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.

Subretinal injections of viral vectors provide great benefits but have limited cargo capacity; they induce innate and adaptive immune responses, which may cause damage and preclude repeated injections; and they pose administration risks. As a new biotechnology, suprachoroidal injections of biodegradable nanoparticles (NPs) containing a reporter plasmid induce reporter expression in rat photoreceptors and RPE throughout the entire eye and maintain expression for at least 8 months. Multiple injections markedly increase expression. Suprachoroidal injection of NPs containing a VEGF expression plasmid caused severe subretinal neovascularization progressing to subretinal fibrosis, similar to what occurs in untreated patients with neovascular age-related macular degeneration, providing a new model and proof of concept for level and duration of expression. Suprachoroidal injection of NPs containing a VEGF-binding protein expression plasmid significantly suppressed VEGF-induced vascular leakage and neovascularization demonstrating therapeutic potential. These data suggest that nonviral NP suprachoroidal gene transfer may provide a noninvasive, repeatable alternative to subretinal injection of viral vectors.

Neovascular age-related macular degeneration and diabetic retinopathy are prevalent causes of vision loss requiring frequent intravitreous injections of VEGF-neutralizing proteins, and under-treatment is common and problematic. Here we report incorporation of sunitinib, a tyrosine kinase inhibitor that blocks VEGF receptors, into a non-inflammatory biodegradable polymer to generate sunitinib microparticles specially formulated to self-aggregate into a depot. A single intravitreous injection of sunitinib microparticles potently suppresses choroidal neovascularization in mice for six months and in another model, blocks VEGF-induced leukostasis and retinal nonperfusion, which are associated with diabetic retinopathy progression. After intravitreous injection in rabbits, sunitinib microparticles self-aggregate into a depot that remains localized and maintains therapeutic levels of sunitinib in retinal pigmented epithelium/choroid and retina for more than six months. There is no intraocular inflammation or retinal toxicity. Intravitreous injection of sunitinib microparticles provides a promising approach to achieve sustained suppression of VEGF signaling and improve outcomes in patients with retinal vascular diseases.

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