Disease-specific variant interpretation highlighted the genetic findings in 2325 Japanese patients with retinitis pigmentosa and allied diseases.

BACKGROUND: As gene-specific therapy for inherited retinal dystrophy (IRD) advances, unified variant interpretation across institutes is becoming increasingly important. This study aims to update the genetic findings of 86 retinitis pigmentosa (RP)-related genes in a large number of Japanese patients with RP by applying the standardised variant interpretation guidelines for Japanese patients with IRD (J-IRD-VI guidelines) built upon the American College of Medical Genetics and Genomics and the Association for Molecular Pathology rules, and assess the contribution of these genes in RP-allied diseases. METHODS: We assessed 2325 probands with RP (n=2155, including n=1204 sequenced previously with the same sequencing panel) and allied diseases (n=170, newly analysed), including Usher syndrome, Leber congenital amaurosis and cone-rod dystrophy (CRD). Target sequencing using a panel of 86 genes was performed. The variants were interpreted according to the J-IRD-VI guidelines. RESULTS: A total of 3564 variants were detected, of which 524 variants were interpreted as pathogenic or likely pathogenic. Among these 524 variants, 280 (53.4%) had been either undetected or interpreted as variants of unknown significance or benign variants in our earlier study of 1204 patients with RP. This led to a genetic diagnostic rate in 38.6% of patients with RP, with EYS accounting for 46.7% of the genetically solved patients, showing a 9% increase in diagnostic rate from our earlier study. The genetic diagnostic rate for patients with CRD was 28.2%, with RP-related genes significantly contributing over other allied diseases. CONCLUSION: A large-scale genetic analysis using the J-IRD-VI guidelines highlighted the population-specific genetic findings for Japanese patients with IRD; these findings serve as a foundation for the clinical application of gene-specific therapies.

Gene Augmentation for Autosomal Dominant CRX-Associated Retinopathies.

The cone-rod homeobox (CRX) protein is a key transcription factor essential for photoreceptor function and survival. Mutations in human CRX gene are linked to a wide spectrum of blinding diseases ranging from mild macular dystrophy to severe Leber congenital amaurosis (LCA), cone-rod dystrophy (CRD), and retinitis pigmentosa (RP). These diseases are still incurable and mostly inherited in an autosomal dominant form. Dysfunctional mutant CRX protein interferes with the function of wild-type CRX protein, demonstrating the dominant negative effect. At present, gene augmentation is the most promising treatment strategy for hereditary diseases. This study aims to review the pathogenic mechanisms of various CRX mutations and propose two therapeutic strategies to rescue sick photoreceptors in CRX-associated retinopathies, namely, Tet-On-hCRX system and adeno-associated virus (AAV)-mediated gene augmentation. The outcome of proposed studies will guide future translational research and suggest guidelines for therapy evaluation in terms of treatment safety and efficacy.

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.

Retinal Organoids from an AIPL1 CRISPR/Cas9 Knockout Cell Line Successfully Recapitulate the Molecular Features of LCA4 Disease.

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is expressed in photoreceptors where it facilitates the assembly of phosphodiesterase 6 (PDE6) which hydrolyses cGMP within the phototransduction cascade. Genetic variations in AIPL1 cause type 4 Leber congenital amaurosis (LCA4), which presents as rapid loss of vision in early childhood. Limited in vitro LCA4 models are available, and these rely on patient-derived cells harbouring patient-specific AIPL1 mutations. While valuable, the use and scalability of individual patient-derived LCA4 models may be limited by ethical considerations, access to patient samples and prohibitive costs. To model the functional consequences of patient-independent AIPL1 mutations, CRISPR/Cas9 was implemented to produce an isogenic induced pluripotent stem cell line harbouring a frameshift mutation in the first exon of AIPL1. Retinal organoids were generated using these cells, which retained AIPL1 gene transcription, but AIPL1 protein was undetectable. AIPL1 knockout resulted in a decrease in rod photoreceptor-specific PDE6α and ß, and increased cGMP levels, suggesting downstream dysregulation of the phototransduction cascade. The retinal model described here provides a novel platform to assess functional consequences of AIPL1 silencing and measure the rescue of molecular features by potential therapeutic approaches targeting mutation-independent pathogenesis.

Current perspectives in Leber congenital amaurosis type 8 mouse modeling.

Mutations in the CRB1 (Crumbs homolog 1) cause rare retinal diseases like retinitis pigmentosa type 12 (RP12) and Leber congenital amaurosis type 8 (LCA8). RP12 results in progressively worsening peripheral vision, whereas LCA8 causes severe visual impairment at birth or in early life. While several mouse models have been proposed for RP12, few replicate the full spectrum of human LCA8 pathology, such as disorganized retinal layering, abnormal retinal thickening, pigmentary defects, hyperreflective lesions, and severely attenuated electroretinogram responses at birth. Six models have been proposed utilizing the Cre-loxP system to delete candidate genes in specific retinal cell types and developmental stages. The model ablating Crb1 and its homolog Crb2 (using mRx-Cre) from the beginning of the eye development is the most complete as it shows blindness during the eye-opening stage, pigmentary defects in the RPE, ganglion cell layer heterotopia, disruption of retinal lamination, and acellular patches. LCA8 represents a unique type of retinal dystrophy among LCA subtypes, driven by dysfunctional retinal progenitor cells during eye development. In contrast, other LCA types and RP12 are caused by photoreceptor defects. Therefore, the most accurate LCA8-like mouse model must target both alleles of the Crb1 and Crb2 genes in the optic vesicle or earlier.

An Alu-mediated duplication in NMNAT1, involved in NAD biosynthesis, causes a novel syndrome, SHILCA, affecting multiple tissues and organs.

We investigated the genetic origin of the phenotype displayed by three children from two unrelated Italian families, presenting with a previously unrecognized autosomal recessive disorder that included a severe form of spondylo-epiphyseal dysplasia, sensorineural hearing loss, intellectual disability and Leber congenital amaurosis (SHILCA), as well as some brain anomalies that were visible at the MRI. Autozygome-based analysis showed that these children shared a 4.76 Mb region of homozygosity on chromosome 1, with an identical haplotype. Nonetheless, whole-exome sequencing failed to identify any shared rare coding variants, in this region or elsewhere. We then determined the transcriptome of patients' fibroblasts by RNA sequencing, followed by additional whole-genome sequencing experiments. Gene expression analysis revealed a 4-fold downregulation of the gene NMNAT1, residing indeed in the shared autozygous interval. Short- and long-read whole-genome sequencing highlighted a duplication involving 2 out of the 5 exons of NMNAT1 main isoform (NM_022787.3), leading to the production of aberrant mRNAs. Pathogenic variants in NMNAT1 have been previously shown to cause non-syndromic Leber congenital amaurosis (LCA). However, no patient with null biallelic mutations has ever been described, and murine Nmnat1 knockouts show embryonic lethality, indicating that complete absence of NMNAT1 activity is probably not compatible with life. The rearrangement found in our cases, presumably causing a strong but not complete reduction of enzymatic activity, may therefore result in an intermediate syndromic phenotype with respect to LCA and lethality.

Gene Augmentation and Readthrough Rescue Channelopathy in an iPSC-RPE Model of Congenital Blindness.

Pathogenic variants of the KCNJ13 gene are known to cause Leber congenital amaurosis (LCA16), an inherited pediatric blindness. KCNJ13 encodes the Kir7.1 subunit that acts as a tetrameric, inwardly rectifying potassium ion channel in the retinal pigment epithelium (RPE) to maintain ionic homeostasis and allow photoreceptors to encode visual information. We sought to determine whether genetic approaches might be effective in treating blindness arising from pathogenic variants in KCNJ13. We derived human induced pluripotent stem cell (hiPSC)-RPE cells from an individual carrying a homozygous c.158G>A (p.Trp53∗) pathogenic variant of KCNJ13. We performed biochemical and electrophysiology assays to confirm Kir7.1 function. We tested both small-molecule readthrough drug and gene-therapy approaches for this "disease-in-a-dish" approach. We found that the LCA16 hiPSC-RPE cells had normal morphology but did not express a functional Kir7.1 channel and were unable to demonstrate normal physiology. After readthrough drug treatment, the LCA16 hiPSC cells were hyperpolarized by 30 mV, and the Kir7.1 current was restored. Similarly, we rescued Kir7.1 channel function after lentiviral gene delivery to the hiPSC-RPE cells. In both approaches, Kir7.1 was expressed normally, and there was restoration of membrane potential and the Kir7.1 current. Loss-of-function variants of Kir7.1 are one cause of LCA. Using either readthrough therapy or gene augmentation, we rescued Kir7.1 channel function in iPSC-RPE cells derived from an affected individual. This supports the development of precision-medicine approaches for the treatment of clinical LCA16.

Deletion of M-Opsin Prevents M Cone Degeneration in a Mouse Model of Leber Congenital Amaurosis.

Mutations in retinoid isomerase (RPE65) or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal synthesis and cause Leber congenital amaurosis (LCA). Despite the success of recent RPE65 gene therapy, follow-up studies show that patients continue to experience photoreceptor degeneration and lose vision benefit over time. In Lrat-/- mouse model, mislocalized medium (M)-wavelength opsin was degraded, whereas mislocalized short (S)-wavelength opsin accumulated before the onset of cone degeneration. The mechanism for the foveal M/long-wavelength cone photoreceptor degeneration in LCA is unknown. By crossing Lrat-/- mice with a proteasome reporter mouse strain, this study showed that M-opsin-enriched dorsal cones in Lrat-/- mice exhibit proteasome stress because of the degradation of large amounts of M-opsin. Deletion of M-opsin relieves the proteasome stress and completely prevents M cone degeneration in Lrat-/-Opn1sw-/- mice (a pure M cone LCA model, Opn1sw encoding S-opsin) for at least 12 months. These results suggest that M-opsin degradation-associated proteasome stress plays a major role in M cone degeneration in Lrat-/- model. This finding may represent a general mechanism for M cone degeneration in multiple forms of cone degeneration because of M-opsin mislocalization and degradation. These results have important implications for the current gene therapy strategy for LCA that emphasizes the need for combinatorial therapies to both improve vision and slow photoreceptor degeneration.

New Insights on the Genetic Basis Underlying SHILCA Syndrome: Characterization of the NMNAT1 Pathological Alterations Due to Compound Heterozygous Mutations and Identification of a Novel Alternative Isoform.

This study aims to genetically characterize a two-year-old patient suffering from multiple systemic abnormalities, including skeletal, nervous and developmental involvements and Leber congenital amaurosis (LCA). Genetic screening by next-generation sequencing identified two heterozygous pathogenic variants in nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) as the molecular cause of the disease: c.439+5G>T and c.299+526_*968dup.This splice variant has never been reported to date, whereas pathogenic duplication has recently been associated with cases displaying an autosomal recessive disorder that includes a severe form of spondylo-epiphyseal dysplasia, sensorineural hearing loss, intellectual disability and LCA (SHILCA), as well as some brain anomalies. Our patient presented clinical manifestations which correlated strongly with this reported syndrome. To further study the possible transcriptional alterations resulting from these mutations, mRNA expression assays were performed in the patient and her father. The obtained results detected aberrant alternative transcripts and unbalanced levels of expression, consistent with severe systemic involvement. Moreover, these analyses also detected a novel NMNAT1 isoform, which is variably expressed in healthy human tissues. Altogether, these findings represent new evidence of the correlation of NMNAT1 and SHILCA syndrome, and provide additional insights into the healthy and pathogenic expression of this gene.

Targeted deletion of Crb1/Crb2 in the optic vesicle models key features of leber congenital amaurosis 8.

The Crb1 and 2 (Crumbs homolog 1 & 2) genes encode large, single-pass transmembrane proteins essential for the apicobasal polarity and adhesion of epithelial cells. Crb1 mutations cause degenerative retinal diseases in humans, including Leber congenital amaurosis type 8 (LCA8) and retinitis pigmentosa type 12 (RP12). In LCA8, impaired photoreceptor development and/or survival is thought to cause blindness during early infancy, whereas, in RP12, progressive photoreceptor degeneration damages peripheral vision later in life. There are multiple animal models of RP12 pathology, but no experimental model of LCA8 recapitulates the full spectrum of its pathological features. To generate a mouse model of LCA8 and identify the functions of Crb1/2 in developing ocular tissues, we used an mRx-Cre driver to generate allelic combinations that enabled conditional gene ablation from the optic vesicle stage. In this series only Crb1/2 double knockout (dKO) mice exhibited characteristics of human LCA8 disease: locally thickened retina with spots devoid of cells, aberrant positioning of retinal cells, severely disrupted lamination, and depigmented retinal-pigmented epithelium. Retinal defects antedated E12.5, which is far earlier than the stage at which photoreceptor cells mainly differentiate. Most remarkably, Crb1/Crb2 dKO showed a severely attenuated electroretinogram at the eye opening stage. These results suggest that human LCA8 can be modeled in the mouse by simultaneously ablating Crb1/2 from the beginning of eye development. Importantly, they also indicate that LCA8 is caused by malfunction of retinal progenitor cells during early ocular development rather than by defective photoreceptor-Muller glial interaction, a mechanism proposed for RP12.

Clinical and molecular findings in a cohort of 152 Brazilian severe early onset inherited retinal dystrophy patients.

Leber congenital amaurosis (LCA) and early-onset retinal dystrophy (EORD) are severe inherited retinal dystrophy that can cause deep blindness childhood. They represent 5% of all retinal dystrophies in the world population and about 10% in Brazil. Clinical findings and molecular basis of syndromic and nonsyndromic LCA/EORD in a Brazilian sample (152 patients/137 families) were studied. In this population, 15 genes were found to be related to the phenotype, 38 new variants were detected and four new complex alleles were discovered. Among 123 variants found, the most common were CEP290: c.2991+1655A>G, CRB1: p.Cys948Tyr, and RPGRIP1: exon10-18 deletion.

Molecular and phenotypic investigation of a New Zealand cohort of childhood-onset retinal dystrophy.

Inherited retinal diseases are clinically heterogeneous and are associated with nearly 300 different genes. In this retrospective, observational study of a consecutive cohort of 159 patients (134 families) with childhood-onset (<16 years of age) retinal dystrophy, molecular investigations, and in-depth phenotyping were performed to determine key clinical and molecular characteristics. The most common ocular phenotype was rod-cone dystrophy in 40 patients. Leber Congenital Amaurosis, the most severe form of retinal dystrophy, was present in 10 patients, and early onset severe retinal dystrophy in 22 patients. Analysis has so far identified 131 pathogenic or likely pathogenic variants including 22 novel variants. Molecular diagnosis was achieved in 112 of 134 families (83.6%) by NGS gene panel investigation in 60 families, Sanger sequencing in 27 families, and Asper microarray in 25 families. An additional nine variants of uncertain significance were also found including three novel variants. Variants in 36 genes have been identified with the most common being ABCA4 retinopathy in 36 families. Five sporadic retinal dystrophy patients were found to have variants in dominant and X-linked genes (CRX, RHO, RP2, and RPGR) resulting in more accurate genetic counseling of inheritance for these families. Variants in syndromic associated genes including ALMS1, SDCCAG8, and PPT1 were identified in eight families enabling directed systemic care.

Sensing through Non-Sensing Ocular Ion Channels.

Ion channels are membrane-spanning integral proteins expressed in multiple organs, including the eye. In the eye, ion channels are involved in various physiological processes, like signal transmission and visual processing. A wide range of mutations have been reported in the corresponding genes and their interacting subunit coding genes, which contribute significantly to an array of blindness, termed ocular channelopathies. These mutations result in either a loss- or gain-of channel functions affecting the structure, assembly, trafficking, and localization of channel proteins. A dominant-negative effect is caused in a few channels formed by the assembly of several subunits that exist as homo- or heteromeric proteins. Here, we review the role of different mutations in switching a "sensing" ion channel to "non-sensing," leading to ocular channelopathies like Leber's congenital amaurosis 16 (LCA16), cone dystrophy, congenital stationary night blindness (CSNB), achromatopsia, bestrophinopathies, retinitis pigmentosa, etc. We also discuss the various in vitro and in vivo disease models available to investigate the impact of mutations on channel properties, to dissect the disease mechanism, and understand the pathophysiology. Innovating the potential pharmacological and therapeutic approaches and their efficient delivery to the eye for reversing a "non-sensing" channel to "sensing" would be life-changing.

Seven novel variants expand the spectrum of RPE65-related Leber congenital amaurosis in the Chinese population.

Purpose: To screen RPE65 in 187 families with Leber congenital amaurosis (LCA). Methods: Sanger sequencing and/or targeted exome sequencing was employed to identify mutations in the RPE65 gene, and intrafamilial cosegregation analysis if DNA was available. In silico analyses and splicing assay were used to evaluate the variants' pathogenicity. Results: Genetic analysis revealed 15 mutations in RPE65 in 14 pedigrees, including one splice-site mutation, one frameshift mutation, three nonsense mutations, and ten missense mutations. Of the mutations identified in RPE65, seven are novel associated with LCA, including five missense variants (c.124C>T, c.149T>C, c.340A>C, c.425A>G, and c.1399C>G) and two indel (insertions or deletions) variants (c.858+1delG and c.1181_1182insT). In vitro splicing assay was performed to evaluate the functional impact on RNA splicing of novel mutations if two of three in silico analyses were predicated to be non-pathogenic at the protein level. Among these 15 variants, 14 were classified as 'pathogenic variants,' and a variant (c.124C>T) was 'variants with uncertain significance' according to the standards and guidelines of the American College of Medical Genetics and Genomics. Conclusions: Mutations in RPE65 were responsible for 11 of the cohort of 187 Chinese families with LCA, which expands the spectrum of RPE65-related LCA in the Chinese population and potentially facilitates its clinical implementation.

Targeted next generation sequencing reveals genetic defects underlying inherited retinal disease in Iranian families.

Purpose: Inherited retinal diseases (IRDs) are clinically and genetically heterogeneous showing progressive retinal cell death which results in vision loss. IRDs include a wide spectrum of disorders, such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), cone-rod dystrophy (CRD), and Stargardt disease (STGD1). Methods: In this study, we performed targeted next-generation sequencing based on molecular inversion probes (MIPs) that allowed the sequence analysis of 108 IRD-associated genes in 50 Iranian IRD probands. Results: The sequencing and variant filtering led to the identification of putative pathogenic variants in 36 out of 50 (72%) probands. Among 36 unique variants, we identified 20 novel variants in 15 genes. Four out of 36 probands carry compound heterozygous variants, and 32 probands carry homozygous variants. Conclusions: Employing a cost-effective targeted next-generation sequencing procedure, we identified the genetic causes of different retinal disorders in the majority of Iranian families in this study.

Missense variants in the conserved transmembrane M2 protein domain of KCNJ13 associated with retinovascular changes in humans and zebrafish.

Mutations in KCNJ13 are associated with two retinal disorders; Leber congenital amaurosis (LCA) and snowflake vitreoretinal degeneration (SVD). We describe a novel fibrovascular proliferation in the retina of two affected members of a KCNJ13-related LCA family with a homozygous c.458C > T, p.(Thr153Ile) missense mutation. Optical coherence tomography retinal imaging of the kcnj13 mutant zebrafish (obelixtd15 c.502T > C, p.[Phe168Leu]) revealed a late onset retinal degeneration at 12 months, with retinal thinning and associated retinovascular changes, including increased vessel calibre and vitreous deposits. Both human and zebrafish variants are missense and located within the conserved transmembrane M2 protein domain, suggesting that disruption of this region may contribute to retinovascular changes as an additional feature to the previously described LCA phenotype. Close monitoring of other patients with similar mutations may be required to minimise the ensuing retinal damage.

How Areal Specification Shapes the Local and Interareal Circuits in a Macaque Model of Congenital Blindness.

There is little understanding of the structural underpinnings of the functional reorganization of the cortex in the congenitally blind human. Taking advantage of the extensive characterization of the macaque visual system, we examine in macaque the influence of congenital blindness resulting from the removal of the retina during in utero development. This effectively removes the normal influence of the thalamus on cortical development leading to an induced hybrid cortex (HC) combining features of primary visual and extrastriate cortex. Retrograde tracers injected in HC reveal a local, intrinsic connectivity characteristic of higher order areas and show that the HC receives a uniquely strong, purely feedforward projection from striate cortex but no ectopic inputs, except from subiculum, and entorhinal cortex. Statistical modeling of quantitative connectivity data shows that HC is relatively high in the cortical hierarchy and receives a reinforced input from ventral stream areas while the overall organization of the functional streams are conserved. The directed and weighted anophthalmic cortical graph from the present study can be used to construct dynamic and structural models. These findings show how the sensory periphery governs cortical phenotype and reveal the importance of developmental arealization for understanding the functional reorganization in congenital blindness.

CRB2 Loss in Rod Photoreceptors Is Associated with Progressive Loss of Retinal Contrast Sensitivity.

Variations in the Crumbs homolog-1 (CRB1) gene are associated with a wide variety of autosomal recessive retinal dystrophies, including early onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). CRB1 belongs to the Crumbs family, which in mammals includes CRB2 and CRB3. Here, we studied the specific roles of CRB2 in rod photoreceptor cells and whether ablation of CRB2 in rods exacerbates the Crb1-disease. Therefore, we assessed the morphological, retinal, and visual functional consequences of specific ablation of CRB2 from rods with or without concomitant loss of CRB1. Our data demonstrated that loss of CRB2 in mature rods resulted in RP. The retina showed gliosis and disruption of the subapical region and adherens junctions at the outer limiting membrane. Rods were lost at the peripheral and central superior retina, while gross retinal lamination was preserved. Rod function as measured by electroretinography was impaired in adult mice. Additional loss of CRB1 exacerbated the retinal phenotype leading to an early reduction of the dark-adapted rod photoreceptor a-wave and reduced contrast sensitivity from 3-months-of-age, as measured by optokinetic tracking reflex (OKT) behavior testing. The data suggest that CRB2 present in rods is required to prevent photoreceptor degeneration and vision loss.

Leber congenital amaurosis in a young female.

Leber congenital amaurosis is a rare congenital disorder caused by a mutation in any of several different genes that causes rod-cone dystrophy and may eventually lead to blindness. Characteristic findings on fundoscopic examination include retinal pigment migration and macular atrophy. Confirmation can be obtained using electroretinograms and the specific gene can be identified with genetic testing. No treatment is available, but multiple clinical trials are underway.

In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery.

Leber congenital amaurosis (LCA) is a severe disorder resulting in visual impairment usually starting in the first year of life. The most frequent genetic cause of LCA is an intronic mutation in CEP290 (c.2991 + 1655A > G) that creates a cryptic splice donor site resulting in the insertion of a pseudoexon (exon X) into CEP290 mRNA. Previously, we showed that naked antisense oligonucleotides (AONs) effectively restored normal CEP290 splicing in patient-derived lymphoblastoid cells. We here explore the therapeutic potential of naked and adeno-associated virus (AAV)-packaged AONs in vitro and in vivo In both cases, AON delivery fully restored CEP290 pre-mRNA splicing, significantly increased CEP290 protein levels and rescued a ciliary phenotype present in patient-derived fibroblast cells. Moreover, administration of naked and AAV-packaged AONs to the retina of a humanized mutant Cep290 mouse model, carrying the intronic mutation, showed a statistically significant reduction of exon X-containing Cep290 transcripts, without compromising the retinal structure. Together, our data highlight the tremendous therapeutic prospective of AONs for the treatment of not only CEP290-associated LCA but potentially many other subtypes of retinal dystrophy caused by splicing mutations.