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Sequence-specific potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7

Abstract

Short interfering RNA (siRNA) is used in RNA interference technology to avoid non-target-related induction of type I interferon (IFN) typical for long double-stranded RNA. Here we show that in plasmacytoid dendritic cells (PDC), an immune cell subset specialized in the detection of viral nucleic acids and production of type I IFN, some siRNA sequences, independent of their GU content, are potent stimuli of IFN-α production. Localization of the immunostimulatory motif on the sense strand of a potent IFN-α-inducing siRNA allowed dissection of immunostimulation and target silencing. Injection into mice of immunostimulatory siRNA, when complexed with cationic liposomes, induced systemic immune responses in the same range as the TLR9 ligand CpG, including IFN-α in serum and activation of T cells and dendritic cells in spleen. Immunostimulation by siRNA was absent in TLR7-deficient mice. Thus sequence-specific TLR7-dependent immune recognition in PDC needs to be considered as an additional biological activity of siRNA, which then should be termed immunostimulatory RNA (isRNA).

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Figure 1: In HEK293 cells siRNA inhibits TLR9 expression without inducing a type I IFN response.
Figure 2: Induction of IFN-α in PDC by siRNA is based on single-strand motif recognition.
Figure 3: A nine-base motif of siRNA9.2 sense strand is responsible for immunostimulation.
Figure 4: LNA modifications of the sense and the anti-sense strand reveal IFN-α induction and silencing as two independent activities.
Figure 5: Induction of immune responses by siRNA are TLR7 dependent.

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Acknowledgements

This study was supported by research funding from the Deutsche Forschungsgemeinschaft (DFG) Ha 2780/4-1 and the Sonderforschungsbereich (SFB) 571, from the Bundesministerium für Bildung und Forschung (BMBF, Biofuture 0311896) and from the Human Science Foundation of Japan to G.H. and from DFG En 169/7-1 to S.E. This work is part of the dissertations of M. Guenthner-Biller and of A. Ablasser at the Ludwig-Maximilians-University, Munich, Germany. Lastly, the authors would like to acknowledge R. Meyers for her assistance.

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Correspondence to Gunther Hartmann.

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Anne Noronha, Muthiah Manoharan and Antonin dr Fougerolles are employees of Alnylam Pharmaceuticals, Cambridge.

Supplementary information

Supplementary Fig. 1

PDC produce IFN-α in response to TLR7 and TLR9 ligands but not upon transfection with long dsRNA. (PDF 54 kb)

Supplementary Fig. 2

Induction of IFN-α in plasmacytoid dendritic cells by siRNA is dose dependent and can be achieved by delivery of siRNA via cationic lipids or electroporation. (PDF 123 kb)

Supplementary Table 1

siRNA sequences (PDF 23 kb)

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Hornung, V., Guenthner-Biller, M., Bourquin, C. et al. Sequence-specific potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med 11, 263–270 (2005). https://doi.org/10.1038/nm1191

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  • DOI: https://doi.org/10.1038/nm1191

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