Rna interference: applications in vertebrates (2003)

One of the most unexpected findings in recent years has been the discovery of RNA interference (RNAi). The story started with paradoxical results from manipulations of model organisms such as plants and worms. For example, addition of supernumerary copies of a pigment gene to a petunia, designed to increase the color intensity in the flower, in fact had the opposite of the desired effect—flowers became less colorful instead of more so [1de Lang P. van Blokland R. Kooter J.M. Mol J.N. Suppression of flavonoid flower pigmentation genes in Petunia hybrida by the introduction of antisense and sense genes.Curr. Top Microbiol. Immunol. 1995; 197: 57-75PubMed Google Scholar]. In the worm C. elegans, attempts to modulated genes with DNA antisense were at most partially successful, but a control using double-stranded RNA yielded very efficient suppression of gene expression [2Fire A. RNA-triggered gene silencing.Trends Genet. 1999; 15: 358-363Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar]. Follow-up experiments established that double-stranded RNA molecules could somehow program the selective degradation of sequence-matched mRNAs (for recent reviews, see [3Sharp P.A. RNA interference-2001.Genes and Dev. 2001; 15: 485-490Crossref PubMed Scopus (653) Google Scholar, 4Marx J. Interfering with gene expression.Science. 2000; 288: 1370-1372Crossref PubMed Scopus (38) Google Scholar, 5Hutvagner G. Zamore P.D. RNAi nature abhors a double-strand.Curr. Opin. Genet. Dev. 2002; 12: 225-232Crossref PubMed Scopus (420) Google Scholar]). The mechanistic picture has been considerably clarified by beautiful studies in vivo and in vitro in a host of laboratories, mostly working with model organisms. Long double-stranded RNAs, presumably derived from genes of invaders such as viruses or transposons, are initially cleaved into roughly 20bp RNA duplexes by the dicer nuclease, a member of the RNAaseIII family of nucleases [6Bernstein E. Caudy A.A. Hammond S.M. Hannon G.J. Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature. 2001; 409: 363-366Crossref PubMed Scopus (3704) Google Scholar]. These “silencing inducing RNAs” (siRNAs) are then recruited into the “RNA-induced silencing complex” (RISC), which has been found to contain dicer and the cellular proteins related to the EIF2C1 and/or EIF2C2 translation initiation factors [6Bernstein E. Caudy A.A. Hammond S.M. Hannon G.J. Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature. 2001; 409: 363-366Crossref PubMed Scopus (3704) Google Scholar, 7Hammond S.M. Boettcher S. Caudy A.A. Kobayashi R. Hannon G.J. Argonaute2, a link between genetic and biochemical analyses of RNAi.Science. 2001; 293: 1146-1150Crossref PubMed Scopus (1158) Google Scholar, 8Martinez J. Patkaniowska A. Urlaub H. Luhrmann R. Tuschl T. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi.Cell. 2002; 110: 563-574Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar]. The double stranded RNAs are melted by cellular helicases, freeing the message-complementary strand to bind the targeted mRNA. This complex then binds the targeted mRNA and directs its degradation. At first it seemed doubtful that RNA interference would operate in vertebrates. Double-stranded RNA has long been known to induce the interferon response in vertebrate cells [9Isaacs A. Lindenmann J. The Interferon.Proc. R. Soc. Lond. (Biol). 1957; 147: 258-267Crossref PubMed Google Scholar]. Cells respond to dsRNA by activating the PKR, RNAaseL, and Mx pathways, which close down cellular protein synthesis. This blocks viral replication and protects neighboring cells from infection. Production of interferon is also induced, which primes cells to induce the above response upon detection of virus-derived double-stranded RNA. A crucial insight came from Thomas Tuschl and coworkers, who found that the nature of the cellular response could be controlled by the length of the double stranded RNA used as inducer. Upon transfection into cultured mammalian cells, long dsRNAs (hundreds of nucleotides) induced the interferon response, but short dsRNAs (roughly 20 bp) induced RNAi only [10Elbashir S.M. et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature. 2001; 411: 494-498Crossref PubMed Scopus (8018) Google Scholar]. This ignited an explosion of work on RNAi in mammalian cells. A technical trick struck on by many groups has made gene therapy by RNAi convenient. For many applications, it may be complicated to introduce short dsRNAs directly into cells. However, many groups have now shown that appropriately designed DNA molecules containing inverted repeat sequences can be transcribed into RNA molecules that form RNA hairpins. If the sequences are chosen correctly, these are processed by the dicer nuclease to form siRNAs (e.g., [11Brummelkamp T.R. Bernards R. Agami R. A system for stable expression of short interfering RNAs in mammalian cells.Science. 2002; 296: 550-553Crossref PubMed Scopus (3928) Google Scholar, 12Zeng Y. Wagner E.J. Cullen B.R. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells.Mol. Cell. 2002; 9: 1327-1333Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar, 13Jacque J.M. Triques K. Stevenson M. Modulation of HIV-1 replication by RNA interference.Nature. 2002; 418: 435-438Crossref PubMed Scopus (763) Google Scholar]). Thus all the methods derived for delivering genes into cells can in principle be used to deliver siRNAs as well. One potentially promising application of RNAi therapy is the prevention and treatment of viral infection. The development of methods for inducing RNAi in cultured vertebrate cells was quickly followed by a series of papers documenting RNAi-based inhibition of poliovirus, oncoretroviruses, and HIV [13Jacque J.M. Triques K. Stevenson M. Modulation of HIV-1 replication by RNA interference.Nature. 2002; 418: 435-438Crossref PubMed Scopus (763) Google Scholar, 14Hu W.Y. Meyers C.P. Kilzer J.M. Pfaff S.L. Bushman F.D. Inhibition of retroviral pathogenesis by RNA interference.Curr. Biol. 2002; 12 ([2002]): 1301-1311Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 15Novina C.D. et al.siRNA-directed inhibition of HIV-1 infection.Nat. Med. 2002; 8: 681-686Crossref PubMed Scopus (731) Google Scholar, 16Gitlin L. Karelsky S. Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells.Nature. 2002; 418: 430-434Crossref PubMed Scopus (516) Google Scholar, 17Coburn G.A. Cullen B.R. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference.J. Virol. 2002; 76: 9225-9231Crossref PubMed Scopus (381) Google Scholar]. Studies of avian retroviruses revealed that viral pathogenesis could even be inhibited by RNAi in an intact chick embryo [14Hu W.Y. Meyers C.P. Kilzer J.M. Pfaff S.L. Bushman F.D. Inhibition of retroviral pathogenesis by RNA interference.Curr. Biol. 2002; 12 ([2002]): 1301-1311Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar]. In the poliovirus study, low-level poliovirus replication could take place in the presence of the RNAi response, allowing selection of mutant viruses that could grow more efficiently. Sequence analysis revealed that these showed changes in the viral genome in the sequences complementary to the region targeted by the added siRNA [16Gitlin L. Karelsky S. Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells.Nature. 2002; 418: 430-434Crossref PubMed Scopus (516) Google Scholar]. Thus, altering the viral target site conferred resistance, confirming an important aspect of the proposed mechanism. This experiment and others bear on a further issue. Remarkably, in plants and worms, RNAi can actually be shown to spread between cells [2Fire A. RNA-triggered gene silencing.Trends Genet. 1999; 15: 358-363Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, 4Marx J. Interfering with gene expression.Science. 2000; 288: 1370-1372Crossref PubMed Scopus (38) Google Scholar, 5Hutvagner G. Zamore P.D. RNAi nature abhors a double-strand.Curr. Opin. Genet. Dev. 2002; 12: 225-232Crossref PubMed Scopus (420) Google Scholar]. In plants, it is possible to induce RNAi in plant tissue, then graft on a new branch and show the RNAi effect spread into the graft. In worms, it is possible to feed dsRNA to the animals and observe the spread of the RNAi response from gut cells to the whole animal. The RNAi effect can even be heritable in the worm model [18Grishok A. Tabara H. Mello C.C. Genetic requirements for inheritance of RNAi in C. elegans.Science. 2000; 287: 2494-2497Crossref PubMed Scopus (355) Google Scholar]. It seems so far, however, that this spreading effect is not seen in vertebrate models. Spreading in plants and worms is thought to require action of RNA-dependent RNA polymerase (RdRP). The siRNA strand that binds the viral RNA target can become extended by RdRP, producing more double stranded RNA that then provides further substrates for the dicer nuclease [19Sijen T. et al.On the role of RNA amplification in dsRNA-triggered gene silencing.Cell. 2001; 107: 465-476Abstract Full Text Full Text PDF PubMed Scopus (999) Google Scholar]. Recruitment of these new siRNAs by further RISC complexes then widens the RNAi response to further target RNA sequences. This amplification correlates with the spreading of the RNAi effect between cells. RdRP, while present in plants and worms, appears to be absent from the vertebrate genome sequences. In the poliovirus experiment, if the initial RNAi strand mediating inhibition could also act as a primer for extension by RdRP, then a single point mutation should not have conferred escape, because other viral sequences would have yielded siRNAs. The fact that a point mutation allowed the virus to escape suppression argues against amplification and spreading in the animal cells studied. The lack of spreading may well limit the applications of RNAi to human disease. It would be great if treatment of patients with a double-stranded RNA 20-mer could induce an enduring systemic effect, but this hope does not seem to be realistic. Rather, double-stranded RNA will probably need to be delivered directly to its site of action in vertebrate cells, for example by encapsulating double- stranded RNA in liposomes or by delivery of siRNA-encoding DNAs using a gene therapy approach. Oddly, despite the intense studies of RNAi, it is still unclear whether RNAi is normally a system of defense against viral infection in vertebrates. It is clear that RNAi controls viral infection in plants [20Voinnet O. Pinto Y.M. Baulcombe D.C. Suppression of gene silencing a general strategy used by diverse DNA and RNA viruses of plants.Proc. Natl. Acad. Sci. USA. 1999; 96: 14147-14152Crossref PubMed Scopus (818) Google Scholar, 21Voinnet O. Lederer C. Baulcombe D.C. A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana.Cell. 2000; 103: 157-167Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 22Thomas C.L. Jones L. Baulcombe D.C. Maule A.J. Size constraints for targeting post-transcriptional gene silencing and for RNA-directed methylation in Nicotiana benthamiana using a potato virus X vector.Plant J. 2001; 25: 417-425Crossref PubMed Google Scholar]. For a number of viruses, mutation of the RNAi system can be shown to increase the severity of viral pathogenesis. In addition, there are genes of plant viruses that inactivate the RNAi response, and these promote replication of the virus in its host. Vertebrate viruses devote many genes to the battle with the host interferon and adaptive immune systems, but no genes are yet known in vertebrate viruses that antagonize the RNAi system. So why, then, should vertebrates bother to encode the RNAi system at all? If it is not for defense against pathogens, what does it do? Again studies in model organisms provide a likely answer. In worms, parts of the RNAi machinery are important not just for suppressing replication of transposons but also for forming small RNAs—microRNAs (miRNAs) or small temporal RNAs (stRNAs)—that are important for developmental gene control. Analysis of the populations of small RNAs in vertebrate cells reveal many probable miRNAs, and analysis of sequenced vertebrate genomes reveals many likely precursor genes. Thus, parts of the RNAi machinery appear to play a dual role in worms—control of pathogens and control of development—but possibly only the function devoted to developmental gene control is conserved in vertebrates [23Pasquinelli A.E. MicroRNAs deviants no longer.Trends Genet. 2002; 18: 171-173Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 24Lagos-Quintana M. Rauhut R. Lendeckel W. Tuschl T. Identification of novel genes coding for small expressed RNAs.Science. 2001; 294: 853-858Crossref PubMed Scopus (3856) Google Scholar, 25Hutvagner G. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.Science. 2001; 293: 834-838Crossref PubMed Scopus (2130) Google Scholar]. However, even within these constraints it is possible to foresee quite promising applications of RNAi-based biotechnology in vertebrates. RNAi should be quite handy for validation of candidate drug targets, since the loss-of- function phenotype of a gene can be induced using RNAi and analyzed prior to developing small molecule inhibitors. Hairpin DNAs encoding siRNAs should be useful in diverse forms of gene therapy. Use of an encoded RNA as an effector molecule in gene therapy instead of a protein should have a further advantage. The immune response against newly introduced proteins is often a problem in gene therapy, and this may be minimized by introducing an effector RNA molecule instead. Perhaps most promising is the prospect of engineering plants and animals of agricultral importance for resistance to pathogens. Could installing an appropriate RNAi construct in the germ line of a farm animal render it insensitive to a viral disease? For example, could RNAi contructs targeting foot and mouth disease virus render cows insensitive to infection? According to what we now know, this seems quite plausible. Five years from now perhaps all farm animals will harbor diverse RNAi-inducing DNA constructs in their germ lines that render them insensitive to agriculturally important viral diseases.