The promises and pitfalls of RNA-interference-based therapeutics

Abstract

The discovery that gene expression can be controlled by the Watson-Crick base-pairing of small RNAs with messenger RNAs containing complementary sequence - a process known as RNA interference - has markedly advanced our understanding of eukaryotic gene regulation and function. The ability of short RNA sequences to modulate gene expression has provided a powerful tool with which to study gene function and is set to revolutionize the treatment of disease. Remarkably, despite being just one decade from its discovery, the phenomenon is already being used therapeutically in human clinical trials, and biotechnology companies that focus on RNA-interference-based therapeutics are already publicly traded.

Figure 1. Mechanisms of cellular gene silencing

a, Primary microRNAs (pri-miRNAs) are, in plants and animals, processed by Drosha and its partner DGCR8 into precursor miRNAs (pre-miRNAs) and then transported to the cytoplasm by exportin 5 (XPO5). In the cytoplasm, they are bound by a Dicer-containing pre-RISC and processed to yield the guide sequence that is loaded into the holo-RISC, which contains all the components required for gene silencing. AGO2 is the catalytic core of the RISC (present but not shown in the schematically drawn holo-RISC). The guide sequence binds to the corresponding target sequences in the 3′ UTRs of cellular mRNAs. If the miRNA guide sequence is fully complementary to its target site (left pathway), it triggers site-specific cleavage and degradation of the mRNA through the catalytic domain of AGO2. If the base-pairing is incomplete (right pathway) but includes pairing of the seed region (nucleotides 2–8 of the miRNA) with the target, translational inhibition occurs, and this can be accompanied by non-sequence-specific degradation of the mRNA in P bodies. b, Similarly to miRNAs, artificially transcribed shRNAs (in this case from a plasmid) are transported to the cytoplasm by XPO5. The dsRNA in the cytoplasm is recognized and processed by Dicer into ~21–25-nucleotide siRNA fragments that are loaded into the RISC. The siRNAs can target complementary sequences of cellular mRNAs and trigger their degradation through AGO2-mediated cleavage. c, When siRNAs are present in the nucleus and are complementary to promoter regions, they can trigger chromatin remodelling and histone modifications that result in transcriptional gene silencing. In mammalian cells, the details of this mechanism are still under investigation but are known to include Argonaute-family proteins. Accessory proteins indicated in the figure are TRBP (HIV tar-RNA-binding protein; also known as TRBP2P) and PACT (activator of protein kinase PKR; also known as PRKRA). m⁷G, 7-methylguanosine.

Figure 2. In vivo delivery strategies for therapeutic siRNAs

a, Cholesterol groups can be linked to modified siRNAs to enhance their stability before systemic delivery. The most common siRNA modifications are 2′-O-methyluridine or 2′-fluorouridine substitutions (blue circles) combined with phosphorothioate linkages. b, Polycation nanoparticles can direct delivery of the siRNAs to specific cells through the use of surface ligands (such as transferrin) that bind to receptors on target cells. c, SNALPs encapsulate modified siRNAs into cationic or neutral lipid bilayers coated with diffusible PEG–lipid conjugates. SNALPs allow siRNAs to be taken up by cells and released by endosomes. d, Masked endosomolytic agent (MEA)–Dynamic PolyConjugates (DPCs) are similar to SNALPS but smaller, and contain a ligand that allows targeted cell delivery. The release of the siRNA from the endosome is also improved by the inclusion of a pH-labile bond in the MEA–DPC particles. e, Tagging specific antibodies with protamine or other positive charges allows the delivery of siRNAs to specific cell types via receptor-mediated uptake. f, Chemically linking or co-transcribing siRNAs with RNA aptamers allows the targeted delivery of the siRNAs to cells expressing the appropriate receptor.