PRPF8 Mutation-Induced Defects in Human iPSC-Derived RPE Are Rescued by Adenine Base Editing.

PURPOSE: The pathological effects of pre-mRNA processing factor 8 (PRPF8) mutations on the retinal pigment epithelium (RPE) are not fully understood. We aimed to identify disease-specific cellular and molecular phenotypes in PRPF8 retinitis pigmentosa (RP) patient-derived induced pluripotent stem cell (iPSC)-RPE and to test whether adenine base editing (ABE), which corrects the PRPF8 mutation in iPSCs, can reverse abnormal RPE phenotypes. METHODS: We obtained patient-derived iPSCs with the heterozygous PRPF8 (c.5792C>T) mutation and created an induced mutation iPSC line by introducing the same mutation into wild-type iPSCs using CRISPR/Cas9. These cells were differentiated into RPE cells. We measured PRPF8 expression, barrier integrity, and apicobasal polarity. Electron microscopy examined apical microvilli and pigment granules. RNA sequencing quantified splicing events and affected pathways. ABE corrected the PRPF8 mutation in patient iPSCs, and the corrected clones were re-differentiated into RPE cells for evaluation. RESULTS: PRPF8-mutant RPE cells exhibited decreased PRPF8 mRNA and protein levels, weakened barrier function, and disrupted cell polarity. Ultrastructural analysis showed loss of apical microvilli and pigment granules. Transcriptomic analysis identified abnormal splicing events, with enrichment in cilium assembly and melanosome pathways. ABE correction restored PRPF8 expression, normalized barrier integrity, apicobasal polarity, and rescued the defects in apical microvilli and pigment granules. CONCLUSIONS: PRPF8 mutations in patient-derived iPSC RPE cause functional and ultrastructural defects driven by splicing abnormalities. ABE correction of the PRPF8 mutation in iPSCs can restore PRPF8 expression and alleviate cellular and molecular defects in RPE and highlights the therapeutic potential of precise gene editing correction strategies for RP.