Sequence- and target-independent angiogenesis suppression by siRNA via TLR3 - PubMed (original) (raw)

. 2008 Apr 3;452(7187):591-7.

doi: 10.1038/nature06765. Epub 2008 Mar 26.

Kiyoshi Yamada, Atsunobu Takeda, Vasu Chandrasekaran, Miho Nozaki, Judit Z Baffi, Romulo J C Albuquerque, Satoshi Yamasaki, Masahiro Itaya, Yuzhen Pan, Binoy Appukuttan, Daniel Gibbs, Zhenglin Yang, Katalin Karikó, Balamurali K Ambati, Traci A Wilgus, Luisa A DiPietro, Eiji Sakurai, Kang Zhang, Justine R Smith, Ethan W Taylor, Jayakrishna Ambati

Affiliations

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

Mark E Kleinman et al. Nature. 2008.

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-alpha/beta 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-gamma 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.

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Figures

Figure 1

Figure 1. Sequence-independent CNV suppression by siRNA through TLR3

a, siRNAs targeting Gfp, Luc, random sequences (RS1, RS2), Bglap1, Cdh16, or Sftpb, and siRNA incapable of RNA-induced silencing (RISC-free), suppressed CNV in wild-type mice. n = 8–24; asterisk, P < 0.05 compared to no injection, phosphate buffered saline (PBS) and siRNA buffer. b, Representative examples of CNV in wild-type eye injected with vehicle (buffer) or Luc siRNA (1 μg). c, Gfp siRNA or Luc siRNA suppressed CNV in Tlr3+/+ but not _Tlr3_−/− mice. n = 16–18; asterisk, P < 0.05 compared to vehicle (buffer). d, Representative examples of CNV in _Gfp_-siRNA-injected (1 μg) Tlr3+/+ and _Tlr3_−/− eyes. e, CNV suppression in wild-type mice by Luc siRNA (0.25 μg) was abrogated by soluble TLR3 (sTLR3) but not soluble TLR4 or heat-denatured (hd) soluble TLR3 (all 2 μg). n = 8. f, CNV suppression in wild-type mice by Luc siRNA (1 μg) was abrogated by neutralizing anti-TLR3 antibodies (Ab; 0.2 μl) but not control IgG (0.2 μl) or chloroquine (Cq; 30 ng). n = 6–8; asterisk, P < 0.05. NS, not significant. Vehicle, buffer. All error bars indicate mean ± s.e.m. Scale bars in b, d are 100 μm.

Figure 2

Figure 2. CNV suppression by siRNA is mediated by IFN-γ and IL-12

a, Luc siRNA (1 μg) suppressed CNV in wild-type and _Ifnar1_−/− mice but not _Ifng_−/− or _Il12_−/− mice. n = 5–10. b, IFN-γ and IL-12 levels in RPE and choroid at 24 h after laser injury were higher in wild-type mouse eyes injected with Luc siRNA (1 μg) compared to vehicle-injected eyes. n = 12. c, Recombinant IFN-γ or IL-12 reduced CNV in wild-type mice. n = 8. Asterisk, P < 0.05 compared to vehicle (siRNA buffer). All error bars indicate mean ± s.e.m.

Figure 3

Figure 3. CNV suppression by targeted siRNAs is mediated by TLR3

a, hVegfa siRNA and Vegfr1 siRNA suppressed CNV in Tlr3+/+ but not _Tlr3_−/− mice. b, _Vegfr1_-siRNA–chol suppressed CNV in Tlr3+/+ but not _Tlr3_−/− mice. n = 5–6. c, hVegfa siRNA and Vegfr1 siRNA, whose anti-sense (as) strands were modified with 5′-methoxy (CH3O) substitution, preventing incorporation into RISC, suppressed CNV in Tlr3+/+ but not _Tlr3_−/− mice. n = 9–14. d, siRNAs targeting mouse (m) or human (h) Vegfa reduced CNV in Tlr3+/+ but not in _Tlr3_−/− mice. _mVegfa_-siRNA–chol and _hVegfa-_siRNA–chol suppressed CNV in Tlr3+/+ mice; however, _mVegfa_-siRNA–chol but not _hVegfa_-siRNA–chol suppressed CNV in _Tlr3_−/− mice. n = 8–10. siRNAs were at 1 μg; asterisk, P < 0.05 compared to vehicle (siRNA buffer). All error bars indicate mean ± s.e.m.

Figure 4

Figure 4. Minimum length for TLR3 activation

a, 21-nucleotide or 23-nucleotide Luc siRNA but not truncated versions suppressed CNV in wild-type mice. n = 8–11; asterisk, P < 0.05 compared to vehicle (buffer); mean ± s.e.m. Equimolar amounts to 1 μg of 21-nucleotide Luc siRNA. nt, nucleotide. b, c, Orthogonal views of a model of TLR3 ectodomain dimer (green and cyan subunits shown as backbone ribbons) with computer-docked 21-nucleotide dsRNA. Protein modules interact via C-terminal domains to form a highly symmetrical dimer. The model incorporated potential interactions involving a larger set of TLR3 residues considered important in RNA binding (displayed as purple and red side chains on ectodomain subunits); these residues were within 4.5 Å of RNA. d, TLR3 dimer with docked 19-nucleotide dsRNA shows proximity to fewer putative RNA-binding residues. Binding of 19-nucleotide dsRNA was less favourable than 21-nucleotide dsRNA (Δ_E_ = &minus308 kcal mol−1; 95% confidence interval: 261–355; n = 5; P = 0.008).

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