Silencing unhealthy alleles naturally

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| Research Focus

Silencing unhealthy alleles naturally Eric G. Moss Cell and Developmental Biology, Fox Chase Cancer Center, Philadelphia, PA 19111, USA

Recently, it has been reported that small interfering RNAs can silence mutant alleles of genes while not affecting wild-type alleles. This high specificity should also limit off-target effects when these molecules are used in drug-target validation or as therapeutics. Small RNA technology is both extremely versatile, because virtually any expressed gene can be inhibited, and effective, because it exploits natural cellular machinery that evolved to use small RNAs to regulate gene expression. Disease-causing alleles of some genes differ from their wild-type counterparts by only a point mutation. The wildtype version is often necessary for health, so any strategy that inhibits both alleles would cause severe toxicity. Recent reports [1,2] show that a technology based on very small RNAs is capable of distinguishing between the two alleles and silencing only the harmful one. This approach holds promise for developing highly individualized therapeutics and identifying candidate drug targets for more conventional approaches. The very small RNAs, called small interfering RNAs (siRNAs), are about 25 nucleotides in length, just long enough to select a unique sequence among all expressed genes in a cell [3]. siRNAs act as guides for an endogenous ribonuclease complex that cleaves mRNA containing a complementary sequence. siRNAs can be delivered as synthetic oligonucleotides or generated in vivo from DNA vectors that produce short RNA hairpins, which are then processed into the active form by cellular components (Fig. 1). The recent explosion in our knowledge of siRNAs and how cells use them to control gene expression is largely the result of work carried out in the model organisms Caenorhabditis elegans and Drosophila. The basic science focused on these organisms has uncovered the essential properties of siRNAs. Even so, there are significant differences in how the technology is adapted for use in mammalian cells. However, importantly, siRNA technology is significantly more effective than a traditional antisense approach [4,5], in part because it taps into an elaborate catalytic machinery that is already present to regulate specific genes and curb aberrant expression. Endogenous mechanisms of RNA interference Cells can silence unwanted gene expression through a highly evolved mechanism that is initiated by doublestranded RNA (dsRNA). A ribonuclease known as Dicer cleaves the dsRNA to yield short RNAs, about 21 –25 nucleotides long, with a 50 -phosphate group and a Corresponding author: Eric G. Moss ([email protected]). http://tibtec.trends.com

30 -hydroxyl group – these are the siRNAs [6]. The siRNAs interact with a complex of cellular proteins, including a ribonuclease, which is then guided by the siRNAs to specific sequences by virtue of complementarity. The complex, known as an RNA-induced silencing complex (RISC), cleaves the target mRNA, thereby inhibiting its translation into protein [7]. The entire process is known as RNA interference (RNAi) [8] (Fig. 1). In C. elegans, one can introduce long dsRNAs by several different means to initiate RNAi. However, in most mammalian cells, long dsRNAs trigger the interferon response, which effectively shuts down all gene expression, making the specific targeting of a gene irrelevant. Two general methods for getting siRNAs into mammalian cells have been developed, each of which could be useful for different applications. One method is to transfect synthetic siRNAs as naked, duplexed molecules. This has been shown in many cases to be highly effective for silencing gene expression [3]. However, the effect of transfected siRNAs is necessarily transient, as they persist in the cell for perhaps a few days if not continually applied. The other method, developed independently by several investigators (e.g. [9]), is to introduce a gene encoding a short RNA hairpin, a dsRNA that is only ,30 nucleotides long, too short to trigger the interferon response. When such a gene is transcribed, the hairpin is processed by Dicer into siRNAs. This method has the advantage of being stable because the hairpin can be expressed constitutively. Perhaps its only disadvantage occurs where virus-mediated gene delivery has inherent problems, such as in a therapeutic setting. Discrimination of a single point mutation As a technology for inhibiting gene expression, RNAi has the advantage of being exquisitely specific because it relies on perfect complementarity between the siRNAs and their targets. A single point mutation can prevent an siRNA from guiding cleavage of a target [10,11]. Recently, investigators have taken advantage of this fact and shown that these agents can specifically target the mutant allele of a cancer-causing gene. Brummelkamp et al. [1] targeted an oncogenic form of K-RAS (K-RASV12) using a retroviral vector to deliver a gene encoding a small RNA hairpin (Fig. 1). The level of expression of the oncogenic form was significantly reduced whereas the wild-type form was unaffected by the presence of the siRNAs. Importantly, the reduction of the expression of oncogenic RAS by this strategy tremendously reduced growth of RAS-transformed cells in soft agar, and the ability of these cells to form tumors in nude mice. Similarly, Martinez et al. have demonstrated the same

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(a)

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(b) Vector is introduced into cells that express mutant K-RAS

5'-GUUGGAGCUGUUGGCGUAGUUC A 3'-UUCAACCUCGACAACCGCAUCA GA GA Precursor is processed by Dicer into siRNA 3'-CAACCUCGACAACCGCAUC-5'

DNA vector Transcription of vector produces siRNA precursor Mutant K-RASV12 mRNA

RNA hairpin Nucleus

5'...UGUCGUAGUUGGAGCUGUUGGCGUAGGCAA...3' 3'-CAACCUCGACAACCGCAUC-5'

siRNA guides RISC to cleave mRNA RNA Cytoplasm

Normal K-RAS mRNA

SiRNA

5'...UGUCGUAGUUGGAGCUGGUGGCGUAGGCAA...3' 3'-CAACCUCGACAACCGCAUC-5' Mismatch prevents targeting

TRENDS in Biotechnology

Fig. 1. (a) A viral vector delivers a gene encoding a small interfering RNA (siRNA) to silence the mutant allele of a cancer-causing gene. The vector encodes a short RNA hairpin, which is processed in the cytoplasm by the ribonuclease Dicer into the siRNA. (b) The siRNA acts as a sequence-specific guide for the RNA-induced silencing complex (RISC) to target cleavage of the mRNA from a specific gene, in this case, the mutant allele of an oncogene. Perfect complementarity is required for cleavage, such that a single base pair mismatch is enough to prevent targeting the mRNA from the wild-type allele. Based on [1].

degree of specificity for siRNAs directed against the mRNA encoding mutant p53 [2]. They transfected cells with synthetic siRNAs and showed that the expression of mutant p53 was reduced whereas the wild-type p53 level remained unchanged. These examples of the exquisite specificity of siRNAs raise real hopes that the use of this class of molecules can be broadly applied with minimal side effects. Nevertheless, our understanding of how the cell normally uses siRNAs in gene silencing has prompted some concerns about the potential for off-target effects of RNAi. Potential off-target effects Does amplification diminish selectivity? A quality of RNAi in C. elegans is its remarkable ability to spread from the original site of occurrence and even to pass on to subsequent generations [12]. Investigation into the mechanism of this phenomenon revealed the importance of RNA synthesis by an RNA-dependent RNA polymerase (RdRp) and that the siRNAs act as primers for production of more dsRNA [13]. Evidently, although amplification is a powerful and useful feature of RNAi in C. elegans, it does cause problems when trying to assign a role to a gene of unknown function, especially if there are related genes in the genome. There is strong evidence that indicates that amplification does not occur in mammalian cells and therefore will not decrease the selectivity of siRNAs [14,15]. For example, Zamore and colleagues demonstrated that the 30 -hydroxyl group of siRNAs is not essential for their effect [15]. A free 30 hydroxyl would be required if the siRNAs were ligated together or used as primers for a polymerase. There is also empirical evidence that targeting the mutant alleles of p53 and RAS did not have an effect on the wildtype alleles, which would happen if siRNAs were generated from the sequence neighboring the original siRNA target site. Thus it appears that each siRNA can be counted on to silence only those genes to which it is perfectly complementary. http://tibtec.trends.com

MicroRNAs and other mechanisms of repression A major recent discovery is that the human genome contains hundreds of genes encoding small RNAs like siRNAs, called microRNAs (miRNAs) [16]. These genes generate hairpins that are processed by Dicer into singlestranded RNAs of 21 – 25 nucleotides. Only a few of these have been studied in detail, mostly in C. elegans, so the mechanisms of other miRNAs can only be inferred. What is known for the best studied miRNAs is that they do not require strict complementarity to repress their specific targets and this repression does not result in cleavage of the mRNA. So what are the implications of miRNAs for siRNA technology? Could any introduced small RNA affect gene expression based on partial mismatches? Some data suggest that this could be possible [17] but data from C. elegans indicate that a simple miRNA – target interaction alone is not sufficient to cause an effect [18]. Further investigation is needed into how miRNAs work. It might be that siRNAs and miRNAs are as different as restriction enzymes and transcription factors; the former can act at essentially any binding site, the latter require a specific context to produce an effect. Concluding remarks Although the greatest immediate impact of siRNAs will be in basic research and drug target validation, they can be seriously considered for clinical situations, such as viral infections and cancer. In an experimental setting, transfection is the method of delivery of siRNAs and genes encoding siRNA precursors. Delivering the precursor genes by viral vectors expands this technology both to cells that are generally resistant to transfection and to tissues in the body [1,19]. Because the gene-silencing specificity can be built into the siRNAs, the infection of normal cells need not be a concern. In addition to silencing unwanted gene expression, the targets of therapeutic siRNAs will probably include mRNAs of pathogenic viruses.

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Although we do not yet fully understand how the cell produces siRNAs and uses them to cleave complementary targets, we do know that organisms have been using such mechanisms for eons for essentially these purposes. For this reason, siRNAs could be the most versatile and widely applicable new technology for affecting cellular processes at the level of gene expression. References 1 Brummelkamp, T.R. et al. (2002) Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2, 243 – 247 2 Martinez, L.A. et al. (2002) Synthetic small inhibiting RNAs: efficient tools to inactivate oncogenic mutations and restore p53 pathways. Proc. Natl. Acad. Sci. U. S. A. 99, 14849 – 14854 3 McManus, M.T. and Sharp, P.A. (2002) Gene silencing in mammals by small interfering RNAs. Nat. Rev. Genet. 3, 737 – 747 4 Caplen, N.J. et al. (2001) Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. U. S. A. 98, 9742– 9747 5 Bertrand, J.R. et al. (2002) Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem. Biophys. Res. Commun. 296, 1000 – 1004 6 Bernstein, E. et al. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363 – 366 7 Hammond, S.M. et al. (2000) An RNA-directed nuclease mediates posttranscriptional gene silencing in Drosophila cells. Nature 404, 293 – 296 8 Hannon, G.J. (2002) RNA interference. Nature 418, 244 – 251

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9 Sui, G. et al. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. U. S. A. 99, 5515– 5520 10 Elbashir, S.M. et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494 – 498 11 Brummelkamp, T.R. et al. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550– 553 12 Grishok, A. and Mello, C.C. (2002) RNAi (nematodes: Caenorhabditis elegans). Adv. Genet. 46, 339 – 360 13 Nishikura, K. (2001) A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell 107, 415– 418 14 Chiu, Y.L. and Rana, T.M. (2002) RNAi in human cells: Basic structural and functional features of small interfering RNA. Mol. Cell 10, 549 – 561 15 Schwarz, D.S. et al. (2002) Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol. Cell 10, 537 – 548 16 Moss, E.G. (2002) MicroRNAs: Hidden in the genome. Curr. Biol. 12, R138– R140 17 Zeng, Y. et al. (2002) Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9, 1327– 1333 18 Seggerson, K. et al. (2002) Two genetic circuits repress the C. elegans heterochronic gene lin-28 after translation initiation. Dev. Biol. 243, 215– 225 19 Devroe, E. and Silver, P.A. (2002) Retrovirus-delivered siRNA. BMC Biotechnol. 2, 15 0167-7799/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0167-7799(03)00088-X

‘Super bugs’ for bioremediation Kensuke Furukawa Department of Bioscience and Biotechnology, Kyushu University, Fukuoka 812-8581, Japan

Chlorinated organic compounds are among the most significant pollutants in the world. Sequential use of anaerobic halorespiring bacteria, which are the key players in biological dehalogenation processes, and aerobic bacteria whose oxygenases are modified by directed evolution could lead to efficient and total degradation of highly chlorinated organic pollutants. Recently three interesting papers on halorespiration and polychlorinated biphenyl biodegradation were published. There is great concern over chlorinated organic compounds because of their toxicity, persistence and bioaccumulation. Among these compounds, polychlorinated biphenyls (PCBs) and chlorinated organic solvents such as trichloroethene (TCE), tetrachloroethene (PCE) and 1,1,1-trichloroethane (TCA) are the major targets for bioremediation. TCE, PCE and TCA were widely used and are recognized as serious environmental contaminants in soil, groundwater and the atmosphere. An increasing number of bacteria has been isolated that can couple reductive dehalogenation of these chlorinated solvents with energy conservation [1]. A halorespiratory process would therefore be Corresponding author: Kensuke Furukawa ([email protected]). http://tibtec.trends.com

effective for in situ bioremediation of these chlorinated solvents. Microbial degradation of PCBs has been extensively documented in terms of the biodegradability and molecular characteristics of enzymes and genes from a variety of soil bacteria [2]. Because PCBs are complicated mixtures containing up to ten chlorine atoms on a biphenyl molecule, microbial degradation is highly dependent on chlorine substitution and is highly strain dependent. Recently, attempts have been made to enhance PCB biodegradation by modifying oxygenases [3]. One of the most efficient methods of biological degradation consists of sequential anaerobic –aerobic treatment for highly chlorinated compounds. Recent biochemical and genetic engineering approaches for dehalogenases and oxygenases could lead to ‘super bugs’ that could be used for the bioremediation of chlorinated environmental pollutants.

Microbial halorespiration An increasing number of bacteria has been isolated that can couple the reductive dehalogenation of various chlorinated compounds to energy conservation by electrontransport-coupled phosphorylation [1]. This process is referred to as halorespiration, or dehalorespiration. Recent studies indicate that halorespiring bacteria have