Fernando, Owen; (2025) Retinal organoid modelling and therapeutic approach for cone dysfunction disorders caused by OPN1LW and OPN1MW interchange haplotypes. Doctoral thesis (Ph.D), UCL (University College London).

Text Fernando_10213570_Thesis.pdf Access restricted to UCL open access staff until 1 April 2027. Download (13MB)

Abstract

X-linked cone dysfunction disorders (CDDs) ranging from Blue Cone Monochromacy (BCM) to cone dystrophy are caused by mutations resulting in non-functional long-wave sensitive (L) and medium-wave sensitive (M) cone opsins. Currently, there are no therapies for these blinding diseases. Interchange haplotypes (ICHs), with rare combinations of single nucleotide polymorphisms (SNPs) in exon 3 of the OPN1LW and OPN1MW genes, have emerged as a common cause of disease that are predicted to alter exon splice enhancer (ESE) and exon splice silencer (ESS) sites. An in vitro mini-gene assay was established to investigate the effects of different interchange haplotypes. The mini-gene assay revealed CDD-associated ICHs (LIAVA, LIVVA, LVAVA, MIAVA, LVVVA and LIAVS) caused aberrant splicing and skipping of exon 3 to varying degrees. Antisense oligonucleotides (AONs) were designed to promote exon 3 inclusion targeting different sequences and with different chemistries. A lead mutation agnostic AON was identified, named AON+11MOE, that targets intronic splice silencers (ISSs), suppressing exon 3 skipping in all ICHs tested in the mini-gene assay. Fibroblasts were isolated from skin biopsies from three patients with different gene compositions of the cone opsin array and three different exon 3 ICHs (LVAVA, LIVVA and LVVVA). Patient fibroblasts were reprogrammed to induced pluripotent stem cells (iPSCs) and differentiated to retinal organoids (ROs). ROs were harvested at different time points of differentiation and characterised using immunohistochemistry, RT-PCR and qPCR. For the first time, exon 3 skipping was observed as a result of CDD-associated ICHs in human retinal photoreceptors in all patient ROs. AON+11MOE successfully suppressed exon 3 skipping and increased the level of full-length transcripts in patient ROs. AON+11MOE is ICH agnostic and represents a promising potential therapeutic for these blinding conditions. This research demonstrates the utility of ROs to study developmental mechanisms of cone photoreceptor fate determination and cone opsin mediated CDDs.

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