Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA (original) (raw)

Abstract

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 applications 1. Many nucleic acids, including double-(dsRNA) 2 and single-stranded RNA (ssRNA) 3-5 , can stimulate innate cytokine responses in mammals. Despite this, few studies have questioned whether siRNA may have a similar effect on the immune system 6,7. 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.

Figures (6)

Protiva Biotherapeutics, 100-3480 Gilmore Way, Burnaby, British Columbia V5G 4Y1, Canada. Correspondence should be addressed to I.M. (ian@protivabio.com) Luc control) induced substantial, dose-dependent interferon-« (IFN-«) responses in outbred ICR mice (Fig. la,b). This was accompanied by the production of inflammatory cytokines including interleukin-¢ (IL-6) and tumor necrosis factor-« (TNF-a) (Fig. Ic), suggesting general activation of the innate immune response. We confirmed the immunostimulatory activity of siRNA in several inbred mouse strains and in experiments using alternate liposomal compositions (Supplementary Fig. 2 online). Innate immune activation by siRNA

Protiva Biotherapeutics, 100-3480 Gilmore Way, Burnaby, British Columbia V5G 4Y1, Canada. Correspondence should be addressed to I.M. (ian@protivabio.com) Luc control) induced substantial, dose-dependent interferon-« (IFN-«) responses in outbred ICR mice (Fig. la,b). This was accompanied by the production of inflammatory cytokines including interleukin-¢ (IL-6) and tumor necrosis factor-« (TNF-a) (Fig. Ic), suggesting general activation of the innate immune response. We confirmed the immunostimulatory activity of siRNA in several inbred mouse strains and in experiments using alternate liposomal compositions (Supplementary Fig. 2 online). Innate immune activation by siRNA

Figure 3 |mmunostimulatory activity of siRNA is regulated by distinct sequence motifs. (a,b) IFN-o induction by B-gal control siRNA or its constituent sense (GU-rich) and antisense ssRNA oligonucleotides. Human PBMC were cultured for 24 h with equimolar concentrations of duplexed siRNA or ssRNA oligonucleotides formulated into (a) liposomes or (b) PEI polyplexes. (c) Modified siRNA sequences used in these studies. B-gal control (highly stimulatory), B-gal Mod1 (single base substitution) and B-gal Mod2 (double base substitution). BP1 control (low stimulatory), BP1 Mod1 (single base substitution) and BP1 Mod2 (double base substitution). Base substitutions are underlined. (d,e) Sequence modifications within a putative motif regulate the immunostimulatory activity of sIRNA on human cells. IFN-« induction from human PBMC treated with (d) encapsulated B-gal control or (e) BP1 control series of sequence-modified duplexes. Mean + s.d. triplicate cultures; in vitro data representative of at least three experiments. (f) Sequence modifications to siRNAs regulates the cytokine response /n vivo. IFN-« and IL-6 in mouse serum 6 h after i.v. administration of 50 pg lipid encapsulated siRNA (mean + s.d. of 4 mice). (g,h) Amelioration of systemic toxicities by sequence modification. (g) Platelet and (h) WBC counts in mice 48 h after i.v. administration of encapsulated B-gal control, Mod1 and Mod2 siRNA. B-gal control siRNA caused a significant drop in both platelet and WBC numbers (**P < 0.001 and P < 0.01, respectively; ttest). Although less severe, B-gal Mod1 and Mod2 sequences also caused a drop in WBC counts as compared to PBS-treated animals (*P = 0.03 and 0.06, respectively). See Supplementary Figure 5 for equivalent data for modified BP1 siRNA.

Figure 3 |mmunostimulatory activity of siRNA is regulated by distinct sequence motifs. (a,b) IFN-o induction by B-gal control siRNA or its constituent sense (GU-rich) and antisense ssRNA oligonucleotides. Human PBMC were cultured for 24 h with equimolar concentrations of duplexed siRNA or ssRNA oligonucleotides formulated into (a) liposomes or (b) PEI polyplexes. (c) Modified siRNA sequences used in these studies. B-gal control (highly stimulatory), B-gal Mod1 (single base substitution) and B-gal Mod2 (double base substitution). BP1 control (low stimulatory), BP1 Mod1 (single base substitution) and BP1 Mod2 (double base substitution). Base substitutions are underlined. (d,e) Sequence modifications within a putative motif regulate the immunostimulatory activity of sIRNA on human cells. IFN-« induction from human PBMC treated with (d) encapsulated B-gal control or (e) BP1 control series of sequence-modified duplexes. Mean + s.d. triplicate cultures; in vitro data representative of at least three experiments. (f) Sequence modifications to siRNAs regulates the cytokine response /n vivo. IFN-« and IL-6 in mouse serum 6 h after i.v. administration of 50 pg lipid encapsulated siRNA (mean + s.d. of 4 mice). (g,h) Amelioration of systemic toxicities by sequence modification. (g) Platelet and (h) WBC counts in mice 48 h after i.v. administration of encapsulated B-gal control, Mod1 and Mod2 siRNA. B-gal control siRNA caused a significant drop in both platelet and WBC numbers (**P < 0.001 and P < 0.01, respectively; ttest). Although less severe, B-gal Mod1 and Mod2 sequences also caused a drop in WBC counts as compared to PBS-treated animals (*P = 0.03 and 0.06, respectively). See Supplementary Figure 5 for equivalent data for modified BP1 siRNA.

Plasmacytoid DC are activated by GU-rich ssRNAs through TLR7 and TLR8 located within endosomes*%, and it has been suggested that siRNA duplexes may be a ligand for TLR7 (and presumably TLR8 in humans’) if endocytosed and denatured in situ into their constituent oligonucleotides'. The finding that immunostimulatory activity of an siRNA was attributable to its constituent GU-rich oligonucleotide and not the complementary strand lends support to this hypothesis.

Plasmacytoid DC are activated by GU-rich ssRNAs through TLR7 and TLR8 located within endosomes*%, and it has been suggested that siRNA duplexes may be a ligand for TLR7 (and presumably TLR8 in humans’) if endocytosed and denatured in situ into their constituent oligonucleotides'. The finding that immunostimulatory activity of an siRNA was attributable to its constituent GU-rich oligonucleotide and not the complementary strand lends support to this hypothesis.

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