Current prospects for RNA interference-based therapies

Abstract

RNA interference (RNAi) is a powerful approach for reducing expression of endogenously expressed proteins. It is widely used for biological applications and is being harnessed to silence mRNAs encoding pathogenic proteins for therapy. Various methods - including delivering RNA oligonucleotides and expressing RNAi triggers from viral vectors - have been developed for successful RNAi in cell culture and in vivo. Recently, RNAi-based gene silencing approaches have been demonstrated in humans, and ongoing clinical trials hold promise for treating fatal disorders or providing alternatives to traditional small molecule therapies. Here we describe the broad range of approaches to achieve targeted gene silencing for therapy, discuss important considerations when developing RNAi triggers for use in humans, and review the current status of clinical trials.

Figure 1. The miRNA and siRNA pathways of RNAi in mammals.

Primary microRNAs (pri-miRNAs) are transcribed by RNA polymerases^,, and are trimmed by the microprocessor complex (comprising Drosha and microprocessor complex subunit DCGR8) into ~70 nucleotide precursors, called pre-miRNAs^,, (left side of the figure). miRNAs can also be processed from spliced short introns (known as mirtrons). pre-miRNAs contain a loop and usually have interspersed mismatches along the duplex. pre-miRNAs associate with exportin 5 and are exported to the cytoplasm^,, where a complex that contains Dicer, TAR RNA-binding protein (TRBP; also known as TARBP2) and PACT (also known as PRKRA) processes the pre-miRNAs into miRNA–miRNA* duplexes^,,. The duplex associates with an Argonaute (AGO) protein within the precursor RNAi-induced silencing complex (pre-RISC). One strand of the duplex (the passenger strand) is removed. The mature RISC contains the guide strand, which directs the complex to the target mRNA for post-transcriptional gene silencing. The 'seed' region of an miRNA is indicated; in RNAi trigger design, the off-target potential of this sequence needs to be considered. Long dsRNAs (right side of the figure) are processed by Dicer, TRBP and PACT into small interfering RNAs (siRNAs). siRNAs are 20–24-mer RNAs and harbour 3′OH and 5′ phosphate (PO₄) groups, with 3′ dinucleotide overhangs^,,. Within the pre-RISC complex, an AGO protein cleaves the passenger siRNA strand. Then, the mature RISC, containing an AGO protein and the guide strand, associates with the target mRNA for cleavage. The inset shows the properties of siRNAs. The thermodynamic stability of the terminal sequences will direct strand loading. Like naturally occurring or artificially engineered miRNAs, the potential 'seed' region can be a source for miRNA-like off-target silencing. shRNA, short hairpin RNA.

Figure 2. Workflow for testing therapeutic RNAi triggers.

a | Cartoon depicting a luciferase reporter system that is used to confirm that the appropriate strand of small interfering RNAs (siRNAs) or stem–loop platforms from RNAi expression systems is loaded into the RNA-induced silencing complex (RISC). A plasmid with a luciferase reporter that harbours sequences complementary to the guide strand in the 3′ UTR is cotransfected with the RNAi system, and if the appropriate guide strand is loaded, luciferase activity will diminish. When a reporter that contains sequences complementary to the passenger strand is cotransfected, luciferase activity should not be reduced. Because silencing is based on a microRNA (miRNA)-like mechanism, inhibition of luciferase activity will indicate RISC loading, independent of the sequence's ability to induce target cleavage. b | Northern blot analysis can be used to evaluate RNAi triggers expressed from vectors. If the short hairpin RNA (shRNA) or primary miRNA (pri-miRNA) mimics are poorly processed but expressed efficiently, build-up of shRNAs may occur (lane 1). Appropriate processing should yield readily detectable mature, processed siRNAs with minimal levels of unprocessed material (lane 2). Northern blots with probes for the passenger strand can also be used to assess RISC loading of the unintended strand (not shown). c | Small RNA quantitative PCR to quantify the mature product will yield information about overall levels of mature product, which is important to know to understand dosing. The figure shows an example of results obtained from effectively or poorly processed RNA precursors. Cloning and sequencing of the mature small RNAs can be used to assess the silencing RNAs in more detail (not shown). Ct, threshold cycle.