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Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA


Short interfering RNAs (siRNAs) that mediate specific gene silencing through RNA interference (RNAi) are widely used to study gene function and are also being developed for therapeutic applications1. Many nucleic acids, including double- (dsRNA)2 and single-stranded RNA (ssRNA)3,4,5, can stimulate innate cytokine responses in mammals. Despite this, few studies have questioned whether siRNA may have a similar effect on the immune system6,7. This could significantly influence the in vivo application of siRNA owing to off-target effects and toxicities associated with immune stimulation. Here we report that synthetic siRNAs formulated in nonviral delivery vehicles can be potent inducers of interferons and inflammatory cytokines both in vivo in mice and in vitro in human blood. The immunostimulatory activity of formulated siRNAs and the associated toxicities are dependent on the nucleotide sequence. We have identified putative immunostimulatory motifs that have allowed the design of siRNAs that can mediate RNAi but induce minimal immune activation.

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Figure 1: Sequence-dependent induction of cytokines by encapsulated siRNA in mice.
Figure 2: Formulated siRNAs induce cytokine responses from human PBMCs in vitro.
Figure 3: Immunostimulatory activity of siRNA is regulated by distinct sequence motifs.
Figure 4: siRNAs can be designed that are active in mediating RNAi and have minimal capacity to activate cytokine responses.


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The authors would like to acknowledge Lorne Palmer for technical assistance and James Heyes for lipid synthesis. D.F. was supported by a Canadian National Science and Engineering Research Council research award.

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Corresponding author

Correspondence to Ian MacLachlan.

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Competing interests

All authors were employed by Protiva Biotherapeutics at the time the work was performed.

Supplementary information

Supplementary Fig. 1

In vitro and In vivo properties of lipid encapsulated siRNA. (PDF 73 kb)

Supplementary Fig. 2

IFN-α responses to encapsulated siRNA in different mouse strains and with alternate liposomal compositions. (PDF 75 kb)

Supplementary Fig. 3

Inflammatory cytokine production by monocytes in response to siRNA. (PDF 90 kb)

Supplementary Fig. 4

Immune stimulatory activity of siRNA is not due to contaminating ssRNA. (PDF 78 kb)

Supplementary Fig. 5

Sequence modifications to stimulatory siRNA can ameliorate systemic toxicities. (PDF 85 kb)

Supplementary Fig. 6

Stimulatory siRNA do not activate HEK293 Cells transfected with human TLR3, TLR7, TLR8 or TLR9. (PDF 66 kb)

Supplementary Fig. 7

PAGE purification of siRNA does not affect its immunostimulatory activity. (PDF 69 kb)

Supplementary Table 1

siRNA sequences used in these studies. (PDF 72 kb)

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Judge, A., Sood, V., Shaw, J. et al. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol 23, 457–462 (2005).

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