Subretinal and intravitreal injection of retinal gene therapy is associated with serious adverse events and poor efficacy. We sought to improve retinal gene therapy delivery by developing fibrin hydrogel encapsulated adeno-associated virus (FE-AAV). Here we investigate conditions for storage and stability for FE-AAV. FE-AAV containing 1.9 × 109 genome copies of AAV2/2-CMV-GFP was manufactured using fibrinogen reconstituted in 0.01 M sodium citrate, pH 7 (NaC) or phosphate-buffered saline containing 0.001% (v/v) Pluronic F68 (F68). Samples were stored at either −80 °C or 4 °C for up to 16 weeks. Changes in transduction efficiency, and mechanical and physical properties were evaluated. In vitro transduction was significantly (p < 0.05) reduced for FE-AAV manufactured with NaC. In contrast, we observed no change in transduction through 16 weeks for FE-AAV made with F68. Physical changes occurred in FE-AAV stored at −80 °C. In contrast to FE-AAV formulated with NaC, FE-AAV formulated with F68 and stored at 4 °C for 16 weeks was essentially equivalent to freshly made FE-AAV and retained the ability to transduce retinal pigment epithelial (RPE) cells in the pig eye. We conclude that FE-AAV formulated with F68 and stored at 4 °C is stable and shows potential for retinal gene therapy for at least 4 months following manufacture.

Subretinal injection of adeno-associated virus (AAV) is generally more efficacious and less inflammatory than intravitreal injection for retinal gene therapy. However, adverse events (e.g., chorioretinal atrophy) have been reported in many patients receiving subretinal injection of Luxturna (voretigene neparvovec-rzyl) and experimental gene therapies. Subretinal injection confines transduction to the treated area. To address this, we manufactured high-concentration fibrin hydrogels encapsulating AAV2-green fluorescent protein (AAV2-GFP). Gels had homogeneous AAV distribution, desired mechanical properties, and retained infectivity. Epiretinal placement of fibrin-AAV2-GFP (n = 11) was compared to subretinal (n = 5) and intravitreal AAV2-GFP injection (n = 3). The subretinal group exhibited inconsistent retinal pigment epithelium (RPE) transduction restricted to the injection region with severe atrophy in two cases. The intravitreal group had weak transduction and inflammation. In contrast, epiretinal hydrogels degraded within days and led to broad transduction of RPE without atrophy or inflammation. This technology could advance gene therapy for retinal degenerations and other ocular or systemic disorders.

BACKGROUND:To explore the prevalence of serious adverse events (SAEs) associated with retinal viral gene therapy and to examine trends influencing SAE occurrences in human gene therapy surgeries and pre-clinical animal trials. METHODS:Literature review was performed to identify peer-reviewed human and animal studies relevant to viral gene therapy, subretinal injections, and intravitreal injections. For clinical trials and post-approval LUXTURNA studies, only those that examined SAEs were included. RESULTS:Of included clinical trial studies (n = 31), SAEs were recorded in 51 out of 438 eyes (11.6%) that received subretinal injections and in 11 out of 348 eyes (3.2%) that received intravitreal injections. There were fewer intravitreal-related SAEs and less vision loss compared to subretinal gene therapy. Inflammation was the predominant SAE following intravitreal injections, whereas unexplained vision loss was the most common for subretinal injections. Clinical trials utilising subretinal injections met primary or secondary efficacy endpoints more than intravitreal trials. Eighteen studies (429 eyes) of post-approval LUXTURNA were reviewed, and SAEs were reported in 24.7% of eyes, retinal degeneration being most common (20.7%). For 58 animal studies, SAEs were recorded in 17.3% of eyes that received subretinal injections and 8.7% of eyes that received intravitreal injections. CONCLUSIONS:Subretinal injections show higher efficacy than intravitreal injections but are associated with more serious adverse events. Consistent adverse event patterns in humans and large animals highlight their predictive value for safety. There is a need for optimised delivery methods, refined dosing protocols, and improved post-treatment monitoring to improve safety and effectiveness in gene therapy.

Purpose: The goal of this study was to develop a lot release assay for iPSC residuals following directed differentiation of iPSCs to retinal pigment epithelial (RPE) cells. Methods: RNA Sequencing (RNA Seq) of iPSCs and RPE derived from them was used to identify pluripotency markers downregulated in RPE cells. Quantitative real time PCR (qPCR) was then applied to assess iPSC residuals in iPSC-derived RPE. The limit of detection (LOD) of the assay was determined by performing spike-in assays with known quantities of iPSCs serially diluted into an RPE suspension. Results: ZSCAN10 and LIN28A were among 8 pluripotency markers identified by RNA Seq as downregulated in RPE. Based on copy number and expression of pseudogenes and lncRNAs ZSCAN10 and LIN28A were chosen for use in qPCR assays for residual iPSCs. Reverse transcription PCR indicated generally uniform expression of ZSCAN10 and LIN28A in 21 clones derived from 8 iPSC donors with no expression of either in RPE cells derived from 5 donor lines. Based on qPCR, ZSCAN10, and LIN28A expression in iPSCs was generally uniform. The LOD for ZSCAN10 and LIN28A in qPCR assays was determined using spike in assays of RPE derived from 2 iPSC lines. Analysis of ΔΔCt found the limit of detection to be <0.01% of cells, equivalent to <1 iPSC/10,000 RPE cells in both iPSC lines. Conclusions: qPCR for ZSCAN10 and LIN28A detects <1 in 10,000 residual iPSCs in a population of iPSC-derived RPE providing an adequate LOD of iPSC residuals for lot release testing.

Purpose:No large-mammal surgical models exist for geographic atrophy (GA), choroidal neovascularization (CNV), and pachychoroidal vascular remodeling. Our goal was to develop a porcine RPE debridement model of advanced macular degeneration to study photoreceptor cell loss and choroidal remodeling. Methods:Seven 2-month-old female domestic pigs were used for this study. After 25G vitrectomy, the area centralis was detached via subretinal bleb. A nitinol wire (Finesse Flex Loop) was used to debride RPE cells across a 3- to 5-mm diameter region. Fluid-air exchange was performed, and 20% SF6 gas injected. Animals underwent fundus photography, fluorescein angiography, optical coherence tomography (OCT), and OCT-angiography (OCTA) at 2 weeks, 1 month, 2 months, 3 months, and 6 months postoperatively. Retinal histology was obtained at euthanasia, 2 months (n = 3), 3 months (n = 2), or 6 months (n = 2) after surgery. Results:RPE debridement resulted in GA with rapid loss of choriocapillaris, progressive loss of photoreceptors, and pachychoroidal changes in Sattler's and Haller's layers in all seven eyes undergoing debridement within 2 months. OCT and histological findings included subretinal disciform scar with overlying outer retinal atrophy; outer retinal tubulations and subretinal hyper-reflective material. OCTA revealed type 2 CNV (n = 4) at the edges of the debridement zone by 2 months, but there was no significant exudation noted at any time point. Conclusions:Surgical debridement of the RPE results in GA, CNV, and pachychoroid and reproduced all forms of advanced macular degeneration. This surgical model may be useful in examining the role of RPE and other cell replacement in treating advanced macular disease.

To investigate downstream molecular changes caused by mitogen-activated protein kinase (MEK) inhibitor treatment and further explore the impact of direct knockdown of early growth response-1 (EGR1) in melanoma cell culture. RNA-sequencing (RNA-Seq) was performed to determine gene expression changes with MEK inhibitor treatment. Treatment with MEK inhibitor (trametinib) was then assessed in two cutaneous (MEL888, MEL624) and one conjunctival (YUARGE 13-3064) melanoma cell line. Direct knockdown of EGR1 was accomplished using lentiviral vectors containing shRNA. Cell viability was measured using PrestoBlueHS Cell Viability Reagent. Total RNA and protein were assessed by qPCR and SimpleWestern. RNA-Seq demonstrated a profound reduction in EGR1 with MEK inhibitor treatment, prompting further study of melanoma cell lines. Following trametinib treatment of melanoma cells, viability was reduced in both cutaneous (MEL888 26%, P < 0.01; MEL624 27%, P < 0.001) and conjunctival (YUARGE 13-3064 33%, P < 0.01) melanoma compared with DMSO control, with confirmed EGR1 knockdown to 0.04-, 0.01-, and 0.16-fold DMSO-treated levels (all P < 0.05) in MEL888, MEL624, and YUARGE 13-3064, respectively. Targeted EGR1 knockdown using shRNA reduced viability in both cutaneous (MEL624 78%, P = 0.05) and conjunctival melanoma (YUARGE-13-3064 67%, P = 0.02). RNA-Sequencing in MEK inhibitor-treated cells identified EGR1 as a candidate effector molecule of interest. In a malignant melanoma cell population, MEK inhibition reduced viability in both cutaneous and conjunctival melanoma with a profound downstream reduction in EGR1 expression. Targeted knockdown of EGR1 reduced both cutaneous and conjunctival melanoma cell viability independent of MEK inhibition, suggesting a key role for EGR1 in melanoma pathobiology.

Purpose: We investigated whether a clinically used carbonic anhydrase inhibitor (CAIs) can modulate intraocular pressure (IOP) through soluble adenylyl cyclase (sAC) signaling.Methods: IOP was measured 1 h after topical treatment with brinzolamide, a topically applied and clinically used CAIs, using direct cannulation of the anterior chamber in sAC knockout (KO) mice or C57BL/6J mice in the presence or absence of the sAC inhibitor (TDI-10229).Results: Mice treated with the sAC inhibitor TDI-10229 had elevated IOP. CAIs treatment significantly decreased increased intraocular pressure (IOP) in wild-type, sAC KO mice, as well as TDI-10229-treated mice.Conclusions: Inhibiting carbonic anhydrase reduces IOP independently from sAC in mice. Our studies suggest that the signaling cascade by which brinzolamide regulates IOP does not involve sAC.

Purpose To improve outcomes for subretinal implantation surgery in pigs. Methods Analysis of variables affecting the success of subretinal implantation surgery was performed on videos of 37 surgeries. Ex vivo experiments were conducted to measure intraocular pressure (IOP) and test various prototyped implanters for effectiveness at maintaining IOP. Results A video analysis revealed a prolonged sclerotomy open time owing to a combination of uncontrolled bleeding and excessive fluid outflow often resulting in retinal prolapse. Precauterization of the choroid before full-thickness sclerotomy (n = 10) resulted in a reduced incidence of uncontrolled bleeding from 39.1% (9/23) versus 0% (0/10) (P = 0.005) and improved implantation success from 73% to 90%. An ex vivo analysis of the IOP revealed a mean decrease in the IOP from 30.2 ± 3.0 mm Hg to 5.0 ± 2.1 mm Hg after a fully penetrating sclerotomy. To address this situation, we produced a series of plugs that integrated with a custom implant insertion device to seal the sclerotomy during implantation. The use of the plugs was cumbersome, however, and so we opted instead to increase the width of the inserter tip to fill the open sclerotomy. This improved device restored and maintained IOP during implantation (27.1 ± 1.9 mm Hg). Combined with precauterization the improved inserter resulted in 100% successful implantation (n = 4). Conclusions For subretinal implantation in pigs, a modified procedure to precauterize the choroid before sclerotomy combined with an instrument that better fills the scleral opening decreases bleeding, hypotony, and open sclerotomy time, improving the success rate. Translational Relevance Better management of IOP and bleeding from a sclerotomy will improve implant-based therapies.

Fibrin is a degradable biopolymer with an excellent clinical safety profile. Use of higher mechanical strength fibrin hydrogels is limited by the rapid rate of fibrin polymerization. We recently demonstrated the use of higher mechanical strength (fibrinogen concentrations >30 mg/ml) fibrin scaffolds for surgical implantation of cells. The rapid polymerization of fibrin at fibrinogen concentrations impaired our ability to scale production of these fibrin scaffolds. We serendipitously discovered that the azo dye Trypan blue (TB) slowed fibrin gelation kinetics allowing for more uniform mixing of fibrinogen and thrombin at high concentrations. A screen of closely related compounds identified similar activity for Evans blue (EB), an isomer of TB. Both TB and EB exhibited a concentration dependent increase in clot time, though EB had a larger effect. While gelation time was increased by TB or EB, overall polymerization time was unaffected. Scanning electron microscopy showed similar surface topography, but transmission electron microscopy showed a higher cross-linking density for gels formed with TB or EB versus controls. Based on these data we conclude that addition of TB or EB during thrombin mediated fibrin polymerization slows the initial gelation time permitting generation of larger more uniform fibrin hydrogels with high-mechanical strength.

PURPOSE:To report a case of adult-onset vitelliform macular dystrophy in a patient who was found to have a previously unreported variant of the IMPG2 gene.METHODS:Case report.RESULTS:A 65-year-old white woman with no significant medical or ocular history presented with a complaint of persistent wavy vision for 10 months. On funduscopic examination, bilateral vitelliform lesions of approximately 1 mm in the right eye and 0.5 mm in the left eye were evident, with no choroidal neovascularization in either eye. The patient was diagnosed with adult-onset vitelliform macular dystrophy. Genetic testing revealed a single likely pathogenic variant of the IMPG2 gene that may explain the examination findings.CONCLUSION:Adult-onset vitelliform macular dystrophy is a common and relatively benign condition occurring in approximately 1 in 8,000 individuals. Although vitelliform lesions can be a manifestation of systemic diseases or be idiopathic, in a minority of patients, genetic predisposition may play a role. Mutations in four particular genes BEST1, PRPH2, IMPG1, and IMPG2 have been associated with some cases of adult-onset vitelliform macular dystrophy, with this particular gene variant of IMPG2 being previously unreported.

Retinal pigment epithelium (RPE) transplantation for the treatment of macular degeneration has been studied for over 30 years. Human clinical trials have demonstrated that RPE monolayers exhibit improved cellular engraftment and survival compared to single cell suspensions. The use of a scaffold facilitates implantation of a flat, wrinkle-free, precisely placed monolayer. Scaffolds currently being investigated in human clinical trials are non-degradable which results in the introduction of a chronic foreign body. To improve RPE transplant technology, a degradable scaffold would be desirable. Using human fibrin, we have generated scaffolds that support the growth of an RPE monolayer in vitro. To determine whether these scaffolds are degraded in vivo, we developed a surgical approach that delivers a fibrin hydrogel implant to the sub-retinal space of the pig eye and determined whether and how fast they degraded. Using standard ophthalmic imaging techniques, the fibrin scaffolds were completely degraded by postoperative week 8 in 5 of 6 animals. Postmortem histologic analysis confirmed the absence of the scaffold from the subretinal space at 8 weeks, and demonstrated the reattachment of the neurosensory retina and a normal RPE-photoreceptor interface. When mechanical debridement of a region of native RPE was performed during implantation surgery degradation was accelerated and scaffolds were undetectable by 4 weeks. These data represent the first in situ demonstration of a fully biodegradable scaffold for use in the implantation of RPE and other cell types for treatment of macular degeneration and other retinal degenerative diseases.