Ciliopathy pathways and biomarkers in IRD variant characterisation and therapy steps in retinal organoids

Abstract

Purpose : Genetic variant characterisation in the inherited retinal dystrophies (IRDs) is critical in understanding pathological disease pathways to inform potential therapy approaches and for classification of variants of uncertain significance (VUS). While there are a number of avenues for investigation of potential splice-site abnormalities, missense VUS provide particular challenges in resolution. To address this, we used patient-derived and CRISPR-edited human induced pluripotent stem cells differentiated to retinal organoids for investigation of ciliopathy disease genes affecting the photoreceptor outer segments.

Methods : Patient- derived and control iPSC lines were generated, along with CRISPR-edited iPSC lines of missense VUS under investigation in RPGRIP1 and PDE6B. Targeted long-read sequencing of cDNA amplicons was undertaken in patient-derived organoids for VUS investigation, where phase was not known. The iPSC lines were differentiated to retinal organoids. Bulk and single cell RNA sequencing, along with morphological and immunofluorescence studies were used to assess key impacted pathways and biomarkers of disease in the retinal organoids.

Results : Retinal organoids with clear-cut pathogenic variants in RPGRIP1 and PDE6B showed changes in ciliary trafficking and/or photoreceptor outer segment morphology compared with isogenic controls, as did missense VUS under investigation. Long-read sequencing of cDNA amplicons showed VUS in trans with a confirmed pathogenic variant, adding to pathogenicity scoring of the VUS. Transcriptomic analysis indicated key signature pathways as markers of the disease phenotype with single cell RNA sequencing providing additional information regarding cell types impacted.

Conclusions : Disease-causing variants in the IRD genes, RPGRIP1 and PDE6B, cause abnormal phenotypes in retinal organoids along with key pathway abnormalities identified on bulk and single cell RNA sequencing. These biomarkers in the retinal organoids, along with long-read sequencing of VUS requiring phase determination in retinal cells, contribute to VUS resolution. These approaches assist definitive genetic management for patients and families, and confirm therapy impact of novel genetic therapy approaches.

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