Sanchez Pintado, Beatriz; (2022) Development of an iPSC-derived corneal epithelial in vitro model for the study and treatment of TGFBI-associated corneal dystrophies with antisense oligonucleotides. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

TGFBI-associated corneal dystrophies are autosomal dominant diseases caused by missense mutations in the TGFBI gene, that lead to protein aggregation and accumulation in the corneal epithelium and stroma, often accompanied by painful erosions and impaired vision. Surgical interventions are the only treatments currently available to restore vision, however, due to the high degree of recurrence of corneal opacity alternative therapeutic approaches are needed. Since animal models have largely failed to recapitulate the human disease, in vitro models represent an alternative approach to study the mechanism of disease and investigate novel treatments. A genetic study of a patient cohort (n=120) at Moorfields Eye Hospital demonstrated over half of the TGFBI corneal dystrophy cases were caused by mutations at the p.R555 hotspot. Fibroblasts from five individuals with heterozygous p.R555W and p.R555Q mutations with Granular Dystrophy 1 and Thiel-Behnke corneal dystrophies respectively, were reprogrammed to iPSC. In addition, wild type (WT) and CRISPR/Cas9-edited TGFBI knockout iPSC lines were generated alongside. Characterised cells revealed features of pluripotency, demonstrated by the endogenous expression of the stem cell markers NANOG, OCT4, SSEA-4, TRA-1-80 and TRA-1-60. Once the iPSC biobank was established, a differentiation protocol was optimised for the development of corneal epithelial-like cells (CEpC). These cell cultures were characterised for transcript and protein expression of corneal epithelial markers, such as E-Cadherin, P63, K14 and K3 throughout the differentiation protocol, verifying the differentiation of CEpC cells for all mutant and control lines. An experimental strategy was designed to suppress expression of the mutant allele with antisense oligonucleotides (ASOs) in the differentiated CEpC lines. ASOs complementary to the mutant TGFBI allele were designed to trigger RNAse H-mediated degradation of the transcript, and prevent translation of the mutant protein. The lead ASOs achieved a reduction of 50% of the total TGFBI transcript and TGFBI protein. Furthermore, next generation sequence analysis demonstrated a shift in WT:mutant transcript ratios in favour of WT, indicating allele-specificity. In summary, human in vitro models of TGFBI dystrophies differentiated to corneal epithelial-like cells were successfully developed from patient, control and gene edited iPSC, providing a platform to study corneal dystrophies and develop novel therapeutics. ASOs targeted to the p.R555 hotspot mutations were effective at targeting the mutant allele and reducing the levels of TGFBI, and therefore represent a promising targeted therapeutic approach for autosomal dominant corneal dystrophies.

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