Advanced Drug Delivery Reviews 61 (2009) 668–671

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Advanced Drug Delivery Reviews
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d rCommentary

Short interfering RNA-mediated gene silencing; towards successful application inhuman patients☆
Mikiko C. Siomi
Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
JST, CREST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japana r t i c l e

i n f oArticle history:
Received 16 March 2009
Accepted 14 April 2009
Available online 23 April 2009
Keywords:
RNAi
siRNA
Argonaute
miRNA
DICER

a b s t r a c t
Gene silencing mechanisms that are mediated by small RNAs of 20-30 nucleotides(nt) are collectively called
RNA silencing. The representative mechanism is RNA interference (RNAi), in which ~ 21-nt small RNAs (shortinterfering RNAs or siRNAs)efﬁciently trigger cleavage of target gene transcripts. As a result, proteins are nolonger made from the targeted mRNAs. RNAi is tremendously speciﬁc and efﬁcient with regard to recognizing
target gene and disrupting their expression. The triggering molecules, double-stranded (ds) RNAs, are quite
stable in vivo and are basically indistinguishable from natural endogenous RNAs; thus, RNAi intrinsically has
great potential for therapeutic use. In fact, RNAi-based clinical trials for treating human diseases are currently
ongoing worldwide. However, optimization of the protocols still requires substantial investigation; indeed,
relevant studies have been undertaken internationally by many researchers. Given the growing anticipationof RNAi as a disease therapy, re-examining the RNAi mechanism and machinery from a biochemical
perspective might provide a way to advance its successful application in the human body; this, therefore, isthe focus of this Commentary.
© 2009 Elsevier B.V. All rights reserved.

1. Discovery of RNAi; a powerful tool for gene silencing
RNAi was discovered a decade ago through basic research in
C. elegans as a model system [1]. It promptly attracted a large numberof researchers worldwide because of its methodological potential,
including great silencing efﬁciency and a high degree of target
transcript speciﬁcity amidst the astonishing numbers of transcripts incells. The RNAi system can be easily manipulated. For example, in
C. elegans, feeding the animals dsRNAs or soaking them in a dsRNA
solution easily induces RNAi. In addition, the fact that dsRNAs aremuch more effective than single-stranded (ss) RNAs in gene silencing
is remarkable and should be widely appreciated.
Before the discovery of RNAi in nematodes, plant scientists had
noticed a gene silencing phenomenon similar to RNAi in the plant
kingdom, which was originally referred to as “co-suppression” [2,3].
Researchers in the fungal ﬁeld termed the phenomenon “quelling” [3].
These observations lead us to anticipate that RNAi was not restricted
to C. elegans, but highly conserved in divergent organisms. Indeed,
many species, including ﬂies, were shown to have RNAi [4–6]. Suchstrong conservation implied that RNAi was not only a powerful tool forsilencing speciﬁc genes, but that it also has an intrinsically important
physiological function in living creatures. Indeed, it has been shown☆ This commentary is part of the Advanced Drug Delivery Reviews theme issue on“Towards Therapeutic Application of RNA-mediated Gene Regulation”.
E-mail address: siomim@sc.itc.keio.ac.jp.
0169-409X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2009.04.008

that in some species, such as Drosophila, RNAi functions as an innate
immune system for defending against infectious viruses and otherinvasive elements [7–9].
2. RNAi pathways and factors
Research on RNAi factors and their functions has been conductedworldwide. Drosophila was chosen as a model system because RNAi is
inducible and effective in Drosophila cultured cells and embryos [4–6],
and both biochemical and genetic approaches are applicable in thissystem. In parallel, cultured human cell lines were also employedbecause RNAi without undesirable side effects such as apoptosis could
be induced when siRNAs, the minimum size of RNAi triggers, were
utilized as the inducers [10,11].
After being introduced into living cells, dsRNAs are processed by an
RNaseIII-domain containing nuclease, Dicer (in humans [12,13];
Dicer2 in ﬂies [14,15]), into siRNA duplexes of ~ 21 nt (for review,see [16,17]). During this primary step, Dicer forms a complex with thedsRNA-binding domain containing protein, TRBP (TAR RNA-binding
proteins, also known as TARBP2 [18,19]; R2D2 in ﬂies [20])or PACT
(also known as PRKRA [21]). In addition to its siRNA processing
activity, the DICER complex also functions at the effector step in theRNAi process [22–24]; the loading of siRNA onto RISC (RNA-induced
silencing complex) [25,26]. Within RISC, ss-siRNA (termed ‘guidesiRNA’ [27]) is associated with Argonaute2 (AGO2in both humans andﬂies) [28–30], a member of the Argonaute family of proteins [17,31].

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AGO2 contains a PAZ domain, which is required for siRNA binding [16].
The PAZ domain is located around the center of the protein and isconserved in all members of Argonaute family in all species [15]. AGO2
also serves as the catalytic factor in RNAi (i.e. it is the enzyme
responsible for cleaving target RNAs) [28–30]. The C-terminal PIWI
domain of AGO2 is required for endonuclease activity (or Slicer
activity) [16,32–34].This domain is also conserved in all Argonaute
proteins [31].
After excising siRNAs, the Dicer-TRBP (or -PACT) heterodimer
apparently releases the products and then binds the tiny dsRNA
molecules once again to determine which strand of the duplex should
be loaded onto RISC. Previous studies in Drosophila have indicatedthat R2D2 (the ﬂy ortholog of TRBP or PACT) binds preferentially tothe more thermodynamically stable end of the siRNA duplex[27,35,36]. As a result, Dicer2 is inevitably positioned close to theless stable end. Following this thermodynamically driven dsRNA end
recognition, AGO2 supposedly receives the duplex from the Dicer2R2D2 heterodimer. The mechanism for this transfer is not yet
understood. Next, AGO2 cleaves one particular strand (termed the‘passenger strand’ [27]),as if AGO2 cleaves target RNAs base-pairingwith guide siRNAs [37–39]. It has been speculated that Dicer2 on the
duplex would be displaced by AGO2 prior to this siRNA duplex receipt
and passenger strand cleavage step. This is based on the observationthat one strand of an siRNA duplex, whose 5′ end faces towards Dicer2,
has the tendency to be loaded onto RISC.

them, like siRNAs. AGO proteins associated with miRNAs appear torepress translation of target mRNAs by interfering with translation
initiation, translation elongation or by some other means, but not bycleaving the transcripts [44]. In this sense, the functions of miRNAs aredifferent from those of siRNAs. mRNAs targeted by miRNAs aresequestered and actively degraded in cytoplasmic foci (processing
bodies or P-bodies) where various factors that function in mRNAdegradation, such as decapping enzymes and nucleases, are concentrated [45]. This means that even mRNAs targeted by AGO proteins
with no Slicer activity are eventually (actively) degraded in cells. In
this regard, it can be noted that AGO proteins that do not have Slicer
activity (namely AGO1, AGO3, and AGO4) are able to function in thesiRNA-mediated gene silencing (RNAi) pathway. Indeed, it waspreviously shown that siRNAs can function as miRNAs [46]. The only
concern would be that the turnover rates of AGO1 (or AGO3, or AGO4)
and of AGO2 might be different. Speculatively, the turnover of AGO1siRNA (or AGO3-siRNA, or AGO4-siRNA) complexes must be slower
compared to that of AGO2-siRNA complexes because before entering
the next round of silencing, anAGO2-siRNA complex can bypass the Pbodies (although AGO2 is detectable in P-bodies [42]) whereas theother AGO protein complexes have to be sequestered into P-bodies torelease their target mRNAs.

3. RNAi in mammalian cells

In any single cell, multiple physiological pathways are coordinatedwith other pathways and such coordination is maintained at a steadystate, even when some pathways are constantly turned on and off. To
successfully apply RNAi therapeutically, small exogenous RNAs must
be introduced and activate RNA silencing machineries without
disturbing the well-coordinated cellular environment. The questionof how to achieve this, however, remains. From a biochemical
perspective, one strategy would be to drastically minimize thedsRNA triggers (in amount) to be introduced into the cells. In thisstrategy, the optimization of siRNA loading onto AGO proteins would
be crucial. Excess siRNAs, or their precursors, would cause severe
unexpected problems in the cells. Indeed, artiﬁcially overexpressed
siRNA precursors, which structurally mimicked miRNA precursors in
mice, have previously been shown to be toxic to the animals [47]. The
precursors occupied a large proportion of Exportin 5 [43], the factor
necessary for exporting pre-miRNAs from the nucleus to thecytoplasm and thus severely disturbed endogenous miRNA activities.
In the worst cases, the transgenic mice died [47]. To avoid such a
serious problem, siRNAs could be directly introduced in mice as in an
siRNA duplex form. However, even in this case, we can predict that
excess siRNAs would mostly occupy the Dicer-TRBP (or -PACT)
complexes and might block the miRNA activities.
One molecule of AGO protein binds one guide siRNA molecule [16].
This means that exogenous siRNA that has been successfully
introduced into living cells may not ﬁnd an AGO recipient, if the
AGO proteins were mostly occupied by endogenous small RNAs
(miRNAs and endogenous siRNAs (endo-siRNAs); the latter wasrecently discovered in mouse oocytes) [48,49]. In this case, theexogenous siRNAs would be degraded without exerting their functions. In this regard, we can conclude that the RNAi efﬁcacy in cells
would largely depend on the availability of AGO proteins for thesiRNAs of interest. The levels of AGO proteins in any given cell or tissue
could be examined, for example, by western blotting utilizing speciﬁc
antibodies against them. Currently, however, there is no means toassess what proportion of cellular AGO proteins are bound or unboundto endogenous small RNAs and under what circumstances the ratio of
the bound form over the unbound form is altered in cells. We do noteven have a clear answer for the basic questions of how much AGO2,
or other AGO proteins, are expressed in any cell or tissue in the humanbody or how their expression changes throughout development.

Many researchers suspected that RNAi could not be used in
mammalian cells or tissues because synthetic dsRNAs are notorious
for inducing interferon responses, which further induce apoptotic
responses [40]. However, papers published in 2001 showed that evenin mammalian cells, RNAi can be activated without adverse side
effects [10,11]. This ﬁnding had a tremendous impact on researchers in
the RNA silencing ﬁeld. The key was to utilize siRNA duplexes rather
than long dsRNA precursors as RNAi inducers. Further investigation
revealed that 27-nt dsRNAs might be more potent triggers than 21-nt
siRNAs for mammalian RNAi [41]. 27 nt dsRNAs can serve as Dicernuclease substrates; the key to this solution might be that Dicernuclease activity from the precursors accelerates the effector step. The
mechanism by which such excision accelerates the effector step
remains unclear however.
4. RNAi machineries in mammalian cells
AGO2 has three distinct homologs in humans, AGO1, AGO3, andAGO4, all of which are known to be ubiquitous [17,31]. Strictly
speaking, four additional Argonaute proteins (PIWIL1 to PIWIL4) canbe detected in humans, but they are expressed exclusively in germline
cells. To distinguish the two subgroups, ubiquitous Argonaute proteinsare called AGO proteins, whereas germline-speciﬁc Argonautes arecalled PIWI proteins [17,31].
All four human AGO proteins (endogenously or exogenously
expressed in cultured cells)can be loaded with siRNAs, but only
AGO2 exhibits Slicer activity [28,29,42]. The amino acids necessary for
Slicer activity, the D (Asp)-D (Asp)-H (His) triad residing in the PIWI
domain, are conserved in all four AGO proteins [31].The cause of thefunctional differences between these proteins remains unclear. An
obvious question now is whether or not AGO1, AGO3 and AGO4, whichbind siRNAs but display no Slicer activity, act as “inhibitors” in theRNAi pathway.
All four AGO proteins can be loaded not only with siRNAs, but alsowith microRNAs (miRNAs) [28,29,42]. miRNAs are a subset of small
RNAs that are thought to be endogenously expressed in all humancells [43]. Their size is very similar to that of siRNA (~ 21–23 nt), andthey guide AGO proteins to their targets by directly associating with

5. Possible new strategy for successful therapeutic
application of RNAi

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M.C. Siomi / Advanced Drug Delivery Reviews 61 (2009) 668–671

Hopefully, the answers to these important questions will be determined experimentally.
What if it was discovered that ‘empty’ AGO proteins were largely
unavailable in the cellsor tissues where you were considering applying
exogenous RNAi? A possible strategy would be to raise the cells’
expression rates to increase the supply of ‘empty’ AGO proteinsavailable for exogenous siRNAs. Indeed, it has been shown thatoverexpression of AGO2 enhances RNAi effects in mammalian cells[50,51]. However, currently it remains unknown how the expressionof AGO proteins is regulated in vivo or how the expression can be
manipulated. Clearly, further research on RNAi machineries will beneeded to answer this type of question.

6. Final remarks
Clinical trials using ‘RNAi’ to treat human diseases were ﬁrst
launched in 2004. Several trials are currently ongoing worldwide (forreview, see [52]). However, the various protocols involved have notyet been optimized. Current issues that need to be addressed so thattherapeutic strategies can be optimized include; how to improve thesystems that deliver synthetic siRNAs or their expression vectors to
the targeted cells (this theme is focused on by Y. Takakura and otherauthors in the reviews); how to modify siRNAs for longer durability;and how to minimize off-target effects to eliminate side effects. A wide
range of studies have demonstrated that certain kinds of RNA
modiﬁcations, such as a 2′-O-methyl in the 2′ ribose at somenucleotides in siRNAs, convey an increased resistance to ribonucleases[53]. Another modiﬁcation was effective for reducing off-target effects.
Conjugation of cholesterol to siRNAs, or siRNA capsulation by a lipid
layer or polycation showed an increased uptake by living cells [54–56].
These non-viral siRNA delivery and viral delivery issues (the latter isfor siRNA-expression vectors) remain ‘hot’ topics, and continuous,
intensive investigation will certainly be needed to optimize them(refer to the reviews in this issue). As mentioned above, even newer
strategies, such as manipulating AGO expression in vivo andassembling RISC with minimal exogenous siRNA levels, will also be
helpful. Clearly, further investigations, from both basic and appliedscience perspectives, need to be pursued to achieve the successful
application of RNAi in disease therapy.

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