Elucidating the Mechanism of PRPF-linked Retinitis Pigmentosa

Abstract

Purpose : Splicing is essential for most basic cellular processes. It is not known why mutations in the largest and most conserved component of the spliceosome (PRPF8) cause Retinitis Pigmentosa (RP13). We hypothesised that mis-splicing affects those processes critical for vision such as retinal development and cilia function[ML1] . This study investigates using iPSCs derived from four affected individuals and isogenic controls generated using CRISPR/Cas9. Retinal and non-retinal tissues were differentiated and analysed to make clear the tissue specific phenotype and identify similarities to other types of PRPF-linked RP.

Methods : Fibroblasts from one de novo and three familial patients with RP13 were reprogrammed to iPSCs via RNA-based transduction. The missense mutation in PRPF8 was then edited using CRISPR/Cas9 to generate paired isogenic control lines (n = 8). All lines were differentiated in parallel to retinal pigment epithelium (RPE), retinal organoids (RO) and kidney organoids using published protocols. Isogenic pairs were cultured, collected, and processed in parallel for a range of experiments including qPCR, immunofluorescence microscopy, and electron microscopy. Statistical analysis was performed using two-tailed paired t test, results shown as mean ± SEM.

Results : Expression of the mutant PRPF8 transcript was detected by qPCR in RP13 cells but not the RP13-Cas9 controls (p < .001 for all cell types). We investigated whether the mutation affects localisation to intranuclear domains enriched with splicing factors, known as nuclear speckles. The colocalization of PRPF8 and nuclear speckle marker SC35 was reduced in RP13 iPSCs, RPE and photoreceptors (p < 0.05 for all cell types). In addition, cilia of RP13 RPE were 10% longer (p < 0.05) and a fraction of these had swollen axonemes, but cilia frequency was unaffected. To gain insight into possible effects on tissue function we carried out a quantitative ultrastructural analysis. A significantly reduced number of mitochondria were found in RP13 RPE (10 ± 1 vs 17 ± 1, p < 0.01) and photoreceptors (57 ± 13 vs 107 ± 11, p < 0.05).

Conclusions : These results suggest that retinal and non-retinal tissues exhibit a splicing phenotype in RP13. Retinal cells exhibit ciliary defects and a possible metabolic shift that warrant further investigation. The combined splicing and ciliary phenotype presented resembles that caused by PRPF31 mutations.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.