Download Silencing unhealthy alleles naturally

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TRENDS in Biotechnology
Vol.21 No.5 May 2003
185
| 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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186
TRENDS in Biotechnology
(a)
Vol.21 No.5 May 2003
(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.
Update
TRENDS in Biotechnology
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,
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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
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10 Elbashir, S.M. et al. (2001) Duplexes of 21-nucleotide RNAs mediate
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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