Misinterpreting the therapeutic effects of small interfering RNA caused by immune stimulation.

Activation of innate immunity has direct effects in modulating viral replication, tumor growth, angiogenesis, and inflammatory and other immunological processes. It is now established that unmodified siRNA can activate this innate immune response and therefore there is real potential for siRNA to elicit nonspecific therapeutic effects in a wide range of disease models. Here we demonstrate that in a murine model of influenza infection, the antiviral activity of siRNA is due primarily to immune stimulation elicited by the active siRNA duplexes and is not the result of therapeutic RNA interference (RNAi) as previously reported. We show that the misinterpretation stems from the use of a particular control green fluorescent protein (GFP) siRNA that we identify as having unusually low immunostimulatory activity compared with the active anti-influenza siRNA. Curiously, this GFP siRNA has served as a negative control for a surprising number of groups reporting therapeutic effects of siRNA. The inert immunologic profile of the GFP sequence was unique among a broad panel of published siRNAs, all of which could elicit significant interferon induction from primary immune cells. This panel included eight active siRNAs against viral, angiogenic, and oncologic targets, the reported therapeutic efficacy of which was based on comparison with the nonimmunostimulatory GFP siRNA. These results emphasize the need for researchers to anticipate, monitor, and adequately control for siRNA-mediated immune stimulation and calls into question the interpretation of numerous published reports of therapeutic RNAi in vivo. The use of chemically modified siRNA with minimal immunostimulatory capacity will help to delineate more accurately the mechanism of action underlying such studies.

[1]  J. Lieberman,et al.  Small interfering RNA targeting Fas protects mice against renal ischemia-reperfusion injury. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[2]  G. Sen,et al.  Double-stranded RNA Signaling by Toll-like Receptor 3 Requires Specific Tyrosine Residues in Its Cytoplasmic Domain* , 2003, The Journal of Biological Chemistry.

[3]  S. Akira,et al.  Sequence-specific potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7 , 2005, Nature Medicine.

[4]  W. Cioffi,et al.  Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis. , 2005, The American journal of pathology.

[5]  S. Akira,et al.  Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8 , 2004, Science.

[6]  J. Bennett,et al.  Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model. , 2003, Molecular vision.

[7]  J. Lieberman,et al.  An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection , 2006, Nature.

[8]  Michael T. McManus,et al.  RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[9]  C. Chung,et al.  In vivo gene silencing (with siRNA) of pulmonary expression of MIP‐2 versus KC results in divergent effects on hemorrhage‐induced, neutrophil‐mediated septic acute lung injury , 2005, Journal of leukocyte biology.

[10]  M. Sioud.  Induction of Inflammatory Cytokines by Double- stranded and Single-stranded siRNAs is Sequence- dependent and Requires Endosomal Localization , 2005 .

[11]  S. Akira,et al.  Nucleic acid agonists for Toll‐like receptor 7 are defined by the presence of uridine ribonucleotides , 2006, European journal of immunology.

[12]  M. Behlke.  Progress towards in Vivo Use of siRNAs , 2006, Molecular Therapy.

[13]  S. Ohashi,et al.  Sequence‐specific gene silencing in murine muscle induced by electroporation‐mediated transfer of short interfering RNA , 2004, The journal of gene medicine.

[14]  Matthias John,et al.  Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs , 2004, Nature.

[15]  S. Barik,et al.  Inhibition of respiratory viruses by nasally administered siRNA , 2005, Nature Medicine.

[16]  A. Lee,et al.  Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo. , 2006, Molecular therapy : the journal of the American Society of Gene Therapy.

[17]  B. Mahon,et al.  Efficient delivery of small interfering RNA for inhibition of IL-12p40 expression in vivo , 2004, Journal of Inflammation.

[18]  Justine R. Smith,et al.  Sequence- and target-independent angiogenesis suppression by siRNA via TLR3 , 2008, Nature.

[19]  A. Judge,et al.  Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA , 2005, Nature Biotechnology.

[20]  B. Williams,et al.  Identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays , 1998 .

[21]  T. Tumpey,et al.  Protection against lethal influenza virus challenge by RNA interference in vivo. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Judy Lieberman,et al.  RNA interference targeting Fas protects mice from fulminant hepatitis , 2003, Nature Medicine.

[23]  A. Judge,et al.  Overcoming the innate immune response to small interfering RNA. , 2008, Human gene therapy.

[24]  Keith Bowman,et al.  Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs , 2005, Nature Biotechnology.

[25]  J. Church,et al.  siRNA-DIRECTED INHIBITION OF HIV-1 INFECTION , 2003, Pediatrics.

[26]  N. Zhong,et al.  Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque , 2005, Nature Medicine.

[27]  Qing Ge,et al.  Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[28]  M. Manns,et al.  Caspase 8 small interfering RNA prevents acute liver failure in mice , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Qing Ge,et al.  Inhibition of influenza virus production in virus-infected mice by RNA interference. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[30]  L. Doughty,et al.  In vivo delivery of caspase-8 or Fas siRNA improves the survival of septic mice. , 2005, Blood.

[31]  Roger E Bumgarner,et al.  A Comprehensive View of Regulation of Gene Expression by Double-stranded RNA-mediated Cell Signaling* , 2001, The Journal of Biological Chemistry.

[32]  Judy Lieberman,et al.  Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors , 2005, Nature Biotechnology.