We present the results of the first study of a large cohort of patients with inherited retinal dystrophies (IRD) performed for the Polish population using whole-exome sequencing (WES) in the years 2016-2019. Moreover, to facilitate such diagnostic analyses and enable future application of gene therapy and genome editing for IRD patients, a Polish genomic reference database (POLGENOM) was created based on whole-genome sequences of healthy Polish Caucasian nonagenarians and centenarians. The newly constructed database served as a control, providing a comparison for variant frequencies in the Polish population. The diagnostic yield for the selected group of IRD patients reached 64.9%. The study uncovered the most common pathogenic variants in ABCA4 and USH2A in the European population, along with several novel causative variants. A significant frequency of the ABCA4 complex haplotype p.(Leu541Pro; Ala1038Val) was observed, as well as that of the p.Gly1961Glu variant. The first VCAN causative variant NM_004385.5:c.4004-2A>G in Poland was found and described. Moreover, one of the first patients with the RPE65 causative variants was identified, and, in consequence, could receive the dedicated gene therapy. The availability of the reference POLGENOM database enabled comprehensive variant characterisation during the NGS data analysis, confirming the utility of a population-specific genomic database for enhancing diagnostic accuracy. Study findings suggest the significance of genetic testing in elder patients with unclear aetiology of eye diseases. The combined approach of NGS and the reference genomic database can improve the diagnosis, management, and future treatment of IRDs.

Gene-based therapies for inherited retinal dystrophies (IRDs) are upcoming. Treatment before substantial vision loss will optimize outcomes. It is crucial to identify common phenotypes and causative genes in children. This study investigated the frequency of these in pediatric IRD with the aim of highlighting relevant groups for future therapy.
Diagnostic, genetic, and demographic data, collected from medical charts of patients with IRD aged up to 20 years (n = 624, 63% male), registered in the Dutch RD5000 database, were analyzed to determine frequencies of phenotypes and genetic causes. Phenotypes were categorized as nonsyndromic (progressive and stationary IRD) and syndromic IRD. Genetic causes, mostly determined by whole-exome sequencing (WES), were examined. Additionally, we investigated the utility of periodic reanalysis of WES data in genetically unresolved cases.
Median age at registration was 13 years (interquartile range, 9-16). Retinitis pigmentosa (RP; n = 123, 20%), Leber congenital amaurosis (LCA; n = 97, 16%), X-linked retinoschisis (n = 64, 10%), and achromatopsia (n = 63, 10%) were the most frequent phenotypes. The genetic cause was identified in 76% of the genetically examined patients (n = 473). The most frequently disease-causing genes were RS1 (n = 32, 9%), CEP290 (n = 28, 8%), CNGB3 (n = 21, 6%), and CRB1 (n = 17, 5%). Diagnostic yield after reanalysis of genetic data increased by 7%.
As in most countries, RP and LCA are the most prominent pediatric IRDs in the Netherlands, and variants in RS1 and CEP290 were the most prominent IRD genotypes. Our findings can guide therapy development to target the diseases and genes with the greatest needs in young patients.

Conventional gene therapy involving supplementation only treats loss-of-function diseases and is limited by viral packaging sizes, precluding therapy of large genes. The discovery of CRISPR/Cas has led to a paradigm shift in the field of genetic therapy, with the promise of precise gene editing, thus broadening the range of diseases that can be treated. The initial uses of CRISPR/Cas have focused mainly on gene editing or silencing of abnormal variants via utilising Cas endonuclease to trigger the target cell endogenous non-homologous end joining. Subsequently, the technology has evolved to modify the Cas enzyme and even its guide RNA, leading to more efficient editing tools in the form of base and prime editing. Further advancements of this CRISPR/Cas technology itself have expanded its functional repertoire from targeted editing to programmable transactivation, shifting the therapeutic focus to precise endogenous gene activation or upregulation with the potential for epigenetic modifications. In vivo experiments using this platform have demonstrated the potential of CRISPR-activators (CRISPRa) to treat various loss-of-function diseases, as well as in regenerative medicine, highlighting their versatility to overcome limitations associated with conventional strategies. This review summarises the molecular mechanisms of CRISPRa platforms, the current applications of this technology in vivo, and discusses potential solutions to translational hurdles for this therapy, with a focus on ophthalmic diseases.

Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) is an age-related macular degeneration-like (AMD-like) retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production in retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus that enabled simple, sensitive, and high-throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix, reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium-derived factor). In vivo, treatment of 8-month-old R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is an important demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of retinal degenerative diseases, including potentially AMD itself.

UNASSIGNED: Biallelic pathogenic variants in the gene encoding the ATP-binding cassette transporter ABCA4 are the leading cause of irreversible vision loss in inherited retinal dystrophies (IRDs). Interpretation of ABCA4 variants is challenging, due to cis-modifying and hypomorphic variants. We have previously detected 10 missense variants of unknown significance (VUS) in patients with suspected ABCA4-retinal dystrophies (ABCA4-RDs) in Norway. In this study, we functionally characterized the VUS to aid interpretation of the variants and to determine if they are associated with the disease.
UNASSIGNED: The ABCA4 VUS were expressed in HEK293T cells and the ABCA4 expression level and ATPase activity were determined and correlated with the patients\' phenotype. The functional data further used for reclassification of the VUS following the American College of Medical Genetics and Genomics (ACMG) guidelines.
UNASSIGNED: Of the 10 VUSs, 2 variants, Cys205Phe and Asn415Thr, were categorized as functionally severe. The age at presentation in the 2 patients carrying these variants was divergent and seemed to be driven by the patients\' second pathogenic variants Gly1961Glu and c.5461-10T>C, respectively. Three variants, Val643Gly, Pro799Leu, and Val1433Ile were categorized as functionally moderate, and were found in patients with intermediate/late age at presentation. The remaining five variants were categorized as functionally normal/mild. Based on our data, c.614G>T p.(Cys205Phe), c.1244A>C p.(Asn415Thr), and c.2396C>T p.(Pro799Leu) were reclassified to (likely) pathogenic, while 4 of the functionally normal/mild variants could be reclassified to likely benign.
UNASSIGNED: Functional analyses of ABCA4 variants are a helpful tool in variant classification and enable us to better predict the disease severity in patients with ABCA4-RDs.

Accurate interpretation of sequence variants in inherited retinal dystrophy (IRD) is vital given the significant genetic heterogeneity observed in this disorder. To achieve consistent and accurate diagnoses, establishment of standardized guidelines for variant interpretation is essential. The American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for variant interpretation serve as the global \"cross-disease\" standard for classifying variants in Mendelian hereditary disorders. These guidelines propose a systematic approach for categorizing variants into 5 classes based on various types of evidence, such as population data, computational data, functional data, and segregation data. However, for clinical genetic diagnosis and to ensure standardized diagnosis and treatment criteria, additional specifications based on features associated with each disorder are necessary. In this context, we present a comprehensive framework outlining the newly specified ACMG/AMP rules tailored explicitly to IRD in the Japanese population on behalf of the Research Group on Rare and Intractable Diseases (Ministry of Health, Labour and Welfare of Japan). These guidelines consider disease frequencies, allele frequencies, and both the phenotypic and the genotypic characteristics unique to IRD in the Japanese population. Adjustments and modifications have been incorporated to reflect the specific requirements of the population. By incorporating these IRD-specific factors and refining the existing ACMG/AMP guidelines, we aim to enhance the accuracy and consistency of variant interpretation in IRD cases, particularly in the Japanese population. These guidelines serve as a valuable resource for ophthalmologists and clinical geneticists involved in the diagnosis and treatment of IRD, providing them with a standardized framework to assess and classify genetic variants.

Inherited retinal diseases (IRDs) are extremely heterogeneous with at least 350 causative genes, complicating the process of genetic diagnosis. We analyzed samples of 252 index cases with IRDs using the Blueprint Genetics panel for \"Retinal Dystrophy\" that includes 351 genes. The cause of disease could be identified in 55% of cases. A clear difference was obtained between newly recruited cases (74% solved) and cases that were previously analyzed by panels or whole exome sequencing (26% solved). As for the mode of inheritance, 75% of solved cases were autosomal recessive (AR), 10% were X-linked, 8% were autosomal dominant, and 7% were mitochondrial. Interestingly, in 12% of solved cases, structural variants (SVs) were identified as the cause of disease. The most commonly identified genes were ABCA4, EYS and USH2A, and the most common mutations were MAK-c.1297_1298ins353 and FAM161A-c.1355_1356del. In line with our previous IRD carrier analysis, we identified heterozygous AR mutations that were not the cause of disease in 36% of cases. The studied IRD panel was found to be efficient in gene identification. Some variants were misinterpreted by the pipeline, and therefore, multiple analysis tools are recommended to obtain a more accurate annotation of potential disease-causing variants.

OBJECTIVE: To report a case of macular hole and detachment occurring after the subretinal injection of Voretigene Neparvovec (VN) in a patient affected by atypical RPE65 retinal dystrophy with high myopia and its successful surgical management.
METHODS: We report a case of a 70-year-old man treated with VN in both eyes. The best corrected visual acuity (BCVA) was 0.7 LogMar in the right eye (RE) and 0.92 LogMar in the left eye (LE). Axial length was 29.60 mm in the RE and 30.28 mm in the LE. Both eyes were pseudophakic. In both eyes, fundus examination revealed high myopia, posterior staphyloma, and extended retinal atrophy areas at the posterior pole, circumscribing a central island of surviving retina. Both eyes were treated with VN subretinal injection, but a full-thickness macular hole and retinal detachment occurred in the LE three weeks after surgery. The patient underwent 23-gauge vitrectomy with internal limiting membrane (ILM) peeling and the inverted flap technique with sulfur hexafluoride (SF6) 20% tamponade. Postoperative follow-up showed that the macular hole was closed and the BCVA was maintained.
CONCLUSIONS: Our experience suggests that patients with atypical RPE65 retinal dystrophy and high myopia undergoing VN subretinal injection require careful management to minimize the risk of macular hole and detachment occurrence and promptly detect and address these potential complications.

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There is growing evidence that the pathogenesis of retinal diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD) have a significant chronic inflammatory component. A vital part of the inflammatory cascade is through the activation of pattern recognition receptors (PRR) such as toll-like receptors (TLR). Here, we reviewed the past and current literature to ascertain the cumulative knowledge regarding the effect of TLRs on the development and progression of retinal diseases. There is burgeoning research demonstrating the relationship between TLRs and risk of developing retinal diseases, utilising a range of relevant disease models and a few large clinical investigations. The literature confirms that TLRs are involved in the development and progression of retinal diseases such as DR, AMD, and ischaemic retinopathy. Genetic polymorphisms in TLRs appear to contribute to the risk of developing AMD and DR. However, there are some inconsistencies in the published reports which require further elucidation. The evidence regarding TLR associations in retinal dystrophies including retinitis pigmentosa is limited. Based on the current evidence relating to the role of TLRs, combining anti-VEGF therapies with TLR inhibition may provide a longer-lasting treatment in some retinal vascular diseases.