Abstract
Purpose:
Inflammation in the retina is a contributing factor in ocular diseases such as uveitis, diabetic retinopathy, and dry-age related macular degeneration (AMD). The M013 protein from the myxoma virus has been shown to interfere with both the inflammasome and NF-κB signaling, modulating the synthesis and secretion of the pro-inflammatory cytokine interleukin-1β (IL-1β). The goal of this project is to develop an adeno-associated virus (AAV) vector that delivers a secretable and cell penetrating form of the M013 gene into the retina to decrease inflammation.
Methods:
Stable THP-1 and ARPE-19 cells expressing the TatM013 protein were generated using lentiviral vectors. Inflammasome activation was induced with either LPS or 4-hydroxynonenal and IL-1β concentration was measured by ELISA. Expression and secretion of sGFP-TatM013 was observed in transfected HEK293T cells by fluorescent microscopy and western blot. The anti-inflammatory effect of TatM013 was evaluated in an Endotoxin Induced Uveitis (EIU) animal model by the intravitreal injection of C57BL/6J mice with AAV2 viral particles delivering either sGFP-TatM013 or GFP followed by injection of LPS one month thereafter. Expression was determined by fluorescence fundoscopy and anti-inflammatory effect was measured by counting the number of infiltrative cells present in the vitreous in H&E stained paraffin sections of enucleated eyes.
Results:
The expression of TatM013 reduced the induced IL-1β secretion in THP-1 and ARPE-19 cells. Cellular distribution of sGFP-TatM013 was punctuate compared to GFP in transfected cells. The secretion and cleavage of the sGFP-TatM013 construct was inferred by detection of the fused sGFP-TatM013 protein in the cell lysate and the free form of sGFP in the conditioned media. A reduction in the concentration of secreted induced IL-1β was observed in THP-1 and RAW264.7 cells exposed to conditioned media from sGFP-TatM013 transfected HEK293T cells. The quantity of infiltrative cells in the vitreous was significantly lower in the eyes injected with sGFP-TatM013 than with GFP injected eyes in the EIU mouse model.
Conclusions:
The expression of TatM013 inhibits the induced secretion of IL-1β in vitro. The expression of a secretable and cell penetrating TatM013 in the retina reduces the inflammatory response in the EIU mouse model. Our vector could be a potential therapy for ocular diseases with an inflammatory component such as uveitis.
Keywords: 538 gene transfer/gene therapy •
557 inflammation •
746 uveitis-clinical/animal model