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Cristhian J Ildefonso, Henrique Jaime, Qiuhong Li, Alfred S Lewin; Gene Therapy with the Caspase Activation and Recruitment Domain (CARD) Reduced Inflammation in the Endotoxin Induced Uveitis (EIU) Mouse Model. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3303.
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Uveitis is an inflammatory response within different tissues of the eye. The current treatment involves prolonged use of steroids which elevates the risk of glaucoma. Our goal is to test anti-inflammatory genes delivered by an adeno-associated virus (AAV) vector as potential treatments for uveitis.
We developed a secretable and cell penetrating form (TatCARD) of the CARD domain from the ASC gene that inhibits the inflammasome mediated activation of caspase-1. The characteristics of TatCARD were validated in cell culture by western blot and ELISA. AAV viral particles were produced for tests in the EIU mouse model. 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 was quantified by two independent investigators.
The TatCARD protein was demonstrated to bind to caspase-1 in ARPE-19 cells stably expressing the TatCARD protein. In these cells as well as in THP-1 cells the expression of TatCARD resulted in a significant decrease in the concentration of secreted IL-1β. A secretable version of the TatCARD was designed and validated by demonstrating the presence of TatCARD in the conditioned media of cell cultures. Furthermore, exposure to this TatCARD-conditioned media was sufficient to significantly inhibit the secretion of IL-1β from stimulated ARPE-19 cells, thus suggesting the transfer of the TatCARD inhibitory activity. 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.
Taken together, these results suggest that the use of anti-inflammatory genes such as CARD could potentially be used to treat inflammatory diseases such as uveitis. This gene therapy may provide therapeutic benefits with a single injection without the side effects of steroids.
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