Statistical Evaluation of ERG Responses: A New Method to Validate Cycle-by-Cycle Recordings in Advanced Retinal Degenerations.

Purpose: To describe and evaluate a novel method to determine the validity of measurements made using cycle-by-cycle (CxC) recording techniques in patients with advanced retinal degenerations (RD) having low-amplitude flicker electroretinogram (ERG) responses. Methods: The method extends the original CxC recording algorithm introduced by Sieving et al., retaining the original recording setup and the preliminary analysis of raw data. Novel features include extended use of spectrum analysis, reduction of errors due to known sources, and a comprehensive statistical assessment using three different tests. The method was applied to ERG recordings from seven patients with RD and two patients with CNGB3 achromatopsia. Results: The method was implemented as a Windows application to processes raw data obtained from a commercial ERG system, and it features a computational toolkit for statistical assessment of ERG recordings with amplitudes as low as 1 µV, commonly found in advanced RD patients. When recorded using conditions specific for eliciting cone responses, none of the CNGB3 patients had a CxC validated response, indicating that no signal artifacts were present with our recording conditions. A comparison of the presented method with conventional 30 Hz ERG was performed. Bland-Altman plots indicated good agreement (mean difference, -0.045 µV; limits of agreement, 0.193 to -0.282 µV) between the resulting amplitudes. Within-session test-retest variability was 15%, comparing favorably to the variability of standard ERG amplitudes. Conclusions: This novel method extracts highly reliable clinical recordings of low-amplitude flicker ERGs and effectively detects artifactual responses. It has potential value both as a cone outcome variable and planning tool in clinical trials on natural history and treatment of advanced RDs.

A Literary Pediatric Retina Fellowship With Michael T. Trese, MD.

Michael T. Trese, MD (1946-2022), a vitreoretinal surgeon, made significant contributions to the field of retina. Although most known for his work in pediatric retina surgery, he was a pioneer in areas such as medical retina, translational research, and telemedicine. This article reviews his major contributions to spread his knowledge more widely to vitreoretinal trainees and specialists. We discuss six areas where Trese made a lasting impact: lens-sparing vitrectomy, familial exudative vitreoretinopathy, congenital X-linked retinoschisis, autologous plasmin enzyme, regenerative medicine, and telemedicine. [Ophthalmic Surg Lasers Imaging Retina 2023;54:701-712.].

X-Linked Retinoschisis.

X-linked retinoschisis (XLRS) is an inherited vitreoretinal dystrophy causing visual impairment in males starting at a young age with an estimated prevalence of 1:5000 to 1:25,000. The condition was first observed in two affected brothers by Josef Haas in 1898 and is clinically diagnosed by characteristic intraretinal cysts arranged in a petaloid "spoke-wheel" pattern centered in the macula. When clinical electroretinogram (ERG) testing began in the 1960s, XLRS was noted to have a characteristic reduction of the dark-adapted b-wave amplitude despite normal or usually nearly normal a-wave amplitudes, which became known as the "electronegative ERG response" of XLRS disease. The causative gene, RS1, was identified on the X-chromosome in 1997 and led to understanding the molecular and cellular basis of the condition, discerning the structure and function of the retinoschisin protein, and generating XLRS murine models. Along with parallel development of gene delivery vectors suitable for targeting retinal diseases, successful gene augmentation therapy was demonstrated by rescuing the XLRS phenotype in mouse. Two human phase I/II therapeutic XLRS gene augmentation studies were initiated; and although these did not yield definitive improvement in visual function, they gave significant new knowledge and experience, which positions the field for further near-term clinical testing with enhanced, next-generation gene therapy for XLRS patients.

A Spontaneous Nonhuman Primate Model of Myopic Foveoschisis.

Purpose: Foveoschisis 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. Methods: We 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. Results: Affected 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. Conclusions: This 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.

Advances in understanding the molecular structure of retinoschisin while questions remain of biological function.

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.

XLRS Rat with Rs1-/Y Exon-1-Del Shows Failure of Early Postnatal Outer Retina Development.

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.

Targeted Expression of Retinoschisin by Retinal Bipolar Cells in XLRS Promotes Resolution of Retinoschisis Cysts Sans RS1 From Photoreceptors.

Purpose: Loss 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. Methods: Bipolar 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. Results: Immunohistochemical 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. Conclusions: These 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.

Predominant Founder Effect among Recurrent Pathogenic Variants for an X-Linked Disorder.

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.

Comprehensive variant spectrum of the CNGA3 gene in patients affected by achromatopsia.

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.

Of men and mice: Human X-linked retinoschisis and fidelity in mouse modeling.

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.

X-Linked Retinoschisis: Novel Clinical Observations and Genetic Spectrum in 340 Patients.

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 patients with XLRS for medical history, symptoms, visual acuity (VA), ophthalmoscopy, full-field electroretinography, and retinal imaging (fundus photography, spectral-domain [SD] OCT, fundus autofluorescence). MAIN OUTCOME MEASURES: Age at onset, age at diagnosis, severity of visual impairment, annual visual decline, and electroretinography and imaging findings. RESULTS: Three hundred forty patients were included with a mean follow-up time of 13.2 years (range, 0.1-50.1 years). The median ages to reach mild visual impairment and low vision were 12 and 25 years, respectively. Severe visual impairment and blindness were observed predominantly in patients older than 40 years, with a predicted prevalence of 35% and 25%, respectively, at 60 years of age. The VA increased slightly during the first 2 decades of life and subsequently transitioned into an average annual decline of 0.44% (P < 0.001). No significant difference was found in decline of VA between variants that were predicted to be severe and mild (P = 0.239). The integrity of the ellipsoid zone (EZ) as well as the photoreceptor outer segment (PROS) length in the fovea on SD OCT correlated significantly with VA (Spearman's ρ = -0.759 [P < 0.001] and -0.592 [P = 0.012], respectively). Fifty-three different RS1 variants were found. The most common variants were the founder variant c.214G→A (p.(Glu72Lys)) (101 patients [38.7%]) and a deletion of exon 3 (38 patients [14.6%]). CONCLUSIONS: Large variabilities in phenotype and natural course of XLRS were 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 3 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 end points in future therapeutic studies. No clear genotype-phenotype correlation was found.

Rs1h-/y exon 3-del rat model of X-linked retinoschisis with early onset and rapid phenotype is rescued by RS1 supplementation.

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.

AMPK modulation ameliorates dominant disease phenotypes of CTRP5 variant in retinal degeneration.

Late-onset retinal degeneration (L-ORD) is an autosomal dominant disorder caused by a missense substitution in CTRP5. Distinctive clinical features include sub-retinal pigment epithelium (RPE) deposits, choroidal neovascularization, and RPE atrophy. In induced pluripotent stem cells-derived RPE from L-ORD patients (L-ORD-iRPE), we show that the dominant pathogenic CTRP5 variant leads to reduced CTRP5 secretion. In silico modeling suggests lower binding of mutant CTRP5 to adiponectin receptor 1 (ADIPOR1). Downstream of ADIPOR1 sustained activation of AMPK renders it insensitive to changes in AMP/ATP ratio resulting in defective lipid metabolism, reduced Neuroprotectin D1(NPD1) secretion, lower mitochondrial respiration, and reduced ATP production. These metabolic defects result in accumulation of sub-RPE deposits and leave L-ORD-iRPE susceptible to dedifferentiation. Gene augmentation of L-ORD-iRPE with WT CTRP5 or modulation of AMPK, by metformin, re-sensitize L-ORD-iRPE to changes in cellular energy status alleviating the disease cellular phenotypes. Our data suggests a mechanism for the dominant behavior of CTRP5 mutation and provides potential treatment strategies for L-ORD patients.

Deciphering the genetic architecture and ethnographic distribution of IRD in three ethnic populations by whole genome sequence analysis.

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.

MASSIVE ADVANCING NONEXUDATIVE TYPE 1 CHOROIDAL NEOVASCULARIZATION IN CTRP5 LATE-ONSET RETINAL DEGENERATION: Longitudinal Findings on Multimodal Imaging and Implications for Age-Related Macular Degeneration.

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.

Immune function in X-linked retinoschisis subjects in an AAV8-RS1 phase I/IIa gene therapy trial.

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.

Genetic Rescue of X-Linked Retinoschisis Mouse (Rs1-/y) Retina Induces Quiescence of the Retinal Microglial Inflammatory State Following AAV8-RS1 Gene Transfer and Identifies Gene Networks Underlying Retinal Recovery.

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.

Trans-Ocular Electric Current In Vivo Enhances AAV-Mediated Retinal Transduction in Large Animal Eye After Intravitreal Vector Administration.

Purpose: Electric 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. Methods: We 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). Müller glial reactivity and inner limiting membrane (ILM) were examined by the glial fibrillary acidic protein (GFAP) and vimentin staining in mouse retina, respectively. Results: ECVM 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 Müller cells. Similar trend of improvement was observed in the ECVM-treated monkey eye. The electric micro-current upregulated bFGF expression in Müller cells and vimentin showed ILM structural changes in mouse retina. Conclusions: ECVM promotes the transduction efficiency of AAV8-CMV-GFP in normal rabbit and monkey retinas following intravitreal injection. Translational Relevance: This work has potential translational relevance to human ocular gene therapy by increasing retinal expression of therapeutic vectors given by intravitreal administration.

Advancing Clinical Trials for Inherited Retinal Diseases: Recommendations from the Second Monaciano Symposium.

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.