Cell-autonomous restoration of splicing homeostasis and RP11 phenotype in patient-derived RPE and retinal organoids by PRPF31.AAV gene therapy

Abstract Mutations in the PRPF31 gene cause retinitis pigmentosa type 11 (RP11), a progressive blinding disease triggered by defects in the spliceosome, the cellular machinery responsible for RNA processing. Although gene replacement holds therapeutic promise, it remains unclear whether it can effectively rescue both primary target cell types across different disease stages. Here we show that viral-mediated delivery of PRPF31 to patient-derived retinal pigment epithelium (RPE) cells and 3D retinal organoids restores RNA splicing activity and reverses core cellular pathology. Gene delivery cleared toxic protein aggregates, restored essential RPE functions, including structural polarity, cilia integrity, and cellular waste clearance, and boosted light-evoked activity in photoreceptors. Notably, robust rescue was achievable even in mature cells, whereas combining gene therapy with autophagy-activating drugs offered no added benefit. These findings demonstrate that restoring splicing homeostasis alone drives functional recovery, supporting a broad clinical window for PRPF31 gene therapy in RP11.