Advanced Drug Delivery Reviews 61 (2009) 746759

Contents lists available at ScienceDirect

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 r

siRNA vs. shRNA: Similarities and differences

Donald D. Rao a, John S. Vorhies a, Neil Senzer a,b,c,d, John Nemunaitis a,b,c,d,
a
Gradalis, Inc., Dallas, TX, USA
b
Mary Crowley Cancer Research Centers, Dallas, TX, USA
c
Texas Oncology PA, USA
d
Baylor Sammons Cancer Center, Dallas, TX, USA

a r t i c l e i n f o a b s t r a c t

Article history: RNA interference (RNAi) is a natural process through which expression of a targeted gene can be knocked
Received 23 January 2009 down with high specicity and selectivity. Using available technology and bioinformatics investigators will
Accepted 13 April 2009 soon be able to identify relevant bio molecular tumor network hubs as potential key targets for knockdown
Available online 20 April 2009
approaches. Methods of mediating the RNAi effect involve small interfering RNA (siRNA), short hairpin RNA
(shRNA) and bi-functional shRNA. The simplicity of siRNA manufacturing and transient nature of the effect
Keywords:
per dose are optimally suited for certain medical disorders (i.e. viral injections). However, using the
RNA interference
Bi-functional
endogenous processing machinery, optimized shRNA constructs allow for high potency and sustainable
Cancer effects using low copy numbers resulting in less off-target effects, particularly if embedded in a miRNA
Personalized scaffold. Bi-functional design may further enhance potency and safety of RNAi-based therapeutics. Remaining
challenges include tumor selective delivery vehicles and more complete evaluation of the scope and scale of
off-target effects. This review will compare siRNA, shRNA and bi-functional shRNA.
2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746
2. Targeted cancer gene therapy and RNA interference. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
2.1. Personalized approach for cancer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
2.2. RNA interference for cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
3. Small interfering RNA (siRNA) and short hairpin RNA (shRNA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
3.1. siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
3.2. shRNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750
3.3. Bi-functional shRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751
3.4. Summary of si/sh/bi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752
4. Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753
5. Off-target effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753
5.1. Specic off-target effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753
5.2. Nonspecic off-target effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754
6. The future outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 756
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 756

This review is part of the Advanced Drug Delivery Reviews theme issue on Towards
Therapeutic Application of RNA-mediated Gene Regulation.
Corresponding author. 1700 Pacic, Suite 1100, Dallas, Texas 75201, USA. Tel.: +1 214
658 1964; fax: +1 214 658 1992.
E-mail addresses: DRao@gradalisinc.com (D.D. Rao), jsvorhies@gmail.com
(J.S. Vorhies), NSenzer@marycrowley.org (N. Senzer), jnemunaitis@marycrowley.org
(J. Nemunaitis).
1. Introduction
Cancer is a disease of genes, whether based on aberrant changes in
sequence or expression (epigenomics). The constellation of genetic
and epigenetic abnormalities characterizing cancer cells present new
and more specic targets for cancer treatment and, hopefully,
prevention. Over the last decade, the mapping of the human genome,

0169-409X/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2009.04.004
 D.D. Rao et al. / Advanced Drug Delivery Reviews 61 (2009) 746759
along with improved understanding of signal transduction and the
pathways responsible for tumor survival, have been transforming
therapeutic oncology from a more promiscuously targeted che-
motherapeutic approach towards a highly selective targeted ther-
apeusis. Indeed, in 1997, antibody-based Herceptin (trastuzumab)
became the rst targeted therapy for breast cancer, specically for
HER2-positive metastatic breast cancer. In 2001, the small molecule
Gleevec (imatinib mesylate), became the rst approved kinase
inhibitor for cancer targeting bcr-abl in chronic myeloid leukemia
(CML), and it has since been approved for the treatment of
gastrointestinal stromal tumors (GIST) targeting c-kit. Over the last
few years, several other targeted cancer therapies have been
approved. Targeted therapeutics directed against amplied genes
and/or over-expressed proteins in malignant cells have proven to be
powerful tools for cancer treatment. A growing understanding and use
of proteomic, genetic, and pharmacogenomic tools are actualizing a
long desired concept of personalized cancer therapy. Genetic
abnormalities of each patient's tumor can be analyzed through a
variety of established means to quantitatively determine both gene
and protein over- and under-expression. Moreover, functional path-
ways can be determined and integrated within the cancer network
allowing for the identication of key molecular relays enabling
experimental testing of target specic therapeutics. Such information
can potentially allow medical care takers to prioritize, if not yet
optimize, treatment for cancer patients, and to uncover surrogate
biomarkers for prognosis, prediction, and therapy assessment.
The recent discovery of RNA interference (RNAi), a natural process
through which the expression of a targeted gene can be knocked down
with high specicity and selectivity, presents an invaluable tool for
personalized cancer therapy. Target specic RNAi agents have the
potential to selectively knockdown key abnormally over- or constitu-
tively expressed molecular targets that are essential for the survival of
each patient's tumor for effective personalized cancer treatment.
Conceptually, target specic RNAi agents can also be applied in
combination with immune modulating agents or small molecules to
improve the efcacy of cancer treatment. Like other new therapeutic
paradigms, there are a multitude of issues which need to be addressed
in order for us to translate RNA interference technology (siRNA,
shRNA, bi-functional RNA) from laboratory to bedside and from
concept to reality. These issues include comparison of each of the RNAi
technologies with respect to effective delivery, possible off-target
effects and the pharmacokinetics and pharmacodynamics. This review
will focus on several issues currently confronting clinical development
of RNAi therapeutics. We will discuss the role of RNAi technology in
personalized cancer gene therapy and address clinical considerations
of appropriate RNAi-based therapeutic agents for cancer.
2. Targeted cancer gene therapy and RNA interference
2.1. Personalized approach for cancer therapy
Most human tumors manifest gene expression patterns that differ
not only from their normal counterparts but, to a lesser extent, even
from each other based on both intrinsic gene modications and
modulated cancer cellmatrix interactions. Such variability in genetic
patterns found between histologically identical tumors arising in
different patients may well explain the widely divergent responses to
the standard treatment regimens most often prescribed for a particular
tumor type. This is supported by recent studies demonstrating that
certain patterns of genetic expression (i.e. expression signatures)
identied in tumor samples from patients with breast cancer are not
only strongly correlated with prognosis [14] but can actually be sub-
classied into differing prognostic categories [5]. The presence of
functional redundancy in a robust, predominantly scale-free network
such as cancer buffers the effect of any single gene/target modication
on the malignant process, with rare exception (e.g., CML). The hierarchy
747
of cancer scale-free networks does not have a threshold for single target
disintegration insofar as random pathway component failure predomi-
nantly affects targets with low connectivity within the network, thereby
having limited functional impact. However, highly connected informa-
tion-transfer targets do allow for attack vulnerability. In other words,
the disordered circuitry characteristic of malignancy results in a change,
such that the otherwise robust oncogenic process can become, almost
paradoxically, more highly dependent on a specic rewired pathway
(i.e., pathway addiction). Conceptually, knockout of those rewired
tumor specic oncogenic pathways should produce a lethal effect on
cancer cells yet not signicantly perturb normal cell functionality.
Genetic diversity of cancer, pathway addiction and targeted therapy are
not the subject of this review and have been extensively reviewed
elsewhere [68]; here, we discuss how RNAi-based therapeutics can be
best applied in light of these mechanisms.
For example, we harvested tumor and normal cells from the lymph
nodes of a melanoma cancer patient for molecular proling by
microarray and proteomic analysis [9]. Expression proles for
malignant versus normal tissue were compared at the mRNA and
protein levels. The goal was to identify a group of gene and protein
doublets differentially over-expressed in that individual's malignancy.
Fig. 1 is a comparative analysis of protein expression prole by the
two-dimensional difference gel electrophoresis (2D-DIGE) analysis.
2D-DIGE can very effectively identify several over-expressed proteins
in the patient's tumor. Correlated DNA/RNA over-expression can then
be conrmed with microarray data. The resulting data is further
analyzed by a modeling and simulation computational system
developed by our team specically for clinical application including,
but not limited to, gene set enrichment analysis and network
inference modeling platforms (Fig. 2). Grouped gene expression
patterns that are highly correlated with the pathway phenotype in an
individual patient allow target genes to be selected, and prioritized
based on connectivity and vector-driven criteria developed analytic
network algorithms. Once this individual cancer ngerprint is
created, it serves as the template for the design, synthesis, and
subsequent validation of individualized therapeutic RNAi molecules
with knockdown activity against these high-degree hub genes for a
personalized and targeted cancer gene therapy.
2.2. RNA interference for cancer
The concept of antisense oligodeoxynucleotides as modulators of
gene expression and their application in targeted cancer gene therapy
was developed more than 25 years ago (for review, [10]). By processes
still unknown, the antisense nucleic acid (ASNA) strand and the mRNA
target come into proximity leading to the destruction of the mRNA
target either by endogenous nucleases, such as RNase H [11,12] that
are recruited into the mRNAASNA duplex or by intrinsic enzymatic
activity engineered into the ASNA sequence, as is the case with
ribozymes [13,14] and DNAzymes [15,16]. The discovery of an
evolutionarily conserved gene silencing mechanism whereby small
sequences of extrinsic dsRNA or intrinsic microRNA inhibit comple-
mentary post-transcriptional mRNA (siRNA) or suppress translation
(miRNA), respectively, ignited strong hope that the natural gene
silencing process would be specic and robust. The silencing process
occurs following interaction of the RNA effector precursors with the
RNase III enzymes Drosha (for miRNA) and Dicer (for miRNA and
siRNA) and subsequent formation of the RNA-interfering silencing
complex (RISC) [17]. Endonucleolytic cleavage of the target mRNA
occurs at a single site ~10 nucleotides from the 5 end of the guide
(antisense) siRNA sequence [18,19]. RNAi offers several advantages
over antisense and ribozyme approaches, including ease of synthesis
[20] and greater activity [18,2124].
Preclinical studies conrm that RNAi techniques can be used to
silence cancer-related targets [2535]. In vivo studies have also shown
favorable outcomes by RNAi targeting of components critical for
748 D.D. Rao et al. / Advanced Drug Delivery Reviews 61 (2009) 746759

Fig. 1. Analysis of the 2-D DIGE images by DeCyder Software and mass spectrometry protein identication. The upper panel shows on the left the protein expression pattern of a
normal lymph node from patient RW following 2-D gel electrophoresis. In the middle of the upper panel the protein expression pattern of a malignant lymph node from patient RW is
shown and to the right is an overlaid image with proteins from the normal lymph node labeled with Cy3 (green) and proteins from the malignant lymph node labeled with Cy5 (red).
Circles indicate protein spots with signicant expression level changes. The upper right panel shows the fold-of-change distribution curve and the level of change for the spots of
interest. The middle panel shows the 3D view of one protein spot change between the normal and malignant lymph nodes. Mass spectrometry (lower panel) subsequently (following
robotic spot picking) identied this protein as RACK1.

tumor cell growth [26,3639], metastasis [4042], angiogenesis
[43,44], and chemoresistance [4547].
3. Small interfering RNA (siRNA) and short hairpin RNA (shRNA)
The applications of RNAi can be mediated through two types of
molecules; the chemically synthesized double-stranded small interfer-
ing RNA (siRNA) or vector based short hairpin RNA (shRNA). Effective
RNAi was initially demonstrated by the application of synthetic siRNA
[48]; later, siRNA produced in vitro by T7 RNA polymerase was found to
be active and it was soon demonstrated that active siRNA consists of a
hairpin structure can be transcribed in cells from an RNA polymerase III
promoter on a plasmid construct [49,50]. Although siRNA and shRNA
can be applied to achieve similar functional outcomes, siRNA and shRNA
are intrinsically different molecules. Therefore, the molecular mechan-
isms of action, the RNA interference pathways, the off-target effects and
the applications can also be different.
3.1. siRNA
Fluorescent labeled siRNA has been used to trace the fate of
delivered siRNA. A uorescent label can either be tagged onto the 5
end or the 3 end of the siRNA. Using a uorescence resonance energy
transfer (FRET)-based visualization method, the intact siRNA can be
observed to be translocated into the nucleus within 15 min of the
delivery and then disseminated into the cytoplasm within the next 4 h
both in intact and dissociated form [51]. The initial accumulation of
siRNA in the nuclei is similar to observations made on the behavior of
antisense olignucleotides [52]. The translocation of antisense oligo-
nucleotide into the nuclei was not dependent on either the ATP pool or
temperature and thus may not involve the active import transport
system of the nuclear pore [52]; it is not clear whether siRNA
translocate into the nuclei using the same mechanism as antisense
olignucleotides. Using HeLa cells and targeting 7SK snRNA which is
exclusively located in the nucleus, Robb showed the efciency of
siRNA mediated silencing to be greater than that effected by antisense
7SK DNA [53]. Berezhna et al. observed nuclear localization of siRNA
targeted against small nuclear RNA (snRNA) and cytoplasmic
localization of siRNA targeted against viral mRNA suggesting selective
localization and compartmentalization of siRNA based on its intended
target [54]. Ago1 and Ago2 containing RISC were found both in the
cytoplasm and nucleus [5557]. A recent study using uorescence
correlation spectroscopy and uorescence cross-correlation spectro-
scopy (FCS/FCCS) to correlate the presence of siRNA with Ago2
 D.D. Rao et al. / Advanced Drug Delivery Reviews 61 (2009) 746759
Fig. 2. (A) Nearest neighbor proteinprotein (rst order) interactions of the 6 prioritized proteins in VisualCell. Second order interactions for Stathmin1 (SDCBP) (B) and RACK1
(GNB2L1) (C).
protein, indicated a shuttling of RISC between nucleus and cytoplasm
[58]. Importin 8 (Imp8) binds to all Ago proteins in a Ran-dependent
manner, but independently of RNA [59]. Knockdown of Imp8 results in
a shift of Ago2 from the nucleus to the cytoplasm without affecting the
total quantity of Ago2. However, although Imp8 is not required for
target mRNA cleavage it is necessary for Ago2 binding to miRNA
targets. In Caenorhabditis elegans, the argonaute protein NRDE-3 is
essential for binding nuclear RNAs and appears to interact with
cytoplasmic siRNAs generated by RNA-dependent RNA polymerase
(RdRP) followed by redistribution to the nucleus [60]. The nuclear
RISC (nRISC) is a complex that is 20 smaller in size than the cyto-
plasmic RISC (cRISC). The nucleus may be the check point controlling
distribution as either the nuclear acting siRNA or the cytoplasmic
acting siRNA.
Dynamically, siRNA steadily increases its accumulation in cells for
4 h before plateau [61]. The steady-state nuclear distribution of siRNA
was mainly found in the nucleolus region and was excluded from the
nucleoplasm [62]. The cytoplasmic distribution of siRNA appears to be
in the perinuclear region forming a ring-like pattern around the
nucleus [63]. The nucleolus and perinuclear regions are possibly the
main site for RNAi. However, Ohrt et al. labeled siRNA with uorescent
dye at the 3 end of either strand of siRNA and did not nd
accumulation of siRNA at the perinuclear region, but rather evenly
distributed throughout the cytoplasm [62]. This discrepancy may be
the result of the uorescent tagging process. At 48 h post injection, the
majority of siRNA appears to have been degraded with only 1%
uorescence remaining in the cell. The spatial and temporal distribu-
tion of siRNA within the cell is in accord with the observed kinetics of
749
siRNA mediated RNA interference activity which peaks around 24 h
post delivery and diminishes within 48 h.
The life-cycle of siRNA inside transfected cells is diagrammatically
illustrated in Fig. 3. In Drosophila, double-stranded RNA-binding
proteins (dsRBPs), such as R2D2 and Loquacious (Loqs), function in
tandem with Dicer (Dcr) enzymes in RNA interference (RNAi) [64
66]. Dcr-1/Loqs and Dcr-2/R2D2 complexes generate microRNAs
(miRNAs) and small interfering RNAs (siRNAs), respectively. Thus,
Loqs and R2D2 represent two distinct functional modes for dsRBPs in
the RNAi pathways [67]. In mammalian cells, only one Dicer gene has
thus far been identied [68]. Human Dicer is an integral component of
the RNA interference pathway. Dicer processes pre-microRNA and
double-strand RNA (dsRNA) to mature miRNA and siRNA, respec-
tively, and transfers the processed products to the RISC [69,70]. Since
there is only one Dicer in the human, the RNA-interfering pathway for
siRNA and for miRNA may not be as compartmentalized as for
Drosophila. Dicer is a multi-domain RNase III-related endonuclease
responsible for processing dsRNA to siRNAs [71]. Dicer preferentially
binds to the 5 phosphate of 2 nt 3 overhang and cleaves dsRNAs into
21 to 22 nucleotide siRNAs [72,73]. Mammalian Dicer interacts with
the double-stranded TatRNA-binding protein (TRBP) or PACT (PKR
activating protein) to mediate RNA interference and miRNA proces-
sing. TRBP and PACT are structurally related but exert opposite
regulatory activities on RNA-dependent protein kinase (PKR). Knock-
down of both TRBP and PACT in cultured cells leads to signicant
inhibition of gene silencing mediated by short hairpin RNA but not by
siRNA, suggesting that TRBP and PACT function primarily at the step of
siRNA production [74]. Human TRBP and PACT directly interact with
750 D.D. Rao et al. / Advanced Drug Delivery Reviews 61 (2009) 746759
Fig. 3. Schematic of the siRNA mediated RNA interference pathway. After entry into the cytoplasm, siRNA is either loaded onto RISC directly or utilize a Dicer mediated process. After
RISC loading, the passenger strand departs, thereby commencing the RNA interference process via target mRNA cleavage and degradation. siRNA loaded RISCs are also found to be
associated with nucleolus region and maybe shuttled in and out of nucleus through an yet unidentied process.
each other and associate with Dicer to stimulate the cleavage of
double-stranded or short hairpin RNA to siRNA [74]. Dicer knockout ES
cells can effectively load processed siRNA onto RISC and carry out RNA
interference as efciently as Dicer+ ES cells [68]. So, it appears that in
mammalian cells, a perfectly processed siRNA can be effectively
loaded onto RISC for RNAi without the help of the TRBP/PACT/Dicer
complex. The TRBP/PACT/Dicer complex, however, is required to
process either shRNA or long dsRNA to appropriate size and form for
their loading onto RISC.
Duplex siRNA in association with holo-RISC, composed of at least
Ago-2, Dicer and TRBP, is identied as the RISC loading complex (RLC)
[75]. In the RLC, the two strands of the duplex are separated, resulting in
the departure of the passenger strand [7678]. The passenger strand is
cleaved by the RNase-H like activity of Ago-2, provided there are
thermodynamically favorable conditions for passenger strand depar-
ture. This is referred to as the cleavage-dependent pathway [79]. There is
also a cleavage-independent by-pass pathway, in which the passenger
strand with mismatches is induced to unwind and depart by an ATP
dependent helicase activity [76,79,80]. The RISC with single-stranded
guide strand siRNA is then able to execute multiple rounds of RNA
interference. ATP is not required for shRNA processing, RISC assembly,
cleavage-dependent pathway, or multiple rounds of target-RNA
cleavage [8183]. Single-stranded siRNA (containing 5-phosphates)
and pre-miRNA can be loaded on RISC, but not duplex siRNA [84].
3.2. shRNA
shRNAs, as opposed to siRNAs, are synthesized in the nucleus of
cells, further processed and transported to the cytoplasm, and then
incorporated into the RISC for activity [85]. The life-cycle of shRNA
inside of transfected cells is diagrammatically illustrated in Fig. 4. To
be effective, the shRNA are designed to follow the rules predicated by
the specics of the cellular machinery and are presumably processed
similar to the microRNA maturation pathways. Thus, studies on the
synthesis and maturation of miRNAs have provided the groundwork
for the synthesis of shRNA [86], particularly the miR-30 based shRNAs
[87].
shRNA can be transcribed by either RNA polymerase II or III
through RNA polymerase II or III promoters on the expression cassette.
The primary transcript generated from RNA polymerase II promoter
contains a hairpin like stem-loop structure that is processed in the
nucleus by a complex containing the RNase III enzyme Drosha and
the double-stranded RNA-binding domain protein DGCR8 [88]. The
complex measures the hairpin and allows precise processing of
the long primary transcripts into individual shRNAs with a 2 nt 3
overhang [89]. The processed primary transcript is the pre-shRNA
molecule. It is transported to the cytoplasm by exportin 5, a Ran-GTP-
dependent mechanism [90,91]. In the cytoplasm the pre-shRNA is
loaded onto another RNase III complex containing the RNase III
enzyme Dicer and TRBP/PACT where the loop of the hairpin is
processed off to form a double-stranded siRNA with 2 nt 3 overhangs
[9294]. The Dicer containing complex then coordinates loading onto
the Ago2 protein containing RISC as described earlier for siRNA. Pre-
shRNA has been found to be part of the RLC; thus, pre-shRNA may
potentially directly associate with RLC rather than through a two steps
process via a different Dicer/TRBP/PACT complex [95].
After loading onto RLC and passenger strand departure; both siRNA
and shRNA in the RISC, in principle, should behave the same. The
argonaute family of proteins is the major component of RISC [96,97].
Within the Argonaute family of proteins, only Ago2 contains the
endonuclease activity necessary to cleave and release the passenger
strand of the double-stranded stem [76,77,79]. The remaining three
members of Argonaute family, Ago1, Ago3 and Ago4, which do not
have identiable endonuclease activity, are also assembled into RISC
and presumably function through a cleavage-independent manner.
Thus, RISC can be further classied as cleavage-dependent and
cleavage-independent [79].
The argonaute family of proteins in RISCs are not only involved in the
loading of siRNA or miRNA, but also implicated in both transcriptional
(targeting heterochromatin) and post-transcriptional gene silencing.
Ago protein complexes loaded with passenger strandless siRNA or
 D.D. Rao et al. / Advanced Drug Delivery Reviews 61 (2009) 746759 751

Fig. 4. Schematic of the shRNA mediated RNA interference pathway. After delivery of the shRNA expression vector into the cytoplasm, the vector needs to be transported into the
nucleus for transcription. The primary transcripts (pre-shRNA) follow a similar route as discovered for the primary transcripts of microRNA. The primary transcripts are processed by
the Drosha/DGCR8 complex and form pre shRNAs. Pre-shRNAs are transported to the cytoplasm via exportin 5, to be loaded onto the Dicer/TRBP/PACT complex where they are
further processed to mature shRNA. Mature shRNA in the Dicer/TRBP/PACT complex are associated with Argonaute protein containing RISC and provide RNA interference function
either through mRNA cleavage and degradation, or through translational suppression via p-bodies.

miRNA seeks out complementary target sites in mRNAs, where
endonucleolytically active Ago-2 cleaves mRNA to initiate mRNA
degradation [98,99]. Other Ago protein containing complexes without
endonucleolytic activity predominantly bind to partially complemen-
tary target sites located at the 3 UTR for translation repression through
mRNA sequestration in processing bodies (p-bodies) [100102]. The
detailed mechanism of mRNA sequestration in p-bodies and later
release from p-bodies is still a debated issue; deadenylation of the target
mRNA which leads to destabilization of the mRNA was also observed to
occur in p-bodies [103,104]. Coimmunoprecipitation experiments
determined that RISCs are also strongly associated with polyribosomes
or the small subunit ribosomes [95] and Ago-2 (actually identied as
elF2c2), strongly suggesting that RISC surveillance is compartmenta-
lized with translational machinery of the cell. Details of the mechanism
involving mRNA scanning and target mRNA identication are still
largely unknown. Whatever the scanning or surveillance mechanism
may be, once the target mRNA is identied, the target mRNA is either
cleaved or conformationally changed following which both types of
structures are routed to the p-body for either sequestration or
degradation [103,104]. The active siRNA or miRNA loaded complex is
then released for additional rounds of gene silencing activity.
3.3. Bi-functional shRNA
There is, however, a third unique RNAi option in development
called bi-functional shRNA. shRNA can potentially be manipulated to
take advantage of the gene silencing machinery within the cells to
improve its efciency and durability of action. Conceptually, targeted
shRNAs can be designed so as to effectively load shRNA onto both the
cleavage-dependent and the cleavage-independent RISCs. This differ-
ential processing is mediated by two pathways primarily dependent
on strand complementarity and/or access to RNase-H cleavage and,
presumably, for nal target effect, on interaction with Imp8.
Simultaneous expression of both types of shRNAs (i.e. the bi-
functional shRNA) in cells should achieve a higher level of efcacy,
greater durability compared to siRNA, and a more rapid onset of gene
expression silencing (the rate dependent on mRNA turnover and
protein kinetics) compared to shRNA as illustrated on Fig. 5. The bi-
functional shRNA, by virtue of loading onto multiple types of RISCs, is
thus able to simultaneously induce degradation of target mRNA and
also inhibit translation through mRNA sequestration. This bi-func-
tional design should be, in principal, much more efcient for two
reasons; rst, the bi-functional will promote loading the guide strand
onto at least two types of RISCs to increase activity; second, by loading
onto both cleavage-dependent RISC and cleavage-independent RISC,
target mRNA can be silenced both through mRNA degradation and
translational inhibition or sequestration.
The design of the bi-functional shRNA expression unit consists of two
stem-loop shRNA structures; one stem-loop structure composed of fully
matched passenger and guide strand for cleavage-dependent RISC
loading, the second stem-loop structure composed of mis-matched
passenger strand (at the positions 912) for cleavage-independent RISC
loading.
There are several experimental observations that support this
approach. In Drosophila, Ago1 preferentially binds to miRNAs that
have been excised from imperfectly paired hairpin precursors, whereas
those miRNAs that have near-perfectly paired hairpin precursors are
bound by Ago2 [105108]. In HEK293 cells transfected with tagged-Ago
proteins, coimmunoprecipitation found similar sets of about 600
transcripts to be bound to Ago1, 2, 3 or 4 [95], suggest all four
mammalian Ago protein containing RISCs are involved in the RNAi
function. Insofar as most mRNA have multiple miRNA target sites (with
distance constraints) at their 3 UTR, the miRNA mediated RNAi system
appears to be redundant for the targeted mRNAs allowing for
cooperative downregulation to ensure target mRNA knockdown. The
bi-functional shRNA approach mimics the natural process by mediating
target mRNA knockdown through multiple RNAi pathways and
complexes.
752 D.D. Rao et al. / Advanced Drug Delivery Reviews 61 (2009) 746759
Fig. 5. Schematic of the bi-functional shRNA concept. The bi-functional concept is to design two shRNAs for each targeted mRNA; one with perfect match, one with mismatches at the
central location (bases 912). The purpose of the bi-functional design is to promote loading of mature shRNAs onto both cleavage-dependent and cleavage-independent RISCs, so that
the expression of target mRNA can be more effectively and efciently shut down both through target mRNA degradation and through translational repression.
In C. elegans, structural features of small RNA precursors determine
Argonaute loading [109]. Recently, Azuma-Mukai et al. observed
miRNAs associated with hAgo-2 and hAgo-3 have some overlaps;
however, some are discriminately loaded onto hAgo-2 or hAgo-3
[110]. Further work is needed to resolve the specicity of miRNA
loading onto different Ago containing RISCs. Although most miRNA
target sites have been identied to be located at the 3-UTR region,
recent systemic identication of mRNAs recruited to hAgo-2 have
identied many mRNAs with target sites located at the coding region
and some at the 5-UTR [111]. hAgo-2 could initiate the target mRNA
degradation with its slicing activity in the coding region. Tay et al.
recently found many naturally occurring miRNA targets are located in
the coding region of embryonic regulated genes to modulate
embryonic stem cell differentiation [112], further support that
miRNA can act through mRNA regions other than 3-UTR.
3.4. Summary of si/sh/bi
In summary, exogenously introduced siRNA with appropriate
length (1921 nt) and 2 nt 3 overhang, can be loaded onto RISC for
RNAi function without interacting with either Dicer, TRBP or PACT;
however, the loading process is10 less efcient than shRNA.
Increasing the length of the siRNA duplex to 2930 nt with a 2 nt 3
overhang only at one end of the duplex (specically antisense [113])
appears to improve efcacy [114]. If so, it is possibly because
increasing the length of an siRNA duplex with an unprocessed end
forces directionality as a result of imposed thermodynamic instability
determining the guide strand motif and thereby enhancing its
association with Dicer/TRBP/PACT complex for more efcient loading
onto RLC [114]. shRNA, on the other hand, assimilates into the
endogenous miRNA pathway and in so doing is signicantly more
efcient [115117]. Additionally, uorescent tagged siRNA tracing
indicated high degradation and turnover of exogenously introduced
siRNA. Less than 1% of the introduced duplex remains in the cell 48 h
after administration. shRNA can be continuously synthesized by the
host cell, therefore, its effect should be much more durable.
Concentrations necessary for effective knockdown are usually in the
low nM range for most siRNAs, while less than 5 copies of shRNA
integrated in the host genome is sufcient to provide continual gene
knockdown effect (Cleary M, personal communication). The higher
dose required for siRNA can further contribute to the off-target effects
to be discussed later.
Understanding the mechanism and the dynamics of siRNA and
shRNA at the cellular and molecular level greatly enhanced the effort
in developing therapeutic siRNAs for various diseases. As a result,
modications can be made to improve the efcacy and stability of
RNAi agents. Chemically synthesized siRNA is easier to modify
through chemistry; however, bulk manufacturing of complex struc-
tures such as modied siRNA is more expensive. Vector based shRNA
relies on the host machinery for expression, but, on the other hand, is
more difcult to modify. Modication can only be achieved through
manipulating expression strategy (e.g. bi-functional shRNA), rede-
signing shRNA structure, or by varying promoter regulation. The
shRNA expression units can be incorporated into varieties of plasmids
and viral vectors for delivery and integration. In addition, vector based
shRNA expression can also be regulated or induced [118120].
Numerous siRNAs have been demonstrated to be effective for in-
vivo tumor growth modulations via intratumoral, ex-vivo, or systemic
routes of application (For review see [121123]. Vector based shRNA
has, likewise, demonstrated in-vivo effectiveness (For review see
[121,123,124]. In-vivo studies employ a variety of delivery methods
and may not ensure equivalency of strand biasing; therefore, it is hard
to perform direct comparison between siRNA and shRNA. McAnuff et
al., using a luciferase expression system, compared the potency of
siRNA versus shRNA mediated knockdown in vivo; they found that
siRNA and shRNA are equivalent in potency at 10 mg dose; however,
on a molar basis, the shRNA was 250 fold more effective than the
siRNA [115]. In an effort to assess the potential of RNAi as a therapeutic
for hepatitis C (HCV), siRNANS5B was targeted against the non-
structural protein 5B viral polymerase coding region fused with
luciferase gene [125]. Luciferase expression in vivo was reduced by
75%. Using a cognate shRNANS5B produced a 92.8% average inhibition
over 3 experiments. McCleary et al. incubated shRNA, directed against
rey luciferase and containing 29 mer stems and 2-nt 3 overhangs,
 D.D. Rao et al. / Advanced Drug Delivery Reviews 61 (2009) 746759
with recombinant human Dicer [116]. The resulting 22-nt shRNA
products, with predictable guide strands, were compared to identi-
cally targeted 21 mer siRNA in HeLa cells. More effective inhibition
was seen with the shRNA. In another study of the feasibility of RNAi for
treatment of HCV, 19 and 25 bp shRNAs were compared with 19 and
25 bp siRNA directed against the HCV IRES (internal ribosomal entry
site) using a luciferase reporter in the AVA5 cell line with stable
expression of the HCV subgenomic genotype 1b replicon [117]. The
19 bp shRNAs were more potent than either the 19- or 25-bp siRNAs.
A recent paper evaluated the levels of Dicer and Drosha in cell lines
and in tumor samples from patients with ovarian cancer [126]. The
distribution of Dicer mRNA levels was bimodal and 60% of specimens
had decreased Dicer mRNA. Furthermore, low Dicer levels were found
to be a predictor of reduced disease-specic survival in multivariate
analysis. Of particular interest was the nding that, compared to siRNA
mediated silencing of the galectin-3 gene, poor silencing was achieved
with shRNA in ovarian cancer cell lines with low versus high Dicer
expression. These data will need to be conrmed and evaluated
further. Although there is a global downregulation of miRNA
expression in cancer [127], whether this is, in large part, due to low
Dicer expression or to shortened 3 UTRs with fewer miRNA-binding
sites [128] in highly proliferating tumors with modulated feedback
mechanisms is not known. miRNA functionality has been conrmed in
the three tumor types (ovary, breast, and lung) evaluated in this study
raising questions regarding both qualitative and quantitative issues.
As the authors note, there are data correlating high Dicer expression
with poor prognostic features in other tumor types [129,130]. In their
retrospective evaluation of lung cancer, the role of let-7 as both a
regulator of Dicer and ras and their feedback networks could not be
assessed [131,132]. The Dicer levels in the tumors used for functional
assay of gene silencing are not given nor is there conrmation of the
equivalency of strand biasing between the siRNA and shRNA
constructs.
RNAi therapeutics have been shown to be well tolerated in
numerous animal models allowing for transition into the clinic. At
least 10 RNAi-based drugs are currently in early phase clinical trials
[133], two of which are cancer related; one targeted against the M2
subunit of ribonucleotide reductase (RRM2) [134] and the other
targeted against tenascin-C [135]. Animal studies with siRNA
inhibitors for RRM2 show efcacy [134] and safety in non-human
primates [136]. shRNA for the treatment of hepatitis B was also
approved for clinical trial by FDA.
Both siRNA and shRNA have their respective advantages and
disadvantages from the mechanistic point of view. However, safety of
this new therapeutic paradigm is of the utmost importance. Although
no signicant adverse event involving initial RNAi-based clinical trials
has been reported, there are concerns over the potential off-target
effect of RNAi-based agents which will be discussed separately below.
4. Delivery
Efcacy of an RNAi cancer therapeutic is limited by the quantity of
the oligomer that effectively enters the tumor cells. In the clinical
setting this is primarily dependent on the method of delivery. An ideal
delivery vehicle must be able to selectively and differentially target
tumors versus normal tissue, homogeneously distribute through the
tumor mass and penetrate the tumor cells following systemic
administration. If cell entry is mediated by endocytosis the delivery
vehicle must negotiate endosomal/lysosomal escape and, in the case
of shRNA, the payload must penetrate the nuclear membrane as well.
Viral vectors are popular for laboratory delivery of shRNA because of
their high transfection efciency and effective integration of exogen-
ous DNA, but they have been losing support in recent years because of
concerns over safety and immunogenicity [137,138]. Non-viral poly-
meric delivery systems, in particular those with biodegradable
components, have much better safety proles than their viral
753
counterparts though their transfection efciency is generally lower
[139141]. Non-viral vehicles for delivery of siRNA and shRNA are
typically cationic preparations. Their positive charge facilitates
complexation with negatively charged nucleic acids and also binding
to the negatively charged glycocalyx on external cell membranes
promoting endocytosis. Both tumor targeting and cell entry can be
enhanced by decoration or complexation of the vehicle with targeting
moieties, such as monoclonal antibodies, peptides, small molecule
ligands, and aptamers to recognize cell surface markers [124,142].
Once endocytosed, the vehicle's positive charge facilitates early escape
from the endosome [143,144]. Though the positive charge of these
vehicles improves their transfection efciency, it is also associated
with increased toxicity [140,145].
A wide variety of potential vehicles are being developed to address
the different issues associated with the delivery of shRNA and siRNA.
There are three major classes of non-viral delivery vehicle systems:
synthetic polymers, natural/biodegradable polymers, and lipids;
many of the vehicles that are showing promise are actually hybrids
of these classes. For instance, there is a cyclodextrin-based cationic
polymer which has been used successfully to deliver siRNA targeted to
RRM2 in various in vivo cancer models [134,146]. This preparation is
currently in Phase I clinical trials. Lipid based nanoparticles are
showing potential for the delivery of shRNA and siRNA [147]. Protiva
Biotherapeutics and Alnaylam have developed nanoparticles com-
posed of a lipidPEG conjugate that is capable of encapsulating and
protecting nucleic acids for the purpose of systemic delivery. These
stable nucleic acid lipid particles (SNALPs) were used in the rst
successful administration of siRNAs to a non-human primate
[148,149]. Silence Therapeutics has developed a lipid-based delivery
vehicle specically designed for siRNA delivery to endothelial cells.
This vehicle, called AtuPLEX, contains a mix of cationic and fusogenic
lipids [150,151]. This vehicle has been used effectively to knockdown
protein kinase N3 in murine prostate and pancreatic cancer models,
inhibiting cancer progression [152,153]. More detailed discussions of
delivery vehicles for shRNA [124,154] and siRNA [141,155157] as well
as general discussions of organ and tissue specic RNAi delivery
[138,158,159] may be found elsewhere.
The magnitude of cytokine induction associated with in vivo
delivery of siRNA has been noted to vary widely based on the delivery
vehicle used [160]. The well recognized conundrum in cationic non-
viral nucleic delivery is that transfection efciency usually correlates
with toxicity [161,162]. Effective strategies being pursued to break this
correlation include molecular modications to shield positive charge
and the use of biodegradable polymers [154,163].
5. Off-target effects
Despite initial results showing excellent specicity in RNAi
mediated gene silencing, over the past 5 years many studies have
shown that there are multiple specic and nonspecic mechanisms
through which siRNA and shRNA can cause effects other than the
intended mRNA suppression. Unintended effects on gene expression
mediated by RNAi are termed off-target effects. Specic off-target
effects are mediated by partial sequence complementarity of the RNAi
construct to mRNAs other than the intended target. Nonspecic off-
target effects include a wide variety of immune and toxicity related
effects that are intrinsic to the RNAi construct itself or its delivery
vehicle. The following provides a brief review to the mechanisms
surrounding off-target effects and addresses strategies that are being
developed to minimize those effects.
5.1. Specic off-target effects
Expression proling experiments have shown that partial sequence
complementarity in the passenger or guide strands of the RNAi
construct can produce off-target gene suppression. The rst group to
754 D.D. Rao et al. / Advanced Drug Delivery Reviews 61 (2009) 746759
denitively demonstrate this used an unbiased genome-wide micro-
array prole to search for downregulated mRNA immediately after
transfecting a variety of different siRNA constructs directed to different
genes into HeLa cells. This study demonstrated that few off-target genes
were regulated in common. Furthermore, the off-target expression
patterns observed for each individual siRNA were consistent, with
repeated runs revealing the same off-target expression prole. It was
also noted that the number and identity of the off-target transcripts was
unrelated to the ability of the siRNA to silence the target gene [164]. It
has subsequently been shown that both siRNA and shRNA constructs
with complementarity in the seed region can produce the same off-
target expression proles, even across cell lines, independent of delivery
method [165].
Even limited sequence siRNA:mRNA complementarity with the
intended target, which is usually less than optimal, can produce off-
target suppression. Off-target silencing effects have been demon-
strated in transcripts with complementarity as low as 7 nucleotides
with the guide siRNA strand [166]. Due to a variety of known and
unknown mechanisms, not all transcripts with this level of similarity
are silenced. The location of the region of complementarity within the
RNAi construct and the mRNA transcript are important predictors of
potential for suppression. Complementarity within nucleotides 27 at
the 5 end of either the siRNA passenger or guide strands has been
shown to be a key determinant in directing off-target effects [167].
This region of the construct is reminiscent of the seed region within
miRNA, i.e., a heptameric sequence beginning at the rst or second
position from the 5 end of the miRNA that is complementary to sites
in the mRNA 3-UTRs, which guide silencing in endogenous RNAi.
Indeed, RNAi off-targeting seems to be mechanistically related to an
miRNA-like effect in that complementarity between the miRNA seed
hexamer and the 3UTR of the off-targeted mRNA produce effective
suppression of gene expression [103,168,169].
Various in vitro and in silico methods are either available or under
development for analysis of the off-target effects of a given RNAi
construct. Screening can be accomplished effectively using mRNA
expression data from transfected cells. Expression proling for off-
target screening must be temporally controlled to ensure observation
of primary changes in mRNA levels and not secondary changes as a
result of downregulation of target protein expression. Results can then
be correlated to qRT-PCR data and to protein expression data through
Western blots. Methods for microarray-based off-target screening are
reviewed in detail elsewhere [169,170].
Computational methods are also being developed so that RNAi
constructs may be more effectively screened prior to in vitro testing.
Global complementarity search algorithms such as BLASTn and
SmithWaterman have been shown to be poor measures of off-target
potential in an RNAi construct [168]. Methods for in silico screening of
RNAi constructs must be optimized to consider seed complementarity
in the constructs as well as 3 UTR complementarity in the
transcriptome. Several groups are working to develop algorithms
and some are freely available online [171174].
The specic off-target effects of a given construct can be mitigated by
several methods. siRNAs with an asymmetric unilateral 2-nt-overhang
on the antisense strand have greater potency than conventional siRNAs
as well as reduced off-target effects due to preferential strand selection
[113]. Sequence based modications designed to reduce specic off-
target effects are likely to benet both siRNA and shRNA approaches.
Single and double base mismatches between an RNAi construct and its
target transcript are often tolerated without reducing the potency of
suppression [168,175177]. This allows for the optimization of the
construct sequence to minimize complementarity with 3UTRs of
unintended targets.
Although the vector-driven shRNA approach to RNAi does not
permit specic chemical modication of the silencing construct, siRNA
oligomers can be chemically modied in order to reduce direct off-
target effects. These modications must be carefully applied as
reductions in off-target effects are sometimes accompanied by
deceases in the overall potency of suppression. [178180].
Chemical modications can also be asymmetrically applied to the
passenger strand of the siRNA construct in order to specically inhibit its
participation in silencing. Annealing of the guide strand to a passenger
strand composed of two segments has been shown to reduce off-target
effects mediated by passenger strand complementarity [181]. The
addition of other chemical moieties to one or both strands can also
limit specic off-target effects. The 5-phosphorylation status of the
siRNA strands has been shown to be a determinant of which strand is
involved in silencing, and thus replacement of the 5 phosphate by of an
siRNA strand with a methyl group has been shown to reduce its
participation in silencing [182].
Chemical modications to nucleotides within the seed region of
the passenger or guide strand can reduce unintended entry of siRNA
constructs into the endogenous miRNA gene silencing pathway by
inhibiting the interaction of the RISC complex with mRNA. It has been
shown that 2-O-methyl ribosyl substitution at position 2 in the siRNA
guide strand can reduce off-target silencing of transcripts with
complementarity to the seed region of the siRNA guide strand [165].
Asymmetric replacement of seed region nucleotides with DNA bases
has also been shown to reduce off-targeting as a result of seed region
complementarity within the passenger strand [183].
Recent in vitro studies have shown that shRNA produces fewer off-
target effects than siRNA. In one study shRNA and siRNA of the same
core sequence directed towards TP53 were applied to HCT-116 colon
carcinoma cells in concentrations necessary to achieve comparable
levels of target knockdown. Microarray proling demonstrated a
much higher degree of up- and downregulation of off-target
transcripts in the siRNA transfected cells (M. Mehaffey, T. Ward, and
M. Cleary, in prep.). It has been suggested that these differences arise
from the fact that shRNA is transcribed in the nucleus and is therefore
subject to endogenous processing and regulatory mechanisms.
Additionally, siRNA is more susceptible to degradation in the
cytoplasm, which may also lead to off-target silencing [184].
5.2. Nonspecic off-target effects
Nonspecic off-target effects are those unintended perturbations
in gene expression not resulting from the direct interaction of an RNAi
construct with an mRNA transcript. Included in this category are
interferon (IFN) and other immune mediated responses to exogenous
RNAi, cellular toxicities due to the nucleotide construct, and effects
related to the delivery vehicle. There is strong reason to believe that
shRNA and siRNA would have very different proles of nonspecic off-
target effects because of the mechanistic differences between the two
approaches.
Introduction of dsRNA longer than 2930 bp into mammalian cells
results in a potent induction of the innate immune system via PKR,
similar to the mammalian cell defense mechanism against viral
infection [185]. Activation of the innate immune response by receptors
sensitive to exogenous nucleic acids leads to global degradation of
mRNA and thus broad inhibition of translation as well as global
upregulation of IFN-stimulated gene expression. Although shorter
siRNA constructs have been shown to avoid receptor activation and
were initially considered to be non-immunogenic [18], subsequent in
vitro data have shown that the introduction of both synthetic siRNA
oligomers and shRNA can induce a partial interferon response [186
188] some of which are sequence dependent (e.g. GU-dependent, 5-
UGUGU-3 and GU-independent, 5-GUCCUUCAA-3) and, therefore,
avoidable. Activation of the innate immune system in the case of
exogenous RNAi is likely mediated through several cytoplasmic and
endosomal mechanisms attuned to recognize exogenous nucleic acids
from infectious agents. The relevant mediators of nucleic acid-
stimulated immunoactivation at the level of the endosome are Toll-
like receptors (TLRs) 7 and 8 (typically activated by ssRNA), TLR 9 (via
 D.D. Rao et al. / Advanced Drug Delivery Reviews 61 (2009) 746759
unmethylated CpG activation) and TLR 3 (via dsRNA) [189193].
Immune activation by nucleic acids at the cytoplasmic level is
mediated through RNA sensing receptors such as RIG-I and MDA-5
[114,194]. Mechanisms surrounding immune activation by RNAi are
reviewed more thoroughly elsewhere [160,195].
Recently, naked siRNA has been shown to activate TLR-3 on the
surface of vascular endothelial cells and trigger the release of IFN-
and IL-12 that mediate nonspecic anti-angiogenic effects in-vivo
[196]. Immune stimulation often is largely responsible for the
observed therapeutic effects of siRNA rather than the direct targeted
effect [197]. Misinterpreting the therapeutic effect of siRNA needs to
be carefully monitored.
Immune activation at the endosomal level is more readily avoidable
by shRNA constructs because the construct is presented on a DNA
plasmid obviating dsRNA activation of TLR 3. However, TLR 9, as noted, is
activated by unmethylated CpG motifs, which are typically found in
bacterial DNA [198,199]. Careful shRNA-encoding plasmid design,
avoiding unmethylated CpG motifs, can effectively attenuate if not
eliminate TLR 9 mediated endosomal immunoactivation [200].
There is also reason to believe that shRNA is less likely to induce an
inammatory response through cytoplasmic dsRNA receptors in vivo
because shRNA is spliced by endogenous mechanisms. It has been
suggested that the 5 ends of the endogenous-dicer spliced RNA
oligomers are less immunogenic than the 5 ends of exogenous siRNA
oligomers [114,194]. This has been supported in vitro in an experiment
that compared liposome delivered siRNAs versus Pol III promoter
expressed shRNAs of the same sequence in primary CD34+ progenitor
derived hematopoietic cells. In this study it was found that siRNA
induced IFN-alpha and type I IFN genes, while the shRNA of the same
sequence did not induce an immune response [201].
Sequence modications can be made to shRNA or siRNA in order to
reduce immunogenicity. It has been shown that endosomal immu-
noactivation by siRNA through TLR7 and TLR8 can be sequence
dependant [189,190,202]. Some simple sequence modications, such
as the introduction of G:U mismatches into the sequence also seem to
lower the IFN response in vitro [203,204]. One recent study showed
that a marked reduction in the expression of the interferon-stimulated
gene oligoadenylate synthetase 1 (Oas1) could be achieved by
modifying the shRNA to contain features of the naturally occurring
microRNA-30 (miR-30) precursor [205].
As in the case of specic off-target effects, chemical modication to
siRNA oligomers can make them less immunostimulatory, however
these modications must be ne-tuned so as not to negatively affect
the potency of intended target silencing. Suppression of the TLR
mediated immune response has been achieved by substituting the 2-
hydroxyl uridines in the construct with 2-uoro, 2-deoxy, or 2-O-
methyl uridines [206208], the products of which do retain target-
silencing potency while reducing immunogenicity.
Usage of endogenous processing systems gives shRNA an advan-
tage over siRNA in terms of its propensity for induction of IFN but its
over-saturation of these systems has been shown to have other
consequences that are more easily avoided by siRNA. In a key in vivo
study of the safety effects of long term expression of shRNA in the
livers of adult mice, a type 8 adeno-associated virus with a Pol III
promoter was used to drive expression of 49 different shRNAs of
different lengths and sequences directed against six targets. 36 of the
constructs tested resulted in a dose dependant liver injury that was
determined to be associated with the downregulation of critical
endogenous miRNAs. The degree of miRNA downregulation was
related neither to the shRNA sequence nor to the degree of
downregulation of the target mRNA [209]. Subsequent transfection
studies suggested the degree of miRNA downregulation to result from
a competitive bottleneck in shared miRNA/shRNA processing, most
likely at the level of exportin-5 (the nuclear membrane export protein
used to transfer pre-miRNAs into the cytoplasm) and the RISC
component, Argonaute-2 [210]. A similar in vivo study of hepatic
755
toxicity of siRNAs in mice and hamsters showed that systemic
introduction of synthetic siRNAs does not result in the suppression
of endogenous miRNA levels. In this study siRNAs targeting two
hepatocyte-specic genes (apolipoprotein B and factor VII) and a
scramble control were administered to mice and one siRNA targeting
the hepatocyte-expressed gene Scap was administered to hamsters.
Robust suppression of target genes was achieved in all cases. No
changes in levels of the hepatocyte endogenously expressed miR-122
were observed in the mice or the hamsters and no changes in the
broadly expressed miRNAs, miR-16 and let-7a, were observed in the
mice [211].
Though siRNA likely does not compete with endogenous miRNA
for processing proteins, care must be taken when using shRNA as an
effector of RNAi in order to minimize the potential for damage
mediated by over-saturation of exportin-5. In one in vitro study, over-
expression of the exportin-5 protein has been shown to eliminate the
nuclear export bottleneck and allow cells to tolerate higher dosages of
shRNA without toxicity [212]. Another study showed that an adeno-
associated virus construct using a Pol-II promoter was able to achieve
stable target gene suppression at high shRNA doses for over 1 year
after the initial dosing [213]. In order to minimize the risk of toxicity
from over-saturation of miRNA professing systems, data to date
suggest both selective promoter integration (e.g. pol II versus pol III)
and limiting the dosage of shRNA so as to stay below the threshold of
competitive inhibition of the endogenous miRNA biogenesis
machinery.
6. The future outlook
The ability to precisely and differentially target functionally bio-
relevant molecular signals in patient's cancers will establish a new
paradigm in cancer management; one which focuses on dening the
uniqueness of each patient's tumor and tumor-host processes and
interactions following rational target prioritization using computa-
tional systems biology algorithms. This, then, would allow for
exploitation of the attack vulnerability of the rewired cancer
network by deconstructing essential hubs and linkages, multiply
targeting and eliminating them. Both siRNA and shRNA effectors are
attractive opportunities. The capability of potentiating activity using a
bi-functional design may further enhance safety and efcacy. The
simplicity of siRNA manufacturing and the transient nature of the
effect per dose may be optimal for certain medical disorders in which
high vector doses are required, e.g. some of the viral infections,
however, by using the endogenous processing machinery, optimized
shRNA constructs allow for high potency sustainable effects using
low-copy numbers resulting in less off-target effects (particularly if
embedded in an miRNA scaffold) thereby ensuring greater safety.
Though shRNA seems ideal for cancer-related therapeutic develop-
ment, new technology such as bi-functional RNA interference may
provide an even greater opportunity for enhancement in potency as
well as heightening safety thereby increasing the opportunities for
multiple target therapy. This, of course, is contingent on optimization
of delivery and minimization of off-target effect which will need to be
established through early clinical testing.
7. Conclusions
Our understanding and application of RNAi has dramatically
advanced over the last 5 years. Despite limitations in developing
effective delivery vehicles and concerns regarding potential off-target
activity, clinical development has been initiated. As the science of this
edgling technology advances, it is evident that issues such as target
selection, effector potency, delivery vehicle design, and off-target
effects will continue to be addressed and resolved. Bi-functional RNAi
products are evolving components in this transition to clinically
effective and safer therapeutics.
756 D.D. Rao et al. / Advanced Drug Delivery Reviews 61 (2009) 746759
Acknowledgements
The authors would like to acknowledge Brenda Marr and Susan
Mill for their competent and knowledgeable assistance in the
preparation of this manuscript, and M. Cleary for providing the data
before publication.
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