RNA interference: from gene silencing to gene-specific therapeutics

Abstract

In the past 4 years, RNA interference (RNAi) has become widely used as an experimental tool to analyse the function of mammalian genes, both in vitro and in vivo. By harnessing an evolutionary conserved endogenous biological pathway, first identified in plants and lower organisms, double-stranded RNA (dsRNA) reagents are used to bind to and promote the degradation of target RNAs, resulting in knockdown of the expression of specific genes. RNAi can be induced in mammalian cells by the introduction of synthetic double-stranded small interfering RNAs (siRNAs) 21-23 base pairs (bp) in length or by plasmid and viral vector systems that express double-stranded short hairpin RNAs (shRNAs) that are subsequently processed to siRNAs by the cellular machinery. RNAi has been widely used in mammalian cells to define the functional roles of individual genes, particularly in disease. In addition, siRNA and shRNA libraries have been developed to allow the systematic analysis of genes required for disease processes such as cancer using high throughput RNAi screens. RNAi has been used for the knockdown of gene expression in experimental animals, with the development of shRNA systems that allow tissue-specific and inducible knockdown of genes promising to provide a quicker and cheaper way to generate transgenic animals than conventional approaches. Finally, because of the ability of RNAi to silence disease-associated genes in tissue culture and animal models, the development of RNAi-based reagents for clinical applications is gathering pace, as technological enhancements that improve siRNA stability and delivery in vivo, while minimising off-target and nonspecific effects, are developed.

Fig. 1

Mechanisms of siRNA-mediated gene silencing. Long (> 30 bp) dsRNA (e.g., viral), vector-based shRNAs, and endogenous miRNAs are processed by the Dicer complex to form 21–23 bp siRNA duplexes with symmetric 3′-overhangs and 5′-phosphate groups. The duplexes then associate with RISC, which contains a helicase that unwinds the duplex. Each siRNA strand incorporated RISC is then presented to the cytoplasmic mRNA pool, where it associates with its complementary mRNA strand and, depending on its complementarity, results in either targeted cleavage of the mRNA or translational arrest. In theory, the antisense strand of the siRNA duplex will target the desirable mRNA for destruction, whereas the RISC that incorporates the sense strand can potentially associate with other unintended mRNAs and cause off-target activities. More details on the mechanism can be found at the following website: http://www.nature.com/nature/focus/rnai/index.html.

Fig. 2

The design of an effective siRNA molecule. Modified from Mittal (2004).

Fig. 3

Tandem and hairpin-type expression vectors for RNAi. In tandem-type vectors, the sense and antisense strands are expressed by separate polymerase III promoters and the strands anneal inside the cells to form duplex siRNA molecules. In the hairpin-type vectors, sense and antisense strands are connected by a loop and are expressed as a single molecule, which rapidly forms a hairpin structure with a stem and a loop that are processed to siRNA by Dicer.

Fig. 4

Strategies for the delivery of RNAi molecules in vivo.