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M. G. Rich, M. Kleinman, H. Kaneko, R. Albuquerque, K. Saitio, W. Cho, J. Baffi, J. Ambati; Retinotoxicity as a Result Of siRNA Activation of Toll-like Receptor-3. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6155.
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Small interfering RNA (siRNA) has revolutionized both basic and clinical scientific approaches to targeted gene therapies. We recently reported (Kleinman et al. Nature 2008) that siRNA functionality as an inhibitor of abnormal blood vessel growth in neovascular age-related macular degeneration is not due to targeted knockdown of pro-vascular mediators but instead relies on activation of the innate immune system through toll-like receptor-3 (TLR3). TLR3 is a double-stranded RNA (dsRNA) receptor that binds to 21-nucleotide and longer RNA duplexes thereby inducing pro-apoptotic pathways. We hypothesized that intraocular siRNA results in retinal-cell caspase cleavage, apoptosis and retinotoxicity.
To investigate this, non-targeted siRNA or buffer was administered, in doses equivalent to those used in human trials, by intravitreous injection to wild-type mice. Retinal architecture and function were evaluated using TUNEL staining, flow cytometry, fundus imaging, and electroretinography.
At 24 hours after siRNA administration, retinal caspase-3 activity was elevated, and at 48 hours, a significant increase in apoptotic cells was observed by TUNEL staining. At 1 week after treatment, color fundus photographs demonstrated changes consistent with retinal pigment epithelial cell loss and diminished electroretinograms from baseline measurements were indicative of retinal dysfunction.
These data strongly suggest that siRNA imparts a significant retinotoxic side-effect profile due to TLR3 activation. We are currently studying a novel inhibitor of TLR3 in order to protect the retina during siRNA administration.
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