Gene silencing below the immune radar

Abstract

In vertebrates, the detection of viral nucleic acids is the first step toward innate and subsequent adaptive antiviral immune responses. A sophisticated,protein receptor-based sensor system has evolved to recognize viral nucleic acids and to trigger a variety of antiviral defense mechanisms. The more we learn about this elaborate sensor system, the more it becomes evident how difficult it is to introduce exogenous nucleic acids such as siRNA into cells without triggering antiviral immunoreceptors. In this issue of the JCI, Judge and colleagues provide evidence that siRNA can be designed and delivered in a way that allows specific and successful silencing of target genes in tumor cells in vivo, leading to tumor cell death and prolonged survival of tumor-bearing mice in the absence of immune activation. This study represents a major technological advance, setting new standards for well-controlled siRNA applications in vivo, and has the potential to guide clinical development toward siRNA therapeutics with well-defined and selective gene-silencing activities.

Figure 1. Antiviral and gene regulatory function of RNAi.

Long, viral dsRNA in the cytoplasm is cleaved to short, viral dsRNA (siRNA) by Dicer. MicroRNA (miRNA), a class of noncoding RNA molecules involved in gene regulation, is generated by Drosha-mediated cleavage from pri-miRNA transcribed in the nucleus, exported by Exportin-5 to the cytoplasm, and then cleaved by Dicer, resulting in siRNA. Similarly to physiological pri-miRNA, shRNA introduced to cells via gene transfer enters the same pathway (Drosha, Dicer), resulting in the release of the corresponding siRNA. siRNA produced by both pathways binds to RNA-induced silencing complex (RISC), and the sense strand is released from the RISC complex. Complementary binding of the antisense strand to target sequences guides RISC to cleave the target sequence (resulting in mRNA degradation) or, in the absence of full complementarity to the target sequence, inhibits its translation.

Figure 2. Immunorecognition of RNA.

RNA delivered to the endosome is detected by TLR3, in the case of long dsRNA or its mimic poly(I:C) and short dsRNA, or by TLR7, in the case of siRNA and single-stranded RNA (ssRNA). RNA delivered to the cytosol can be recognized by cytosolic helicases. The RNA helicase RIG-I detects RNA carrying a triphosphate group at the 5′ end (5′-PPP) and possibly blunt-end short RNA and intermediate dsRNA. Melanoma differentiation-associated protein-5 (MDA-5) detects long dsRNA. Long dsRNA also binds to RNA-binding PKR, but activation of IRF3 and IRF7 and subsequent type I IFN induction by PKR is controversial (as indicated by question mark). TLRs signal via TIR domain–containing adapter inducing IFN-β (TRIF) (in the case of TLR3) to activate IRF3 and NF-κB or via MyD88 (in the case of TLR7) to activate IRF7 and NF-κB. RIG-I and MDA-5 signal via IFN-β promoter stimulator-1 (IPS-1) to induce IRF7 and NF-κB. Depending on the cell type and its receptor expression pattern (e.g., ECs, myeloid DCs), recognition of RNA leads to the production of type I IFNs (e.g., IFN-α/β), and of proinflammatory cytokines or may lead to the modulation of cell-specific functions. In the study reported by Judge et al. (18) in this issue of the JCI, immunorecognition of siRNA is avoided by chemical modification and by the specific mode of delivery.