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