Characterization of a ß-carotene isomerase from the cyanobacterium Cyanobacteria aponinum.

Carotenoids are essential components of the photosynthetic apparatus and precursors of plant hormones, such as strigolactones (SLs). SLs are involved in various aspects of plant development and stress-response processes, including the establishment of root and shoot architecture. SL biosynthesis begins with the reversible isomerization of all-trans-carotene into 9-cis-ß-carotene, catalysed by DWARF27 ß-carotene isomerase (D27). Sequence comparisons have revealed the presence of D27-related proteins in photosynthetic eukaryotes and cyanobacteria lacking SLs. To gain insight into the evolution of SL biosynthesis, we characterized the activity of a cyanobacterial D27 protein (CaD27) from Cyanobacterim aponinum, using carotenoid-accumulating Escherichia coli cells and in vitro enzymatic assays. Our results demonstrate that CaD27 is an all-trans/cis and cis/cis-ß-carotene isomerase, with a cis/cis conversion preference. CaD27 catalysed 13-cis/15-cis-, all-trans/9-cis-ß-carotene, and neurosporene isomerization. Compared with plant enzymes, it exhibited a lower 9-cis-/all-trans-ß-carotene conversion ratio. A comprehensive genome survey revealed the presence of D27 as a single-copy gene in the genomes of 20 out of 200 cyanobacteria species analysed. Phylogenetic and enzymatic analysis of CaD27 indicated that cyanobacterial D27 genes form a single orthologous group, which is considered an ancestral type of those found in photosynthetic eukaryotes. This article is part of the theme issue 'The evolution of plant meta|bolism'.

A novel mutation in non-constitutive lycopene beta cyclase (CstLcyB2a) from Crocus sativus modulates carotenoid/apocarotenoid content, biomass and stress tolerance in plants.

MAIN CONCLUSION: Mutation at A126 in lycopene-ß-cyclase of Crocus (CstLcyB2a) sterically hinders its binding of δ-carotene without affecting lycopene binding, thereby diverting metabolic flux towards ß-carotene and apocarotenoid biosynthesis. Crocus sativus, commonly known as saffron, has emerged as an important crop for research because of its ability to synthesize unique apocarotenoids such as crocin, picrocrocin and safranal. Metabolic engineering of the carotenoid pathway can prove a beneficial strategy for enhancing the quality of saffron and making it resilient to changing climatic conditions. Here, we demonstrate that introducing a novel mutation at A126 in stigma-specific lycopene-ß-cyclase of Crocus (CstLcyB2a) sterically hinders its binding of δ-carotene, but does not affect lycopene binding, thereby diverting metabolic flux towards ß-carotene formation. Thus, A126L-CstLcyB2a expression in lycopene-accumulating bacterial strains resulted in enhanced production of ß-carotene. Transient expression of A126L-CstLcyB2a in C. sativus stigmas enhanced biosynthesis of crocin. Its stable expression in Nicotiana tabacum enhanced ß-branch carotenoids and phyto-hormones such as abscisic acid (ABA) and gibberellic acids (GA's). N. tabacum transgenic lines showed better growth performance and photosynthetic parameters including maximum quantum efficiency (Fv/Fm) and light-saturated capacity of linear electron transport. Exogenous application of hormones and their inhibitors demonstrated that a higher ratio of GA4/ABA has positive effects on biomass of wild-type and transgenic plants. Thus, these findings provide a platform for the development of new-generation crops with improved productivity, quality and stress tolerance.

Partial rescue of the full-field electroretinogram in patients with RPE65-related retinal dystrophy following gene augmentation therapy with voretigene neparvovec-rzyl.

PURPOSE: To present a series of patients with RPE65-related retinal dystrophy showing a partial rescue of the full-field electroretinogram (ERG) following gene replacement therapy with voretigene neparovec-rzyl (Luxturna®). METHODS: This retrospective chart review examined 17 patients treated with voretigene neparovec-rzyl (VN) at the Casey Eye Institute (2018-2022). The last pre-operative ERG and all available post-operative ERGs were analyzed to identify subjects with functional rescue. Measurements of amplitudes and implicit times were compared to data from age-matched controls and the attenuation relative to the lower limit of normal (LLN) was calculated. For comparison with other functional exams, the last pre-operative and all post-treatment best-corrected visual acuity (BCVA) data, visual field (VF) tests and full-field threshold stimulus tests (FST) were also described. RESULTS: Of patients who underwent ERGs, most had unrecordable ERGs that did not change after treatment. However, we identified three patients, treated bilaterally, who demonstrated partial rescue of the full-field ERG in both eyes which was sustained during the course of the study. CONCLUSIONS: This is the largest series of patients treated with VN showing a partial rescue of the ERG. This is also the first report of bilateral ERG rescue, as well as the first description of ERG recovery occurring in non-pediatric subjects. Full-field ERG could be used in combination with other psychophysical tests and imaging modalities to detect and deepen our understanding of the response to this gene therapy approach.

Whole-Exome Analysis for Polish Caucasian Patients with Retinal Dystrophies and the Creation of a Reference Genomic Database for the Polish Population.

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.

Synchronized Photoactivation of T4K Rhodopsin Causes a Chromophore-Dependent Retinal Degeneration That Is Moderated by Interaction with Phototransduction Cascade Components.

Multiple mutations in the Rhodopsin gene cause sector retinitis pigmentosa in humans and a corresponding light-exacerbated retinal degeneration (RD) in animal models. Previously we have shown that T4K rhodopsin requires photoactivation to exert its toxic effect. Here we further investigated the mechanisms involved in rod cell death caused by T4K rhodopsin in mixed male and female Xenopus laevis In this model, RD was prevented by rearing animals in constant darkness but surprisingly also in constant light. RD was maximized by light cycles containing at least 1 h of darkness and 20 min of light exposure, light intensities >750 lux, and by a sudden light onset. Under conditions of frequent light cycling, RD occurred rapidly and synchronously, with massive shedding of ROS fragments into the RPE initiated within hours and subsequent death and phagocytosis of rod cell bodies. RD was minimized by reduced light levels, pretreatment with constant light, and gradual light onset. RD was prevented by genetic ablation of the retinal isomerohydrolase RPE65 and exacerbated by ablation of phototransduction components GNAT1, SAG, and GRK1. Our results indicate that photoactivated T4K rhodopsin is toxic, that cell death requires synchronized photoactivation of T4K rhodopsin, and that toxicity is mitigated by interaction with other rod outer segment proteins regardless of whether they participate in activation or shutoff of phototransduction. In contrast, RD caused by P23H rhodopsin does not require photoactivation of the mutant protein, as it was exacerbated by RPE65 ablation, suggesting that these phenotypically similar disorders may require different treatment strategies.

A combination treatment based on drug repurposing demonstrates mutation-agnostic efficacy in pre-clinical retinopathy models.

Inherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6ßrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.

Myopic Macular Hole and Detachment after Gene Therapy in Atypical RPE65 Retinal Dystrophy: A Case Report.

PURPOSE: 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. CASE DESCRIPTION: 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.

Perimacular Atrophy Following Voretigene Neparvovec-Rzyl Treatment in the Setting of Previous Contralateral Eye Treatment With a Different Viral Vector.

Purpose: To report on cases of unilateral perimacular atrophy after treatment with voretigene neparvovec-rzyl, in the setting of previous contralateral eye treatment with a different viral vector. Design: Single-center, retrospective chart review. Methods: In this case series, four patients between the ages of six and 11 years old with RPE65-related retinopathy were treated unilaterally with rAAV2-CB-hRPE65 as part of a gene augmentation clinical trial (NCT00749957). Six to 10 years later the contralateral eyes were treated with the Food and Drug Administration-approved drug, voretigene neparvovec-rzyl. Best-corrected visual acuity (BCVA), fundus photos, ocular coherence tomography, two-color dark-adapted perimetry, full field stimulus threshold testing (FST), and location of subretinal bleb and chorioretinal atrophy were evaluated. Results: Three out of four patients showed unilateral perimacular atrophy after treatment with voretigene, ranging from five to 22 months after treatment. Areas of robust visual field improvement were followed by areas of chorioretinal atrophy. Despite perimacular changes, BCVA, FST, and subjective improvements in vision and nyctalopia were maintained. Perimacular atrophy was not observed in the first eye treated with the previous viral vector. Conclusions: We observed areas of robust visual field improvement followed by perimacular atrophy in voretigene treated eyes, as compared to the initially treated contralateral eyes. Translational Relevance: Caution is advised when using two different viral vectors between eyes in gene therapy. This may become an important issue in the future with increasing gene therapy clinical trials for inherited retinal dystrophies.

Ablation of Fatty Acid Transport Protein-4 Enhances Cone Survival, M-cone Vision, and Synthesis of Cone-Tropic 9-cis-Retinal in rd12 Mouse Model of Leber Congenital Amaurosis.

The canonical visual cycle employing RPE65 as the retinoid isomerase regenerates 11-cis-retinal to support both rod- and cone-mediated vision. Mutations of RPE65 are associated with Leber congenital amaurosis that results in rod and cone photoreceptor degeneration and vision loss of affected patients at an early age. Dark-reared Rpe65-/- mouse has been known to form isorhodopsin that employs 9-cis-retinal as the photosensitive chromophore. The mechanism regulating 9-cis-retinal synthesis and the role of the endogenous 9-cis-retinal in cone survival and function remain largely unknown. In this study, we found that ablation of fatty acid transport protein-4 (FATP4), a negative regulator of 11-cis-retinol synthesis catalyzed by RPE65, increased the formation of 9-cis-retinal, but not 11-cis-retinal, in a light-independent mechanism in both sexes of RPE65-null rd12 mice. Both rd12 and rd12;Fatp4-/- mice contained a massive amount of all-trans-retinyl esters in the eyes, exhibiting comparable scotopic vision and rod degeneration. However, expression levels of M- and S-opsins as well as numbers of M- and S-cones surviving in the superior retinas of rd12;Fatp4-/ - mice were at least twofold greater than those in age-matched rd12 mice. Moreover, FATP4 deficiency significantly shortened photopic b-wave implicit time, improved M-cone visual function, and substantially deaccelerated the progression of cone degeneration in rd12 mice, whereas FATP4 deficiency in mice with wild-type Rpe65 alleles neither induced 9-cis-retinal formation nor influenced cone survival and function. These results identify FATP4 as a new regulator of synthesis of 9-cis-retinal, which is a "cone-tropic" chromophore supporting cone survival and function in the retinas with defective RPE65.

Voretigene neparvovec for inherited retinal dystrophy due to RPE65 mutations: a scoping review of eligibility and treatment challenges from clinical trials to real practice.

Biallelic mutations in the RPE65 gene affect nearly 8% of Leber Congenital Amaurosis and 2% of Retinitis Pigmentosa cases. Voretigene neparvovec (VN) is the first gene therapy approach approved for their treatment. To date, real life experience has demonstrated functional improvements following VN treatment, which are consistent with the clinical trials outcomes. However, there is currently no consensus on the characteristics for eligibility for VN treatment. We reviewed relevant literature to explore whether recommendations on patient eligibility can be extrapolated following VN marketing. We screened 166 papers through six research questions, following scoping reviews methodology, to investigate: (1) the clinical and genetic features considered in VN treatment eligibility; (2) the psychophysical tests and imaging modalities used in the pre-treatment and follow-up; (3) the potential correlations between visual function and retinal structure that can be used to define treatment impact on disease progression; (4) retinal degeneration; (5) the most advanced testing modalities; and (6) the impact of surgical procedure on treatment outcomes. Current gaps concerning patients' eligibility in clinical settings, such as pre-treatment characteristics and outcomes are not consistently reported across the studies. No upper limit of retinal degeneration can be defined as the univocal factor in patient eligibility, although evidence suggested that the potential for function rescue is related to the preservation of photoreceptors before treatment. In general, paediatric patients retain more viable cells, present a less severe disease stage and show the highest potential for improvements, making them the most suitable candidates for treatment.

Functional Characterization of a CruP-Like Isomerase in Dunaliella.

Dunaliella bardawil is a marine unicellular green algal that produces large amounts of ß-carotene and is a model organism for studying the carotenoid synthesis pathway. However, there are still many mysteries about the enzymes of the D. bardawil lycopene synthesis pathway that have not been revealed. Here, we have identified a CruP-like lycopene isomerase, named DbLyISO, and successfully cloned its gene from D. bardawil. DbLyISO showed a high homology with CruPs. We constructed a 3D model of DbLyISO and performed molecular docking with lycopene, as well as molecular dynamics testing, to identify the functional characteristics of DbLyISO. Functional activity of DbLyISO was also performed by overexpressing gene in both E. coli and D. bardawil. Results revealed that DbLyISO acted at the C-5 and C-13 positions of lycopene, catalyzing its cis-trans isomerization to produce a more stable trans structure. These results provide new ideas for the development of a carotenoid series from engineered bacteria, algae, and plants.

Generation of two induced pluripotent stem cell lines (LVPEIi004-A and LVPEIi005-A) from probands with Leber Congenital Amaurosis 2 (LCA2) and harboring mutations in RPE65.

Leber Congenital Amaurosis 2 is an early onset retinal dystrophy that occurs due to mutation in RPE65 gene. Here, we report the generation of two patient specific induced pluripotent stem cell lines harboring nonsense mutations in exon 7 (c.646A > T) and exon 9 (c.992G > A) of RPE65 gene, respectively, which leads to premature translational termination and formation of defective protein. These lines were generated by the reprogramming of human dermal fibroblast cells using integration-free, episomal constructs expressing stemness genes. The stable lines maintained a normal karyotype, expressed the key stemness factors, underwent trilineage differentiation, and maintained their genetic identity and genomic integrity.

RPE65-Associated Retinal Dystrophies: Phenotypes and Treatment Effects with Voretigene Neparvovec.

Retinal dystrophies linked to the RPE65 gene are mostly fast-progressing retinal diseases, with childhood onset of night blindness and progressive visual loss up to the middle adult age. Rare phenotypes linked to this gene are known with congenital stationary night blindness or slowly progressing retinitis pigmentosa, as well as an autosomal dominant c.1430A>G (p.Asp477Gly) variant. This review gives an overview of the current knowledge of the clinical phenotypes, as well as experience with the efficacy and safety of the approved gene augmentation therapy voretigene neparvovec.

Central visual pathways affected by degenerative retinal disease before and after gene therapy.

Genetic diseases affecting the retina can result in partial or complete loss of visual function. Leber's congenital amaurosis (LCA) is a rare blinding disease, usually inherited in an autosomally recessive manner, with no cure. Retinal gene therapy has been shown to improve vision in LCA patients caused by mutations in the RPE65 gene (LCA2). However, little is known about how activity in central visual pathways is affected by the disease or by subsequent gene therapy. Functional MRI (fMRI) was used to assess retinal signal transmission in cortical and subcortical visual structures before and 1 year after retinal intervention. The fMRI paradigm consisted of 15-s blocks of flickering (8 Hz) black and white checkerboards interleaved with 15 s of blank (black) screen. Visual activation in the brain was assessed using the general linear model, with multiple comparisons corrected using the false discovery rate method. Response to visual stimulation through untreated eyes of LCA2 patients showed heightened fMRI responses in the superior colliculus and diminished activities in the lateral geniculate nucleus (LGN) compared to controls, indicating a shift in the patients' visual processing towards the retinotectal pathway. Following gene therapy, stimuli presented to the treated eye elicited significantly stronger fMRI responses in the LGN and primary visual cortex, indicating some re-engagement of the geniculostriate pathway (GS) pathway. Across patients, the post-treatment LGN fMRI responses correlated significantly with performance on a clinical test measuring light sensitivity. Our results demonstrate that the low vision observed in LCA2 patients involves a shift in visual processing toward the retinotectal pathway, and that gene therapy partially reinstates visual transmission through the GS pathway. This selective boosting of retinal output through the GS pathway and its correlation to improved visual performance, following several years of degenerative retinal disease, is striking. However, while retinal gene therapy and other ocular interventions have given hope to RPE65 patients, it may take years before development of therapies tailored to treat the diseases in other low vision patients are available. Our demonstration of a shift toward the retinotectal pathway in these patients may spur the development of new tools and rehabilitation strategies to help maximize the use of residual visual abilities and augment experience-dependent plasticity.

RPE65 mutations in Leber congenital amaurosis, early-onset severe retinal dystrophy, and retinitis pigmentosa from a tertiary eye care center in India.

INTRODUCTION: Mutations in the retinal pigment epithelial 65 kilodalton protein (RPE65) gene are associated with various inherited retinal diseases (IRDs), including Leber congenital amaurosis (LCA), early-onset severe retinal dystrophy (EOSRD), and retinitis pigmentosa (RP). We screened for mutations in RPE65 in a series of Indian patients with these IRDs to determine the frequency/types of mutations and to describe the associated phenotypes. MATERIALS AND METHODS: Diagnosis of LCA, EOSRD, and RP was made by standard and pre-defined criteria. Patients were evaluated by clinical, retinal imaging, and electrophysiological parameters. Genomic DNA from patients and available family members were used for identifying mutations by direct Sanger sequencing of the RPE65 gene or targeted NGS gene panel for IRDs covering 260+ genes. Variations detected were tested in healthy control populations and for co-segregation with the disease in available family members. RESULTS: Mutations were found in eight patients, out of 220 total cases screened, all homozygous for the respective mutant alleles. Seven patients had mutations leading to premature termination codons and one patient had a missense change. The onset of visual loss ranged from birth to <2 years of life. At presentation, RPE mottling in the background retina was present in all cases with macular involvement in five cases with or without vascular attenuation and optic disc pallor. CONCLUSION: RPE65 mutations in this series were found in 3.6% of cases associated with severe, early-onset disease, with consistent RPE mottling and variable manifestations with regard to the extent of disc pallor, arteriolar attenuation, and appearance of the macula.

Carotenoid cleavage enzymes evolved convergently to generate the visual chromophore.

The retinal light response in animals originates from the photoisomerization of an opsin-coupled 11-cis-retinaldehyde chromophore. This visual chromophore is enzymatically produced through the action of carotenoid cleavage dioxygenases. Vertebrates require two carotenoid cleavage dioxygenases, ß-carotene oxygenase 1 and retinal pigment epithelium 65 (RPE65), to form 11-cis-retinaldehyde from carotenoid substrates, whereas invertebrates such as insects use a single enzyme known as Neither Inactivation Nor Afterpotential B (NinaB). RPE65 and NinaB couple trans-cis isomerization with hydrolysis and oxygenation, respectively, but the mechanistic relationship of their isomerase activities remains unknown. Here we report the structure of NinaB, revealing details of its active site architecture and mode of membrane binding. Structure-guided mutagenesis studies identify a residue cluster deep within the NinaB substrate-binding cleft that controls its isomerization activity. Our data demonstrate that isomerization activity is mediated by distinct active site regions in NinaB and RPE65-an evolutionary convergence that deepens our understanding of visual system diversity.

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.

Multi-luminance mobility testing after gene therapy in the context of retinal functional diagnostics.

BACKGROUND: Voretigene neparvovec (Luxturna®) is the first approved gene therapy for RPE65-linked Leber congenital amaurosis (LCA). Though individual effects are highly variable, most recipients report improved vision in everyday life. To describe such effects, visual navigation tests are now frequently used in clinical trials. However, it is still unclear how their results should be interpreted compared to conventional parameters of visual function. METHODS: Seven LCA patients underwent a multi-luminance visual navigation test (Ora-VNCTM) before and 3 months after receiving Luxturna gene therapy. Their performance was rated based on the luminance level at which they passed the course. Differences between the first and second test were correlated to changes in visual acuity, full-field stimulus thresholds, chromatic pupil campimetry, and dark-adapted perimetry. RESULTS: A few patients displayed notable improvements in conventional measures of visual function whereas patients with advanced retinal degeneration showed no relevant changes. Independent of these results, almost all participants improved in the visual navigation task by one or more levels. The improvement in the mobility test was best correlated to the change in full-field stimulus thresholds. Other measures of visual functions showed no clear correlation with visual navigation. DISCUSSION: In patients who passed the test's more difficult levels, improved visual navigation can be attributed to the reactivation of rods. However, the performance of patients with low vision seemed to depend much more on confounding factors in the easier levels. In sum, such tests might only be meaningful for patients with better preserved visual functions.