Leber Congenital Amaurosis Patients Research Articles

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.

Qualifications for Retinitis Pigmentosa and Leber Congenital Amaurosis Patients for Adeno-Associated Viral Gene-Replacement Therapy Clinical Trials

Introduction: This study identifies Retinitis Pigmentosa (RP) and Leber Congenital Amaurosis (LCA) patients at an Inherited Retinal Disease (IRD) clinic that qualify for ongoing or FDA-approved Adeno-Associated Virus (AAV) gene-replacement therapies. The goal is to demonstrate the benefits of genetic testing at the initial evaluation. Method: A database for RP and LCA patients was curated and clinicaltrials.gov was used to search all ongoing or approved gene-replacement therapies between 1 January 2022 – 1 January 2023. Patients were evaluated for qualification based on the inclusion/exclusion criteria set by each trial. Results: 199 RP and 31 LCA patients were included in the study. Our team identified six AAV gene-replacement therapy clinical trials and the FDA-approved Luxturna®. One hundred fifty-five patients underwent genetic testing and 89 patients had a pathogenic variant identified. A total of 15 patients qualified for one of the proposed trials. Three patients had a biallelic RPE65 mutation and two of them qualified for Luxturna®. All 11 patients with an RPGR mutation qualified for one of the three clinical trials that focused on this gene. Three patients had a c.2991+1655A>G mutation in CEP290 and two of them qualified one of two clinical trials for this gene. Conclusion: Overall, ~10% of patients who had genetic testing qualified for one of the reviewed therapies. A total of 15 patients qualified for an AAV gene-replacement therapy. This study highlights the importance of promoting genetic testing for IRD patients, the need for earlier disease evaluation and the value of continual monitoring of disease progression.

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

BackgroundVoretigene 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.MethodsSeven 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.ResultsA 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.DiscussionIn 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.

Genetic and Clinical Profile of Retinopathies Due to Disease-Causing Variants in Leber Congenital Amaurosis (LCA)-Associated Genes in a Large German Cohort.

To report the spectrum of Leber congenital amaurosis (LCA) associated genes in a large German cohort and to delineate their associated phenotype. Local databases were screened for patients with a clinical diagnosis of LCA and for patients with disease-causing variants in known LCA-associated genes independent of their clinical diagnosis. Patients with a mere clinical diagnosis were invited for genetic testing. Genomic DNA was either analyzed in a diagnostic-genetic or research setup using various capture panels for syndromic and non-syndromic IRD (inherited retinal dystrophy) genes. Clinical data was obtained mainly retrospectively. Patients with genetic and phenotypic information were eventually included. Descriptive statistical data analysis was performed. A total of 105 patients (53 female, 52 male, age 3-76 years at the time of data collection) with disease-causing variants in 16 LCA-associated genes were included. The genetic spectrum displayed variants in the following genes: CEP290 (21%), CRB1 (21%), RPE65 (14%), RDH12 (13%), AIPL1 (6%), TULP1 (6%), and IQCB1 (5%), and few cases harbored pathogenic variants in LRAT, CABP4, NMNAT1, RPGRIP1, SPATA7, CRX, IFT140, LCA5, and RD3 (altogether accounting for 14%). The most common clinical diagnosis was LCA (53%, 56/105) followed by retinitis pigmentosa (RP, 40%, 42/105), but also other IRDs were seen (cone-rod dystrophy, 5%; congenital stationary night blindness, 2%). Among LCA patients, 50% were caused by variants in CEP290 (29%) and RPE65 (21%), whereas variants in other genes were much less frequent (CRB1 11%, AIPL1 11%, IQCB1 9%, and RDH12 7%, and sporadically LRAT, NMNAT1, CRX, RD3, and RPGRIP1). In general, the patients showed a severe phenotype hallmarked by severely reduced visual acuity, concentric narrowing of the visual field, and extinguished electroretinograms. However, there were also exceptional cases with best corrected visual acuity as high as 0.8 (Snellen), well-preserved visual fields, and preserved photoreceptors in spectral domain optical coherence tomography. Phenotypic variability was seen between and within genetic subgroups. The study we are presenting pertains to a considerable LCA group, furnishing valuable comprehension of the genetic and phenotypic spectrum. This knowledge holds significance for impending gene therapeutic trials. In this German cohort, CEP290 and CRB1 are the most frequently mutated genes. However, LCA is genetically highly heterogeneous and exhibits clinical variability, showing overlap with other IRDs. For any therapeutic gene intervention, the disease-causing genotype is the primary criterion for treatment access, but the clinical diagnosis, state of the retina, number of to be treated target cells, and the time point of treatment will be crucial.

Molecular background of Leber congenital amaurosis in a Polish cohort of patients—novel variants discovered by NGS

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophies and the most frequent cause of congenital blindness in children. To date, 25 genes have been implicated in the pathogenesis of this rare disorder. Performing an accurate molecular diagnosis is crucial as gene therapy is becoming available. This study aimed to report the molecular basis of Leber congenital amaurosis, especially novel and rare variants in 27 Polish families with a clinical diagnosis of LCA fully confirmed by molecular analyses. Whole exome sequencing or targeted next-generation sequencing (NGS) of inherited retinal dystrophies-associated (IRD) genes was applied to identify potentially pathogenic variants. Bidirectional Sanger sequencing and quantitative PCR (qPCR) were carried out for validation and segregation analysis of the variants identified within the families. We identified 28 potentially pathogenic variants, including 11 novel, in 8 LCA genes: CEP290, CRB1, GUCY2D, NMNAT1, RPGRIP1, CRX, LRAT1, and LCA5. This study expands the mutational spectrum of the LCA genes. Moreover, these results, together with the conclusions from our previous studies, allow us to point to the most frequently mutated genes and variants in the Polish cohort of LCA patients.

Mobility test to assess functional vision in dark-adapted patients with Leber congenital amaurosis

BackgroundInherited retinal degenerations (IRDs) affect daylight and night vision to different degrees. In the current work, we devise a method to quantify mobility under dark-adapted conditions in patients with severe childhood blindness due to Leber congenital amaurosis (LCA). Mobility thresholds from two different LCA genotypes are compared to dark-adapted vision measurements using the full-field stimulus test (FST), a conventional desktop outcome measure of rod vision.MethodsA device consisting of vertical LED strips on a plane resembling a beaded curtain was programmed to produce a rectangular pattern target defining a ‘door’ of varying luminance that could appear at one of three positions. Mobility performance was evaluated by letting the subject walk from a fixed starting position ~ 4 m away from the device with instructions to touch the door. Success was defined as the subject touching within the ‘door’ area. Ten runs were performed and the process was repeated for different levels of luminance. Tests were performed monocularly in dark-adapted and dilated eyes. Results from LCA patients with the GUCY2D and CEP290 genotypes and normal subjects were analyzed using logistic regression to estimate the mobility threshold for successful navigation. The relation of thresholds for mobility, FST and visual acuity were quantified using linear regression.ResultsNormal subjects had mobility thresholds near limits of dark-adapted rod vision. GUCY2D-LCA patients had a wide range of mobility thresholds from within 1 log of normal to greater than 8 log abnormal. CEP290-LCA patients had abnormal mobility thresholds that were between 5 and 6 log from normal. Sensitivity loss estimates using FST related linearly to the mobility thresholds which were not correlated with visual acuity.ConclusionsThe mobility task we developed can quantify functional vision in severely disabled patients with LCA. Taken together with other outcome measures of rod and cone photoreceptor-mediated vision, dark-adapted functional vision should provide a more complete understanding of the natural history and effects of treatment in patients with LCA.

PacMAGI: A pipeline including accurate indel detection for the analysis of PacBio sequencing data applied to RPE65

Third generation sequencing methods, like PacBio, provide information about structural variants, introns, enhancers and promoters. We developed an automated pipeline, called PacMAGI, including quality control, alignment, SNV, INDELs, structural variant calling, phasing, annotation and variant interpretation, for the analysis of PacBio data for any target region. Bi-allelic mutations in the RPE65 gene are associated with different inherited retinal dystrophies, such as Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Diagnostic panel-based NGS analysis is performed on coding regions and intron/exon junctions of genes. To obtain a more conclusive diagnosis, we applied PacMAGI to obtain a second hit on RPE65 in LCA or RP patients who showed a single heterozygous variant by NGS. We used PacBio to sequence the full gene and identify putative second-hits in intronic, problematic and promoter regions. All variants identified in the diagnostic setting with NGS were correctly detected by the pipeline, and thanks to our custom algorithm for INDELs, a previously undetected ‘Pathogenic’ frameshift variant was found in a RP patient already identified to carry a ‘Likely Pathogenic’ variant.

In vivo base editing rescues cone photoreceptors in a mouse model of early-onset inherited retinal degeneration

Leber congenital amaurosis (LCA) is the most common cause of inherited retinal degeneration in children. LCA patients with RPE65 mutations show accelerated cone photoreceptor dysfunction and death, resulting in early visual impairment. It is therefore crucial to develop a robust therapy that not only compensates for lost RPE65 function but also protects photoreceptors from further degeneration. Here, we show that in vivo correction of an Rpe65 mutation by adenine base editor (ABE) prolongs the survival of cones in an LCA mouse model. In vitro screening of ABEs and sgRNAs enables the identification of a variant that enhances in vivo correction efficiency. Subretinal delivery of ABE and sgRNA corrects up to 40% of Rpe65 transcripts, restores cone-mediated visual function, and preserves cones in LCA mice. Single-cell RNA-seq reveals upregulation of genes associated with cone phototransduction and survival. Our findings demonstrate base editing as a potential gene therapy that confers long-lasting retinal protection.

Clinical exome sequencing facilitates the understanding of genetic heterogeneity in Leber congenital amaurosis patients with variable phenotype in southern India

BackgroundLeber congenital amaurosis (LCA), primarily characterized by retinal degeneration is the most severe form of inherited retinal dystrophy (IRD) responsible for congenital blindness. The presence of phenotypic heterogeneity makes the diagnosis of LCA challenging, especially in the absence of pronounced disease pathognomonic, yet it can be well comprehended by employing molecular diagnosis. Therefore, the present study aimed to reveal the causative mutations in ten LCA patients with variable phenotypes using clinical exome sequencing (CES).MethodsCES was performed in ten unrelated LCA patients. Ophthalmic information and family history of all patients were obtained to make a meaningful interpretation. The clinical exome data was analyzed and prioritized using a bioinformatics pipeline to identify mutations, which was further validated by Sanger sequencing. Segregation analysis was also performed on available family members.ResultsCES led to the identification of causative mutations in nine LCA patients. Seven patients harbored a mutation in six LCA candidate genes, including RPE65, LCA5 (n = 2), CRX, PRPH2, CEP290, and ALMS1, while two patients possess a mutation in IFT80 and RP1, known to cause other diseases. Three novel mutations in LCA5 (c.1823del), CRX (c.848del) and CEP290 (c.2483G > T) were identified. The current study reports for the first time, a mutation in PRPH2, CEP290, and ALMS1 from the Indian population. Additionally, we observed a novel association of LCA phenotype with IFT80 known to cause Jeune syndrome. Based on the genetic finding, the patient AS09, who harbored a mutation in the RP1 gene, was re-diagnosed with early-onset retinitis pigmentosa.ConclusionIn conclusion, the results underline the importance of CES in clinically diagnosed LCA patients with variable phenotypes. The correlation between mutations in candidate genes and clinical phenotypes, helps to refine the clinical diagnosis. However, molecular evaluation with a larger cohort of LCA patients is needed for better understanding of the mutational spectrum in southern India.

Genetically-Caused Blindness Reversed with Single Injection of Antisense Therapeutic

Clinical OMICsVol. 8, No. 3 NewsFree AccessGenetically-Caused Blindness Reversed with Single Injection of Antisense TherapeuticPublished Online:18 Jun 2021https://doi.org/10.1089/clinomi.08.03.04AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail A patient with a genetic form of childhood blindness gained vision for more than a year after receiving a single injection of an experimental antisense oligonucleotide, sepofarsen, into the eye.The clinical trial was conducted by researchers at the University of Pennsylvania. According to their study, which appeared in Nature Medicine, the treatment led to marked changes at the fovea, the most important locus of human central vision.Almost a dozen antisense treatments have been approved by the FDA to date. Almost all of these are for orphan diseases, including the drug used in this study. It targets Leber congenital amaurosis (LCA), but is specifically designed for LCA patients with the CEP290 mutation – one of the more commonly implicated gene variants. Patients with this form of LCA suffer from severe visual impairment, typically beginning in infancy.In an international clinical trial led by Penn Medicine researchers Artur Cideciyan, Ph.D., and Samuel Jacobson, M.D., Ph.D., participants received an intraocular injection of sepofarsen. This oligonucleotide works by increasing normal CEP290 protein levels in the eye's photoreceptors.In a previous study, Cideciyan, Jacobson, and collaborators found that injections of sepofarsen every three months resulted in continued vision gains in ten patients.The eleventh patient, whose results are reported in this study, had only one injection and was examined over a 15-month period. Prior to treatment, the patient had reduced visual acuity, small visual fields, and no night vision.After a single injection, more than a dozen measurements of visual function and retinal structure showed large improvements supporting a biological effect from the treatment, which were retained at month 15. The patient's vision reached a peak effect after month two.FiguresReferencesRelatedDetails Volume 8Issue 3May 2021 InformationCopyright © GEN PublishingTo cite this article:Clinical OMICs.May 2021.5-5.http://doi.org/10.1089/clinomi.08.03.04Published in Volume: 8 Issue 3: June 18, 2021PDF download

Whole-exome sequencing identifies two novel ALMS1 mutations in Indian patients with Leber congenital amaurosis

Leber congenital amaurosis (LCA) is a severe autosomal recessive retinal degenerative disease. The current study describes exome sequencing results for two unrelated Indian LCA patients carrying novel nonsense p.(Glu636*) and frameshift p.(Pro2281Leufs*63) mutations in the ALMS1 gene. Although ALMS1 gene mutations are associated with Alstrom syndrome (AS), the current patients did not exhibit typical syndromic features of AS. These data suggest that ALMS1 should be included in the candidate gene panel for LCA to improve diagnostic efficiency.

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.

Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids.

SummaryMutations in the photoreceptor transcription factor gene cone-rod homeobox (CRX) lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs48 mutation, we established an in vitro model of CRX-LCA in retinal organoids that showed defective photoreceptor maturation by histology and gene profiling, with diminished expression of visual opsins. Adeno-associated virus (AAV)-mediated CRX gene augmentation therapy partially restored photoreceptor phenotype and expression of phototransduction-related genes as determined by single-cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying K88N mutation revealed the loss of opsin expression as a common phenotype, which was alleviated by AAV-mediated augmentation of CRX. Our studies provide a proof-of-concept for developing gene therapy of dominant CRX-LCA and other CRX retinopathies.

Comprehensive structure-function analysis of causative variants in retinal pigment epithelium specific 65kDa protein associated Leber Congenital Amaurosis.

A recent study published to screen RPE65 in 187 families with Leber Congenital Amaurosis (LCA) by Zilin Zhong in 2019. There are seven novel variants were identified in RPE65, which was associated with LCA, but among only five were missense mutations [(c.124C > T, p.(Leu42Phe), c.149T > C, p. (Phe50Ser), c.340A > C, p.(Asn114His), c.425A > G, p.(Asp142Gly) and c.1399C > G, p.(Pro467Ala)] in the Chinese population and potentially facilitates its clinical implementation. Further in-continuation of this study to the target of five novel missense mutations were the analysis of both structural and functional impact by the molecular dynamics and simulation. The result of five missense mutations might in critical structural alterations of RPE65 protein, disrupt its membrane association or rescue the activity of enzyme due to thermodynamics stability, and for this reason impair its isomerohydrolase activity, resulting in retinal dystrophy. These observations suggest that the reduced protein stability and altered subcellular localization of RPE65 might signify a mechanism for these mutations to lead to vision loss in LCA patients.

Generation and Characterization of Induced Pluripotent Stem Cells and Retinal Organoids From a Leber's Congenital Amaurosis Patient With Novel RPE65 Mutations.

RPE65-associated Leber congenital amaurosis (LCA) is one of highly heterogeneous, early onset, severe retinal dystrophies with at least 130 gene mutation sites identified. Their pathogenicity has not been directly clarified due to lack of diseased cells. Here, we generated human-induced pluripotent stem cells (hiPSCs) from one putative LCA patient carrying two novel RPE65 mutations with c.200T>G (p.L67R) and c.430T>C (p.Y144H), named RPE65-hiPSCs, which were confirmed to contain the same mutations. The RPE65-hiPSCs presented typical morphological features with normal karyotype, expressed pluripotency markers, and developed teratoma in NOD-SCID mice. Moreover, the patient hiPSCs were able to differentiate toward retinal lineage fate and self-form retinal organoids with layered neural retina. All major retinal cell types including photoreceptor and retinal pigment epithelium (RPE) cells were also acquired overtime. Compared to healthy control, RPE cells from patient iPSCs had lower expression of RPE65, but similar phagocytic activity and VEGF secretion level. This study provided the valuable patient specific, disease targeted retinal organoids containing photoreceptor and RPE cells, which would facilitate the study of personalized pathogenic mechanisms of disease, drug screening, and cell replacement therapy.

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

PurposeTo 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.MethodsPatients 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.ResultsThe 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.ConclusionsCone 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.

Gene and Cell Therapy for AIPL1-Associated Leber Congenital Amaurosis: Challenges and Prospects.

Leber congenital amaurosis (LCA) caused by AIPL1 mutations is one of the most severe forms of inherited retinal degeneration (IRD). The rapid and extensive photoreceptor degeneration challenges the development of potential treatments. Nevertheless, preclinical studies show that both gene augmentation and photoreceptor transplantation can regenerate and restore retinal function in animal models of AIPL1-associated LCA. However, questions regarding long-term benefit and safety still remain as these therapies advance towards clinical application. Ground-breaking advances in stem cell technology and genome editing are examples of alternative therapeutic approaches and address some of the limitations associated with previous methods. The continuous development of these cutting-edge biotechnologies paves the way towards a bright future not only for AIPL1-associated LCA patients but also other forms of IRD.

Generation of a human iPSC line from a patient with Leber congenital amaurosis caused by mutation in AIPL1

The human induced pluripotent stem cell (hiPSC) line, derived from dermal fibroblasts from Leber congenital amaurosis patient with homozygous mutation c.265 T > C, p.Cys89Arg in aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) was generated by Sendai virus reprogramming. The generated hiPSC line was free of Sendai virus genes, had stable karyotype, carried the homozygous mutation, was immunopositive to pluripotency markers and able to generate all three germ layers upon embryoid body formation.Resource table.Unlabelled TableUnique stem cell line identifierCIPFi001-AAlternative name(s) of stem cell lineLCA –FiPS4F1InstitutionResearch Center Principe Felipe, Eduardo Primo Yufera 3, Valencia, SpainContact information of distributorDunja Lukovic dlukovic@cipf.esType of cell lineiPSCOriginHumanAdditional origin infoAge:31Sex: FemaleEthnicity if known: CaucasianCell SourceDermal fibroblastsClonalityClonalMethod of reprogrammingSendai virusGenetic modificationN/AType of modificationN/AAssociated diseaseLeber congenital amaurosisGene/locusAIPL1 (NM_014336.3), Chr17: g.6337250A > G (hg19); Ex.2 c.[265 T > C];[265 T > C], p.[Cys89Arg];[Cys89Arg]Method of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateApril 2017Cell line repository/bankhttp://www.isciii.es/ISCIII/es/contenidos/fd-el-instituto/fd-organizacion/fd-estructura-directiva/fd-subdireccion-general-investigacion-terapia-celular-medicina-regenerativa/fd-centros-unidades/fd-banco-nacional-lineas-celulares/fd-lineas-celulares-disponibles/lineas-de-celulas-iPS.shtmlEthical approvalEthics Review Board-competent authority approval obtained by the Valencian Authority for Stem Cell Research (Approval number: S:177–15)

Mutation in the Zebrafish cct2 Gene Leads to Abnormalities of Cell Cycle and Cell Death in the Retina: A Model of CCT2-Related Leber Congenital Amaurosis.

PurposeThe compound heterozygous mutations in the β subunit of chaperonin containing TCP-1 (CCT), encoded by CCT2, lead to the Leber congenital amaurosis (LCA). In this study, a cct2 mutant line of zebrafish was established to investigate the role of CCT2 mutations in LCA in vertebrates.MethodsA cct2 mutant zebrafish line was produced using the CRISPR-Cas9 system. Changes in the eyes of developing wild-type and mutant larvae were monitored using microscopy, immunostaining, TUNEL, and EdU assays. Phenotypic rescue of mutant phenotype was investigated by injection of CCT2 RNA into zebrafish embryos.ResultsThe cct2 mutation (L394H-7del) led to the synthesis of a mutated cctβ protein with the L394H replacement and deletion of 7 amino acid residues (positions 395–401). The homozygous cct2-L394H-7del mutant exhibited a small eye phenotype at 2 days post fertilization (dpf) and was embryonically lethal after 5 dpf. In homozygous cct2-L394H-7del mutants, the retinal ganglion cell differentiation was attenuated, retinal cell cycle was affected, and the neural retinal cell death was significantly increased at 2 dpf compared with wild-type. Injection of RNA encoding wild-type human CCTβ rescued the small eye phenotype, reduced retinal cell death, and restored the levels of CCTβ protein and the major client protein Gβ1 that were significantly reduced in the homozygous cct2-L394H-7del mutant compared with wild-type. These results indicate that cct2 plays an essential role in retinal development by regulating the cell cycle.ConclusionsThe retinal pathology observed in the homozygous cct2-L394H-7del mutants resembles the retinal pathology of human LCA patients.

Rpgrip1 is required for rod outer segment development and ciliary protein trafficking in zebrafish

Mutations in the RPGR-interacting protein 1 (RPGRIP1) gene cause recessive Leber congenital amaurosis (LCA), juvenile retinitis pigmentosa (RP) and cone-rod dystrophy. RPGRIP1 interacts with other retinal disease-causing proteins and has been proposed to have a role in ciliary protein transport; however, its function remains elusive. Here, we describe a new zebrafish model carrying a nonsense mutation in the rpgrip1 gene. Rpgrip1homozygous mutants do not form rod outer segments and display mislocalization of rhodopsin, suggesting a role for RPGRIP1 in rhodopsin-bearing vesicle trafficking. Furthermore, Rab8, the key regulator of rhodopsin ciliary trafficking, was mislocalized in photoreceptor cells of rpgrip1 mutants. The degeneration of rod cells is early onset, followed by the death of cone cells. These phenotypes are similar to that observed in LCA and juvenile RP patients. Our data indicate RPGRIP1 is necessary for rod outer segment development through regulating ciliary protein trafficking. The rpgrip1 mutant zebrafish may provide a platform for developing therapeutic treatments for RP patients.