Unravelling the Role of EYS gene in Retinitis Pigmentosa Type 25 Progression Using Retinal Organoids

Abstract

Purpose : Retinitis pigmentosa type 25 (RP25), caused by mutations in the EYS gene, is one of the most common forms of autosomal recessive RP across various ethnicities. Despite its prevalence, various obstacles have hampered therapeutic developments including the large size of the EYS gene, lack of understanding of its function, and absence in common animal models (mouse, rat, guinea pig). This project aims to better understand EYS function by modeling RP25 disease, utilizing patients-derived pluripotent stem cells (PSC) and retinal organoids (RO) as an in vitro model of the disease.

Methods : Induced PSCs were generated from urine cells obtained from a patient diagnosed with RP25 carrying the homozygous mutation in the EYS (c.8897G>A in exon 43) and her daughters -carriers but asymptomatic- as controls. Additionally, another hiPSC carrying a mutation in both alleles of the EYS at c.3567delA in exon 23 and c.4829-4832delCATT in exon 26 was acquired from the BNLC (ISCIII; Spain) for comparative analysis. RO from the lines were generated and characterised (N=3-6) at various maturation stages. Structural, gene expression and functional differences among the groups were studied using histology, immunohistochemistry, PCR, qPCR, and calcium imaging techniques. Statistical significance was determined using 2-way ANOVA with Bonferroni post-test.

Results : The hiPSC lines generated displayed pluripotency capacity through PCR, IF, and differentiation to three germ layers. At early stages of retinal differentiation, RP25 lines exhibited disorganised neuroectoderm and surface ectoderm, with decreased expression of key ocular genes (SIX6 and CHX10) and increased expression of PAX6, PAX3 and TYR genes. This disorganisation persisted during RO formation and maturation. RP25-derived RO exhibited reduced retinal thickness, disorganization of bipolar and photoreceptor cells, higher pigmentation levels and immature photoreceptors with decreased outer segment formation. Furthermore, different functional patterns were observed in calcium imaging dynamics of retinal neural cells.

Conclusions : Distinctive differences between RP25 and controls in the in vitro RO model were evident at both structural and functional levels and reproducible in more than one line with mutations in the EYS gene. Our research sheds light on the intricate role of EYS in RP25 disease, providing valuable insights for potential therapeutic strategies.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.