There has been great progress in ocular gene therapy, but delivery of viral vectors to the retinal pigmented epithelium (RPE) and retina can be challenging. Subretinal injection, the preferred route of delivery for most applications, requires a surgical procedure that has risks. Herein we report a novel gene therapy delivery approach, suprachoroidal injection of AAV8 vectors, which is less invasive and could be done in an outpatient setting. Two weeks after suprachoroidal injection of AAV8.GFP in rats, GFP fluorescence covered 18.9% of RPE flat mounts and extended entirely around sagittal and transverse sections in RPE and photoreceptors. After 2 suprachoroidal injections of AAV8.GFP, GFP fluorescence covered 30.5% of RPE flat mounts. Similarly, widespread expression of GFP occurred in nonhuman primate and pig eyes after suprachoroidal injection of AAV8. GFP. Compared with subretinal injection in rats of RGX-314, an AAV8 vector expressing an anti-VEGF Fab, suprachoroidal injection of the same dose of RGX-314 resulted in similar expression of anti-VEGF Fab and similar suppression of VEGFinduced vascular leakage. Suprachoroidal AAV8 vector injection provides a noninvasive outpatient procedure to obtain widespread transgene expression in retina and RPE.

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Acriflavine, a fluorescent drug previously used for bacterial and trypanosomal infections, reduces hypoxia-inducible factor-1 (HIF-1) and HIF-2 transcriptional activity. In mice with oxygen-induced ischemic retinopathy, intraocular or intraperitoneal injections of acriflavine caused dose-dependent suppression of retinal neovascularization (NV) and significantly reduced expression of HIF-1-responsive genes. Intraocular injection of 100 ng caused inner retina fluorescence within 1 hour that was seen throughout the entire retina between 1 and 5 days, and at 7 days after injection, strongly suppressed choroidal NV at Bruch’s membrane rupture sites. After suprachoroidal injection of 300 ng in rats, there was retinal fluorescence in the quadrant of the injection at 1 hour that spread throughout the entire retina and choroid by 1 day, was detectable for 5 days, and dramatically reduced choroidal NV 14 days after rupture of Bruch’s membrane. After topical administration of acriflavine in mice, fluorescence was seen in the retina and retinal pigmented epithelium within 5 minutes and was detectable for 6–12 hours. Administration of 0.5% drops to the cornea twice a day, significantly reduced choroidal NV in mice. Electroretinographic b-wave amplitudes were normal 7 days after intravitreous injection of 100 ng of acriflavine in mice, showed mild threshold reductions at highest stimulus intensities after injection of 250 ng, and more extensive changes after injection of 500 ng. These data provide additional evidence for an important role for HIF-1 in retinal and choroidal NV and suggest that acriflavine can target HIF-1 through a variety of modes of administration and has good potential to provide a novel therapy for retinal and choroidal vascular diseases.

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