Targeted and Non-Targeted Small Interfering RNAs Equally Suppress Choroidal Neovascularization Through Activation of Toll-Like Receptor 3

Abstract

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: choroid: neovascularization • immunomodulation/immunoregulation • drug toxicity/drug effects