Abstract
The SiRNA strategy is a potent and versatile method for modulating expression of any gene in various species for investigational or therapeutic purposes. Clinical translation of SiRNA-based approaches proved challenging, mainly due to the difficulty of targeted SiRNA delivery into cells of interest and the immunogenic side effects of oligonucleotide reagents. However, the intrinsic sensitivity of immune cells to nucleic acids can be utilized for the delivery of SiRNAs designed for the purpose of cancer immunotherapy. We have demonstrated that synthetic ligands for the intracellular receptor TLR9 can serve as targeting moiety for cell-specific delivery of SiRNAs. Chemically synthesized CpG-SiRNA conjugates are quickly internalized by TLR9-positive cells in the absence of transfection reagents, inducing target gene silencing. The CpG-SiRNA strategy allows for effective targeting of TLR9-positive cells in vivo after local or systemic administration of these oligonucleotides into mice.
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References
Snead NM, Rossi JJ (2012) RNA interference trigger variants: getting the most out of RNA for RNA interference-based therapeutics. Nucleic Acid Ther 22:139–146
Rettig GR, Behlke MA (2012) Progress toward in vivo use of siRNAs-II. Mol Ther 20:483–512
Davidson BL, McCray PB Jr (2011) Current prospects for RNA interference-based therapies. Nat Rev Genet 12:329–340
Zimmermann TS, Lee AC, Akinc A, Bramlage B, Bumcrot D, Fedoruk MN, Harborth J, Heyes JA, Jeffs LB, John M, Judge AD, Lam K, McClintock K, Nechev LV, Palmer LR, Racie T, Rohl I, Seiffert S, Shanmugam S, Sood V, Soutschek J, Toudjarska I, Wheat AJ, Yaworski E, Zedalis W, Koteliansky V, Manoharan M, Vornlocher HP, MacLachlan I (2006) RNAi-mediated gene silencing in non-human primates. Nature 441:111–114
Davis ME, Zuckerman JE, Choi CH, Seligson D, Tolcher A, Alabi CA, Yen Y, Heidel JD, Ribas A (2010) Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464:1067–1070
Whitehead KA, Langer R, Anderson DG (2009) Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 8:129–138
Kortylewski M, Swiderski P, Herrmann A, Wang L, Kowolik C, Kujawski M, Lee H, Scuto A, Liu Y, Yang C, Deng J, Soifer HS, Raubitschek A, Forman S, Rossi JJ, Pardoll DM, Jove R, Yu H (2009) In vivo delivery of siRNA to immune cells by conjugation to a TLR9 agonist enhances antitumor immune responses. Nat Biotechnol 27:925–932
Krieg AM (2012) CpG still rocks! Update on an accidental drug. Nucleic Acid Ther 22:77–89
Kawai T, Akira S (2010) The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 11:373–384
Amarzguioui M, Lundberg P, Cantin E, Hagstrom J, Behlke MA, Rossi JJ (2006) Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nat Protoc 1:508–517
Zhang Q, Hossain DM, Nechaev S, Kozlowska A, Zhang W, Liu Y, Kowolik CM, Swiderski P, Rossi JJ, Forman S, Pal S, Bhatia R, Raubitschek A, Yu H, Kortylewski M (2013) TLR9-mediated siRNA delivery for targeting of normal and malignant human hematopoietic cells in vivo. Blood 121:1304–1315
Nechaev S, Gao C, Moreira D, Swiderski P, Jozwiak A, Kowolik CM, Zhou J, Armstrong B, Raubitschek A, Rossi JJ, Kortylewski M (2013) Intracellular processing of immunostimulatory CpG-siRNA: toll-like receptor 9 facilitates siRNA dicing and endosomal escape. J Control Release 170:307–315
Stalder L, Heusermann W, Sokol L, Trojer D, Wirz J, Hean J, Fritzsche A, Aeschimann F, Pfanzagl V, Basselet P, Weiler J, Hintersteiner M, Morrissey DV, Meisner-Kober NC (2013) The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing. EMBO J 32:1115–1127
Hossain DM, Dos Santos C, Zhang Q, Kozlowska A, Liu H, Gao C, Moreira D, Swiderski P, Jozwiak A, Kline J, Forman S, Bhatia R, Kuo YH, Kortylewski M (2014) Leukemia cell-targeted STAT3 silencing and TLR9 triggering generate systemic antitumor immunity. Blood 123:15–25
Deng J, Liu Y, Lee H, Herrmann A, Zhang W, Zhang C, Shen S, Priceman SJ, Kujawski M, Pal SK, Raubitschek A, Hoon DS, Forman S, Figlin RA, Liu J, Jove R, Yu H (2012) S1PR1-STAT3 signaling is crucial for myeloid cell colonization at future metastatic sites. Cancer Cell 21:642–654
Moreira D, Zhang Q, Hossain DM, Nechaev S, Li H, Kowolik CM, D’Apuzzo M, Forman S, Jones J, Pal SK, Kortylewski M (2015) TLR9 signaling through NF-κB/RELA and STAT3 promotes tumor-propagating potential of prostate cancer cells. Oncotarget 6:17302–17313
Acknowledgments
This work was supported by the Department of Defense grant number W81XWH-12-1-0132, Prostate Cancer Foundation, STOP CANCER Allison Tovo-Dwyer Memorial Career Development Award and by the National Cancer Institute of the National Institutes of Health under grant numbers R01CA155367 (to M.K.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
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Hossain, D.M.S., Moreira, D., Zhang, Q., Nechaev, S., Swiderski, P., Kortylewski, M. (2016). TLR9-Targeted SiRNA Delivery In Vivo. In: Shum, K., Rossi, J. (eds) SiRNA Delivery Methods. Methods in Molecular Biology, vol 1364. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3112-5_15
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DOI: https://doi.org/10.1007/978-1-4939-3112-5_15
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3111-8
Online ISBN: 978-1-4939-3112-5
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