Abstract
Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/− mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/− mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical – basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.
Highlights
Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause nonsyndromic retinal disease
Autosomal-dominant inheritance accounts for about 40% of Retinitis pigmentosa (RP), with an estimated 15% of cases of this RP inheritance type caused by mutations in premRNA processing factors (PRPFs)
To gain insights into RP pathomechanisms, we characterised the cellular phenotypes and splicing programmes of retinal pigment epithelium (RPE) and retinal organoids in comparison to fibroblasts and induced pluripotent stem cells (iPSCs) derived from RP11 patients with PRPF31 mutations
Summary
Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause nonsyndromic retinal disease. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/− mouse retinae and RPE. Functional primary cilia are required for maturation of the retinal pigment epithelium (RPE)[13] These data suggest that the precise regulation of splicing programmes for photoreceptor-specific transcripts, including those that are involved in ciliogenesis, are essential for retinal development. Large-scale transcriptome analyses identified mis-splicing of cell type and patient-specific target genes affected by PRPF31 mutations, providing unprecedented molecular characterisation of splicing-factor RP clinical phenotypes. CRISPR/Cas[9] correction of a PRPF31 mutation in cells derived from an RP11 patient with very severe RP, resulted in the rescue of molecular and cellular phenotypes, providing proof-of-concept evidence for the effectiveness of in situ gene correction
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