Abstract

Purpose: Age-related macular degeneration (AMD) is associated with oxidative stress and pro-inflammatory signals within the retina. Our goal is to test anti-inflammatory genes delivered by an adeno-associated virus (AAV) vector as potential treatments for AMD.

Methods: We developed a secretable and cell penetrating form (TatCARD) of the CARD domain from the ASC gene that inhibits inflammasome mediated activation of caspase-1. C57BL/6J mice were injected intravitreally with AAV2 vector delivering either GFP control or sGFP-TatCARD. Gene expression was determined one month after injection by fluorescence fundoscopy. Mice were then injected intravitreally in both eyes with 25 ng of lipopolysaccharide. The eyes of these mice were harvested 24 hrs later and fixed, embedded in paraffin, sectioned and stained with H&E for histological analysis. The number of infiltrative cells within the vitreous and anterior chamber were quantified using Image J. The effects of our vector on oxidative damage were studied by its intravitreal injection followed by a systemic injection of 25 mg/kg of NaIO₃. Mice were evaluated 1 and 4 weeks after NaIO₃ by ERG.

Results: When injected intravitreally, our secretable TatCARD vector fused to sGFP showed a diffused pattern of fluorescence in the fundus microscope. Eyes injected with the AAV vector had a significantly lower number of infiltrating cells than eyes injected with a similar AAV vector delivering GFP only or with saline. Furthermore, there was a significant decrease in the concentration of IL-1β in eyes treated with the sGFP-TatCARD vector. In mice that received NaIO₃, all the animals had a decrease in a-, b-, and c-wave amplitudes one week after systemic injection. However, eyes injected with the sGFP-TatCARD vector had a significant increase in all three amplitudes of the ERG response after 4 weeks of the NaIO₃ injection.

Conclusions: Taken together, these results suggest that the use of anti-inflammatory genes such as CARD could potentially be used to treat ocular diseases with inflammation and oxidative stress such as AMD. This gene therapy may provide therapeutic benefits with a single injection without the side effects of steroids or other immunosuppressant drugs.