Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

In vivo delivery of siRNA to immune cells by conjugation to a TLR9 agonist enhances antitumor immune responses

Abstract

Efficient delivery of small interfering (si)RNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We show that siRNA synthetically linked to a CpG oligonucleotide agonist of toll-like receptor (TLR)9 targets and silences genes in TLR9+ myeloid cells and B cells, both of which are key components of the tumor microenvironment. When a CpG-conjugated siRNA that targets the immune suppressor gene Stat3 is injected in mice either locally at the tumor site or intravenously, it enters tumor-associated dendritic cells, macrophages and B cells. Silencing of Stat3 leads to activation of tumor-associated immune cells and ultimately to potent antitumor immune responses. Our findings demonstrate the potential of TLR agonist–siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure and function of the CpG–Stat3 siRNA conjugate.
Figure 2: Uptake and gene silencing by CpG–Stat3 siRNA in vitro. (a) Targeted delivery: splenocytes were incubated for 3 h with CpG–Stat3 siRNA or for 24 h with unconjugated Stat3 siRNA labeled with FITC (bottom left panel), without any transfection reagents.
Figure 3: Treatment with CpG–Stat3 siRNA leads to cell-specific gene silencing in vivo. (a) In vivo uptake by myeloid cells of peritumorally injected CpG–Stat3 siRNA.
Figure 4: Both local and systemic treatments with CpG–Stat3 siRNA inhibit tumor growth.
Figure 5: In vivo administration of CpG–Stat3 siRNA induces innate immunity.
Figure 6: Targeting Stat3 using CpG–Stat3 siRNA augments innate and adaptive antitumor immunity.

Similar content being viewed by others

References

  1. Song, E. et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol. 23, 709–717 (2005).

    Article  CAS  Google Scholar 

  2. McNamara, J.O. II et al. Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nat. Biotechnol. 24, 1005–1015 (2006).

    Article  CAS  Google Scholar 

  3. Kumar, P. et al. Transvascular delivery of small interfering RNA to the central nervous system. Nature 448, 39–43 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Li, B.J. et al. Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque. Nat. Med. 11, 944–951 (2005).

    Article  CAS  Google Scholar 

  6. Zimmermann, T.S. et al. RNAi-mediated gene silencing in non-human primates. Nature 441, 111–114 (2006).

    Article  CAS  Google Scholar 

  7. Bui, J.D. & Schreiber, R.D. Cancer immunosurveillance, immunoediting and inflammation: independent or interdependent processes? Curr. Opin. Immunol. 19, 203–208 (2007).

    Article  CAS  Google Scholar 

  8. Koebel, C.M. et al. Adaptive immunity maintains occult cancer in an equilibrium state. Nature 450, 903–907 (2007).

    Article  CAS  Google Scholar 

  9. Shankaran, V. et al. IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature 410, 1107–1111 (2001).

    Article  CAS  Google Scholar 

  10. Kortylewski, M. et al. Inhibiting Stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity. Nat. Med. 11, 1314–1321 (2005).

    Article  CAS  Google Scholar 

  11. Zou, W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat. Rev. Cancer 5, 263–274 (2005).

    Article  CAS  Google Scholar 

  12. Kujawski, M. et al. Stat3 mediates myeloid cell-dependent tumor angiogenesis in mice. J. Clin. Invest. 118, 3367–3377 (2008).

    Article  CAS  Google Scholar 

  13. Yu, C.L. et al. Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. Science 269, 81–83 (1995).

    Article  CAS  Google Scholar 

  14. Bromberg, J.F. et al. Stat3 as an oncogene. Cell 98, 295–303 (1999).

    Article  CAS  Google Scholar 

  15. Yu, H. & Jove, R. The STATs of cancer–new molecular targets come of age. Nat. Rev. Cancer 4, 97–105 (2004).

    Article  CAS  Google Scholar 

  16. Darnell, J.E. Jr. Transcription factors as targets for cancer therapy. Nat. Rev. Cancer 2, 740–749 (2002).

    Article  CAS  Google Scholar 

  17. Yu, H., Kortylewski, M. & Pardoll, D. Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment. Nat. Rev. Immunol. 7, 41–51 (2007).

    Article  CAS  Google Scholar 

  18. Wang, T. et al. Regulation of the innate and adaptive immune responses by Stat-3 signaling in tumor cells. Nat. Med. 10, 48–54 (2004).

    Article  Google Scholar 

  19. Kortylewski, M. & Yu, H. Role of Stat3 in suppressing anti-tumor immunity. Curr. Opin. Immunol. 20, 228–233 (2008).

    Article  CAS  Google Scholar 

  20. Kortylewski, M. et al. Regulation of the IL-23 and IL-12 balance by Stat3 signaling in the tumor microenvironment. Cancer Cell 15, 114–123 (2009).

    Article  CAS  Google Scholar 

  21. Bollrath, J. et al. gp130-mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis. Cancer Cell 15, 91–102 (2009).

    Article  CAS  Google Scholar 

  22. Grivennikov, S. et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer. Cancer Cell 15, 103–113 (2009).

    Article  CAS  Google Scholar 

  23. Lee, H. et al. Persistently activated Stat3 maintains constitutive NF-kappaB activity in tumors. Cancer Cell 15, 283–293 (2009).

    Article  CAS  Google Scholar 

  24. Wang, L. et al. IL-17 is pro-carcinogenic through an IL-6/Stat3 signaling pathway. J. Exp. Med. 206, 1457–1464 (2009).

    Article  CAS  Google Scholar 

  25. Kortylewski, M. et al. Toll-like receptor 9 activation of signal transducer and activator of transcription 3 constrains its agonist-based immunotherapy. Cancer Res. 69, 2497–2505 (2009).

    Article  CAS  Google Scholar 

  26. Iwasaki, A. & Medzhitov, R. Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5, 987–995 (2004).

    Article  CAS  Google Scholar 

  27. Kanzler, H., Barrat, F.J., Hessel, E.M. & Coffman, R.L. Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nat. Med. 13, 552–559 (2007).

    Article  CAS  Google Scholar 

  28. Barchet, W., Wimmenauer, V., Schlee, M. & Hartmann, G. Accessing the therapeutic potential of immunostimulatory nucleic acids. Curr. Opin. Immunol. 20, 389–395 (2008).

    Article  CAS  Google Scholar 

  29. Krieg, A.M. Toll-like receptor 9 (TLR9) agonists in the treatment of cancer. Oncogene 27, 161–167 (2008).

    Article  CAS  Google Scholar 

  30. Klinman, D.M., Currie, D., Gursel, I. & Verthelyi, D. Use of CpG oligodeoxynucleotides as immune adjuvants. Immunol. Rev. 199, 201–216 (2004).

    Article  CAS  Google Scholar 

  31. Klinman, D.M., Yi, A.K., Beaucage, S.L., Conover, J. & Krieg, A.M. CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma. Proc. Natl. Acad. Sci. USA 93, 2879–2883 (1996).

    Article  CAS  Google Scholar 

  32. Krieg, A.M. et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374, 546–549 (1995).

    Article  CAS  Google Scholar 

  33. Kim, D.H. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23, 222–226 (2005).

    Article  CAS  Google Scholar 

  34. Rose, S.D. et al. Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res. 33, 4140–4156 (2005).

    Article  CAS  Google Scholar 

  35. Hemmi, H. et al. Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 (2000).

    Article  CAS  Google Scholar 

  36. Latz, E. et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. Immunol. 5, 190–198 (2004).

    Article  CAS  Google Scholar 

  37. Chendrimada, T.P. et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436, 740–744 (2005).

    Article  CAS  Google Scholar 

  38. Samarasinghe, R. et al. Induction of an anti-inflammatory cytokine, IL-10, in dendritic cells after toll-like receptor signaling. J. Interferon Cytokine Res. 26, 893–900 (2006).

    Article  CAS  Google Scholar 

  39. Cao, Y.A. et al. Shifting foci of hematopoiesis during reconstitution from single stem cells. Proc. Natl. Acad. Sci. USA 101, 221–226 (2004).

    Article  CAS  Google Scholar 

  40. Benkhart, E.M., Siedlar, M., Wedel, A., Werner, T. & Ziegler-Heitbrock, H.W. Role of Stat3 in lipopolysaccharide-induced IL-10 gene expression. J. Immunol. 165, 1612–1617 (2000).

    Article  CAS  Google Scholar 

  41. Xie, T.X. et al. Stat3 activation regulates the expression of matrix metalloproteinase-2 and tumor invasion and metastasis. Oncogene 23, 3550–3560 (2004).

    Article  CAS  Google Scholar 

  42. Takeda, K. et al. Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils. Immunity 10, 39–49 (1999).

    Article  CAS  Google Scholar 

  43. Welte, T. et al. STAT3 deletion during hematopoiesis causes Crohn's disease-like pathogenesis and lethality: a critical role of STAT3 in innate immunity. Proc. Natl. Acad. Sci. USA 100, 1879–1884 (2003).

    Article  CAS  Google Scholar 

  44. Dhodapkar, M.V., Steinman, R.M., Krasovsky, J., Munz, C. & Bhardwaj, N. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J. Exp. Med. 193, 233–238 (2001).

    Article  CAS  Google Scholar 

  45. Bui, J.D., Uppaluri, R., Hsieh, C.S. & Schreiber, R.D. Comparative analysis of regulatory and effector T cells in progressively growing versus rejecting tumors of similar origins. Cancer Res. 66, 7301–7309 (2006).

    Article  CAS  Google Scholar 

  46. Klinman, D., Shirota, H., Tross, D., Sato, T. & Klaschik, S. Synthetic oligonucleotides as modulators of inflammation. J. Leukoc. Biol. 84, 958–964 (2008).

    Article  CAS  Google Scholar 

  47. Sica, A. & Bronte, V. Altered macrophage differentiation and immune dysfunction in tumor development. J. Clin. Invest. 117, 1155–1166 (2007).

    Article  CAS  Google Scholar 

  48. Tan, T.T. & Coussens, L.M. Humoral immunity, inflammation and cancer. Curr. Opin. Immunol. 19, 209–216 (2007).

    Article  CAS  Google Scholar 

  49. Spaner, D.E., Foley, R., Galipeau, J. & Bramson, J. Obstacles to effective Toll-like receptor agonist therapy for hematologic malignancies. Oncogene 27, 208–217 (2008).

    Article  CAS  Google Scholar 

  50. Clarke, P., Mann, J., Simpson, J.F., Rickard-Dickson, K. & Primus, F.J. Mice transgenic for human carcinoembryonic antigen as a model for immunotherapy. Cancer Res. 58, 1469–1477 (1998).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to the Light Microscopy Imaging and Flow Cytometry Cores and Animal Facilities of Beckman Research Institute at City of Hope Medical Center for technical support and assistance and X. Li of the Department of Biomedical Informatics for consultation on statistical analyses. We thank C.H. Contag of Stanford University School of Medicine for providing the original luciferase mice. Mouse dendritic DC2.4 cells were originally from K. Rock (University of Massachusetts Medical School). The highly metastatic clone of K1735 melanoma (C4) was kindly provided by S. Huang and J. Fidler of M.D. Anderson Cancer Center. The stably transduced A20-Luc cell line was provided by D. Zheng (City of Hope). This work is funded in part by grants from the board of governors for City of Hope Medical Center, Harry Lloyd Charitable Trust and Keck Foundation, in addition to National Institutes of Health grants (R01CA122976, R01CA115815, R01CA115674, P50CA107399).

Author information

Authors and Affiliations

Authors

Contributions

H.Y. and M. Kortylewski conceived the project, designed the majority of the experiments, analyzed the data and wrote the paper. M. Kortylewski also carried out many of the key experiments and was instrumental in the design of the CpG-siRNA construct. P.S. contributed to the design of the construct and synthesized all the CpG-siRNA constructs. A.H. performed imaging and EMSA experiments. L.W. did A20 tumor cell experiments and luciferase CpG-siRNA experiments in vivo. C.K. tested siRNA sequences and carried out all the real-time PCR experiments. M. Kujawski and Y.L. performed some in vivo experiments. H.L. and C.Y. did western blot analysis. A.S. and J.D. tested CpG-siRNA in B-cell malignant cells. H.S.S. suggested the in vitro Dicer experiment. A.R. contributed to the MC38-CEA tumor experiment. J.J.R. and S.F. provided helpful discussion. J.J.R. was also helpful in the siRNA design. D.M.P. contributed to immunological experimental design, provided insightful discussion and assisted in writing the manuscript. R.J. contributed to the concept of the project and the Stat3 siRNA design.

Corresponding author

Correspondence to Hua Yu.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–13 and Supplementary Table 1 (PDF 1609 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kortylewski, M., Swiderski, P., Herrmann, A. et al. In vivo delivery of siRNA to immune cells by conjugation to a TLR9 agonist enhances antitumor immune responses. Nat Biotechnol 27, 925–932 (2009). https://doi.org/10.1038/nbt.1564

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.1564

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing