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Luke A. Wiley, Emily E. Kaalberg, Chunhua Jiao, Robert F Mullins, Edwin M Stone, Budd Tucker, Ian Han; Characterizing the immune response to helper-dependent adenovirus in the rat retina.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3408.
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While gene augmentation in the retina has been promising, many disease-causing genes, like ABCA4 or USH2A, are too large for packaging into adeno-associated viruses. One option for delivery of large retinal genes is helper-dependent adenoviruses (HDAds) which efficiently transduce post-mitotic neurons, and unlike lentiviruses, will not integrate into the host genome. However, the immune tolerance to HDAds in the retina is unknown. The purpose of this study was to characterize the immune response to the HDAd serotype 5 (HDAd5) in the rat retina.
For this study we utilized RNU+/- and RNU-/- rat lines. RNU+/- rats possess intact wildtype immune systems, whereas the RNU-/- rats are athymic (i.e., deficient of T cells) and therefore cannot mount an adaptive response, but retain an intact innate immune system. Ten microliters of either viral particles (5 x 1010 vg/mL) of HDAd5 carrying enhanced Green Fluorescent Protein (eGFP) driven by the cytomegalovirus promoter (CMVp; HDAd5-CMVp-eGFP) or 3% sucrose buffer were subretinally injected. Eyes were enucleated and either processed for histological analysis using immunohistochemistry and confocal microscopy or the retinas dissected and subjected to cytokine quantibody array analysis.
At one-week post-injection, both RNU+/- (N=11) and RNU-/- (N=11) retinas that received HDAd5-CMVp-eGFP displayed significantly-elevated levels of the proteins IL-1b, MCP-1 and TIMP-1 compared to sucrose buffer-injected control eyes (p<0.05). By two-weeks post-injection, a 2.5-fold increase in the number of Iba1-postive microglia and macrophages was observed in RNU+/- retinas (N=6, p<0.05), even in the outer nuclear layer, suggesting an influx of circulating monocytes from the retinal vasculature into the retina and activation of resident retinal microglia. At 4-weeks post injection in RNU+/- retinas, HDAd5-treated eyes also displayed a 3-fold increase (N=5, p<0.05) in the number of CD8a-positive cytotoxic T-cells in the neural retina. Together, these data suggest both an innate and an adaptive immune response to HDAd5 in the neural retina.
Subretinal injection of HDAd5 elicited cytokine and cellular markers indicative of both an innate and adaptive immune response in the retinae of RNU+/- rats. Future studies will aim to characterize these immune responses further with the goal of modulating them to enable HDAd5-mediated gene transfer in the retina.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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