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.

PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics.

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.

Compound heterozygous mutations in a mouse model of Leber congenital amaurosis reveal the role of CCT2 in photoreceptor maintenance.

TRiC/CCT is a chaperonin complex required for the folding of cytoplasmic proteins. Although mutations in each subunit of TRiC/CCT are associated with various human neurodegenerative diseases, their impact in mammalian models has not yet been examined. A compound heterozygous mutation in CCT2 (p.[Thr400Pro]; p.[Arg516His]) is causal for Leber congenital amaurosis. Here, we generate mice carrying each mutation and show that Arg516His (R516H) homozygosity causes photoreceptor degeneration accompanied by a significant depletion of TRiC/CCT substrate proteins in the retina. In contrast, Thr400Pro (T400P) homozygosity results in embryonic lethality, and the compound heterozygous mutant (T400P/R516H) mouse showed aberrant cone cell lamination and died 2 weeks after birth. Finally, CCDC181 is identified as a interacting protein for CCTß protein, and its localization to photoreceptor connecting cilia is compromised in the mutant mouse. Our results demonstrate the distinct impact of each mutation in vivo and suggest a requirement for CCTß in ciliary maintenance.

Four Unique Genetic Variants in Three Genes Account for 62.7% of Early-Onset Severe Retinal Dystrophy in Chile: Diagnostic and Therapeutic Consequences.

Leber congenital amaurosis (LCA)/early-onset severe retinal dystrophy (EOSRD) stand as primary causes of incurable childhood blindness. This study investigates the clinical and molecular architecture of syndromic and non-syndromic LCA/EOSRD within a Chilean cohort (67 patients/60 families). Leveraging panel sequencing, 95.5% detection was achieved, revealing 17 genes and 126 variants (32 unique). CRB1, LCA5, and RDH12 dominated (71.9%), with CRB1 being the most prevalent (43.8%). Notably, four unique variants (LCA5 p.Glu415*, CRB1 p.Ser1049Aspfs*40 and p.Cys948Tyr, RDH12 p.Leu99Ile) constituted 62.7% of all disease alleles, indicating their importance for targeted analysis in Chilean patients. This study underscores a high degree of inbreeding in Chilean families affected by pediatric retinal blindness, resulting in a limited mutation repertoire. Furthermore, it complements and reinforces earlier reports, indicating the involvement of ADAM9 and RP1 as uncommon causes of LCA/EOSRD. These data hold significant value for patient and family counseling, pharmaceutical industry endeavors in personalized medicine, and future enrolment in gene therapy-based treatments, particularly with ongoing trials (LCA5) or advancing preclinical developments (CRB1 and RDH12).

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.

Human CRB1 and CRB2 form homo- and heteromeric protein complexes in the retina.

Crumbs homolog 1 (CRB1) is one of the key genes linked to retinitis pigmentosa and Leber congenital amaurosis, which are characterized by a high clinical heterogeneity. The Crumbs family member CRB2 has a similar protein structure to CRB1, and in zebrafish, Crb2 has been shown to interact through the extracellular domain. Here, we show that CRB1 and CRB2 co-localize in the human retina and human iPSC-derived retinal organoids. In retina-specific pull-downs, CRB1 was enriched in CRB2 samples, supporting a CRB1-CRB2 interaction. Furthermore, novel interactors of the crumbs complex were identified, representing a retina-derived protein interaction network. Using co-immunoprecipitation, we further demonstrate that human canonical CRB1 interacts with CRB1 and CRB2, but not with CRB3, which lacks an extracellular domain. Next, we explored how missense mutations in the extracellular domain affect CRB1-CRB2 interactions. We observed no or a mild loss of CRB1-CRB2 interaction, when interrogating various CRB1 or CRB2 missense mutants in vitro. Taken together, our results show a stable interaction of human canonical CRB2 and CRB1 in the retina.

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.

Characteristics of Eyes With CRB1-Associated EOSRD/LCA: Age-Related Changes.

PURPOSE: To evaluate ocular and retinal features of CRB1-associated early onset severe retinal dystrophy/Leber congenital amaurosis (EOSRD/LCA) for age-related changes. DESIGN: Retrospective cohort study. METHODS: Sixteen pediatric patients with biallelic CRB1 EOSRD/LCA who had been followed for up to 18 years were reviewed. Results of comprehensive ophthalmic examinations-including visual acuity, refractive error, dark-adapted visual threshold, Goldmann perimetry, and macular optical coherence tomography (OCT)-were analyzed for significant age-related changes using mixed-effects models. RESULTS: Visual acuity dark-adapted visual sensitivity, and area of seeing visual field (all subnormal from the earliest ages recorded) declined with increasing age. Hyperopia was stable through childhood and adolescence. In CRB1 EOSRD/LCA, OCT extrafoveal inner and outer laminar thicknesses exceeded those in controls but varied little with age, and foveal metrics (depth, breadth, thickness at rim) differed significantly from those in controls, but variations in foveal metrics were not associated with declines in acuity. CONCLUSIONS: From the youngest ages, retinal and visual function is significantly subnormal and becomes progressively compromized. A goal of future therapies should be intervention at young ages, when there is more function to be rescued.

Genotype-Phenotype of CRB1-Associated Early-Onset Retinal Dystrophy: Novel Insights on Retinal Architecture and Therapeutic Window for Clinical Trials.

Purpose: The purpose of this study was to investigate the genotypic and phenotypic characteristics of CRB1-associated early onset retinal dystrophy (CRB1-eoRD) and retinal architecture by swept-source optical coherence tomography (SS-OCT). Methods: Eleven probands with CRB1-eoRD were recruited. Clinical information, genetic analysis, and comprehensive ophthalmic examinations including SS-OCT and SS-OCT angiography (SS-OCTA) were conducted. Results: A total of 81.8% (9/11) of CRB1-eoRD presented as Leber congenital amaurosis (LCA). Common clinical manifestations included coin-like yellow-white retinal spots (20/22, 90.9%) and para-arteriolar retinal pigment epithelial retention (12/22, 54.5%). Nineteen different CRB1 variants were detected in our case series, including 12 missense, 3 frameshifts, 3 nonsense, and 1 splicing. Of them, 12 variants had been reported, and 7 were novel. SS-OCT showed thinner central macula (the LCA group, P < 0.0001), thicker total retina (P < 0.0001), thinner outer retina (P < 0.05), and thicker inner retina (P < 0.0001) compared with the healthy control. The inner/outer (I/O) retina thickness ratio of CRB1-eoRD was 3.0, higher than the healthy control of 1.2 and other inherited retinal diseases (IRDs) of 2.2 (P < 0.0001 and P = 0.0027, respectively). SS-OCTA revealed an increased vascular density and perfusion area of the superficial vascular complex and deep vascular complex in CRB1-eoRD. Conclusions: LCA emerges as a frequently occurring phenotype in CRB1-eoRD. The unique features of SS-OCT and SS-OCTA are illustrated, and the novel biomarker, I/O ratio, may facilitate early diagnosis. The insights gained from this study hold significant value in determining the treatment window for potential forthcoming CRB1 gene therapy.

Autozygome-guided exome-first study in a consanguineous cohort with early-onset retinal disease uncovers an isolated RIMS2 phenotype and a retina-enriched RIMS2 isoform.

Leber congenital amaurosis (LCA) and early-onset retinal degeneration (EORD) are inherited retinal diseases (IRD) characterized by early-onset vision impairment. Herein, we studied 15 Saudi families by whole exome sequencing (WES) and run-of-homozygosity (ROH) detection via AutoMap in 12/15 consanguineous families. This revealed (likely) pathogenic variants in 11/15 families (73%). A potential founder variant was found in RPGRIP1. Homozygous pathogenic variants were identified in known IRD genes (ATF6, CRB1, CABP4, RDH12, RIMS2, RPGRIP1, SPATA7). We established genotype-driven clinical reclassifications for ATF6, CABP4, and RIMS2. Specifically, we observed isolated IRD in the individual with the novel RIMS2 variant, and we found a retina-enriched RIMS2 isoform conserved but not annotated in mouse. The latter illustrates potential different phenotypic consequences of pathogenic variants depending on the particular tissue/cell-type specific isoforms they affect. Lastly, a compound heterozygous genotype in GUCY2D in one non-consanguineous family was demonstrated, and homozygous variants in novel candidate genes ATG2B and RUFY3 were found in the two remaining consanguineous families. Reporting these genes will allow to validate them in other IRD cohorts. Finally, the missing heritability of the two unsolved IRD cases may be attributed to variants in non-coding regions or structural variants that remained undetected, warranting future WGS studies.

Generation of Leber congenital amaurosis, type 12 patient-specific induced pluripotent stem cell line (LVPEIi006-A), harboring a homozygous mutation in RD3.

Leber congenital amaurosis (LCA) is a congenital, early onset, autosomal recessive inherited retinal disease (IRD). This report describes an LCA12 patient-specific iPSC line (LVPEIi006-A), generated by the reprogramming of dermal fibroblasts using integration-free episomal plasmids.This disease-specific iPSC model carries a homozygous point mutation in RD3, within the donor splice site at the end of exon 2 (c.296 + 1G > A). The stable line at passage 15 has displayed a normal colony morphology, expressed multiple stemness and pluripotency markers, lost all transgenes, differentiated into cell types of all three germ layers, and maintained a normal karyotype.

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.

CRB1-associated retinal degeneration is dependent on bacterial translocation from the gut.

The Crumbs homolog 1 (CRB1) gene is associated with retinal degeneration, most commonly Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Here, we demonstrate that murine retinas bearing the Rd8 mutation of Crb1 are characterized by the presence of intralesional bacteria. While normal CRB1 expression was enriched in the apical junctional complexes of retinal pigment epithelium and colonic enterocytes, Crb1 mutations dampened its expression at both sites. Consequent impairment of the outer blood retinal barrier and colonic intestinal epithelial barrier in Rd8 mice led to the translocation of intestinal bacteria from the lower gastrointestinal (GI) tract to the retina, resulting in secondary retinal degeneration. Either the depletion of bacteria systemically or the reintroduction of normal Crb1 expression colonically rescued Rd8-mutation-associated retinal degeneration without reversing the retinal barrier breach. Our data elucidate the pathogenesis of Crb1-mutation-associated retinal degenerations and suggest that antimicrobial agents have the potential to treat this devastating blinding disease.

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.

The Clinical Findings, Pathogenic Variants, and Gene Therapy Qualifications Found in a Leber Congenital Amaurosis Phenotypic Spectrum Patient Cohort.

This retrospective study examines the clinical characteristics and underlying genetic variants that exist in a Leber congenital amaurosis (LCA) patient cohort evaluated at the inherited retinal disease (IRD) clinic at the University of Minnesota (UMN)/M Health System. Our LCA cohort consisted of 33 non-syndromic patients and one patient with Joubert syndrome. We report their relevant history, clinical findings, and genetic testing results. We monitored disease presentation utilizing ocular coherence tomography (OCT) and fundus autofluorescence (FAF). Electroretinogram testing (ERG) was performed in patients when clinically indicated. Next-generation sequencing (NGS) and genetic counseling was offered to all evaluated patients. Advanced photoreceptor loss was noted in 85.7% of the subjects. All patients who underwent FAF had findings of either a ring of macular hypo/hyper AF or peripheral hypo-AF. All patients had abnormal ERG findings. A diagnostic genetic test result was identified in 74.2% of the patients via NGS single-gene testing or panel testing. Two patients in our cohort qualified for Luxturna® and both received treatment at the time of this study. These data will help IRD specialists to understand the genetic variants and clinical presentations that characterize our patient population in the Midwest region of the United States.

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.

Comparison of Worldwide Disease Prevalence and Genetic Prevalence of Inherited Retinal Diseases and Variant Interpretation Considerations.

One of the considerations in planning the development of novel therapeutic modalities is disease prevalence that is usually defined by studying large national/regional populations. Such studies are rare and might suffer from inaccuracies and challenging clinical characterization in heterogeneous diseases, such as inherited retinal diseases (IRDs). Here we collected reported disease prevalence information on various IRDs in different populations. The most common IRD, retinitis pigmentosa, has an average disease prevalence of ∼1:4500 individuals, Stargardt disease ∼1:17,000, Usher syndrome ∼1:25,000, Leber congenital amaurosis ∼1:42,000, and all IRDs ∼1:3450. We compared these values to genetic prevalence (GP) calculated based on allele frequency of autosomal-recessive IRD mutations. Although most values did correlate, some differences were observed that can be explained by discordant, presumably null mutations that are likely to be either nonpathogenic or hypomorphic. Our analysis highlights the importance of performing additional disease prevalence studies and to couple them with population-dependent allele frequency data.

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.

Clinical and Genetic Characterization of RDH12-Retinal Dystrophy in a South American Cohort.

PURPOSE: To characterize the largest cohort of individuals with retinol dehydrogenase 12 (RDH12)-retinal dystrophy to date, and the first one from South America. DESIGN: Retrospective multicenter international study. SUBJECTS: Seventy-eight patients (66 families) with an inherited retinal dystrophy and biallelic variants in RDH12. METHODS: Review of clinical notes, ophthalmic images, and molecular diagnosis. MAIN OUTCOME MEASURES: Visual function, retinal imaging, and characteristics were evaluated and correlated. RESULTS: Thirty-seven individuals self-identified as Latino (51%) and 34 as White (47%). Sixty-nine individuals (88%) had Leber congenital amaurosis (LCA)/early-onset severe retinal dystrophy. Macular and midperipheral atrophy were seen in all patients from 3 years of age. A novel retinal finding was a hyperautofluorescent ring in 2 young children with LCA. Thirty-nine patients (50%) had subsequent visits, with mean follow-up of 6.8 ± 7.3 (range, 0-29) years. Eight variants (21%) were previously unreported, and the most frequent variant was c.295C>A, p.Leu99Ile, present in 52 alleles of 32 probands. Individuals with LCA homozygous for p.Leu99Ile (31%) had a later age of onset, a slower rate of best-corrected visual acuity decrease, the largest percentage of patients with mild visual impairment, and were predicted to reach legal blindness at an older age than the rest of the cohort. CONCLUSIONS: By describing the largest molecularly confirmed cohort to date, improved understanding of disease progression was possible. Our detailed characterization aims to support research and the development of novel therapies that may have the potential to reduce or prevent vision loss in individuals with RDH12-associated retinal dystrophy. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures.

Update on gene therapies in pediatric ophthalmology.

Rare eye diseases encompass a broad spectrum of genetic anomalies with or without additional extraocular manifestations. Genetic eye disorders in pediatric patients often lead to severe visual impairments. Therefore, a challenge of gene therapy is to provide better vision to these affected children. In recent years, inherited retinal diseases, inherited optic neuropathies, and corneal dystrophies have dominated discussions to establish gene and cell replacement therapies for these diseases. Gene therapy involves the transfer of genetic material to remove, replace, repair, or introduce a gene, or to overexpress a protein, whose activity would have a therapeutic impact. For the majority of anterior segment diseases, these studies are still emerging at a preclinical stage; however, for inherited retinal disorders, translation has been reached, leading to the introduction of the first gene therapies into clinical practice. In the past decade, the first gene therapy for biallelic RPE65-mediated inherited retinal dystrophy has been developed and the FDA and EMA both approved ocular gene therapy. Other promising approaches by intravitreal injection have been investigated such as in CEP290-Leber congenital amaurosis. Various techniques of gene therapies include gene supplementation, CRISPR-based genome editing, as well as RNA modulation and optogenetics. Optogenetic therapies deliver light-activated ion channels to surviving retinal cell types in order to restore photosensitivity. Beyond retinal function, ataluren, a nonsense mutation suppression therapy, enables ribosomal read-through of mRNA containing premature termination codons, resulting in the production of a full-length protein. An ophthalmic formulation was recently evaluated with the aim of repairing corneal damage, pending new clinical studies. However, various congenital disorders exhibit severe developmental defects or cell loss at birth, limiting the potential for viral gene therapy. Therefore mutation-independent strategies seem promising for maintaining the survival of photoreceptors or for restoring visual function. Restoring vision in children with gene therapy continues to be a challenge in ophthalmology. © 2023 Published by Elsevier Masson SAS on behalf of French Society of Pediatrics.