Objective Outcomes to Evaluate Voretigene Neparvovec Treatment Effects in Clinical Practice.

PURPOSE: To assess the efficacy of voretigene neparvovec (VN) treatment by objective fixation stability and chromatic pupillometry testing in clinical practice. DESIGN: Retrospective cohort study with longitudinal follow-up. SUBJECTS: Twelve patients (aged 7-34 years) with RPE65-related inherited retinal dystrophies were treated at the same center with VN in both eyes. METHODS: Patients treated at the same center with VN were evaluated over a 12-month posttreatment follow-up by subjective and objective tests. Furthermore, patients treated with VN who developed atrophy were compared with those who did not. MAIN OUTCOME MEASURES: Best-corrected visual acuity (BCVA), full-field stimulus threshold test (FST), semiautomated kinetic visual field (SKVF), microperimetry, and chromatic pupillometry over a 12-month follow-up. RESULTS: Significant improvements of BCVA (P < 0.001), SKVF (P < 0.05), and FST (P < 0.001) were already observed 45 days after treatment and were maintained at the 12-month timepoint. Fixation stability, assessed by microperimetry, improved significantly (P < 0.05) after treatment. Chromatic pupillometry showed significant improvements (P < 0.05) at the 6- and 12-month timepoints. The increase in maximum pupillary constriction significantly (P < 0.001) correlated with higher retinal sensitivity in FST. Four patients developed multifocal retinal atrophy in both eyes, detected at the 6-month timepoint, but this atrophy was not generally associated with worse visual function outcomes. CONCLUSIONS: This study explores objective outcomes in order to demonstrate the efficacy of VN treatment in addition to the tests normally performed in clinical practice. Our findings show a significant improvement of retinal function both in subjective assessments, such as BCVA, SKVF, and FST, and in objective measurements of fixation stability and maximum pupillary constriction. Moreover, the significant correlation between maximum pupillary constriction and light sensitivity thresholds corroborates the introduction of chromatic pupillometry as an objective test to better assess treatment outcomes in patients with inherited retinal dystrophies. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references in the Footnotes and Disclosures at the end of this article.

Generation of Zebrafish Models of Human Retinitis Pigmentosa Diseases Using CRISPR/Cas9-Mediated Gene Editing System.

Generating animal models can explore the role of new candidate genes in causing diseases and the pathogenicity of a specific mutation in the underlying genes. These animals can be used to identify new pharmaceutical or genetic therapeutic methods. In the present experiment, we developed a rpe65a knock out (KO) zebrafish as a retinitis pigmentosa (RP) disease model. Using the CRISPR/Cas9 system, the rpe65a gene was KO in zebrafish. Two specific single-guide RNAs (sgRNAs) were designed for the zebrafish rpe65a gene. SgRNAs were cloned into the DR274 plasmid and synthesized using in vitro transcription method. The efficiency of Ribonucleoprotein (synthesized sgRNA and recombinant Cas9) was evaluated by in vitro digestion experiment. Ribonucleoprotein complexes were microinjected into one to four-celled eggs of the TU zebrafish strain. The effectiveness of sgRNAs in KO the target gene was determined using the Heteroduplex mobility assay (HMA) and Sanger sequencing. Online software was used to determine the percent of mosaicism in the sequenced samples. By examining the sequences of the larvae that showed a mobility shift in the HMA method, the presence of indels in the binding region of sgRNAs was confirmed, so the zebrafish model for RP disease established. Zebrafish is an ideal animal model for the functional study of various diseases involving different genes and mutations and used for evaluating different therapeutic approaches in human diseases. This study presents the production of rpe65a gene KO zebrafish models using CRISPR/Cas9 technology. This model can be used in RP pathophysiology studies and preclinical gene therapy experiments.

Voretigene Neparvovec for the Treatment of RPE65-associated Retinal Dystrophy: Consensus and Recommendations from the Korea RPE65-IRD Consensus Paper Committee.

Mutations in the RPE65 gene, associated with Leber congenital amaurosis, early-onset severe retinal dystrophy, and retinitis pigmentosa, gained growing attention since gene therapy for patients with RPE65-associated retinal dystrophy is available in clinical practice. RPE65 gene accounts for a very small proportion of patients with inherited retinal degeneration, especially Asian patients. Because RPE65-associated retinal dystrophy shares common clinical characteristics, such as early-onset severe nyctalopia, nystagmus, low vision, and progressive visual field constriction, with retinitis pigmentosa by other genetic mutations, appropriate genetic testing is essential to make a correct diagnosis. Also, fundus abnormalities can be minimal in early childhood, and the phenotype is highly variable depending on the type of mutations in RPE65-associated retinal dystrophy, which makes a diagnostic difficulty. The aim of this paper is to review the epidemiology of RPE65-associated retinal dystrophy, mutation spectrum, genetic diagnosis, clinical characteristics, and voretigene neparvovec, a gene therapy product for the treatment of RPE65-related retinal dystrophy.

The Predicted Splicing Variant c.11+5G>A in RPE65 Leads to a Reduction in mRNA Expression in a Cell-Specific Manner.

Pathogenic variants in RPE65 lead to retinal diseases, causing a vision impairment. In this work, we investigated the pathomechanism behind the frequent RPE65 variant, c.11+5G>A. Previous in silico predictions classified this change as a splice variant. Our prediction using novel software's suggested a 124-nt exon elongation containing a premature stop codon. This elongation was validated using midigenes-based approaches. Similar results were observed in patient-derived induced pluripotent stem cells (iPSC) and photoreceptor precursor cells. However, the splicing defect in all cases was detected at low levels and thereby does not fully explain the recessive condition of the resulting disease. Long-read sequencing discarded other rearrangements or variants that could explain the diseases. Subsequently, a more relevant model was employed: iPSC-derived retinal pigment epithelium (RPE) cells. In patient-derived iPSC-RPE cells, the expression of RPE65 was strongly reduced even after inhibiting a nonsense-mediated decay, contradicting the predicted splicing defect. Additional experiments demonstrated a cell-specific gene expression reduction due to the presence of the c.11+5G>A variant. This decrease also leads to the lack of the RPE65 protein, and differences in size and pigmentation between the patient and control iPSC-RPE. Altogether, our data suggest that the c.11+5G>A variant causes a cell-specific defect in the expression of RPE65 rather than the anticipated splicing defect which was predicted in silico.

Frequency of RPE65 Gene Mutation in Patients with Hereditary Retinal Dystrophy.

Objectives: Hereditary retinal dystrophies are a rare group of diseases which are heterogeneous in genotype and phenotype and result in total blindness. One of the genetic defects that cause hereditary retinal dystrophy is mutation of the RPE65 gene. Genetic therapy studies in hereditary retinal dystrophies have increased in number recently, and important developments have been reported in these studies. Voretigene neparvovec-rzyl (Luxturna, Spark Therapeutics), a gene therapy drug for retinal dystrophy associated with RPE65 mutation, received Food and Drug Administration approval in 2017. This study aimed to investigate the frequency and clinical findings of patients with RPE65 gene defects, which may be amenable to genetic treatment. Materials and Methods: The data of patients diagnosed with hereditary retinal dystrophy who were followed up between 2017 and 2021 were retrospectively reviewed. Of these, 460 patients with genetic analysis results were included in the study. The clinical findings of patients with homozygous (biallelic) RPE65 mutation were screened. Results: RPE65 homozygous gene mutation was detected in only 11 of 460 cases (2.39%). Genetic results of the cases were presented in detail. The inheritance patterns of the cases were autosomal recessive. The demographic data and clinical findings were defined. Conclusion: RPE65 gene mutation is a very rare disorder. Genetic screening has gained importance with the emergence of gene therapy alternatives. New treatment methods are promising in cases for which there was no chance of a cure to date.

The first gene therapy for RPE65 biallelic dystrophy with voretigene neparvovec-rzyl in Brazil.

PURPOSE: To report the first Brazilian patient with RPE65 deficiency-inherited retinal dystrophy (RPE65-IRD) treated with voretigene neparvovec-rzyl (VN). METHODS: An adult patient with Leber congenital amaurosis-2 with a homozygous mutation in the RPE65 gene (p.Phe83Leu) was treated bilaterally with VN. The clinical and surgical aspects are described. The baseline and 4-month postoperative ophthalmologic examinations included measurement of the best-corrected visual acuity (BCVA), full-field stimulus threshold (FST) test, Octopus 900 semiautomated kinetic visual fields (VFs), and microperimetry. RESULTS: No complications developed in this patient. The BCVA remained stable. The full-field stimulus threshold test (FST) and VFs showed clinically significant improvements bilaterally. The patient reported significant improvements in the ability to perform daily activities, mainly for those requiring the VFs and vision in a low-luminescence environment. CONCLUSIONS: The treatments were beneficial for this patient who was homozygous for RPE65 p.Phe83Leu. The first VN treatments in an adult Brazilian patient in clinical practice showed measurable improvements in visual outcomes that were meaningful for the patient's daily activities. TRANSLATIONAL RELEVANCE: This case reinforces the clinical trial results and proves that the procedure is feasible in countries such as Brazil.

Epidemiology of Mutations in the 65-kDa Retinal Pigment Epithelium (RPE65) Gene-Mediated Inherited Retinal Dystrophies: A Systematic Literature Review.

INTRODUCTION: Inherited retinal dystrophies (IRDs) represent a genetically diverse group of progressive, visually debilitating diseases. Adult and paediatric patients with vision loss due to IRD caused by biallelic mutations in the 65-kDa retinal pigment epithelium (RPE65) gene are often clinically diagnosed as retinitis pigmentosa (RP), and Leber congenital amaurosis (LCA). This study aimed to understand the epidemiological landscape of RPE65 gene-mediated IRD through a systematic review of the literature, as the current evidence base for its epidemiology is very limited. METHODS: Medline, Embase, and other databases were searched for articles on the epidemiology of RPE65 gene-mediated IRDs from inception until June 2021. Studies were included if they were original research articles reporting the epidemiology of RP and LCA and/or proportion of RPE65 gene mutations in these clinically diagnosed or molecularly confirmed IRDs patients. RESULTS: A total of 100 studies with relevant data were included in this systematic review. The range for prevalence of LCA and RP in the literature was 1.20-2.37 and 11.09-26.43 per 100,000, respectively. The proportion of RPE65 mutations in clinically diagnosed patients with LCA was found to be between ~ 2-16% within the US and major European countries (France, Germany, Italy, Spain, and the UK). This range was also comparable to our findings in the Asian region for RPE65-LCA (1.26-16.67%). Similarly, for these European countries, RPE65-RP was estimated between 0.23 and 1.94%, and RPE65-IRD range was 1.2-14%. Further, in the Americas region, mutations in RPE65 were reported to cause 1-3% of RP and 0.8-3.7% of IRD cases. Lastly, the RPE65-IRD range was 4.81-8% in the Middle East region. CONCLUSIONS: There are significant variations in reporting of RPE65 proportions within countries as well as regions. Generating robust epidemiological evidence on RPE65 gene-mediated IRDs would be fundamental to support rare disease awareness, timely therapeutic intervention, and public health decision-making.

[Diagnostics of inherited retinal degenerations by gene therapy]. / Diagnostika nasledstvennykh distrofii setchatki s pozitsii gennoi terapii.

The article presents recent worldwide achievements in the area of diagnosis and treatment of inherited retinal degenerations (IRDs) from the standpoint of ophthalmic genetics. Clinical studies conducted in patients with Leber congenital amaurosis and retinitis pigmentosa caused by biallelic mutations in the RPE65 gene have provided the basis for future genes studies associated with IRDs. The conducted studies highlight the importance of fundamental understanding of function of the gene, timely diagnosis and study of natural history of the disease. Currently, surgical techniques are being improved for the efficient delivery of gene preparations to target cells, as well as the criteria for evaluating treatment outcomes.

Analysis of an NGS retinopathy panel detects chromosome 1 uniparental isodisomy in a patient with RPE65-related leber congenital amaurosis.

Purpose: This study aims to demonstrate the possibility of detecting segmental uniparental isodisomy (iUPD) using a next-generation sequencing gene panel by reporting a Leber congenital amaurosis (LCA) case caused by a homozygous pathogenic variant in RPE65 (c.1022 T > C:p.Leu341Ser) inherited exclusively from the proband's mother.Methods: Samples from the trio (proband, mother, and father) were sequenced with a next-generation sequencing (NGS) retinopathy gene panel (224 genes) and the VCF file containing all variants was used in order to determine single nucleotide variant (SNV) counts from each sample across all chromosomes.Results: Trio analysis showed that of 81 Chr1 inherited variants 41 were exclusively maternal, including 21 homozygous. The other 40 variants were common to both parents. On remaining autosomal chromosomes (Chr2-22) 645 inherited variants were found, 147 of them were exclusively maternal and 132 exclusively paternal. Based on these NGS data, it was possible to note that the proband's chromosomes 1 are more similar to his mother's chromosome 1 than his father's, suggesting the pathogenic homozygous variant found in this patient was inherited exclusively from the mother due to uniparental maternal isodisomy.Conclusions: This study presents a secondary analysis pipeline to identify responsible variants for a phenotype and the correct inheritance pattern, which is a critical step to the proper and accurate genetic counseling of all family members. In addition, this approach could be used to determine iUPD in different Mendelian disorders if the sequencing panel identifies variants spread throughout the genome.

Frequency and phenotypic characteristics of RPE65 mutations in the Chinese population.

BACKGROUND: The retinoid isomerohydrolase RPE65 has received considerable attention worldwide since a successful clinical gene therapy was approved in 2017 as the first treatment for vision loss associated with RPE65-mediated inherited retinal disease. Identifying patients with RPE65 mutations is a prerequisite to assessing the patients' eligibility to receive RPE65-targeted gene therapies, and it is necessary to identify individuals who are most likely to benefit from gene therapies. This study aimed to investigate the RPE65 mutations frequency in the Chinese population and to determine the genetic and clinical characteristics of these patients. RESULTS: Only 20 patients with RPE65 mutations were identified, and RPE65 mutations were determined to be the 14th most common among all patients with genetic diagnoses. Ten novel variants and two hotspots associated with FAP were identified. A literature review revealed that a total of 57 patients of Chinese origin were identified with pathogenic mutations in the RPE65 gene. The mean best Snellen corrected visual acuity was worse (mean 1.3 ± 1.3 LogMAR) in patients older than 20 years old than in those younger than 15 years old (0.68 ± 0.92 LogMAR). Bone spicule-like pigment deposits (BSLPs) were observed in six patients; they were older than those without BSLP and those with white-yellow dots. Genotype-phenotype analysis revealed that truncating variants seem to lead to a more severe clinical presentation, while best corrected visual acuity testing and fundus changes did not correlate with specific RPE65 variants or mutation types. CONCLUSIONS: This study provides a detailed clinical-genetic assessment of patients with RPE65 mutations of Chinese origin. These results may help to elucidate RPE65 mutations in the Chinese population and may facilitate genetic counseling and the implementation of gene therapy in China.

[Implementation of a new gene therapy in ophthalmology: Regulatory and organizational issues]. / Mise en place d'une nouvelle thérapie génique en ophtalmologie : aspects réglementaires et organisationnels.

Voretigene neparvovec (VN) is the first gene therapy in ophthalmology for patients with RPE65-mediated hereditary retinal dystrophy. It has recently obtained European market approval, which is subject to strict regulatory and organizational conditions for its use. Here, we analyze the main studies supporting the authorization of this new therapy and describe the necessary steps to take at a hospital level for optimal administration to patients following current regulations.

Compensatory Cross-Modal Plasticity Persists After Sight Restoration.

Sensory deprivation prompts extensive structural and functional reorganizations of the cortex resulting in the occupation of space for the lost sense by the intact sensory systems. This process, known as cross-modal plasticity, has been widely studied in individuals with vision or hearing loss. However, little is known on the neuroplastic changes in restoring the deprived sense. Some reports consider the cross-modal functionality maladaptive to the return of the original sense, and others view this as a critical process in maintaining the neurons of the deprived sense active and operational. These controversial views have been challenged in both auditory and vision restoration reports for decades. Recently with the approval of Luxturna as the first retinal gene therapy (GT) drug to reverse blindness, there is a renewed interest for the crucial role of cross-modal plasticity on sight restoration. Employing a battery of task and resting state functional magnetic resonance imaging (rsfMRI), in comparison to a group of sighted controls, we tracked the functional changes in response to auditory and visual stimuli and at rest, in a group of patients with biallelic mutations in the RPE65 gene ("RPE65 patients") before and 3 years after GT. While the sighted controls did not present any evidence for auditory cross-modal plasticity, robust responses to the auditory stimuli were found in occipital cortex of the RPE65 patients overlapping visual responses and significantly elevated 3 years after GT. The rsfMRI results showed significant connectivity between the auditory and visual areas for both groups albeit attenuated in patients at baseline but enhanced 3 years after GT. Taken together, these findings demonstrate that (1) RPE65 patients present with an auditory cross-modal component; (2) visual and non-visual responses of the visual cortex are considerably enhanced after vision restoration; and (3) auditory cross-modal functions did not adversely affect the success of vision restitution. We hypothesize that following GT, to meet the demand for the newly established retinal signals, remaining or dormant visual neurons are revived or unmasked for greater participation. These neurons or a subset of these neurons respond to both the visual and non-visual demands and further strengthen connectivity between the auditory and visual cortices.

Pathogenicity Reclasssification of RPE65 Missense Variants Related to Leber Congenital Amaurosis and Early-Onset Retinal Dystrophy.

A challenge in molecular diagnosis and genetic counseling is the interpretation of variants of uncertain significance. Proper pathogenicity classification of new variants is important for the conclusion of molecular diagnosis and the medical management of patient treatments. The purpose of this study was to reclassify two RPE65 missense variants, c.247T>C (p.Phe83Leu) and c.560G>A (p.Gly187Glu), found in Brazilian families. To achieve this aim, we reviewed the sequencing data of a 224-gene retinopathy panel from 556 patients (513 families) with inherited retinal dystrophies. Five patients with p.Phe83Leu and seven with p.Gly187Glu were selected and their families investigated. To comprehend the pathogenicity of these variants, we evaluated them based on the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) classification guidelines. Initially, these RPE65 variants met only three pathogenic criteria: (i) absence or low frequency in the population, (ii) several missense pathogenic RPE65 variants, and (iii) 15 out of 16 lines of computational evidence supporting them as damaging, which together allowed the variants to be classified as uncertain significance. Two other pieces of evidence were accepted after further analysis of these Brazilian families: (i) p.Phe83Leu and p.Gly187Glu segregate with childhood retinal dystrophy within families, and (ii) their prevalence in Leber congenital amaurosis (LCA)/early-onset retinal dystrophy (EORD) patients can be considered higher than in other inherited retinal dystrophy patients. Therefore, these variants can now be classified as likely pathogenic according to ACMG/AMP classification guidelines.

Generation and Characterization of Induced Pluripotent Stem Cells and Retinal Organoids From a Leber's Congenital Amaurosis Patient With Novel RPE65 Mutations.

RPE65-associated Leber congenital amaurosis (LCA) is one of highly heterogeneous, early onset, severe retinal dystrophies with at least 130 gene mutation sites identified. Their pathogenicity has not been directly clarified due to lack of diseased cells. Here, we generated human-induced pluripotent stem cells (hiPSCs) from one putative LCA patient carrying two novel RPE65 mutations with c.200T>G (p.L67R) and c.430T>C (p.Y144H), named RPE65-hiPSCs, which were confirmed to contain the same mutations. The RPE65-hiPSCs presented typical morphological features with normal karyotype, expressed pluripotency markers, and developed teratoma in NOD-SCID mice. Moreover, the patient hiPSCs were able to differentiate toward retinal lineage fate and self-form retinal organoids with layered neural retina. All major retinal cell types including photoreceptor and retinal pigment epithelium (RPE) cells were also acquired overtime. Compared to healthy control, RPE cells from patient iPSCs had lower expression of RPE65, but similar phagocytic activity and VEGF secretion level. This study provided the valuable patient specific, disease targeted retinal organoids containing photoreceptor and RPE cells, which would facilitate the study of personalized pathogenic mechanisms of disease, drug screening, and cell replacement therapy.