John Wiley & Sons, Ltd.

Current Protocols in Nucleic Acid Chemistry 16.1.1-16.1.10, March 2009

Published online March 2009 in Wiley Interscience (www.interscience.wiley.com).

DOI: 10.1002/0471142700.nc1601s36

Copyright © 2009 John Wiley & Sons, Inc.

UNIT 16.1

Overview of Gene Silencing by RNA Interference

Daniel H. Kim1 and John J. Rossi2

1Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston, Massachusetts

2Graduate School of Biological Sciences and Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California

ABSTRACT

The potential of harnessing RNA interference (RNAi) for sequence-specific gene silencing has generated much excitement and progress in the field. Recent advances in RNAi technology suggest that RNAi-based approaches may soon become an effective therapeutic strategy against a myriad of diseases. This overview provides a brief description of important considerations when designing an RNAi-based method for gene silencing and therapeutic development: (a) mechanistic aspects of RNAi-mediated gene silencing in mammalian cells; (b) structural requirements for potent siRNA duplexes; (c) off-target effects and interferon responses; and (d) effective delivery of RNAi-inducing agents. Promising therapeutic applications of RNAi that are currently in the developmental pipeline are also described. Curr. Protoc. Nucleic Acid Chem. 36:16.1.1-16.1.10. © 2009 by John Wiley & Sons, Inc.

Keywords: RNAi • siRNA • microRNA • shRNA • transcriptional gene silencing

INTRODUCTION

Discovery of the mechanistic aspects of RNA interference (RNAi) has progressed rapidly since the phenomenon was first described in C. elegans (Fire et al., 1998). Three years after the initial observation of RNAi in worms, double-stranded small interfering RNAs (siRNAs) of ~21 nt in length were shown to be the effector molecules that trigger RNAi in mammalian cells (Elbashir et al., 2001a). This finding and other subsequent studies in mammalian cells opened up possibilities for using RNAi-mediated gene silencing against various human diseases (Kim and Rossi, 2007). The use of RNAi is a particularly advantageous therapeutic approach, given the sequence specificity and potency in knocking down gene expression of target genes whose sequences are known. Testing the efficacy of an RNAi-based approach is also amenable to a shorter turnaround time in drug development, and even the gene products of newly emerging foreign pathogens can readily be targeted once their genomes are sequenced. These potential applications of RNAi across a broad spectrum of diseases have accelerated the development of RNAi-based treatments.

The demonstrated effectiveness of RNAi-based approaches in silencing gene expression has led to the initiation of several clinical trials. Among the first diseases that have been targeted by RNAi in the clinic are wet, age-related macular degeneration (AMD; McFarland et al., 2004; Check, 2005) and respiratory syncytial virus (RSV) infection (Bitko et al., 2005). RNAi-based preclinical studies are also being done for various cancers (Pai et al., 2006), neurodegenerative disorders (Raoul et al., 2006), and additional viral diseases (Dykxhoorn and Lieberman, 2006; Rossi, 2006). These advances in the preclinical development of RNAi-based therapies have resulted from studies demonstrating the enhanced potency and/or longevity of diverse RNAi effector molecules. Unraveling the mechanistic aspects of RNAi pathways has yielded greater insights into the design of RNA structures that effectively trigger gene silencing. Various siRNAs and short hairpin RNAs (shRNAs; Amarzguioui et al., 2005) have been constructed to optimize the levels of silencing for a given type of gene and related disease. However, issues of concern that have also arisen from studies of RNAi include the induction of type 1 interferon responses (de Veer et al., 2005) and the saturation of key components in the RNAi pathway (Grimm et al., 2006). Additionally, the challenge of delivery to a specific tissue or cell type has been an important issue to resolve. New studies suggest that these hurdles are beginning to be overcome through innovative technological approaches to using RNAi (Behlke, 2006).

RNAi MECHANISMS

siRNA-Mediated Post-Transcriptional Gene Silencing

Small RNAs are the guide molecules for directing sequence-specific gene silencing. siRNAs mediate cleavage of mRNA targets with which they exhibit full complementarity (Zamore et al., 2000). Along with microRNAs (miRNAs), siRNAs effect post-transcriptional gene silencing (PTGS) predominantly in the cytoplasm of mammalian cells. The 3′ ends of siRNAs contain a 2-nt overhang, characteristic of double-stranded RNAs processed by the RNase III enzyme Dicer (Bernstein et al., 2001). In PTGS, siRNAs load into an effector complex known as the RNA-induced silencing complex (RISC). One strand of the siRNA in particular, referred to as the antisense or guide strand, targets the corresponding mRNA for degradation (Martinez et al., 2002). The other strand, known as the sense or passenger strand, is destroyed (Matranga et al., 2005; Rand et al., 2005) when cleaved by the RNase H–like PIWI domain of the endonuclease Argonaute 2 (AGO2; Liu et al., 2004). AGO2 is part of the Argonaute family of proteins that includes four members of the AGO subfamily and four additional members of the PIWI subfamily (Carmell et al., 2002), which are expressed predominantly in the germline. Of the ubiquitously expressed AGO subfamily members, only AGO2 appears to exhibit “slicer” endonuclease activity (Liu et al., 2004), although AGO3 also contains the DDH “slicer” motif in its catalytic PIWI domain. AGO proteins and Dicer also possess a PIWI-Argonaute-Zwille (PAZ) domain, which recognizes the 2-nt 3′ overhang structures of siRNAs and miRNAs (Ma et al., 2004). Along with the HIV-1 TAR RNA-binding protein (TRBP), AGO2 and Dicer comprise RISC (Preall and Sontheimer, 2005), which can direct multiple rounds of mRNA “slicing” (Hutvagner and Zamore, 2002), taking place between the 10th and 11th base relative to the 5′ end of the siRNA guide strand (Elbashir et al., 2001b).

siRNA-Mediated Transcriptional Gene Silencing

RNAi-mediated gene silencing has also been observed in the nucleus of mammalian cells in a process known as transcriptional gene silencing (TGS; Zaratiegui et al., 2007). This nuclear pathway has been characterized in fission yeast, plants, flies, and worms, and recent work has begun to reveal mechanistic insights in mammals as well. siRNAs with sequence complementarity to gene promoter regions serve to trigger TGS in mammalian cells by directing epigenetic modifications to targeted regions of the genome (Morris et al., 2004; Ting et al., 2005; Kim et al., 2006; Weinberg et al., 2006; Han et al., 2007). In particular, studies of siRNA-directed TGS in mammalian cells have shown that promoter sequences proximal to transcription start sites (TSS) are amenable to silencing. Most findings have reported targeting a promoter region within 200 bp of the TSS, suggesting that this region is especially well-suited to targeting by siRNAs. Additionally, shRNAs have been shown to mediate TGS of endogenous gene expression (Castanotto et al., 2005; Kim et al., 2007). Promoter-targeting small RNAs load into an AGO-containing complex (Janowski et al., 2006; Kim et al., 2006) and repress RNA polymerase II activity through directed epigenetic changes (Fig. 16.1.1).

Figure 16.1.1 Overview of mechanisms for transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). AGO1 and AGO2, Argonaute 1 and 2; K9me2, lysine 9 dimethylation; K27me3, lysine 27 trimethylation; TRBP, TAR RNA-binding protein.

TGS in mammalian cells induces epigenetic modifications such as histone and DNA methylation, although some findings indicate that transcriptional repression can occur in the absence of both silencing marks (Janowski et al., 2005, 2006). Inhibitors of DNA methyltransferase (DNMT) activity, such as 5-azacytidine, attenuate TGS (Morris et al., 2004), indicating a potential role for DNMTs, and, in particular, de novo DNMT3a (Weinberg et al., 2006). In plants, this phenomenon is referred to as RNA-directed DNA methylation (RdDM) and involves the AGO4 protein (Zilberman et al., 2003). siRNA-directed TGS also induces changes in the local chromatin structure, leading to the formation of facultative heterochromatin, such as that found on the inactive X chromosome (Zaratiegui et al., 2007). As evidenced by the induction of histone H3 lysine 9 dimethylation (H3K9me2; Ting et al., 2005; Kim et al., 2006; Weinberg et al., 2006; Han et al., 2007) and lysine 27 trimethylation (H3K27me3; Kim et al., 2006; Weinberg et al., 2006; Han et al., 2007), siRNAs silence transcription by effecting changes in the histone code (Jenuwein and Allis, 2001). Histone deacetylase inhibitors such as trichostatin A reverse the effects of siRNA-mediated TGS (Morris et al., 2004), further supporting the role of siRNAs in chromatin remodeling and gene...