Objective: To explore the effect of patient's age, baseline visual acuity, and intraoperative foveal detachment on outcomes of subretinal voretigene neparvovec-rzyl (VN, Luxturna®) therapy and to assess patients' perceptions of the treatment effect.

Design: Multicenter, retrospective, consecutive case series and cross-sectional prospective survey.

Subjects: All 41 consecutive patients treated with VN after FDA approval at three institutions between January 2018 and May 2020.

Methods: Retrospective chart review of operative reports, clinical notes, ancillary testing and complications, comparing data at baseline and 1, 2-3, 6-9, and 10-15 months after subretinal surgery. A survey was administered to adult patients and parents of pediatric patients.

Main outcome measures: Changes in BCVA and retinal morphology, and patients' perceptions.

Results: 77 eyes of 41 patients (16 adults and 25 children, range 2-44 years, mean follow-up 10 months, range 1 week to 18.5 months) were analyzed. There was no statistically significant vision change for the adults, whereas there was a trend of improvement for children that reached statistical significance for some time points but not all. At the last follow-up, 14/48 (29%) pediatric and 3/26 (12%) adult eyes improved ≥2 lines (p=0.15). Baseline VA did not have an effect on post-therapy VA (p=0.23). Central foveal thickness decreased mildly in both children and adults, without significant difference between the populations. The fovea was detached by VN in 62 eyes (81%). Inner segment-outer segment junction remained unchanged in 91% of 54 eyes with gradable OCTs, with or without foveal detachment. Thirty-two patients (78%) were reached for the survey an average of 1.15±0.50 years (range 0.31 to 2.31) after surgery in the first eye. Improvement in night, day, and/or color vision was reported by 23 (72%), 22 (69%), and 18 (56%) patients respectively.

Conclusions: This study is limited by large variability in follow-up time. There were no persistent statistical significant vision changes. A decrease in foveal thickness was noted in most eyes. The long-term significance of this remains to be determined.

Keywords: FST; Leber congenital amaurosis; Luxturna; OCT; RPE65; full-field light sensitivity threshold testing; gene therapy; optical coherence tomography; subretinal injection; visual acuity; voretigene neparvovec-rzyl.

Purpose: This study aimed to systematically review and summarize gene therapy treatment for monogenic retinal and optic nerve diseases.

Methods: This review was prospectively registered (CRD42021229812). A comprehensive literature search was performed in Ovid MEDLINE, Ovid Embase, Cochrane Central, and clinical trial registries (February 2021). Clinical studies describing DNA-based gene therapy treatments for monogenic posterior ocular diseases were eligible for inclusion. Risk of bias evaluation was performed. Data synthesis was undertaken applying Synthesis Without Meta-analysis guidelines.

Results: This study identified 47 full-text publications, 50 conference abstracts, and 54 clinical trial registry entries describing DNA-based ocular gene therapy treatments for 16 different genetic variants. Study summaries and visual representations of safety and efficacy outcomes are presented for 20 unique full-text publications in RPE65-mediated retinal dystrophies, choroideremia, Leber hereditary optic neuropathy, rod-cone dystrophy, achromatopsia, and X-linked retinoschisis. The most common adverse events were related to lid/ocular surface/cornea abnormalities in subretinal gene therapy trials and anterior uveitis in intravitreal gene therapy trials.

Conclusion: There is a high degree of variability in ocular monogenic gene therapy trials with respect to study design, statistical methodology, and reporting of safety and efficacy outcomes. This review improves the accessibility and transparency in interpreting gene therapy trials to date.

Keywords: Gene therapy; Inherited retinal disease; Luxturna; Retinal dystrophy; Systematic review.

Inherited retinal dystrophies (IRD) have been studied since their recognition by Franz Donders and Albrecht von Graefe. It nevertheless took 100 years for a causal therapy to take shape in the form of gene therapy: The approval of Voretigen Neparvovec (VN) for the treatment of hereditary retinal dystrophies due to RPE65 mutations was thus a significant milestone - for the era of personalised medicine in general and ophthalmology in particular. The clinical management around gene therapy applications is complex and requires the cooperation of various experts as a multidisciplinary team. This article describes the requirements, challenges, approaches, and open questions regarding the surgical aspects of gene therapy for retinal dystrophies. The first part outlines the standard surgical treatment. Based on this standard, alternative approaches are indicated for each individual step and their value discussed. Knowledge gaps are defined and in the outlook we speculate on future developments.

Erbliche Netzhautdystrophien (engl. inherited retinal dystrophies, IRD) wurden bereits von Franz Donders und Albrecht von Graefe diagnostiziert und waren seitdem Gegenstand intensiver wissenschaftlicher Auseinandersetzung. Dennoch hat es 100 Jahre gedauert, bis mit der Gentherapie eine kausale Therapie heranreifte: Die Zulassung von Voretigen Neparvovec (VN) zur Behandlung erblicher Netzhautdystrophien aufgrund von RPE65-Mutationen war somit ein bedeutsamer Meilenstein – für die Ära der personalisierten Medizin insgesamt und die Augenheilkunde im Besonderen. Die Indikationsstellung, Behandlung und Nachsorge rund um gentherapeutische Anwendungen sind komplex und erfordern eine Zusammenarbeit verschiedener Experten. In diesem Artikel werden die Anforderungen, Herausforderungen, Lösungsansätze und offene Fragen zu den chirurgischen Aspekten der Gentherapie von Netzhautdystrophien beschrieben. Im 1. Teil wird der Standard zur chirurgischen Behandlung skizziert. Ausgehend von diesem Standard werden für die einzelnen Schritte Alternativen aufgezeigt und deren Stellenwert diskutiert. Es werden offene Fragen definiert, deren Lösung zu einer optimierten Anwendung führen sollen; ein Ausblick auf zukünftige Entwicklungen bildet den Schlusspunkt.

Within the last decade, continuous advances in molecular biological techniques have made it possible to develop causative therapies for inherited retinal disorders (IRDs). Some of the most promising options are gene-specific approaches using adeno-associated virus-based vectors to express a healthy copy of the disease-causing gene in affected cells of a patient. This concept of gene supplementation therapy is already advocated for the treatment of retinal dystrophy in RPE65-linked Leber's congenital amaurosis (LCA) patients. While the concept of gene supplementation therapy can be applied to treat autosomal recessive and X-linked forms of IRD, it is not sufficient for autosomal dominant IRDs, where the pathogenic gene product needs to be removed. Therefore, for autosomal dominant IRDs, alternative approaches that utilize CRISPR/Cas9 or antisense oligonucleotides to edit or deplete the mutant allele or gene product are needed. In recent years, research retinal gene therapy has intensified and promising approaches for various forms of IRD are currently in preclinical and clinical development. This review article provides an overview of current clinical trials for the treatment of IRDs.

Stetige Fortschritte in molekularbiologischen und genetischen Techniken ermöglichten es innerhalb der letzten Jahre, die Entwicklung ursächlicher Therapien für erbliche Netzhauterkrankungen (IRD) voranzubringen. Zu den vielversprechendsten Ansätzen gehört die Gensupplementierungstherapie, bei der mittels Adeno-assoziierten Viren (AAV) eine gesunde Kopie des krankheitsverursachenden Gens in die betroffenen Zellen eines Patienten eingeschleust wird. Dieses Therapiekonzept ist bereits bei RPE65-assoziierten Netzhautdystrophien, wie beispielsweise einer Form der Leberʼschen kongenitalen Amaurose (LCA2) als Therapie zugelassen. Während das Konzept der Gensupplementierungstherapie zur Behandlung autosomal-rezessiver und X-chromosomaler Formen von IRD angewendet werden kann, muss bei autosomal-dominanten IRDs, zusätzlich zur Gensupplementation, das pathogene Genprodukt entfernt werden. Daher sind für autosomal-dominante IRDs alternative Ansätze erforderlich, die CRISPR/Cas9- oder Antisense-Oligonukleotide verwenden, um das mutierte Allel oder Genprodukt gezielt zu eliminieren. In den letzten Jahren wurden die Forschungsaktivitäten auf dem Gebiet der retinalen Gentherapie intensiviert und etliche, vielversprechende Ansätze für verschiedene Formen der IRD befinden sich derzeit in der präklinischen und klinischen Entwicklung. Mit diesem Übersichtsartikel möchten wir einen Überblick über aktuelle Studien zur Behandlung von IRDs bieten und einen Ausblick auf zukünftige Entwicklungen geben.

Ageing presents adverse effects on the retina and is the primary risk factor for age-related macular degeneration (AMD). We report the first RNA-seq analysis of age-related transcriptional changes in the human retinal pigment epithelium (RPE), the primary site of AMD pathogenesis. Whole transcriptome sequencing of RPE from human donors ranging in age from 31 to 93 reveals that ageing is associated with increasing transcription of main RPE-associated visual cycle genes (including LRAT, RPE65, RDH5, RDH10, RDH11; pathway enrichment BH-adjusted P = 4.6 × 10^-6 ). This positive correlation is replicated in an independent set of 28 donors and a microarray dataset of 50 donors previously published. LRAT expression is positively regulated by retinoid by-products of the visual cycle (A2E and all-trans-retinal) involving modulation by retinoic acid receptor alpha transcription factor. The results substantiate a novel age-related positive feedback mechanism between accumulation of retinoid by-products in the RPE and the up-regulation of visual cycle genes.

Keywords: A2E; LRAT; RDH10; RDH11; RDH5; RNA-seq; RPE65; ageing; macular degeneration; retinal pigment epithelium; retinol metabolism; visual cycle.

Purpose: Previously, we reported that the intravenous injection of bone marrow-derived cells (BMDC) infected with lentivirus expressing the human RPE65 gene resulted in the programming of BMDC to promote visual recovery in a mouse model of age-related macular degeneration (AMD). The aim of this study was to characterize the spatial and temporal recruitment of these programmed BMDC to the retinal pigment epithelial (RPE) layer.

Methods: C57BL/6J female mice received a subretinal injection of AAV1-SOD2 ribozyme to knock down (KD) superoxide dismutase 2 (SOD2) and induce AMD-like pathology. BMDC were isolated from GFP⁺ mice and infected with a lentivirus expressing RPE65. One month after SOD2 KD, fifty thousand GFP⁺ RPE65-BMDC were injected in the mouse tail vein. Animals were terminated at different time points up to 60 min following cell administration, and localization of GFP⁺ cells was determined by fluorescence microscopy of neural retina and RPE flat mounts and tissue sections.

Results: GFP⁺ RPE65- BMDC were observed in SOD2 KD neural retina and RPE as early as 1 min following administration. With increasing time, the number of cells in the neural retina decreased, while those in the RPE increased. While the number of cells in peripheral and central retina remained similar at each time point, the number of BMDC recruited to the central RPE increased in a time-dependent manner up to a maximum by 60 min post administration. Immunohistochemistry of cross-sections of the RPE layer confirmed the incorporation of donor GFP⁺ BMDC into the RPE layer and that these GFP⁺ human RPE65 expressing cells co-localized with murine RPE65. No GFP⁺ cells were observed in the neural retina or RPE layer of normal uninjured control eyes.

Conclusions: Our study shows that systemically administered GFP⁺ RPE65-BMDC can reach the retina within minutes and that the majority of these BMDC are recruited to the injured RPE layer by 60 min post injection.

Keywords: Age-related macular degeneration; Bone marrow-derived cells; Cell recruitment; RPE65; Retinal pigment epithelium.

Inherited retinal diseases (IRDs) are a heterogeneous group of conditions that include retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) and early-onset severe retinal dystrophy (EO[S]RD), which differ in severity and age of onset. IRDs are caused by mutations in >250 genes. Variants in the RPE65 gene account for 0.6-6% of RP and 3-16% of LCA/EORD cases. Voretigene neparvovec is a gene therapy approved for the treatment of patients with an autosomal recessive retinal dystrophy due to confirmed biallelic RPE65 variants (RPE65-IRDs). Therefore, the accurate molecular diagnosis of RPE65-IRDs is crucial to identify 'actionable' genotypes-i.e., genotypes that may benefit from the treatment-and is an integral part of patient management. To date, hundreds of RPE65 variants have been identified, some of which are classified as pathogenic or likely pathogenic, while the significance of others is yet to be established. In this review, we provide an overview of the genetic diagnostic workup needed to select patients that could be eligible for voretigene neparvovec treatment. Careful clinical characterization of patients by multidisciplinary teams of experts, combined with the availability of next-generation sequencing approaches, can accelerate patients' access to available therapeutic options.

Keywords: RPE65; genetic counseling; genetic testing; inherited retinal diseases; next-generation sequencing; variants of uncertain significance.

Background and objectives: Retinal stem cells (RSCs) resided in ciliary epithelium have shown to possess a high capacity to self-renew and differentiate into retinal cells. RSCs could be induced to differentiate when they are exposed to stimuli like natural compounds and suitable contexts such as biomaterials. The aim of this study was to examine the effects of Retinol and alginate/gelatin-based scaffolds on differentiation potential of mesenchymal stem cells (MSCs) originated from mouse ciliary epithelium.

Methods and results: MSCs were extracted from mouse ciliary epithelium, and their identity was verified by detecting specific surface antigens. To provide a three-dimensional in vitro culture system, 2% alginate, 0.5% gelatin and the mixed alginate-gelatin hydrogels were fabricated and checked by SEM. Retinol treatment was performed on MSCs expanded on alginate/gelatin hydrogels and the survival rate and the ability of MSCs to differentiate were examined through measuring expression alterations of retina-specific genes by ICC and qPCR. The cell population isolated from ciliary epithelium contained more than 93.4% cells positive for MSC-specific marker CD105. Alginate/gelatin scaffolds showed to provide an acceptable viability (over 70%) for MSC cultures. Retinol treatment could induce a high expression of rhodopsin protein in MSCs expanded in alginate and alginate-gelatin mixtures. An elevated presentation of Nestin, RPE65 and Rhodopsin genes was detected in retinol-treated cultures expanded on alginate and alginate-gelatin scaffolds.

Conclusions: The results presented here elucidate that retinol treatment of MSCs grown on alginate scaffolds would promote the mouse ciliary epithelium-derived MSCs to differentiate towards retinal neurons.

Keywords: Alginate; Cell differentiation; Gelatin; Mesenchymal stem cell; Retinal neurons; Scaffolds.

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.

В статье представлены современные мировые достижения в области диагностики и лечения наследственных дистрофий сетчатки (НДС) с позиции офтальмогенетики. Клинические исследования, проведенные у пациентов с врожденным амаврозом Лебера и пигментным ретинитом, вызванными биаллельными мутациями в гене RPE65, обеспечили основу для будущих исследований других генов и ассоциированных с ними НДС. Проведенные исследования обозначили важность фундаментального понимания функции гена, своевременной диагностики и изучения естественной истории течения болезни. В настоящее время совершенствуются хирургические техники для эффективной доставки генных препаратов к клеткам-мишеням и критерии оценки полученных результатов лечения.

Keywords: Inherited retinal degenerations; Leber congenital amaurosis; RPE65 gene; retinitis pigmentosa.

Aim: To explore the temporal mitochondrial characteristics of retinal pigment epithelium (RPE) cells obtained from human embryonic stem cells (hESC)-derived retinal organoids (hEROs-RPE), to verify the optimal period for using hEROs-RPE as donor cells from the aspect of mitochondria and to optimize RPE cell-based therapeutic strategies for age-related macular degeneration (AMD).

Methods: RPE cells were obtained from hEROs and from spontaneous differentiation (SD-RPE). The mitochondrial characteristics were analyzed every 20d from day 60 to 160. Mitochondrial quantity was measured by MitoTracker Green staining. Transmission electron microscopy (TEM) was adopted to assess the morphological features of the mitochondria, including their distribution, length, and cristae. Mitochondrial membrane potentials (MMPs) were determined by JC-1 staining and evaluated by flow cytometry, reactive oxygen species (ROS) levels were evaluated by flow cytometry, and adenosine triphosphate (ATP) levels were measured by a luminometer. Differences between two groups were analyzed by the independent-samples t-test, and comparisons among multiple groups were made using one-way ANOVA or Kruskal-Wallis H test when equal variance was not assumed.

Results: hEROs-RPE and SD-RPE cells from day 60 to 160 were successfully differentiated from hESCs and expressed RPE markers (Pax6, MITF, Bestrophin-1, RPE65, Cralbp). RPE features, including a cobblestone-like morphology with tight junctions (ZO-1), pigments and microvilli, were also observed in both hEROs-RPE and SD-RPE cells. The mitochondrial quantities of hEROs-RPE and SD-RPE cells both peaked at day 80. However, the cristae of hEROs-RPE mitochondria were less mature and abundant than those of SD-RPE mitochondria at day 80, with hEROs-RPE mitochondria becoming mature at day 100. Both hEROs-RPE and SD-RPE cells showed low ROS levels from day 100 to 140 and maintained a normal MMP during this period. However, hEROs-RPE mitochondria maintained a longer time to produce high levels of ATP (from day 120 to 140) than SD-RPE cells (only day 120).

Conclusion: hEROs-RPE mitochondria develop more slowly and maintain a longer time to supply high-level energy than SD-RPE mitochondria. From the mitochondrial perspective, hEROs-RPE cells from day 100 to 140 are an optimal cell source for treating AMD.

Keywords: human embryonic stem cells, retinal degenerative diseases; mitochondria; retinal organoids; retinal pigment epithelium cells.

Inherited retinal degenerative diseases (IRDs) are rare neurodegenerative disorders with mutations in hundreds of genes leading to vision loss, primarily owing to photoreceptor cell death. This genetic diversity is impeding development of effective treatment options. Gene-based therapies have resulted in the first FDA-approved drug (Luxturna) for RPE65-specific IRD. Although currently explored in clinical trials, genomic medicines are mutation-dependent, hence suitable only for patients harboring a specific mutation. Better understanding of the pathways leading to photoreceptor degeneration may help to determine common targets and develop mutation-independent therapies for larger groups of patients with IRDs. In this review, we discuss the key pathways involved in photoreceptor cell death studied by transcriptomics, proteomics, and metabolomics techniques to identify potential therapeutic targets in IRDs.

RPE65-associated inherited retinal dystrophy (RPE65-IRD) is an early-onset retinal degeneration. The aim of this study was to describe the clinical features and natural course of this disease in a Chinese patient cohort with RPE65 biallelic variants. Thirty patients from 29 unrelated families with biallelic disease-causing RPE65 variants underwent full ophthalmic examinations. Thirteen were followed up over time. An additional 57 Chinese cases from 49 families were retrieved from the literature to analyze the relationship between best-corrected visual acuity (BCVA) and age. Our 30 patients presented age-dependent phenotypic characteristics. Multiple white dots were a clinical feature of young patients, while maculopathy, epiretinal membrane, and bone spicules were common in adult patients. Among the 84 patients, BCVA declined with age in a nonlinear, positive-acceleration relationship (p < 0.001). All patients older than 40 years met the WHO standard for low vision. Longitudinal observation revealed a slower visual acuity loss in patients younger than 20 years than those in their third or fourth decade of life. Our study detailed the clinical features and natural course of disease in Chinese patients with RPE65-IRD. Our results indicated that these patients have a relatively stable BCVA in childhood and adolescence, but eyesight deteriorates rapidly in the third decade of life. These findings may facilitate the implementation of gene therapy in China.

Keywords: RPE65 variants; RPE65-associated inherited retinal dystrophy; best-corrected visual acuity; clinical features; genotype.

The objective of this report was to perform a systematic review of the beneficial and harmful effects of voretigene neparvovec administered as a single dose of 1.5 × 10¹¹ vector genomes in each eye via subretinal injection for the treatment of adult and pediatric patients with vision loss due to inherited retinal dystrophy caused by confirmed biallelic RPE65 mutations and who have sufficient viable retinal cells.

Non-exudative age-related macular degeneration (NE-AMD), the main cause of blindness in people above 50 years old, lacks effective treatments at the moment. We have developed a new NE-AMD model through unilateral superior cervical ganglionectomy (SCGx), which elicits the disease main features in C57Bl/6J mice. The involvement of oxidative stress in the damage induced by NE-AMD to the retinal pigment epithelium (RPE) and outer retina has been strongly supported by evidence. We analysed the effect of enriched environment (EE) and visual stimulation (VS) in the RPE/outer retina damage within experimental NE-AMD. Exposure to EE starting 48 h post-SCGx, which had no effect on the choriocapillaris ubiquitous thickness increase, protected visual functions, prevented the thickness increase of the Bruch's membrane, and the loss of the melanin of the RPE, number of melanosomes, and retinoid isomerohydrolase (RPE65) immunoreactivity, as well as the ultrastructural damage of the RPE and photoreceptors, exclusively circumscribed to the central temporal (but not nasal) region, induced by experimental NE-AMD. EE also prevented the increase in outer retina/RPE oxidative stress markers and decrease in mitochondrial mass at 6 weeks post-SCGx. Moreover, EE increased RPE and retinal brain-derived neurotrophic factor (BDNF) levels, particularly in Müller cells. When EE exposure was delayed (dEE), starting at 4 weeks post-SCGx, it restored visual functions, reversed the RPE melanin content and RPE65-immunoreactivity decrease. Exposing animals to VS protected visual functions and prevented the decrease in RPE melanin content and RPE65 immunoreactivity. These findings suggest that EE housing and VS could become an NE-AMD promising therapeutic strategy.

Introduction: Biallelic pathogenic RPE65 variants are related to a spectrum of clinically overlapping inherited retinal dystrophies (IRD). Most affected individuals progress to severe disease, with 50% of patients becoming legally blind by 20 years of age. Deeper knowledge of the mutational spectrum and the phenotype-genotype correlation in RPE65-related IRD is needed.

Patients and methods: Forty-five affected subjects from 27 unrelated families with a clinical diagnosis of RPE65-related IRD were included. Clinical evaluation consisted of self-reported ophthalmological history and objective ophthalmological examination. Patients' genotype was classified according to variant class (truncating or missense) or to variant location at different protein domains. The main phenotypic outcome measure was age at onset (AAO) of symptomatic disease and a Kaplan-Meier analysis of disease symptom event-free survival was performed.

Results: Twenty-nine different RPE65 variants were identified in our cohort, 7 of them novel. Patients carrying two missense alleles showed a later disease onset than those with 1 or 2 truncating variants (log-rank test p < 0.05). While 60% of patients carrying a missense/missense genotype presented symptoms before or during the first year of life, almost all patients with at least 1 truncating allele (91%) had an AAO ≤1 year (p < 0.05).

Conclusion: Our findings suggest an association between the type of RPE65 variant carried and AAO. These findings provide useful data on RPE65-associated IRD phenotypes and may help improve clinical and therapeutic management of these patients.

Keywords: Early-onset retinitis pigmentosa; Genotype-phenotype correlation; Inherited retinal dystrophy; Leber congenital amaurosis; Pathogenic variant; RPE65.

Inherited retinal diseases (IRDs) are chronic, hereditary disorders that lead to progressive degeneration of the retina. Disease etiology originates from a genetic mutation-inherited or de novo-with a majority of IRDs resulting from point mutations. Given the plethora of IRDs, to date, mutations that cause these dystrophies have been found in approximately 280 genes. However, there is currently only one FDA-approved gene augmentation therapy, Luxturna (voretigene neparvovec-rzyl), available to patients with RPE65-mediated retinitis pigmentosa (RP). Although clinical trials for other genes are underway, these techniques typically involve gene augmentation rather than genome surgery. While gene augmentation therapy delivers a healthy copy of DNA to the cells of the retina, genome surgery uses clustered regularly interspaced short palindromic repeats (CRISPR)-based technology to correct a specific genetic mutation within the endogenous genome sequence. A new technique known as prime editing (PE) applies a CRISPR-based technology that possesses the potential to correct all twelve possible transition and transversion mutations as well as small insertions and deletions. EDIT-101, a CRISPR-based therapy that is currently in clinical trials, uses double-strand breaks and nonhomologous end joining to remove the IVS26 mutation in the CEP290 gene. Preferably, PE does not cause double-strand breaks nor does it require any donor DNA repair template, highlighting its unparalleled efficiency. Instead, PE uses reverse transcriptase and Cas9 nickase to repair mutations in the genome. While this technique is still developing, with several challenges yet to be addressed, it offers promising implications for the future of IRD treatment.

Keywords: CRISPR/Cas9 systems; Ophthalmology; adeno-associated viral (AAV) vectors; gene editing; inherited retinal diseases (IRD); prime editing; retinal degeneration.

Purpose: This study evaluates whether topical ketotifen fumarate (KTF) can prevent geographic atrophy (GA)-like phenotypes in a rat model.

Methods: Pharmacokinetics (PKs) of KTF after topical administration twice daily for 5 days was analyzed in rat retina, retinal pigment epithelium (RPE)/choroid/sclera, and in plasma by an liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Rats were then given hydrogel implants +/- 48/80 in the superior subconjunctival space and topically treated with 1% and 0.25% of KTF or phosphate buffer saline (PBS) twice daily. Rats were euthanized at 1, 2, 4, and 8 weeks postinjection. Choroidal mast cells (MCs) were stained with nonspecific esterase and the RPE monolayer was labeled with RPE65 and ZO-1 in whole mount choroids. Retinal and choroidal areas were determined in cryosections stained with picrosirius red. Dark-adapted electroretinogram (ERG) was also performed to evaluate retinal function.

Results: PK results showed the highest level of KTF (average 5.6 nM/mg) in the RPE/choroid/sclera in rats given topical 1% KTF. Topical 1% KTF significantly reduced choroidal MC degranulation at 1 week and 2 weeks (both P < 0.001) and RPE loss at 4 weeks (P < 0.001) as well as retinal and choroidal thinning (both P < 0.001) and reduction in ERG amplitude at 8 weeks (P < 0.05) compared to PBS. Similar results were obtained with 0.25% KTF.

Conclusions: Both 1% and 0.25% KTF eye drops effectively reduced MC degranulation, RPE loss, and retinal and choroidal thinning while preventing the decline of ERG amplitude in a GA-like rat model. These data suggest that topical KTF might be a new therapeutic drug for treating GA.

Translational relevance: The results of this study demonstrate that topical KTF successfully reduced GA-like phenotypes in a rat model and may provide a novel therapy for GA.

Purpose: Melanotic cells with large spherical melanosomes, thought to originate from retinal pigment epithelium (RPE), are found in eyes with neovascular age-related macular degeneration (nvAMD). To generate hypotheses about RPE participation in fibrosis, we correlate histology to clinical imaging in an eye with prominent black pigment in fibrotic scar secondary to nvAMD.

Methods: Macular findings in a white woman with untreated inactive subretinal fibrosis due to nvAMD in her right eye were documented over 9 years with color fundus photography (CFP), fundus autofluorescence (FAF) imaging, and optical coherence tomography (OCT). After death (age 90 years), this index eye was prepared for light and electron microscopy to analyze 7 discrete zones of pigmentation in the fibrotic scar. In additional donor eyes with nvAMD, we determined the frequency of black pigment (n = 36 eyes) and immuno-labeled for retinoid, immunologic, and microglial markers (RPE65, CD68, Iba1, TMEM119; n = 3 eyes).

Results: During follow-up of the index eye, black pigment appeared and expanded within a hypoautofluorescent fibrotic scar. The blackest areas correlated to melanotic cells (containing large spherical melanosomes), some in multiple layers. Pale areas had sparse pigmented cells. Gray areas correlated to cells with RPE organelles entombed in the scar and multinucleate cells containing sparse large spherical melanosomes. In 94% of nvAMD donor eyes, hyperpigmentation was visible. Certain melanotic cells expressed some RPE65 and mostly CD68. Iba1 and TMEM119 immunoreactivity, found both in retina and scar, did not co-localize with melanotic cells.

Conclusion: Hyperpigmentation in CFP results from both organelle content and optical superimposition effects. Black fundus pigment in nvAMD is common and corresponds to cells containing numerous large spherical melanosomes and superimposition of cells containing sparse large melanosomes, respectively. Melanotic cells are molecularly distinct from RPE, consistent with a process of transdifferentiation. The subcellular source of spherical melanosomes remains to be determined. Detailed histology of nvAMD eyes will inform future studies using technologies for spatially resolved molecular discovery to generate new therapies for fibrosis. The potential of black pigment as a biomarker for fibrosis can be investigated in clinical multimodal imaging datasets.

Keywords: Age-related macular degeneration; Color fundus photography; Fibrosis; Histopathology; Melanosomes; Optical coherence tomography; Retinal pigment epithelium.

Purpose: To investigate real-world safety and efficacy of voretigene neparvovec gene therapy administration in pediatric patients with biallelic RPE65 disease-causing variants.

Methods: A retrospective study of 27 eyes of 14 patients with RPE65-associated Leber congenital amaurosis examined postoperative complications and longitudinal changes in photoreceptor function following treatment with subretinal injection of voretigene neparvovec. Full-field stimulus threshold testing (FST), Goldmann visual fields (GVF), best-corrected visual acuity (BCVA), and central subfield thickness (CST) on optical coherence tomography (OCT) scans were collected preoperatively and up to 12 months posttreatment.

Results: Baseline through 6-12 month follow-up FST and GVF data were obtained for 13 eyes of 7 patients. FST improved for each eye after treatment with a mean improvement of 2.1 log-units (P < 0.001) and GVF improved for each eye with a mean improvement of 221 sum degrees (P < 0.001). BCVA improved from logMAR 0.98 at baseline to logMAR 0.83 at last follow-up (P < 0.001). Across 19 eyes of 10 patients included in CST analysis, there was a small but statistically significant 9-μ decrease in mean CST from baseline to last follow-up (P < 0.001). The most common postoperative issues included elevation in intraocular pressure (59%), persistent intraocular inflammation (15%), and vitreous opacities (26%) that resolved over a period of months.

Conclusions: This report provides some of the earliest longitudinal real-world evidence of the pediatric safety and efficacy of voretigene neparvovec using multiple functional and structural measures of the retina. Outcomes demonstrate significant improvements in visual function consistent with clinical trial results.

Keywords: FST; GVF; Gene therapy; Retinal degeneration; Retinal dystrophy.

Our ability to see flicker has an upper frequency limit above which flicker is invisible, known as the "critical flicker frequency" (CFF), that typically grows with light intensity (I). The relation between CFF and I, the focus of nearly 200 years of research, is roughly logarithmic, i.e., CFF ∝ log(I)-a relation called the Ferry-Porter law. However, why this law should occur, and how it relates to the underlying physiology, have never been adequately explained. Over the past two decades we have measured CFF in normal observers and in patients with retinal gene defects. Here, we reanalyse and model our data and historical CFF data. Remarkably, CFF-versus-I functions measured under a wide range of conditions in patients and in normal observers all have broadly similar shapes when plotted in double-logarithmic coordinates, i.e., log(CFF)-versus-log(I). Thus, the entire dataset can be characterised by horizontal and vertical logarithmic shifts of a fixed-shape template. Shape invariance can be predicted by a simple model of visual processing built from a sequence of low-pass filters, subtractive feedforward stages and gain adjustment (Rider, Henning & Stockman, 2019). It depends primarily on the numbers of visual processing stages that approach their power-law region at a given intensity and a frequency-independent gain reduction at higher light levels. Counter-intuitively, the CFF-versus-I relation depends primarily on the gain of the visual response rather than its speed-a conclusion that changes our understanding and interpretation of human flicker perception. The Ferry-Porter "law" is merely an approximation of the shape-invariant template.

Keywords: CFF; Clinical vision; Critical flicker fusion; Flicker acuity; GNAT2; GRM6; GUCA1A; GUCY2D; Gene defects; KCNV2; LRAT; Light adaptation; Linear systems; Molecular loss; NR2E3; OPA1; OPN1LW; OPN1MW; RGS9; RPE65; Temporal processing; Visual psychophysics.

Pharmacotherapies targeting vascular endothelial growth factor (VEGF) have revolutionized the management for neovascular retinal disorders including diabetic retinopathy and neovascular age-related macular degeneration. However, the burden of frequent injections, high cost, and treatment resistance in some patients remain unresolved. To overcome these challenges, newer generations of anti-angiogenic biological therapies, engineered proteins, implantable delivery systems, and biopolymers are currently being developed to enable more sustained, longer-lasting treatments. The use of gene therapies for pathologic angiogenesis has garnered renewed interests since the first FDA-approval of a gene therapy to treat inherited retinal diseases associated with biallelic RPE65 mutations. Newer generations of viral vectors and novel methods of intraocular injections helped overcome ocular barriers, improving the efficiency of transduction as well as safety profile. In addition, unlike current anti-VEGF gene therapy strategies which employ a biofactory approach to mimic existing pharmacotherapies, novel genome editing strategies that target pro-angiogenic factors at the DNA level offer a unique and distinct mechanistic approach that can potentially be more precise and lead to a permanent cure. Here, we review current anti-VEGF therapies and newer pharmacologic agents under development, examine technologies and progress in adapting anti-VEGF gene therapies, and explore the future application of CRISPR-Cas9 technology to suppress ocular angiogenesis.

Keywords: Angiogenesis; gene therapy; neovascular age-related macular degeneration (nAMD); neovascularization; vascular endothelial growth factor (VEGF).

Inherited retinal diseases (IRDs) are a genetically variable collection of devastating disorders that lead to significant visual impairment. Advances in genetic characterization over the past two decades have allowed identification of over 260 causative mutations associated with inherited retinal disorders. Thought to be incurable, gene supplementation therapy offers great promise in treating various forms of these blinding conditions. In gene replacement therapy, a disease-causing gene is replaced with a functional copy of the gene. These therapies are designed to slow disease progression and hopefully restore visual function. Gene therapies are typically delivered to target retinal cells by subretinal (SR) or intravitreal (IVT) injection. The historic Food and Drug Administration (FDA) approval of voretigene neparvovec for RPE65-associated Leber's congenital amaurosis (LCA) spurred tremendous optimism surrounding retinal gene therapy for various other monogenic IRDs. Novel disease-causing mutations continue to be discovered annually, and targeted genetic therapy is now under development in clinical and preclinical models for many IRDs. Numerous clinical trials for other IRDs are ongoing or have recently completed. Disorders being targeted for genetic therapy include retinitis pigmentosa (RP), choroideremia (CHM), achromatopsia (ACHM), Leber's hereditary optic neuropathy, usher syndrome (USH), X-linked retinoschisis, and Stargardt disease. Here, we provide an update of completed, ongoing, and planned clinical trials using gene supplementation strategies for retinal degenerative disorders.

Keywords: Inherited retinal disease (IRD); clinical trial; gene therapy.

Purpose: To present visual outcomes of the first two Italian patients with RPE65-related inherited retinal dystrophy (RPE65-IRD) treated with voretigene neparvovec (VN).

Methods: Two pediatric patients with RPE65-IRD were treated with VN in both eyes. Patients were evaluated by best-corrected visual acuity (BCVA), full-field stimulus threshold (FST) test, semiautomated kinetic visual field (SKVF), microperimetry, and chromatic pupillometry over 6 months.

Results: No complications occurred in the first patient, whereas in the second a subretinal hemorrhage was observed in the first treated eye, and excessive resistance to drug injection occurred during treatment of the second eye. BCVA improved by at least one Early Treatment Diabetic Retinopathy Study line in all treated eyes. The FST test and SKVF showed clinically significant improvements in all eyes (i.e., change of light sensitivity > 10 decibels; area enlargement of at least 20%). Moreover, microperimetry showed better fixation stability. Finally, chromatic pupillometry showed increases in pupillary constriction that ranged from 10% to 20%. All visual changes remained stable during follow-up.

Conclusions: The first VN treatments in two pediatric Italian patients in clinical practice showed significant improvements in visual outcomes, even in the case of surgical complications, which spontaneously recovered without sequelae.

Translational relevance: These findings with VN in patients with RPE65-IRD confirm the results of clinical trials.

Highly expressed in the retinal pigment epithelium (RPE), the RPE-specific 65-kDa (RPE65) enzyme is indispensable to generate 11-cis-retinal (11cRAL), a chromophore for rhodopsin and cone photopigments. RPE65 deficiency can lead to Leber congenital amaurosis type 2 (LCA2), in which the isomerization of photobleached all-trans-retinal into photosensitive 11cRAL is blocked, ultimately causing severe retinal dysfunction and degeneration. The related mouse models, which are constructed through gene knockout or caused by spontaneous mutations, morphologically present with early-onset and rapid retinal cone cells degeneration, including loss of short-wavelength-sensitive cone opsins (S-opsins) and mislocalization of medium-wavelength-sensitive cone opsins (M-opsins). Studies have shown that routine Rpe65 gene replacement therapy, mediated by an adeno-associated virus (AAV) vector, can restore RPE65 protein. However, AAV transfection and Rpe65 transgene expression require at least one to two weeks, and the treatment cannot fully block the early-onset cone degeneration. To determine the feasibility of delaying cone degeneration before gene therapy, we investigated the impact of 11cRAL treatment in an early-age LCA2 retinal degeneration 12 (rd12) mouse model. Similar to human patients, the mouse model carries a spontaneous mutation in the Rpe65 gene, which results in disrupted endogenous 11cRAL regeneration. We found that RPE65 deficiency did not notably affect rodent retinal vessels. Under red light illumination, the rd12 mice were intraperitoneally injected with exogenous 11cRAL from postnatal day (P) 14 to P21. Three days after the last injection, a notable recovery of retinal function was observed using scotopic and photopic electroretinograms. Using optical coherence tomography and histological analyses of the deficient retinas, we found changes in the thickness of the photoreceptor outer segment (OS); this change could be rescued by early 11cRAL treatment. In addition, the treatment notably preserved M- and S-opsins, both of which maintained appropriate localization inside cone cells, as shown by the wild-type mice. In contrast, the age-matched untreated rd12 mice were characterized by retinal S-opsin loss and M-opsin mislocalization from the photoreceptor OS to the inner segment, outer nuclear layer, or outer plexiform layer. Notably, 11cRAL treatment could not maintain retinal function for a long time. Ten days after the last injection, the rod and M-cone electroretinograms significantly decreased, and S-cone responses almost extinguished. Our findings suggest that early 11cRAL treatment is useful for restoring retinal function and rescuing morphology in the rd12 mouse model, and the early-onset and rapid cone degeneration can be delayed before gene therapy.

Keywords: Chromophore; Cone visual pigments; Leber congenital amaurosis; Mice; Retina.