Generating in vitro human optic vesicles to dissect the genetic modifiers affecting ocular maldevelopment

Model in vitro 3D-human optic vesicles using iPSC technology from patient skin fibroblasts

to investigate the molecular pathology of ocular maldevelopment. Patient skin and blood samples will be reprogrammed to iPSCs, characterised and differentiated to 3D retinal organoids. Molecular and phenotypic analysis using a wide range of techniques, measurements and developmental markers will be used to compare this development to human foetal development.

Using CRISPR/Cas9 gene editing to correct the mutation in the proband's iPSC to observe reversal of the phenotype in the human model to determine whether the mutation is causative or not. The mutation identified in the patient will be corrected using CRISPR/Cas9 gene editing operating through homologous-directed repair that will be validated prior to this in HEK-293 cells. Optic vesicles used in this step will be derived according to the differentiation process previously established in the project.

Comparative analysis of the transcriptome in optic vesicles to identify genetic modifiers. Changes in expression of any genes in the array tested will be identified and results corroborated from various approaches to accurately measure these changes with the aim of identifying genetic modifiers influencing the genotype/phenotype relationship.