siRNAs: a new wave of RNA-based therapeutics

Glen A. Coburn and Bryan R. Cullen*

Howard Hughes Medical Institute, Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27701, USA

Keywords: antiviral, RNA, interference


    Introducing silence
 Top
 Introducing silence
 RNAi-mediated inhibition of...
 Conclusions and future...
 References
 
In 1998, Fire et al.1 made the startling discovery that double-stranded RNA (dsRNA) could induce a potent silencing effect on homologous genes in the nematode Caenorhabditis elegans. This technique, termed RNA interference (RNAi), has proved to be a powerful tool with which to dissect gene function in plants, C. elegans and Drosophila. Although RNAi is evolutionarily conserved among plants and animals, silencing of specific genes in mammalian cells has been difficult because of the induction of the interferon response by dsRNAs of >=30 nt.2,3 This non-specific response to dsRNA leads to global changes in cellular gene expression and apoptosis, masking any specific silencing effect by RNAi in mammalian cells. Recently, however, it was shown that potent and specific gene silencing could be achieved in human cells transfected with small interfering RNAs (siRNA) of 21–23 nt, a key intermediate in the RNAi pathway.4 This landmark discovery by Tuschl and co-workers has led to routine RNA interference in mammalian cell culture and may have finally opened the door to tractable genetic analysis in mammalian cells.

Current models of RNAi suggest that the pathway involves a five-step process (for an excellent review see reference 2). First, dsRNAs (>26 bp) are cleaved by an RNase III-like enzyme, termed Dicer, to generate 21–23 nt fragments (step 1).57 These short duplexed siRNAs have two unusual properties that are critical for their function: they contain 2 nt 3'-overhangs and 5'-phosphate groups.8 It is presumed that an ATP-dependent RNA helicase recognizes these short duplexes and resolves the siRNA duplex into two single-stranded RNAs (step 2).9 One strand is then incorporated into a high-molecular-weight protein complex termed RISC (RNA-induced silencing complex) (step 3),9 where it serves as guide RNA to direct the cleavage of homologous RNA sequences by an as yet unidentified endonucleolytic component of RISC (step 4).8,10 Finally, RISC is liberated from the cleaved mRNA and is recycled to perform multiple rounds of catalysis (step 5). This property of RISC is responsible for the potent nature of the silencing effect.

An intriguing facet of the RNAi pathway is the tight link between RNAi and developmental timing in C. elegans.11 The enzyme Dicer is required for both induction of RNAi and processing of two small temporal RNAs (stRNAs), lin-4 and let-7 from ~70 nt structured precursor RNAs.6,7 Interestingly, the 21 nt single-stranded stRNAs, also referred to as microRNAs (miRNAs), are identical in length to siRNAs; however, unlike siRNAs that trigger mRNA degradation, stRNAs bind to multiple sites within the 3' UTRs of target mRNAs and inhibit their translation by a mechanism that remains poorly understood.12 The finding that repressed mRNAs are associated with polysomes suggests that translation initiation is not affected.12

Additional observations have further strengthened the connection between the RNAi and miRNA pathways. Purification of miRNP complexes from HeLa extracts led to the isolation of >40 unique miRNAs.13 The miRNP complex isolated from human cells is the same approximate size as Drosophila RISC (~500 kDa), and one member of the complex, eIF2C2, an orthologue of Argonaute-2, is a known component of Drosophila RISC.14 Secondly, and more importantly, the recent discovery that the endogenous human let-7 miRNA not only directs mRNA cleavage but also co-purifies with human RISC,15 implies that RISC mediates both silencing processes. The choice of RISC action is probably dependent on the degree of complementarity between the guide RNA and the target sequence, with mRNA cleavage activity requiring almost 100% complete base pairing between the siRNA and target sequence.15 Moreover, the finding that at least one plant miRNA can function as an siRNA, directing the cleavage of an endogenous mRNA in Arabidopsis,16 provides a clear demonstration that at least some miRNAs can enter the RNAi pathway. The discovery of this new class of RNA, with >200 distinct miRNAs now catalogued in nematodes,17,18 fruit flies,19 humans13 and plants,16,20 suggests that miRNAs and RNAi may play a more general role in regulating gene expression than hitherto imagined.


    RNAi-mediated inhibition of viral replication
 Top
 Introducing silence
 RNAi-mediated inhibition of...
 Conclusions and future...
 References
 
In plants, it has long been recognized that post-transcriptional gene silencing and RNAi play critical roles in genome surveillance, protecting the cell from inappropriate expression of repetitive sequences, transposable elements and viruses.21 In fact, certain plant viruses, including potyviruses, potato virus X and cucumber mosaic virus, have evolved proteins that antagonize the RNAi pathway, providing some of the strongest evidence that RNAi can serve as an innate cellular antiviral mechanism (reviewed in 21 and references therein). The finding that an animal virus, flock house virus (a small, positive-strand RNA virus, belonging to the Novaviridae, that infects insect cells and is morphologically similar to some plant viruses), also encodes an RNAi antagonist (the B2 protein) and is subject to RNAi in Drosophila in the absence of B2 expression,22 clearly demonstrates that the antiviral properties of the RNAi pathway are conserved between the plant and animal kingdoms. The potential ability to tap into this native antiviral pathway, as a therapeutic strategy to target viruses and viral gene expression, has generated great excitement among many researchers. Several initial studies, which test the potential application of synthetic siRNAs as antiviral agents, have shown significant promise. To date, RNAi has been used effectively to inhibit the replication of several different pathogenic viruses in culture, including: RSV (respiratory syncytial virus),23 poliovirus24 and HIV-1.2527 In the case of HIV-1, several specific mRNAs have been successfully targeted for siRNA-mediated silencing, including those that encode Gag, Pol, Vif and the small regulatory proteins Tat and Rev. These studies show that RNAi can effectively trigger the degradation of not only viral mRNAs, but also genomic RNAs at both the pre- and post-integration stages of the viral lifecycle.2527 In addition to targeting viruses directly, alternative strategies have employed siRNAs that silence the expression of essential host factors including Tsg101, required for vacuolar sorting and efficient budding of HIV-1 progeny,28 and the chemokine receptor CCR5, required as a co-receptor for HIV-1 cell entry.29

One impediment to utilizing RNAi technology for therapeutic benefit in humans remains the development of efficient delivery systems for siRNAs. Previous methods relied on harsh lipid-based transfection reagents to introduce siRNAs into cells in culture and are either inefficient and/or unsuitable for use in animals. An additional caveat is that, unlike in plants and lower eukaryotes, RNAi-mediated gene silencing is not long lasting in mammals. In cell culture, gene silencing effects can disappear within three to four generations, with the targeted protein returning to normal levels quickly thereafter.4 An enormous step forward in addressing these complications is the finding that natural or designed siRNAs and miRNAs can be expressed in vivo.3033 Short hairpin and pre-miRNA-based precursor RNAs can be transcribed from either RNA polymerase III (H1 or U6)30,32 or RNA polymerase II promoters33 in vivo, and processed by Dicer to release functional siRNAs. Importantly, siRNAs expressed from DNA templates can silence gene expression as effectively as exogenously introduced synthetic siRNAs.30,31,33 As an extension of these studies, many groups have begun constructing the first generation of retroviral-,34 adenoviral-35 and lentiviral-based36 gene therapy vectors that are capable of expressing siRNAs in a stable manner in virtually any cell and tissue type. Already progress has been made, with the demonstration that murine retroviral vectors expressing siRNAs directed against a mutant allele of the human K-Ras proto-oncogene have the ability to reverse tumorigenicity in mice.34 The further development of these delivery systems will push siRNA technology quickly from the ‘proof of principle’ phase into animal studies of important human diseases.


    Conclusions and future challenges
 Top
 Introducing silence
 RNAi-mediated inhibition of...
 Conclusions and future...
 References
 
It is striking how rapidly the field has moved from an initial discovery phase to a stage where implementation of siRNA technology in a therapeutic setting appears not only foreseeable, but imminent. Currently, however, our understanding of the biological mechanisms underlying RNAi lags behind the movement to apply this technology to human diseases such as cancer and infectious diseases such as HIV-1 and hepatitis C virus. Clearly the objectives, in the short term, are to improve viral delivery systems with the goal of maximizing siRNA expression. Presumably, this would involve optimization of promoters and siRNA precursor design. At present, information pertaining to endogenous miRNA promoter usage and important sequence and RNA structural constraints for Dicer processing are entirely lacking. A better understanding of the fundamental biochemistry of the RNAi pathway would certainly lead to improved target site selection and better overall siRNA design.

The appearance of a resistant strain of poliovirus in cultures treated with polio-specific siRNAs24 is a strong reminder that a single nucleotide mutation could render viruses immune to a single specific siRNA. Although it was concluded that the mutation was already present in the primary viral isolate, emergence of siRNA resistance is a major concern that will need to be addressed, particularly for viruses encoding error-prone polymerases such as HIV-1. Thus, in future, siRNA expression cassettes will require a higher degree of sophistication, probably encoding tandem arrays of highly expressed siRNAs that target several conserved viral sequences simultaneously.

Much remains to be accomplished, but RNAi will continue to play an important role in determining cellular gene function and shows a great deal of promise as a therapeutic agent. The next few years of research will indicate whether RNAi technology will realize its potential as the ‘next wave of therapeutic molecules’.3


    Footnotes
 
* Corresponding author. Tel: +1-919-684-3369; Fax: +1-919-681-8979; E-mail: culle002{at}mc.duke.edu Back


    References
 Top
 Introducing silence
 RNAi-mediated inhibition of...
 Conclusions and future...
 References
 
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