Modeling PRPF31 retinitis pigmentosa using iPSC-derived retinal organoids and retinal pigmented epithelium and rescue by gene augmentation strategy
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ABSTRACT:
PROVIDER: PRJNA851279 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
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| SRR19761400_1.fastq.gz | Fastqsanger.gz | |||
| SRR19761400_2.fastq.gz | Fastqsanger.gz | |||
| SRR19761401_1.fastq.gz | Fastqsanger.gz | |||
| SRR19761401_2.fastq.gz | Fastqsanger.gz | |||
| SRR19761402_1.fastq.gz | Fastqsanger.gz |
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Project description:Mutations in the ubiquitously expressed pre-mRNA processing factor (PRPF) 31, one of the most common cause of dominant form of Retinitis Pigmentosa (RP), lead to retina-specific phenotype. It is uncertain which retinal cell types are affected and animal models do not clearly present the RP phenotype observed in PRPF31 patients. Retinal organoids and retinal pigmented epithelial (RPE) cells derived from human induced pluripotent stem cells (iPSCs) provide potential opportunities for studying human PRPF31-related RP. We demonstrated that RPE cells carrying PRPF31 mutations present important morphological and functional changes and that PRPF31-mutated retinal organoids recapitulate the human RP phenotype, with a rod photoreceptor cell death followed by a loss of cones. The low level of PRPF31 expression may explain the defective phenotypes of PRPF31-mutated RPE and photoreceptor cells, which were not observed in cells derived from asymptomatic patients or after correction of the pathogenic mutation by CRISPR/Cas9. Transcriptome profiles revealed differentially expressed and mis-spliced genes belonging to pathways in line with the observed defective phenotypes. The rescue of RPE and photoreceptor defective phenotypes by PRPF31 gene augmentation, provide the proof of concept for future therapeutic strategies.
2022-08-01 | GSE206529 | GEO
Project description:Purpose Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterised by progressive degeneration of the mid-peripheral retina, leading to night blindness and eventual loss of visual acuity. RP11, caused by mutations in the pre-mRNA processing factor 31 (PRPF31) gene, involved in pre-mRNA splicing as a key component of the spliceosome, accounts for approximately 10% of autosomal dominant RP (adRP) cases. Previous studies have shown that PRPF31 mutations disrupt pre-mRNA splicing of other splicing-related genes, resulting in global spliceosome dysregulation, reduced spliceosome activity, leading to accumulation of insoluble cytoplasmic aggregates, cell dysfunction and ultimately retinal cell death. The pathogenic mechanism is consistent with haploinsufficiency, as the mutant PRPF31 is excluded from the active spliceosome and resulting levels of the gene are insufficient. Therefore, PRPF31 gene augmentation via adeno-associated virus (AAV) makes a promising therapeutic strategy. This study aims to assess the efficiency of PRPF31.AAV gene therapy in restoring PRPF31 expression and function in RPE and photoreceptors, focusing on restoring spliceosome dysregulation, reduction of cytoplasmic aggregates and rescuing cellular phenotypes. Approach Using induced pluripotent stem cells (iPSC) derived from patients with severe RP11, we generated retinal organoids and RPE cells models. Both photoreceptors and RPE cells were treated with ShH10.CMV.PRPF31.AAV to enhance PRPF31 expression. The therapeutic impact of PRPF31.AAV was assessed through qPCR and immunoblotting for gene and protein expression of PRPF31 and immunofluorescence analysis of splicing markers, cilia and cytoplasmic aggregates. Lastly, the phagocytotic function of AAV-treated RPE cells was assessed using fluorescently labelled photoreceptor outer segments (POS). Results PRPF31.AAV treatment resulted in a nearly two-fold increase of total PRPF31 expression in RPE cells and a ten-fold increase in photoreceptors, compared to GFP.AAV controls, as assessed by qPCR. Quantification of protein levels confirmed a significant elevated PRPF31 levels in PRPF31.AAV treated photoreceptors. Moreover, PRPF31 localisation was corrected, and nuclear splicing speckles were increased, indicative of restored nuclear speckle morphology and splicing functions in RPE cells. A significant reduction in cytoplasmic aggregates of degradation-related protein HSPB1 and retinal-specific protein RLBP1 was observed in AAV treated RP11-RPE cells and photoreceptors, when compared to controls. A significant increase in both cilia length and incidence was observed in RPE cells upon AAV augmentation. Finally, the phagocytic capacity of RPE cells was restored following PRPF31.AAV treatment, indicating restoration of RPE function. Conclusions Our findings demonstrate that PRPF31.AAV gene augmentation therapy effectively restores critical functions in both RPE and photoreceptors. This highlights the therapeutic potential of an AAV-based gene therapy for patients with PRPF31-related adRP.
2026-08-16 | GSE309908 | GEO
Project description:Pathogenic variants in splicing factors are the second most common cause of autosomal dominant retinitis pigmentosa (RP), with mutations in PRPF31 being the most prevalent. Here, we characterize a novel intronic variant in PRPF31 (c.1074-11C>G) that creates a cryptic 3′ splice site, resulting in an aberrantly spliced transcript predicted to encode a protein with an altered C-terminus. However, the pathogenic protein was not detected in patient-derived induced pluripotent stem cells (iPSCs). In addition, expression of PRPF31 protein was reduced in patient iPSC-derived retinal pigment epithelium (RPE). To correct the splicing defect, we designed a panel of antisense oligonucleotides (ASOs) targeting putative RNA-binding sites in exon 10 and intron 10 and identified a candidate that corrects PRPF31 splicing in a minigene reporter system as well as in patient-derived iPSCs and RPE. Finally, we showed that ASO treatment enhances PRPF31 protein expression in patient iPSC-derived RPE, supporting its potential as a therapeutic approach to restore PRPF31 expression in RP patients.
2026-06-12 | PXD071251 | Pride
Project description:Mutations in pre-mRNA processing factors (PRPFs) cause autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause retinal disease. We have generated 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.
2018-08-20 | PXD010821 | Pride
Project description:Dysfunction of splicing factors often result in abnormal cell differentiation and apoptosis, especially in neural tissues. Mutations in pre-mRNAs processing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration disease. The transcriptome-wide splicing events specifically regulated by PRPF31 and their biological roles in the development and maintenance of retina are still unclear. Here, we showed that the differentiation and viability of retinal progenitor cells (RPCs) are severely perturbed in prpf31 knockout zebrafish when compared with other tissues at an early embryonic stage. At the cellular level, significant mitotic arrest and DNA damage were observed. These defects could be rescued by the wild-type human PRPF31 rather than the disease-associated mutants. Further bioinformatic analysis and experimental verification uncovered that Prpf31 deletion predominantly causes the skipping of exons with a weak 5′ splicing site. Moreover, genes necessary for DNA repair and mitotic progression are most enriched among the differentially spliced events, which may explain the cellular and tissular defects in prpf31 mutant retinas. This is the first time that Prpf31 is demonstrated to be essential for the survival and differentiation of RPCs during retinal neurogenesis by specifically modulating the alternative splicing of genes involved in DNA repair and mitosis.
2021-01-06 | GSE151273 | GEO
Project description:Retinitis pigmentosa (RP) encompasses a group of inherited retinal disorders characterized by progressive degeneration of rod and cone photoreceptors, leading to vision loss. Among RP subtypes, RP11 is linked to mutations in PRPF31, a key spliceosome component, resulting in retinal cell dysfunction. Although PRPF31 is ubiquitously expressed, its mutations predominantly impact retinal cells, leading to the progressive loss of photoreceptors. Human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) offer a powerful in vitro model for studying RP pathophysiology and therapeutic development. Despite significant progress, studies focused on photoreceptor and retinal pigment epithelium dysfunction in late disease stages, leaving early molecular events and the involvement of other retinal cell types unresolved. Moreover, comprehensive single-cell analyses capturing dynamic transcriptional changes across all retinal populations at early and late differentiation stages are still lacking. Using patient-derived ROs, this study investigates the developmental trajectory of PRPF31-RP11 mutation through single-cell RNA sequencing, highlighting early Müller glial expansion, retinal ganglion cell stress, and progressive photoreceptor degeneration. Findings identify dysregulated molecular pathways associated with phototransduction, oxidative stress, and inflammation, providing insights into RP11 pathogenesis and potential therapeutic targets.
2026-03-10 | GSE307923 | GEO
Project description:Mutations in RP2 lead to a severe form of X-linked retinitis pigmentosa (XLRP). RP2 functions as a GTPase activating protein (GAP) for the small GTPase ARL3, which is essential for cilia function and for photoreceptor development and maintenance. The mechanisms of RP2 associated retinal degeneration in humans are poorly understood, and genetically engineered animal models of RP2 XLRP present with differing retinal phenotypes and slow degeneration suggesting potential species differences. Here, we developed CRISPR gene edited isogenic RP2 knock-out (RP2 KO) induced pluripotent stem cells (iPSC) and RP2 patient derived iPSC to produce 3D retinal organoids as a human retinal disease model. The isogenic RP2 KO retinal organoids and two unrelated RP2 patient iPSC lines produced retinal organoids with a defined photoreceptor cell layer (ONL) containing rod and cone photoreceptors. Strikingly the RP2 KO and RP2 patient derived organoids showed a thinning of the ONL by 180 days (D180) of culture, which was associated with a spike in cell death in the ONL at D150 of differentiation. RNA sequencing confirmed induction of cell death pathways in the RP2 null organoids at this stage. Photoreceptor cell death and ONL thinning was attributed to cells undergoing terminal rod cell differentiation characterized by reduced RHO expression and fewer rhodopsin immunoreactive photoreceptors. Gene augmentation with a human RP2 transgene in an AAV2/5 vector efficiently transduced RP2 KO organoids and led to high levels of RP2 expression in both rods and cones. Importantly, the viral transduction significantly increased ONL thickness and restored rhodopsin expression, suggesting rescue of the RP2 degeneration phenotype. These data show that 3D retinal organoids can be used to model molecular defects associated with inherited retinal disease, photoreceptor cell death and also to test potential therapies targeted to prevent photoreceptor degeneration.
2020-07-08 | GSE148300 | GEO
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Project description:A hallmark of inherited retinal degenerative diseases such as Retinitis Pigmentosa (RP) is progressive structural and functional remodeling of the remaining retinal cells as photoreceptors degenerate. Extensive remodeling of the retina stands as a barrier for the successful implementation of strategies to restore vision. To understand the molecular basis of remodeling, we performed analyses of single-cell transcriptome data from adult Zebrafish retina of wild-type and a P23H mutant rhodopsin transgenic model of RP with continuous degeneration and regeneration. We provide a benchmark atlas of retinal cell type transcriptomes in Zebrafish and find changes in all retinal cell types in the P23H model. These include widespread oxidative stress, changes in reliance on oxidative metabolism and glycolysis, widespread synaptic remodeling, and changes in circadian rhythm regulation. This comprehensive transcriptomic analysis provides a molecular road map to understand how the retina remodels in the context of chronic retinal degeneration with ongoing regeneration.
2023-11-10 | GSE234435 | GEO