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

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.

We are grateful to Retina UK (GR # 601, #GR584, #GR595), Fight for Sight UK (1456/1457), MRC UK (MR/T017503/1), and EPSRC/ERC (EP/Y031016/1) for funding this work. The purchase of IncuCyte used in this study was supported by a UKRI MRC Capital Funding for World Class Labs Award (MR/X012360/1). H.U. was supported by the German Research Society (DFG) within the CRC SFB1289 (project number 317475864).

We would also like to thank the Newcastle University FMS Bioimaging Unit, flow cytometry, and genomics core facilities for their continued support during this project. We would like to thank LeedsOmics at the University of Leeds for help with technical assays and RNA-seq analysis, and the University of Leeds alumnus funding from Peter & Sue Cheney. These authors jointly supervised this work: Sina Mozaffari-Jovin, Robin R.

Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK Maria Elia, Maria Georgiou, Robert Atkinson & Majlinda Lako Centre for Gene Therapy & Regenerative Medicine, Faculty of Life Sciences & Medicine, King’s College London, London, UK Valda Pauzuolyte, Mark Basche, Alexander J. Ali Department of Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany Max-Planck-Institute for Multidisciplinary Sciences, Göttingen, Germany Carina Hansohn, Henning Urlaub & Sina Mozaffari-Jovin Leeds Omics, University of Leeds, Leeds, UK Department of Informatics, University of Bergen, Bergen, Norway Leeds Institute of Medical Research, Faculty of Medicine & Health, University of Leeds, Leeds, UK School of Geography and Natural Sciences, Northumbria University, Newcastle upon Tyne, UK Correspondence to Sina Mozaffari-Jovin, Robin R. The authors declare no competing interests.

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