Widespread Interference
Ian F. Dunn, Robert Max Friedlander · Neurosurgery · 2008
Since its first detailed description by Fire and Mello (Nature 391:806–811, 1998)—which would later garner the Nobel Prize—RNA interference (RNAi) has become a central tool for the investigation of gene function. Sequence- specific gene silencing by RNAi also holds substantial promise as a therapeutic approach in disorders characterized by pathological expression of disease-causing genes. Indeed, early-phase clinical trials have involved the vitreal injection of small interfering (si) RNAs targeting vascular endothelial growth factor (VEGF)-A and VEGF receptor 1 (VEGFR1) in age-related macular degeneration. Until now, the assumption has been that injected siRNAs enter target cells and engage the cell's RNAi machinery to suppress the expression of the angiogenic target genes in a sequence-specific manner. Kleinman et al. have shown that this assumption is too simplistic. In a recent article in Nature (452:591–597, 2008), they show in murine models of ocular neovascularization that angiogenesis can be inhibited by essentially any siRNA in a sequence-independent manner provided that it is greater than 21 nucleotides in length. siRNAs targeting even nonmammalian genes suppressed angiogenesis to a degree comparable to that of siRNAs targeting VEGF-A and VEGFR1. Essentially, antiangiogenesis appears to be a generic property of siRNAs regardless of sequence and target. In investigating the mechanism of siRNA-mediated antiangiogenesis, the authors first showed that it is target-independent: numerous synthetic siNRAs, e.g., those against jellyfish green fluorescent protein, nonocular genes, or random genomic fragments, were able to suppress neovascularization comparable to siRNAs against VEGF and VEGFR1 when injected intraocularly. Next, they elegantly showed that this effect is mediated by the immune system regulator Toll-like receptor 3 (TLR3) on the cell surface and that the effect is absent in TLR3-deficient mice. TLR3 has a well-known role in the recognition of virus-derived double-stranded RNA. To demonstrate the fact that siRNA-mediated antiangiogenesis is TLR3-specific, they showed that soluble TLR3 reverses the antiangiogenic phenotype. Further work showed that interleukin-12 and interferon-g are induced by siRNA-mediated TLR3 activation and are antiangiogenic. The authors' study of TLR3 single nucleotide polymorphisms with a view toward establishing whether siRNA therapy may have differential effects on patients with different TLR3 alleles, and, indeed, variable responses to TLR3 activation by siRNAs in different genotypes led them to suggest that pharmacogenetic testing may be considered before siRNA therapy initiation in patients. In the same issue of Nature (452:543–545, 2008), Kalluri and Kanasaki diagrammed the proposed mechanism of RNA interference's generic block on angiogenesis (Fig.).FIGUREThis information is important for several reasons. First, the authors have demonstrated that the premise of a large clinical trial was erroneous. The authors used a murine model for siRNA therapy in clinical trials—siRNA against VEGF for choroidal neovascularization in macular degeneration—and showed that the antiangiogenic response has little to do with the siRNA sequence targeting VEGF. It appears that any 21-nucleotide siRNA regardless of sequence can inhibit angiogenesis. Second, they provided the biological basis for cellular engagement of siRNAs: TLR3. Last, as these data show that siRNAs activate TLR3, a widely expressed receptor with pleiotropic biological effects, this study suggests that more stringent preclinical testing be carried out before additional siRNAs are brought to clinical trial. An enhanced understanding of siRNA biology will be important as more RNAi-based therapies are investigated for neurosurgical disorders (Neurosurgery 60:3–16, 2007). IAN F. DUNN, MD ROBERT M. FRIEDLANDER, MD BASIC SCIENCE RESEARCH