Exploring the onset of retinal degeneration in Joubert Syndrome using retinal organoids

Abstract

Purpose : Pathogenic variants in both INPP5E and NPHP1 genes are known to cause the syndromic ciliopathy Joubert Syndrome (JS). Patients with JS can present with a variety of phenotypes including retinal degeneration, polydactyly, and brain abnormalities visible upon MRI (known as the molar tooth sign). While NPHP1 and INPP5E are known to play a role in the primary cilium, the actual function of these proteins in the photoreceptor sensory cilium has remained elusive to date. Here, we utilize retinal organoids to study the role of these JS-associated genes in retinal development.

Methods : Retinal organoids were derived by differentiating human induced pluripotent stem cells (iPSCs) containing either INPP5E^D477N/D477N loss of function or NPHP1^-/- knock out mutations generated previously using CRISPR/Cas9 approaches. Differentiation was continued for 250 days and phenotypic changes to organoid structure and development was analyzed at d35, d90, d120, d180, and d230 by immunofluorescence (photoreceptors – Rho, OPSIN; photoreceptor precursors – CRX, RCVN; ciliary markers – INPP5E, ARL13B) and qPCR analysis (CRX, RCVN, Rho). One-way ANOVA was used for statistical analysis.

Results : Preliminary data of retinal organoid structural analysis (n=4-8 organoids per time point) shows that retinal organoids derived from INPP5E^D477N/D477N iPSCs have a mislocalization of INPP5E in the primary cilium and connecting cilium of photoreceptors (p<0.0001) and a delay in the expression of photoreceptor progenitor markers CRX and RCVN (p=0.0004 at d230) throughout development. In addition, organoids show reduced outer segment length (p=0.0001 at d230) and a slight decrease in the number of photoreceptors at d180 and d230 (p=0.0007). NPHP1^-/- organoids show a severely reduced outer nuclear layer (p<0.0001), little to no RCVN expression during development and no photoreceptor development at d180.

Conclusions : The loss of INPP5E function in retinal organoids leads to a delay in photoreceptor generation and altered outer segment development, while NPHP1 knock outs showed no photoreceptor generation and severe morphological changes in retinal organoid structure. Together, these findings provide insight into the functions of INPP5E and NPHP1 in the retina and furthers our understanding of how pathogenic variants in these genes lead to retinal degeneration in JS, facilitating future studies towards therapy.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.