RNAi and RNAi-based Therapeutics

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Since its discovery in the mid-1990s [45, 46], RNAi has rapidly transformed from a curious phenomenon in worms to an invaluable tool in the study of functional genomics. The significance of RNAi technology was underlined by the award of the 2006 Nobel Prize in Physiology or Medicine to Fire and Mello [47].

RNAi describes the cellular process that occurs in various organisms, including mammals, plants and nematodes, whereby double stranded (ds) RNAs mediate specific and potent gene silencing [48]. RNAi is believed to have evolved from an early immune mechanism against viruses and transposable elements [49]. Foreign dsRNAs are recognised and processed by Dicer RNases into 21-24 nucleotide fragments, known as small interfering RNA (siRNA), then loaded onto an Argonaute-containing RNA-induced silencing complex (RISC). One strand of the siRNA (passenger strand) is degraded, whilst the other (guide strand) in complex with RISC searches cytoplasmic RNA for complementary sequences. Once located, Argonaute triggers cleavage of the targeted RNA, thereby silencing expression of the foreign gene. This RNAi pathway forms an important component of innate antiviral immunity in plants, nematodes, fungi and arthropods [49].

Another RNAi pathway, the endogenous microRNA (miRNA) pathway, enables post-transcriptional regulation of gene expression in animals and plants [50]. This pathway commences with the transcription of ~1,000 nucleotide primary miRNAs (pri-miRNA) from the host genome. These transcripts are typically excised by a microprocessor complex into 65-70 nucleotide precursor miRNAs (pre-miRNA), which are then exported to the cytoplasm by exportin-5 and Ran GTP [51]. Similar to the siRNA pathway, precursor miRNAs are processed by Dicer to form 21-26 nucleotide mature miRNAs, which can be loaded onto RISC, termed miRISC. Again, the passenger strand is degraded whist the guide strand targets RISC to a specific mRNA for silencing. Compared to siRNAs, miRNAs only partially base pair with their target sequences in the 3’-untranslated regions (3’-UTRs) of mRNA, mainly via 7-8 consecutive base pairs of the so-called seed region. Binding of miRISCs to 3’-UTRs inhibits 5’-cap dependent translational initiation and can trigger mRNA degradation. Individual miRNAs usually have several different mRNAs as targets.

To date, 1,872 miRNA sequences have been identified within the human genome (http://microrna.sanger.ac.uk; accessed March 6, 2014) which regulate almost a third of protein-encoding genes [52]. Unsurprisingly, endogenous RNAi plays a critical role in regulating numerous vital processes, including cell growth, cell proliferation, apoptosis and tissue differentiation [53].

  

Cellular RNAi can be exploited to silence a gene of interest by introducing exogenous sxRNA analogues, either siRNA or shRNA, that target its mRNA. SiRNAs are competent for RISC loading, and may directly enter the RNAi pathway once delivered to the cytoplasm (see ‘Non-viral RNAi Delivery’). For shRNAs, viral vectors are typically used to deliver shRNA-encoding genes into cells for expression (see ‘Viral RNAi Delivery’). Expressed shRNA undergoes processing to form siRNA, which can then be loaded onto RISC complexes. Many different sxRNA libraries now exist that cover the entire genomes of both mice and humans, enabling high-throughput loss of function analyses and the identification of essential genes for virtually any cellular process [54].

RNAi technology has played a key role in the identification of many (non-)oncogene-dependent cancers, and the oncogenes on which they rely. One example of this is the identification of the oncogene, I Kappa B Kinase ε (IKBKE), in breast cancers. A large shRNA library targeting 1,200 genes was used to screen the breast cancer cell line, MCF-7, in which three shRNAs targeting IKBKE were able to reduce proliferation and viability of MCF-7 cells, indicating their dependence on IKBKE for maintenance and survival [55]. These findings not only added to the accumulating body of evidence for oncogene dependence in cancers, but also highlighted the therapeutic potential of sxRNA-mediated RNAi against them.

SxRNAs have several advantages as novel therapeutic agents. They are synthetic and relatively easy to produce compared to protein-based therapeutics, can be rationally designed to target any (non-)oncogene, and can achieve a level of specificity far higher than that of traditional cancer therapeutics. Furthermore, as sxRNAs target (non-)oncogenes at the mRNA level, they may exhibit synergistic effects when used with drugs that target at the protein level. For instance, siRNA-mediated silencing of BCR-ABL and the gene encoding multidrug resistance protein 1 has been shown to sensitise CML cells to imatinib treatment [56, 57], highlighting the potential of such combinatorial therapies.

However, several barriers have severely hindered the progress of RNAi therapeutics towards clinical approval, including the short plasma half-lives of RNAs, their poor cellular uptake, and the lack of tumour-specific targeting. To overcome these barriers, various sxRNA delivery systems have been developed, which aim to (1) protect the RNA from nuclease degradation, (2) evade immune surveillance, (3) prevent rapid renal clearance, (4) promote its accumulation in tumour tissues, (5) deliver the RNA to cancer cells in a highly specific manner, and (6) interact with cellular trafficking pathways to deliver the RNA to appropriate locations within the cell.

A diverse range of RNAi delivery systems have been explored, these can generally be categorised as viral or non-viral. Selected examples that have shown the most promise in the laboratory or clinical trials are discussed in detail below, but several others exist [58].