Novel RNA-based strategies for therapeutic gene silencing

Abstract

The past decade has seen intense scientific interest in non-coding RNAs. In particular, the discovery and subsequent exploitation of gene silencing via RNA interference (RNAi) has revolutionized the way in which gene expression is now studied and understood. It is now well established that post-transcriptional gene silencing (PTGS) by the microRNA (miRNA) and other RNAi-associated pathways represents an essential layer of complexity to gene regulation. Gene silencing using RNAi additionally demonstrates huge potential as a therapeutic strategy for eliminating pathogenic gene expression. Yet despite the early promise and excitement of gene-specific silencing, several critical hurdles remain to be overcome before widespread clinical adoption. These include off-target effects, toxicity due to saturation of the endogenous RNAi functions, limited duration of silencing, and effective targeted delivery. In recent years, a range of novel strategies for producing RNA-mediated silencing have been developed that can circumvent many of these hurdles, including small internally segmented interfering RNAs, tandem hairpin RNAs, and pri-miRNA cluster mimics. This review discusses RNA-mediated silencing in light of this recent research, and highlights the benefits and limitations conferred by these novel gene-silencing strategies.

Figure 1

Endogenous and synthetic methods of RNA induced gene silencing. (a) In the canonical mammalian miRNA pathway, pri-miRNA transcripts are processed by the microprocessor complex into pre-miRNA hairpins that are recognized by exportin-5 for nuclear export. Further processing by Dicer, recruitment into RISC and strand selection result in a mature antisense capable of directing translational repression of imperfectly matched targets, or cleavage of perfectly matched targets. (b) The commonly employed synthetic RNAi effectors (siRNAs, shRNAs, and pri-miRNA mimics) imitate different precursors of the endogenous miRNA pathway. (c) Novel “second-generation” approaches improve on existing strategies. Asymmetric interfering RNAs, small internally segmented interfering RNAs, and RNA-DNA chimeric duplexes reduce off-target effects of siRNAs by limiting sense strand activity and/or off-target effects of antisense seed regions. Trans-kingdom shRNAs and tRNA-shRNAs limit exportin-5-mediated toxicity of shRNAs by using alternate delivery mechanisms of shRNAs to the cytosol. Tandem siRNAs, tandem hairpin RNAs, and miRNA cluster mimics can be used when targeting multiple sites in the same gene or when simultaneously targeting multiple targets is desired. RNAi effectors may be additionally used to induce transcriptional gene silencing that offers the ability to induce long-lasting silencing through epigenetic changes with minimal repeated delivery.

Figure 2

Problems associated with synthetic RNAi approaches. (a) DNA-based approaches offer long-term induction of RNAi, whereas siRNAs are short-lived effectors requiring repeat delivery. (b) Promoter and/or viral specificity can allow spatially restricted control of DNA-encoded approaches to areas not readily accessible by siRNAs due to restricted access. (c) DNA-based approaches can compete with endogenous miRNAs for different components of the miRNA pathway, including exportin-5-mediated nuclear export, leading to associated toxicities. (d) In addition to desired silencing of targets, off-target effects can result from antisense or sense strands binding nonspecifically to other mRNAs.