, however, the underlying mechanism of these conditions are poorly understood. TGFBI CDs are characterised by the accumulation of TGFBI positive protein deposits in the sub-epithelial, Bowman and/or stromal layers of the cornea, presenting with painful corneal erosions and visual impairment. Current surgical treatments do not target the underlying genetic cause of the disease and symptoms commonly reoccur; therefore, alternative therapeutic approaches are required. In order to facilitate the investigation of TGFBI CD pathogenesis and provide an appropriate platform for therapeutic screening, patient-derived in vitro models offer a compelling approach. A genetic study of TGFBI CD cases at the Moorfields Eye Hospital patient cohort (n=120) demonstrated that around 1/3 of cases have mutations at the p.R124 hotspot of the TGFBI gene. Fibroblasts from four individuals with heterozygous p.R124C or p.R124H mutations, presenting with Lattice Corneal Dystrophy I and Granular Corneal Dystrophy II, respectively, were obtained and reprogrammed into iPSC. Wild type (WT) and a CRISPR/Cas9-edited TGFBI knockout iPSC cell lines were cultured in parallel. The endogenous expression of the pluripotency markers Oct4, Nanog, SSEA4 and Tra-1-81 was confirmed in all iPSC lines. All iPSC lines were differentiated to corneal epithelial-like cells and characterised for the transcript and protein expression of corneal epithelial markers such as K14, K3, P63 and PAX6 throughout key differentiation time points. Bulk RNA sequencing was carried out on day 21 of differentiation to investigate potential mechanisms of dysregulated transcription underlying disease, and provided insight into potential disease mechanisms. Antisense oligonucleotides (ASOs) were designed to specifically reduce the expression of TGFBI in a mutant-specific manner by triggering RNase H-mediated degradation of the mutant transcript. ASO treatment resulted in a 20-40% decrease of total TGFBI transcript levels. Targeted next generation sequencing demonstrated allele specificity of the ASOs and confirmed their efficacy. In summary, patient-derived iPSC-derived corneal epithelial-like models have been developed and characterised for the investigation of molecular mechanisms underlying TGFBI CDs and to test therapeutic approaches. ASOs targeting the p.R124 hotspot mutations effectively reduced the expression of the mutant allele of TGFBI, therefore, representing a promising targeted therapeutic approach for TGFBI CDs.

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