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Purpose To describe the natural course, phenotype and genotype of patients with X-linked retinoschisis (XLRS). Design Retrospective cohort study. Participants Three hundred forty patients with XLRS from 178 presumably unrelated families. Methods This multicenter, retrospective cohort study reviewed medical records of XLRS patients for medical history, symptoms, visual acuity (VA), ophthalmoscopy, full-field electroretinography and retinal imaging (fundus photography, spectral-domain optical coherence tomography (SD-OCT), fundus autofluorescence). Main Outcome Measures Age at onset, age of diagnosis, severity of visual impairment, annual visual decline, electroretinogram and imaging findings. Results In total, 340 patients were included with a mean follow-up time of 13.2 years (range, 0–50.1 years). The median age to reach mild visual impairment and low vision was 12 and 25 years, respectively. Severe visual impairment and blindness were predominantly observed in patients above 40 years old, with a predicted prevalence of 35% and 25% at the age of 60, respectively. The VA increased slightly in the first two decades of life, and subsequently transitioned in to an average annual decline of 0.44% (P A (p.(Glu72Lys) (101 subjects, 29.7%) and a deletion of exon 3 (38 subjects, 11.2%). Conclusion A large variability in phenotype and natural course of XLRS was seen in this study. In most patients, XLRS showed a slow deterioration starting in the second decade of life, suggesting an optimal window of opportunity for treatment within the first three decades of life. The integrity of EZ as well as the PROS length on SD-OCT may be important in choosing optimal candidates for treatment, and as potential structural endpoints in future therapeutic studies. No clear genotype-phenotype correlation was found.

PurposeFoveoschisis involves the pathologic splitting of retinal layers at the fovea, which may occur congenitally in X-linked retinoschisis (XLRS) or as an acquired complication of myopia. XLRS is attributed to functional loss of the retinal adhesion protein retinoschisin 1 (RS1), but the pathophysiology of myopic foveoschisis is unclear due to the lack of animal models. Here, we characterized a novel nonhuman primate model of myopic foveoschisis through clinical examination and multimodal imaging followed by morphologic, cellular, and transcriptional profiling of retinal tissues and genetic analysis.MethodsWe identified a rhesus macaque with behavioral and anatomic features of myopic foveoschisis, and monitored disease progression over 14 months by fundus photography, fluorescein angiography, and optical coherence tomography (OCT). After necropsy, we evaluated anatomic and cellular changes by immunohistochemistry and transcriptomic changes using single-nuclei RNA-sequencing (snRNA-seq). Finally, we performed Sanger and whole exome sequencing with focus on the RS1 gene.ResultsAffected eyes demonstrated posterior hyaloid traction and progressive splitting of the outer plexiform layer on OCT. Immunohistochemistry showed increased GFAP expression in Müller glia and loss of ramified Iba-1+ microglia, suggesting macro- and microglial activation with minimal photoreceptor alterations. SnRNA-seq revealed gene expression changes predominantly in cones and retinal ganglion cells involving chromatin modification, suggestive of cellular stress at the fovea. No defects in the RS1 gene or its expression were detected.ConclusionsThis nonhuman primate model of foveoschisis reveals insights into how acquired myopic traction leads to phenotypically similar morphologic and cellular changes as congenital XLRS without alterations in RS1.

PurposeLoss of retinoschisin (RS1) function underlies X-linked retinoschisis (XLRS) pathology. In the retina, both photoreceptor inner segments and bipolar cells express RS1. However, the loss of RS1 function causes schisis primarily in the inner retina. To understand these cell type-specific phenotypes, we decoupled RS1 effects in bipolar cells from that in photoreceptors.MethodsBipolar cell transgene RS1 expression was achieved using two inner retina-specific promoters: (1) a minimal promoter engineered from glutamate receptor, metabotropic glutamate receptor 6 gene (mini-mGluR6/ Grm6) and (2) MiniPromoter (Ple155). Adeno-associated virus vectors encoding RS1 gene under either the mini-mGluR6 or Ple-155 promoter were delivered to the XLRS mouse retina through intravitreal or subretinal injection on postnatal day 14. Retinal structure and function were assessed 5 weeks later: immunohistochemistry for morphological characterization, optical coherence tomography and electroretinography (ERG) for structural and functional evaluation.ResultsImmunohistochemical analysis of RS1expression showed that expression with the MiniPromoter (Ple155) was heavily enriched in bipolar cells. Despite variations in vector penetrance and gene transfer efficiency across the injected retinas, those retinal areas with robust bipolar cell RS1 expression showed tightly packed bipolar cells with fewer cavities and marked improvement in inner retinal structure and synaptic function as judged by optical coherence tomography and electroretinography, respectively.ConclusionsThese results demonstrate that RS1 gene expression primarily in bipolar cells of the XLRS mouse retina, independent of photoreceptor expression, can ameliorate retinoschisis structural pathology and provide further evidence of RS1 role in cell adhesion.

We generated a Long Evans transgenic rat with targeted deletion of the whole Rs1 exon-1 and evaluated the pathological retinal phenotype of this Rs1-/Y rat model of X-linked retinoschisis (XLRS). The Rs1−/Y rat exhibited very early onset and rapidly progressive photoreceptor degeneration. The outer limiting membrane (OLM) was disrupted and discontinuous by post-natal day (P15) and allowed photoreceptor nuclei to dislocate from the outer nuclear layers (ONL) into the sub-retinal side of the OLM. Dark-adapted electroretinogram (ERG) a-wave and b-wave amplitudes were considerably reduced to only 20–25% of WT by P17. Microglia and Müller glial showed cell marker activation by P7. Intravitreal application of AAV8-RS1 at P5–6 induced RS1 expression by P15 and rescued the inner nuclear layer (INL) and outer plexiform layer (OPL) cavity formation otherwise present at P15, and the outer-retinal structure was less disrupted. This Rs1−/Y exon-1-del rat model displays substantially faster rod cell loss compared to the exon-1-del Rs1-KO mouse. Most unexpected was the rapid appearance of schisis cavities between P7 and P15, and then cavities rapidly disappeared by P21/P30. The rat model provides clues on the molecular and cellular mechanisms underlying XLRS pathology in this model and points to a substantial and early changes to normal retinal development.

Animal models of X-linked juvenile retinoschisis (XLRS) are valuable tools for understanding basic biochemical function of retinoschisin (RS1) protein and to investigate outcomes of preclinical efficacy and toxicity studies. In order to work with an eye larger than mouse, we generated and characterized an Rs1h−/y knockout rat model created by removing exon 3. This rat model expresses no normal RS1 protein. The model shares features of an early onset and more severe phenotype of human XLRS. The morphologic pathology includes schisis cavities at postnatal day 15 (p15), photoreceptors that are misplaced into the subretinal space and OPL, and a reduction of photoreceptor cell numbers by p21. By 6 mo age only 1–3 rows of photoreceptors nuclei remain, and the inner/outer segment layers and the OPL shows major changes. Electroretinogram recordings show functional loss with considerable reduction of both the a-wave and b-wave by p28, indicating early age loss and dysfunction of photoreceptors. The ratio of b-/a-wave amplitudes indicates impaired synaptic transmission to bipolar cells in addition. Supplementing the Rs1h−/y exon3-del retina with normal human RS1 protein using AAV8-RS1 delivery improved the retinal structure. This Rs1h−/y rat model provides a further tool to explore underlying mechanisms of XLRS pathology and to evaluate therapeutic intervention for the XLRS condition.

We generated a Long Evans transgenic rat with targeted deletion of the whole Rs1 exon-1 and evaluated the pathological retinal phenotype of this

Intravitreal administration for human adeno-associated vector (AAV) delivery is easier and less traumatic to ocular tissues than subretinal injection, but it gives limited retinal transduction. AAV vectors are large (about 4,000 kDa) compared with most intraocular drugs, such as ranibizumab (48 kDa), and the large size impedes diffusion to reach the retina from the usual injection site in the anterior/mid-vitreous. Intuitively, a preferred placement for the vector would be deep in the vitreous near the retina, which we term “para-retinal” delivery. We explored the consequences of para-retinal intravitreal delivery in the rabbit eye and in non-human primate (NHP) eye. 1 h after para-retinal administration in the rabbit eye, the vector concentration near the retina remained four times greater than in the anterior vitreous, indicating limited vector diffusion through the gelatinous vitreous matrix. In NHP, para-retinal placement showed greater transduction in the fovea than vector applied in the mid-vitreous. More efficient retinal delivery translates to using lower vector doses, with reduced risk of ocular inflammatory exposure. These results indicate that para-retinal delivery yields more effective vector concentration near the retina, thereby increasing the potential for better retinal transduction in human clinical application., Graphical Abstract, The authors explored an alternate approach to standard intravitreal administration called “para-retinal” delivery where AAV vectors were placed deep into the vitreous near the retina surface. This modification increased vector concentration in the posterior vitreous in rabbit eyes, minimized vector losses, and enhanced foveal transgene expression in NHP eyes.

For disorders with X-linked inheritance, variants may be transmitted through multiple generations of carrier females before an affected male is ascertained. Pathogenic RS1 variants exclusively cause X-linked retinoschisis (XLRS). While RS1 is constrained to variation, recurrent variants are frequently observed in unrelated probands. Here, we investigate recurrent pathogenic variants to determine the relative burden of mutational hotspot and founder allele events to this phenomenon. A cohort RS1 variant analysis and standardized classification, including variant enrichment in the XLRS cohort and in RS1 functional domains, were performed on 332 unrelated XLRS probands. A total of 108 unique RS1 variants were identified. A subset of 19 recurrently observed RS1 variants were evaluated in 190 probands by a haplotype analysis, using microsatellite and single nucleotide polymorphisms. Fourteen variants had at least two probands with common variant-specific haplotypes over ~1.95 centimorgans (cM) flanking RS1. Overall, 99/190 of reportedly unrelated probands had 25 distinct shared haplotypes. Examination of this XLRS cohort for common RS1 haplotypes indicates that the founder effect plays a significant role in this disorder, including variants in mutational hotspots. This improves the accuracy of clinical variant classification and may be generalizable to other X-linked disorders.

Achromatopsia (ACHM) is a congenital cone photoreceptor disorder characterized by impaired color discrimination, low visual acuity, photosensitivity, and nystagmus. To date, six genes have been associated with ACHM (CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6), the majority of these being implicated in the cone phototransduction cascade. CNGA3 encodes the CNGA3 subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors and is one of the major disease-associated genes for ACHM. Herein, we provide a comprehensive overview of the CNGA3 variant spectrum in a cohort of 1060 genetically confirmed ACHM patients, 385 (36.3%) of these carrying "likely disease-causing" variants in CNGA3. Compiling our own genetic data with those reported in the literature and in public databases, we further extend the CNGA3 variant spectrum to a total of 316 variants, 244 of which we interpreted as "likely disease-causing" according to ACMG/AMP criteria. We report 48 novel "likely disease-causing" variants, 24 of which are missense substitutions underlining the predominant role of this mutation class in the CNGA3 variant spectrum. In addition, we provide extensive in silico analyses and summarize reported functional data of previously analyzed missense, nonsense and splicing variants to further advance the pathogenicity assessment of the identified variants.

PURPOSE To describe longitudinal multimodal imaging findings of nonexudative choroidal neovascularization in CTRP5 late-onset retinal degeneration. METHODS Four patients with CTRP5-positive late-onset retinal degeneration underwent repeated ophthalmoscopic examination and multimodal imaging. All four patients (two siblings and their cousins, from a pedigree described previously) had the heterozygous S163R mutation. RESULTS All four patients demonstrated large subretinal lesions in the mid-peripheral retina of both eyes. The lesions were characterized by confluent hypercyanescence with hypocyanescent borders on indocyanine green angiography, faintly visible branching vascular networks with absent/minimal leakage on fluorescein angiography, Type 1 neovascularization on optical coherence tomography angiography, and absent retinal fluid, consistent with nonexudative choroidal neovascularization. The neovascular membranes enlarged substantially over time and the birth of new membranes was observed, but all lesions remained nonexudative/minimally exudative. Without treatment, all involved retinal areas remained free of atrophy and subretinal fibrosis. CONCLUSION We report the existence of massive advancing nonexudative Type 1 choroidal neovascularization in CTRP5 late-onset retinal degeneration. These findings have implications for age-related macular degeneration. They provide a monogenic model system for studying the mechanisms underlying the distinct events of choroidal neovascularization development, enlargement, progression to exudation, and atrophy in age-related macular degeneration. They suggest that choroidal hypoperfusion precedes neovascularization and that nonexudative neovascularization may protect against atrophy.

Patients with inherited retinal dystrophies (IRDs) were recruited from two understudied populations: Mexico and Pakistan as well as a third well-studied population of European Americans to define the genetic architecture of IRD by performing whole-genome sequencing (WGS). Whole-genome analysis was performed on 409 individuals from 108 unrelated pedigrees with IRDs. All patients underwent an ophthalmic evaluation to establish the retinal phenotype. Although the 108 pedigrees in this study had previously been examined for mutations in known IRD genes using a wide range of methodologies including targeted gene(s) or mutation(s) screening, linkage analysis and exome sequencing, the gene mutations responsible for IRD in these 108 pedigrees were not determined. WGS was performed on these pedigrees using Illumina X10 at a minimum of 30X depth. The sequence reads were mapped against hg19 followed by variant calling using GATK. The genome variants were annotated using SnpEff, PolyPhen2, and CADD score; the structural variants (SVs) were called using GenomeSTRiP and LUMPY. We identified potential causative sequence alterations in 61 pedigrees (57%), including 39 novel and 54 reported variants in IRD genes. For 57 of these pedigrees the observed genotype was consistent with the initial clinical diagnosis, the remaining 4 had the clinical diagnosis reclassified based on our findings. In seven pedigrees (12%) we observed atypical causal variants, i.e. unexpected genotype(s), including 4 pedigrees with causal variants in more than one IRD gene within all affected family members, one pedigree with intrafamilial genetic heterogeneity (different affected family members carrying causal variants in different IRD genes), one pedigree carrying a dominant causative variant present in pseudo-recessive form due to consanguinity and one pedigree with a de-novo variant in the affected family member. Combined atypical and large structural variants contributed to about 20% of cases. Among the novel mutations, 75% were detected in Mexican and 50% found in European American pedigrees and have not been reported in any other population while only 20% were detected in Pakistani pedigrees and were not previously reported. The remaining novel IRD causative variants were listed in gnomAD but were found to be very rare and population specific. Mutations in known IRD associated genes contributed to pathology in 63% Mexican, 60% Pakistani and 45% European American pedigrees analyzed. Overall, contribution of known IRD gene variants to disease pathology in these three populations was similar to that observed in other populations worldwide. This study revealed a spectrum of mutations contributing to IRD in three populations, identified a large proportion of novel potentially causative variants that are specific to the corresponding population or not reported in gnomAD and shed light on the genetic architecture of IRD in these diverse global populations., Author summary The study was performed to identify the underlying cause of inherited retinal degeneration (IRD) in 409 individuals from 108 families. Primarily, these families were recruited from three different geographic regions: Mexico, Pakistan and European Americans from the United States. Blood samples were collected from all individuals for genome analysis. This analysis detected causative variants in 61 out of the 108 pedigrees. A total of 93 gene variants were found in the 61 families. Among these, 54 were previously reported as causative variants and the remaining 39 have not been reported in IRD pedigrees. Interestingly, 54% of these novel variants were not listed in gnomAD. In addition to these findings, complex causative genotypes were observed in 20% of pedigrees. Overall, causative variants were detected in 63% Mexican, 60% Pakistani and 45% European American pedigrees. This study revealed the distribution of IRD causative variants in pedigrees with diverse ethnic and geographic backgrounds.

Recombinant adeno-associated virus (rAAV) has been widely used for gene delivery in animal models and successfully applied in clinical trials for treating inherited retinal disease. Although subretinal delivery of AAVs can effectively transduce photoreceptors and/or retinal pigmental epithelium (RPE), cells most affected by inherited retinal diseases, the procedure is invasive and complicated, and only delivers the gene to a limited retinal area. AAVs can also be delivered intravitreally to the retina, a much less invasive nonsurgical procedure. However, intravitreal administration of non-modified AAV serotypes tends to transduce only ganglion cells and inner nuclear layer cells. To date, most non-modified AAV serotypes that have been identified are incapable of efficiently transducing photoreceptors and/or RPE when delivered intravitreally. In this study, we investigate the retinal tropism of AAVrh10 vector administered by intravitreal injection to mouse, rat, and rabbit eyes. Our results demonstrate that AAVrh10 is capable of transducing not only inner retinal cells, but also outer retinal cells in all three species, though the transduction efficiency in rabbit was low. In addition, AAVrh10 preferentially transduced outer retinal cells in mouse models of retinal disease. Therefore, AAVrh10 vector could be a useful candidate to intravitreally deliver genes to photoreceptor and RPE cells.

Adeno-associated virus (AAV) vector-mediated gene delivery is a promising approach for therapy, but implementation in the eye currently is hampered by the need for delivering the vector underneath the retina, using surgical application into the subretinal space. This limits the extent of the retina that is treated and may cause surgical injury. Vector delivery into the vitreous cavity would be preferable because it is surgically less invasive and would reach more of the retina. Unfortunately, most conventional, non-modified AAV vector serotypes penetrate the retina poorly from the vitreous; this limits efficient transduction and expression by target cells (retinal pigment epithelium and photoreceptors). We developed a method of applying a small and safe electric current across the intact eye in vivo for a brief period following intravitreal vector administration. This significantly improved AAV-mediated transduction of retinal cells in wild-type mice following intravitreal delivery, with gene expression in retinal pigment epithelium and photoreceptor cells. The low-level current had no adverse effects on retinal structure and function. This method should be generally applicable for other AAV serotypes and may have broad application in both basic research and clinical studies.

Retinoschisin (RS1) is a secreted protein that is essential for maintaining integrity of the retina. Numerous mutations in RS1 cause X-linked retinoschisis (XLRS), a progressive degeneration of the retina that leads to vision loss in young males. A key manifestation of XLRS is the formation of cavities (cysts) in the retina and separation of the layers (schisis), disrupting synaptic transmission. There are currently no approved treatments for patients with XLRS. Strategies using adeno-associated viral (AAV) vectors to deliver functional copies of RS1 as a form of gene augmentation therapy, are under clinical evaluation. To improve therapeutic strategies for treating XLRS, it is critical to better understand the secretion of RS1 and its molecular function. Immunofluorescence and immunoelectron microscopy show that RS1 is located on the surfaces of the photoreceptor inner segments and bipolar cells. Sequence homology indicates a discoidin domain fold, similar to many other proteins with demonstrated adhesion functions. Recent structural studies revealed the tertiary structure of RS1 as two back-to-back octameric rings, each cross-linked by disulfides. The observation of higher order structures in vitro suggests the formation of an adhesive matrix spanning the distance between cells (∼100 nm). Several studies indicated that RS1 readily binds to other proteins such as the sodium-potassium ATPase (NaK-ATPase) and extracellular matrix proteins. Alternatively, RS1 may influence fluid regulation via interaction with membrane proteins such as the NaK-ATPase, largely inferred from the use of carbonic anhydrase inhibitors to shrink the typical intra-retinal cysts in XLRS. We discuss these models in light of RS1 structure and address the difficulty in understanding the function of RS1.

The suprachoroid is a potential space located between the sclera and choroid of the eye, which provides a novel route for ocular drug or viral vector delivery. Suprachoroidal injection of adeno-associated virus (AAV)8 using transscleral microneedles enables widespread transgene expression in eyes of nonhuman primates, but may cause intraocular inflammation. We characterized the host humoral and cellular immune responses after suprachoroidal delivery of AAV8 expressing green fluorescent protein (GFP) in rhesus macaques, and found that it can induce mild chorioretinitis that resolves after systemic corticosteroid administration, with recovery of photoreceptor morphology, but persistent immune cell infiltration after 3 months, corresponding to a loss of GFP expression from retinal pigment epithelial cells, but persistent expression in scleral fibroblasts. Suprachoroidal AAV8 triggered B cell and T cell responses against GFP, but only mild antibody responses to the viral capsid compared to intravitreal injections of the same vector and dose. Systemic biodistribution studies showed lower AAV8 levels in liver and spleen after suprachoroidal injection compared with intravitreal delivery. Our findings suggest that suprachoroidal AAV8 primarily triggers host immune responses to GFP, likely due to sustained transgene expression in scleral fibroblasts outside the blood-retinal barrier, but elicits less humoral immune reactivity to the viral capsid than intravitreal delivery due to lower egress into systemic circulation. As GFP is not native to primates and not a clinically relevant transgene, suprachoroidal AAV delivery of human transgenes may have significant translational potential for retinal gene therapy.

To understand RS1 gene interaction networks in the X-linked retinoschisis (XLRS) mouse retina (Rs1−/y), we analyzed the transcriptome by RNA sequencing before and after in vivo expression of exogenous retinoschisin (RS1) gene delivered by AAV8. RS1 is a secreted cell adhesion protein that is critical for maintaining structural lamination and synaptic integrity of the neural retina. RS1 loss-of-function mutations cause XLRS disease in young boys and men, with splitting (“schisis”) of retinal layers and synaptic dysfunction that cause progressive vision loss with age. Analysis of differential gene expression profiles and pathway enrichment analysis of Rs1-KO (Rs1−/y) retina identified cell surface receptor signaling and positive regulation of cell adhesion as potential RS1 gene interaction networks. Most importantly, it also showed massive dysregulation of immune response genes at early age, with characteristics of a microglia-driven proinflammatory state. Delivery of AAV8-RS1 primed the Rs1-KO retina toward structural and functional recovery. The disease transcriptome transitioned toward a recovery phase with upregulation of genes implicated in wound healing, anatomical structure (camera type eye) development, metabolic pathways, and collagen IV networks that provide mechanical stability to basement membrane. AAV8-RS1 expression also attenuated the microglia gene signatures to low levels toward immune quiescence. This study is among the first to identify RS1 gene interaction networks that underlie retinal structural and functional recovery after RS1 gene therapy. Significantly, it also shows that providing wild-type RS1 gene function caused the retina immune status to transition from a degenerative inflammatory phenotype toward immune quiescence, even though the transgene is not directly linked to microglia function. This study indicates that inhibition of microglial proinflammatory responses is an integral part of therapeutic rescue in XLRS gene therapy, and gene therapy might realize its full potential if delivered before microglia activation and photoreceptor cell death. Clinical Trials. gov Identifier NTC 02317887.

Animal models of X-linked juvenile retinoschisis (XLRS) are valuable tools for understanding basic biochemical function of retinoschisin (RS1) protein and to investigate outcomes of preclinical efficacy and toxicity studies. In order to work with an eye larger than mouse, we generated and characterized an Rs1h

X-linked Retinoschisis (XLRS) is an early-onset transretinal dystrophy, often with a prominent macular component, that affects males and generally spares heterozygous females because of X-linked recessive inheritance. It results from loss-of-function RS1 gene mutations on the X-chromosome. XLRS causes bilateral reduced acuities from young age, and on clinical exam and by ocular coherence tomography (OCT) the neurosensory retina shows foveo-macular cystic schisis cavities in the outer plexiform (OPL) and inner nuclear layers (INL). XLRS manifests between infancy and school-age with variable phenotypic presentation and without reliable genotype-phenotype correlations. INL disorganization disrupts synaptic signal transmission from photoreceptors to ON-bipolar cells, and this reduces the electroretinogram (ERG) bipolar b-wave disproportionately to photoreceptor a-wave changes. RS1 gene expression is localized mainly to photoreceptors and INL bipolar neurons, and RS1 protein is thought to play a critical cell adhesion role during normal retinal development and later for maintenance of retinal structure. Several independent XLRS mouse models with mutant RS1 were created that recapitulate features of human XLRS disease, with OPL-INL schisis cavities, early onset and variable phenotype across mutant models, and reduced ERG b-wave to a-wave amplitude ratio. The faithful phenotype of the XLRS mouse has assisted in delineating the disease pathophysiology. Delivery to XLRS mouse retina of an AAV8-RS1 construct under control of the RS1 promoter restores the retinal structure and synaptic function (with increase of b-wave amplitude). It also ameliorates the schisis-induced inflammatory microglia phenotype toward a state of immune quiescence. The results imply that XLRS gene therapy could yield therapeutic benefit to preserve morphological and functional retina particularly when intervention is conducted at earlier ages before retinal degeneration becomes irreversible. A phase I/IIa single-center, open-label, three-dose-escalation clinical trial reported a suitable safety and tolerability profile of intravitreally administered AAV8-RS1 gene replacement therapy for XLRS participants. Dose-related ocular inflammation occurred after dosing, but this resolved with topical and oral corticosteroids. Systemic antibodies against AAV8 increased in dose-dependent fashion, but no antibodies were observed against the RS1 protein. Retinal cavities closed transiently in one participant. Technological innovations in methods of gene delivery and strategies to further reduce immune responses are expected to enhance the therapeutic efficacy of the vector and ultimate success of a gene therapy approach.

This study explored systemic immune changes in 11 subjects with X-linked retinoschisis (XLRS) in a phase I/IIa adeno-associated virus 8 (AAV8)-RS1 gene therapy trial (ClinicalTrials.gov: NCT02317887). Immune cell proportions and serum analytes were compared to 12 healthy male controls. At pre-dosing baseline the mean CD4/CD8 ratio of XLRS subjects was elevated. CD11c(+) myeloid dendritic cells (DCs) and the serum epidermal growth factor (EGF) level were decreased, while CD123(+) plasmacytoid DCs and serum interferon (IFN)-γ and tumor necrosis factor (TNF)-α were increased, indicating that the XLRS baseline immune status differs from that of controls. XLRS samples 14 days after AAV8-RS1 administration were compared with the XLRS baseline. Frequency of CD11b(+)CD11c(+) DCc was decreased in 8 of 11 XLRS subjects across all vector doses (1e9–3e11 vector genomes [vg]/eye). CD8(+)human leukocyte antigen-DR isotype (HLA-DR)(+) cytotoxic T cells and CD68(+)CD80(+) macrophages were upregulated in 10 of 11 XLRS subjects, along with increased serum granzyme B in 8 of 11 XLRS subjects and elevated IFN-γ in 9 of 11 XLRS subjects. The six XLRS subjects with ocular inflammation after vector application gave a modestly positive correlation of inflammation score to their respective baseline CD4/CD8 ratios. This exploratory study indicates that XLRS subjects may exhibit a proinflammatory, baseline immune phenotype, and that intravitreal dosing with AAV8-RS1 leads to systemic immune activation with an increase of activated lymphocytes, macrophages, and proinflammatory cytokines.

Major advances in the study of inherited retinal diseases (IRDs) have placed efforts to develop treatments for these blinding conditions at the forefront of the emerging field of precision medicine. As a result, the growth of clinical trials for IRDs has increased rapidly over the past decade and is expected to further accelerate as more therapeutic possibilities emerge and qualified participants are identified. Although guided by established principles, these specialized trials, requiring analysis of novel outcome measures and endpoints in small patient populations, present multiple challenges relative to study design and ethical considerations. This position paper reviews recent accomplishments and existing challenges in clinical trials for IRDs and presents a set of recommendations aimed at rapidly advancing future progress. The goal is to stimulate discussions among researchers, funding agencies, industry, and policy makers that will further the design, conduct, and analysis of clinical trials needed to accelerate the approval of effective treatments for IRDs, while promoting advocacy and ensuring patient safety.

Author(s): Wright, Charles; Mazzucco, Anna E; Becker, Steven M; Sieving, Paul A; Tumminia, Santa J | Abstract: PurposeTo review past and current National Eye Institute (NEI)-supported age-related macular degeneration (AMD) activities and initiatives and preview upcoming coordinated efforts for studying AMD.MethodsWe conducted and summarized a portfolio analysis and literature review of NEI intramural and extramural AMD activities.ResultsThe NEI supports a broad range of AMD research, both by individual independent investigators as well as through networks and consortia. The International AMD Genomics Consortium, Age-Related Eye Disease Study, Age-Related Eye Disease Study 2 (AREDS2), and Comparison of AMD Treatments Trial legacy work probed the complex genetics, clinical presentation, and standards of patient care, respectively. The NEI AMD Pathobiology Working Group identified gaps and opportunities for future research efforts. The AMD Ryan Initiative Study and clinical trials testing the efficacies of minocycline to modulate retinal microglia activity and induced pluripotent stem cells-derived retinal pigmented epithelium (RPE) patch implants to rescue photoreceptor cell death are among the future directions for NEI-supported AMD research. Finally, NEI commissioned the creation of AREDS2 participant-derived induced pluripotent stem cell (iPSC) lines linked to their associated genomic and phenotypic datasets. These datasets will also be linked to the data obtained using their associated iPSC-derived cells (RPE, retina, choroid) and made publicly available.ConclusionsInvestments by NEI for AMD research will continue to provide invaluable resources to investigators committed to addressing this complex blinding disease and other retinal degenerative diseases.Translational relevanceNEI now stands poised to expand the resources available to clinical investigators to uncover disease mechanisms and move experimental therapies into clinical trials.

Purpose To explore the experiences of parents of sons with X-linked retinoschisis (XLRS). Design Mixed methods—qualitative interviews with quantitative survey. Methods Parents of sons with XLRS who were evaluated at the National Eye Institute between December 2017 and January 2019 were eligible for this study. Participation involved engaging in a semistructured interview and completing a survey assessing optimism, anxiety, personality traits, and sociodemographics using valid and reliable scales. Interview transcripts were coded and analyzed thematically, and scales were scored and used descriptively. Results Eleven mothers and 8 fathers from 13 families participated. Optimism, anxiety, and personality traits fell into the normative ranges for the scales. Parents described a process of continuous adaptation to their son's condition. The initial diagnosis was characterized by shock, grief, and “devastation” for most parents. Maternal guilt was common, but usually lessened over time. As parents adjusted to life postdiagnosis, they attempted to achieve a state of normalcy while balancing a desire to protect their sons. Significant sources of stress included decisions around sports participation and driving. Among all parents, the fear of retinal detachment was an ongoing concern. Most parents did identify perceived benefits from their experiences, such as feelings of gratitude or family cohesion. Conclusions Most parents viewed XLRS as a significant challenge in their sons' lives, but one that could be overcome. Clinical encounters may be enhanced for families with XLRS by providing accurate information, preparing families for potential challenges, anticipating stressful decisions, and meeting other families with XLRS.

International audience; Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide–gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3–/–) mice to obtain triallelic Cnga3+/– Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.

Within the field of glaucoma research, neuroprotection is defined as slowing the functional loss in glaucoma by a mechanism independent of lowering of intraocular pressure. There is currently a great potential for research surrounding neuroprotection as it relates to glaucoma. Anatomical targets for neuroprotection should focus on upstream rather than downstream factors, and could include any part of the retinal ganglion cell, the glia, especially astrocytes or Muller cells, and vasculature. The great number of anatomical targets is exceeded only by the number of possible biochemical pathways and potential treatments. Successful treatment may be accomplished through the targeting of one or even a combination of multiple pathways. Once a treatment is shown effective in vitro, it should be evaluated in vivo with carefully chosen animal models and studied in sufficient numbers to detect statistically and clinically significant effects. Such a drug should have few systemic side effects and its delivery should be optimized so as to encourage compliance. There are still a multitude of possible screens available to test the efficacy of a neuroprotective drug and a single gold standard is ideal for the accurate assessment and comparison of new drugs. Future studies in neuroprotection should investigate the genetic component of the disease, novel pharmaceutical agents for new or known pathways, modulations of scleral biomechanics, and relation to research of other complex disorders of the central nervous system.

In this study, we describe efforts by the National Eye Institute (NEI) and National Center for Advancing Translational Science (NCATS) to catalyze advances in 3-dimensional (3-D) ocular organoid and microphysiological systems (MPS). We reviewed the recent literature regarding ocular organoids and tissue chips. Animal models, 2-dimensional cell culture models, and postmortem human tissue samples provide the vision research community with insights critical to understanding pathophysiology and therapeutic development. The advent of induced pluripotent stem cell technologies provide researchers with enticing new approaches and tools that augment study in more traditional models to provide the scientific community with insights that have previously been impossible to obtain. Efforts by the National Institutes of Health (NIH) have already accelerated the pace of scientific discovery, and recent advances in ocular organoid and MPS modeling approaches have opened new avenues of investigation. In addition to more closely recapitulating the morphologies and physiological responses of in vivo human tissue, key breakthroughs have been made in the past year to resolve long-standing scientific questions regarding tissue development, molecular signaling, and pathophysiological mechanisms that promise to provide advances critical to therapeutic development and patient care. 3-D tissue culture modeling and MPS offer platforms for future high-throughput testing of therapeutic candidates and studies of gene interactions to improve models of complex genetic diseases with no well-defined etiology, such as age-related macular degeneration and Fuchs' dystrophy.

Author(s): Li, Lin; Chen, Yabin; Jiao, Xiaodong; Jin, Chongfei; Jiang, Dan; Tanwar, Mukesh; Ma, Zhiwei; Huang, Li; Ma, Xiaoyin; Sun, Wenmin; Chen, Jianjun; Ma, Yan; M'hamdi, Oussama; Govindarajan, Gowthaman; Cabrera, Patricia E; Li, Jiali; Gupta, Nikhil; Naeem, Muhammad Asif; Khan, Shaheen N; Riazuddin, Sheikh; Akram, Javed; Ayyagari, Radha; Sieving, Paul A; Riazuddin, S Amer; Hejtmancik, J Fielding | Abstract: PurposeThe Pakistan Punjab population has been a rich source for identifying genes causing or contributing to autosomal recessive retinal degenerations (arRD). This study was carried out to delineate the genetic architecture of arRD in the Pakistani population.MethodsThe genetic origin of arRD in a total of 144 families selected only for having consanguineous marriages and multiple members affected with arRD was examined. Of these, causative mutations had been identified in 62 families while only the locus had been identified for an additional 15. The remaining 67 families were subjected to homozygosity exclusion mapping by screening of closely flanking microsatellite markers at 180 known candidate genes/loci followed by sequencing of the candidate gene for pathogenic changes.ResultsOf these 67 families subjected to homozygosity mapping, 38 showed homozygosity for at least one of the 180 regions, and sequencing of the corresponding genes showed homozygous cosegregating mutations in 27 families. Overall, mutations were detected in approximately 61.8 % (89/144) of arRD families tested, with another 10.4% (15/144) being mapped to a locus but without a gene identified.ConclusionsThese results suggest the involvement of unmapped novel genes in the remaining 27.8% (40/144) of families. In addition, this study demonstrates that homozygosity mapping remains a powerful tool for identifying the genetic defect underlying genetically heterogeneous arRD disorders in consanguineous marriages for both research and clinical applications.

Author(s): Zeng, Yong; Petralia, Ronald S; Vijayasarathy, Camasamudram; Wu, Zhijian; Hiriyanna, Suja; Song, Hongman; Wang, Ya-Xian; Sieving, Paul A; Bush, Ronald A | Abstract: PurposeSpectral-domain optical coherence tomography (SD-OCT) was used to characterize the retinal phenotype, natural history, and treatment responses in a mouse model of X-linked retinoschisis (Rs1-KO) and to identify new structural markers of AAV8-mediated gene therapy outcome.MethodsOptical coherence tomography scans were performed on wild-type and Rs1-KO mouse retinas between 1 and 12 months of age and on Rs1-KO mice after intravitreal injection of AAV8-scRS/IRBPhRS (AAV8-RS1). Cavities and photoreceptor outer nuclear layer (ONL) thickness were measured, and outer retina reflective band (ORRB) morphology was examined with age and after AAV8-RS1 treatment. Outer retina reflective band morphology was compared to immunohistochemical staining of the outer limiting membrane (OLM) and photoreceptor inner segment (IS) mitochondria and to electron microscopy (EM) images of IS.ResultsRetinal cavity size in Rs1-KO mice increased between 1 and 4 months and decreased thereafter, while ONL thickness declined steadily, comparable to previous histologic studies. Wild-type retina had four ORRBs. In Rs1-KO, ORRB1was fragmented from 1 month, but was normal after 8 months; ORRB2 and ORRB3 were merged at all ages. Outer retina reflective band morphology returned to normal after AAV-RS1 therapy, paralleling the recovery of the OLM and IS mitochondria as indicated by anti-β-catenin and anti-COX4 labeling, respectively, and EM.ConclusionsSpectral-domain OCT is a sensitive, noninvasive tool to monitor subtle changes in retinal morphology, disease progression, and effects of therapies in mouse models. The ORRBs may be useful to assess the outcome of gene therapy in the treatment of X-linked retinoschisis patients.

Mutations in the essential retinal protein retinoschisin (RS1) cause a form of macular degeneration. Heymann et al. use cryo-EM to show that RS1 assembles into branched networks that may play a stabilizing role in maintaining the integrity of the retina., Mutations in the retinal protein retinoschisin (RS1) cause progressive loss of vision in young males, a form of macular degeneration called X-linked retinoschisis (XLRS). We previously solved the structure of RS1, a 16-mer composed of paired back-to-back octameric rings. Here, we show by cryo–electron microscopy that RS1 16-mers can assemble into extensive branched networks. We classified the different configurations, finding four types of interaction between the RS1 molecules. The predominant configuration is a linear strand with a wavy appearance. Three less frequent types constitute the branch points of the network. In all cases, the “spikes” around the periphery of the double rings are involved in these interactions. In the linear strand, a loop (usually referred to as spike 1) occurs on both sides of the interface between neighboring molecules. Mutations in this loop suppress secretion, indicating the possibility of intracellular higher-order assembly. These observations suggest that branched networks of RS1 may play a stabilizing role in maintaining the integrity of the retina., Graphical Abstract

Author(s): Song, Hongman; Zeng, Yong; Sardar Pasha, Sheik Pran Babu; Bush, Ronald A; Vijayasarathy, Camasamudram; Qian, Haohua; Wei, Lisa; Wiley, Henry E; Wu, Zhijian; Sieving, Paul A | Abstract: PurposeElectric micro-current has been shown to enhance penetration and transduction of adeno-associated viral (AAV) vectors in mouse retina after intravitreal administration. We termed this: "electric-current vector mobility (ECVM)." The present study considered whether ECVM could augment retinal transduction efficiency of intravitreal AAV8-CMV-EGFP in normal rabbit and nonhuman primate (NHP) macaque. Potential mechanisms underlying enhanced retinal transduction by ECVM were also studied.MethodsWe applied an electric micro-current across the intact eye of normal rabbit and monkey in vivo for a brief period immediately after intravitreal injection of AAV8-CMV-EGFP. Retinal GFP expression was evaluated by fundus imaging in vivo. Retinal immunohistochemistry was performed to assess the distribution of retinal cells transduced by the AAV8-EGFP. Basic fibroblast growth factor (bFGF) was analyzed by quantitative RT-polymerase chain reaction (PCR). Muller glial reactivity and inner limiting membrane (ILM) were examined by the glial fibrillary acidic protein (GFAP) and vimentin staining in mouse retina, respectively.ResultsECVM significantly increased the efficiency of AAV reaching and transducing the rabbit retina following intravitreal injection, with gene expression in inner nuclear layer, ganglion cells, and Muller cells. Similar trend of improvement was observed in the ECVM-treated monkey eye. The electric micro-current upregulated bFGF expression in Muller cells and vimentin showed ILM structural changes in mouse retina.ConclusionsECVM promotes the transduction efficiency of AAV8-CMV-GFP in normal rabbit and monkey retinas following intravitreal injection.Translational relevanceThis work has potential translational relevance to human ocular gene therapy by increasing retinal expression of therapeutic vectors given by intravitreal administration.

The NIH Blueprint for Neuroscience Research, a group of 14 NIH Institutes, Centers, and Offices (ICOs) that support research on the nervous system, recently released a Request for Information ( ) on the Proposed Funding Priorities

IMPORTANCE: Understanding factors associated with attaining independent research funding by ophthalmology clinician-scientists receiving National Eye Institute career development awards (K08 or K23) in ophthalmology can be important to maintaining the pipeline of clinician-scientists. OBJECTIVE: To provide continued follow-up of a cohort of ophthalmology clinician-scientists who received National Institutes of Health (NIH) K career development grants. DESIGN, SETTING, AND PARTICIPANTS: Cohort study from an electronic database review of ophthalmologists who have received either a K08 or K23 career development grant from the NIH. Data were analyzed between December 30, 2015, and December 30, 2017. MAIN OUTCOMES AND MEASURES: Receipt of an NIH R01 grant. RESULTS: We previously characterized a group of more than 100 ophthalmologists who received K awards from 1996 to 2010, of whom 29 were awarded R01 grants. In follow-up of this cohort in 2017, 27 additional K awardees of this initial cohort were awarded an R01 from 2011 to 2017, leading to a total of 62 of 128 ophthalmologists receiving an R01. The mean time to receiving an R01 grant after the K award ended was 2.8 years. The data did not identify a definitive association with sex, having a PhD degree, or research tier of university in obtaining an R01 grant in this cohort. CONCLUSIONS AND RELEVANCE: In comparison with our previous report of the same cohort, there was a 93% increase in the number of K awardees who have received an R01 award, with the mean time to award being nearly 3 years after completing their K grant. This suggests that most K awardees in ophthalmology are successful in obtaining R01 grants, but one should recognize this may be several years after their K grant has ended.

The following review summarizes the state of the art in representative aspects of gene therapy/translational medicine and evolves from a symposium held at the School of Veterinary Medicine, University of Pennsylvania on November 16, 2017 honoring Dr. Gustavo Aguirre, recipient of ARVO's 2017 Proctor Medal. Focusing on the retina, speakers highlighted current work on moving therapies for inherited retinal degenerative diseases from the laboratory bench to the clinic.

Background on the National Institutes of Health BRAIN Initiative and neuroethics The National Institutes of Health (NIH) works tirelessly to support the best science and ensure that its funded research adheres to the highest ethical standards. From its inception, the NIH Brain Research through

We identified a homozygous missense alteration (c.75C>A, p.D25E) in CLCC1, encoding a presumptive intracellular chloride channel highly expressed in the retina, associated with autosomal recessive retinitis pigmentosa (arRP) in eight consanguineous families of Pakistani descent. The p.D25E alteration decreased CLCC1 channel function accompanied by accumulation of mutant protein in granules within the ER lumen, while siRNA knockdown of CLCC1 mRNA induced apoptosis in cultured ARPE-19 cells. TALEN KO in zebrafish was lethal 11 days post fertilization. The depressed electroretinogram (ERG) cone response and cone spectral sensitivity of 5 dpf KO zebrafish and reduced eye size, retinal thickness, and expression of rod and cone opsins could be rescued by injection of wild type CLCC1 mRNA. Clcc1+/-KO mice showed decreased ERGs and photoreceptor number. Together these results strongly suggest that intracellular chloride transport by CLCC1 is a critical process in maintaining retinal integrity, and CLCC1 is crucial for survival and function of retinal cells.

Author(s): Cukras, Catherine A; Huryn, Laryssa A; Jeffrey, Brett G; Turriff, Amy; Sieving, Paul A | Abstract: PurposeTo examine the symmetry of structural and functional parameters between eyes in patients with X-linked retinoschisis (XLRS), as well as changes in visual acuity and electrophysiology over time.MethodsThis is a single-center observational study of 120 males with XLRS who were evaluated at the National Eye Institute. Examinations included best-corrected visual acuity for all participants, as well as ERG recording and optical coherence tomography (OCT) on a subset of participants. Statistical analyses were performed using nonparametric Spearman correlations and linear regression.ResultsOur analyses demonstrated a statistically significant correlation of structural and functional measures between the two eyes of XLRS patients for all parameters. OCT central macular thickness (n = 78; Spearman r = 0.83, P l 0.0001) and ERG b/a ratio (n = 78; Spearman r = 0.82, P l 0.0001) were the most strongly correlated between a participant's eyes, whereas visual acuity was less strongly correlated (n = 120; Spearman r = 0.47, P l 0.0001). Stability of visual acuity was observed with an average change of less than one letter (n = 74; OD -0.66 and OS -0.70 letters) in a mean follow-up time of 6.8 years. There was no statistically significant change in the ERG b/a ratio within eyes over time.ConclusionsAlthough a broad spectrum of clinical phenotypes is observed across individuals with XLRS, our study demonstrates a significant correlation of structural and functional findings between the two eyes and stability of measures of acuity and ERG parameters over time. These results highlight the utility of the fellow eye as a useful reference for monocular interventional trials.

To test the effect of docosahexanoic acid (DHA) dietary supplementation on macular function in patients with Stargardt disease.A single center, double-masked, randomized placebo-controlled trial of 11 subjects (2 males, 9 females) with Stargardt disease in a crossover design (NCT00060749). Six participants were randomized to two sequences of three month periods of DHA supplementation (2000 mg/day) followed by three months of placebo. Five participants were randomized to the opposite sequence. All participants were evaluated with a food frequency and NEI-VF25 questionnaires, complete ophthalmic examination, multifocal electroretinography (ERG, primary outcome), 30-Hz flicker ERG, Humphrey 10-2 visual field, D15 color tests and serum lipid analysis.During periods of DHA supplementation, serum rose and then fell with transition to periods of placebo. None of the participants experienced greater than 20% change from baseline values of the mfERG during periods of DHA supplementation or placebo, while the average change in peak amplitude and phase angle of the flicker ERG remained similar at all visits. No significant change was observed for any of the secondary outcome measures. Eight adverse events occurred but these were not considered to be due to the treatment.No perceived effect of DHA supplementation on macular function was observed in a small sample of Stargardt patients who were compliant with the protocol as estimated by changes in serum DHA. This study will help design future studies of the effect of DHA supplementation on retinal function in cohorts with retinal dystrophies.

Retinal dystrophies are a phenotypically and genetically complex group of conditions. Because of this complexity, it can be challenging in many families to determine the inheritance based on pedigree analysis alone. Clinical examinations were performed and blood samples were collected from a North American (M1186) and a consanguineous Pakistani (PKRD168) pedigree affected with two different retinal dystrophies (RD). Based on the structure of the pedigrees, inheritance patterns in the families were difficult to determine. In one family, linkage analysis was performed with markers on X-chromosome. In the second family, whole-exome sequencing (WES) was performed. Subsequent Sanger sequencing of genes of interest was performed. Linkage and haplotype analysis localized the disease interval to a 70 Mb region on the X chromosome that encompassed RP2 and RPGR in M1186 . The disease haplotype segregated with RD in all individuals except for an unaffected man (IV:3) and his affected son (V:1) in this pedigree. Subsequent analysis identified a novel RPGR mutation (p. Lys857Glu fs221X) in all affected members of M1186 except V:1. This information suggests that there is an unidentified second cause of retinitis pigmentosa (RP) within the family. A novel two-base-pair deletion (p. Tyr565Ter fsX) in CHM (choroideremia) was found to segregate with RD in PKRD168. This paper highlights the challenges of interpreting family history in families with RD and reports on the identification of novel mutations in two RD families.

Author(s): Allahdina, Ali M; Stetson, Paul F; Vitale, Susan; Wong, Wai T; Chew, Emily Y; Ferris, Fredrick L; Sieving, Paul A; Cukras, Catherine | Abstract: Purpose:As optical coherence tomography (OCT) minimum intensity (MI) analysis provides a quantitative assessment of changes in the outer nuclear layer (ONL), we evaluated the ability of OCT-MI analysis to detect hydroxychloroquine toxicity. Methods:Fifty-seven predominantly female participants (91.2% female; mean age, 55.7 ± 10.4 years; mean time on hydroxychloroquine, 15.0 ± 7.5 years) were enrolled in a case-control study and categorized into affected (i.e., with toxicity, n = 19) and unaffected (n = 38) groups using objective multifocal electroretinographic (mfERG) criteria. Spectral-domain OCT scans of the macula were analyzed and OCT-MI values quantitated for each subfield of the Early Treatment Diabetic Retinopathy Study (ETDRS) grid. A two-sample U-test and a cross-validation approach were used to assess the sensitivity and specificity of toxicity detection according to OCT-MI criteria. Results:The medians of the OCT-MI values in all nine of the ETDRS subfields were significantly elevated in the affected group relative to the unaffected group (P l 0.005 for all comparisons), with the largest difference found for the inner inferior subfield (P l 0.0001). The receiver operating characteristic analysis of median MI values of the inner inferior subfields showed high sensitivity and high specificity in the detection of toxicity with area under the curve = 0.99. Conclusions:Retinal changes secondary to hydroxychloroquine toxicity result in increased OCT reflectivity in the ONL that can be detected and quantitated using OCT-MI analysis. Analysis of OCT-MI values demonstrates high sensitivity and specificity for detecting the presence of hydroxychloroquine toxicity in this cohort and may contribute additionally to current screening practices.

Strategies aimed at invoking synaptic plasticity have therapeutic potential for several neurological conditions. The human retinal synaptic disease X-linked retinoschisis (XLRS) is characterized by impaired visual signal transmission through the retina and progressive visual acuity loss, and mice lacking retinoschisin (RS1) recapitulate human disease. Here, we demonstrate that restoration of RS1 via retina-specific delivery of adeno-associated virus type 8-RS1 (AAV8-RS1) vector rescues molecular pathology at the photoreceptor-depolarizing bipolar cell (photoreceptor-DBC) synapse and restores function in adult Rs1-KO animals. Initial development of the photoreceptor-DBC synapse was normal in the Rs1-KO retina; however, the metabotropic glutamate receptor 6/transient receptor potential melastatin subfamily M member 1-signaling (mGluR6/TRPM1-signaling) cascade was not properly maintained. Specifically, the TRPM1 channel and G proteins Gαo, Gβ5, and RGS11 were progressively lost from postsynaptic DBC dendritic tips, whereas the mGluR6 receptor and RGS7 maintained proper synaptic position. This postsynaptic disruption differed from other murine night-blindness models with an electronegative electroretinogram response, which is also characteristic of murine and human XLRS disease. Upon AAV8-RS1 gene transfer to the retina of adult XLRS mice, TRPM1 and the signaling molecules returned to their proper dendritic tip location, and the DBC resting membrane potential was restored. These findings provide insight into the molecular plasticity of a critical synapse in the visual system and demonstrate potential therapeutic avenues for some diseases involving synaptic pathology.

This study evaluated the safety and tolerability of ocular RS1 adeno-associated virus (AAV8-RS1) gene augmentation therapy to the retina of participants with X-linked retinoschisis (XLRS). XLRS is a monogenic trait affecting only males, caused by mutations in the RS1 gene. Retinoschisin protein is secreted principally in the outer retina, and its absence results in retinal cavities, synaptic dysfunction, reduced visual acuity, and susceptibility to retinal detachment. This phase I/IIa single-center, prospective, open-label, three-dose-escalation clinical trial administered vector to nine participants with pathogenic RS1 mutations. The eye of each participant with worse acuity (≤63 letters; Snellen 20/63) received the AAV8-RS1 gene vector by intravitreal injection. Three participants were assigned to each of three dosage groups: 1e9 vector genomes (vg)/eye, 1e10 vg/eye, and 1e11 vg/eye. The investigational product was generally well tolerated in all but one individual. Ocular events included dose-related inflammation that resolved with topical and oral corticosteroids. Systemic antibodies against AAV8 increased in a dose-related fashion, but no antibodies against RS1 were observed. Retinal cavities closed transiently in one participant. Additional doses and immunosuppressive regimens are being explored to pursue evidence of safety and efficacy (ClinicalTrials.gov: NCT02317887).

Whole genome sequencing (WGS) was performed to identify the variants responsible for inherited retinal degeneration (IRD) in a Caucasian family. Segregation analysis of selected rare variants with pathogenic potential identified a set of compound heterozygous changes p.Arg266*:c.796C>T and p.Ala568Thr:c.1702G>A in the intraflagellar transport protein-88 (IFT88) gene segregating with IRD. Expression of IFT88 with the p.Arg266* and p.Ala568Thr mutations in mIMDC3 cells by transient transfection and in HeLa cells by introducing the mutations using CRISPR-cas9 system suggested that both mutations result in the formation of abnormal ciliary structures. The introduction of the IFT88 p.Arg266* variant in the homozygous state in HeLa cells by CRISPR-Cas9 genome-editing revealed that the mutant transcript undergoes nonsense-mediated decay leading to a significant depletion of IFT88 transcript. Additionally, abnormal ciliogenesis was observed in these cells. These observations suggest that the rare and unique combination of IFT88 alleles observed in this study provide insight into the physiological role of IFT88 in humans and the likely mechanism underlying retinal pathology in the pedigree with IRD.