AAV gene therapy rescues photoreceptor degeneration and ciliopathies in retinal organoids of PRPF6-associated retinitis pigmentosa

Mutations in the PRPF6 gene cause autosomal dominant retinitis pigmentosa (RP), an inherited retinal disease resulting in the severe loss of retinal photoreceptor cells. Herein, we generated retinal organoids (ROs) from patient-derived induced pluripotent stem cells (iPSCs), CRISPR/Cas9-edited PRPF6 knock-in iPSCs, and gene-corrected iPSCs. PRPF6 knock-in and PRPF6 patient-derived ROs showed decreased retinal outer layer thickness, impaired photoreceptor outer segment, increased apoptosis, and reduced cilium incidence and length. These phenotypes were significantly improved by CRISPR/Cas9-mediated correction of the PRPF6 mutation. Bulk and single-cell transcriptomic analyses revealed defects in photoreceptors and cilia, particularly in rod cells. Significantly, we demonstrated that adeno-associated virus (AAV)-mediated PRPF6 gene augmentation therapy could reverse photoreceptor degeneration and ciliopathies in patient-derived ROs. These findings showed that human iPSC-derived ROs recapitulated the disease phenotypes of PRPF6 -related RP and were rescued by AAV-mediated gene therapy. Consequently, this work lays the foundation for clinical trials of gene therapy for PRPF6 -related RP diseases.