Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro-and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-a/b activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-c and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3-RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.Therapeutic application of long, double-stranded (ds)RNAmediated RNAi and sequence-specific gene silencing through RNAi by short synthetic RNA duplexes is challenging because mammalian cells do not uptake 'naked' siRNA (whether chemically modified or not) without cell-permeating entities [1][2][3][4] . To minimize systemic exposure, initial clinical trials of siRNA were launched using intraocular injection in patients with CNV. CNV, wherein the retina is invaded by choroidal vessels beneath the retinal pigmented epithelium (RPE), is a late stage of age-related macular degeneration that afflicts 30-50 million people globally. The preclinical bases for trials of naked VEGFA siRNA (Bevasiranib) or VEGFR1 siRNA (AGN211745/ siRNA-027) were single reports in mice 5,6 that such siRNAs suppressed laser-injury-induced CNV, a model predictive of efficacy in humans 7,8 . These findings were interpreted as anomalous examples of local delivery surmounting the impediment to intracellular entry 9-11 . Instead, we show in two animal models that suppression of neovascularization is a generic property of siRNAs independent of sequence, target and internalization.Sequence-independent angiogenesis suppression by siRNA Numerous synthetic non-targeted 21-nucleotide duplex siRNAs from multiple vendors, when injected into the vitreous humour of wild-type mice, uniformly and dose-dependently suppressed CNV (Fig. 1a, b and Supplementary Fig. 1). siRNAs targeting jellyfish green fluorescent ...

Atorvastatin effectively inhibited laser-induced CNV in mice and was associated with downregulation of CCL2/MCP-1 and VEGF and reduced macrophage infiltration into the RPE/choroid.

Retinal scatter laser photocoagulation induced upregulation of VEGF in the sensory retina and RPE-choroid at an early period. The authors speculate that the major source of VEGF in the retina after retinal scatter laser photocoagulation is the recruited monocytes.

PURPOSE. To investigate the effect of nontargeted siRNAs on vascular leakage and vascular endothelial growth factor (VEGF)-A expression in the development of choroidal neovascularization (CNV). METHODS. Nontargeted siRNAs were 21-nt (nucleotides) siRNA-Luc (Luciferase) or 16-nt siRNA-Luc. Targeted 21-nt siRNA-Vegfa or phosphate-buffered saline (PBS) was used for comparison. Laser photocoagulation was used to induce CNV in wild-type C57BL/6J mice; 7 days later, vascular leakage was determined by fluorescein angiography, and CNV volumes were measured by confocal microscopy. Expression of VEGF-A in the retinal pigment epithelium (RPE)/choroid was quantified by ELISA 3 days after photocoagulation. RESULTS. Pathologically significant leakage developed in most of the 16nt-siRNA-Luc- or PBS-injected mice but in significantly fewer 21nt-siRNA-Luc- and 21nt-siRNA-Vegfa-injected mice (P = 0.0004, P = 0.0001, respectively). CNV volume in 21-nt siRNA-Luc- and 21nt-siRNA-Vegfa-injected eyes was significantly lower than in PBS-injected eyes (P = 0.0124, P = 0.0040, respectively). CNV volume was not suppressed by 16-nt siRNA-Luc injection (P = 0.7700). The mean VEGF protein level decreased significantly in the 21nt-siRNA-Luc- and 21nt-siRNA-Vegfa-injected eyes compared with PBS-injected eyes 3 days after laser photocoagulation (P = 0.0011, P = 0.0063, respectively). The 16nt-siRNA-Luc-injected eyes did not show VEGF-A suppression 3 days after laser photocoagulation (P = 0.3177). Between 21-nt siRNA-Luc- and 21nt-siRNA-Vegfa-injected eyes, there were no significant differences in CNV volume, the VEGF-A level, or pathologic leakage detected by fluorescein. CONCLUSIONS. These data suggest that nontarget 21nt-siRNA can suppress laser-induced choroidal neovascularization anatomically and functionally through VEGF suppression.

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