Recessive Retinitis Pigmentosa Research Articles

The temporal progression of retinal degeneration and early-stage idebenone treatment in the Pde6brd1/rd1 mouse model of retinal dystrophy

Photoreceptor cell death, primarily through apoptosis, related to retinal disorders like retinitis pigmentosa (RP), would result in vision loss. The pathological processes and crucial mutant conditions preceding photoreceptor cell demise are not well understood. This study aims to conduct an in-depth examination of early-stage changes in the widely utilized Pde6brd1/rd1 (rd1) mouse model, which has Pde6b gene mutations representing autosomal recessive RP disorder. We investigated the morphology and ultrastructure of retinal cells, including second-order neurons, during the initial phase of disease progression. Our findings revealed that mitochondrial alterations in rod photoreceptors were present as a predeath mutant state as early as postnatal day 3 (P3). The bipolar and horizontal cells from the rd1 mouse retina exhibited significant morphological changes in response to loss of photoreceptor cells, indicating that second-order neurons rely on these cells for their structures. Subsequent oral administration of idebenone, a mitochondria-protective agent, enhanced retinal function and promoted both photoreceptor cell survival and inner retinal second-order synaptogenesis in rd1 mice at P14. Our findings offer a mechanistic framework, suggesting that mitochondrial damage acts as an early driver for photoreceptor cell death in retinal degeneration.

RETINITIS PIGMENTOSA ASSOCIATED WITH THE EYS C2139Y VARIANT : An Important Cause of Blindness in East Asian Populations.

The study aimed to describe the phenotypic features of retinitis pigmentosa (RP) associated with the previously described EYS C2139Y variant in Singaporeans and establish the importance of this variant as a prevalent cause of RP among East Asians. A clinical phenotyping and exome-sequencing study was conducted on consecutive patients with nonsyndromic RP. Epidemiological analysis was performed using Singaporean and global population-based genetic data. A study of 150 consecutive unrelated individuals with nonsyndromic RP found that 87 (58%) of cases had plausible genotypes. A previously described missense variant in the EYS gene, 6416G>A (C2139Y), occurred heterozygously or homozygously in 17 of 150 families (11.3%), all with autosomal recessive RP. Symptom onset in EYS C2139Y-related RP ranged from 6 to 45 years, with visual acuity ranging from 20/20 at 21 years to no light perception by 48 years. C2139Y-related RP had typical findings, including sectoral RP in cases with EYS E2703X in trans . The median age at presentation was 45 years and visual fields declined to less than 20° (Goldmann V4e isopter) by age 65 years. Intereye correlation for visual acuity, fields, and ellipsoid band width was high (r 2 = 0.77-0.95). Carrier prevalence was 0.66% (allele frequency of 0.33%) in Singaporean Chinese and 0.34% in East Asians, suggesting a global disease burden exceeding 10,000 individuals. The EYS C2139Y variant is common in Singaporean RP patients and other ethnic Chinese populations. Targeted molecular therapy for this single variant could potentially treat a significant proportion of RP cases worldwide.

Successful large gene augmentation of USH2A with non-viral episomal vectors

USH2A mutations are a common cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome, for which there are currently no approved treatments. Gene augmentation is a valuable therapeutic strategy for treating many inherited retinal diseases; however, conventional adeno-associated virus (AAV) gene therapy cannot accommodate cDNAs exceeding 4.7 kb, such as the 15.6-kb-long USH2A coding sequence. In the present study, we adopted an alternative strategy to successfully generate scaffold/matrix attachment region (S/MAR) DNA plasmid vectors containing the full-length human USH2A coding sequence, a GFP reporter gene, and a ubiquitous promoter (CMV or CAG), reaching a size of approximately 23 kb. We assessed the vectors in transfected HEK293 cells and USH2A patient-derived dermal fibroblasts in addition to ush2au507 zebrafish microinjected with the vector at the one-cell stage. pS/MAR-USH2A vectors drove persistent transgene expression in patient fibroblasts with restoration of usherin. Twelve months of GFP expression was detected in the photoreceptor cells, with rescue of Usher 2 complex localization in the photoreceptors of ush2au507 zebrafish retinas injected with pS/MAR-USH2A. To our knowledge, this is the first reported vector that can be used to express full-length usherin with functional rescue. S/MAR DNA vectors have shown promise as a novel non-viral retinal gene therapy, warranting further translational development.

Molecular Genetic Analysis of the Autosomal Recessive Non-Syndromic Inherited Retinitis Pigmentosa.

90% of visually impaired people live in developing countries. There are various types of vision impairment, but the focus of the current study is retinitis pigmentosa (RP). Up to now, 150 mutations have been reported that are linked with RP. Healthy and affected members from two Pakistani families (RP01 and RP02) segregating autosomal recessive RP were selected for DNA extraction. PCR was conducted, and the amplified PCR products were analyzed using Polyacrylamide Gel Electrophoresis (PAGE) andvisualized in the Gel Doc systemfor linkage analysis.The Gene Hunter 2.1r5 tool in the Simple Linkage v5.052 beta software suite was used to conduct multipoint parametric linkage analysis on the two consanguineous families examined on the 6K Illumina array. Exons and intron-exon borders of all known arRP genes found in homozygous areas were sequenced in the matching probands using a 3130 automated sequencer and the Big Dye Terminator Cycle Sequencing Kit v3.1. The mutation study was carried out using the AlaMut 1.5 program. In both families, linkage analysis was performed using microsatellite marker DIS422 for genecrumbs homolog 1 (CRB1)and microsatellite marker D8S2332 for geneRetinitis Pigmentosa 1 (RP1). Multipoint linkage analysis identifies genomic regions that could potentially contain the genetic defect. In family RP01, only a single peak with a maximal multipoint LOD score of 3.00 was identified on chromosome 1, whereas in family RP02, multiple peaks with multipoint LOD scores of 1.80 were identified on chromosome 8. Analysis of theCRB1gene revealed a homozygous substitution of glycine for valine (c.1152T>G; p.V243G), whereas theRP1gene demonstrated that leucine was substituted for proline as a result of cytosine to thymine transfer (c.3419C>T; p. P1035L). Conclusion: Homozygosity mapping is a powerful method for finding genetic abnormalities that are both precise and comprehensive for identifying harmful variations in consanguineous families. This method is invaluable for providing accurate clinical diagnostic and genetic advice in remote regions of Pakistanwhile also increasing knowledge about autosomal recessive diseases and the dangers of mixing.

Mutational Spectrum, Ocular and Olfactory Phenotypes of CNGB1-Related RP-Olfactory Dysfunction Syndrome in a Multiethnic Cohort.

CNGB1 gene mutations are a well-known cause of autosomal recessive retinitis pigmentosa (RP), which was recently associated with olfactory dysfunction. The purpose of this study was to report the molecular spectrum and the ocular and olfactory phenotypes of a multiethnic cohort with CNGB1-associated RP. A cross-sectional case series was conducted at two ophthalmic genetics referral centers. Consecutive patients with molecularly confirmed CNGB1-related RP were included. All patients underwent a complete ophthalmological examination complemented by psychophysical olfactory evaluation. Fifteen patients (10 families: 8 Portuguese, 1 French, and 1 Turkish), mean aged 57.13 ± 15.37 years old (yo), were enrolled. Seven disease-causing variants were identified, two of which are reported for the first time: c.2565_2566del and c.2285G > T. Although 11/15 patients reported onset of nyctalopia before age 10, diagnosis was only established after 30 yo in 9/15. Despite widespread retinal degeneration being present in 14/15 probands, a relatively preserved visual acuity was observed throughout follow-up. Olfactory function was preserved in only 4/15 patients, all of whom carried at least one missense variant. Our study supports previous reports of an autosomal recessive RP-olfactory dysfunction syndrome in association with certain disease-causing variants in the CNGB1 gene and expands the mutational spectrum of CNGB1-related disease by reporting two novel variants.

Novel compound heterozygous variants in the USH2A gene associated with autosomal recessive retinitis pigmentosa without hearing loss.

Background: Retinitis pigmentosa (RP) is a group of progressive inherited retinal dystrophies characterized by the primary degeneration of rod photoreceptors and the subsequent loss of cone photoreceptors because of cell death. It is caused by different mechanisms, including inflammation, apoptosis, necroptosis, pyroptosis, and autophagy. Variants in the usherin gene (USH2A) have been reported in autosomal recessive RP with or without hearing loss. In the present study, we aimed to identify causative variants in a Han-Chinese pedigree with autosomal recessive RP. Methods: A six-member, three-generation Han-Chinese family with autosomal recessive RP was recruited. A full clinical examination, whole exome sequencing, and Sanger sequencing, as well as co-segregation analysis were performed. Results: Three heterozygous variants in the USH2A gene, c.3304C>T (p.Q1102*), c.4745T>C (p.L1582P), and c.14740G>A (p.E4914K), were identified in the proband, which were inherited from parents and transmitted to the daughters. Bioinformatics analysis supported the pathogenicity of the c.3304C>T (p.Q1102*) and c.4745T>C (p.L1582P) variants. Conclusions: Novel compound heterozygous variants in the USH2A gene, c.3304C>T (p.Q1102*) and c.4745T>C (p.L1582P), were identified as the genetic causes of autosomal recessive RP. The findings may enhance the current knowledge of the pathogenesis of USH2A-associated phenotypes, expand the spectrum of the USH2A gene variants, and contribute to improved genetic counseling, prenatal diagnosis, and disease management.

In vivo base editing rescues photoreceptors in a mouse model of retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of retinal diseases that cause the progressive death of retinal photoreceptor cells and eventually blindness. Mutations in the β-domain of the phosphodiesterase 6 (Pde6b) gene are the most identified causes of autosomal recessive RP. Clinically, there is no effective treatment so far that can stop the progression of RP and restore the vision. Here, we report a base editing approach in which adeno-associated virus (AAV)-mediated adenine base editor (ABE) delivering to postmitotic photoreceptors was conducted to correct the Pde6b mutation in a retinal degeneration 10 (rd10) mouse model of RP. Subretinal delivery of AAV8-ABE corrected Pde6b mutation with averaging up to 20.79% efficiency at the DNA level and 54.97% efficiency at the cDNA level without bystanders, restored PDE6B expression, preserved photoreceptors, and rescued visual function. RNA-seq revealed the preservation of genes associated with phototransduction and photoreceptor survival. Our data have demonstrated that base editing is a potential gene therapy that could provide durable protection against RP.

A Novel Intronic Deletion in PDE6B Causes Autosomal Recessive Retinitis Pigmentosa by Interfering with RNA Splicing

Introduction: Retinitis pigmentosa (RP) is a rare degenerative retinal disease caused by mutations in approximately seventy genes. Currently, despite the availability of large-scale DNA sequencing technologies, ∼30–40% of patients still cannot be diagnosed at the molecular level. In this study, we investigated a novel intronic deletion of PDE6B, encoding the beta subunit of phosphodiesterase 6 in association with recessive RP. Methods: Three unrelated consanguineous families were recruited from the northwestern part of Pakistan. Whole exome sequencing was performed for the proband of each family, and the data were analyzed according to an in-house computer pipeline. Relevant DNA variants in all available members of these families were assessed through Sanger sequencing. A minigene-based splicing assay was also performed. Results: The clinical phenotype for all patients was compatible with rod cone degeneration, with the onset during childhood. Whole exome sequencing revealed a homozygous 18 bp intronic deletion (NM_000283.3:c.1921-20_1921-3del) in PDE6B, which co-segregated with disease in 10 affected individuals. In vitro splicing tests showed that this deletion causes aberrant RNA splicing of the gene, leading to the in-frame deletion of 6 codons and, likely, to disease. Conclusion: Our findings further expand the mutational spectrum of the PDE6B gene.

Prime Editing for the Installation and Correction of Mutations Causing Inherited Retinal Disease: A Brief Methodology.

Inherited retinal diseases (IRDs) encompass a large heterogeneous group of rare blinding disorders whose etiology originates from mutations in the 280 genes identified to date. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems represent a promising avenue for the treatment of IRDs, as exemplified by FDA clinical trial approval of EDIT-101 (AGN-151587), which removes a deep intronic variant in the CEP290 gene that causes Leber congenital amaurosis (LCA) type 10. Prime editing is a novel double-strand break (DSB) independent CRISPR/Cas system which has the potential to correct all 12 possible transition and transversion mutations in addition to small deletions and insertions. Here, as a proof-of-concept study, we describe a methodology using prime editing for the in vitro installation and correction of the classical Pde6brd10 c.1678C>T (p.Arg560Cys) mutation which causes autosomal recessive retinitis pigmentosa (RP) in mice.

Generation and characterization of human USH2A retinal disease model

AbstractPurpose: Mutations in USH2A are the most common cause of autosomal recessive retinitis pigmentosa (RP) and syndromic RP in combined deaf‐blindness, Usher syndrome type 2 (USH2). In both cases, the effective treatment is lacking. The failure of retinal phenotype in available animal models warrants the generation of human tissue mimics in which to explore the underlying molecular mechanism and test available therapeutic modalities. To model this disease in vitro, we generated iPSC‐derived retinal organoids from the USH2 patient and isogenic control. We studied cellular and morphological alterations in retinal organoids and the effectiveness of gene editing to rescue retinal cell phenotypes. We describe the precise gene editing approach to genetically correct the p.Cys759Phe mutation in USH2A and hence generate the isogenic iPSC line.Methods: The editing machinery was introduced by delivery of recombinant Cas9 and synthetic crRNA into the iPSCs derived from the patient bearing p.Cys759Phe mutation in USH2A. A single stranded oligonucleotide donor (ssODN) template carrying the wild‐type sequence was used to drive the mutation correction. The single cell clones were obtained and their edited genotype confirmed. Patient and isogenic control were subjected toward retinal organoid differentiation and analysed by fluorescence microscopy throughout the protocol, stage specific retinal cell markers were identified.Results: The CRISPR/Cas9 gene editing approach generated homo‐and heterozygously corrected clones with acceptable efficiency. Patient and isogenic control lines were induced toward retinal organoids with all mayor retinal cell types. We observe reduced efficiency in neural retina formation and paucity in cellular laminar arrangement of patients' cells. The isogenic control was able to revert disease related cellular phenotypes and subcellular features.Conclusions: Our study corroborates the feasibility of CRISPR/Cas9 editing of genetic lesion in monogenic disease bearing hiPSCs further supporting the applicability of CRISPR/Cas9 machinery in in vivo settings. We have identified retinal cell phenotype in USH2A p.Cys759Phe disease background which allows us to study disease mechanism and test novel treatment options.

Characterization of a novel Pde6b-deficient rat model of retinal degeneration and treatment with adeno-associated virus (AAV) gene therapy.

In humans, mutations in the beta subunit of cGMP-phosphodiesterase type 6 (PDE6B) cause autosomal recessive retinitis pigmentosa (RP), which typically has an aggressive clinical course of early-onset severe vision loss due to rapid photoreceptor degeneration. In this study, we describe the generation of a novel Pde6b-deficient rat model using CRISPR-Cas9 genome editing. We characterize the model at multiple time points using clinical imaging modalities as well as histology with immunohistochemistry to show rapid photoreceptor degeneration compared to wild-type and heterozygous animals. We describe the manufacture of two different adeno-associated viral (AAV) vectors (AAV2/1, AAV2/5) under current Good Manufacturing Practices (cGMP) and demonstrate their ability to drive human PDE6B expression in vivo. We further demonstrate the ability of AAV-mediated subretinal gene therapy to delay photoreceptor loss in Pde6b-deficient rats compared to untreated controls. However, severe progressive photoreceptor loss was noted even in treated eyes, likely due to the aggressive nature of the disease. These data provide useful preclinical data to guide the development of potential human gene therapy for PDE6B-associated RP. In addition, the rapid photoreceptor degeneration of the Pde6b-deficient rat with intact inner retina may provide a useful model for the study of cell replacement strategies.

Genotypes Influence Clinical Progression in EYS-Associated Retinitis Pigmentosa.

PurposeThe purpose of this study was to investigate the genetic and clinical characteristics of eyes shut homolog (EYS)-associated retinitis pigmentosa (RP).MethodsThis was a retrospective cross-sectional observational study of 36 patients with EYS-associated autosomal recessive RP (arRP).ResultsThe gene sequencing results revealed that c.6416G>A (p.Cys2139Tyr) and c.7228+1G>A were the two most predominant variants in our cohort and that variants near the C-terminus, which contains alternating laminin and epidermal growth factor (EGF) domains, accounted for the majority of the allele counts (58 of a total of 72) and relative allele frequencies (81%). Over half of the patients presented with pericentral-type RP (n = 19, 60%), which frequently occurred in combination with macular lesions (n = 10, 52%). Patients having both variants within the alternating laminin and EGF domains near the C-terminus had a more severe disease progression (average 0.045 logMAR increase per year) than those having one variant in the N-terminus and the other in the C-terminus (average 0.001 logMAR increase per year).ConclusionsPericentral RP was the major phenotype in patients with EYS-associated arRP. There was also a statistically significant relationship between the location of the variants and the severity of the disease.Translational RelevanceThis study may aid patients with EYS-associated arRP to predict future vision acuity based on their genetic and clinical features.

The Natural History of CNGB1-Related Retinopathy: A Longitudinal Phenotypic Analysis.

Cyclic nucleotide-gated channel β 1 (CNGB1) encodes a subunit of the rod cyclic nucleotide-gated channel. Pathogenic variants in CNGB1 are responsible for 4% of autosomal recessive retinitis pigmentosa (RP). Several treatment strategies show promise for treating inherited retinal degenerations, however relevant metrics of progression and sensitive clinical trial endpoints are needed to assess therapeutic efficacy. This study reports the natural history of CNGB1-related RP with a longitudinal phenotypic analysis of 33 molecularly-confirmed patients with a mean follow-up period of 4.5 ± 3.9 years (range 0–17). The mean best corrected visual acuity (BCVA) of the right eye was 0.31 ± 0.43 logMAR at baseline and 0.47 ± 0.63 logMAR at the final visit over the study period. The ellipsoid zone (EZ) length was measurable in at least one eye of 23 patients and had a mean rate of constriction of 178 ± 161 µm per year (range 1.0–661 µm), with 57% of patients having a decrease in EZ length of greater than 250 µm in a simulated two-year trial period. Hyperautofluorescent outer ring (hyperAF) area was measurable in 17 patients, with 10 patients not displaying a ring phenotype. The results support previous findings of CNGB1-related RP being a slowly progressive disease with patients maintaining visual acuity. Prospective deep phenotyping studies assessing multimodal retinal imaging and functional measures are now required to determine clinical endpoints to be used in a trial.

Ultra-widefield fundus autofluorescence imaging in patients with autosomal recessive retinitis pigmentosa reveals a genotype-phenotype correlation.

To analyze the genotype-phenotype correlation in patients with retinitis pigmentosa (RP) caused by mutations in the FAM161A, DHDDS, or MAK genes using ultra-widefield fundus autofluorescence (UWF-FAF) imaging. Retrospective case series of patients with autosomal recessive RP (ARRP) with confirmed causative genetic mutations and available UWF-FAF imaging data. The UWF-FAF data were graded in a blinded fashion using the following criteria: the pattern of macular abnormalities on FAF, the presence or absence of horizontal linear hyperautofluorescence, the extent of decreased autofluorescence (DAF), the shape of DAF, and the presence of hyperautofluorescence at the optic disk. A total of 43 patients (mean age of 47 ± 16years, ranging from 17 to 79years) with ARRP (86 eyes) were included in our analysis. Genotyping data revealed biallelic mutations in the FAM161A, DHDDS, and MAK genes in 20, 12, and 11 patients, respectively. We found significant differences between the three groups with respect to the pattern of macular abnormalities on FAF (p = 0.001), DAF configuration (p = 0.007), and extent of DAF (p = 0.037). The largest difference between groups was found for macular abnormalities on FAF, with DHDDS patients differing significantly from the MAK and FAM161A groups (p = 0.001). Specifically, DHDDS patients had a more abnormal macular FAF pattern and more widespread decrease in peripheral autofluorescence. No other parameters differed significantly between the three groups. Patients with ARRP can present with specific UWF-FAF patterns based on the underlying causative gene. Future studies are warranted in order to expand this analysis to include additional genes, mutations, and patients as well as assessment of disease progression by following patients over longer periods of time.

Ex vivo and In vivo Assays for Drug Discovery and Detection of Photoreceptor Survival Factors

Retinitis pigmentosa (RP) is one of the most common inherited diseases of the retina (retinopathies) that is estimated to affect 1 in 4,000 people in the United States and worldwide. This collection of genetic diseases leads to progressive photoreceptor cell degeneration and ultimately vision loss, with more than 60 genes implicated in molecular pathways towards photoreceptor cell death. Currently, there are no treatments available and, due to its vast diverse genetic background, a putative gene‐based therapy is challenging. Consequently, mutation‐independent strategies to protect photoreceptors against continued damage and progressive vision loss are appealing approaches. One of them is the use of survival, neurotrophic and neuroprotective factors that target common signaling pathways of cell death in the different types of RP. The aim of this study is to optimize methods to evaluate the efficacy of compounds to protect photoreceptor cells against death.We used PSVue® to optimize the cell death read‐out in cultured retinal explants and in retinas of a mouse model for a human inherited autosomal recessive RP, the rd10 mouse. Retinal degeneration in the rd10 mice is caused by a spontaneous mutation in the gene for the β‐subunit of phosphodiesterase. PSVue® is a robust probe for detecting phosphatidyl serine on the surface of cells undergoing death. We used retinal tissue explanted from wild type mouse eyes, and cultured it ex vivo. Induction of photoreceptor cell death was achieved with Zaprinast, an inhibitor of phosphodiesterase, to mimic the rd10 photoreceptor cell death. Low probe concentrations and short incubation times resulted in a reproducible detection of photoreceptor death in the retina by confocal super resolution microscopy. The PSVue® detection method yielded lower fluorescence background than the TUNEL detection method. Pigment epithelium‐derived factor (PEDF), a photoreceptor survival protein, was used as control to prevent photoreceptor cell death induced by Zaprinast. PEDF supplementation prior to Zaprinast treatment decreased cell death detection, as expected. We used the mouse model rd10 to evaluate photoreceptor death in vivo. PSVue® eye‐drops were administered to the left eye of rd10 mice of P17‐P25 postnatal ages, and fluorescence was detected by fundoscopy using MICRON III a day later. HBBS eye‐drops were administered to the right eye as a control reference. The fluorescence of the probe detected in the back of mouse eyes increased between ages P17 and P23 relative to that of HBBS‐treated eyes. The results revealed a peak of photoreceptor cell degeneration that occurred at P25 days of age in living rd10 mouse models.We have optimized methods to determine photoreceptor cell death in the ex vivo and in vivo experimental systems described here using a fluorescent probe. The methodologies are simple, fast, and complementary, and together they will prove to be suitable for screening factors and drugs against RP.

Evaluation of photoreceptor-directed fibroblasts derived from retinitis pigmentosa patients with defects in the EYS gene: a possible cost-effective cellular model for mechanism-oriented drug

BackgroundThe most common gene responsible for autosomal recessive retinitis pigmentosa (RP) is EYS. The manner of decay of genetically defective EYS gene transcripts varies depending on the type of mutation using our cellular model, which consists of induced photoreceptor-directed fibroblasts from EYS-RP patients (EYS-RP cells). However, disease-specific profiles have not been clarified in EYS-RP cells. Herein we investigated comprehensive gene expression patterns and restoration of altered expression by low molecular weight molecules in EYS-RP cells.MethodsUsing induced photoreceptor-like cells by CRX, RAX, NeuroD, and OTX2, we employed qRT-PCR and DNA microarray analysis to compare expression levels of disease-related genes in EYS-RP cells. We investigated the effect of antiapoptotic or anti-endoplasmic reticulum (ER) stress/antioxidant reagents on the restoration of altered gene expression.ResultsExpression levels of phototransduction-related genes (blue opsin, rhodopsin, S-antigen, GNAT1, GNAT2) were lower in EYS-RP cells. CRYGD was extracted by global gene expression analysis, as a downregulated, retina-related and apoptosis-, endoplasmic reticulum (ER) stress- or aging-related gene. Pathway enrichment analysis suggested that “complement and coagulation cascades,” “ECM-receptor interaction” and “PI3K-Akt signaling pathway” could be involved in EYS-RP-associated pathogenesis. Among the matching/overlapping genes involved in those pathways, F2R was suggested as an EYS-RP-associated gene. The downregulation of CRYGD and F2R was completely restored by additional 4-PBA, an inhibitor of ER stress, and partially restored by metformin or NAC. In addition, 4-PBA normalized the expression level of cleaved caspase-3.ConclusionsOur cellular model may reflect the ER stress-mediated degenerative retina and serve as a pathogenesis-oriented cost-effective rescue strategy for RP patients.

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.

Identification of a novel compound heterozygous CYP4V2 variant in a patient with autosomal recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) belongs to a family of retinal disorders that is characterized by the progressive degeneration of rod and cone photoreceptors. The aim of the present study was to screen for possible disease-causing genetic variants in a non-consanguineous Chinese family with non-syndromic autosomal recessive RP. Whole-exome sequencing (WES) was performed in samples from the affected individual (the proband) and those from the two children of the proband. A novel compound heterozygous variant of c.C958T (p.R320X) and c.G1355A (p.R452H) in the Cytochrome P450 family 4 subfamily V member 2 (CYP4V2) gene was identified through WES. Subsequently, this variant was validated by direct Sanger sequencing. This compound heterozygous variant was found to be absent from other unaffected family members and 400 ethnically-matched healthy control individuals. In addition, this compound variant was co-segregated with the RP phenotype in an autosomal recessive manner. In silico analysis revealed that both c.C958T (p.R320X) and c.G1355A (p.R452H) could compromise the protein function of CYP4V2. These results strongly suggest this compound variant to be a disease-causing variant, which expands upon the spectrum of currently known CYP4V2 genetic variants associated with retinal diseases.

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.

Clinical and Genetic Correlations of Inherital Retinal Disease with Mutations in the ABCA4 Gene by Patients of the Russian Population

Aim: to study genotype-phenotype correlations in patients with inherited retinal diseases with mutations in ABCA4 gene in Russian Federation.Patients and methods. 21 patients from Russian population aged from 7 to 51 years old (mean age 20 ± 11 years with best-corrected visual acuity from 0,02 to 0,6 (0,14 ± 0,11) with ABCA4-associated retinopathy, verified by molecular genetics methods. All patients besides standard ophthalmic examination and photodocumentation were performed Spectral-Domain OCT and fundus autofluorescence on Spectralis ®HRA+OCT (Heidelberg Engineering, Germany). Full-field electroretinogram (ERG), 30-Hz flicker ERG and macular chromatic ERG (MERG) to red stimulus were recorded on electroretinographic system MBN (MBN, Russia). (Russia) Molecular genetic studies were performed using Next Generation Sequencing (NGS) and Sandger direct sequencing. Results: In ABCA4-associated Stargardt disease 1 type (STGD1) genotype [p.L541P, p.A1038V] of «frequent» mutations was revealed in 9 patients, in 2 cases in was associated another “frequent” mutation p.G1961E. In 4 patients with genotype [p.L541P, p.A1038V] “severe” phenotype of Stargardt disease was found: with large defect of the ellipsoid zone and large zone of central reduced autofluorescence, severely subnormal macular ERG (MERG) to red stimulus and subnormal 30 Hz flicker and full-field maximal ERG. In one patient with these mutations in homozygous state ABCA4-associated cone-rod dystrophy (CORD3, clinically looking alike secondary retinal dystrophy is diagnosed. In 2 patients with genotype [p.L541P, p.A1038V] and mutation p.G1961E was found mild phenotype. One patient with homozygous mutation p.R653C autosomal recessive ABCA4-associated retinitis pigmentosa (RP19) was diagnosed. Clinical picture and autofluorescence were polymorphic in all patients.Conclusions. Our study with ophthalmological, molecular genetics and instrumental methods widens the spectrum of clinical signs of inherited eye diseases associated with mutations in АВСА4 gene, widens the spectrum mutations in Russian Federation and reveals clinicо-genetic genotype-phenotype correlations.