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M. E. Kleinman, K. Yamada, A. Takeda, M. Nozaki, R. Albuquerque, J. Smith, E. W. Taylor, J. Ambati; Targeted and Non-Targeted Small Interfering RNAs Equally Suppress Choroidal Neovascularization Through Activation of Toll-Like Receptor 3. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3298.
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siRNA targeting non-mammalian genes, non-expressed genes, non-genomic sequences, both pro- and anti-angiogenic genes, or RNAi-incompetent siRNA were intravitreously injected into wild-type mice and mice deficient in TLR3 or its downstream mediators after laser photocoagulation. CNV volumes were assessed at 7 days. Spatial trafficking of fluorescent siRNA was monitored by immunofluorescence and flow cytometry (FACS). Interferon responses after siRNA treatment were analyzed by ELISA, and mRNA levels of anti-angiogenic factors were measured with real-time RT-PCR. Primary human choroidal endothelial cells (CECs) were interrogated for surface expression of TLR3 using FACS and confocal microscopy. Structural determinants of siRNA:TLR3 interaction were identified by docking simulations and molecular dynamics.
Non-targeted siRNA reduced CNV in wild-type mice as effectively as siRNA-Vegfa or siRNA-Vegfr1 without entering cells. CNV suppression by any siRNA required activation of surface TLR3, its adaptor TRIF, and induction of interferon-γ and interleukin-12. siRNA shorter than 21 nucleotides did not inhibit CNV; modelling studies suggested that this resulted from their inability to bridge effectively between dsRNA-binding residues of two TLR3 monomers. In vitro, surface TLR3 was found on all human CEC isolates tested.
These results demonstrate that targeted siRNAs currently in clinical trials reduce CNV solely via TLR3 activation, that non-targeted siRNA are equally as effective in suppressing laser induced CNV, and that siRNA therapeutics may cause unanticipated vascular or immune effects due to the innate host response.
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