Gene silencing by double-stranded RNA, denoted RNA interference, represents a new paradigm for rational drug design1. However, the transformative therapeutic potential of short interfering RNA (siRNA) has been stymied by a key obstacle—safe delivery to specified target cells in vivo2. Macrophages are particularly attractive targets for RNA interference therapy because they promote pathogenic inflammatory responses in diseases such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease and diabetes3. Here we report the engineering of β1,3-d-glucan-encapsulated siRNA particles (GeRPs) as efficient oral delivery vehicles that potently silence genes in mouse macrophages in vitro and in vivo. Oral gavage of mice with GeRPs containing as little as 20 μg kg-1 siRNA directed against tumour necrosis factor α (Tnf-α) depleted its messenger RNA in macrophages recovered from the peritoneum, spleen, liver and lung, and lowered serum Tnf-α levels. Screening with GeRPs for inflammation genes revealed that the mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) is a previously unknown mediator of cytokine expression. Importantly, silencing Map4k4 in macrophages in vivo protected mice from lipopolysaccharide-induced lethality by inhibiting Tnf-α and interleukin-1β production. This technology defines a new strategy for oral delivery of siRNA to attenuate inflammatory responses in human disease.
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Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans . Nature 391, 806–811 (1998)
Grimm, D. & Kay, M. A. Therapeutic application of RNAi: is mRNA targeting finally ready for prime time? J. Clin. Invest. 117, 3633–3641 (2007)
Duffield, J. S. The inflammatory macrophage: a story of Jekyll and Hyde. Clin. Sci. (Lond.) 104, 27–38 (2003)
Beier, R. & Gebert, A. Kinetics of particle uptake in the domes of Peyer’s patches. Am. J. Physiol. 275, G130–G137 (1998)
Herre, J., Gordon, S. & Brown, G. D. Dectin-1 and its role in the recognition of β-glucans by macrophages. Mol. Immunol. 40, 869–876 (2004)
Vazquez-Torres, A. et al. Extraintestinal dissemination of Salmonella by CD18-expressing phagocytes. Nature 401, 804–808 (1999)
Soto, E. R. & Ostroff, G. R. Characterization of multilayered nanoparticles encapsulated in yeast cell wall particles for DNA delivery. Bioconjug. Chem. 19, 840–848 (2008)
Tang, X. et al. An RNA interference-based screen identifies MAP4K4/NIK as a negative regulator of PPARγ, adipogenesis, and insulin-responsive hexose transport. Proc. Natl Acad. Sci. USA 103, 2087–2092 (2006)
Tesz, G. J. et al. Tumor necrosis factor α (TNFα) stimulates Map4k4 expression through TNFα receptor 1 signaling to c-Jun and activating transcription factor 2. J. Biol. Chem. 282, 19302–19312 (2007)
Bouzakri, K. & Zierath, J. R. MAP4K4 gene silencing in human skeletal muscle prevents tumor necrosis factor-α-induced insulin resistance. J. Biol. Chem. 282, 7783–7789 (2007)
Lu, Y. C., Yeh, W. C. & Ohashi, P. S. LPS/TLR4 signal transduction pathway. Cytokine 42, 145–151 (2008)
Frank-Kamenetsky, M. et al. Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates. Proc. Natl Acad. Sci. USA 105, 11915–11920 (2008)
Barthel, R. et al. Regulation of tumor necrosis factor alpha gene expression by mycobacteria involves the assembly of a unique enhanceosome dependent on the coactivator proteins CBP/p300. Mol. Cell. Biol. 23, 526–533 (2003)
Tsytsykova, A. V. et al. Post-induction, stimulus-specific regulation of tumor necrosis factor mRNA expression. J. Biol. Chem. 282, 11629–11638 (2007)
Zhou, Y., Yang, Y., Warr, G. & Bravo, R. LPS down-regulates the expression of chemokine receptor CCR2 in mice and abolishes macrophage infiltration in acute inflammation. J. Leukoc. Biol. 65, 265–269 (1999)
Endo, Y. et al. Enhancement by galactosamine of lipopolysaccharide (LPS)-induced tumour necrosis factor production and lethality: its suppression by LPS pretreatment. Br. J. Pharmacol. 128, 5–12 (1999)
Okusawa, S., Gelfand, J. A., Ikejima, T., Connolly, R. J. & Dinarello, C. A. Interleukin 1 induces a shock-like state in rabbits. Synergism with tumor necrosis factor and the effect of cyclooxygenase inhibition. J. Clin. Invest. 81, 1162–1172 (1988)
Silverstein, R. D-galactosamine lethality model: scope and limitations. J. Endotoxin Res. 10, 147–162 (2004)
Filleur, S. et al. SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res. 63, 3919–3922 (2003)
McCaffrey, A. P. et al. RNA interference in adult mice. Nature 418, 38–39 (2002)
Peer, D., Zhu, P., Carman, C. V., Lieberman, J. & Shimaoka, M. Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1. Proc. Natl Acad. Sci. USA 104, 4095–4100 (2007)
Song, E. et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nature Biotechnol. 23, 709–717 (2005)
Soutschek, J. et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, 173–178 (2004)
Wesche-Soldato, D. E. et al. In vivo delivery of caspase-8 or Fas siRNA improves the survival of septic mice. Blood 106, 2295–2301 (2005)
Zimmermann, T. S. et al. RNAi-mediated gene silencing in non-human primates. Nature 441, 111–114 (2006)
Sorensen, D. R., Leirdal, M. & Sioud, M. Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J. Mol. Biol. 327, 761–766 (2003)
Shealy, D. J. & Visvanathan, S. Anti-TNF antibodies: lessons from the past, roadmap for the future. Handb. Exp. Pharmacol. 181, 101–29 (2008)
Ferrante, A. W. Obesity-induced inflammation: a metabolic dialogue in the language of inflammation. J. Intern. Med. 262, 408–414 (2007)
Hansson, G. K. & Libby, P. The immune response in atherosclerosis: a double-edged sword. Nature Rev. Immunol. 6, 508–519 (2006)
Shoda, L. K. et al. A comprehensive review of interventions in the NOD mouse and implications for translation. Immunity 23, 115–126 (2005)
We appreciate critical reading of the manuscript and suggestions by C. Mello, V. Ambros, G. Hannon, S. Corvera and J. Sullivan. We thank P. Zamore for advice on methods. We also appreciate the technical help of P. Furcinitti, A. Burkhart and A. Goller. This work was supported by The University of Massachusetts Diabetes and Endocrinology Center (DK 32520), including its Genomics, Bioinformatics and Imaging Cores, the Diabetes Genome Anatomy Project (DK 60837), Commonwealth Medicine and NIH grant DK 30898.
Author Contributions G.R.O., M.A. and M.P.C. initially conceptualized the study. M.A., G.J.T., M.W., M.C. and S.M.N. performed experiments, and all authors participated in designing experiments, and analysing and interpreting data. M.A. and G.J.T. contributed equally to this work. G.R.O. and E.S. provided the β1,3-d-glucan-shell-encapsulated cationic materials and they with M.A. and G.J.T. developed the GeRP formulations used in these studies. M.A., G.R.O. and M.P.C. wrote the manuscript.
[Competing interests: After the completion of this work and submission of this manuscript, the authors’ institution licensed the reported technology to RXi Pharmaceutials, Inc. M.P.C. served as a member of the Scientific Advisory Board of RXi during the reported work and G.R.O. currently receives funding from the company through a sponsored research agreement to his institution. G.R.O. is also a consultant for Biotech Pharmacon ASA and Eden Research plc.]
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Aouadi, M., Tesz, G., Nicoloro, S. et al. Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation. Nature 458, 1180–1184 (2009). https://doi.org/10.1038/nature07774
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