Further insight into the properties that render RNA molecules stimulatory to the immune system is driven by siRNA technology. Based on studies by Tuschl and colleagues (Elbashir et al. 2001), siRNA is now used worldwide as a robust tool for target-specific gene silencing in cell lines and human primary cells. However, depending on the mode of synthesis and the sequences used to generate siRNA, also nonspecific, so-called nonspecific off-target effects of siRNAs were observed.
To overcome limitations with the transfection of synthetic siRNA, vector-based (e.g., lentiviral) expression systems for the introduction of short hairpin siRNAs (shRNA) mimicking siRNAs were developed (Brummelkamp et al. 2002; Harborth et al. 2003; Paddison et al. 2002). The most commonly used shRNA expression system consists of a RNA-polymerase III dependent promoter driving the expression of two complementary 19- to 29-bp RNA sequences linked by a short loop of 4-10 nt. The resulting transcript is exported to the cytoplasm and processed by dicer. Lentiviral vectors haboring the Pol III-shRNA expression cassette (Li et al. 2003; Rubinson et al. 2003; Tiscornia et al. 2003) allow RNAi-mediated gene silencing via siRNA in cells that are otherwise difficult to transfect.
Sequence specificity of gene silencing by such shRNA was questioned by Bridge et al. (Bridge et al. 2003), who demonstrated that infection of human lung fibroblasts with Pol III-shRNA containing lentivirus directed against the gene MORF4L1 not only silenced MORF4L1 but also stimulated interferon-inducible genes such as 2',5'-OAS, an indicator of type I interferon. The IFN-inducing effect was dependent on the sequence and the dose of the vector; seven of 23 shRNAs targeting different genes exhibited IFN induction. In contrast, transfection of synthetic siRNA with the same putative IFN-inducing sequences led to sequence-specific silencing without triggering an IFN response. Northern blot analysis of shRNA showed that the majority of shRNA transcripts were correctly processed to 20 nt transcripts. The authors speculated that remaining unprocessed transcripts could be detected by cytosolic RNA sensing receptors. In the follow-up paper, the group of Iggo (Pebernard and Iggo 2004), further correlated the U6 promoter sequence with OAS induction. This study revealed that the region between -2 (the end of the promoter) and +2 (the start of RNA transcript) is crucial for the immune stimulatory effect, which was lost when they used the endogenous human sequence (CCGA). Further mutations leading to a partial mismatch in the shRNA (predicted to create a 14-bp duplex) suggested that stimulation required more than a 14-bp duplex.
William's group (Sledz et al. 2003) described the induction of IFN target genes by transfection of synthetic siRNAs into a human glioblastoma cell line (T98G) or a renal carcinoma cell line (RCC). When comparing the two studies from Bridge and colleagues and from Sledz and colleagues, it is important to note that different cell lines (Bridge, human lung fibroblasts; Sledz, RCC and T98G) and different ways of siRNA generation (Bridge, synthetic siRNAs and shRNAs; Sledz, synthetic siRNA and T7-phage-polymerase siRNA) were used.
Using mouse embryonic fibroblasts (MEFs) with different gene deficiencies related to the IFN response system, Sledz et al. proposed that PKR was the interferon-inducing receptor for siRNAs. Later, the same group postulated a different siRNA receptor (RIG-I, see below) in T98G cells (Marques et al. 2006). Of note, in the two studies published by Bridge et al. (2003) and Sledz et al. (2003), type I interferon was not analyzed at the protein level.
Kariko et al. (2004a) suggested that TLR3 was responsible for the induction of type I IFN by siRNA. These data are based on keratinocyte (HaCaT) and HEK 293 cells, which responded to synthetic siRNA but not to the single-stranded components (ssRNA) by secretion of low amounts of IFN-P that was comparable to stimulation with poly I:C. Overexpression of TLR3 in HEK 293 cells resulted in fourfold higher induction of type I IFN secretion in response to transfected siRNA. However, overexpression of NF-KB-inducing receptors such as TLR3 may also contribute indirectly to the enhanced type I IFN response induced by siRNA, for example by upregulating IFN-inducible cytosolic RNA receptors. For example, TLR3 overexpressing HEK 293 cells secrete more IL-8 than empty vector or TLR9 overexpressing HEK 293 cells (Kariko et al. 2005); consequently, such studies do not necessarily provide evidence for a direct interaction between siRNA and TLR3.
Kim et al. (2004) showed that the induction of type I IFN by siRNA depended on the use of T7-RNA polymerase (T7 RNAP) for siRNA generation. In contrast to Bridge et al. (2003) and Sledz et al. (2003), in the study by Kim and colleagues, type I IFN was measured at the protein level, which is less sensitive than measuring IFN-dependent responses on the transcriptional level and thus underscores the magnitude of the IFN response they reported. In their study, Kim and colleagues examined siRNAs targeting the early ICP4 gene of HSV-1. Only T7 RNAP-derived transcripts but not synthetic siRNA elicited a potent antiviral activity when transfected into HEK 293 cells. The same antiviral activity was observed by transfection of T7 transcripts with unrelated sequences. Analysis of supernatants revealed the presence of substantial amounts of IFN-a and IFN-P protein. These results were reproduced in HeLa cells, as well as K562, CEM, and Jurkat cells. It is well known that unlike capped mammalian mRNA, the 5' ends of T7 transcripts harbor a triphosphate GTP-nucleotide. Treatment of T7 transcripts with RNase T1 (with the 5' end p-GGG removed, which was single-stranded in their case) and alkaline phosphatase was sufficient to completely abrogate interferon-inducing activity. Additional experiments using T3 and Sp6 phage RNA polymerases demonstrated similar induction of type I IFN. The examination of multiple cell lines by Kim et al. (2004) pointed to a powerful ubiquitously expressed sensor for short triphosphate RNA.
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