Sequence- and target-independent angiogenesis suppression by siRNA via TLR3

Abstract

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 ...

Loss-of-function of inositol polyphosphate-4-phosphatase reversibly increases the severity of allergic airway inflammation

“…5g-j). Similar to naive mice, siRNA delivery to mice with AAI did not induce typeI IFN response, such as release of IFN-α or IFN-β, or toll-like receptor-3-dependent upregulation of IFN-γ and IL-12 23 ( Supplementary Fig. S4 a-d).…”

“…To investigate whether administration of siRNA in mice led to the non-specific induction of interferon (IFN)-α, IFN-β, IFN-γ and IL-12, we measured the levels of these cytokines in the lungs of siRNA-administered mice. Importantly, administration of siRNA to naive mice did not induce type-I IFN responses, such as release of IFN-α or IFN-β, or toll-like receptor-3-dependent upregulation of IFN-γ and IL-12 23 ( Supplementary Fig. S2a-d), excluding non-specific effects.…”

Special delivery: targeted therapy with small RNAs

“…Indeed, some of the early demonstrations of siRNA therapeutic effects in small animals may have been due to off-target effects. 8,9 However, chemical modifications of the RNA can largely eliminate off-target effects without compromising target-gene knockdown.…”

Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

“…RNAi can cause expression suppression in genes homologous to the desired gene, and produce effects which were not the result of the silencing of the target gene. In early tests of siRNA therapies in small animals, the effects produced by the therapies may not have been from the target knock-down at all, but from the off-target effects [82,113]. Additionally, RNA sensors in the innate immune system can prohibit effective RNAi by triggering the release of interferons.…”

“…However, entering the cell from the bloodstream is still a hurdle. Because of their negative charge and high molecular weight, siRNAs cannot cross the plasma membrane [82,113]. Cells can uptake siRNAs through endocytosis, but then the siRNA is trapped within an endosome, and cannot produce gene knock-downs.…”