Epiretinal placement of fibrin hydrogel-encapsulated adeno-associated virus transduces retinal pigment epithelium in the domestic pig

Abstract

Purpose : Gene therapy for retinal transduction often involves subretinal injection of viral suspension, commonly with adeno-associated virus (AAV). Subretinal blebs require detachment of the retina, and complications include chorioretinal atrophy, macular hole, and choroidal neovascularization. These risks necessitate development of innovative surgical approaches for gene therapy delivery that avoid damage to the retina. We posit that implanting a virus-embedded fibrin hydrogel on the epiretinal surface will result in transduced RPE.

Methods : Fibrin hydrogels were cast with AAV2-GFP at 5.7e11 vector genomes (vg)/cm³. These gels were characterized using immunofluorescence and electron microscopy and a diffusion assay. Round 3mm gels were placed on cultured ARPE19 cells to determine infectivity. Ovoid 1.5mm x 5mm gels were implanted on the epiretinal surface of right eyes in six domestic pigs after vitrectomy. Nine additional pigs received AAV2-GFP via subretinal bleb alone (N=3), fibrin-AAV gels placed in the subretinal space (N=3), or intravitreal injection (N=3) at comparable titers. At month 1, eyes were enucleated and examined for GFP expression using FITC imaging on retina whole mounts and using histological analysis with and without fixation. PCR was performed to determine AAV presence in various tissues.

Results : Gels had homogenous virus distribution. Approximately 50% viral diffusion occurred after 48 hours. GFP signal was detected by day 4 in cultured cells. RPE transduction was observed in all pigs that received virus-loaded gels in the subretinal and epiretinal space. The epiretinal fibrin-AAV implant, which degraded within five days of surgery, resulted in more diffuse RPE transduction compared to the confined transduction of the subretinal groups. The intravitreal group had minimal RPE transduction. No complications were noted.

Conclusions : We developed a surgical approach that leverages fibrin hydrogels as a vehicle for gene therapy vectors. Data demonstrate that gels function as a “time release” device releasing virus with retained infectivity of the RPE both in vitro and in vivo. Epiretinal placement of degradable fibrin hydrogels encapsulating gene therapy may enable precise targeting and enhance transduction efficiency of the RPE and retina without the risks associated with subretinal blebs.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Epiretinal fibrin-AAV hydrogels transduce the RPE in pigs.