Phenotyping and genotyping inherited retinal diseases: Molecular genetics, clinical and imaging features, and therapeutics of macular dystrophies, cone and cone-rod dystrophies, rod-cone dystrophies, Leber congenital amaurosis, and cone dysfunction syndromes.

Inherited retinal diseases (IRD) are a leading cause of blindness in the working age population and in children. The scope of this review is to familiarise clinicians and scientists with the current landscape of molecular genetics, clinical phenotype, retinal imaging and therapeutic prospects/completed trials in IRD. Herein we present in a comprehensive and concise manner: (i) macular dystrophies (Stargardt disease (ABCA4), X-linked retinoschisis (RS1), Best disease (BEST1), PRPH2-associated pattern dystrophy, Sorsby fundus dystrophy (TIMP3), and autosomal dominant drusen (EFEMP1)), (ii) cone and cone-rod dystrophies (GUCA1A, PRPH2, ABCA4, KCNV2 and RPGR), (iii) predominant rod or rod-cone dystrophies (retinitis pigmentosa, enhanced S-Cone syndrome (NR2E3), Bietti crystalline corneoretinal dystrophy (CYP4V2)), (iv) Leber congenital amaurosis/early-onset severe retinal dystrophy (GUCY2D, CEP290, CRB1, RDH12, RPE65, TULP1, AIPL1 and NMNAT1), (v) cone dysfunction syndromes (achromatopsia (CNGA3, CNGB3, PDE6C, PDE6H, GNAT2, ATF6), X-linked cone dysfunction with myopia and dichromacy (Bornholm Eye disease; OPN1LW/OPN1MW array), oligocone trichromacy, and blue-cone monochromatism (OPN1LW/OPN1MW array)). Whilst we use the aforementioned classical phenotypic groupings, a key feature of IRD is that it is characterised by tremendous heterogeneity and variable expressivity, with several of the above genes associated with a range of phenotypes.

A Systematic Review and Meta-Analyses of Interventional Clinical Trial Studies for Gene Therapies for the Inherited Retinal Degenerations (IRDs).

IRDs are one of the leading causes of visual loss in children and young adults. Mutations in over 271 genes lead to retinal dysfunction, degeneration and sight loss. Though no cure exists, gene augmentation therapy has brought hope to the field. This systematic review sought to assess the efficacy of available gene therapy treatments for IRDs. Databases and public resources were searched for randomised controlled trials (RCTs) and non-randomised studies of interventions (NRSIs). Standard methodological procedures were used, including a risk-of-bias assessment. One RCT and five NRSIs were assessed, all for adeno-associated virus two (AAV2)-mediated treatment of RPE-specific 65 kDa (RPE65)-associated LCA (Leber congenital amaurosis). Five outcomes were reported for meta-analyses. Modest improvements in visual acuity, ambulatory navigation/mobility testing or central retinal thickness was observed. There was significant improvement in red and blue light full-field stimulus testing (FST) (red light risk ratio of 1.89, treated v control, p = 0.04; and blue light risk ratio of 2.01, treated v control, p = 0.001). Study design assessment using a ROBIN-I tool (Cochrane Library) showed risk-of-bias judgement to be "low/moderate", whilst there were "some concerns" for the RCT using a RoB-2 tool (Cochrane Library). Although comparison by meta-analysis is compromised by, amongst other issues, a variable amount of vector delivered in each trial, FST improvements demonstrate a proof-of-principle for treating IRDs with gene therapy.

Molecular genetics with clinical characteristics of Leber congenital amaurosis in the Han population of western China.

Purpose: Leber congenital amaurosis (LCA) is one of the earliest inherited retinal dystrophies (IRD) that leads to blindness. To date, there have been 25 LCA-associated genes reported in China as well as other countries. The current study aimed to present the dominant molecular genetics and clinical features of LCA in the Han population of western China.Methods: Our study comprised 37 patients with strictly defined Leber congenital amaurosis in a cohort of IRD (2009-2019). The mutations were detected by targeted next-generation sequencing (NGS), Sanger sequencing, and segregation analysis. The patients underwent comprehensive clinical examinations, analysis of phenotypes and genotypes.Results: Out of the 37 patients, 34 harbored known LCA genes; the detection rate of mutations was 91.9%. Forty-seven different alleles incorporated 21 novel mutations; 8 were known LCA-associated genes. The three most frequently mutated genes included CRB1 (27.0%), RDH12 (24.3%), and RPGRIP1 (18.9%). The CRB1-associated LCA showed a pigmented fundus; the RDH12-associated LCA featured macular atrophy. Our results revealed that CRB1 and RPGRIP1 genes occupied a greater proportion in the western Chinese population. The proportion of these two genes was similar in other regions of China as well. The difference existed in a larger proportion of RDH12-associated LCA in the western Chinese population.Conclusions: The new findings in our study group polished the spectrum of the novel mutations and phenotypes of LCA with regional and ethnic variations. This comprehensive database can provide essential information for gene therapies.

Durable vision improvement after a single treatment with antisense oligonucleotide sepofarsen: a case report.

Leber congenital amaurosis due to CEP290 ciliopathy is being explored by treatment with the antisense oligonucleotide (AON) sepofarsen. One patient who was part of a larger cohort (ClinicalTrials.gov NCT03140969 ) was studied for 15 months after a single intravitreal sepofarsen injection. Concordant measures of visual function and retinal structure reached a substantial efficacy peak near 3 months after injection. At 15 months, there was sustained efficacy, even though there was evidence of reduction from peak response. Efficacy kinetics can be explained by the balance of AON-driven new CEP290 protein synthesis and a slow natural rate of CEP290 protein degradation in human foveal cone photoreceptors.

Leber Congenital Amaurosis Due to GUCY2D Mutations: Longitudinal Analysis of Retinal Structure and Visual Function.

Gene augmentation therapy is being planned for GUCY2D-associated Leber congenital amaurosis (LCA). To increase our understanding of the natural history of GUCY2D-LCA, patients were evaluated twice with an interval of 4 to 7 years between visits using safety and efficacy outcome measures previously determined to be useful for monitoring this disorder. In this group of molecularly-identified LCA patients (n = 10; ages 7-37 years at first visit), optical coherence tomography (OCT) was used to measure foveal cone outer nuclear layer (ONL) thickness and rod ONL at a superior retinal locus. Full-field stimulus testing (FST) with chromatic stimuli in dark- and light-adapted states was used to assay rod and cone vision. Changes in OCT and FST over the interval were mostly attributable to inter-visit variability. There were no major negative changes in structure or function across the cohort and over the intervals studied. Variation in severity of disease expression between patients occurs; however, despite difficulties in quantifying structure and function in such seriously visually impaired individuals with nystagmus, the present work supports the use of OCT as a safety outcome and FST as an efficacy outcome in a clinical trial of GUCY2D-LCA. A wide age spectrum for therapy was confirmed, and there was relative stability of structure and function during a typical time interval for clinical trials.

SENIOR-LØKEN SYNDROME: A Case Series and Review of the Renoretinal Phenotype and Advances of Molecular Diagnosis.

PURPOSE: To report genetic and clinical findings in a case series of 10 patients from eight unrelated families diagnosed with Senior-Løken syndrome. METHODS: A retrospective study of patients with Senior-Løken syndrome. Data collected included clinical findings electroretinography and ocular imaging. Genetic analysis was based on molecular inversion probes, whole-exome sequencing (WES), and Sanger sequencing. RESULTS: All patients who underwent electrophysiology (8/10) had widespread photoreceptor degeneration. Genetic analysis revealed two mutations in NPHP1, two mutations in NPHP4, and two mutations in IQCB1 (NPHP5). Five of the six mutations identified in the current study were found in a single family each in our cohort. The IQCB1-p.R461* mutation has been identified in 3 families. Patients harboring mutations in IQCB1 were diagnosed with Leber congenital amaurosis, while patients with NPHP4 and NPHP1 mutations showed early and sector retinitis pigmentosa, respectively. Full-field electroretinography was extinct for 6 of 10 patients, moderately decreased for two, and unavailable for another 2 subjects. Renal involvement was evident in 7/10 patients at the time of diagnosis. Kidney function was normal (based on serum creatinine) in patients younger than 10 years. Mutations in IQCB1 were associated with high hypermetropia, whereas mutations in NPHP4 were associated with high myopia. CONCLUSION: Patients presenting with infantile inherited retinal degeneration are not universally screened for renal dysfunction. Modern genetic tests can provide molecular diagnosis at an early age and therefore facilitate early diagnosis of renal disease with recommended periodic screening beyond childhood and family planning.

Ciliopathies and the Kidney: A Review.

Primary cilia are specialized sensory organelles that protrude from the apical surface of most cell types. During the past 2 decades, they have been found to play important roles in tissue development and signal transduction, with mutations in ciliary-associated proteins resulting in a group of diseases collectively known as ciliopathies. Many of these mutations manifest as renal ciliopathies, characterized by kidney dysfunction resulting from aberrant cilia or ciliary functions. This group of overlapping and genetically heterogeneous diseases includes polycystic kidney disease, nephronophthisis, and Bardet-Biedl syndrome as the main focus of this review. Renal ciliopathies are characterized by the presence of kidney cysts that develop due to uncontrolled epithelial cell proliferation, growth, and polarity, downstream of dysregulated ciliary-dependent signaling. Due to cystic-associated kidney injury and systemic inflammation, cases result in kidney failure requiring dialysis and transplantation. Of the handful of pharmacologic treatments available, none are curative. It is important to determine the molecular mechanisms that underlie the involvement of the primary cilium in cyst initiation, expansion, and progression for the development of novel and efficacious treatments. This review updates research progress in defining key genes and molecules central to ciliogenesis and renal ciliopathies.

Inverse correlation between fatty acid transport protein 4 and vision in Leber congenital amaurosis associated with RPE65 mutation.

Fatty acid transport protein 4 (FATP4), a transmembrane protein in the endoplasmic reticulum (ER), is a recently identified negative regulator of the ER-associated retinal pigment epithelium (RPE)65 isomerase necessary for recycling 11-cis-retinal, the light-sensitive chromophore of both rod and cone opsin visual pigments. The role of FATP4 in the disease progression of retinal dystrophies associated with RPE65 mutations is completely unknown. Here we show that FATP4-deficiency in the RPE results in 2.8-fold and 1.7-fold increase of 11-cis- and 9-cis-retinals, respectively, improving dark-adaptation rates as well as survival and function of rods in the Rpe65 R91W knockin (KI) mouse model of Leber congenital amaurosis (LCA). Degradation of S-opsin in the proteasomes, but not in the lysosomes, was remarkably reduced in the KI mouse retinas lacking FATP4. FATP4-deficiency also significantly rescued S-opsin trafficking and M-opsin solubility in the KI retinas. The number of S-cones in the inferior retinas of 4- or 6-mo-old KI;Fatp4-/- mice was 7.6- or 13.5-fold greater than those in age-matched KI mice. Degeneration rates of S- and M-cones are negatively correlated with expression levels of FATP4 in the RPE of the KI, KI;Fatp4+/- , and KI;Fatp4-/- mice. Moreover, the visual function of S- and M-cones is markedly preserved in the KI;Fatp4-/- mice, displaying an inverse correlation with the FATP4 expression levels in the RPE of the three mutant lines. These findings establish FATP4 as a promising therapeutic target to improve the visual cycle, as well as survival and function of cones and rods in patients with RPE65 mutations.

Treatment Potential for LCA5-Associated Leber Congenital Amaurosis.

Purpose: To determine the therapeutic window for gene augmentation for Leber congenital amaurosis (LCA) associated with mutations in LCA5. Methods: Five patients (ages 6-31) with LCA and biallelic LCA5 mutations underwent an ophthalmic examination including optical coherence tomography (SD-OCT), full-field stimulus testing (FST), and pupillometry. The time course of photoreceptor degeneration in the Lca5gt/gt mouse model and the efficacy of subretinal gene augmentation therapy with AAV8-hLCA5 delivered at postnatal day 5 (P5) (early, n = 11 eyes), P15 (mid, n = 14), and P30 (late, n = 13) were assessed using SD-OCT, histologic study, electroretinography (ERG), and pupillometry. Comparisons were made with the human disease. Results: Patients with LCA5-LCA showed a maculopathy with detectable outer nuclear layer (ONL) in the pericentral retina and at least 4 log units of dark-adapted sensitivity loss. The Lca5gt/gt mouse has a similarly severe and rapid photoreceptor degeneration. The ONL became progressively thinner and was undetectable by P60. Rod- and cone-mediated ERGs were severely reduced in amplitudes at P30 and became nondetectable by P60. Subretinal AAV8-hLCA5 administered to Lca5gt/gt mice at P5 and P15, but not at P30, resulted in structural and functional rescue. Conclusions: LCA5-LCA is a particularly severe form of LCA that was recapitulated in the Lca5gt/gt mouse. Gene augmentation resulted in structural and functional rescue in the Lca5gt/gt mouse if delivered before P30. Retained photoreceptors were visible within the central retina in all patients with LCA5-LCA, at a level equivalent to that observed in rescued Lca5gt/gt mice, suggesting a window of opportunity for the treatment of patients with LCA5-LCA.

Transient pupillary light reflex in CEP290- or NPHP5-associated Leber congenital amaurosis: Latency as a potential outcome measure of cone function.

Mutations in photoreceptor cilium genes CEP290 and NPHP5 cause a form of Leber congenital amaurosis (LCA) which typically lacks rods but retains central cones. The current study evaluated the transient pupillary light reflex (TPLR) as an objective outcome measure to assess efficacy of ongoing and future therapies. Eleven eyes of six patients selected for retained cone function were tested with TPLR using full-field stimuli in the dark-adapted state. Stimuli were red or blue with 1 s duration and spanned a 6-log unit dynamic range. TPLR response amplitude was quantified at fixed times of 0.9 and 2 s after stimulus onset and TPLR latency was defined as the time to reach 0.3 mm constriction. Full-field stimulus testing (FST) and static perimetry were used to correlate subjective perception with objective TPLR parameters. TPLR and FST thresholds with both red and blue stimuli were abnormally elevated in patients to near -1.25 log phot-cd·m-2 consistent with the lack of rods. TPLR latencies were delayed on average but showed some differences among patients. Remnant extrafoveal vision was correlated with faster TPLR latencies. Our results support the use of a short TPLR protocol with full-field red stimuli of 0.7 log phot-cd·m-2 or brighter as an objective and convenient outcome measure of cone function in CEP290- and NPHP5-LCA. The latency parameter of the TPLR would be expected to show a detectable change when an intervention modifies cone sensitivity in the extrafoveal region.

GCAP neuronal calcium sensor proteins mediate photoreceptor cell death in the rd3 mouse model of LCA12 congenital blindness by involving endoplasmic reticulum stress.

Loss-of-function mutations in the retinal degeneration 3 (RD3) gene cause inherited retinopathy with impaired rod and cone function and fast retinal degeneration in patients and in the natural strain of rd3 mice. The underlying physiopathology mechanisms are not well understood. We previously proposed that guanylate cyclase-activating proteins (GCAPs) might be key Ca2+-sensors mediating the physiopathology of this disorder, based on the demonstrated toxicity of GCAP2 when blocked in its Ca2+-free form at photoreceptor inner segments. We here show that the retinal degeneration in rd3 mice is substantially delayed by GCAPs ablation. While the number of retinal photoreceptor cells is halved in 6 weeks in rd3 mice, it takes 8 months to halve in rd3/rd3 GCAPs-/- mice. Although this substantial morphological rescue does not correlate with recovery of visual function due to very diminished guanylate cyclase activity in rd3 mice, it is very informative of the mechanisms underlying photoreceptor cell death. By showing that GCAP2 is mostly in its Ca2+-free-phosphorylated state in rd3 mice, we infer that the [Ca2+]i at rod inner segments is permanently low. GCAPs are therefore retained at the inner segment in their Ca2+-free, guanylate cyclase activator state. We show that in this conformational state GCAPs induce endoplasmic reticulum (ER) stress, mitochondrial swelling, and cell death. ER stress and mitochondrial swelling are early hallmarks of rd3 retinas preceding photoreceptor cell death, that are substantially rescued by GCAPs ablation. By revealing the involvement of GCAPs-induced ER stress in the physiopathology of Leber's congenital amaurosis 12 (LCA12), this work will aid to guide novel therapies to preserve retinal integrity in LCA12 patients to expand the window for gene therapy intervention to restore vision.

Possible dual contribution of a novel GUCY2D mutation in the development of retinal degeneration in a consanguineous population.

Molecular characterization of novel mutations in Leber Congenital Amaurosis (LCA) disease improves the disease diagnosis and contributes to the development of preventive and therapeutic approaches. We studied an isolated inbred population in Iran with a high prevalence of retinal degeneration with clinical variability. The clinical examinations were performed on eight patients belonging to three consanguineous families. The identical-by-descent (IBD) mapping technique was employed to identify the shared loci in patients. Subsequently, Sanger sequencing of the GUCY2D gene, in silico analysis, as well as segregation study were conducted. The whole-exome sequencing method was applied for negative cases of GUCY2D mutation, followed by segregation study in suspected variants among families. A novel deletion mutation in the GUCY2D gene can explain the emergence of LCA-1 in most patients but not all. Besides, a heterozygous variant of uncertain significance (VUS) was observed in the BEST1 gene in some healthy and participant patients. These results further support inter/intra-familial clinical heterogeneity in retinal dystrophy and suggest that screening the GUCY2D gene would be needed for the diagnosis of LCA in Iranian people living in the central regions. The variant in the BEST1 gene might be considered a benign heterozygous variant; however, we hypothesized a possible double heterozygosity in both GUCY2D and BEST1 genes that may cause the pathogenesis of cone-rod dystrophy-6 (CRD-6) disease. This would propose a new scenario for the pathogenesis of a monogenic disorder such as CRD-6 disease in which other genetic elements may be involved in the development of the disease.

CEP290 Mutation Spectrum and Delineation of the Associated Phenotype in a Large German Cohort: A Monocentric Study.

PURPOSE: Gene therapy for Leber congenital amaurosis (LCA) is becoming available, and therefore it is crucial to identify eligible candidates. We report the spectrum and associated phenotype of CEP290 mutations in the largest German cohort observed by a single clinical site. DESIGN: Prospective cohort study. METHODS: Twenty-three patients with mutations in CEP290 were included. Genomic DNA was analyzed by Sanger sequencing or high-throughput sequencing for all retinitis pigmentosa-associated genes in patients, and segregation analysis was done in family members. Patients underwent functional and morphologic examinations, including fundus autofluorescence and spectral-domain optical coherence tomography. RESULTS: The most frequent mutation was c.2991+1655A>G, found in 87% of patients (20/23). Thirty percent of patients (7/23) carried the mutation in an apparent homozygous state and 57% (13/23) in a likely compound heterozygous state. The most common clinical diagnosis was LCA and/or early onset severe retinal dystrophy in 82% (19/23), followed by retinitis pigmentosa in 14% (3/23) and cone-rod dystrophy (4%, 1/23). Best-corrected visual acuity was severely reduced to residual light perception and hand motion vision, with the exception of 3 patients with best-corrected visual acuity of 0.8 (Snellen). The visual field was severely decreased and electroretinogram was undetectable in most cases; however, retinal layers at the fovea appeared to be relatively well preserved. Systemic disorders were not noticed. CONCLUSIONS: c.2991+1655A>G is by far the most important CEP290 mutation, contributing to 87% of patients with the CEP290 mutation in Germany. In our cohort, a homozygous c.2991+1655A>G genotype presented with a more severe phenotype. National studies and further detailed phenotype analysis seem to be important to assess the need for and promise of specific gene therapies.

Utility of In Vitro Mutagenesis of RPE65 Protein for Verification of Mutational Pathogenicity Before Gene Therapy.

Importance: Next-generation sequencing can detect variants of uncertain significance (VUSs), for some of which gene therapy would not be advantageous. Therefore, the pathogenicity of compound heterozygous or homozygous variants should be confirmed before bilateral vitrectomy and administration of voretigene neparvovec-rzyl. Objective: To describe an in vitro mutagenesis assay for assessing the pathogenicity of variants in the RPE65 gene. Design, Setting, and Participants: This case series was conducted at 2 tertiary referral centers. Clinical history, imaging, and electrophysiologic testing results were reviewed from September 5, 2008, to December 31, 2019. Participants were 4 pediatric patients with Leber congenital amaurosis who were evaluated for or met the inclusion criteria for phase 1 to 3 clinical trials or were referred for voretigene neparvovec-rzyl treatment. Main Outcomes and Measures: A functional assay was used to confirm the pathogenicity of novel RPE65 VUSs in 4 patients with Leber congenital amaurosis. Results: Four patients with Leber congenital amaurosis had VUSs in RPE65. Patients 1 and 2 were siblings with the homozygous VUS c.311G>T p.(G104V). Patient 3 was a compound heterozygote with 1 known pathogenic allele, c.1202_1203insCTGG p.(Glu404AlafsTer4), and 1 VUS, c.311G>T p.(G104V), which segregated to separate alleles. Patient 4 was also a compound heterozygote with 1 pathogenic variant, c.11 + 5G>A, and 1 variant in trans, c.1399C>T p.(P467S). In vitro mutagenesis revealed that the G104V and P467S RPE65 proteins were catalytically inactive (0% isomerase activity). Patients 1 and 2 were excluded from participation in a phase 1 trial owing to high Adeno-associated virus 2 capsid-neutralizing antibodies. Patients 3 (G104V) and 4 (P467S) underwent successful surgical gene therapy with voretigene neparvovec-rzyl, and their response to lower white light intensity and visual field increased in fewer than 30 days after gene therapy intervention. Conclusions and Relevance: Findings from this study suggest that, in patients with missense mutations in RPE65, functional assays of protein function can be performed to assess the pathogenicity of variants in both compound heterozygous and homozygous cases. Given the potential risks of gene therapy operations, in vitro RPE65 activity testing should be considered to avoid the possibility of treating a false genotype.

Treatment Potential for Macular Cone Vision in Leber Congenital Amaurosis Due to CEP290 or NPHP5 Mutations: Predictions From Artificial Intelligence.

Purpose: To use supervised machine learning to predict visual function from retinal structure in retinitis pigmentosa (RP) and apply these estimates to CEP290- and NPHP5-associated Leber congenital amaurosis (LCA) to determine the potential for functional improvement. Methods: Patients with RP (n = 20) and LCA due to CEP290 (n = 12) or NPHP5 (n = 6) mutations were studied. A patient with CEP290 mutations but mild retinal degeneration was included. RP patients had cone-mediated macular function. A machine learning technique was used to associate perimetric sensitivities to local structure in RP patients. Models trained on RP data were applied to predict visual function in LCA. Results: The RP and LCA patients had comparable retinal structure. RP patients had peak sensitivity at the fovea surrounded by decreasing sensitivity. Machine learning could successfully predict perimetry results from segmented or unsegmented optical coherence tomography (OCT) input. Application of machine learning predictions to LCA within the residual macular island of photoreceptor structure showed differences between predicted and measured sensitivities defining treatment potential. In patients with retained vision, the treatment potential was 4.6 ± 2.9 dB at the fovea but 16.4 ± 4.4 dB at the parafovea. In patients with limited or no vision, the treatment potential was 17.6 ± 9.4 dB. Conclusions: Cone vision improvement potential in LCA due to CEP290 or NPHP5 mutations is predictable from retinal structure using a machine learning approach. This should allow individual prediction of the maximal efficacy in clinical trials and guide decisions about dosing. Similar strategies can be used in other retinal degenerations to estimate the extent and location of treatment potential.

A high prevalence of biallelic RPE65 mutations in Costa Rican children with Leber congenital amaurosis and early-onset retinal dystrophy.

BACKGROUND: Leber congenital amaurosis (LCA) and early-onset retinal dystrophy (EORD), are primary causes of inherited childhood blindness. Both are autosomal recessive diseases, with mutations in more than 25 genes explaining approximately ~70% of cases. However, the genetic cause for many cases remains unclear. Sequencing studies from genetically isolated populations with increased prevalence of a disorder has proven useful for rare variant studies, making Costa Rica an ideal place to study LCA/EORD genetics. MATERIALS AND METHODS: Twenty-eight affected children (25 LCA, three EORD) and their immediate family members, totaling 52 individuals (30 affected) from 22 families, were sequenced. Whole exome sequencing was performed on all affected individuals. Available parents were analyzed either by whole exome sequencing (WES) or Sanger sequencing to determine transmission. RESULTS: All affected individuals demonstrated compound heterozygous or homozygous mutations in known Inherited Retinal Disease (IRD) associated genes. Twelve variants were identified in at least one individual in three genes, RDH12, RPE65, and USH2A. Four recurrent RPE65 mutations were observed in 97% of individuals and 95% of families. All patients with LCA and two of the three individuals with EORD had biallelic mutations in RPE65; one child with EORD had a homozygous RDH12 mutation. CONCLUSIONS: These data suggest that the majority of LCA/EORD in Costa Rica is due to four founder mutations in RPE65 which have been maintained in this genetically isolated population. This finding is of great clinical significance due to the availability of gene therapy recently approved in the US and European Union for patients with biallelic RPE65 defects.

Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.

Loss of CRB2 in Müller glial cells modifies a CRB1-associated retinitis pigmentosa phenotype into a Leber congenital amaurosis phenotype.

Variations in the human Crumbs homolog-1 (CRB1) gene lead to an array of retinal dystrophies including early onset of retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in children. To investigate the physiological roles of CRB1 and CRB2 in retinal Müller glial cells (MGCs), we analysed mouse retinas lacking both proteins in MGC. The peripheral retina showed a faster progression of dystrophy than the central retina. The central retina showed retinal folds, disruptions at the outer limiting membrane, protrusion of photoreceptor nuclei into the inner and outer segment layers and ingression of photoreceptor nuclei into the photoreceptor synaptic layer. The peripheral retina showed a complete loss of the photoreceptor synapse layer, intermingling of photoreceptor nuclei within the inner nuclear layer and ectopic photoreceptor cells in the ganglion cell layer. Electroretinography showed severe attenuation of the scotopic a-wave at 1 month of age with responses below detection levels at 3 months of age. The double knockout mouse retinas mimicked a phenotype equivalent to a clinical LCA phenotype due to loss of CRB1. Localization of CRB1 and CRB2 in non-human primate (NHP) retinas was analyzed at the ultrastructural level. We found that NHP CRB1 and CRB2 proteins localized to the subapical region adjacent to adherens junctions at the outer limiting membrane in MGC and photoreceptors. Our data suggest that loss of CRB2 in MGC aggravates the CRB1-associated RP-like phenotype towards an LCA-like phenotype.

Effect of an intravitreal antisense oligonucleotide on vision in Leber congenital amaurosis due to a photoreceptor cilium defect.

Photoreceptor ciliopathies constitute the most common molecular mechanism of the childhood blindness Leber congenital amaurosis. Ten patients with Leber congenital amaurosis carrying the c.2991+1655A>G allele in the ciliopathy gene centrosomal protein 290 (CEP290) were treated (ClinicalTrials.gov no. NCT03140969 ) with intravitreal injections of an antisense oligonucleotide to restore correct splicing. There were no serious adverse events, and vision improved at 3 months. The visual acuity of one exceptional responder improved from light perception to 20/400.