Using retinal organoids to model Stargardt’s disease and test therapies

Abstract

Purpose : Stargardt’s disease (STG) is the most common inherited macular dystrophy and it is caused by mutations in the ATP-binding cassette sub-family A member 4 (ABCA4) gene. Mutation in ABCA4 leads to lipofuscin build-up in the RPE and subsequent photoreceptor degeneration. Currently, there are no treatments for STG and the large size of ABCA4 hinders standard adeno-associated virus (AAV) gene replacement. In this study we aimed to further characterise the pathophysiology of STG disease using retinal organoids derived from STG induced pluripotent stem cells (iPSC) lines. The establishment of disease biomarkers is essential to demonstrate efficacy for novel AAV gene therapy for Stargardt’s disease.

Methods : Using an established differentiation protocol, we generated organoids from three STG patients’ iPSC lines with varied disease severity. Mature STG retinal organoids were characterised using single cell RNA sequencing (ScRNA-seq). Some disease biomarkers were then validated by functional assays. Gene therapy using a dual AAV hybrid approach was used to treat 15-17 weeks old retinal organoids. A dose scalation with two titres was tested and organoids evaluated for ABCA4 expression using qPCR, western blots, and immunohistochemistry.

Results : Diseased retinal organoids generated typical photoreceptor cells with outer segment structures showing reduced ABCA4 protein. Cell population analysis using ScRNA-seq data showed a reduced proportion of cone cells, a feature of maculopathies. ScRNA-seq also highlighted potential disease pathways, such as oxidative phosphorylation. Downregulation of mitochondrial-related genes was evident and validated using OXPHOS western blots. Guanosine-3’,5’-cyclic monophosphate (cGMP)-concentration was altered in STG organoids. This characteristic phenotype of many retinal degenerative diseases corroborates dysregulations on phototransduction pathways observed in our ScRNA-seq data. Finally, STG organoids treated with hybrid dual AAV gene therapy showed an increase in ABCA4 expression.

Conclusions : We have demonstrated that retinal organoids are robust models of disease to elucidate pathological mechanisms of retinal degeneration in STG disease. Dual AAV gene therapy in STG retinal organoids increases ABCA4 expression. Further optimisation of dual AAV delivery in vitro promises to establish the effectiveness of Stargardt’s gene therapy.

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