Therapeutic Potential of RNA Interference
http://www.100md.com
《新英格兰医药杂志》
Just when scientists thought they had figured out the fundamental mechanisms through which gene expression is regulated, studies of the nematode Caenorhabditis elegans1 revealed the existence of a pathway, now known as RNA interference (RNAi), that silences gene expression by promoting degradation of RNA. Scientists have discovered ways to control RNAi in order to regulate gene expression in a variety of biologic systems, and they are researching ways to harness RNAi to interrupt disease processes such as those caused by human immunodeficiency virus type 1 (HIV-1), hepatitis viruses, and influenzavirus.
Discovery of RNAi
Events leading up to the discovery of RNAi illustrate the importance of following up on the results of control experiments. Guo and Kemphues2 were using antisense RNA to inhibit gene expression (antisense RNAs or DNAs pair up with their target messenger RNA [mRNA] through sequence complementarity to inhibit the translation of mRNA). They found, unexpectedly, that the sense RNA controls suppressed gene expression as effectively as the antisense RNA. Several years later, Fire and colleagues1 discovered that double-stranded RNA (dsRNA) mixtures were substantially more potent than sense or antisense single strands at silencing gene expression in worms. They called this mechanism RNA interference.
Since only a few molecules of dsRNA were needed to effect gene silencing, Fire and colleagues proposed that a catalytic or amplification component may be involved. Also, the discovery that dsRNA could cause the silencing of homologous genes helped reconcile observations of this phenomenon (called cosuppression) in transgenic plants: the genes were silenced by homologous transgenic sequences, and transgenic plants were subsequently resistant to viral infection.3,4,5 It is now clear that RNAi, cosuppression, and virus-induced interference are all triggered by a mechanism that involves a dsRNA intermediate. Thus, dsRNA provides the trigger for the activation of RNAi, the silencing is sequence-specific (i.e., only genes homologous to the dsRNA trigger are silenced), and silencing involves the degradation of RNA encoded by the targeted gene.6,7
Mechanism of Gene Silencing
RNAi, which can cause the degradation of virtually any RNA, is a simple mechanism (Figure 1). Long dsRNA is processed to short interfering RNAs (siRNAs) by the action of a dsRNA-specific endonuclease known as Dicer.11,12 The resultant siRNAs are 21 to 24 nucleotides in length, are double-stranded, and have 3' overhangs of 2 nucleotides.
Figure 1. Mechanism of Gene Silencing by RNA Interference.
The pathway of RNA interference can be broken down into two main phases. In the first phase, long double-stranded RNA (dsRNA) is recognized and processed by Dicer, an RNase III enzyme, into duplexes of short interfering RNA (siRNA) of 21 to 24 nucleotides (nt) in length. Exogenous synthetic siRNAs or endogenous expressed siRNAs can also be incorporated into the RNA-induced silencing complex (RISC), thereby bypassing the requirement for dsRNA processing by Dicer. In the second phase, siRNAs are incorporated into the multiprotein RISC. A helicase in RISC unwinds the duplex siRNA, which then pairs by means of its unwound antisense strand to messenger RNAs (mRNAs) that bear a high degree of sequence complementarity to the siRNA. An as yet unidentified RNase (Slicer) within RISC proceeds to degrade the mRNA at sites not bound by the siRNA. Cleavage of the target mRNA begins at a single site 10 nucleotides upstream of the 5'-most residue of the siRNA–target mRNA duplex.8 Although the composition of RISC is not completely known, it includes members of the Argonaute family9 that have been implicated in processes directing post-transcriptional silencing. ADP denotes adenosine diphosphate, Pi inorganic phosphate, P phosphate, and OH hydroxyl. The figure was adapted from Stevenson.10
These siRNAs are incorporated into a nuclease complex known as the RNA-induced silencing complex (RISC). ATP-dependent unwinding of the siRNAs activates RISC. RISC is directed by the unwound antisense siRNA strand to homologous target RNAs, which undergo endonucleolytic cleavage. Within RISC, mRNA cutting is affected by Slicer (the actual identity of Slicer is not yet known). There is some evidence that Dicer, in addition to processing long dsRNA to siRNAs, has additional roles in RNAi, since the efficiency of RNAi in mammalian cells is markedly impaired when Dicer itself is also silenced.13
(Glossary)
There are two Dicers (DCR-1 and DCR-2) in drosophila. In drosophila eggs lacking functional DCR-2, there is an impaired response of RNAi to synthetic siRNAs. Since use of synthetic siRNAs bypasses the dsRNA-processing activity of Dicer, this implies an additional role for Dicer, perhaps in the execution phase of RNAi.14 An important advance in the RNAi field was the demonstration that synthetic 21-nucleotide duplexes with the same structure as Dicer-generated siRNAs could also be incorporated into RISC and induce the degradation of homologous target RNA.15
The complementarity between the antisense strand of the unwound siRNA and the target RNA governs the sequence-specificity of gene silencing, a specificity that parallels the specificity governing the antibody–antigen interaction. Indeed, RNAi probably served as an ancient defense mechanism against mobile genetic elements such as RNA viruses and transposons. Research is continually revealing roles for RNAi in basic cellular processes, including gene regulation and the formation of heterochromatin.16 Vestiges of a role for RNAi as an antiviral defense mechanism may still be at work, as indicated by viruses that appear to have evolved strategies to counteract RNAi.
In many regards, RNAi has already exceeded expectations for its application to the study of biologic processes. Experiments involving gene knockouts, which were previously in the domain of model systems such as rodents, flies, worms, and zebrafish, can now be carried out on almost any cell type. RNAi is also a powerful tool for validating the targets for therapeutic drugs.17 For example, the ability of HIV-1 to detach physically from the surface of the infected cell requires the activity of a cellular cofactor called tumor suppressor gene 101 (Tsg101).17,18 Virus detachment and hence dissemination of the virus to other cells are blocked when Tsg101 expression is inhibited by RNAi.17 This provides the rationale for the development of small-molecule inhibitors of Tsg101 as potential drugs for patients with the acquired immunodeficiency syndrome (AIDS). Some cellular proteins can oppose HIV-1 infection, and RNAi showed that TRIM5 (tripartite motif 5 alpha) is one such factor that restricts HIV-1 infection of cells from certain monkey species.19
Therapeutic Applications
The therapeutic applications of RNAi are potentially enormous. The genetic etiology of many disorders has now been defined and, in some cases, has been targeted by RNAi in in vitro and in vivo model systems. Because the specificity of RNAi is governed by sequence complementarity between the siRNA and the target RNA, the most obvious application would be to treat diseases in which genetic polymorphisms within the disease-inducing gene in a particular lesion or tumor can be targeted for degradation without affecting RNA from wild-type alleles.
Oncogenesis
For example, RNAi is being explored as a way to inhibit the expression of genes involved in oncogenesis. The translocation of the Philadelphia chromosome (Ph) generates a fusion gene called BCR-ABL. The translation product of this gene creates a constitutively active protein tyrosine kinase that induces and maintains leukemic transformation in chronic myelogenous leukemia and Ph-positive acute lymphoblastic leukemia. The siRNAs specific for the BCR-ABL transcript have been shown to silence the oncogenic fusion transcripts without affecting expression levels of normal c-ABL and c-BCR transcripts.20,21
Pancreatic and colon carcinomas, in which RAS genes are often mutated, provide another example. In many cases, the RAS oncogenes contain point mutations that differ by a single-base mutation from their normal counterparts. The use of retroviral vectors to introduce interfering RNAs specific for an oncogenic variant of K-RAS (called K-RASV12) reduces the level of K-RASV12 transcripts and effects a loss of anchorage-independent growth and tumorigenicity.22,23 Studies of this kind provide proof-of-concept for RNAi-based strategies aimed at reversing tumorigenesis. A major factor confounding cancer treatment is resistance to chemotherapeutic agents. The siRNAs have been used to decrease the drug resistance of cells in vitro by inhibiting the expression of MDR1, a multidrug transporter with a major role in multidrug resistance.24
Viral Hepatitis
Acute liver failure and subacute liver failure, induced by viral hepatitis, are associated with high mortality rates. In animal models of liver failure, liver toxins and viral hepatitis lead to apoptosis of hepatocytes through cell-death receptors such as Fas. The siRNAs targeted to Fas RNA and delivered by high-pressure injection into the tails of mice were able to reach the liver, inhibit Fas expression, and protect mice from hepatitis.25 Similarly, siRNAs that target CASP8 RNA (encoding caspase 8) prevent acute liver failure induced by Fas activation.26
Infectious Diseases
Perhaps the most promising applications of RNAi are in the treatment of infectious diseases. In these cases, the RNA targets are exogenous and, as such, can be inhibited without affecting cellular functions. Pathogens of major importance to human health worldwide include HIV, influenzaviruses, hepatitis B and C viruses, poliovirus, papillomaviruses, herpesviruses, and West Nile virus. Protozoan parasites cause diseases that profoundly affect human health, and some species exhibit RNAi.27
An alternative strategy to targeting the RNA of the pathogen directly would be to target transcripts of cellular cofactors on which the invasion or pathogenicity of the agent depends (e.g., specific cell-surface receptors). A variety of studies have shown that RNAi can directly target viral transcripts28,29,30 or, in the case of viruses with RNA-based genomes, the viral chromosome itself.31,32 The ability to target the viral RNA chromosome after it enters the cell provides an opportunity to prevent the virus from establishing residence, thereby sterilizing the cell even after infection.
Some studies of HIV suggest that incoming viral RNA can be targeted by RNAi,31,32 whereas others indicate that it is resistant to RNAi.33,34 Perhaps this discrepancy exists because the studies targeted siRNAs to different regions of the viral genome. The viral nucleocapsid of the Rous sarcoma virus shields the incoming viral RNA from RNAi-mediated degradation. Likewise, there may be regions of the HIV genome that are differentially shielded from RNAi by a nucleocapsid.35
In addition to shielding of the viral chromosome by viral proteins, viruses may have evolved other ways to avoid RNAi.36 Infection of drosophila cells by a flock house virus requires the suppression of RNAi by a flock house virus–encoded protein, B2. This defense against silencing seems to have been evolutionarily conserved, since the B2 protein also inhibits silencing in transgenic plants.37 Furthermore, evidence now exists that vaccinia virus and human influenza A, B, and C viruses also encode a protein that inhibits silencing in drosophila and in plants.38,39,40 This supports the notion that RNA silencing is an antiviral defense and that viruses have evolved to counteract it.
Drug resistance is a major obstacle in the treatment of many infectious agents. However, the ability to target RNAi to multiple regions of the viral genome may overcome this obstacle. Similarly, targeting viruses with multiple interfering RNAs reduces the chances of a virus escaping RNAi repression through spontaneous mutation. One might envision a "salvage therapy" role for RNAi that could be used to inhibit viruses that are broadly resistant to conventional antiviral drugs. However, one must bear in mind that RNAi may be saturable41 and that simultaneous introduction of siRNAs with different specificities into cells can reduce the potency of the silencing effect more than siRNAs introduced individually.42 This is likely to be an important issue for future RNAi-based therapeutic strategies against viruses that, because of mutation rate, rapidly overcome drug or immune-mediated pressure.
Prospects for Therapy
The silencing effect of RNAi is highly specific and potent and requires only that the sequence of the target RNA be known. Obstacles must be overcome before RNAi can live up to its potential as a therapeutic method. Experiences with two other RNA-based therapeutic strategies, antisense RNA and ribozymes, illustrate the daunting obstacles that stand in the way of the therapeutic application of RNAi. Ribozymes are RNA molecules with a particular sequence and structure that cause cleavage of target RNA. No ribozyme has been approved for clinical use, and only one antisense agent, the DNA antisense drug fomivirsen (Vitravene, Isis Pharmaceuticals), has been approved by the Food and Drug Administration, for the treatment of cytomegalovirus retinitis in patients with AIDS.43 The RNA, whether the ribozyme, antisense, or siRNA, must interact with its target RNA inside the cell (in the case of RNAi, the mechanism of action is in the cytoplasm)44 and must remain in a biologically active form until it reaches its target. Therefore, the RNA must be stable and nontoxic, cross cell membranes, and not negatively affect the health of the organism. On the basis of the track record of ribozymes and antisense RNA, enthusiasm for RNAi-based therapeutics is, in reality, tempered.
Obstacles to Therapy
Delivery is probably the single biggest obstacle to the development of RNAi-based therapeutic agents. Trigger RNAs (dsRNAs from which siRNAs are derived by the action of Dicer) can be expressed from vectors or delivered as artificial siRNAs. A variety of strategies to express interfering RNAs with the use of plasmid and virus vector-based cassettes have been explored.7,37 Well-documented hazards of inserting foreign vector sequences into chromosomal DNA include insertional activation and inactivation of cellular genes.
Direct (e.g., intravenous) administration of siRNAs would require siRNAs that are modified to be resistant to nucleases and perhaps conjugated with a ligand to target the siRNA to specific tissues. In mice, intravenous introduction of Fas siRNAs leads to specific silencing of Fas mRNA in the liver,25 so in principle, unmodified siRNAs can be taken up by the liver and perhaps other tissues. It is not clear, however, whether there are selective tissue sites for the uptake of siRNAs and whether the lymphoid system or the brain, for instance, is accessible by this route. Furthermore, the silencing effect of siRNAs is short-lived, because the siRNAs eventually decay within the cell.
In addition to the danger of using vectors that integrate into the genome, the expression or injection of siRNAs may also have untoward biologic effects. Researchers are continually finding new cellular processes in which RNAi is involved. Therefore, a stoichiometric excess of a virus-specific siRNA, for example, could saturate RNAi and interrupt the pathway's normal functions in the cell.
Interferons, which form part of the host's defense against viral infection, are activated by long dsRNA (more than 500 bp). It is now apparent that siRNAs45 as well as short hairpin RNAs (short sequences of RNA that make tight hairpin turns and can be used to silence gene expression) expressed from DNA vectors46 can trigger the activation of interferons. However, there is no evidence that the activation of interferons by short RNAs influences the degree or specificity of RNA silencing. In addition, these effects have to be reconciled with the manufacturing in cells of many thousands of copies of pre-micro RNAs47,48,49 that do not appear to activate interferons.
Conclusions
RNAi is attractive as a therapeutic approach owing in part to the diversity of its applications. Given the need for therapeutic agents that are sequence-specific and can maintain pace with the high mutation rate of viruses such as HIV, there is good reason to expect RNAi-based therapeutic agents in the not-too-distant future.
Source Information
From the Program in Molecular Medicine, University of Massachusetts Medical School, Worcester.
Address reprint requests to Dr. Stevenson at the Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St., Suite 319, Worcester, MA 01605, or at mario.stevenson@umassmed.edu.
References
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Guo S, Kemphues KJ. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 1995;81:611-620.
Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 1990;2:279-289.
Lindbo JA, Dougherty WG. Untranslatable transcripts of the tobacco etch virus coat protein gene sequence can interfere with tobacco etch virus replication in transgenic plants and protoplasts. Virology 1992;189:725-733.
Angell SM, Baulcombe DC. Consistent gene silencing in transgenic plants expressing a replicating potato virus X RNA. EMBO J 1997;16:3675-3684.
Hannon GJ. RNA interference. Nature 2002;418:244-251.
Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 2003;4:457-467.
Elbashir SM, Martinez J, Patkaniowska A, Lendeckel W, Tuschl T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J 2001;20:6877-6888.
Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 2001;293:1146-1150.
Stevenson M. Dissecting HIV-1 through RNA interference. Nat Rev Immunol 2003;3:851-858.
Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 2000;404:293-296.
Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001;409:363-366.
Doi N, Zenno S, Ueda R, Ohki-Hamazaki H, Ui-Tei K, Saigo K. Short-interfering-RNA-mediated gene silencing in mammalian cells requires Dicer and eIF2C translation initiation factors. Curr Biol 2003;13:41-46.
Tijsterman M, Plasterk RHA. Dicers at RISC: the mechanism of RNAi. Cell 2004;117:1-3.
Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494-498.
Denli AM, Hannon GJ. RNAi: an ever-growing puzzle. Trends Biochem Sci 2003;28:196-201.
Garrus JE, von Schwedler UK, Pornillos OW, et al. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 2001;107:55-65.
VerPlank L, Bouamr F, LaGrassa TJ, et al. Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). Proc Natl Acad Sci U S A 2001;98:7724-7729.
Stremlau M, Owens CM, Perron MJ, Kiessling M, Autissier P, Sodroski J. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 2004;427:848-853.
Scherr M, Battmer K, Winkler T, Heidenreich O, Ganser A, Eder M. Specific inhibition of bcr-abl gene expression by small interfering RNA. Blood 2003;101:1566-1569.
Wohlbold L, van der Kuip H, Miething C, et al. Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib mesylate (STI571). Blood 2003;102:2236-2239.
Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2002;2:243-247.
Wilda M, Fuchs U, Wossmann W, Borkhardt A. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene 2002;21:5716-5724.
Nieth C, Priebsch A, Stege A, Lage H. Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi). FEBS Lett 2003;545:144-150.
Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 2003;9:347-351.
Zender L, Hutker S, Liedtke C, et al. Caspase 8 small interfering RNA prevents acute liver failure in mice. Proc Natl Acad Sci U S A 2003;100:7797-7802.
Ullu E, Tschudi C, Chakraborty T. RNA interference in protozoan parasites. Cell Microbiol 2004;6:509-519. [CrossRef][ISI][Medline]
Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 2002;20:500-505.
Novina CD, Murray MF, Dykxhoom DM, et al. siRNA-directed inhibition of HIV-1 infection. Nat Med 2002;8:681-686.
Gitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells. Nature 2002;25:430-434.
Jacque J-M, Triques K, Stevenson M. Modulation of HIV-1 replication by RNA interference. Nature 2002;418:435-438.
Coburn GA, Cullen BR. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. J Virol 2002;76:9225-9231.
Hu WY, Myers CP, Kilzer JM, Pfaff SL, Bushman FD. Inhibition of retroviral pathogenesis by RNA interference. Curr Biol 2002;12:1301-1311.
Surabhi RM, Gaynor RB. RNA interference directed against viral and cellular targets inhibits human immunodeficiency virus type 1 replication. J Virol 2002;76:12963-12973.
Bitko V, Barik S. Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses. BMC Microbiol 2001;1:34-34.
Zamore PD. Plant RNAi: how a viral silencing suppressor inactivates siRNA. Curr Biol 2004;14:R198-R200.
Li H, Li WX, Ding SW. Induction and suppression of RNA silencing by an animal virus. Science 2002;296:1319-1321.
Delgadillo MO, Saenz P, Salvador B, Garcia JA, Simon-Mateo C. Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants. J Gen Virol 2004;85:993-999.
Bucher E, Hemmes H, de Haan P, Goldbach R, Prins M. The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants. J Gen Virol 2004;85:983-991.
Li WX, Li H, Lu R, et al. Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc Natl Acad Sci U S A 2004;101:1350-1355.
Holen T, Amarzguioui M, Wiiger MT, Babaie E, Prydz H. Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor. Nucleic Acids Res 2002;30:1757-1766.
McManus MT, Haines BB, Dillon CP, et al. Small interfering RNA-mediated gene silencing in T lymphocytes. J Immunol 2002;15:5754-5760.
Vitravene Study Group. A randomized controlled clinical trial of intravitreous fomivirsen for treatment of newly diagnosed peripheral cytomegalovirus retinitis in patients with AIDS. Am J Ophthalmol 2002;133:467-474.
Zeng Y, Cullen BR. RNA interference in human cells is restricted to the cytoplasm. RNA 2002;8:855-860.
Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 2003;5:834-839.
Bridge AJ, Pebernard S, Ducraux A, Nicoulaz AL, Iggo R. Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet 2003;34:263-264.
Lau NC, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 2001;294:858-862.
Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science 2001;294:862-864.
Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 2001;294:853-858.(Mario Stevenson, Ph.D.)
Discovery of RNAi
Events leading up to the discovery of RNAi illustrate the importance of following up on the results of control experiments. Guo and Kemphues2 were using antisense RNA to inhibit gene expression (antisense RNAs or DNAs pair up with their target messenger RNA [mRNA] through sequence complementarity to inhibit the translation of mRNA). They found, unexpectedly, that the sense RNA controls suppressed gene expression as effectively as the antisense RNA. Several years later, Fire and colleagues1 discovered that double-stranded RNA (dsRNA) mixtures were substantially more potent than sense or antisense single strands at silencing gene expression in worms. They called this mechanism RNA interference.
Since only a few molecules of dsRNA were needed to effect gene silencing, Fire and colleagues proposed that a catalytic or amplification component may be involved. Also, the discovery that dsRNA could cause the silencing of homologous genes helped reconcile observations of this phenomenon (called cosuppression) in transgenic plants: the genes were silenced by homologous transgenic sequences, and transgenic plants were subsequently resistant to viral infection.3,4,5 It is now clear that RNAi, cosuppression, and virus-induced interference are all triggered by a mechanism that involves a dsRNA intermediate. Thus, dsRNA provides the trigger for the activation of RNAi, the silencing is sequence-specific (i.e., only genes homologous to the dsRNA trigger are silenced), and silencing involves the degradation of RNA encoded by the targeted gene.6,7
Mechanism of Gene Silencing
RNAi, which can cause the degradation of virtually any RNA, is a simple mechanism (Figure 1). Long dsRNA is processed to short interfering RNAs (siRNAs) by the action of a dsRNA-specific endonuclease known as Dicer.11,12 The resultant siRNAs are 21 to 24 nucleotides in length, are double-stranded, and have 3' overhangs of 2 nucleotides.
Figure 1. Mechanism of Gene Silencing by RNA Interference.
The pathway of RNA interference can be broken down into two main phases. In the first phase, long double-stranded RNA (dsRNA) is recognized and processed by Dicer, an RNase III enzyme, into duplexes of short interfering RNA (siRNA) of 21 to 24 nucleotides (nt) in length. Exogenous synthetic siRNAs or endogenous expressed siRNAs can also be incorporated into the RNA-induced silencing complex (RISC), thereby bypassing the requirement for dsRNA processing by Dicer. In the second phase, siRNAs are incorporated into the multiprotein RISC. A helicase in RISC unwinds the duplex siRNA, which then pairs by means of its unwound antisense strand to messenger RNAs (mRNAs) that bear a high degree of sequence complementarity to the siRNA. An as yet unidentified RNase (Slicer) within RISC proceeds to degrade the mRNA at sites not bound by the siRNA. Cleavage of the target mRNA begins at a single site 10 nucleotides upstream of the 5'-most residue of the siRNA–target mRNA duplex.8 Although the composition of RISC is not completely known, it includes members of the Argonaute family9 that have been implicated in processes directing post-transcriptional silencing. ADP denotes adenosine diphosphate, Pi inorganic phosphate, P phosphate, and OH hydroxyl. The figure was adapted from Stevenson.10
These siRNAs are incorporated into a nuclease complex known as the RNA-induced silencing complex (RISC). ATP-dependent unwinding of the siRNAs activates RISC. RISC is directed by the unwound antisense siRNA strand to homologous target RNAs, which undergo endonucleolytic cleavage. Within RISC, mRNA cutting is affected by Slicer (the actual identity of Slicer is not yet known). There is some evidence that Dicer, in addition to processing long dsRNA to siRNAs, has additional roles in RNAi, since the efficiency of RNAi in mammalian cells is markedly impaired when Dicer itself is also silenced.13
(Glossary)
There are two Dicers (DCR-1 and DCR-2) in drosophila. In drosophila eggs lacking functional DCR-2, there is an impaired response of RNAi to synthetic siRNAs. Since use of synthetic siRNAs bypasses the dsRNA-processing activity of Dicer, this implies an additional role for Dicer, perhaps in the execution phase of RNAi.14 An important advance in the RNAi field was the demonstration that synthetic 21-nucleotide duplexes with the same structure as Dicer-generated siRNAs could also be incorporated into RISC and induce the degradation of homologous target RNA.15
The complementarity between the antisense strand of the unwound siRNA and the target RNA governs the sequence-specificity of gene silencing, a specificity that parallels the specificity governing the antibody–antigen interaction. Indeed, RNAi probably served as an ancient defense mechanism against mobile genetic elements such as RNA viruses and transposons. Research is continually revealing roles for RNAi in basic cellular processes, including gene regulation and the formation of heterochromatin.16 Vestiges of a role for RNAi as an antiviral defense mechanism may still be at work, as indicated by viruses that appear to have evolved strategies to counteract RNAi.
In many regards, RNAi has already exceeded expectations for its application to the study of biologic processes. Experiments involving gene knockouts, which were previously in the domain of model systems such as rodents, flies, worms, and zebrafish, can now be carried out on almost any cell type. RNAi is also a powerful tool for validating the targets for therapeutic drugs.17 For example, the ability of HIV-1 to detach physically from the surface of the infected cell requires the activity of a cellular cofactor called tumor suppressor gene 101 (Tsg101).17,18 Virus detachment and hence dissemination of the virus to other cells are blocked when Tsg101 expression is inhibited by RNAi.17 This provides the rationale for the development of small-molecule inhibitors of Tsg101 as potential drugs for patients with the acquired immunodeficiency syndrome (AIDS). Some cellular proteins can oppose HIV-1 infection, and RNAi showed that TRIM5 (tripartite motif 5 alpha) is one such factor that restricts HIV-1 infection of cells from certain monkey species.19
Therapeutic Applications
The therapeutic applications of RNAi are potentially enormous. The genetic etiology of many disorders has now been defined and, in some cases, has been targeted by RNAi in in vitro and in vivo model systems. Because the specificity of RNAi is governed by sequence complementarity between the siRNA and the target RNA, the most obvious application would be to treat diseases in which genetic polymorphisms within the disease-inducing gene in a particular lesion or tumor can be targeted for degradation without affecting RNA from wild-type alleles.
Oncogenesis
For example, RNAi is being explored as a way to inhibit the expression of genes involved in oncogenesis. The translocation of the Philadelphia chromosome (Ph) generates a fusion gene called BCR-ABL. The translation product of this gene creates a constitutively active protein tyrosine kinase that induces and maintains leukemic transformation in chronic myelogenous leukemia and Ph-positive acute lymphoblastic leukemia. The siRNAs specific for the BCR-ABL transcript have been shown to silence the oncogenic fusion transcripts without affecting expression levels of normal c-ABL and c-BCR transcripts.20,21
Pancreatic and colon carcinomas, in which RAS genes are often mutated, provide another example. In many cases, the RAS oncogenes contain point mutations that differ by a single-base mutation from their normal counterparts. The use of retroviral vectors to introduce interfering RNAs specific for an oncogenic variant of K-RAS (called K-RASV12) reduces the level of K-RASV12 transcripts and effects a loss of anchorage-independent growth and tumorigenicity.22,23 Studies of this kind provide proof-of-concept for RNAi-based strategies aimed at reversing tumorigenesis. A major factor confounding cancer treatment is resistance to chemotherapeutic agents. The siRNAs have been used to decrease the drug resistance of cells in vitro by inhibiting the expression of MDR1, a multidrug transporter with a major role in multidrug resistance.24
Viral Hepatitis
Acute liver failure and subacute liver failure, induced by viral hepatitis, are associated with high mortality rates. In animal models of liver failure, liver toxins and viral hepatitis lead to apoptosis of hepatocytes through cell-death receptors such as Fas. The siRNAs targeted to Fas RNA and delivered by high-pressure injection into the tails of mice were able to reach the liver, inhibit Fas expression, and protect mice from hepatitis.25 Similarly, siRNAs that target CASP8 RNA (encoding caspase 8) prevent acute liver failure induced by Fas activation.26
Infectious Diseases
Perhaps the most promising applications of RNAi are in the treatment of infectious diseases. In these cases, the RNA targets are exogenous and, as such, can be inhibited without affecting cellular functions. Pathogens of major importance to human health worldwide include HIV, influenzaviruses, hepatitis B and C viruses, poliovirus, papillomaviruses, herpesviruses, and West Nile virus. Protozoan parasites cause diseases that profoundly affect human health, and some species exhibit RNAi.27
An alternative strategy to targeting the RNA of the pathogen directly would be to target transcripts of cellular cofactors on which the invasion or pathogenicity of the agent depends (e.g., specific cell-surface receptors). A variety of studies have shown that RNAi can directly target viral transcripts28,29,30 or, in the case of viruses with RNA-based genomes, the viral chromosome itself.31,32 The ability to target the viral RNA chromosome after it enters the cell provides an opportunity to prevent the virus from establishing residence, thereby sterilizing the cell even after infection.
Some studies of HIV suggest that incoming viral RNA can be targeted by RNAi,31,32 whereas others indicate that it is resistant to RNAi.33,34 Perhaps this discrepancy exists because the studies targeted siRNAs to different regions of the viral genome. The viral nucleocapsid of the Rous sarcoma virus shields the incoming viral RNA from RNAi-mediated degradation. Likewise, there may be regions of the HIV genome that are differentially shielded from RNAi by a nucleocapsid.35
In addition to shielding of the viral chromosome by viral proteins, viruses may have evolved other ways to avoid RNAi.36 Infection of drosophila cells by a flock house virus requires the suppression of RNAi by a flock house virus–encoded protein, B2. This defense against silencing seems to have been evolutionarily conserved, since the B2 protein also inhibits silencing in transgenic plants.37 Furthermore, evidence now exists that vaccinia virus and human influenza A, B, and C viruses also encode a protein that inhibits silencing in drosophila and in plants.38,39,40 This supports the notion that RNA silencing is an antiviral defense and that viruses have evolved to counteract it.
Drug resistance is a major obstacle in the treatment of many infectious agents. However, the ability to target RNAi to multiple regions of the viral genome may overcome this obstacle. Similarly, targeting viruses with multiple interfering RNAs reduces the chances of a virus escaping RNAi repression through spontaneous mutation. One might envision a "salvage therapy" role for RNAi that could be used to inhibit viruses that are broadly resistant to conventional antiviral drugs. However, one must bear in mind that RNAi may be saturable41 and that simultaneous introduction of siRNAs with different specificities into cells can reduce the potency of the silencing effect more than siRNAs introduced individually.42 This is likely to be an important issue for future RNAi-based therapeutic strategies against viruses that, because of mutation rate, rapidly overcome drug or immune-mediated pressure.
Prospects for Therapy
The silencing effect of RNAi is highly specific and potent and requires only that the sequence of the target RNA be known. Obstacles must be overcome before RNAi can live up to its potential as a therapeutic method. Experiences with two other RNA-based therapeutic strategies, antisense RNA and ribozymes, illustrate the daunting obstacles that stand in the way of the therapeutic application of RNAi. Ribozymes are RNA molecules with a particular sequence and structure that cause cleavage of target RNA. No ribozyme has been approved for clinical use, and only one antisense agent, the DNA antisense drug fomivirsen (Vitravene, Isis Pharmaceuticals), has been approved by the Food and Drug Administration, for the treatment of cytomegalovirus retinitis in patients with AIDS.43 The RNA, whether the ribozyme, antisense, or siRNA, must interact with its target RNA inside the cell (in the case of RNAi, the mechanism of action is in the cytoplasm)44 and must remain in a biologically active form until it reaches its target. Therefore, the RNA must be stable and nontoxic, cross cell membranes, and not negatively affect the health of the organism. On the basis of the track record of ribozymes and antisense RNA, enthusiasm for RNAi-based therapeutics is, in reality, tempered.
Obstacles to Therapy
Delivery is probably the single biggest obstacle to the development of RNAi-based therapeutic agents. Trigger RNAs (dsRNAs from which siRNAs are derived by the action of Dicer) can be expressed from vectors or delivered as artificial siRNAs. A variety of strategies to express interfering RNAs with the use of plasmid and virus vector-based cassettes have been explored.7,37 Well-documented hazards of inserting foreign vector sequences into chromosomal DNA include insertional activation and inactivation of cellular genes.
Direct (e.g., intravenous) administration of siRNAs would require siRNAs that are modified to be resistant to nucleases and perhaps conjugated with a ligand to target the siRNA to specific tissues. In mice, intravenous introduction of Fas siRNAs leads to specific silencing of Fas mRNA in the liver,25 so in principle, unmodified siRNAs can be taken up by the liver and perhaps other tissues. It is not clear, however, whether there are selective tissue sites for the uptake of siRNAs and whether the lymphoid system or the brain, for instance, is accessible by this route. Furthermore, the silencing effect of siRNAs is short-lived, because the siRNAs eventually decay within the cell.
In addition to the danger of using vectors that integrate into the genome, the expression or injection of siRNAs may also have untoward biologic effects. Researchers are continually finding new cellular processes in which RNAi is involved. Therefore, a stoichiometric excess of a virus-specific siRNA, for example, could saturate RNAi and interrupt the pathway's normal functions in the cell.
Interferons, which form part of the host's defense against viral infection, are activated by long dsRNA (more than 500 bp). It is now apparent that siRNAs45 as well as short hairpin RNAs (short sequences of RNA that make tight hairpin turns and can be used to silence gene expression) expressed from DNA vectors46 can trigger the activation of interferons. However, there is no evidence that the activation of interferons by short RNAs influences the degree or specificity of RNA silencing. In addition, these effects have to be reconciled with the manufacturing in cells of many thousands of copies of pre-micro RNAs47,48,49 that do not appear to activate interferons.
Conclusions
RNAi is attractive as a therapeutic approach owing in part to the diversity of its applications. Given the need for therapeutic agents that are sequence-specific and can maintain pace with the high mutation rate of viruses such as HIV, there is good reason to expect RNAi-based therapeutic agents in the not-too-distant future.
Source Information
From the Program in Molecular Medicine, University of Massachusetts Medical School, Worcester.
Address reprint requests to Dr. Stevenson at the Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St., Suite 319, Worcester, MA 01605, or at mario.stevenson@umassmed.edu.
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