Novel Types of Small RNA Exhibit Sequence- and Target-dependent Angiogenesis Suppression Without Activation of Toll-like Receptor 3 in an Age-related Macular Degeneration (AMD) Mouse Model

Abstract

RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I-like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.

Figure 1

Novel types of RNAs induced downregulation of VEGF mRNA expression as effectively as siRNA. (a) Structure of novel RNA interference agents. Both nkRNA and PnkRNA were prepared as single-stranded RNA oligomers that underwent self-annealing, as shown. P indicates a proline derivative. (b) 10, 50, 100, and 200 µg of NIRNA, nkRNA, and PnkRNA against VEGF were transfected into mouse endothelial cells. Twenty-four hours after transfection, VEGF expression in the culture medium was measured using a VEGF ELISA kit. siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

Figure 2

nkRNA and PnkRNA induced TLR3 phosphorylation at low levels. (a) Phosphorylated TLR3 was analyzed by western blotting. Ten micrograms of protein was loaded in each lane. The ratio of phosphorylated TLR3 to total TLR3 is shown in the graph. (b) NF-κB activity was measured using an NF-κB reporter in which SEAP cDNA was placed under the control of the κB response element. The numbers on x-axis show RNA concentrations (nM). SEAP, secreted alkaline phosphatase; siRNA, small interfering RNA; TLR, Toll-like receptor.

Figure 3

nkRNA and PnkRNA did not induce an innate immune response, as demonstrated by expression of type I IFNs. (a,b) Levels of IFNα (a) and IFNβ (b) from mouse endothelial cells in culture medium 24 hours after transfection of nkRNA, PnkRNA, NIRNA, or TLR ligands were measured by specific ELISA. The numbers in x-axis showed concentrations (nM) of RNAs. TLR3, TLR7, or TLR9 revealed the ligand against each receptor. IFN, interferon; TLR, Toll-like receptor.

Figure 4

nkRNA and PnkRNA did not induce expression of IFNβ in murine eyes. To evaluate of activation for innate immune in vivo by nkRNA and PnkRNA, eyes of C57BL/6J mice were injected 1 µg of nkRNA, PnkRNA, or NIRNA. (a) IFNα and (b) IFNβ mRNAs from murine eyes 24 hours after administration mRNA were quantitated by real-time PCR. IFN, interferon.

Figure 5

nkRNA and PnkRNA inhibited laser-induced CNV in a mouse model. (a) Fluorescein angiography. Neovascularization was evaluated based on leakage of blood fluid from neovasculature by fluorescein angiography. (b) Confocal micrograph of laser-induced CNV in mice. The retinas of eyes on RNA-treated mice after laser-induced coagulation were used to generate choroidal flatmounts, which were then examined with a scanning laser confocal microscope to visualize vessels. Vessels in the laser-treated area and superficial to this reference plane were judged as CNV. The area of CNV-related fluorescence was measured. CNV, choroidal neovascularization.