ABSTRACT: Purpose Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterised by progressive degeneration of the mid-peripheral retina, leading to night blindness and eventual loss of visual acuity. RP11, caused by mutations in the pre-mRNA processing factor 31 (PRPF31) gene, involved in pre-mRNA splicing as a key component of the spliceosome, accounts for approximately 10% of autosomal dominant RP (adRP) cases. Previous studies have shown that PRPF31 mutations disrupt pre-mRNA splicing of other splicing-related genes, resulting in global spliceosome dysregulation, reduced spliceosome activity, leading to accumulation of insoluble cytoplasmic aggregates, cell dysfunction and ultimately retinal cell death. The pathogenic mechanism is consistent with haploinsufficiency, as the mutant PRPF31 is excluded from the active spliceosome and resulting levels of the gene are insufficient. Therefore, PRPF31 gene augmentation via adeno-associated virus (AAV) makes a promising therapeutic strategy. This study aims to assess the efficiency of PRPF31.AAV gene therapy in restoring PRPF31 expression and function in RPE and photoreceptors, focusing on restoring spliceosome dysregulation, reduction of cytoplasmic aggregates and rescuing cellular phenotypes. Approach Using induced pluripotent stem cells (iPSC) derived from patients with severe RP11, we generated retinal organoids and RPE cells models. Both photoreceptors and RPE cells were treated with ShH10.CMV.PRPF31.AAV to enhance PRPF31 expression. The therapeutic impact of PRPF31.AAV was assessed through qPCR and immunoblotting for gene and protein expression of PRPF31 and immunofluorescence analysis of splicing markers, cilia and cytoplasmic aggregates. Lastly, the phagocytotic function of AAV-treated RPE cells was assessed using fluorescently labelled photoreceptor outer segments (POS). Results PRPF31.AAV treatment resulted in a nearly two-fold increase of total PRPF31 expression in RPE cells and a ten-fold increase in photoreceptors, compared to GFP.AAV controls, as assessed by qPCR. Quantification of protein levels confirmed a significant elevated PRPF31 levels in PRPF31.AAV treated photoreceptors. Moreover, PRPF31 localisation was corrected, and nuclear splicing speckles were increased, indicative of restored nuclear speckle morphology and splicing functions in RPE cells. A significant reduction in cytoplasmic aggregates of degradation-related protein HSPB1 and retinal-specific protein RLBP1 was observed in AAV treated RP11-RPE cells and photoreceptors, when compared to controls. A significant increase in both cilia length and incidence was observed in RPE cells upon AAV augmentation. Finally, the phagocytic capacity of RPE cells was restored following PRPF31.AAV treatment, indicating restoration of RPE function. Conclusions Our findings demonstrate that PRPF31.AAV gene augmentation therapy effectively restores critical functions in both RPE and photoreceptors. This highlights the therapeutic potential of an AAV-based gene therapy for patients with PRPF31-related adRP.
