Correction: Suppression of choroidal neovascularization and epithelial-mesenchymal transition in retinal pigmented epithelium by adeno-associated virus-mediated overexpression of CCN5 in mice.

[This corrects the article DOI: 10.1371/journal.pone.0269937.].

Intravitreal Administration of Retinal Organoids-Derived Exosomes Alleviates Photoreceptor Degeneration in Royal College of Surgeons Rats by Targeting the Mitogen-Activated Protein Kinase Pathway.

Increasing evidence suggests that exosomes are involved in retinal cell degeneration, including their insufficient release; hence, they have become important indicators of retinopathies. The exosomal microRNA (miRNA), in particular, play important roles in regulating ocular and retinal cell functions, including photoreceptor maturation, maintenance, and visual function. Here, we generated retinal organoids (ROs) from human induced pluripotent stem cells that differentiated in a conditioned medium for 60 days, after which exosomes were extracted from ROs (Exo-ROs). Subsequently, we intravitreally injected the Exo-RO solution into the eyes of the Royal College of Surgeons (RCS) rats. Intravitreal Exo-RO administration reduced photoreceptor apoptosis, prevented outer nuclear layer thinning, and preserved visual function in RCS rats. RNA sequencing and miRNA profiling showed that exosomal miRNAs are mainly involved in the mitogen-activated protein kinase (MAPK) signaling pathway. In addition, the expression of MAPK-related genes and proteins was significantly decreased in the Exo-RO-treated group. These results suggest that Exo-ROs may be a potentially novel strategy for delaying retinal degeneration by targeting the MAPK signaling pathway.

Short-term Efficacy and Safety of Intravitreal Brolucizumab Injection for Treatment-Naive Exudate Age-related Macular Degeneration: A Multicenter Study.

PURPOSE: To compare short-term efficacy and safety of intravitreal brolucizumab injection with aflibercept in treatment-naive neovascular age-related macular degeneration (nAMD) patients. METHODS: A total of 59 eyes from 59 treatment-naive nAMD patients in three hospitals were retrospectively reviewed. Of which, 27 patients underwent intravitreal brolucizumab injections and 32 received aflibercept. After monthly consecutive three injections, best-corrected visual acuity (BCVA; in logarithm of minimal angle of resolution [logMAR]), central macular thickness (CMT), dry macula achievement rate, and intraocular inflammation (IOI) incidence were compared. RESULTS: After loading-phase treatment, BCVA was significantly increased from 0.48 ± 0.30 logMAR at baseline to 0.33 ± 0.21 logMAR at 3 months in the brolucizumab group (p = 0.002) and 0.40 ± 0.39 logMAR at baseline to 0.33 ± 0.36 logMAR at 3 months in the aflibercept group (p = 0.007). But there was no significant difference in BCVA improvement at 3 months between the two groups. CMT significantly decreased from 429.67 ± 250.59 µm at baseline to 210.67 ± 93.53 µm at 3 months in the brolucizumab group and from 346.69 ± 159.09 µm to 234.52 ± 83.42 µm in the aflibercept group (both p < 0.001). The amount of CMT reduction was significantly greater in the brolucizumab group after 3 months (p = 0.036). In typical AMD eyes, brolucizumab showed similar BCVA improvement but better CMT reduction at 3 months (p = 0.018). Dry macula achievement rate was not significantly different between the two groups. One IOI was observed in the brolucizumab group. CONCLUSIONS: Intravitreal injections of brolucizumab and aflibercept showed similar anatomical and functional outcomes. But CMT reduction was greater in the brolucizumab group. One IOI was identified, which was tolerable for topical agents. These results suggest that brolucizumab could be a novel first line treatment option for treating naive nAMD patients.

Voretigene Neparvovec for the Treatment of RPE65-associated Retinal Dystrophy: Consensus and Recommendations from the Korea RPE65-IRD Consensus Paper Committee.

Mutations in the RPE65 gene, associated with Leber congenital amaurosis, early-onset severe retinal dystrophy, and retinitis pigmentosa, gained growing attention since gene therapy for patients with RPE65-associated retinal dystrophy is available in clinical practice. RPE65 gene accounts for a very small proportion of patients with inherited retinal degeneration, especially Asian patients. Because RPE65-associated retinal dystrophy shares common clinical characteristics, such as early-onset severe nyctalopia, nystagmus, low vision, and progressive visual field constriction, with retinitis pigmentosa by other genetic mutations, appropriate genetic testing is essential to make a correct diagnosis. Also, fundus abnormalities can be minimal in early childhood, and the phenotype is highly variable depending on the type of mutations in RPE65-associated retinal dystrophy, which makes a diagnostic difficulty. The aim of this paper is to review the epidemiology of RPE65-associated retinal dystrophy, mutation spectrum, genetic diagnosis, clinical characteristics, and voretigene neparvovec, a gene therapy product for the treatment of RPE65-related retinal dystrophy.

Suppression of choroidal neovascularization and epithelial-mesenchymal transition in retinal pigmented epithelium by adeno-associated virus-mediated overexpression of CCN5 in mice.

Choroidal neovascularization (CNV) is a defining characteristic feature of neovascular age-related macular degeneration (nAMD) that frequently results in irreversible vision loss. The current strategies for the treatment of nAMD are mainly based on neutralizing vascular endothelial growth factor (VEGF). However, anti-VEGF therapies are often associated with subretinal fibrosis that eventually leads to damages in macula. In this study, we tested whether an anti-fibrotic and anti-angiogenic protein CCN5 can potentially be an effective and safe therapeutic modality in a mouse model of CNV. Laser photocoagulation was utilized to induce CNV, which was followed by intravitreal injection of recombinant adeno-associated virus serotype 2 encoding CCN5 (rAAV2-CCN5). Our data demonstrated that rAAV2-CCN5, but not a control viral vector, rAAV2-VLP, prominently attenuated both CNV lesions and angiogenesis. Aflibercept, which was utilized as a positive control, exhibited similar effects on CNV lesions and angiogenesis in our experimental settings. Upon laser photocoagulation, retinal pigmented epithelium (RPE) cells underwent significant morphological changes including cellular enlargement and loss of hexagonality. rAAV2-CCN5 significantly normalized these morphological defects. Laser photocoagulation also led to fibrotic deformation in RPE cells through inducing epithelial-mesenchymal transition (EMT), which was completely blocked by rAAV2-CCN5. In a striking contrast, aflibercept as well as rAAV2-VLP failed to exhibit any effects on EMT. Collectively, this study suggest that CCN5 might provide a potential novel strategy for the treatment of nAMD with a capability to inhibit CNV and fibrosis simaultaneously.

Association between chronic kidney disease and open-angle glaucoma in South Korea: a 12-year nationwide retrospective cohort study.

Various non-intraocular pressure factors have been identified as possible risk factors for open-angle glaucoma (OAG). However, there is still controversy around the association between OAG and chronic kidney disease (CKD). In this study, we used a nationwide cohort to investigate the risk of OAG in the 12 years following a diagnosis of CKD. This retrospective cohort study included 1,103,302 subjects from the Korean National Health Insurance Service National Sample Cohort database. The CKD group (n = 1318) included patients who were initially diagnosed with CKD between 2003 and 2008. The subjects in the comparison group were matched at a 1:5 ratio using propensity scores. In multivariate Cox regression analysis, a diagnosis of CKD was significantly associated with an increased incidence of OAG (hazard ratio [HR] = 1.546, 95% confidence interval [CI] 1.363-1.754, p < 0.001). Further analysis revealed that the risk of OAG increased with the severity of CKD (mild to moderate CKD [CKD stage 1-3]: HR = 1.280, 95% CI 1.077-1.521, p = 0.005; advanced CKD [CKD stage 4-5]: HR = 1.861, 95% CI 1.589-2.180, p < 0.001). In subgroup analysis, female CKD patients had a greater risk of developing OAG than males, and subjects with CKD aged ≥ 40 years were more likely to develop OAG compared with those aged < 40 years. Our study demonstrates that CKD is a significant risk factor for OAG and that severe CKD is associated with an increased risk of developing OAG.

Role of mTORC1 activity during early retinal development and lamination in human-induced pluripotent stem cell-derived retinal organoids.

Retinal organoids derived from human-induced pluripotent stem cells (hiPSC) are powerful tools for studying retinal development as they model spatial and temporal differentiation of retinal cell types. Vertebrate retinal development involves a delicate and coordinated process of retinal progenitor cell (RPC) differentiation, and the mammalian target of rapamycin complex 1 (mTORC1) has been reported to play a significant role in this complex process. Herein, using hiPSC-derived retinal organoids, we identify the time-dependent role of mTORC1 in retinal development, specifically in retinal ganglion cell (RGC) differentiation and the retinal lamination process, during the early stages of retinal organoid (RO) development. mTORC1 activity in ROs was the highest at 40 days of differentiation. MHY1485-induced hyperactivation of mTORC1 during this period resulted in a significant increase in the overall size of ROs compared to the untreated controls and rapamycin-treated Ros; there was also a marked increase in proliferative activity within the inner and outer layers of ROs. Moreover, the MHY1485-treated ROs showed a significant increase in the number of ectopic RGCs in the outer layers (indicating disruption of retinal laminar structure), with robust expression of HuC/D-binding proteins in the inner layers. These results demonstrate that mTORC1 plays a critical role in the development of hiPSC-derived ROs, especially during the early stages of differentiation.

Clinical Manifestations and Genetic Analysis of 5 Korean Choroideremia Patients Initially Diagnosed With Retinitis Pigmentosa.

BACKGROUND: To investigate the clinical findings of choroideremia patients and perform genetic analysis by whole-exome sequencing (WES). METHODS: A total of 94 patients initially diagnosed with retinitis pigmentosa (RP) at another hospital, and who visited our hospital for genetic analysis by WES, were included in the study, along with 64 family members. All subjects underwent comprehensive ophthalmic evaluation, including best-corrected visual acuity, slit lamp examination, fundus photography, fundus autofluorescence (FAF), fluorescein angiography (FAG), visual field (VF), electroretinogram (ERG), and optical coherence tomography (OCT). RESULTS: In six male patients with suspected choroideremia, extensive retinal pigment epithelium (RPE) and severe loss of choroid were observed in the fundus, but not in the macula. CHM gene mutation was confirmed in five patients. A novel single nucleotide variant at a splice site was observed in one patient. OCT showed marked thinning of the outernuclear layer and choroid, except in the macula. FAF showed a small area of hyperfluorescence in the posterior pole. In addition, characteristic interlaminar bridges were observed in four patients. On FAG, hypofluorescence was seen up to the far-peripheral retina in five patients. CONCLUSION: Of the 94 patients initially diagnosed with RP, CHM mutation was identified in five (5.3%) by WES. Choroideremia should be considered as a differential diagnosis of RP. WES would be useful for identifying the causes of hereditary retinal disease.

Proteome alterations in the aqueous humor reflect structural and functional phenotypes in patients with advanced normal-tension glaucoma.

Previous reports have shown possible association between altered protein levels in aqueous humor (AH) and normal-tension glaucoma (NTG), but the underlying pathogenetic mechanism as well as specific molecular biomarkers for NTG remains still elusive. Here, we aimed to identify novel biomarkers for advanced NTG by analyzing the proteome of patient-derived AH and their correlation with various functional and structural parameters from the visual field test (VF), optical coherence tomography (OCT), and OCT angiography (OCTA). We determined differentially expressed proteins (DEPs) of the AH of patients with advanced NTG (n = 20) using label-free quantitative (LFQ) proteomics with pooled samples and data-independent acquisition (DIA) analysis with individual samples, and the roles of AH DEPs in biological pathways were evaluated using bioinformatics. We identified 603 proteins in the AH of patients with advanced NTG, and 61 of them were selected as DEPs via global proteome LFQ profiling. Individual DIA analyses identified a total of 12 DEPs as biomarker candidates, seven of which were upregulated, and five were downregulated. Gene ontology enrichment analysis revealed that those DEPs were mainly involved in the immune response. Moreover, IGFBP2, ENO1, C7, B2M, AMBP, DSP, and DCD showed a significant correlation with the mean deviation of VF and with peripapillary and macular parameters from OCT and OCTA. The present study provides possible molecular biomarkers for the diagnosis of advanced NTG.

Wnt signalling inhibits adipogenesis in orbital fibroblasts from patients with Graves' orbitopathy.

BACKGROUND/AIMS: To investigate the role of Wnt signalling in adipogenesis using an in vitro model of Graves' orbitopathy (GO). METHODS: Orbital fat was obtained from patients with GO and non-GO participants for primary orbital fibroblast (OF) culture. Expression levels of Wnt5a, Wnt10b, ß-catenin, phospho-ß-catenin and cyclin D1 were compared between GO and non-GO OFs. These expression levels were also determined during adipogenesis of GO and non-GO OFs. The effects of a stimulator and inhibitor of Wnt signalling on adipogenesis of GO and non-GO OFs were investigated. RESULTS: Western blotting analysis showed significant reductions in ß-catenin and cyclin D1 and significant enhancement of phospho-ß-catenin in OFs from patients with GO, compared with OFs from non-GO participants (p<0.05). Expression of Wnt5a, Wnt10b, ß-catenin and cyclin D1 in OFs was highest on day 0, and then gradually declined after induction of adipogenic differentiation. The expression levels of PPARγ, C/EBPα and C/EBPß were reduced in Wnt stimulator-treated OFs in a dose-dependent manner. Oil red O staining confirmed that a stimulator of Wnt inhibited adipogenesis in GO OFs. CONCLUSION: These results indicate that Wnt signalling inhibits adipogenesis in OFs from patients with GO and non-GO participants. Further studies are required to examine the potential of Wnt signalling as a target for therapeutic strategies.

Characterization of the role of autophagy in retinal ganglion cell survival over time using a rat model of chronic ocular hypertension.

Autophagy is an essential cellular process for the degradation and recycling of cellular components, and its dysregulation has been linked to neuronal cell death and neurodegeneration. In glaucoma, the role of autophagy in retinal ganglion cell (RGC) survival remains contradictory. Moreover, the effects of autophagy modulation at different time-points on RGC survival in a glaucoma model have not been investigated. In this study, we assessed the time-dependent role of autophagy in RGC survival in a circumlimbal suture-induced ocular hypertensive (OHT) rat model. Intraocular pressure (IOP) elevation led to a gradual autophagy induction, which reached a maximum between 1 and 4 weeks after OHT induction. On the other hand, early autophagy was impaired between 1 and 3 days after circumlimbal suturing, indicated by increased p62 levels due to reduced autophagosomal turnover. The intravitreal administration of rapamycin at different time-points after the application of the circumlimbal suture indicated that autophagy induction early during OHT development had potent survival-promoting effects in RGCs. In conclusion, our findings suggest that the role of autophagy in RGCs during OHT development might differ in a time-dependent manner. Modulating autophagy at the appropriate time might serve as a potential therapeutic approach to enhance RGC survival in OHT.

mTOR-dependent dysregulation of autophagy contributes to the retinal ganglion cell loss in streptozotocin-induced diabetic retinopathy.

BACKGROUND: Neurodegeneration, an early event in the pathogenesis of diabetic retinopathy (DR), precedes clinically detectable microvascular damage. Autophagy dysregulation is considered a potential cause of neuronal cell loss, however underlying mechanisms remain unclear. The mechanistic target of rapamycin (mTOR) integrates diverse environmental signals to coordinate biological processes, including autophagy. Here, we investigated the role of mTOR signaling in neuronal cell death in DR. METHODS: Diabetes was induced by a single intraperitoneal injection of streptozotocin and tissue samples were harvested at 1, 2, 3, 4, and 6 months of diabetes. Early-stage of DR was investigated in 1-month-diabetic mice treated with phlorizin (two daily subcutaneous injections at a dose of 200 mg/kg of body weight during the last 7 full days of the experiment and the morning of the 8th day, 3 h before sacrifice) or rapamycin (daily intraperitoneal injections, at a dose of 3 mg/kg for the same period as for phlorizin treatment). The effect of autophagy modulation on retinal ganglion cells was investigated in 3-months-diabetic mice treated with phlorizin (two daily subcutaneous injections during the last 10 full days of the experiment and the morning of the 11th day, 3 h before sacrifice) or MHY1485 (daily i.p. injections, at a dose of 10 mg/kg for the same period as for phlorizin treatment). Tissue samples obtained from treated/untreated diabetic mice and age-matched controls were used for Western blot and histologic analysis. RESULTS: mTOR-related proteins and glucose transporter 1 (GLUT1) was upregulated at 1 month and downregulated in the following period up to 6 months. Diabetes-induced neurodegeneration was characterized by an increase of apoptotic marker-cleaved caspase 3, a decrease of the total number of cells, and NeuN immunoreactivity in the ganglion cell layer, as well as an increase of autophagic protein. Insulin-independent glycemic control restored the mTOR pathway activity and GLUT1 expression, along with a decrease of autophagic and apoptotic proteins in 3-months-diabetic mice neuroretina. However, blockade of autophagy using MHY1485 resulted in a more protective effect on ganglion cells compared with phlorizin treatment. CONCLUSION: Collectively, our study describes the mechanisms of neurodegeneration through the hyperglycemia/ mTOR/ autophagy/ apoptosis pathway. Video Abstract.

Genotype and Long-term Clinical Course of Bietti Crystalline Dystrophy in Korean and Japanese Patients.

PURPOSE: To investigate the genotype and long-term clinical phenotype of patients with Bietti crystalline dystrophy (BCD) in Korea and Japan. DESIGN: Retrospective case series. PARTICIPANTS: We analyzed 62 patients with clinical features of BCD who harbor pathogenic biallelic CYP4V2 variants in their homozygote or compound heterozygote. METHODS: Data were collected from patient charts, including age, best-corrected visual acuity (BCVA), Goldmann perimetry results, fundus photography, OCT findings, fundus autofluorescence results, and electroretinography findings. We compared the clinical course of the patients with homozygous c.802-8_810de117insGC [exon7del], the most common mutation in the East Asian population, with those of the patients with other genotypes. MAIN OUTCOME MEASURES: Best-corrected visual acuity, visual field (VF), and their changes during follow-up. RESULTS: The mean age at the first visit was 55.2 years, with a mean follow-up of 7.1 years. The mean BCVAs at the first and last visits were 0.28 logarithm of the minimum angle of resolution (logMAR) and 0.89 logMAR, respectively. In genetic testing, c.802-8_810de117insGC was detected in 86 of 124 alleles of the patients, and 36 patients were homozygous for this mutation. The age, BCVA, VF area, central foveal thickness, and abnormal hypoautofluorescent area at either the first or last visit were not different between the exon7del homozygotes and the others. The mean BCVA changes per year were 0.089 logMAR in the exon7del homozygotes and 0.089 logMAR in the others. An age- and gender-adjusted linear regression analysis showed no association between the exon7del homozygote status and the rate of vision loss. Characteristic crystalline deposits in the posterior pole were generally observed in younger patients and disappeared over time along with progressive retinochoroidal atrophy. CONCLUSIONS: Patients with BCD and a homozygote for c.802-8_810de117insGC accounted for more than 50% of this cohort of Korean and Japanese patients, and the clinical effect of this deleterious variant was not severe in the spectrum of CYP4V2 retinopathy.

Peripapillary Intravitreal Injection Improves AAV-Mediated Retinal Transduction.

The intravitreal (IVT) injection method is a choice when targeting the inner retina for gene therapy. However, the transduction efficiency of adeno-associated virus (AAV) vectors administered by the IVT route is usually low and may be affected by several factors. To improve the transduction efficiency, we developed a novel illuminated long-needle attached injection system and injected AAV2-CMV (cytomegalovirus)-EGFP in front of the retina in rabbit eyes. Ophthalmological examinations were performed and the levels of pro-inflammatory cytokines in the aqueous humor were assessed at the baseline and 1 month, and the results were compared with those of the conventional injection method. Retinal tissues were used for immunohistochemistry. In the ophthalmological examinations, no significant inflammatory signs were detected in both groups, except for transient, mild hyperemia. In the tissues of the rabbits in the peripapillary injection group, significantly increased GFP expression was detected at the ganglion cell and the inner nuclear layers (p < 0.01). There were no differences between groups in glial activation and expressions of interleukin (IL)-6 and IL-8. These results suggest that peripapillary IVT injection in front of the retina would be safe and efficiently transduce viral vectors into the retina of large animals and is considered as a potential method for use in clinical trials.

Morphologic and electrophysiologic findings of retinal degeneration after intravitreal sodium iodate injection following vitrectomy in canines.

We developed and characterized a canine model of outer retinal degeneration induced by sodium iodate (SI) intravitreal injection after vitrectomy. In the preliminary study, we repeatedly injected SI intravitreally into the eyes of three canines to develop outer retinal degeneration two weeks after vitrectomy. Based on the preliminary study, a single dose of either 1.2 mg or 1.0 mg SI/0.05 mL was also injected (1.2 mg in n = 5 canines, 1.0 mg in n = 2 canines). Spectral domain-optical coherence tomography (OCT), electroretinography (ERG), and histological examinations were performed at baseline and following intravitreal injection. In the preliminary study, after a 0.5-mg SI injection and a 1.0-mg SI injection and after two 0.8-mg SI injections, retinal degeneration with retinal thinning was observed on OCT imaging. In the second study, after a single 1.0- or 1.2-mg SI injection, outer retinal degeneration was induced. All eyes showed diffuse outer retinal degeneration on OCT and a loss of both cone and rod responses in ERG. Histological examination also showed the loss of outer retinal layer. Intravitreally injected SI (1.0-1.2 mg) in a vitrectomized canine model induced outer retinal degeneration effectively, and could be evaluated through in vivo ophthalmic examination.

The effects of intravitreal sodium iodate injection on retinal degeneration following vitrectomy in rabbits.

We sought to develop and characterize outer retinal degeneration induced by intravitreal injection of sodium iodate (SI) after vitrectomy in rabbits. To determine the effective dose of SI, the right eyes of 19 male New Zealand white rabbits received an intravitreal injection of SI or sham. Based on the dose-dependence results, 0.4 mg of SI in 0.05 mL of total volume was injected into the right eyes of 10 rabbits at two weeks after vitrectomy. In the dose-dependence study, localized retinal atrophy was observed with 0.3- and 0.4-mg SI injections without vitrectomy. Severe and diffuse retinal atrophy was identified by spectral-domain optical coherence tomography (SD-OCT) at one month after a 0.5-mg SI injection following vitrectomy. In the second experiment, 0.4 mg of SI in 0.05 mL was injected, and the severity of outer retinal degeneration was graded as one of two types according to electroretinography (ERG) response change. There was no response on ERG in complete retinal degeneration, 30% of all 10 rabbits. Intravitreal injection of 0.4 mg of SI into vitrectomized rabbit eyes induces diffuse outer retinal degeneration, and the degree of retinal degeneration can be evaluated through in vivo ophthalmic examination.

Transduction Pattern of AAVs in the Trabecular Meshwork and Anterior-Segment Structures in a Rat Model of Ocular Hypertension.

Adeno-associated viruses (AAVs) are the vector of choice for gene therapy in the eye, and self-complementary AAVs (scAAVs), which do not require second-strand DNA synthesis, can be transduced into cells of the trabecular meshwork (TM). The scAAV transduction patterns in the anterior segment of normotensive eyes have been investigated previously, but those in ocular hypertensive (OHT) eyes have not. We assessed the transduction efficiencies of AAV serotypes 2, 5, and 8 in the anterior-segment structures of the eyes of Sprague-Dawley rats with OHT by circumlimbal suturing, followed 3 days later by intracameral injection of scAAV serotype 2 (scAAV2), scAAV5, or scAAV8 packaged with EGFP. The transduction of scAAV2 and scAAV5 in the TM of OHT rats was markedly enhanced after 1 month, and transduction of scAAV5 was more efficient than that of scAAV2; transduction of scAAV8 into the TM did not occur. The transduction of scAAV2, scAAV5, and scAAV8 was enhanced in the ciliary body, iris, and corneal endothelium of the OHT eyes for 3 months. The expression levels of receptors for scAAV2 and scAAV5 were significantly increased in the OHT compared with control eyes. The results demonstrated that scAAV2 and scAAV5 target the ciliary body and TM in OHT eyes, and that the OHT-related changes in anterior-segment structures enhance scAAV transduction.

Effects of Stuffer DNA on the Suppression of Choroidal Neovascularization by a rAAV Expressing a mTOR-Inhibiting shRNA.

Choroidal neovascularization (CNV) is the defining characteristic of the wet subtype of age-related macular degeneration (AMD), which is a rapidly growing global health problem. Previously, we had demonstrated the therapeutic potential of gene therapy against CNV using short hairpin RNA (shRNA) delivered via recombinant adeno-associated virus (rAAV), which abrogates mammalian-to-mechanistic (mTOR) activity in a novel manner by simultaneously inhibiting both mTOR complexes. Both the target and use of gene therapy represent a novel treatment modality against AMD. Here, the xenogeneic GFP gene used as a reporter in previous studies was removed from the virus vector to further develop the therapeutic for clinical trials. Instead, a stuffer DNA derived from the 3' UTR of the human UBE3A gene was used to ensure optimal viral genome size for efficient rAAV assembly. The virus vector containing the stuffer DNA, rAAV2-shmTOR-SD, positively compares to one encoding the shRNA and a GFP expression cassette in terms of reducing CNV in a laser-induced mouse model, as determined by fundus fluorescein angiography. These results were confirmed via immunohistochemistry using anti-CD31, while a TUNEL assay showed that rAAV2-shmTOR-SD possesses anti-apoptotic properties as well. The qualities exhibited by rAAV2-shmTOR-SD demonstrate its potential as a human gene therapeutic for the treatment of wet AMD.

mTORC1 and mTORC2 are differentially engaged in the development of laser-induced CNV.

BACKGROUND: The mechanistic target of rapamycin (mTOR) pathway is a potential target to inhibit pathologic processes in choroidal neovascularization. However, the exact role of mTOR signaling in the development of CNV remains obscure. In this study, we assessed the role of mTORC1 and mTORC2 as well as the effect of rapamycin (sirolimus) on choroidal neovascularization (CNV) in a laser-induced mouse model. METHODS: In experiment A, we observed the natural course of CNV development and the dynamics of mTOR-related proteins during the 12 days after the laser injury. The expression of mTOR-related proteins was evaluated using Western blot (WB). Cryosections of CNV-induced mice were immunostained for the visualization of the vascular and extravascular components of the CNV. Experiment B was performed to confirm the critical period of mTOR signaling in the development of laser-induced CNV, we administered rapamycin before and/or during the active period of mTOR complexes. WB and immunofluorescence staining was performed to evaluate the mode of action and the effect of mTOR inhibition on CNV development. RESULTS: In experiment A, we detected high levels of p-mTOR S2448 and p-mTOR S2481 from the 5th to 12th day of laser injury. Immunofluorescence imaging of cryosections of mice sacrificed on day 7 revealed greater co-immunoreactivity of p-mTOR S2448 positive cells with CD11b and F4/80, while p-mTOR S2481 positive cells showed colocalization with CD31, α-SMA, and cytokeratin. In experiment B, rapamycin injection during the active period of mTOR signaling demonstrated near-complete inhibition of CNV lesion as well as significant induction of autophagy. CONCLUSION: Our study suggests the mTOR as a critical player during CNV development in laser-induced mouse model through differentially acting with the mTORC1 and mTORC2. mTORC1 activity was high predominantly in inflammatory cells in CNV lesion, while mTORC2 activity was higher in vascular components and the RPE.

Development of a Post-vitrectomy Injection of N-methyl-N-nitrosourea as a Localized Retinal Degeneration Rabbit Model.

Since genetic models for retinal degeneration (RD) in animals larger than rodents have not been firmly established to date, we sought in the present study to develop a new rabbit model of drug-induced RD. First, intravitreal injection of N-methyl-N-nitrosourea (MNU) without vitrectomy in rabbits was performed with different doses. One month after injection, morphological changes in the retinas were identified with ultra-wide-field color fundus photography (FP) and fundus autofluorescence (AF) imaging as well as spectral-domain optical coherence tomography (OCT). Notably, the degree of RD was not consistently correlated with MNU dose. Then, to check the effects of vitrectomy on MNU-induced RD, the intravitreal injection of MNU after vitrectomy in rabbits was also performed with different doses. In OCT, while there were no significant changes in the retinas for injections up to 0.1 mg (i.e., sham, 0.05 mg, and 0.1 mg), outer retinal atrophy and retinal atrophy of the whole layer were observed with MNU injections of 0.3 mg and 0.5 mg, respectively. With this outcome, 0.2 mg MNU was chosen to be injected into rabbit eyes (n=10) at two weeks after vitrectomy for further study. Six weeks after injection, morphological identification with FP, AF, OCT, and histology clearly showed localized outer RD - clearly bordered non-degenerated and degenerated outer retinal area - in all rabbits. We suggest our post-vitrectomy MNU-induced RD rabbit model could be used as an interim animal model for visual prosthetics before the transition to larger animal models.