Targeting ON-bipolar cells by AAV gene therapy stably reverses LRIT3-congenital stationary night blindness.
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ABSTRACT:
SUBMITTER: Miyadera K
PROVIDER: S-EPMC9060458 | biostudies-literature | 2022 Mar
REPOSITORIES: biostudies-literature
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Targeting ON-bipolar cells by AAV gene therapy stably reverses <i>LRIT3</i>-congenital stationary night blindness.
Miyadera Keiko K Santana Evelyn E Roszak Karolina K Iffrig Sommer S Visel Meike M Iwabe Simone S Boyd Ryan F RF Bartoe Joshua T JT Sato Yu Y Gray Alexa A Ripolles-Garcia Ana A Dufour Valérie L VL Byrne Leah C LC Flannery John G JG Beltran William A WA Aguirre Gustavo D GD
Proceedings of the National Academy of Sciences of the United States of America 20220322 13
SignificanceCanine models of inherited retinal diseases have helped advance adeno-associated virus (AAV)-based gene therapies targeting specific cells in the outer retina for treating blinding diseases in patients. However, therapeutic targeting of diseases such as congenital stationary night blindness (CSNB) that exhibit defects in ON-bipolar cells (ON-BCs) of the midretina remains underdeveloped. Using a leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (<i>LRIT3</i>) mutant ...[more]
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Project description:Congenital stationary night blindness (CSNB) is a group of inherited retinal diseases in which either rod-to-ON-bipolar cell (ON-BC) signaling, or rod function is affected leading to impaired vision under low light conditions. One type of CSNB is associated with defects in genes (NYX, GRM6, TRPM1, GPR179, and LRIT3) involved in the mGluR6 signaling cascade at the ON-BC dendritic tips. We have previously characterized a canine model of LRIT3-CSNB and demonstrated short-term safety and efficacy of an ON-BC targeting AAV-LRIT3 (AAVK9#4-shGRM6-cLRIT3-WPRE) gene therapy. Herein, we demonstrate long-term functional recovery and molecular restoration following subretinal injection of the ON-BC targeting AAV-LRIT3 vector in all eight treated eyes for up to 32 months. Following subretinal administration of the therapeutic vector, expression of the LRIT3 transgene, as well as restoration of mGluR6 signaling cascade member TRPM1, were confirmed in the outer plexiform layer (OPL) of the treated area. However, further investigation of the transgene LRIT3 transcript expression by RNA in situ hybridization (RNA-ISH) revealed off-target expression in non-BCs including the photoreceptors, inner nuclear, and ganglion cell layers, despite the use of a mutant AAVK9#4 capsid and an improved mGluR6 promoter designed to specifically transduce and promote expression in ON-BCs. While the long-term therapeutic potential of AAVK9#4-shGRM6-cLRIT3-WPRE is promising, we highlight the necessity for further optimization of AAV-LRIT3 therapy in the canine CSNB model prior to its clinical application.
| S-EPMC10524691 | biostudies-literature
Project description:Mutations in LRIT3, coding for a Leucine-Rich Repeat, immunoglobulin-like and transmembrane domains 3 protein lead to autosomal recessive complete congenital stationary night blindness (cCSNB). The role of the corresponding protein in the ON-bipolar cell signaling cascade remains to be elucidated. Here we genetically and functionally characterize a commercially available Lrit3 knock-out mouse, a model to study the function and the pathogenic mechanism of LRIT3. We confirm that the insertion of a Bgeo/Puro cassette in the knock-out allele introduces a premature stop codon, which presumably codes for a non-functional protein. The mouse line does not harbor other mutations present in common laboratory mouse strains or in other known cCSNB genes. Lrit3 mutant mice exhibit a so-called no b-wave (nob) phenotype with lacking or severely reduced b-wave amplitudes in the scotopic and photopic electroretinogram (ERG), respectively. Optomotor tests reveal strongly decreased optomotor responses in scotopic conditions. No obvious fundus auto-fluorescence or histological retinal structure abnormalities are observed. However, spectral domain optical coherence tomography (SD-OCT) reveals thinned inner nuclear layer and part of the retina containing inner plexiform layer, ganglion cell layer and nerve fiber layer in these mice. To our knowledge, this is the first time that SD-OCT technology is used to characterize an animal model for CSNB. This phenotype is noted at 6 weeks and at 6 months. The stationary nob phenotype of mice lacking Lrit3, which we named nob6, confirms the findings previously reported in patients carrying LRIT3 mutations and is similar to other cCSNB mouse models. This novel mouse model will be useful for investigating the pathogenic mechanism(s) associated with LRIT3 mutations and clarifying the role of LRIT3 in the ON-bipolar cell signaling cascade.
| S-EPMC3943948 | biostudies-literature
Project description:Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disorder. Two forms can be distinguished clinically: complete CSNB (cCSNB) and incomplete CSNB. Individuals with cCSNB have visual impairment under low-light conditions and show a characteristic electroretinogram (ERG). The b-wave amplitude is severely reduced in the dark-adapted state of the ERG, representing abnormal function of ON bipolar cells. Furthermore, individuals with cCSNB can show other ocular features such as nystagmus, myopia, and strabismus and can have reduced visual acuity and abnormalities of the cone ERG waveform. The mode of inheritance of this form can be X-linked or autosomal recessive, and the dysfunction of four genes (NYX, GRM6, TRPM1, and GPR179) has been described so far. Whole-exome sequencing in one simplex cCSNB case lacking mutations in the known genes led to the identification of a missense mutation (c.983G>A [p.Cys328Tyr]) and a nonsense mutation (c.1318C>T [p.Arg440(?)]) in LRIT3, encoding leucine-rich-repeat (LRR), immunoglobulin-like, and transmembrane-domain 3 (LRIT3). Subsequent Sanger sequencing of 89 individuals with CSNB identified another cCSNB case harboring a nonsense mutation (c.1151C>G [p.Ser384(?)]) and a deletion predicted to lead to a premature stop codon (c.1538_1539del [p.Ser513Cysfs(?)59]) in the same gene. Human LRIT3 antibody staining revealed in the outer plexiform layer of the human retina a punctate-labeling pattern resembling the dendritic tips of bipolar cells; similar patterns have been observed for other proteins implicated in cCSNB. The exact role of this LRR protein in cCSNB remains to be elucidated.
| S-EPMC3542465 | biostudies-literature
Project description:Congenital stationary night blindness (CSNB), in the complete form, is caused by dysfunctions in ON-bipolar cells (ON-BCs) which are secondary neurons of the retina. We describe the first disease causative variant associated with CSNB in the dog. A genome-wide association study using 12 cases and 11 controls from a research colony determined a 4.6 Mb locus on canine chromosome 32. Subsequent whole-genome sequencing identified a 1 bp deletion in LRIT3 segregating with CSNB. The canine mutant LRIT3 gives rise to a truncated protein with unaltered subcellular expression in vitro. Genetic variants in LRIT3 have been associated with CSNB in patients although there is limited evidence regarding its apparently critical function in the mGluR6 pathway in ON-BCs. We determine that in the canine CSNB retina, the mutant LRIT3 is correctly localized to the region correlating with the ON-BC dendritic tips, albeit with reduced immunolabelling. The LRIT3-CSNB canine model has direct translational potential enabling studies to help understand the CSNB pathogenesis as well as to develop new therapies targeting the secondary neurons of the retina.
| S-EPMC6775105 | biostudies-literature
Project description:Congenital stationary night blindness (CSNB) is an inherited retinal disease (IRD) that causes night blindness in childhood with heterogeneous genetic, electrophysical, and clinical characteristics. The development of sequencing technologies and gene therapy have increased the ease and urgency of diagnosing IRDs. This study describes seven Taiwanese patients from six unrelated families examined at a tertiary referral center, diagnosed with CSNB, and confirmed by genetic testing. Complete ophthalmic exams included best corrected visual acuity, retinal imaging, and an electroretinogram. The effects of identified novel variants were predicted using clinical details, protein prediction tools, and conservation scores. One patient had an autosomal dominant CSNB with a RHO variant; five patients had complete CSNB with variants in GRM6, TRPM1, and NYX; and one patient had incomplete CSNB with variants in CACNA1F. The patients had Riggs and Schubert-Bornschein types of CSNB with autosomal dominant, autosomal recessive, and X-linked inheritance patterns. This is the first report of CSNB patients in Taiwan with confirmed genetic testing, providing novel perspectives on molecular etiology and genotype-phenotype correlation of CSNB. Particularly, variants in TRPM1, NYX, and CACNA1F in our patient cohort have not previously been described, although their clinical significance needs further study. Additional study is needed for the genotype-phenotype correlation of different mutations causing CSNB. In addition to genetic etiology, the future of gene therapy for CSNB patients is reviewed and discussed.
| S-EPMC9740538 | biostudies-literature
Project description:Cav1.4 L-type Ca2+ channels are predominantly expressed in retinal neurons, particularly at the photoreceptor terminals where they mediate sustained Ca2+ entry needed for continuous neurotransmitter release at their ribbon synapses. Cav1.4 channel gating properties are controlled by accessory subunits, associated regulatory proteins, and also alternative splicing. In humans, mutations in the CACNA1F gene encoding for Cav1.4 channels are associated with X-linked retinal disorders such as congenital stationary night blindness type 2. Mutations in the Cav1.4 protein result in a spectrum of altered functional channel activity. Several mouse models broadened our understanding of the role of Cav1.4 channels not only as Ca2+ source at retinal synapses but also as synaptic organizers. In this review, we highlight different structural and functional phenotypes of Cav1.4 mutations that might also occur in patients with congenital stationary night blindness type 2. A further important yet mostly neglected aspect that we discuss is the influence of alternative splicing on channel dysfunction. We conclude that currently available functional phenotyping strategies should be refined and summarize potential specific therapeutic options for patients carrying Cav1.4 mutations. Importantly, the development of new therapeutic approaches will permit a deeper understanding of not only the disease pathophysiology but also the physiological function of Cav1.4 channels in the retina.
| S-EPMC8370969 | biostudies-literature
Project description:PurposeComplete congenital stationary night blindness (cCSNB) is an incurable inherited retinal disorder characterized by an ON-bipolar cell (ON-BC) defect. GRM6 mutations are the third most prevalent cause of cCSNB. The Grm6-/- mouse model mimics the human phenotype, showing no b-wave in the electroretinogram (ERG) and a loss of mGluR6 and other proteins of the same cascade at the outer plexiform layer (OPL). Our aim was to restore protein localization and function in Grm6-/- adult mice targeting specifically ON-BCs or the whole retina.MethodsAdeno-associated virus-encoding Grm6 under two different promoters (GRM6-Grm6 and CAG-Grm6) were injected intravitreally in P15 Grm6-/- mice. ERG recordings at 2 and 4 months were performed in Grm6+/+, untreated and treated Grm6-/- mice. Similarly, immunolocalization studies were performed on retinal slices before or after treatment using antibodies against mGluR6, TRPM1, GPR179, RGS7, RGS11, Gβ5, and dystrophin.ResultsFollowing treatment, mGluR6 was localized to the dendritic tips of ON-BCs when expressed with either promoter. The relocalization efficiency in mGluR6-transduced retinas at the OPL was 2.5% versus 11% when the GRM6-Grm6 and CAG-Grm6 were used, respectively. Albeit no functional rescue was seen in ERGs, relocalization of TRPM1, GPR179, and Gβ5 was also noted using both constructs. The restoration of the localization of RGS7, RGS11, and dystrophin was more obvious in retinas treated with GRM6-Grm6 than in retinas treated with CAG-Grm6.ConclusionsOur findings show the potential of treating cCSNB with GRM6 mutations; however, it appears that the transduction rate must be improved to restore visual function.
| S-EPMC7980044 | biostudies-literature
Project description:Adeno-associated virus (AAV) effectively targets therapeutic genes to photoreceptors, pigment epithelia, Müller glia and ganglion cells of the retina. To date, no one has shown the ability to correct, with gene replacement, an inherent defect in bipolar cells (BCs), the excitatory interneurons of the retina. Targeting BCs with gene replacement has been difficult primarily due to the relative inaccessibility of BCs to standard AAV vectors. This approach would be useful for restoration of vision in patients with complete congenital stationary night blindness (CSNB1), where signaling through the ON BCs is eliminated due to mutations in their G-protein-coupled cascade genes. For example, the majority of CSNB1 patients carry a mutation in nyctalopin (NYX), which encodes a protein essential for proper localization of the TRPM1 cation channel required for ON BC light-evoked depolarization. As a group, CSNB1 patients have a normal electroretinogram (ERG) a-wave, indicative of photoreceptor function, but lack a b-wave due to defects in ON BC signaling. Despite retinal dysfunction, the retinas of CSNB1 patients do not degenerate. The Nyx(nob) mouse model of CSNB1 faithfully mimics this phenotype. Here, we show that intravitreally injected, rationally designed AAV2(quadY-F+T-V) containing a novel 'Ple155' promoter drives either GFP or YFP_Nyx in postnatal Nyx(nob) mice. In treated Nyx(nob) retina, robust and targeted Nyx transgene expression in ON BCs partially restored the ERG b-wave and, at the cellular level, signaling in ON BCs. Our results support the potential for gene delivery to BCs and gene replacement therapy in human CSNB1.
| S-EPMC4612567 | biostudies-literature
Project description:Congenital Stationary Night Blindness (CSNB) is a retinal disorder caused by a signal transmission defect between photoreceptors and bipolar cells. CSNB can be subdivided in CSNB2 (rod signal transmission reduced) and CSNB1 (rod signal transmission absent). The present study is the first in which night vision problems are assessed in CSNB patients in a systematic way, with the purpose of improving rehabilitation for these patients. We assessed the night vision problems of 13 CSNB2 patients and 9 CSNB1 patients by means of a questionnaire on low luminance situations. We furthermore investigated their dark adapted visual functions by the Goldmann Weekers dark adaptation curve, a dark adapted static visual field, and a two-dimensional version of the "Light Lab". In the latter test, a digital image of a living room with objects was projected on a screen. While increasing the luminance of the image, we asked the patients to report on detection and recognition of objects. The questionnaire showed that the CSNB2 patients hardly experienced any night vision problems, while all CSNB1 patients experienced some problems although they generally did not describe them as severe. The three scotopic tests showed minimally to moderately decreased dark adapted visual functions in the CSNB2 patients, with differences between patients. In contrast, the dark adapted visual functions of the CSNB1 patients were more severely affected, but showed almost no differences between patients. The results from the "2D Light Lab" showed that all CSNB1 patients were blind at low intensities (equal to starlight), but quickly regained vision at higher intensities (full moonlight). Just above their dark adapted thresholds both CSNB1 and CSNB2 patients had normal visual fields. From the results we conclude that night vision problems in CSNB, in contrast to what the name suggests, are not conspicuous and generally not disabling.
| S-EPMC3643903 | biostudies-other
Project description:Importance:Congenital stationary night blindness (CSNB) implies a stable condition, with the major symptom being nyctalopia present at birth. Pediatric clinical presentation and the course of different genetic subtypes of CSNB have not, to our knowledge, been well described in the era of molecular genetic diagnosis. Objective:To describe the presentation and longitudinal clinical characteristics of pediatric patients with molecularly confirmed TRPM1-associated complete CSNB (cCSNB). Design, Setting, Participants:This study was conducted at the University of Iowa from January 1, 1990, to July 1, 2015, and was a retrospective, longitudinal case series of 7 children (5 [71.4%] female) with TRPM1-associated cCSNB followed up for a mean (SD) of 11.1 (2.8) years. Main Outcomes and Measures:History, ophthalmologic examination findings, full-field electroretinogram (ffERG) results, full-field stimulus threshold testing results, Goldmann visual field results, optical coherence tomography results, and molecular genetic results were evaluated. Presenting symptoms and signs, the correlation of refractive error with electroretinography, and clinical evolution were analyzed. Results:Seven patients (5 [71.4%] female) presented early in childhood with strabismus (n = 6 [86%]), myopia (n = 5 [71%]), and/or nystagmus (n = 3 [43%]). The mean (SD) age at presentation was 8 (4) months and for receiving a diagnosis by ffERG was 7.3 years, with molecular diagnosis at 9.7 years. The mean (SD) length of follow-up was 11 (2.8) years. The best-corrected visual acuity at the most recent visit averaged 20/30 in the better-seeing eye (range, 20/20-20/60). The mean (SD) initial refraction was -2.80 (4.42) diopters (D) and the mean refraction at the most recent visit was -8.75 (3.53) D (range, -4.00 to -13.75 D), with the greatest rate of myopic shift before age 5 years. Full-field electroretinogram results were electronegative, consistent with cCSNB, without a significant change in amplitude over time. No patient or parent noted night blindness at presentation; however, subjective nyctalopia was eventually reported in 5 of 7 patients (71%). The full-field stimulus threshold testing results were moderately subnormal (-29.7 [3.8] dB; normal -59.8 [4.0] dB). Goldmann visual field results were significant for full I-4e, but constricted I-2e isopter. Eight different mutations or rare variants in TRPM1 predicted to be pathogenic were detected, with 3 novel variants. Conclusions and Relevance:Children with TRPM1-associated cCSNB presented before school age with progressive myopia as well as strabismus and nystagmus (but not nyctalopia), with stable, electronegative ffERG results, mildly subnormal full-field stimulus threshold testing results, and a constricted I2e isopter on perimetry. These findings suggest that ffERG and cCSNB genetic testing should be considered for children who present with early-onset myopia, especially in the presence of strabismus and/or nystagmus, and that TRPM1-associated cCSNB is a channelopathy that may present without complaints of night blindness in childhood.
| S-EPMC5876850 | biostudies-literature