Autosomal Dominant Retinitis Pigmentosa Research Articles

Progressive accumulation of cytoplasmic aggregates in PRPF31 retinal pigment epithelium cells interferes with cell survival

Progressive accumulation of cytoplasmic aggregates in PRPF31 retinal pigment epithelium cells interferes with cell survival

A 69 kb Deletion in chr19q13.42 including PRPF31 Gene in a Chinese Family Affected with Autosomal Dominant Retinitis Pigmentosa.

We aimed to identify the genetic cause of autosomal dominant retinitis pigmentosa (adRP) and characterize the underlying molecular mechanisms of incomplete penetrance in a Chinese family affected with adRP. All enrolled family members underwent ophthalmic examinations. Whole-genome sequencing (WGS), multiplex ligation-dependent probe amplification (MLPA), linkage analysis and haplotype construction were performed in all participants. RNA-seq was performed to analyze the regulating mechanism of incomplete penetrance among affected patients, mutation carriers and healthy controls. In the studied family, 14 individuals carried a novel heterozygous large deletion of 69 kilobase (kb) in 19q13.42 encompassing exon 1 of the PRPF31 gene and five upstream genes: TFPT, OSCAR, NDUFA3, TARM1, and VSTM1. Three family members were sequenced and diagnosed as non-penetrant carriers (NPCs). RNA-seq showed significant differential expression of genes in deletion between mutation carriers and healthy control. The RP11 pedigree in this study was the largest pedigree compared to other reported RP11 pedigrees with large deletions. Early onset in all affected members in this pedigree was considered to be a special phenotype and was firstly reported in a RP11 family for the first time. Differential expression of PRPF31 between affected and unaffected subjects indicates a haploinsufficiency to cause the disease in the family. The other genes with significant differential expression might play a cooperative effect on the penetrance of RP11.

Comprehensive analysis of the PRPF31 gene in retinitis pigmentosa patients: Four novel Alu-mediated copy number variations at the PRPF31 locus.

Retinitis pigmentosa (RP) is a monogenic disease characterized by irreversible degeneration of the retina. PRPF31, the second most common causative gene of autosomal dominant RP, frequently harbors copy number variations (CNVs), but the underlying mechanism is unclear. In this study, we summarized the phenotypic and genotypic characteristics of 18 RP families (F01-F18) with variants in PRPF31. The prevalence of PRPF31 variants in our cohort of Chinese RP families was 1.7% (18/1024). Seventeen different variants in PRPF31 were detected, including eight novel variants. Notably, four novel CNVs encompassing PRPF31, with a proportion of 22.2% (4/18), were validated to harbor gross deletions involving Alu/Alu-mediated rearrangements (AAMRs) in the same orientation. Among a total of 12 CNVs of PRPF31 with breakpoints mapped on nucleotide-resolution, 10 variants (83.3%) were presumably mediated by Alu elements. Furthermore, we described the correlation between the genotypes and phenotypes in PRPF31-related RP. Our findings expand the mutational spectrum of the PRPF31 gene and provide strong evidence that Alu elements of PRPF31 probably contribute to the susceptibility to genomic rearrangement in this locus.

REPRODUCTIVE OPHTHALMOLOGY: The Intersection of Inherited Eye Diseases and Reproductive Technologies.

To propose a working framework for patients with inherited eye diseases presenting to ophthalmologists who are interested in assisted reproductive technology and preimplantation genetic testing. Retrospective chart review and case series of three families with inherited eye diseases who successfully underwent preimplantation genetic testing, in vitro fertilization, and birth of unaffected children. Preimplantation genetic testing was performed for three families with different inherited eye diseases, which included autosomal dominant retinitis pigmentosa, autosomal recessive achromatopsia, and X-linked Goltz syndrome. Preimplantation genetic testing led to the identification of unaffected embryos, which were then selected for in vitro fertilization and resulted in the birth of unaffected children. A close collaboration between patients, families, ophthalmologists, reproductive genetic counselors, and reproductive endocrinology and infertility specialists is the ideal model for taking care of patients interested in preimplantation genetic testing for preventing the transmission of inherited eye diseases.

Anatomical and functional correlates of cystic macular edema in retinitis pigmentosa.

Cystoid macular edema (CME) is a major cause of central visual deterioration in retinitis pigmentosa. The exact reason for CME and its prognostic significance in this patient population is unknown. We seek to find clues to answer these questions by examining the anatomical correlations between retinal cysts and retinal morphometric parameters in a cohort of patients with retinitis pigmentosa and CME. For this reason, 103 patients (196 eyes) with untreated cystoid macular edema (CME) were identified from a pool of 578 genotyped patients with retinitis pigmentosa. Image analyses were conducted using three central horizontal OCT scans of these patients to calculate cross-sectional areas of the retinal nerve fiber layer, outer retinal, inner retinal, cysts, and total retinal areas. Lengths of the ellipsoid zone and outer limiting membrane were also measured. Best-fit curves were derived for analyzing the factors playing a role in the size of the retinal cysts and the patients’ visual acuity. Generalized Estimating Equation and multivariate linear regression analyses were conducted to determine the correlations between visual acuity, morphometric and clinical data, and the significant cyst size and visual acuity determinants. Twenty-five percent of the screened patients (103/578) had CME. Patients with autosomal dominant retinitis pigmentosa had the highest incidence of CME (43.6%, p<0.001) but also had the best visual acuity (20/34±20/30, p = 0.02). The total cyst area was 0.14±0.18 mm2. Outer retinal area (B = 0.214; p = 0.008), age (B = -0.003; p<0.001) and retinal nerve fiber area (B = 0.411; p = 0.005) were main determinants of the (r = 0.44; p<0.001) cyst size. Cysts resolved with progressing retinal degeneration. Length of the intact ellipsoid zone (B = -5.16E-5; p<0.001), the inheritance pattern (B = 0.04; p = 0.028) and retinal nerve fiber area (B = 0.751; p<0.001) were the main determinants of visual acuity. In patients with retinitis pigmentosa and cystoid macular edema, retinal nerve fiber layer thickness is associated with decreasing visual acuity and cyst size. This finding suggests that intraretinal cysts may compress retinal axons and cause subsequent visual loss in retinitis pigmentosa.

Pre- and postsynaptic alterations in the visual cortex of the P23H-1 retinal degeneration rat model.

P23H rats express a variant of rhodopsin with a mutation that leads to loss of visual function with similar properties as human autosomal dominant retinitis pigmentosa (RP). The advances made in different therapeutic strategies to recover visual system functionality reveal the need to know whether progressive retina degeneration affects the visual cortex structure. Here we are interested in detecting cortical alterations in young rats with moderate retinal degeneration, and in adulthood when degeneration is severer. For this purpose, we studied the synaptic architecture of the primary visual cortex (V1) by analyzing a series of pre- and postsynaptic elements related to excitatory glutamatergic transmission. Visual cortices from control Sprague Dawley (SD) and P23H rats at postnatal days 30 (P30) and P230 were used to evaluate the distribution of vesicular glutamate transporters VGLUT1 and VGLUT2 by immunofluorescence, and to analyze the expression of postsynaptic density protein-95 (PSD-95) by Western blot. The amount and dendritic spine distribution along the apical shafts of the layer V pyramidal neurons, stained by the Golgi-Cox method, were also studied. We observed that at P30, RP does not significantly affect any of the studied markers and structures, which suggests in young P23H rats that visual cortex connectivity seems preserved. However, in adult rats, although VGLUT1 immunoreactivity and PSD-95 expression were similar between both groups, a narrower and stronger VGLUT2-immunoreactive band in layer IV was observed in the P23H rats. Furthermore, RP significantly decreased the density of dendritic spines and altered their distribution along the apical shafts of pyramidal neurons, which remained in a more immature state compared to the P230 SD rats. Our results indicate that the most notable changes in the visual cortex structure take place after a prolonged retinal degeneration period that affected the presynaptic thalamocortical VGLUT2-immunoreactive terminals and postsynaptic dendritic spines from layer V pyramidal cells. Although plasticity is more limited at these ages, future studies will determine how reversible these changes are and to what extent they can affect the visual system’s functionality.

Aberrant Retinal Pigment Epithelial Cells Derived from Induced Pluripotent Stem Cells of a Retinitis Pigmentosa Patient with the PRPF6 Mutation.

Pre-mRNA processing factors (PRPFs) are vital components of the spliceosome and are involved in the physiological process necessary for pre-mRNA splicing to mature mRNA. As an important member, PRPF6 mutation resulting in autosomal dominant retinitis pigmentosa (adRP) is not common. Recently, we reported the establishment of an induced pluripotent stem cells (iPSCs; CSUASOi004-A) model by reprogramming the peripheral blood mononuclear cells of a PRPF6-related adRP patient, which could recapitulate a consistent disease-specific genotype. In this study, a disease model of retinal pigment epithelial (RPE) cells was generated from the iPSCs of this patient to further investigate the underlying molecular and pathological mechanisms. The results showed the irregular morphology, disorganized apical microvilli and reduced expressions of RPE-specific genes in the patient’s iPSC-derived RPE cells. In addition, RPE cells carrying the PRPF6 mutation displayed a decrease in the phagocytosis of fluorescein isothiocyanate-labeled photoreceptor outer segments and exhibited impaired cell polarity and barrier function. This study will benefit the understanding of PRPF6-related RPE cells and future cell therapy.

Gene Therapy for Rhodopsin Mutations.

Mutations in RHO, the gene for rhodopsin, account for a large fraction of autosomal-dominant retinitis pigmentosa (adRP). Patients fall into two clinical classes, those with early onset, pan retinal photoreceptor degeneration, and those who experience slowly progressive disease. The latter class of patients are candidates for photoreceptor-directed gene therapy, while former may be candidates for delivery of light-responsive proteins to interneurons or retinal ganglion cells. Gene therapy for RHO adRP may be targeted to the mutant gene at the DNA or RNA level, while other therapies preserve the viability of photoreceptors without addressing the underlying mutation. Correcting the RHO gene and replacing the mutant RNA show promise in animal models, while sustaining viable photoreceptors has the potential to delay the loss of central vision and may preserve photoreceptors for gene-directed treatments.

CRISPR DNA Base Editing Strategies for Treating Retinitis Pigmentosa Caused by Mutations in Rhodopsin.

Retinitis pigmentosa (RP) is the most common group of inherited retinal degenerations and pathogenic variants in the Rhodopsin (RHO) gene are major cause for autosomal dominant RP (adRP). Despite extensive attempts to treat RHO-associated adRP, standardized curative treatment is still lacking. Recently developed base editors offer an exciting opportunity to correct pathogenic single nucleotide variants and are currently able to correct all transition variants and some transversion variants. In this study, we analyzed previously reported pathogenic RHO variants (n = 247) for suitable PAM sites for currently available base editors utilizing the Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9) or the KKH variant of SaCas9 (KKH-SaCas9) to assess DNA base editing as a treatment option for RHO-associated adRP. As a result, 55% of all the analyzed variants could, in theory, be corrected with base editors, however, PAM sites were available for only 32% of them and unwanted bystander edits were predicted for the majority of the designed guide RNAs. As a conclusion, base editing offers exciting possibilities to treat RHO-associated adRP in the future, but further research is needed to develop base editing constructs that will provide available PAM sites for more variants and that will not introduce potentially harmful bystander edits.

A precise and efficient adenine base editor

Adenine base editors (ABEs) are novel genome-editing tools, and their activity has been greatly enhanced by eight additional mutations, thus named ABE8e. However, elevated catalytic activity was concomitant with frequent generation of bystander mutations. This bystander effect precludes its safe applications required in human gene therapy. To develop next-generation ABEs that are both catalytically efficient and positionally precise, we performed combinatorial engineering of NG-ABE8e. We identify a novel variant (NG-ABE9e), which harbors nine mutations. NG-ABE9e exhibits robust and precise base-editing activity in human cells, with more than 7-fold bystander editing reduction at some sites, compared with NG-ABE8e. To demonstrate its practical utility, we used NG-ABE9e to correct the frequent T17M mutation in Rhodopsin for autosomal dominant retinitis pigmentosa. It reduces bystander editing by ∼4-fold while maintaining comparable efficiency. NG-ABE9e possesses substantially higher activity than NG-ABEmax and significantly lower bystander editing than NG-ABE8e in rice. Therefore, this study provides a versatile and improved adenine base editor for genome editing.

In Through the Out Door: Editas' CEOs Grasp Opportunities Amidst Challenges

In Through the Out Door: Editas' CEOs Grasp Opportunities Amidst Challenges

Autosomal Dominant Retinitis Pigmentosa-Associated TOPORS Protein Truncating Variants Are Exclusively Located in the Region of Amino Acid Residues 807 to 867.

PurposeHeterozygous truncating variants of TOPORS have been reported to cause autosomal dominant retinitis pigmentosa (adRP). The purpose of this study was to investigate whether all heterozygous truncating variants, including copy number variants (CNVs), are pathogenic.Methods TOPORS truncating variants were collected and reviewed through an in-house dataset and existing databases. Individuals with truncating variants underwent ophthalmological evaluation.ResultsSix truncating variants were detected in seven families. Three N-terminus truncating variants were detected in three families without RP, and the other three were identified in four unrelated families with typical RP. Based on the in-house dataset and published literature, 17 truncating variants were identified in 47 families with RP. All RP-associated truncating alleles, except one, were distributed in the last exon of TOPORS and clustered in amino acid residues 807 to 867 (46/47, 97.9%). Conversely, in the gnomAD database, only one truncating allele (1/27, 3.7%) was in this region, and the others were outside (26/27, 96.3%), suggesting that the pathogenic truncating variants were significantly clustered in residues 807 to 867 (χ2 = 65.6, P = 1.1 × 10–17). Additionally, three CNVs involving the N-terminus of TOPORS were recorded in control populations but were absent in affected patients.ConclusionsThis study suggests that all pathogenic truncating variants of TOPORS were clustered in residues 807 to 867, whereas the truncating variants outside this region and the CNVs involving the N-terminus were not associated with RP. A dominant-negative effect, rather than haploinsufficiency, is speculated to be the underlying pathogenesis. These findings provide valuable information for interpreting variation in TOPORS and other genes in similar situations, especially for CNVs.

Uncovering Pre‐messenger RNA Splicing Mechanisms in Retinitis Pigmentosa

Disruptions of pre‐messenger RNA splicing is a common mechanism found in human diseases, like autosomal dominant Retinitis Pigmentosa (adRP). adRP affects ~1 in 4,000 people and is associated with the breakdown and loss of cells in the peripheral retina. Genetic analyses of patients harboring adRP have linked eight proteins that are associated with the spliceosome to adRP. Currently, there are two working hypotheses for how mutations in these splicing proteins contribute to adRP. First, splicing defects are occurring ubiquitously in all tissues but are more noticeable in the retinal tissue. Alternatively, that these splicing defects are strictly happening in the retina. This study focuses on understanding how mutations in one of these splicing proteins, PRPF31, impact splicing and cellular growth. In this study, we use a mouse retinal ganglion precursor‐like cell line model to identify various mRNAs effects of adRP mutants splicing function. We are examining six prpf31 mutants adRP mutants selected from patients with adRP. Expression constructs have been prepared for wildtype and our adRP mutants and successfully transfected into the mouse cell lines. We have also established a splicing assay for our mouse photoreceptor cell studies utilizing Pladienolide B, a splicing inhibitor as a model, and will examine the PRPF31 mutants using this assay. Our ongoing work will be described.

Subcellular localization of mutant P23H rhodopsin in an RFP fusion knock-in mouse model of retinitis pigmentosa.

ABSTRACTThe P23H mutation in rhodopsin (Rho), the rod visual pigment, is the most common allele associated with autosomal-dominant retinitis pigmentosa (adRP). The fate of misfolded mutant Rho in rod photoreceptors has yet to be elucidated. We generated a new mouse model, in which the P23H-Rho mutant allele is fused to the fluorescent protein Tag-RFP-T (P23HhRhoRFP). In heterozygotes, outer segments formed, and wild-type (WT) rhodopsin was properly localized, but mutant P23H-Rho protein was mislocalized in the inner segments. Heterozygotes exhibited slowly progressing retinal degeneration. Mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. Quantification of mRNA for markers of ER stress and the unfolded protein response revealed little or no increases. mRNA levels for both the mutant human rhodopsin allele and the WT mouse rhodopsin were reduced, but protein levels revealed selective degradation of the mutant protein. These results suggest that the mutant rods undergo an adaptative process that prolongs survival despite unfolded protein accumulation in the ER. The P23H-Rho-RFP mouse may represent a useful tool for the future study of the pathology and treatment of P23H-Rho and adRP. This article has an associated First Person interview with the first author of the paper.

Therapeutic homology-independent targeted integration in retina and liver

Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs.

AAV-CRISPR/Cas9 Gene Editing Preserves Long-Term Vision in the P23H Rat Model of Autosomal Dominant Retinitis Pigmentosa.

Retinitis pigmentosa (RP) consists of a group of inherited, retinal degenerative disorders and is characterized by progressive loss of rod photoreceptors and eventual degeneration of cones in advanced stages, resulting in vision loss or blindness. Gene therapy has been effective in treating autosomal recessive RP (arRP). However, limited options are available for patients with autosomal dominant RP (adRP). In vivo gene editing may be a therapeutic option to treat adRP. We previously rescued vision in neonatal adRP rats by the selective ablation of the Rhodopsin S334ter transgene following electroporation of a CRISPR/Cas9 vector. However, the translational feasibility and long-term safety and efficacy of ablation therapy is unclear. To this end, we show that AAV delivery of a CRISPR/Cas9 construct disrupted the Rhodopsin P23H transgene in postnatal rats, which rescued long-term vision and retinal morphology.

Intravitreal Administration of Stanniocalcin-1 Rescues Photoreceptor Degeneration with Reduced Oxidative Stress and Inflammation in a Porcine Model of Retinitis Pigmentosa

To investigate the effect of stanniocalcin-1 (STC-1), a secreted polypeptide exhibiting multiple functions in cell survival and death, on photoreceptor degeneration in a porcine model of retinitis pigmentosa (RP). P23H transgenic pigs (TG P23H) and wild-type hybrid littermates were obtained from the National Swine Resource and Research Center. Human recombinant STC-1 was injected intravitreally every 2 weeks from postnatal day 15 (P15) to P75. The contralateral eye was injected with balanced salt solution as a control. Electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed to evaluate retinal function and morphology in vivo at P90. Retinal tissue was collected for histologic analysis and molecular assays to evaluate the antioxidative and anti-inflammatory mechanisms by which STC-1 may rescue photoreceptor degeneration. Intravitreal injection of STC-1 improved retinal function in TG P23H pigs with increased photopic and flicker ERG a- and b-wave amplitudes. Greater integrity of the ellipsoid zone (EZ) band on SD-OCT and morphologic rescue with preservation of cone photoreceptors were observed in STC-1-treated TG P23H pigs. STC-1 altered gene expression in TG P23H pig retina on microarray analysis and increased photoreceptor specific gene expression by reverse transcription-polymerase chain reaction analysis. STC-1 significantly decreased oxidative stress and the expressions of NLRP3 inflammasome, cleaved caspase-1, and IL-1β in TG P23H pig retina. Intravitreal administration of STC-1 enhances cone photoreceptor function, improves EZ integrity, and reduces retinal degeneration through antioxidative and anti-inflammatory effects in a large animal (pig) model of the most common form of autosomal dominant RP in the United States.

The Potential Role of Epigenetic Mechanisms in the Development of Retinitis Pigmentosa and Related Photoreceptor Dystrophies.

Retinitis pigmentosa and related photoreceptor dystrophies (RPRPD) are rare retinal diseases caused by hereditary gene mutations resulting in photoreceptor death, followed by vision loss. While numerous genes involved in these diseases have been identified, many cases have still not been associated with any gene, indicating that new mechanisms may be involved in the pathogenesis of these photoreceptor dystrophies. Many genes associated with RPRPD regulate photoreceptor specification and maturation in the developing retina. Since retinal development begins with a population of equivalent, proliferating retinal progenitor cells (RPCs) having a specific “competence” in generating all types of retinal neurons, including cone and rod photoreceptors, we tested the epigenetic changes in promoters of genes required for photoreceptor development and genes associated with RPRPD during RPC differentiation into cone and rod photoreceptors. We found that promoters of many of these genes are epigenetically repressed in RPCs but have no epigenetic restrictions in photoreceptors. Our findings also suggest that DNA methylation as an epigenetic mark, and DNA demethylation as a process, are more important than other epigenetic marks or mechanisms in the pathogenesis of these diseases. Most notably, irregularities in the DNA demethylation process during the RPC-to-photoreceptor transition may significantly contribute to retinitis pigmentosa (RP) pathogenesis since genes with hypermethylated promoters in RPCs account for at least 40% of autosomal recessive RP cases and at least 30% of autosomal dominant RP cases. Thus, we proposed an epigenetic model according to which unsuccessful demethylation of regulatory sequences (e.g., promoters, enhancers) of genes required for photoreceptor development, maturation, and function during the RPC-to-photoreceptor transition may reduce or even eliminate their activity, leading to RPRPD without any inheritable mutations in these genes.

Rod genesis driven by mafba in an nrl knockout zebrafish model with altered photoreceptor composition and progressive retinal degeneration.

Neural retina leucine zipper (NRL) is an essential gene for the fate determination and differentiation of the precursor cells into rod photoreceptors in mammals. Mutations in NRL are associated with the autosomal recessive enhanced S-cone syndrome and autosomal dominant retinitis pigmentosa. However, the exact role of Nrl in regulating the development and maintenance of photoreceptors in the zebrafish (Danio rerio), a popular animal model used for retinal degeneration and regeneration studies, has not been fully determined. In this study, we generated an nrl knockout zebrafish model via the CRISPR-Cas9 technology and observed a surprising phenotype characterized by a reduced number, but not the total loss, of rods and over-growth of green cones. We discovered two waves of rod genesis, nrl-dependent and -independent at the embryonic and post-embryonic stages, respectively, in zebrafish by monitoring the rod development. Through bulk and single-cell RNA sequencing, we characterized the gene expression profiles of the whole retina and each retinal cell type from the wild type and nrl knockout zebrafish. The over-growth of green cones and mis-expression of green-cone-specific genes in rods in nrl mutants suggested that there are rod/green-cone bipotent precursors, whose fate choice between rod versus green-cone is controlled by nrl. Besides, we identified the mafba gene as a novel regulator of the nrl-independent rod development, based on the cell-type-specific expression patterns and the retinal phenotype of nrl/mafba double-knockout zebrafish. Gene collinearity analysis revealed the evolutionary origin of mafba and suggested that the function of mafba in rod development is specific to modern fishes. Furthermore, the altered photoreceptor composition and abnormal gene expression in nrl mutants caused progressive retinal degeneration and subsequent regeneration. Accordingly, this study revealed a novel function of the mafba gene in rod development and established a working model for the developmental and regulatory mechanisms regarding the rod and green-cone photoreceptors in zebrafish.

Activation of autophagy reverses progressive and deleterious protein aggregation in PRPF31 patient-induced pluripotent stem cell-derived retinal pigment epithelium cells.

IntroductionMutations in pre‐mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri‐snRNP complex, cause autosomal‐dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri‐snRNP proteins result in retina‐specific disorders, and so far, the underlying mechanism of splicing factors‐related RP is poorly understood.MethodsWe used the induced pluripotent stem cell (iPSC) technology to generate retinal organoids and RPE models from four patients with severe and very severe PRPF31‐adRP, unaffected individuals and a CRISPR/Cas9 isogenic control.ResultsTo fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells were carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle‐related pathways to be significantly affected. Strikingly, the patient‐derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin‐conjugated proteins including key visual cycle and other RP‐linked tri‐snRNP proteins, which accumulated progressively with time. The mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wild‐type levels led to tri‐snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin reduced cytoplasmic aggregates, leading to improved cell survival.ConclusionsOur data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31‐initiated mis‐splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31‐adRP patients.