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5′-triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma


Genetic and epigenetic plasticity allows tumors to evade single-targeted treatments. Here we direct Bcl2-specific short interfering RNA (siRNA) with 5′-triphosphate ends (3p-siRNA) against melanoma. Recognition of 5′-triphosphate by the cytosolic antiviral helicase retinoic acid–induced protein I (Rig-I, encoded by Ddx58) activated innate immune cells such as dendritic cells and directly induced expression of interferons (IFNs) and apoptosis in tumor cells. These Rig-I–mediated activities synergized with siRNA-mediated Bcl2 silencing to provoke massive apoptosis of tumor cells in lung metastases in vivo. The therapeutic activity required natural killer cells and IFN, as well as silencing of Bcl2, as evidenced by rescue with a mutated Bcl2 target, by site-specific cleavage of Bcl2 messenger RNA in lung metastases and downregulation of Bcl-2 protein in tumor cells in vivo. Together, 3p-siRNA represents a single molecule–based approach in which Rig-I activation on both the immune- and tumor cell level corrects immune ignorance and in which gene silencing corrects key molecular events that govern tumor cell survival.

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Figure 1: 3p-2.2 siRNA potently silences Bcl2 expression and reduces metastatic growth of B16 melanoma cells in the lungs.
Figure 2: Activation of type I IFNs and NK cells are necessary for the antitumor activity of Bcl2-specific 3p-siRNA in vivo.
Figure 3: Bcl-2-specific 3p-siRNA induces cell type specific innate immune responses and apoptosis in vitro.
Figure 4: Bcl2-specific gene silencing and activation of the innate immune system synergistically promote tumor cell apoptosis in vivo.
Figure 5: Bcl2-specific gene silencing contributes to Bcl2-specific 3p-siRNA–induced inhibition of tumor growth and apoptosis.
Figure 6: Bcl2-specific 3p-siRNA is effective in other models of tumorigenesis and in human melanoma cells.


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This study was supported by grant Biofuture 0311896 of the Bundesministerium für Bildung und Forschung and by grants SFB 704, SFB 670, KFO115 and KFO177 of the Deutsche Forschungsgemeinschaft to G. Hartmann; by grants Tu90-6/1 of the Deutsche Forschungsgemeinschaft and 10741 of the Deutsche Krebshilfe to T.T.; by grant 107805 of the Deutsche Krebshilfe to R.B.; by Graduiertenkolleg 1202 to C.M., M.B. and T.S.; by LMUexcellent (CIPSM 114, research professorship), and by SFB-TR 36 and En 169/7-2 of the Deutsche Forschungsgemeinschaft to S.E. and C. Bourquin. This work is part of the theses of C.M. and M.B. at the University of Munich and of M.R. and J.L. at the University of Bonn. We thank A. Dann for excellent technical assistance, T. Maniatis (Harvard University) and J. Chen (University of Texas Southwestern Medical Center) for providing IFN-β–Luc reporter plasmids and wild-type pPME-myc NS3-4A (NS3-4A), respectively, T. Fujita Institute for Virus Research, Kyoto University for providing Rig-I and the empty control vector and C. Borner (Institute of Molecular Medicine and Cell Research, University of Freiburg) for providing wild-type mouse Bcl-2 (mBcl-2/pcDNA).

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Authors and Affiliations



H.P., R.B., T.T. and G. Hartmann designed the research; H.P., C.M., R.B., D.T., S.S.M., M.R., S.K., E.G., J.L., J.H., A.S., M.B., T.S., D.A., M.N., M.P., C. Berking and T.T. performed experiments; H.P., R.B., S.E., C. Bourquin, G. Häcker, C. Berking, R.M., V.H., S.R., T.T. and G. Hartmann conducted the data analyses; E.K., U.K., D.B., J.R., H.K. and S.A. provided genetically deficient mice and key research tools; H.P., C.M., R.B. and T.T. prepared the figures; and H.P., T.T. and G. Hartmann wrote the manuscript.

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Correspondence to Thomas Tüting or Gunther Hartmann.

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Competing interests

S.S.M. and R.M., as employees of Alnylam Pharmaceuticals, have options to purchase shares of Alnylam stock, and R.M. owns Alnylam stock.

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Supplementary Tables 1–3, Supplementary Figs. 1–10 and Supplementary Methods (PDF 2137 kb)

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Poeck, H., Besch, R., Maihoefer, C. et al. 5′-triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma. Nat Med 14, 1256–1263 (2008).

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