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

Area of Science:

• Molecular Biology
• Immunology
• Ophthalmology

Background:

• Current clinical trials for blinding choroidal neovascularization (CNV) use small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1, relying on intracellular RNA interference (RNAi).
• The efficacy of siRNA in these trials is presumed to be due to specific gene silencing of VEGFA or VEGFR1.

Purpose of the Study:

• To investigate the mechanism by which siRNA inhibits CNV.
• To determine if siRNA's effect on CNV is dependent on targeting specific genes like Vegfa or Vegfr1.
• To explore alternative pathways for siRNA-mediated inhibition of neovascularization.

Main Methods:

• Administered various siRNAs (targeting non-mammalian genes, non-expressed genes, non-genomic sequences, RNAi-incompetent sequences) to mice with CNV.
• Assessed CNV suppression and compared it to siRNAs targeting Vegfa or Vegfr1.
• Investigated the role of cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induced cytokines (interferon-gamma, interleukin-12) in siRNA-mediated CNV inhibition.
• Evaluated siRNA's effect on dermal neovascularization.
• Determined the minimum siRNA length required for neovascularization inhibition.
• Examined the expression of surface TLR3 in human endothelial cells and the effect of a TLR3 variant (412FF) on siRNA-induced cytotoxicity.

Main Results:

• siRNA-mediated CNV inhibition was observed with various non-specific and RNAi-incompetent siRNAs, comparable to Vegfa/Vegfr1-targeted siRNAs.
• CNV suppression was mediated by cell-surface TLR3, TRIF, and induction of interferon-gamma and interleukin-12, independent of off-target RNAi or interferon-alpha/beta activation.
• siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, suggesting a direct interaction with TLR3.
• Non-targeted siRNA effectively suppressed dermal neovascularization.
• Human endothelial cells express surface TLR3, and a specific TLR3 variant (412FF) conferred refractoriness to siRNA-induced cytotoxicity.

Conclusions:

• CNV inhibition by siRNA is a class effect mediated by cell-surface TLR3 activation, not intracellular RNA interference targeting specific genes.
• Generic siRNAs, by activating TLR3, may offer a therapeutic strategy for angiogenic disorders.
• The findings suggest potential for individualized pharmacogenetic therapy based on TLR3 variants and highlight the possibility of unanticipated vascular or immune effects from siRNA treatments.