Abstract
Purpose :
Mutations in RP2 lead to a severe form of X-linked retinitis pigmentosa (XLRP). RP2 functions as a GTPase activating protein (GAP) for the small GTPase Arl3, which is essential for cilia function and for photoreceptor development and maintenance. The mechanisms of RP2 associated retinal degeneration are poorly understood with animal models presenting with differing retinal phenotypes including defects in trafficking of prenylated cargo into photoreceptor outer segments. We used patient derived induced pluripotent stem cells (iPSC) differentiated into 3D retinal organoids to study XLRP in a human retinal cell model and to create a tool for testing therapies.
Methods :
iPSC were derived from two patients with stop mutations in RP2 (R120X) and CRISPR/CAS9 RP2 knockout iPSC lines were developed. The iPSC were differentiated into mature retinal organoids (5-6 months) in parallel with the un-edited control parent line. Retinal organoid composition and gene expression were assessed by IHC on organoid cryosections, qPCR and RNAseq.
Results :
Control and R120X IPSC retinal organoids consisted of a laminated structure containing all the major retinal cells types arranged in their appropriate layers including rod and cone photoreceptors in a compacted ONL. Control retinal organoids took 5-6 months to develop mature opsin expressing photoreceptors and to begin to develop nascent outer segments. RP2 deficient patient and CRISPR/CAS9 RP2 knockout retinal organoids consistently presented with significantly reduced numbers of rhodopsin expressing photoreceptors (n=6-9, p<0.005) and a thinner ONL at 6 months (p<0.01). There was a significant increase in photoreceptor cell death (TUNEL) at month 5; a time point at which rhodopsin cell differentiation was increasing in control cell lines. This correlated with an up regulation in cell death pathways and a decrease in rod photoreceptor gene expression by RNAseq and qPCR.
Conclusions :
iPSC retinal organoids have been used to model XLRP. A significant increase in rod photoreceptor cell death was observed in two independent RP2 patient lines and CRISPR/CAS9 RP2 knockout retinal organoids suggesting that this effect is a specific consequence of the RP2 depletion. In addition to giving insight into disease mechanisms this system provides an ideal platform for drug screening in which efficacy can be measured in human retinal cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.