The Road towards Gene Therapy for X-Linked Juvenile Retinoschisis: A Systematic Review of Preclinical Gene Therapy in Cell-Based and Rodent Models of XLRS.

X-linked juvenile retinoschisis (XLRS) is an early-onset progressive inherited retinopathy affecting males. It is characterized by abnormalities in the macula, with formation of cystoid retinal cavities, frequently accompanied by splitting of the retinal layers, impaired synaptic transmission of visual signals, and associated loss of visual acuity. XLRS is caused by loss-of-function mutations in the retinoschisin gene located on the X chromosome (RS1, MIM 30083). While proof-of-concept studies for gene augmentation therapy have been promising in in vitro and rodent models, clinical trials in XLRS patients have not been successful thus far. We performed a systematic literature investigation using search strings related to XLRS and gene therapy in in vivo and in vitro models. Three rounds of screening (title/abstract, full text and qualitative) were performed by two independent reviewers until consensus was reached. Characteristics related to study design and intervention were extracted from all studies. Results were divided into studies using (1) viral and (2) non-viral therapies. All in vivo rodent studies that used viral vectors were assessed for quality and risk of bias using the SYRCLE's risk-of-bias tool. Studies using alternative and non-viral delivery techniques, either in vivo or in vitro, were extracted and reviewed qualitatively, given the diverse and dispersed nature of the information. For in-depth analysis of in vivo studies using viral vectors, outcome data for optical coherence tomography (OCT), immunohistopathology and electroretinography (ERG) were extracted. Meta-analyses were performed on the effect of recombinant adeno-associated viral vector (AAV)-mediated gene augmentation therapies on a- and b-wave amplitude as well as the ratio between b- and a-wave amplitudes (b/a-ratio) extracted from ERG data. Subgroup analyses and meta-regression were performed for model, dose, age at injection, follow-up time point and delivery method. Between-study heterogeneity was assessed with a Chi-square test of homogeneity (I2). We identified 25 studies that target RS1 and met our search string. A total of 19 of these studies reported rodent viral methods in vivo. Six of the 25 studies used non-viral or alternative delivery methods, either in vitro or in vivo. Of these, five studies described non-viral methods and one study described an alternative delivery method. The 19 aforementioned in vivo studies were assessed for risk of bias and quality assessments and showed inconsistency in reporting. This resulted in an unclear risk of bias in most included studies. All 19 studies used AAVs to deliver intact human or murine RS1 in rodent models for XLRS. Meta-analyses of a-wave amplitude, b-wave amplitude, and b/a-ratio showed that, overall, AAV-mediated gene augmentation therapy significantly ameliorated the disease phenotype on these parameters. Subgroup analyses and meta-regression showed significant correlations between b-wave amplitude effect size and dose, although between-study heterogeneity was high. This systematic review reiterates the high potential for gene therapy in XLRS, while highlighting the importance of careful preclinical study design and reporting. The establishment of a systematic approach in these studies is essential to effectively translate this knowledge into novel and improved treatment alternatives.

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.

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.

Carbonic anhydrase inhibition in X-linked retinoschisis: An eye on the photoreceptors.

The retinoschisin protein is encoded on the short arm of the X-chromosome by RS1, is expressed abundantly in photoreceptor inner segments and in bipolar cells, and is secreted as an octamer that maintains the structural integrity of the retina. Mutations in RS1 lead to X-linked retinoschisis (XLRS), a disease characterized by the formation of cystic spaces between boys' retinal layers that frequently present in ophthalmoscopy as a "spoke-wheel" pattern on their maculae and by progressively worsening visual acuity (VA). There is no proven therapy for XLRS, but there is mixed evidence that carbonic anhydrase inhibitors (CAIs) produce multiple beneficial effects, including improved VA and decreased volume of cystic spaces. Consequently, linear mixed-effects (LME) models were used to evaluate the effects of CAI therapy on VA and central retinal thickness (CRT, a proxy for cystic cavity volume) in a review of 19 patients' records. The mechanism of action of action of CAIs is unclear but, given that misplaced retinoschisin might accumulate in the photoreceptors, it is possible-perhaps even likely-that CAIs act to benefit the function of photoreceptors and the neighboring retinal pigment epithelium by acidification of the extracellular milieu; patients on CAIs have among the most robust photoreceptor responses. Therefore, a small subset of five subjects were recruited for imaging on a custom multimodal adaptive optics retinal imager for inspection of their parafoveal cone photoreceptors. Those cones that were visible, which numbered far fewer than in controls, were enlarged, consistent with the retinoschisin accumulation hypothesis. Results of the LME modeling found that there is an initial benefit to both VA and CRT in CAI therapy, but these wane, in both cases, after roughly two years. That said, even a short beneficial effect of CAIs on the volume of the cystic spaces may give CAI therapy an important role as pretreatment before (or immediately following) administration of gene therapy.

Two cases of diabetic macular edema complicated by an atypical macular hole.

BACKGROUND: Here we report two patients who developed an atypical macular hole (MH) during the treatment course for diabetic macular edema (DME). CASE PRESENTATIONS: Patient 1 was a 73-year-old male. Optical coherence tomography (OCT) revealed perifoveal retinoschisis (RS) in addition to cystoid macular edema and serous retinal detachment (SRD) in his left eye, and that an MH had developed during the clinical course. A convex surface was formed at the MH margin toward the vitreous cavity, and granular shadows were observed in the fluid cuff. Intraoperative findings revealed a thin epiretinal macular membrane (ERM) around the MH. Patient 2 was a 79-year-old male. Although the patient underwent pars plana vitrectomy (PPV) for proliferative diabetic retinopathy (PDR) in both eyes, RS and a thin ERM in addition to SRD was observed in his left eye after surgery, and an MH developed during the clinical course. As in Patient 1, a convex surface was formed at the fluid cuff margin toward the vitreous cavity. CONCLUSIONS: Both patients had persistent DME, SRD, RS, and a thin ERM before the development of the MH. OCT revealed the formation of a convex surface at the MH margin toward the vitreous cavity, suggesting that the fragility of the layered structure of the retina combined with tangential retinal traction may have been involved in the atypical MH form.

Retinal AAV8-RS1 Gene Therapy for X-Linked Retinoschisis: Initial Findings from a Phase I/IIa Trial by Intravitreal Delivery.

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).

An ex vivo gene therapy approach in X-linked retinoschisis.

PURPOSE: X-linked retinoschisis (XLRS) is juvenile-onset macular degeneration caused by haploinsufficiency of the extracellular cell adhesion protein retinoschisin (RS1). RS1 mutations can lead to either a non-functional protein or the absence of protein secretion, and it has been established that extracellular deficiency of RS1 is the underlying cause of the phenotype. Therefore, we hypothesized that an ex vivo gene therapy strategy could be used to deliver sufficient extracellular RS1 to reverse the phenotype seen in XLRS. Here, we used adipose-derived, syngeneic mesenchymal stem cells (MSCs) that were genetically modified to secrete human RS1 and then delivered these cells by intravitreal injection to the retina of the Rs1h knockout mouse model of XLRS. METHODS: MSCs were electroporated with two transgene expression systems (cytomegalovirus (CMV)-controlled constitutive and doxycycline-induced Tet-On controlled inducible), both driving expression of human RS1 cDNA. The stably transfected cells, using either constitutive mesenchymal stem cell (MSC) or inducible MSC cassettes, were assayed for their RS1 secretion profile. For single injection studies, 100,000 genetically modified MSCs were injected into the vitreous cavity of the Rs1h knockout mouse eye at P21, and data were recorded at 2, 4, and 8 weeks post-injection. The control groups received either unmodified MSCs or vehicle injection. For the multiple injection studies, the mice received intravitreal MSC injections at P21, P60, and P90 with data collection at P120. For the single- and multiple-injection studies, the outcomes were measured with electroretinography, optokinetic tracking responses (OKT), histology, and immunohistochemistry. RESULTS: Two lines of genetically modified MSCs were established and found to secrete RS1 at a rate of 8 ng/million cells/day. Following intravitreal injection, RS1-expressing MSCs were found mainly in the inner retinal layers. Two weeks after a single injection of MSCs, the area of the schisis cavities was reduced by 65% with constitutive MSCs and by 83% with inducible MSCs, demonstrating improved inner nuclear layer architecture. This benefit was maintained up to 8 weeks post-injection and corresponded to a significant improvement in the electroretinogram (ERG) b-/a-wave ratio at 8 weeks (2.6 inducible MSCs; 1.4 untreated eyes, p<0.05). At 4 months after multiple injections, the schisis cavity areas were reduced by 78% for inducible MSCs and constitutive MSCs, more photoreceptor nuclei were present (700/µm constitutive MSC; 750/µm inducible MSC; 383/µm untreated), and the ERG b-wave was significantly improved (threefold higher with constitutive MSCs and twofold higher with inducible MSCs) compared to the untreated control group. CONCLUSIONS: These results establish that extracellular delivery of RS1 rescues the structural and functional deficits in the Rs1h knockout mouse model and that this ex vivo gene therapy approach can inhibit progression of disease. This proof-of-principle work suggests that other inherited retinal degenerations caused by a deficiency of extracellular matrix proteins could be targeted by this strategy.

Retinoschisin gene therapy in photoreceptors, Müller glia or all retinal cells in the Rs1h-/- mouse.

X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a putative secreted cell adhesion protein. Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise. We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Müller glia, photoreceptors or multiple cell types throughout the retina. Müller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease. However, photoreceptors, not glia, normally secrete retinoschisin. We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h-/- mouse model of retinoschisis. Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to transport of the protein across the retina. The greatest long-term rescue was observed when photoreceptors produce retinoschisin. Similar rescue was observed with photoreceptor-specific or generalized expression, although photoreceptor secretion may contribute to rescue in the latter case. These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies.

Retinal organoids with X-linked retinoschisis RS1 (E72K) mutation exhibit a photoreceptor developmental delay and are rescued by gene augmentation therapy.

BACKGROUND: X-linked juvenile retinoschisis (XLRS) is an inherited disease caused by RS1 gene mutation, which leads to retinal splitting and visual impairment. The mechanism of RS1-associated retinal degeneration is not fully understood. Besides, animal models of XLRS have limitations in the study of XLRS. Here, we used human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) to investigate the disease mechanisms and potential treatments for XLRS. METHODS: hiPSCs reprogrammed from peripheral blood mononuclear cells of two RS1 mutant (E72K) XLRS patients were differentiated into ROs. Subsequently, we explored whether RS1 mutation could affect RO development and explore the effectiveness of RS1 gene augmentation therapy. RESULTS: ROs derived from RS1 (E72K) mutation hiPSCs exhibited a developmental delay in the photoreceptor, retinoschisin (RS1) deficiency, and altered spontaneous activity compared with control ROs. Furthermore, the delays in development were associated with decreased expression of rod-specific precursor markers (NRL) and photoreceptor-specific markers (RCVRN). Adeno-associated virus (AAV)-mediated gene augmentation with RS1 at the photoreceptor immature stage rescued the rod photoreceptor developmental delay in ROs with the RS1 (E72K) mutation. CONCLUSIONS: The RS1 (E72K) mutation results in the photoreceptor development delay in ROs and can be partially rescued by the RS1 gene augmentation therapy.

Intravitreal Delivery of rAAV2-hSyn-hRS1 Results in Retinal Ganglion Cell-Specific Gene Expression and Retinal Improvement in the Rs1-KO Mouse.

X-linked retinoschisis (XLRS) is a monogenic recessive inherited retinal disease caused by defects in retinoschisin (RS1). It manifests clinically as retinal schisis cavities and a disproportionate reduction of b-wave amplitude compared with the a-wave amplitude. Currently there is no approved treatment. In the last decade, there has been major progress in the development of gene therapy for XLRS. Previous preclinical studies have demonstrated the treatment benefits of hRS1 gene augmentation therapy in mouse models. However, outcomes in clinical trials have been disappointing, and this might be attributed to dysfunctional assembly of RS1 complexes and/or the impaired targeted cells. In this study, the human synapsin 1 gene promoter (hSyn) was used to control the expression of hRS1 to specifically target retinal ganglion cells and our results confirmed the specific expression and functional assembly of the protein. Moreover, our results demonstrated that a single intravitreal injection of rAAV2-hSyn-hRS1 results in architectural restoration of retinal schisis cavities and improvement in vision in a mouse model of XLRS. In brief, this study not only supports the clinical development of the rAAV2-hSyn-hRS1 vector in XLRS patients but also confirms the therapeutic potential of rAAV-based gene therapy in inherited retinal diseases.

Gas tamponade for myopic foveoschisis with foveal detachment.

PURPOSE: To evaluate the effects of gas tamponade without vitrectomy in patients with myopic foveoschisis and foveal detachment. METHODS: We examined 10 eyes of 10 patients with myopic foveoschisis and foveal detachment. All patients received an intravitreal injection of 0.2-0.4 mL C3F8 and remained in the prone position for 5 to 7 days. The patients were followed up for at least 6 months after the procedure. The refractive status, best-corrected visual acuity (BCVA), fundus changes, premacular disorders, and anatomical results before and after the treatment were assessed and recorded. All participants underwent serial optical coherence tomography (OCT). RESULTS: All patients had myopia of more than -7 diopters in the lesion eye. Staphyloma with chorioretinal atrophy was noted in all 10 eyes. The best-corrected visual acuity (BCVA) ranged from logMAR 0.52-2.0 before treatment. After initial gas tamponade, foveal detachment had resolved completely in four eyes and partially in four eyes at the 1-month follow-up visit, and two of the partially resolved cases achieved anatomical success after repeated treatment. Another eye achieved reattachment 15 months later and the other had a persistent but decreased level of detachment. Vitrectomy was subsequently performed in the one eye that showed increased detachment, and complete resolution of the schisis-detachment was achieved. The final BCVA improved in seven eyes and none of the study subjects showed decreased visual acuity. CONCLUSION: Gas tamponade is an alternative treatment for myopic foveoschisis with foveal detachment. More than 50 % cases showed a positive response with increased visual acuity. Further, the procedure did not appear to interfere with subsequent vitrectomy, if this procedure must be performed.

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.

Recent trends in the management of maculopathy secondary to pathological myopia.

BACKGROUND: Pathological myopia is a frequent cause of secondary visual disturbance in young individuals worldwide. Myopic maculopathy describes a spectrum of clinical changes that comprise the main cause of visual loss among highly myopic individuals. Our aim is to describe current trends in the medical and surgical management of maculopathy secondary to pathological myopia. METHODS: The epidemiology, natural history, medical and surgical treatment modalities for choroidal neovascular membrane (CNV) and vitreomacular disorders secondary to pathological myopia (PM) are reviewed and evaluated. RESULTS: The medical and surgical treatment modalities in the management of myopic maculopathy have evolved over time. Laser photocoagulation, photodynamic therapy with verteporfin and other medical treatments have been superseded by the use of anti-vascular endothelial growth factor in the management of CNV secondary to PM. Surgical treatments are beneficial in the treatment of vitreomacular interface disorders such as macular hole retinal detachment and macular traction; however, primary success rates remain lower than those for non-myopic individuals. CONCLUSIONS: This updated clinical perspective demonstrates that CNV and vitreomacular disorders in pathological myopia are treatable conditions. There are numerous medical and surgical interventions that have significantly improved the outcome of myopic maculopathy and several others currently under investigation. Nonetheless, as technology advances, further well-designed studies are necessary to establish a uniform evidence-based approach for classification and treatment.

AAV2/4-RS1 gene therapy in the retinoschisin knockout mouse model of X-linked retinoschisis.

OBJECTIVE: To evaluate efficacy of a novel adeno-associated virus (AAV) vector, AAV2/4-RS1, for retinal rescue in the retinoschisin knockout (Rs1-KO) mouse model of X-linked retinoschisis (XLRS). Brinzolamide (Azopt®), a carbonic anhydrase inhibitor, was tested for its ability to potentiate the effects of AAV2/4-RS1. METHODS: AAV2/4-RS1 with a cytomegalovirus (CMV) promoter (2x1012 viral genomes/mL) was delivered to Rs1-KO mice via intravitreal (N = 5; 1µL) or subretinal (N = 21; 2µL) injections at postnatal day 60-90. Eleven mice treated with subretinal therapy also received topical Azopt® twice a day. Serial full field electroretinography (ERG) was performed starting at day 50-60 post-injection. Mice were evaluated using a visually guided swim assay (VGSA) in light and dark conditions. The experimental groups were compared to untreated Rs1-KO (N = 11), wild-type (N = 12), and Rs1-KO mice receiving only Azopt® (N = 5). Immunofluorescence staining was performed to assess RS1 protein expression following treatment. RESULTS: The ERG b/a ratio was significantly higher in the subretinal plus Azopt® (p<0.0001), subretinal without Azopt® (p = 0.0002), and intravitreal (p = 0.01) treated eyes compared to untreated eyes. There was a highly significant subretinal treatment effect on ERG amplitudes collectively at 7-9 months post-injection (p = 0.0003). Cones showed more effect than rods. The subretinal group showed improved time to platform in the dark VGSA compared to untreated mice (p<0.0001). RS1 protein expression was detected in the outer retina in subretinal treated mice and in the inner retina in intravitreal treated mice. CONCLUSIONS: AAV2/4-RS1 shows promise for improving retinal phenotype in the Rs1-KO mouse model. Subretinal delivery was superior to intravitreal. Topical brinzolamide did not improve efficacy. AAV2/4-RS1 may be considered as a potential treatment for XLRS patients.

Intravitreal Delivery of rAAV2tYF-CB-hRS1 Vector for Gene Augmentation Therapy in Patients with X-Linked Retinoschisis: 1-Year Clinical Results.

PURPOSE: To evaluate the safety and efficacy of rAAV2tYF-CB-hRS1, a recombinant adeno-associated virus vector expressing retinoschisin (RS1), in individuals with retinal disease caused by mutations in the RS1 gene. DESIGN: Open-label, phase I/II dose-escalation clinical trial. SUBJECTS: Twenty-two adults and 5 children with X-linked retinoschisis (XLRS), aged 10 to 79 years, were enrolled. METHODS: The participants received an intravitreal (IVT) injection of rAAV2tYF-CB-hRS1, at 1 of 3 dose levels, in the poorer-seeing eye and were followed up for a minimum of 1 year after treatment. MAIN OUTCOME MEASURES: The primary safety measures were local (ocular) or systemic (nonocular) adverse events (AEs) during the 12-month period after study agent administration. Efficacy was assessed based on measures of best-corrected visual acuity (BCVA), schisis cavity volume, static perimetry visual field testing, and electroretinography (ERG). RESULTS: The IVT administration of rAAV2tYF-CB-hRS1 was generally safe at each of the dose levels. There were no AEs resulting in early termination, and no dose-limiting toxicities were reported. The most common ocular AEs observed were related to ocular inflammation (blurred vision, visual impairment, and the presence of vitreous cells, keratic precipitates, vitreous floaters, anterior chamber cells, and vitreous haze). Ocular inflammation was generally either mild or moderate in severity and responsive to standard immunosuppressive therapy, except in 3 participants (all in the highest-dose group) who developed chronic uveitis, which required prolonged therapy. Two patients experienced retinal detachments. There was no overall improvement in BCVA, visual fields, or ERG in the study eye compared with that in the fellow eye for any dose group. Variable changes in the cystic cavity volume over time were similar in the study and fellow eyes. CONCLUSIONS: Gene augmentation therapy with rAAV2tYF-CB-hRS1 for XLRS was generally safe and well tolerated but failed to demonstrate a measurable treatment effect. The clinical trial is ongoing through 5 years of follow-up to assess its long-term safety.

Clinical manifestation and current therapeutics in X-juvenile retinoschisis.

X-linked juvenile retinoschisis (XLRS) is one of the common early-onset hereditary retinal degenerative diseases in men. The common symptoms of XLRS range from mild to severe central vision loss and radial stripes created by the fovea, the division of the inner layer of the retina in the peripheral retina and the significant decrease in b-wave amplitude (ERG). Retinoschisin, the 224-amino-acid protein product of the retinoschisis 1 (RS1) gene, contains a discoid domain as the primary structural unit, an N-terminal cleavable signal sequence, and an oligomerization-area component. Retinoschisin is a homo-octamer complex with disulfide links that are released by retinal cells. It helps preserve the retina's integrity by binding to the surface of photoreceptors and bipolar cells. As a recessive genetic disease, XLRS was usually treated by prescribing low vision aids in most clinical cases. A gene replacement therapy based on adeno-associated virus vectors was initiated and showed a breakthrough in treating XLRS in 2014. Understanding the revolution of gene therapy for treating XLRS may accelerate its development and make this gene therapy the template for developing therapeutics against other inherited retinal diseases.

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.

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.

Molecular mechanisms leading to null-protein product from retinoschisin (RS1) signal-sequence mutants in X-linked retinoschisis (XLRS) disease.

Retinoschisin (RS1) is a cell-surface adhesion molecule expressed by photoreceptor and bipolar cells of the retina. The 24-kDa protein encodes two conserved sequence motifs: the initial signal sequence targets the protein for secretion while the larger discoidin domain is implicated in cell adhesion. RS1 helps to maintain the structural organization of the retinal cell layers and promotes visual signal transduction. RS1 gene mutations cause X-linked retinoschisis disease (XLRS) in males, characterized by early-onset central vision loss. We analyzed the biochemical consequences of several RS1 signal-sequence mutants (c.1A>T, c.35T>A, c.38T>C, and c.52G>A) found in our subjects. Expression analysis in COS-7 cells demonstrates that these mutations affect RS1 biosynthesis and result in an RS1 null phenotype by several different mechanisms. By comparison, discoidin-domain mutations generally lead to nonfunctional conformational variants that remain trapped inside the cell. XLRS disease has a broad heterogeneity in general, but subjects with the RS1 null-protein signal-sequence mutations are on the more severe end of the clinical phenotype. Results from the signal-sequence mutants are discussed in the context of the discoidin-domain mutations, clinical phenotypes, genotype-phenotype correlations, and implications for RS1 gene replacement therapy.

Intravitreal delivery of AAV8 retinoschisin results in cell type-specific gene expression and retinal rescue in the Rs1-KO mouse.

X-linked juvenile retinoschisis (XLRS) is a neurodevelopmental abnormality caused by retinoschisin gene mutations. XLRS is characterized by splitting through the retinal layers and impaired synaptic transmission of visual signals resulting in impaired acuity and a propensity to retinal detachment. Several groups have treated murine retinoschisis models successfully using adeno-associated virus (AAV) vectors. Owing to the fragile nature of XLRS retina, translating this therapy to the clinic may require an alternative to invasive subretinal vector administration. Here we show that all layers of the retinoschisin knockout (Rs1-KO) mouse retina can be transduced efficiently with AAV vectors administered by simple vitreous injection. Retinoschisin expression was restricted to the neuroretina using a new vector that uses a 3.5-kb human retinoschisin promoter and an AAV type 8 capsid. Intravitreal administration to Rs1-KO mice resulted in robust retinoschisin expression with a retinal distribution similar to that observed in wild-type retina, including the expression by the photoreceptors lying deep in the retina. No off-target expression was observed. Rs1-KO mice treated with this vector showed a decrease in the schisis cavities and had improved retinal signaling evaluated by recording the electroretinogram 11-15 weeks after the application.