Tropism of the Novel AAVBR1 Capsid Following Subretinal Delivery.

A serious limitation of current adeno-associated viral (AAV) capsids employed for subretinal delivery is achieving adequate lateral spread beyond the injection site, required for the efficient delivery of gene therapy to the outer retina and/or RPE. AAVBR1 is a unique AAV with exceptional tropism for CNS microvasculature following systemic delivery. Here, we used in vivo and ex vivo analysis to show that subretinal delivery of AAVBR1.GFP in mice achieves superior tropism to RPE and outer retina than either AAV2.GFP or AAV8.GFP, two of the most common capsids used for subretinal delivery. At a low (5 × 108 vg) subretinal dose, the AAVBR1.GFP signal was visible by 48 h and significantly surpassed peak fluorescence of other AAVs in retina and RPE. The co-injection of AAVBR1.GFP with the AAVBR1-specific heptapeptide, NRGTEWD, significantly blocked the AAVBR1.GFP signal, but had no effect on AAV2.GFP fluorescence, confirming that AAVBR1's enhanced tropism for RPE and outer retina derives from this 7AA modification within the capsid-binding motif. Enhanced dispersal and consequent transduction suggest that AAVBR1 can be employed at a lower dosage than the standard AAV2 capsid to achieve equivalent expression for gene therapy, warranting further evaluation of its utility as a therapeutic vehicle for subretinal delivery.

COMP-Ang1: Therapeutic potential of an engineered Angiopoietin-1 variant.

The Angiopoietin-1/2 system is an opportune target for therapeutic intervention in a wide range of vascular pathologies, particularly through its association with endothelium. The complex multi-domain structure of native human Angiopoietin-1 has hindered its widespread applicability as a therapeutic agent, prompting the search for alternative approaches to mimicking the Ang1:Tie2 signalling axis; a system with highly complex patterns of regulation involving multiple structurally similar molecules. An engineered variant, Cartilage Oligomeric Matrix Protein - Angiopoietin-1 (COMP-Ang1), has been demonstrated to overcome the limitations of the native molecule and activate the Tie2 pathway with several fold greater potency than Ang1, both in vitro and in vivo. The therapeutic efficacy of COMP-Ang1, at both the vascular and systemic levels, is evident from multiple studies. Beneficial impacts on skeletal muscle regeneration, wound healing and angiogenesis have been reported alongside renoprotective, anti-hypertensive and anti-inflammatory effects. COMP-Ang1 has also demonstrated synergy with other compounds to heighten bone repair, has been leveraged for potential use as a co-therapeutic for enhanced targeted cancer treatment, and has received considerable attention as an anti-leakage agent for microvascular diseases like diabetic retinopathy. This review examines the vascular Angiopoietin:Tie2 signalling mechanism, evaluates the potential therapeutic merits of engineered COMP-Ang1 in both vascular and systemic contexts, and addresses the inherent translational challenges in moving this potential therapeutic from bench-to-bedside.

Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy.

A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early-onset Fuchs' endothelial corneal dystrophy (FECD), which progressively impairs vision through the loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 start codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore the phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.

Neovascular Macular Degeneration: A Review of Etiology, Risk Factors, and Recent Advances in Research and Therapy.

Neovascular age-related macular degeneration (exudative or wet AMD) is a prevalent, progressive retinal degenerative macular disease that is characterized by neovascularization of the choroid, mainly affecting the elderly population causing gradual vision impairment. Risk factors such as age, race, genetics, iris color, smoking, drinking, BMI, and diet all play a part in nvAMD's progression, with anti-vascular endothelial growth factor (anti-VEGF) therapy being the mainstay of treatment. Current therapeutic advancements slow the progression of the disease but do not cure or reverse its course. Newer therapies such as gene therapies, Rho-kinase inhibitors, and levodopa offer potential new targets for treatment.

Systemic AAV10.COMP-Ang1 rescues renal glomeruli and pancreatic islets in type 2 diabetic mice.

INTRODUCTION: Diabetic hyperglycemia causes progressive and generalized damage to the microvasculature. In renal glomeruli, this results in the loss of podocytes with consequent loss of constitutive angiopoietin-1 (Ang1) signaling, which is required for stability of the glomerular endothelium. Repeated tail vein injection of adenovirus expressing COMP-Ang1 (a stable bioengineered form of Ang1) was previously reported to improve diabetic glomerular damage despite the liver and lungs being primary targets of adenoviral infection. We thus hypothesized that localizing delivery of sustained COMP-Ang1 to the kidney could increase its therapeutic efficacy and safety for the treatment of diabetes. RESEARCH DESIGN AND METHODS: Using AAVrh10 adeno-associated viral capsid with enhanced kidney tropism, we treated 10-week-old uninephrectomized db/db mice (a model of type 2 diabetes) with a single dose of AAVrh10.COMP-Ang1 delivered via the intracarotid artery, compared with untreated diabetic db/db control and non-diabetic db/m mice. RESULTS: Surprisingly, both glomerular and pancreatic capillaries expressed COMP-Ang1, compensating for diabetes-induced loss of tissue Ang1. Importantly, treatment with AAVrh10.COMP-Ang1 yielded a significant reduction of glycemia (blood glucose, 241±193 mg/dL vs 576±31 mg/dL; glycosylated hemoglobin, 7.2±1.5% vs 11.3±1.3%) and slowed the progression of albuminuria and glomerulosclerosis in db/db mice by 70% and 61%, respectively, compared with untreated diabetic db/db mice. Furthermore, COMP-Ang1 ameliorated diabetes-induced increases of NF-kBp65, nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase-2 (Nox2), p47phox and productions of myeloperoxidase, the inflammatory markers in both renal and pancreatic tissues, and improved beta-cell density in pancreatic islets. CONCLUSIONS: These results highlight the potential of localized Ang1 therapy for treatment of diabetic visceropathies and provide a mechanistic explanation for reported improvements in glucose control via Ang1/Tie2 signaling in the pancreas.

Transscleral Iontophoresis for Noninvasive Ocular Drug Delivery of Macromolecules.

Purpose: The objectives were to investigate the effect of transscleral iontophoresis of macromolecules in vitro and in vivo, to study the importance of electroosmosis on macromolecules of low charge to mass ratio, and to evaluate transscleral iontophoresis efficacy in a choroidal neovascularization (CNV) animal model. Methods: Through in vitro transport experiments, the permeability coefficients of macromolecules [eg, immunoglobulin G (IgG), dextran 70 kDa] were determined under different conditions. The effect of ionic strength formulations and iontophoretic conditions was studied on the distribution of IgG and bevacizumab into the eye in vivo. Magnetic resonance imaging (MRI) was utilized to evaluate in vivo real time distribution of gadolinium-labeled albumin (Galbumin) following iontophoresis. The efficacy between no treatment, intravitreal injection (IVT), and iontophoresis of bevacizumab on a CNV model of subretinal injection of adeno-associated virus encoding human VEGF-165 was investigated. Results: The permeability data suggested a significant effect of ionic strength on the iontophoretic transport of macromolecules. Transscleral iontophoresis of IgG at 4 mA with a low ionic strength formulation was about 600 times greater than passive diffusion and 14-fold over a conventional formulation in vitro. Approximately 0.6 mg of bevacizumab can be delivered into the rabbit eye in vivo with a 20-min treatment of iontophoresis. MRI showed that Galbumin was in the posterior tissues after iontophoresis. In the CNV model, the iontophoresis and IVT methods of bevacizumab delayed retinal neovascularization by 4 and 8 weeks, respectively. Conclusions: Transscleral iontophoresis is capable of delivering macromolecule drugs through the conjunctiva and sclera, eventually exposing the retina/choroid to the drugs.

Chronic Dicer1 deficiency promotes atrophic and neovascular outer retinal pathologies in mice.

Degeneration of the retinal pigmented epithelium (RPE) and aberrant blood vessel growth in the eye are advanced-stage processes in blinding diseases such as age-related macular degeneration (AMD), which affect hundreds of millions of people worldwide. Loss of the RNase DICER1, an essential factor in micro-RNA biogenesis, is implicated in RPE atrophy. However, the functional implications of DICER1 loss in choroidal and retinal neovascularization are unknown. Here, we report that two independent hypomorphic mouse strains, as well as a separate model of postnatal RPE-specific DICER1 ablation, all presented with spontaneous RPE degeneration and choroidal and retinal neovascularization. DICER1 hypomorphic mice lacking critical inflammasome components or the innate immune adaptor MyD88 developed less severe RPE atrophy and pathological neovascularization. DICER1 abundance was also reduced in retinas of the JR5558 mouse model of spontaneous choroidal neovascularization. Finally, adenoassociated vector-mediated gene delivery of a truncated DICER1 variant (OptiDicer) reduced spontaneous choroidal neovascularization in JR5558 mice. Collectively, these findings significantly expand the repertoire of DICER1 in preserving retinal homeostasis by preventing both RPE degeneration and pathological neovascularization.

Intravitreal AAV2.COMP-Ang1 Attenuates Deep Capillary Plexus Expansion in the Aged Diabetic Mouse Retina.

Purpose: We determine whether intravitreal angiopoietin-1 combined with the short coiled-coil domain of cartilage oligomeric matrix protein by adeno-associated viral serotype 2 (AAV2.COMP-Ang1) delivery following the onset of vascular damage could rescue or repair damaged vascular beds and attenuate neuronal atrophy and dysfunction in the retinas of aged diabetic mice. Methods: AAV2.COMP-Ang1 was bilaterally injected into the vitreous of 6-month-old male Ins2Akita mice. Age-matched controls consisted of uninjected C57BL/6J and Ins2Akita males, and of Ins2Akita males injected with PBS or AAV2.REPORTER (AcGFP or LacZ). Retinal thickness and visual acuity were measured in vivo at baseline and at the 10.5-month endpoint. Ex vivo vascular parameters were measured from retinal flat mounts, and Western blot was used to detect protein expression. Results: All three Ins2Akita control groups showed significantly increased deep vascular density at 10.5 months compared to uninjected C57BL/6J retinas (as measured by vessel area, length, lacunarity, and number of junctions). In contrast, deep microvascular density of Ins2Akita retinas treated with AAV2.COMP-Ang1 was more similar to uninjected C57BL/6J retinas for all parameters. However, no significant improvement in retinal thinning or diabetic retinopathy-associated visual loss was found in treated diabetic retinas. Conclusions: Deep retinal microvasculature of diabetic Ins2Akita eyes shows late stage changes consistent with disorganized vascular proliferation. We show that intravitreally injected AAV2.COMP-Ang1 blocks this increase in deep microvascularity, even when administered subsequent to development of the first detectable vascular defects. However, improving vascular normalization did not attenuate neuroretinal degeneration or loss of visual acuity. Therefore, additional interventions are required to address neurodegenerative changes that are already underway.

Antiangiogenesis Effect of Albendazole on the Cornea.

Purpose: To investigate the anti-(lymph)angiogenic and anti-inflammatory effects of albendazole and to study whether these effects are additive with bevacizumab therapy in a murine corneal suture model. Methods: Corneal neovascularization (NV) and lymphangiogenesis (LY) were compared in a corneal suture model after administration of a subconjunctival injection of albendazole, bevacizumab, dexamethasone, or phosphate-buffered saline (PBS). Immunohistochemical staining and analysis were performed in each group. Real-time polymerase chain reaction (RT-PCR) was performed to quantify the expression of inflammatory cytokines (tumor necrosis factor [TNF]-alpha and interleukin-6), vascular endothelial growth factor (VEGF)-A, VEGF-C, vascular endothelial growth factor receptor (VEGFR)-2, and VEGFR-3. To evaluate the additive effect of albendazole, corneal NV and LY were also analyzed in a combined group of albendazole and bevacizumab therapy and the additive effect was compared with that in the group of double dose of bevacizumab. Results: The albendazole group showed less NV and less LY compared with the PBS control group (P < 0.01). When albendazole was combined with bevacizumab therapy, a significant decrease in NV and LY was seen compared with bevacizumab treatment alone, and with albendazole alone (all P values <0.05). The combination group showed better antilymphangiogenesis effect than the group of double dose bevacizumab. The albendazole-treated group showed reduced expression of VEGF-A, VEGF-C, TNF-alpha, and VEGFR-2 compared with corneas from the PBS group (P value <0.05 in all respective comparisons). Conclusion: Albendazole significantly decreased NV and LY in the cornea. This beneficial effect is additively enhanced when combined with bevacizumab treatment.

RNA activating-double stranded RNA targeting flt-1 promoter inhibits endothelial cell proliferation through soluble FLT-1 upregulation.

Short-activating RNA (saRNA), which targets gene promoters, has been shown to increase the target gene expression. In this study, we describe the use of an saRNA (Flt a-1) to target the flt-1 promoter, leading to upregulation of the soluble isoform of Flt-1 and inhibition of angiogenesis. We demonstrate that Flt a-1 increased sFlt-1 mRNA and protein levels, while reducing VEGF expression. This was associated with suppression of human umbilical vascular endothelial cell (HUVEC) proliferation and cell cycle arrest at the G0/G1 phase. HUVEC migration and tube formation were also suppressed by Flt a-1. An siRNA targeting Flt-1 blocked the effects of Flt a-1. Flt a-1 effects were not mediated via argonaute proteins. However, trichostatin A and 5'-deoxy-5'-(methylthio) adenosine inhibited Flt a-1 effects, indicating that histone acetylation and methylation are mechanistically involved in RNA activation of Flt-1. In conclusion, RNA activation of sFlt-1 can be used to inhibit angiogenesis.

Epithelial Keratitis After Cataract Surgery.

PURPOSE: To evaluate the incidence, related perioperative factors, clinical characteristics, and possible etiologies of epithelial keratitis after cataract surgery. METHODS: A retrospective chart review of 666 eyes in 666 patients who underwent cataract surgery was performed to evaluate the incidence of epithelial keratitis and related factors in the postoperative period. RESULTS: Postoperative epithelial keratitis developed in 15 eyes. Eleven of the 15 eyes were diagnosed with herpes simplex keratitis (HSK); 10 of the 11 eyes were diagnosed by polymerase chain reaction, and the remaining 1 eye by clinical diagnosis. All patients diagnosed with HSK had no previous clinical history of the infection before undergoing cataract surgery. Initially, the diagnosis of all 15 eyes was toxic keratitis, but the final diagnosis of 11 of the initial 15 was found to be epithelial herpes keratitis. The incision location was shown to be related to the occurrence of HSK in our study (P < 0.05). CONCLUSIONS: HSK epithelial keratitis after cataract surgery is a relatively uncommon complication and can be misdiagnosed in its early disease course because of its relative rarity. This study explores the possibility that the temporal corneal penetrating incisional approach used in routine cataract surgery interrupts the corneal nerves and subsequently can trigger reactivation of HSK.

Comparative analysis of the safety and efficacy of intracameral cefuroxime, moxifloxacin and vancomycin at the end of cataract surgery: a meta-analysis.

BACKGROUND: Current practice methods are unclear as to the most safe and effective prophylactic pharmacotherapy and method of delivery to reduce postoperative endophthalmitis occurrence. METHODS: A systematic review and meta-analysis using Meta-analysis of Observational Studies in Epidemiology guidelines was performed to compare the efficacy of intracameral cefuroxime, moxifloxacin and vancomycin in preventing postphacoemulsification cataract surgery endophthalmitis. A safety analysis of intracameral antibiotics was concurrently performed. DATA SOURCES: BIOSIS Previews, CINAHL, ClinicalTrials.gov, Cochrane Library, Dissertations & Theses, EMBASE, PubMed, ScienceDirect and Scopus were searched from inception to January 2017. Data were pooled using a random effects model. All articles were individually reviewed and data were extracted by two independent reviewers. Funnel plot, risk of bias and quality of evidence analyses were performed. RESULTS: Seventeen studies with over 900 000 eyes were included, which favoured the use of intracameral antibiotics at the end of cataract surgery (OR 0.20; 95% CI 0.13 to 0.32; P<0.00001). The average weighted postoperative endophthalmitis incidence rates with intracameral cefuroxime, moxifloxacin and vancomycin were 0.0332%, 0.0153% and 0.0106%, respectively. Secondary analyses showed no difference in efficacy between intracameral plus topical antibiotics versus intracameral alone (P>0.3). Most studies had low to moderate risk of bias. The safety analysis showed minimal toxicity for moxifloxacin. Dosing errors led to the majority of toxicities with cefuroxime. Although rare, vancomycin was associated with toxic retinal events. CONCLUSION: Intracameral cefuroxime and moxifloxacin reduced endophthalmitis rates compared with controls with minimal or no toxicity events at standard doses. Additionally, intracameral antibiotics alone may be as effective as intracameral plus topical antibiotics.

Effect of itraconazole on the cornea in a murine suture model and penetrating keratoplasty model.

AIM: To investigate the anti-(lymph)angiogenic and/or anti-inflammatory effect of itraconazole in a corneal suture model and penetrating keratoplasty (PK) model. METHODS: Graft survival, corneal neovascularization, and corneal lymphangiogenesis were compared among itraconazole, amphotericin B, dexamethasone, phosphate buffered saline (PBS) and surgery-only groups following subconjunctival injection in mice that underwent PK and corneal suture. Immunohistochemical staining and analysis were performed in each group. Real-time polymerase chain reaction (RT-PCR) was performed to quantify the expression of inflammatory cytokines (TNF-alpha, IL-6) and vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGFR-2, and VEGFR-3. RESULTS: In the suture model, the itraconazole group showed less angiogenesis, less lymphangiogenesis, and less inflammatory infiltration than the PBS group (all P<0.05). The itraconazole group showed reduced expression of VEGF-A, VEGFR-2, TNF-alpha, IL-6 than the PBS group (all P<0.05). In PK model, the two-month graft survival rate was 28.57% in itraconazole group, 62.50% in dexamethasone group, 12.50% in PBS group, 0 in amphotericin B group and 0 in surgery-only group. Graft survival in the itraconazole group was higher than that in the amphotericin, PBS and surgery-only group (P=0.057, 0.096, 0.012, respectively). The itraconazole group showed less total angiogenesis and lymphangiogenesis than PBS group (all P<0.05). CONCLUSION: Itraconazole decrease neovascularization, lymphangiogenesis, and inflammation in both a corneal suture model and PK model. Itraconazole has anti-(lymph)-angiogenic and anti-inflammatory effects in addition to its intrinsic antifungal effect and is therefore an alternative treatment option in cases where steroids cannot be used.

Effect of sorafenib in a murine high risk penetrating keratoplasty model.

AIM: To evaluate the effect of sorafenib in murine high risk keratoplasty model. METHODS: Graft survival, corneal neovascularization, and corneal lymphangiogenesis were compared among the sorafenib, dexamethasone, dimethyl sulfoxide (DMSO), and phosphate buffered saline (PBS) groups following subconjunctival injection in mice that underwent high risk penetrating keratoplasty (HRPK). Real-time polymerase chain reaction was performed to quantify the expression of inflammatory cytokines and vascular endothelial growth factor (VEGF)-A, VEGF-C, vascular endothelial growth factor receptor (VEGFR)-2, VEGFR-3. RESULTS: The two-month graft survival rate for HRPK was 42.86% in sorafenib group, 37.50% in dexamethasone group, 0 in DMSO group, and 0 in PBS group. Sorafenib significantly increased graft survival compared to the DMSO and PBS group (P<0.05). The sorafenib didn't show significant effect in decreasing neovascularization compared with dexamethsone, DMSO, and PBS group. The sorafenib showed less total lymphangiogenesis than the dexamethasone, DMSO, and PBS group (P=0.011, P<0.001, P<0.001, respectively). The sorafenib group showed reduced expression of VEGF-C, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, VEGFR-2 and VEGFR-3 compared with DMSO group and PBS group (all P<0.05). The sorafenib group didn't show difference in the expression of VEGF-A compared with DMSO, neither with PBS. The sorafenib group showed reduced expression of VEGFR-3 compared with dexamethasone (P=0.051). CONCLUSION: The subconjunctivally administered sorafenib shows significant anti-lymphangiogenic effect, resulting in increased transplant survival in a murine high risk keratoplasty model. We suggest that a close linkage between decreased VEGF-C/VEGFR-2 and -3 signaling and increased corneal graft survival by sorafenib seems to exist.

New capsular tension segment with 2-point fixation for zonular weakness.

We describe clinical applications and surgical techniques for a new type of capsular tension segment (CTS) for use during cataract surgery. The Ambati CTS is distinguished from other CTS devices by having 2 eyelets close to each other, which allows it to distribute tension to 2 points, avoiding too much stress at a single point on the anterior capsulotomy, which prevents peaking of the capsulorhexis, and potentially reducing the risk for anterior capsule tear. Two of these CTS devices could possibly be used to provide 4-point fixation of a capsular bag in eyes with near-complete zonular instability. We describe 4 cases and 2 surgical techniques for implanting the new CTS, 1 technique in an adult patient with zonular weakness secondary to trauma and the other in 3 children with subluxated lenses due to Marfan syndrome.