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

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

Single‐stranded small interfering RNA are more immunostimulatory than their double‐stranded counterparts: A central role for 2′‐hydroxyl uridines in immune responses

“…The observed effects were cell type dependent, thus suggesting the involvement of cellular factors that are differentially expressed by mammalian cells. In this respect, we [19] and others [20,21] have recently demonstrated that PKR and TLR3 do not represent the major mechanisms by which chemically synthesized siRNA activate innate immunity. Moreover, we found that single-stranded (ss) siRNA (sense or antisense strands) and ds siRNA separately could activate innate immunity genes.…”

“…In both cases, internalization of RNA and endosomal maturation is required for immune stimulation, suggesting the involvement of endosomal TLR, in particular TLR7 and TLR8. Indeed, inhibitors of endosomal maturation, such as bafilomycin, abrogated immune activation [19,21]. The data reported by Hornung et al [20] and Judge et al [21] suggest that immunostimulatory motifs are equally recognized within the context of either ss siRNA or siRNA duplexes because the magnitude of cytokines and IFN responses was comparable for both molecules.…”

“…Indeed, inhibitors of endosomal maturation, such as bafilomycin, abrogated immune activation [19,21]. The data reported by Hornung et al [20] and Judge et al [21] suggest that immunostimulatory motifs are equally recognized within the context of either ss siRNA or siRNA duplexes because the magnitude of cytokines and IFN responses was comparable for both molecules. In contrast, we have found that ss siRNA were more potent than siRNA duplexes [19].…”

Approches degene silencingpour le traitement de la maladie de Huntington

“…Cela a déjà été observé dans les organes périphériques, mais reste peu présent dans le système nerveux central du fait de la protection du cerveau par la barrière hémato-encéphalique [36]. Cependant, une quantité anormale de matériel génétique dans le cerveau peut conduire à l'expression par les neurones de protéines reconnues par le système immunitaire, et donc à une destruction des cellules traitées par l'organisme [37].…”

RNAi therapeutics: SNALPing siRNAs in vivo

“…[7][8][9][10] Morrissey et al 4 investigated whether or not SNALP delivered, nonbackbone-modified siRNAs were immunostimulatory, and sure enough they were. The fact that the unmodified siRNAs were immunostimulatory indicates that they are subject to endosomal trafficking, where the RNA-activated Toll-like receptors 3 and 7 reside.…”