Short interfering RNA (siRNA) is used in RNA interference technology to avoid non-target-related induction of type I interferon (IFN) typical for long double-stranded RNA. Here we show that in plasmacytoid dendritic cells (PDC), an immune cell subset specialized in the detection of viral nucleic acids and production of type I IFN, some siRNA sequences, independent of their GU content, are potent stimuli of IFN-alpha production. Localization of the immunostimulatory motif on the sense strand of a potent IFN-alpha-inducing siRNA allowed dissection of immunostimulation and target silencing. Injection into mice of immunostimulatory siRNA, when complexed with cationic liposomes, induced systemic immune responses in the same range as the TLR9 ligand CpG, including IFN-alpha in serum and activation of T cells and dendritic cells in spleen. Immunostimulation by siRNA was absent in TLR7-deficient mice. Thus sequence-specific TLR7-dependent immune recognition in PDC needs to be considered as an additional biological activity of siRNA, which then should be termed immunostimulatory RNA (isRNA).

Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7.

The discovery of RNA interference (RNAi) may well be one of the transforming events in biology in the past decade. RNAi can result in gene silencing or even in the expulsion of sequences from the genome. Harnessed as an experimental tool, RNAi has revolutionized approaches to decoding gene function. It also has the potential to be exploited therapeutically, and clinical trials to test this possibility are already being planned.

Unlocking the potential of the human genome with RNA interference

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.

http://arbutusbio.com/docs/Protiva_Judge_et_al_2005.pdf

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

Since the first description of RNA interference (RNAi) in animals less than a decade ago, there has been rapid progress towards its use as a therapeutic modality against human diseases. Advances in our understanding of the mechanisms of RNAi and studies of RNAi in vivo indicate that RNAi-based therapies might soon provide a powerful new arsenal against pathogens and diseases for which treatment options are currently limited. Recent findings have highlighted both promise and challenges in using RNAi for therapeutic applications. Design and delivery strategies for RNAi effector molecules must be carefully considered to address safety concerns and to ensure effective, successful treatment of human diseases.

Strategies for silencing human disease using RNA interference.

Delivery of small interfering RNAs (siRNAs) into cells is a key obstacle to their therapeutic application. We designed a protamine-antibody fusion protein to deliver siRNA to HIV-infected or envelope-transfected cells. The fusion protein (F105-P) was designed with the protamine coding sequence linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope. Additionally, siRNAs targeted against the HIV-1 capsid gene gag, inhibited HIV replication in hard-to-transfect, HIV-infected primary T cells. Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelope-negative B16 cells; injection of F105-P with siRNAs targeting c-myc, MDM2 and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. This study demonstrates the potential for systemic, cell-type specific, antibody-mediated siRNA delivery.

Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors

RNA interference (RNAi) is a powerful tool used to manipulate gene expression or determine gene function1,2. One technique of expressing the short double-stranded (ds) RNA intermediates required for interference in mammalian systems is the introduction of short-interfering (si) RNAs3,4,5,6. Although RNAi strategies are reliant on a high degree of specificity, little attention has been given to the potential non-specific effects that might be induced. Here, we found that transfection of siRNAs results in interferon (IFN)-mediated activation of the Jak–Stat pathway and global upregulation of IFN-stimulated genes. This effect is mediated by the dsRNA-dependent protein kinase, PKR, which is activated by 21-base-pair (bp) siRNAs and required for upregulation of IFN-β in response to siRNAs. In addition, we show by using cell lines deficient in specific components mediating IFN action that the RNAi mechanism itself is independent of the interferon system. Thus, siRNAs have broad and complicating effects beyond the selective silencing of target genes when introduced into cells. This is of critical importance, as siRNAs are currently being explored for their potential therapeutic use7,8.

Activation of the interferon system by short-interfering RNAs

RNA interference in biology and disease

RNAi (RNA interference) has become a powerful tool to determine gene function. Different methods of expressing the short ds (double-stranded) RNA intermediates required for interference in mammalian systems have been developed, including the introduction of si (short interfering) RNAs by direct transfection or driven from transfected plasmids or lentiviral vectors encoding sh (short hairpin) RNAs. Although RNAi relies upon a high degree of specificity, recent findings suggest that off-target non-specific effects can be encountered. We found that transfection of siRNAs can results in an interferon-mediated activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway and global up-regulation of interferon-stimulated genes. This effect is mediated in part by the dsRNA-dependent protein kinase PKR, as this kinase is activated by the 21 bp siRNA, and is required in response to the siRNAs. However, the transcription factor IRF3 (interferon-regulatory factor 3) is also activated by siRNA as a primary response, resulting in the stimulation of genes independent of an interferon response. In cells deficient in IRF3, this response is blunted, but can be restored by re-introduction of IRF3. Thus siRNAs induce complex signalling responses in target cells, leading to effects beyond the selective silencing of specific genes.

RNA interference and double-stranded-RNA-activated pathways

RNA interference (RNAi) is an exciting technology with applications in basic research, in elucidation and validation of drug targets, and as a direct therapeutic. In mammalian settings, it is based on the introduction or expression of small interfering RNA (siRNA) that guide the cleavage of a complementary target messenger RNA. While siRNA certainly directs specific silencing of genes in mammalian cells, longer RNA typically used to silence genes in other organisms potently activate mammalian cell defence mechanisms leading to a non-specific halt in translation, to activation of transcription and often, to cell death. Recent research has revealed that siRNA in certain settings can also activate these RNA-responsive pathways. With the recent advances in RNAi technology and its first forays into the in vivo setting now coming to light, it is pertinent to review the cellular response to ribonucleic acids typically used in RNAi methods.

Detection of foreign RNA: implications for RNAi.

Extract: Over the past 5 years, the phenomenon of RNA interference (RNAi) has progressed from being considered a mysterious response to double-stranded RNA (dsRNA) in the nematode C. elegans to the latest potential therapeutic tool to silence gene expression in certain diseases such as cancer or viral infections. However, the application of this biological phenomenon as a powerful research tool is not as clear-cut as scientists had initially hoped. dsRNA, the initiator of RNAi, is not made as a result of normal cellular processes, but is considered a warning to the cell of potential danger. Foreign dsRNA molecules can be formed through the transcription of potentially harmful transposons that contain inverted repeat sequences, as well as through the replication process of the majority of viruses during an infection. In organisms, such as the aforementioned nematode C. elegans and Drosophila, that respond to dsRNA-containing challenges through RNAi, the foreign dsRNA is recognized and cleaved into 21-23 nucleotide short interfering (si) RNAs. The siRNA is then incorporated into the RNA Induced Silencing Complex, which targets the mRNA of homologous sequence for degradation.

Carol A Sledz

Papers

Activation of the interferon system by short-interfering RNAs

RNA interference in biology and disease

RNA interference and double-stranded-RNA-activated pathways

Detection of foreign RNA: implications for RNAi.

RNA interference and interferon.