Within the past two decades we have become increasingly aware of the roles that RNAs play in regulation of gene expression. The RNA world was given a booster shot with the discovery of RNA interference (RNAi), a compendium of mechanisms involving small RNAs (less than 30 bases long) that regulate the expression of genes in a variety of eukaryotic organisms. Rapid progress in our understanding of RNAi-based mechanisms has led to applications of this powerful process in studies of gene function as well as in therapeutic applications for the treatment of disease. RNAi-based therapies involve two-dimensional drug designs using only identification of good Watson-Crick base pairing between the RNAi guide strand and the target, thereby resulting in rapid design and testing of RNAi triggers. To date there are several clinical trials using RNAi, and we should expect the list of new applications to grow at a phenomenal rate. This article summarizes our current knowledge about the mechanisms and applications of RNAi.

Introduction

RNA interference (RNAi) is a regulatory mechanism of most eukaryotic cells that uses small double-stranded RNA (dsRNA) molecules as triggers to direct homology-dependent control of gene activity ((Figure 1)) (1). Known as small interfering RNAs (siRNA), these ∼21–22 bp long dsRNA molecules have characteristic 2 nt 3′ overhangs that allow them to be recognized by the enzymatic machinery of RNAi, which eventually leads to homology-dependent degradation of the target mRNA. In mammalian cells, siRNAs are produced from cleavage of longer dsRNA precursors by the RNase III endonuclease Dicer (2), or they can be synthesized by chemical or biochemical methods. Dicer is complexed with RNA-binding proteins, the TAR-RNA-binding protein (TRBP), PACT, and Ago-2, which are involved in the hand-off of siRNAs to the RNA-induced silencing complex (RISC) (3). The core components of RISC are the Argonaute (Ago) family members. In humans there are eight members of this family but only Ago-2 possesses an active catalytic domain for cleavage activity (4,5). While siRNAs loaded into RISC are double-stranded, Ago-2 cleaves and releases the “passenger” strand, leading to an activated form of RISC with a single-stranded “guide” RNA molecule that directs the specificity of the target recognition by intermolecular base pairing (6). Rules that govern selectivity of strand loading into RISC are based on differential thermodynamic stabilities of the ends of the siRNAs (7,8). The less thermodynamically stable end is favored for binding to the PIWI domain of Ago-2.

Figure 1.

MicroRNAs

An important arm of RNAi involves the microRNAs (miRNAs). These are endogenous duplexes that posttranscriptionally regulate gene expression by complexing with RISC and binding to the 3′ untranslated regions (UTRs) of target sequences via short stretches of homology, termed “seed sequences” (9,10). The primary mechanism of action of miRNAs is translational repression, although this can be accompanied by message degradation (11). The miRNA duplexes possess incomplete Watson-Crick base pairing, and the antisense strand cannot be chosen by cleavage of the passenger strand as it is for siRNAs; therefore the antisense strand must be chosen by an alternative mechanism (12,13,14). miRNAs are endogenous substrates for the RNAi machinery. They are initially expressed as long primary transcripts (pri-miRNAs), which are processed within the nucleus into 60–70 bp hairpins by the Microprocessor complex, consisting of Drosha and DGCR8 (15,16) into pre-miRNAs. The pre-miRNAs are further processed in the cytoplasm by Dicer and one of the two strands is loaded into RISC, presumably via interaction with one of the Dicer accessory proteins (3). Importantly, it is possible to exploit this native gene silencing pathway for regulation of gene(s) of choice. If the siRNA effector is delivered to the cell it will “activate” RISC, resulting in potent and specific silencing of the targeted mRNA. Because of the potency and selectivity of RNAi, it has become the methodology of choice for silencing specific gene expression in mammalian cells.

RNAi as a Therapeutic Approach for Treatment of Disease

Control of disease-associated genes makes RNAi an attractive choice for future therapeutics. Basically every human disease caused by activity from one or a few genes should be amenable to RNAi-based intervention. This list includes cancer, autoimmune diseases, dominant genetic disorders, and viral infections. RNAi can be triggered by two different pathways: (i) an RNA-based approach where synthetic effector siRNAs are delivered by various carriers to target cells as preformed 21 base duplexes; or (ii) via DNA-based strategies in which the siRNA effectors are produced by intracellular processing of longer RNA hairpin transcripts (reviewed in References 17, and 18). The latter approach is primarily based on nuclear synthesis of short-hairpin RNAs (shRNAs), which are transported to the cytoplasm via the miRNA export pathway and are processed into siRNAs by Dicer. While direct use of synthetic siRNA effectors is simple and usually results in potent gene silencing, the effect is transient. DNA-based RNAi drugs, on the other hand, have the potential of being stably introduced when used in a gene therapy setting, allowing, in principle, a single treatment of viral vector-delivered shRNA genes.