Self-assembling short immunostimulatory duplex RNAs with broad-spectrum antiviral activity

Abstract

The current coronavirus disease 2019 (COVID-19) pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III). These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique sequence motif (sense strand, 5'-C; antisense strand, 3'-GGG) that mediates end-to-end dimer self-assembly. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory small interfering RNAs (siRNAs), their activity is independent of Toll-like receptor (TLR) 7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with immunostimulant poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad-spectrum inhibition of infections by many respiratory viruses with pandemic potential, including severe acute respiratory syndrome coronavirus (SARS-CoV)-2, SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus (HCoV)-NL63, and influenza A virus in cell lines, human lung chips that mimic organ-level lung pathophysiology, and a mouse SARS-CoV-2 infection model. These short double-stranded RNAs (dsRNAs) can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics.

Keywords: Hoogsteen G-G base pairing; MT: Oligonucleotides; RIG-I; SARS-CoV-2; Therapies and Applications; antiviral therapeutic; immunostimulatory RNA; influenza; interferon; organ on a chip.

Graphical abstract

Figure 1

Discovery of new immunostimulatory RNAs (A) Viral load of A549 cells transfected with RNA-1, RNA-2, or a scrambled duplex RNA control, and 24 h later infected with influenza A/WSN/33 (H1N1) virus (MOI = 0.01). Titers of progeny viruses in medium supernatants collected at 48 h post infection were determined by quantifying plaque-forming units (PFU); data are shown as percentage viral infection measured in the cells treated with the control RNA (data shown are mean ± SD; N = 3; ∗∗∗p < 0.001). (B) Transcriptome by RNA-seq (left) and proteome by TMT mass spectrometry (Mass Spec) of A549 cells after RNA-1 transfection. Differentially expressed genes (DEGs) from RNA-seq or proteins from TMT Mass Spec are shown in volcano plots (top) and results of GO enrichment analysis performed for the DEGs are shown at the bottom (N = 3). (C) qPCR analysis of cellular IFN-β and IFN-α RNA levels at 48 h after A549 cells were transfected with RNA-1, RNA-2, or scrambled dsRNA control (N = 3). (D) RNA-mediated production kinetics of IFN production in wild-type A549-Dual cells that were transfected with RNA-1, RNA-2, or scramble RNA control measured using a Quanti-Luc assay. Optical density (OD) values from cells transfected with the scrambled RNA control were subtracted as background (N = 6). (E) Induction of IFN by different concentrations of RNA-1 and RNA-2 in A549-Dual cells compared with scrambled RNA control measured at 48 h post transfection (control OD values were subtracted as background; N = 6).

Figure 2

Comparison of the immunostimulatory activities of different RNAs IFN luciferase reporter activity of A549-Dual cells transfected with indicated duplex RNAs for 48 h. Data are shown as fold change relative to RNA-1, and the immunostimulatory activity of RNA-1 was set as 1 (N = 6). Data are shown as means ± SD.

Figure 3

Immunostimulatory RNAs induce IFN-I production through RIG-I-IRF3 pathway (A) IFN-β mRNA levels in wild-type (WT) HAP1 cells, IRF3 knockout HAP1 cells, or IRF7 knockout HAP1 cells after transfection with RNA-1 or scrambled RNA control for 48 h. Data are shown as fold change relative to the scrambled RNA control (N = 3). Note that IRF3 knockdown completely abolished the IFN-β response. (B) IRF3 mRNA levels measured in A549 cells transfected with immunostimulatory RNA-4 or a scrambled RNA control (data are shown as fold change relative to the control RNA; N = 3). (C) Total and phosphorylated IRF3 protein levels in A549 cells at 48 h after transfection with RNA-4 or scrambled RNA control. GAPDH was used as a loading control. (D) Immunofluorescence micrographs showing the distribution of phosphorylated IRF3 in A549 cells transfected with RNA-4 or scrambled RNA control at 48 h post transfection (green, phosphorylated IRF3; blue, DAPI-stained nuclei; arrowheads, nuclei expressing phosphorylated IRF3). Scale bar, 20 μm. (E) IFN-β expression in WT A549-Dual cells, RIG-I knockout A549-Dual cells, MDA5 knockout A549-Dual cells, or TLR3 knockout A549 cells at 48 h after transfection with immunostimulatory RNA-4 or a scrambled RNA control. Data are shown as fold change relative to the scrambled RNA control; N = 6. Note that RIG-I knockout abolished the ability of the immunostimulatory RNAs to induce IFN-β. (F) SPR characterization of the binding affinity between cellular RNA sensors (RIG-I, MDA5, and TLR3) and RNA-1. Equilibrium dissociation constant (K_D), association rate constant (K_a), and dissociation rate constant (K_d) are labeled on the graphs. (A, B, and E) Data are shown as means ± SD.

Figure 4

The common motif mediates the formation of duplex RNA dimers via intramolecular G-quadruplex formed by GG overhang (A) The image of native gel electrophoresis showing the formation of RNA-1 dimer; 1 μL of 10 μM RNA samples was loaded. RNA-12 was used as negative control. (B) The structure of end-to-end RNA-1 dimer due to terminal G-G Hoogsteen paring.

Figure 5

Immunostimulatory RNAs elicit responses with a stronger antiviral component and a lower proinflammatory component (A and B) Volcano plots showing significant upregulated genes (red) or downregulated genes (blue) in RNA-1 transfected (A) or poly(I:C) transfected (B) A549 cells. Threshold for fold change = 2, threshold for P_adj = 0.01. (C) Comparison of the immunostimulatory activities of different RNAs. A549-Dual cells were transfected with indicated duplex RNAs at a 5-fold serial dilution from 50 nM to 16 pM for 24 h, and then activation of the IFN pathway and NF-kB pathway was measured by quantifying luciferase reporter activity or alkaline phosphatase activity, respectively. N = 4. Data are shown as means ± SD.

Figure 6

Immunostimulatory RNAs induce IFN-β production in differentiated human lung epithelial and endothelial cells in organ chips and exhibit broad-spectrum inhibition of infection by H3N2 influenza virus, SARS-CoV-2, SARS-CoV-1, MERS-CoV, and HCoV-NL63 (A) Schematic diagram of a cross section through the human lung on chip, which faithfully recapitulate human lung physiology and pathophysiology. (B) IFN-β mRNA in the epithelial or endothelial cells on human lung airway and alveolus chips at 48 h after transfection with RNA-1 or scrambled RNA control by perfusion through both channels of the chip. Data are presented as fold change relative to the RNA control; N = 3; ∗p < 0.05, ∗∗∗p < 0.001. (C) Effects of treatment with RNA-1 or a scrambled control on viral nucleoprotein (NP) mRNA levels in the human lung airway chips or human lung alveolus chips infected with influenza A/HK/8/68 (H3N2) (MOI = 0.1) at 24 h after RNA-1 treatment. Results are shown as fold change relative to RNA control; N = 3; ∗p < 0.05. (D) Viral load of indicated cells at 48 h after infection after transfection with RNA-1, RNA-2, or a scrambled control for 24 h. For infection, influenza A/HK/8/68 (H3N2) (MOI = 0.1), SARS-CoV-2 (MOI = 0.05), SARS-CoV-1 (MOI = 0.01), MERS-CoV (MOI = 0.01), and HCoV-NL63 (MOI = 0.002), respectively. qPCR in cell lysates was used to quantify viral NP gene for H3N2, and the N gene for SARS-CoV-2 and HCoV-NL63, and plaque-forming assay for SARS-CoV and MERS-CoV. All results are shown as fold change relative to RNA control; N = 3; ∗p < 0.05, ∗∗∗p < 0.001. Data are shown as means ± SD.

Figure 7

Viral titers at day 3 after challenge with SARS-CoV-2 virus in the lungs of K18-hACE2 mice treated with indicated RNAs or vehicle (n = 4) Data are plotted for individual mice and overlaid with mean ± SD; ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.