Oncogene dependency and the potential of targeted, RNA
interference-based anti-cancer therapy
Ruiyang Yan*,†, Andrew Hallam*, Peter G. Stockley†,‡, and Joan Boyes*,‡
*Institute
of Molecular and Cellular Biology, University of Leeds, Leeds LS2
9JT, United Kingdom. †Astbury Centre for Structural Molecular Biology,
University of Leeds, Leeds, LS2 9JT, United Kingdom
Corresponding authors:
‡Peter
G. Stockley, Astbury Centre for Structural Molecular Biology,
University of Leeds, Leeds LS2 9JT, UK
Phone: 44 (0) 113 343 3092
Email: p.g.stockley@leeds.ac.uk
‡Joan
Boyes, Institute of Molecular and Cellular Biology, University of Leeds,
Leeds LS2 9JT, United Kingdom.
Phone: 44 (0) 113 343 3147
E-mail: j.m.boyes@leeds.ac.uk
1
Abstract
Cancers arise through the progression of multiple genetic and epigenetic
defects that lead to deregulation of numerous signalling networks. However,
the last decade has seen the development of the concept of ‘oncogene
addiction’, where tumours appear to depend on a single oncogene for
survival. RNA interference (RNAi) has provided an invaluable tool in the
identification of these oncogenes and oncogene-dependent cancers, and also
presents great potential as a novel therapeutic strategy against them.
Although RNAi therapeutics have demonstrated effective killing of oncogenedependent cancers in vitro, their efficacy in vivo is severely limited by effective
delivery systems. Several virus-based RNAi delivery strategies have been
explored but problems arose associated with high immunogenicity, random
genome integration and nonspecific targeting. This has directed efforts
towards non-viral formulations, including delivery systems based on virus-like
particles, liposomes and cationic polymers, which can circumvent some of
these problems by immunomasking and the use of specific tumour-targeting
ligands. This review outlines the prevalence of oncogene-dependent cancers,
evaluates the potential of RNAi-based therapeutics and assesses the relative
strengths and weaknesses of different approaches to targeted RNAi delivery.
Summary statement
This review focuses on the tremendous possibilities brought about
by the emerging concept of oncogene addiction and the use of RNAi
technology in creating a potentially revolutionising way of combating
cancer.
Short title: RNAi therapeutics for oncogene-dependent cancers
Keywords: Cancer, Oncogene addiction, Targeted therapy, RNAi delivery
Abbreviations used: AD, adamantane; CDP, cyclodextrin-containing polymers; CML, chronic
myeloid (or myelogenous) leukemia; ECE-1, endothelin converting enzyme 1; EGFR,
epidermal growth factor receptor; EPR, enhanced permeability and retention; HCC,
hepatocellular carcinoma; IKBKE, I Kappa B Kinase ε; IRF4, interferon regulatory factor 4;
miRNA, microRNA; MM, multiple myeloma; NSCLC, non-small cell lung carcinoma; PEG,
polyethylene glycol; precursor miRNA, pre-miRNA; pri-miRNA, primary miRNA; PSMA,
prostate-specific membrane antigen; RISC, RNA-induced silencing complex; RNAi, RNA
interference; RRM2, ribonucleotide reductase subunit M2; RSV, Rous Sarcoma virus;
SNALPs, stable nucleic acid lipid particles; Tf, transferrin.
2
1. Introduction
Cancer cells are remarkable in that they are able to display an
extraordinary array of properties to promote their survival and growth. Being
able to proliferate unchecked by the immune system, not responding to
apoptotic or anti-growth signals and their ability to harness support from other
cells, such as macrophages are just a few examples [1, 2].
To be able to maintain these properties, a complex collection of
signalling pathways must be affected, both intra- and extracellularly. Exactly
how cancerous cells are able to achieve this was unknown until the discovery
of oncogenes and the subsequent emergence of evidence for oncogene
dependency.
1.1 Discovery of Oncogenes
The detection of the first oncogene, SRC, took decades to achieve. Rous’
preliminary experiments showed that cell filtrates from chicken sarcomas
could cause cancer in healthy chickens, suggesting the presence of cancerinducing agents within the tumour, which were found to be the virus that
became known as the Rous Sarcoma Virus (RSV) [3]. Such tumour viruses
provided the first window in the genetics of cancer biology.
Successive experiments by Baltimore and Temin revealed that RSV had
an RNA genome that could convert back to DNA and integrate itself within the
host’s genome [4, 5]. This discovery identified that a genetic element of the
viral genome was responsible for transforming cells, which was subsequently
narrowed down to a single gene, the SRC oncogene [6]. Homologues of SRC
were then found in several species of bird and later across all vertebrates [7],
suggesting that the viral oncogene had been acquired from host cells during
evolution. This was a crucial development in cancer research as it implied the
presence of oncogenes within the host genome that may cause cancer if
mutated. This realisation led to the term ‘proto-oncogene’, describing a normal
gene present within the genome of the organism, which, when mutated or
overexpressed, can induce tumorigenesis. Since then, more than 40 protooncogenes have been identified within the human genome.
1.2 The Two-hit Hypothesis
The two-hit hypothesis was proposed following the observation in
retinoblastoma patients that two mutations, one in each allele of a tumour
suppressor gene, are needed to trigger retinoblastoma [8]. A tumour
suppressor gene encodes a protein which inhibits cell division when the cell is
under unfavourable stress [9]. Knudson postulated that one faulty copy of a
gene could be inherited but a further somatic mutation in the other allele was
needed to produce the disease; those individuals with no inherited faulty copy
required two somatic mutations [8].
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The two hit hypothesis was further developed to include the first hit being
the activation of a proto-oncogene, which may not be enough to induce
cancer as tumour suppressor genes will counteract its effect; therefore, the
second hit is the loss-of-function of a tumour suppressor [10]. It is now known
that multiple genetic alterations occur to trigger tumorigenesis; indeed, it has
been shown that as many as 7-10 (epi)genetic events are required to bring
about the cancer phenotypes [1, 11, 12].
Originally, this notion of a complex network of interacting pathways in
cancers led to the idea that they may be too complicated for simple effective
treatments [13, 14]. However, it has since been shown that knocking down
just one specific gene can be sufficient to destroy a cancer. This phenomenon
can be classed as oncogene or non-oncogene dependency.
1.3 Oncogene Dependency
First coined by Bernard Weinstein in 2000, the phenomenon of oncogene
addiction has now been identified in numerous cancers [2, 15-17]. This theory
proposes that, despite the plethora of genetic alterations that occur in cancer
progression, some cancers can become ‘addicted’ to a single oncogene on
which they depend for proliferation and survival. Mouse models were initially
used to identify and demonstrate oncogene dependency; for instance, the
inactivation of oncogenic Myc in mice with osteosarcomas or lymphomas led
to significant tumour regression [18, 19].
However, the state of oncogene addiction is not always permanent.
Oncogenic escape has been observed in some tumours, which utilise their
genomic instability to mutate around their addiction, by activating other
oncogenes rendering the original oncogene redundant [20, 21]. In many
cases the new oncoprotein influences the same molecular pathway as its
predecessor, implying that it may be a particular signalling pathway to which
the cancer cells are addicted rather than a specific oncogene per se. This was
demonstrated in c-Myc-addicted mammary adenocarcinomas in mice [22]:
following c-Myc downregulation, full regression was observed in many
tumours. However, a subset of tumours continued to proliferate, in which
activating mutations in KRAS2, an upstream effector of c-MYC in the MAPK
pathway, were identified.
1.4 Non-Oncogene Dependency
Non-oncogene addiction is related to oncogene addiction, but in this case
tumours depend on normal genes for their survival [23]. One of the best
studied examples of this is the dependency of some classes of multiple
myelomas on interferon regulatory factor 4 (IRF4) [2], where IRF4 is
absolutely required for proliferation and survival despite the fact it is not
mutated or amplified [17].
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2. Prevalence of (Non-)Oncogene-dependent Cancers
A diverse range of cancers exhibiting (non-)oncogene dependency
have now been identified, these are summarised in Table 1. Some well
characterised examples, including the oncogene-addicted, non-small cell lung
carcinoma (NSCLC) and chronic myelogenous leukemia (CML), and nononcogene-addicted multiple myelomas (MM), are described in more detail
below.
2.1 Oncogene-Dependent Cancers
2.1.1 EGFR in various cancers
Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase
found on the plasma membrane of epithelial cells that is involved in initiating
growth and proliferation signalling pathways [24]. It is an important protooncogene due to its prevalence in several cancers, including NSCLC, breast,
prostate and ovarian cancers. NSCLC is of particular significance because of
the poor patient survival rates. It has been reported that NSCLC represents
85% of lung cancers [25], which account for one fifth of all cancer-related
deaths [26].
Aberrant EGFR activation in these cancers can arise from mutation or
overexpression. Nearly 90% of oncogenic EGFR mutations occur in the
cytoplasmic kinase domain, and result in its continuous autophosphorylation
[27]. The consequence of this is a constitutively active EGFR signalling
cascade, through which downstream transcription factors are activated and
confer various properties to the cancer phenotype, including angiogenesis,
migration, proliferation, stromal invasion and resistance to apoptosis [28].
These EGFR mutations are mostly restricted to NSCLC, and are rare in other
cancers. Nevertheless, there is still a high incidence of EGFR overexpression
in glioblastomas [29], NSCLC, head and neck squamous cell carcinomas, and
colorectal cancer [27].
Yamazaki and coworkers provided initial evidence for the therapeutic
potential of targeting EGFR in cancers. They demonstrated that a ribozyme,
targeted against an aberrant EGFR, could suppress its expression in the
ERM5-1 cell line and significantly reduce their tumorigenic capacity in nude
mice [30]. This provided evidence of tumour dependence on EGFR, which
was reinforced by the subsequent emergence and clinical success of EGFRtargeted therapies in various cancers [27]. Examples include the small
molecule drugs, gefitinib and erlotinib, which inhibit EGFR signalling by
competing for ATP-binding sites within its intracellular kinase domain, and
initially exhibited great efficacy in NSCLC patients [31, 32]. However, the
success of these drugs was short-lived due to the emergence of drug
resistance, often acquired by cancers via mutations in the drug-binding site of
EGFR [33], thus highlighting the need for new therapeutic strategies against
EGFR-dependent cancers.
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2.1.2 BCR-ABL in chronic myeloid leukaemia
BCR-ABL is an oncogene resulting from a chromosomal translocation
found in 95% of CML [34], and in some acute myeloid and lymphoblastic
leukaemias. The translocation between chromosomes 9 and 22 generates the
Philadelphia chromosome, giving rise to a BCR-ABL fusion gene composed of
exons 1-3 from BCR and all except the first exon from ABL [35]. The
identification of this translocation in CML cells provided the first example of a
chromosomal translocation in cancer [36], though it was not proven until later
that it could induce tumorigenesis. When BCR-ABL was forcibly expressed in
mouse models, development of several hematologic malignancies, particularly
CML, was observed [37].
Wild-type ABL is a highly regulated tyrosine kinase with roles in the cell
cycle, genotoxic stress response and integrin signalling [38]. The fusion of
BCR-ABL results in the loss of the ABL autoinhibition domain, and ultimately
leads to its deregulation [39]. This constitutively active tyrosine kinase hyperphosphorylates a vast range of substrates involved in growth, cell adhesion
and inhibition of apoptosis, which results in the induction of tumorigenesis.
The dependence of CML on BCR-ABL for survival was demonstrated through
the clinical success of BCR-ABL inhibitors, most notably imatinib. Like
gefitinib and erlotinib, imatinib is a small molecule tyrosine kinase inhibitor but
has high specificity for BCR-ABL; it inhibits BCR-ABL by binding in the active
site, locking the kinase in its autoinhibited conformation. The remarkable
efficacy of imatinib in CML patients during clinical trials led to its rapid FDA
approval to treat the disease in the USA [40]. However, it has since emerged
that drug resistance can develop in CML patients that renders them
unresponsive to imatinib treatment, typically through gene amplification of
BCR-ABL or mutations in its catalytic domain [41]. Such drug resistance is
especially prevalent during the later stages of CML, including the accelerated
phase or blast crisis [42].
2.2 Non-oncogene Dependent Cancers
2.2.1 IRF4 in multiple myeloma
IRF4 is a transcription factor that is required at different stages of B cell
development and, in particular, in the differentiation of B cells into plasma
cells. In many MM, the malignant plasma cells are dependent on IRF4 for
maintenance and survival despite the fact that the gene is not always mutated
to an oncogenic form [17]. Hence this dependency is termed non-oncogene
dependency. IRF4 is of notable significance because current treatments for
MM are ineffective and the median survival time is only 3-4 years following
initial treatment [43].
IRF4 is expressed in acutely activated B cells, and directs them to
terminally differentiate into plasma cells by acting as a positive transcriptional
regulator of genes involved in differentiation and proliferation. As many as 308
genes are direct or indirect targets of IRF4, of which 101 have been shown to
6
be upregulated in MM cell lines [17]. This broad regulation stems from the fact
that IRF4 is at the apex in the hierarchy of gene regulators in that it regulates
expression of other transcription factors which then further regulate gene
expression. Moreover MYC, one transcription factor upregulated by IRF4, is a
direct positive regulator of IRF4 itself [17]. Thus, IRF4 may act in a positive
feedback loop to maintain its own expression whilst driving cancer
progression.
Dependence of MM on non-oncogenic IRF4 was demonstrated using
short hairpin RNA (shRNA) screens with retroviral expression vectors [17].
Retroviruses carrying different shRNAs were transfected into MM cell lines
and shRNA expression was induced by addition of doxycycline. Several
shRNAs targeting IRF4 were identified that were able to kill ten different cell
lines, each with a distinct molecular manifestation of MM. One particular
shRNA targeting the 3’ untranslated region of IRF4 mRNA reduced its
expression by 50-75%, and killed MM cells within 3 days.
IRF4 is only expressed in lymphoid and myeloid cells [44]. This tissue
specificity makes IRF4 an attractive therapeutic target for MM as (1) the
uptake of IRF4-targeting drugs by other tissues will be unlikely to induce
significant adverse effects, and (2) healthy blood cells lost due to the action of
these drugs can be regenerated by natural haematopoiesis.
3. RNAi and RNAi-based Therapeutics
Since its discovery in the mid-1990s [45, 46], RNAi has rapidly
transformed from a curious phenomenon in worms to an invaluable tool in the
study of functional genomics. The significance of RNAi technology was
underlined by the award of the 2006 Nobel Prize in Physiology or Medicine to
Fire and Mello [47].
RNAi describes the cellular process that occurs in various organisms,
including mammals, plants and nematodes, whereby double stranded (ds)
RNAs mediate specific and potent gene silencing [48]. RNAi is believed to
have evolved from an early immune mechanism against viruses and
transposable elements [49]. Foreign dsRNAs are recognised and processed
by Dicer RNases into 21-24 nucleotide fragments, known as small interfering
RNA (siRNA), then loaded onto an Argonaute-containing RNA-induced
silencing complex (RISC). One strand of the siRNA (passenger strand) is
degraded, whilst the other (guide strand) in complex with RISC searches
cytoplasmic RNA for complementary sequences. Once located, Argonaute
triggers cleavage of the targeted RNA, thereby silencing expression of the
foreign gene. This RNAi pathway forms an important component of innate
antiviral immunity in plants, nematodes, fungi and arthropods [49].
Another RNAi pathway, the endogenous microRNA (miRNA) pathway,
enables post-transcriptional regulation of gene expression in animals and
plants [50]. This pathway commences with the transcription of primary
miRNAs (pri-miRNA) from the host genome. These transcripts are typically
7
excised by a microprocessor complex into 65-70 nucleotide precursor
miRNAs (pre-miRNA), which are then exported to the cytoplasm by exportin-5
and Ran GTP [51]. Similar to the siRNA pathway, precursor miRNAs are
processed by Dicer to form 21-26 nucleotide mature miRNAs, which can be
loaded onto RISC, termed miRISC. Compared to siRNAs, miRNAs only
partially base pair with their target sequences in the 3’-untranslated regions
(3’-UTRs) of mRNA, mainly via 7-8 consecutive base pairs of the so-called
seed region. Binding of miRISCs to 3’-UTRs inhibits 5’-cap dependent
translational initiation and can trigger mRNA degradation. Individual miRNAs
usually have several different mRNAs as targets.
To date, 1,872 miRNA sequences have been identified within the
human genome (http://microrna.sanger.ac.uk; accessed March 6, 2014) which
regulate almost a third of protein-encoding genes [52]. Unsurprisingly,
endogenous RNAi plays a critical role in regulating numerous vital processes,
including cell growth, cell proliferation, apoptosis and tissue differentiation
[53].
Cellular RNAi can be exploited to silence a gene of interest by
introducing exogenous sxRNA analogues, either siRNA or shRNA, that target
its mRNA. SiRNAs are competent for RISC loading, and may directly enter
the RNAi pathway once delivered to the cytoplasm (see ‘Non-viral RNAi
Delivery’). For shRNAs, viral vectors are typically used to deliver shRNAencoding genes into cells for expression (see ‘Viral RNAi Delivery’).
Expressed shRNA undergoes processing to form siRNA, which can then be
loaded onto RISC complexes. Many different sxRNA libraries now exist that
cover the entire genomes of both mice and humans, enabling high-throughput
loss of function analyses and the identification of essential genes for virtually
any cellular process [54].
RNAi technology has played a key role in the identification of many
(non-)oncogene-dependent cancers, and the oncogenes on which they rely.
One example of this is the identification of the oncogene, I Kappa B Kinase ε
(IKBKE), in breast cancers. A large shRNA library targeting 1,200 genes was
used to screen the breast cancer cell line, MCF-7, in which three shRNAs
targeting IKBKE were able to reduce proliferation and viability of MCF-7 cells,
indicating their dependence on IKBKE for maintenance and survival [55].
These findings not only added to the accumulating body of evidence for
oncogene dependence in cancers, but also highlighted the therapeutic
potential of sxRNA-mediated RNAi against them.
SxRNAs have several advantages as novel therapeutic agents. They
are synthetic and relatively easy to produce compared to protein-based
therapeutics, can be rationally designed to target any (non-)oncogene, and
can achieve a level of specificity far higher than that of traditional cancer
therapeutics. Furthermore, as sxRNAs target (non-)oncogenes at the mRNA
level, they may exhibit synergistic effects when used with drugs that target at
the protein level. For instance, siRNA-mediated silencing of BCR-ABL and the
gene encoding multidrug resistance protein 1 has been shown to sensitise
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CML cells to imatinib treatment [56, 57], highlighting the potential of such
combinatorial therapies.
However, several barriers have severely hindered the progress of RNAi
therapeutics towards clinical approval, including the short plasma half-lives of
RNAs, their poor cellular uptake, and the lack of tumour-specific targeting. To
overcome these barriers, various sxRNA delivery systems have been
developed, which aim to (1) protect the RNA from nuclease degradation, (2)
evade immune surveillance, (3) prevent rapid renal clearance, (4) promote its
accumulation in tumour tissues, (5) deliver the RNA to cancer cells in a highly
specific manner, and (6) interact with cellular trafficking pathways to deliver
the RNA to appropriate locations within the cell.
A diverse range of RNAi delivery systems have been explored, these
can generally be categorised as viral or non-viral. Selected examples that
have shown the most promise in the laboratory or clinical trials are discussed
in detail below, but several others exist [58].
3.1 Viral RNAi Delivery
Evolutionary fine-tuning has given viruses the ability to infiltrate specific
cells and deliver genetic material for expression, making them an attractive
option for delivering RNAi effectors therapeutically. Viral delivery systems
typically carry shRNA expression cassettes [59]. Non-essential regions within
the virus genome can be modified to incorporate a promoter and shRNA of
interest, to achieve long-term shRNA expression and hence repression of
targeted genes in transfected cells. Adenoviruses, adeno-associated viruses,
herpesviruses and lentiviruses are among those that have been tested as
shRNA delivery vectors [60].
Adenoviruses are widely used in gene therapy studies due to their
ability to incorporate large genes and deliver them to the nucleus [61].
Adenoviral delivery of shRNAs has been demonstrated in several human
cancer cell lines, in which delivered shRNAs targeting the mRNA of the
tumour suppressor protein, p53, successfully triggered gene silencing [62].
Despite these encouraging results from in vitro studies, the inherent, high
immunogenicity of adenoviruses has severely limited their use for shRNA
delivery in vivo [63, 64].
Systemic administration of adenoviral vectors to human patients leads
to high liver uptake and activation of innate immunity, causing severe acute
inflammatory responses and even fatalities [65]. Moreover, the development
of neutralising anti-adenoviral antibodies prohibits repeated drug dosing [66].
These findings seriously question the suitability of adenoviral vectors for in
vivo applications. However, work is under way to address some of these
issues, in particular the immunogenicity and poor targeting of adenoviruses.
Various approaches, including addition of polyethylene glycol (PEG) to the
virus surface [67], liposomal modification [68], and genetic incorporation of
tumour-targeting ligands [61], have shown promising results.
9
Lentiviruses, of the Retroviridae family, have also been explored for
therapeutic shRNA delivery. Lentiviruses are single stranded RNA viruses
with the ability to reverse transcribe their RNA genome into DNA, before
inserting the viral DNA into the host genome for expression [69].
Brummelkamp and coworkers successfully used lentiviruses to deliver shRNA
vectors that targeted an activated K-RAS oncogene to pancreatic cancer
cells. Effective knockdown of the oncogene was achieved, resulting in the
elimination of the tumorigenic capacity of transfected cancer cells in mice [12].
Another lentiviral shRNA delivery system is currently under clinical
evaluation for autologous cell therapy against AIDS-related non-Hodgkin’s
lymphoma [70]. This involves ex vivo shRNA delivery. Haematopoietic
progenitor cells are removed from patients via apheresis, transfected with
lentiviral shRNA vectors and then reinfused into the patient. Early results
revealed no short-term toxicity associated with the transfected haematopoietic
progenitor cells, and two of four patients exhibited persistent expression of the
shRNA [71].
The major drawback of retroviral shRNA delivery is that random
retroviral genome insertion may cause major disruptions to the host genome,
resulting in a multitude of problems, including the activation of protooncogenes [72]. This could be particularly dangerous if a large amount of
virus is taken up by untargeted healthy tissues. Thus, retroviruses are not
ideal candidates for shRNA delivery in vivo.
Hong and coworkers demonstrated that Herpesvirus saimiri can deliver
shRNA targeting the endothelin converting enzyme 1 (ECE-1) into prostate
cancer cells [73]. ECE-1 was selected as the target due to its involvement in
the invasion and migration of several cancers, including prostate, lung, breast,
colorectal and ovarian [74]. The herpesvirus vector was tested in vitro in
several cell lines and ex vivo in primary cells from three different forms of
prostate cancer. Significant levels of ECE-1 knockdown were observed in all
cases, which severely limited the capacity of the cancer cells to migrate and
invade [73]. However, much in vivo testing is still needed to evaluate the
safety of herpesviruses for therapeutic applications, especially considering the
well reported tendency of vectors based on herpes simplex virus 1, another
member of the herpesviridae family, to trigger severe inflammatory responses
in the central nervous system [75, 76].
3.2 Non-viral RNAi Delivery
Although viral RNAi delivery has proven effective in vitro, its clinical
application has largely been deterred by problems associated with its
oncogenic potential, lack of tumour-specific targeting, high immunogenicity
and difficulty of large scale production. As a result, non-viral RNAi delivery
systems, which can potentially circumvent or minimise these problems, have
attracted considerable attention in recent years [77].
10
Several important parameters need to be considered in the design of a
non-viral RNAi delivery system, including:
 Size: Particles should be 10-100 nm in diameter to avoid rapid renal
clearance [78], and to accumulate preferentially at disease sites
characterised by increased vascular permeability, such as sites of
infection or tumour [79, 80]. This effect is known as enhanced
permeability and retention (EPR).
 Zeta potential: Zeta potential is a measure of the magnitude of the
electrostatic attraction or repulsion between particles, and is related to
their surface charge. To minimize nonspecific electrostatic interactions
with cells, which typically display a zeta potential within the range of -5
to -20 mV [81, 82], and to reduce macrophage uptake, a small negative
zeta potential is generally desirable for nanoparticles [83].
 Immunomasking: A highly immunogenic delivery system can trigger
potentially lethal inflammatory responses in patients, as seen with
adenoviruses, and also prohibits repeated dosing. This can be
minimised in non-viral delivery systems via surface coverage with PEG
molecules, which mask epitopes from the immune system [84].
 Targeting: One common problem with many conventional cancer drugs
in use today is a lack of specificity. They are readily internalised by
healthy as well as cancer cells, thus producing side effects. To this
end, ligands that bind cancer cell surface markers can be incorporated
into non-viral RNAi delivery systems. These markers are typically
receptors which are overexpressed on cancer cells, such as EGFR,
and receptors for transferrin (Tf) and folate [77]. Targeting agents can
be natural receptor ligands like Tf or synthetic ones like peptide or
nucleic acid aptamers [85]. Aside from preferential binding to cancer
cells, targeting ligands also offer a mechanism of cell entry by receptormediated endocytosis [86].
 Endosomal escape: Following endocytosis, the delivery system and
sxRNA must escape the endosome into the cytosol, where the sxRNA
mediates its effect. This may be achieved by incorporating endosomal
buffering
ligands,
such
as
histidine-rich
peptides
and
polyethyleneimine. These ligands can absorb protons as they are
pumped into the endosome, acting as ‘proton sponges’, and promoting
further proton influx. This in turn is coupled to an increased influx of
chloride ions and water, resulting in the osmotic swelling and eventual
rupture of the endosome, which can then release its contents into the
cytosol [87].
A diverse range of non-viral RNAi delivery systems have been tested
and some are already in advanced stages of clinical trials. A selection of
some of the most promising formulations is highlighted below.
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3.2.1 Virus-like Particles (VLPs)
Similar to viral delivery, RNAi delivery by VLPs relies on capsids for the
encapsidation, nuclease protection and intracellular delivery of RNAs.
However, several important distinctions exist between the two types of
delivery system.
Firstly, VLP delivery utilises capsids of simple viruses, typically
bacteriophages, to deliver siRNAs to cells, rather than complex, geneticallymodified human viruses to deliver shRNA. Because siRNAs can directly enter
the RNAi pathway and do not require expression from a vector, the virus
genome can be removed, avoiding problems associated with host genome
integration, as seen in retroviral shRNA delivery. Secondly, the components of
a VLP delivery system are relatively simple and cheap to prepare. Coat
protein subunits that constitute capsids can be produced in large quantities
and purified from recombinant E. coli expression systems [88, 89]. In vitro
assembly can then be performed to trigger capsid formation and package
sxRNA cargo [90, 91] (Figure 1). Thirdly, VLPs can be surface-modified to
incorporate a range of useful ligands for immunomasking and targeting [77,
89, 90, 92]. Thus, the resultant semi-synthetic particles, whilst retaining
desirable viral attributes for RNAi delivery, exhibit enhanced functionalities.
Research on VLP-mediated siRNA delivery has mostly been based on
bacteriophage MS2 (Figure 1) [90, 91]. MS2 is a well characterised T=3
icosahedral virus, with a 180-subunit capsid encapsidating its single-stranded
RNA genome [93]. The ease of producing MS2 coat proteins, the robust
nature of its capsids, and the abundance of reactive amines on its surface for
multivalent ligand display are some reasons for its selection as a model VLP
delivery system. MS2 capsids are pH-sensitive, readily disassembling at
acidic pH and reassembling upon mixing with a packaging signal, TR, at
neutral pH (Figure 1). This not only enables simple siRNA packaging, but also
provides a mechanism of siRNA release upon entering the acidic environment
of the endosome. With a diameter of 26 nm, MS2 capsids are ideal to benefit
from enhanced permeability and retention (EPR). Further, MS2 capsids have
a zeta potential of -25 mV [94], thus are unlikely to have strong electrostatic
interactions with cells; this may also be adjusted via surface modifications if
required.
MS2 VLPs have recently been used to deliver siRNA targeted against
the anti-apoptotic factor, BCL2, to HeLa cells [90]. Tf was conjugated to the
VLP surface as a targeting ligand. VLPs successfully delivered siRNAs to the
cancer cells in a Tf receptor-dependent manner, with significant levels of
BCL2 knockdown and cell death observed at nanomolar siRNA
concentrations. The efficiency of siRNA delivery by VLPs was comparable to
that by a commercially available liposomal agent, although VLP-delivered
siRNAs appeared to be more active.
MS2 VLPs have also been targeted to hepatocellular carcinoma (HCC)
cells for siRNA delivery [91]. A cocktail of siRNAs targeting the mRNA of
several
cyclins,
whose
overexpression
is
associated
with
12
hepatocarcinogenesis [95], were packaged inside MS2 VLPs. The VLP
surface was then decorated with (1) PEG for immunomasking, (2) SP94, a
targeting peptide previously identified by affinity selection from a phage
display library against HCC cell surface targets [96], and (3) a histidine-rich
peptide to promote endosomal escape. Gene silencing of targeted cyclins,
growth arrest and apoptosis were observed at picomolar siRNA
concentrations in HCC cells, but not in normal hepatocyte cells. This
demonstrated the ability of VLPs to simultaneously deliver different siRNAs to
targeted cells, and to achieve multiple gene knockdowns. Such combination
therapies may play a significant role in the treatment of tumours in which a
diverse set of mutations are present [97].
Preliminary in vivo testing of VLP-mediated RNAi delivery has recently
been carried out [98, 99]. MS2 VLPs packaging a pre-miRNA, pre-miR146a,
were surface-decorated with HIV Tat peptides for cell penetration; no celltargeting ligands were used. Upon intravenous delivery to mice, these VLPs
displayed widespread biodistribution in the plasma, lung, spleen and kidney,
where high levels of mature miR146a were detected and knockdown of known
targets of miR146a was observed. Additionally, no off-target toxicities in VLPtreated mice were reported.
Despite these promising data, more is required, in particular detailed
pharmacokinetic and long-term safety profiles of VLPs in vivo, to fully assess
their suitability for entering clinical trials as an RNAi delivery system.
3.2.2 Cationic Liposomes
Since its first report two decades ago [100], significant progress has
been made towards the use of cationic liposomes for the cellular delivery of
nucleic acids, particularly siRNAs. This delivery system self-assembles with
siRNA via electrostatic interactions between positively charged lipids and the
negatively charged phosphate backbone of siRNA, forming lipoplexes.
Lipoplexes protect encapsulated siRNA from nuclease degradation, and
facilitate their transport across the cell membrane.
Cationic liposomes can be constituted from a variety of lipidic
components, including phospholipids, cholesterols, cationic lipids and various
lipid-like materials. Lipid composition can greatly influence the physical
properties of liposomes [101] and their delivery efficiency [102]. For instance,
cholesterols are commonly included to improve liposomal stability and uptake
[103]. Some cationic derivatives of cholesterol can also facilitate endosomal
escape [104]. PEGylated lipids can be incorporated to confer steric
stabilisation to the nanoparticle, and prolong its blood circulation by evading
the mononuclear phagocyte system [105, 106]. These PEG-displaying
liposomes are termed stable nucleic acid lipid particles (SNALPs). Amino lipid
derivatives containing cleavable ester bonds have recently been developed to
improve biodegradability of liposomes [107]. A wide range of methods for
liposome preparation are available, which can also influence important
properties such as the size and dispersity of liposomes [108].
13
Nucleic acid delivery by cationic liposomes is well established, as
demonstrated by the prevalence of commercially available products such as
Lipofectin, Oligofectamine, Lipofectamine and RNAifect, which are commonly
used in laboratories to enhance siRNA delivery in vitro [109-111].
Furthermore, nearly half of the most promising siRNA drug candidates
currently in clinical trials are formulated with cationic liposome systems for
delivery [112]. For example, Alnylam Pharmaceuticals' SNALP-based drug
ALN-VSP02, which is used to treat liver cancer, delivers siRNAs targeted
against the mRNAs of vascular endothelial growth factor and kinesin spindle
protein, both of which have important roles in tumour proliferation and
survival. ALN-VSP02 has suitable physical properties for delivering siRNA in
vivo, including a diameter of 80-100 nm and a small zeta potential of <6 mV at
pH 7.4. Intravenous administration of ALN-VSP02 in patients with advanced
cancer and liver metastases gave encouraging results, including siRNAmediated cleavage of targeted mRNAs in the liver and pronounced tumour
regression [113]. One potential disadvantage of current liposomal siRNA drug
candidates, including ALN-VSP02, is the lack of a targeting mechanism for
cancer cells. As a result, significant levels of siRNA may be taken up by
healthy cells alongside the cancer cells.
It is notable that liposomes are already clinically approved to deliver
other drugs, such as doxorubicin and daunorubicin, in the treatment of various
cancers [114]. Whether this platform will prove to be the most effective for
delivering siRNA therapeutics remains to be seen.
3.2.3 Cyclodextrin-containing Polymers
Cyclodextrins are cyclic oligosaccharides that are biocompatible,
exhibit low toxicity and immunogenicity in humans, and have strong
resistance to degradation by human enzymes [115]. They can be incorporated
into a cationic linear backbone to form cyclodextrin-containing polymers
(CDPs, Figure 2A), which can package siRNA for intracellular delivery.
One prominent example of CDP-based siRNA delivery is the
RNAi/Oligonucleotide Nanoparticle Delivery, or RONDEL platform [116]. At
the heart of RONDEL are CDPs which consist of 5-6 repeating units of βcyclodextrin coupled to amidine charge centres and terminal imidazoles
(Figure 2A). These CDPs provide several functions to the delivery system: (1)
positively charged amidines enable electrostatic interactions with siRNA, (2)
CDPs condense siRNAs into nanoparticles and protect them from nuclease
degradation, and (3) imidazole groups utilise the proton sponge effect to
promote endosomal escape.
CDPs also serve as a structural scaffold onto which additional
components can be incorporated, including PEG for steric stabilisation [117]
and immunomasking of the delivery system [118], and Tf for preferential
tumour-targeting. By coupling PEG and PEG-Tf to adamantane, a cycloalkane
which forms high affinity inclusion complexes with β-cyclodextrin [119], the
14
CDP-siRNA core can be non-covalently surface decorated with these ligands
(Figure 2B). Assembled nanoparticles are 70 nm in diameter and thus benefit
from EPR, and each contains approximately 10,000 CDPs, 2,000 siRNAs,
4,000 PEG-adamantanes and 100 Tf-PEG-adamatanes [120]. Furthermore,
the zeta potential of RONDEL particles can be adjusted from +15 mV to -25
mV through incorporation of AD-PEGs modified to contain an anionic charge
[121].
RONDEL demonstrated good efficacy when tested in a metastatic
murine model of Ewing’s sarcoma, in which RONDEL was used to deliver
siRNAs that targeted the EWS/Fli1 fusion oncogene, resulting in effective
silencing of the oncogene and significant anti-tumour effects [122]. Further,
good tolerability of the delivery system was shown in cynomolgus monkeys at
dosages of 3 and 9 mg siRNA/kg [118]. These promising results led to the
advancement of RONDEL into clinical trials. Calando Pharmaceuticals’
CALAA-01, a drug candidate currently undergoing phase 1b clinical
evaluation [112], utilises RONDEL to deliver systemically siRNA targeted
against the M2 subunit of ribonucleotide reductase (RRM2) to solid tumours.
Ribonucleotide reductase is required for DNA synthesis and is a key
component in the proliferation of cancer cells [123]. CALAA-01 has so far
demonstrated promising results in human patients with metastatic melanoma
refractory to standard therapies, in which delivered siRNAs localised to
tumours, and levels of RRM2 mRNA decreased in a dose-dependent manner
[124].
3.2.4 Chemical Modification
Whilst polymer-based delivery systems protect siRNAs from serum
nucleases, they do not offer the same protection against intracellular
nucleases upon cytoplasmic delivery. For this reason, chemical modifications
are commonly made to siRNAs to confer resistance to these intracellular
nucleases and hence increase silencing persistence [125]. Modifications
typically involve the addition of phosphorothioate linkages to the phosphate
backbone of siRNA, or substitution of 2′-hydroxyls with 2′-methoxy or 2′-fluoro
groups on ribose sugars [126]. Importantly, these modifications do not reduce
siRNA activity [127]. In fact recent studies have identified 2′-hydroxyl
modifications at defined positions on the guide strand which increase siRNA
potency [128].
Chemical modifications can also enhance other pharmacological
properties of siRNA. For example, 2′-methoxy groups on the passenger
strand, can reduce immunogenicity by preventing siRNA interaction with Tolllike receptors thus averting the type I interferon pathway [129]. Off-target
effects, whereby siRNAs trigger silencing of partially complementary mRNA,
can also be mitigated by 2′-methoxy substitutions at certain positions on the
guide strand [130].
More recently, modifications with locked nucleic acids (LNA) and
unlocked nucleic acids (UNA) have shown to improve several aspects of
15
siRNA function [131]. In LNA, a methylene bridge links the 2′-oxygen and 4′carbon of the ribose, locking the sugar ring in a C3′-endo conformation.
Strategic incorporation of LNA into siRNAs increases their thermostability, and
can increase nuclease resistance [132, 133], reduce immunogenicity [134],
and mitigate off-target effects [135]. Conversely, incorporation of UNA, which
lack a C2′-C3′ bond in their ribose groups, at certain positions in siRNA can
lead to local destabilisation of the duplex [136]. Such modifications have
shown to reduce off-targeting [137] and improve the biostability of siRNAs
[138].
One modification which can promote tumour-targeting of siRNAs is the
covalent attachment of a nucleic acid aptamer [85]. Similar to antibodies,
nucleic acid aptamers can bind to a large number of biomolecules and display
a high degree of specificity and affinity to their targets [139]. Aptamers offer
several advantages over antibodies. They can be selected against any protein
or cell targets via automated in vitro selection. They are amenable to simple
chemical modifications as well as relatively cheap mass production. They
elicit little immune response and their smaller size may facilitate access to
epitopes that are inaccessible to antibodies [140].
The use of aptamer-siRNA chimaeras has been demonstrated by the
Giangrande group [141], who used an aptamer targeting the prostate-specific
membrane antigen (PSMA), an overexpressed surface marker in prostate
cancers, conjugated to siRNAs against BCL2 and polo-like kinase 1. Aptamerconjugated siRNAs were specifically internalised into prostate cancer cells via
PSMA, and induced knockdown of targeted genes as well as cell death. In
contrast, few effects were observed with unmodified siRNAs or when
aptamer-siRNAs were incubated with non-PSMA expressing cells. These
aptamer-siRNA conjugates demonstrated great efficacy when tested in vivo,
which led to significant regression of PSMA-expressing tumours upon
systemic administration in mice [142].
Six of the fourteen most promising siRNA therapeutics in clinical trials
are chemically modified, naked siRNAs [112]. It is notable that despite the
advantages of these modifications, such RNA-only formulations may still
encounter problems such as rapid renal clearance due to their small size, or
undesired electrostatic interactions with serum proteins. Therefore, it seems
likely that it will be ultimately more effective to package chemically modified
siRNAs inside polymer-based delivery systems.
3.3 Problems Associated with RNAi Therapy
Although RNAi holds huge therapeutic promise, it might not be as
effective as it first appeared. Problems associated with off-target effects have
been observed, whereby different mRNAs with siRNA seed matches in their
3-UTR' are bound and repressed by the siRNA/RISC complex [143]. Off16
target effects can also result from aberrant loading of the passenger strand
into the RISC complex, possibly targeting other mRNAs and leading to their
knockdown [144]. These highlight the need for careful sxRNA design to avoid
sequence similarity in other genes to the target mRNA. Algorithms have been
developed to facilitate specific siRNA design to reduce the likelihood of these
off-target effects [145]. Modifications to the passenger strand can also be
used to this end [146, 147].
Exogenous RNA is known to induce cellular stress responses and
cause toxicity in vivo [59]. For sxRNAs, toxicities can arise as a result of the
saturation of endogenous RNAi pathways in cells, in which important natural
miRNAs are outcompeted for interaction with limiting cellular factors. Several
studies have reported hepatotoxic side effects of high shRNA expression in
the liver of mice, which correlated with the downregulation of liver-derived
miRNAs and the upregulation of miRNA-controlled hepatic genes [148, 149].
Further experiments revealed that the silencing capacity of both shRNAs and
miRNAs can be improved by overexpressing exportin-5 in mice [148].
Exportin-5 is a protein of low abundance that is essential for the nuclear
export of shRNAs, miRNAs and tRNAs, hence the data suggests it might be
one limiting factor over which shRNAs and miRNAs compete to exert their
silencing effects.
These studies underline the need for stringent control over sxRNA
dosing for therapeutic applications in vivo. For viral shRNA delivery, the use of
a moderate promoter and limited vector dosages should be considered to
avoid saturating endogenous RNAi pathways and adverse effects in healthy
cells. For non-viral siRNA delivery, as well as limiting dosages, the
incorporation of PEG and tumour-targeting ligands could be considered to
promote preferential siRNA uptake by cancer cells and mitigate uptake by
healthy cells.
4. Conclusion
RNAi technology has undoubtedly developed into a powerful laboratory
tool in the last two decades, but the hypothesis that it can be translated into
an effective therapeutic strategy for cancer remains to be proven. The
development of RNAi-based anti-cancer therapeutics is motivated by several
factors. First, the inability of some conventional cancer treatments, such as
chemotherapy and radiation, to specifically and fully eradicate cancers has
shifted the emphasis towards more effective, targeted therapies. Second,
despite the complex array of processes associated with tumour formation and
maintenance, some cancers appear to be addicted to single gene products for
their survival. This (non-)oncogene dependence has been dubbed the
‘Achilles’ heel’ of cancers [15], potentially yielding effective therapies that
specifically target these genes for silencing. Finally, despite the tremendous
success of drugs such as imatinib and gefitinib in the treatment of various
cancers, one recurring problem has been the emergence of drug resistance,
typically through gene mutations. This emphasises the importance of novel,
targeted therapeutic strategies against cancers.
17
Various strategies have been explored to improve the pharmacological
properties of sxRNA, by enhancing their stability, prolonging their plasma halflives, promoting their cellular uptake, and facilitating tumour-specific targeting.
The natural ability of viruses to deliver genetic material to cells makes them
an attractive option for delivering shRNA expression vectors. However,
problems with high immunogenicity, lack of tumour-specific targeting and the
oncogenic potential of insertional mutagenesis have directed efforts towards
non-viral siRNA delivery, which can circumvent these issues. A diverse range
of promising nanoparticulate delivery systems have emerged from
laboratories, some of which are already in clinical trials. Development of these
platforms will be crucial to making RNAi a viable therapeutic strategy against
cancer.
Funding
R.Y. is grateful for the financial support from the Leeds University Research
Scholarship program.
Conflict of interest
The authors declare no conflict of interest.
18
References
1
Hanahan, D. and Weinberg, R. A. (2000) The hallmarks of cancer. Cell.
100, 57-70
2
Luo, J., Solimini, N. L. and Elledge, S. J. (2009) Principles of cancer
therapy: oncogene and non-oncogene addiction. Cell. 136, 823-837
3
Rous, P. (1911) A sarcoma of the fowl transmissible by an agent from
the tumor cells. Journal of Experimental Medicine. 13, 397-411
4
Baltimore.D. (1970) Viral RNA-dependent DNA Polymerase: RNAdependent DNA Polymerase in Virions of RNA Tumour Viruses. Nature. 226,
1209-1211
5
Temin, H. M. and Mizutani, S. (1970) RNA-dependent DNA
polymerase in virions of Rous sarcoma virus. Nature. 226, 1211-1213
6
Wang, L. H., Duesberg, P. H., Kawai, S. and Hanafusa, H. (1976)
Location of envelope-specific and sarcoma-specific oligonucleotides on RNA
of Schmidt-Ruppin Rous sarcoma virus. Proc. Natl. Acad. Sci. U. S. A. 73,
447-451
7
Stehelin, D., Varmus, H. E., Bishop, J. M. and Vogt, P. K. (1976) DNA
related to the transforming gene(s) of avian sarcoma viruses is present in
normal avian DNA. Nature. 260, 170-173
8
Knudson, A. G. (1971) Mutation and cancer: statistical study of
retinoblastoma. Proc. Natl. Acad. Sci. U. S. A. 68, 820-823
9
Sherr, C. J. (2004) Principles of tumor suppression. Cell. 116, 235-246
10
MacPherson, D. and Dyer, M. A. (2007) Retinoblastoma: From the twohit hypothesis to targeted chemotherapy. Cancer Research. 67, 7547-7550
11
Renan, M. J. (1993) How many mutations are required for
tumorigenesis? Implications from human cancer data. Molecular
Carcinogenesis. 7, 139-146
12
Brummelkamp, T. R., Bernards, R. and Agami, R. (2002) Stable
suppression of tumorigenicity by virus-mediated RNA interference. Cancer
Cell. 2, 243-247
13
Li, R. H., Yerganian, G., Duesberg, P., Kraemer, A., Willer, A., Rausch,
C. and Hehlmann, R. (1997) Aneuploidy correlated 100% with chemical
transformation of Chinese hamster cells. Proc. Natl. Acad. Sci. U. S. A. 94,
14506-14511
14
Hahn, W. C. and Weinberg, R. A. (2002) Modelling the molecular
circuitry of cancer. Nat. Rev. Cancer. 2, 331-341
15
Weinstein, I. B. (2002) Cancer: Addiction to oncogenes - The Achilles
heal of cancer. Science. 297, 63-64
16
Weinstein, I. B. and Joe, A. (2008) Oncogene addiction. Cancer
Research. 68, 3077-3080
17
Shaffer, A. L., Emre, N. C. T., Lamy, L., Ngo, V. N., Wright, G., Xiao,
W. M., Powell, J., Dave, S., Yu, X., Zhao, H., Zeng, Y. X., Chen, B. Z.,
19
Epstein, J. and Staudt, L. M. (2008) IRF4 addiction in multiple myeloma.
Nature. 454, 226-231
18
Pelengaris, S., Littlewood, T., Khan, M., Elia, G. and Evan, G. (1999)
Reversible activation of c-Myc in skin: Induction of a complex neoplastic
phenotype by a single oncogenic lesion. Molecular Cell. 3, 565-577
19
Jain, M., Arvanitis, C., Chu, K., Dewey, W., Leonhardt, E., Trinh, M.,
Sundberg, C. D., Bishop, J. M. and Felsher, D. W. (2002) Sustained loss of a
neoplastic phenotype by brief inactivation of MYC. Science. 297, 102-104
20
Karlsson, A., Giuriato, S., Tang, F., Fung-Weier, J., Levan, G. and
Felsher, D. W. (2003) Genomically complex lymphomas undergo sustained
tumor regression upon MYC inactivation unless they acquire novel
chromosomal translocations. Blood. 101, 2797-2803
21
Gunther, E. J., Moody, S. E., Belka, G. K., Hahn, K. T., Innocent, N.,
Dugan, K. D., Cardiff, R. D. and Chodosh, L. A. (2003) Impact of p53 loss on
reversal and recurrence of conditional Wnt-induced tumorigenesis. Genes
Dev. 17, 488-501
22
D'Cruz, C. M., Gunther, E. J., Boxer, R. B., Hartman, J. L., Sintasath,
L., Moody, S. E., Cox, J. D., Ha, S. I., Belka, G. K., Golant, A., Cardiff, R. D.
and Chodosh, L. A. (2001) c-MYC induces mammary tumorigenesis by
means of a preferred pathway involving spontaneous Kras2 mutations. Nature
medicine. 7, 235-239
23
Solimini, N. L., Luo, J. and Elledge, S. J. (2007) Non-oncogene
addiction and the stress phenotype of cancer cells. Cell. 130, 986-988
24
Kolch, W. and Pitt, A. (2010) Functional proteomics to dissect tyrosine
kinase signalling pathways in cancer. Nat. Rev. Cancer. 10, 618-629
25
Ettinger, D. S., Akerley, W., Bepler, G., Blum, M. G. and Yang, S. C.
(2010) Non Small Cell Lung Cancer. J. Natl. Compr. Cancer Netw. 8, 740-801
26
Ferlay, J., Autier, P., Boniol, M., Heanue, M., Colombet, M. and Boyle,
P. (2007) Estimates of the cancer incidence and mortality in Europe in 2006.
Annals of Oncology. 18, 581-592
27
Ciardiello, F. and Tortora, G. (2008) Drug therapy: EGFR antagonists
in cancer treatment. New England Journal of Medicine. 358, 1160-1174
28
Nair, P. (2005) Epidermal growth factor receptor family and its role in
cancer progression. Current Science. 88, 890-898
29
Mellinghoff, I. K., Cloughesy, T. F. and Mischel, P. S. (2007) PTENmediated resistance to epidermal growth factor receptor kinase inhibitors.
Clin. Cancer Res. 13, 378-381
30
Yamazaki, H., Kijima, H., Ohnishi, Y., Abe, Y., Oshika, Y., Tsuchida, T.,
Tokunaga, T., Tsugu, A., Ueyama, Y., Tamaoki, N. (1998) Inhibition of tumor
growth by ribozymemediated suppression of aberrant epidermal growth factor
receptor gene expression. J. Natl. Cancer Inst. 90, 581–587
31
Lynch, T. J., Bell, D. W., Sordella, R., Gurubhagavatula, S., Okimoto,
R. A., Brannigan, B. W., Harris, P. L., Haserlat, S. M., Supko, J. G., Haluska,
F. G., Louis, D. N., Christiani, D. C., Settleman, J. and Haber, D. A. (2004)
20
Activating mutations in the epidermal growth factor receptor underlying
responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 350,
2129-2139
32
Pao, W., Miller, V., Zakowski, M., Doherty, J., Politi, K., Sarkaria, I.,
Singh, B., Heelan, R., Rusch, V., Fulton, L. (2004) EGF receptor gene
mutations are common in lung cancers from ‘never smokers’ and are
associated with sensitivity of tumors to gefitinib and erlotinib. Proc. Natl. Acad.
Sci. USA. 101, 13306–13311
33
Tartarone, A., Lazzari, C., Lerose, R., Conteduca, V., Improta, G.,
Zupa, A., Bulotta, A., Aieta, M. and Gregorc, V. (2013) Mechanisms of
resistance to EGFR tyrosine kinase inhibitors gefitinib/erlotinib and to ALK
inhibitor crizotinib. Lung Cancer. 81, 328-336
34
Wada, H., Mizutani, S., Nishimura, J., Usuki, Y., Kohsaki, M., Komai,
M., Kaneko, H., Sakamoto, S., Delia, D., Kanamaru, A. and Kakishita, E.
(1995) Establishment and molecular characterization of a novel leukemic cell
line with Philadelphia chromosome expressing p230 BCR/ABL fusion protein.
Cancer Research. 55, 3192-3196
35
Faderl, S., Talpaz, M., Estrov, Z., O'Brien, S., Kurzrock, R. and
Kantarjian, H. M. (1999) Mechanisms of disease - The biology of chronic
myeloid leukemia. New England Journal of Medicine. 341, 164-172
36
Nowell, P. C. and Hungerford, D. A. (1960) A Minute Chromosome in
Human Chronic Granulocytic Leukemia. Science. 132, 1497-1497
37
Daley, G. Q., Vanetten, R. A. and Baltimore, D. (1990) Induction of
chronic myelogenous leukemia in mice by the P210bcr/abl gene of the
Philadelphia chromosome. Science. 247, 824-830
38
Deininger, M. W. N., Goldman, J. M. and Melo, J. V. (2000) The
molecular biology of chronic myeloid leukemia. Blood. 96, 3343-3356
39
Pluk, H., Dorey, K. and Superti-Furga, G. (2002) Autoinhibition of c-Abl.
Cell. 108, 247-259
40
Druker, B. J., Talpaz, M., Resta, D. J., Peng, B., Buchdunger, E., Ford,
J. M., Lydon, N. B., Kantarjian, H., Capdeville, R., Ohno-Jones, S. and
Sawyers, C. L. (2001) Efficacy and safety of a specific inhibitor of the BCRABL tyrosine kinase in chronic myeloid leukemia. New England Journal of
Medicine. 344, 1031-1037
41
Gambacorti-Passerini, C. B., Gunby, R. H., Piazza, R., Galietta, A.,
Rostagno, R. and Scapozza, L. (2003) Molecular mechanisms of resistance to
imatinib in Philadelphia-chromosome-positive leukaemias. Lancet Oncol. 4,
75-85
42
Druker, B. J. (2008) Translation of the Philadelphia chromosome into
therapy for CML. Blood. 112, 4808-4817
43
Raab, M. S., Podar, K., Breitkreutz, I., Richardson, P. G. and
Anderson, K. C. (2009) Multiple myeloma. Lancet. 374, 324-339
21
44
Eisenbeis, C. F., Singh, H. and Storb, U. (1995) Pip, a novel IRF family
member, is a lymphoid-specific, PU.1-dependent transcriptional activator.
Genes Dev. 9, 1377-1387
45
Fire, A., Xu, S. Q., Montgomery, M. K., Kostas, S. A., Driver, S. E. and
Mello, C. C. (1998) Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans. Nature. 391, 806-811
46
Baulcombe, D. C. (1996) RNA as a target and an initiator of posttranscriptional gene silencing in transgenic plants. Plant Mol Biol. 32, 79-88
47
Bernards, R. (2006) [The Nobel Prize in Physiology or Medicine for
2006 for the discovery of RNA interference]. Ned Tijdschr Geneeskd. 150,
2849-2853
48
Wilson, R. C. and Doudna, J. A. (2013) Molecular mechanisms of RNA
interference. Annual review of biophysics. 42, 217-239
49
Obbard, D. J., Gordon, K. H., Buck, A. H. and Jiggins, F. M. (2009) The
evolution of RNAi as a defence against viruses and transposable elements.
Philosophical transactions of the Royal Society of London. Series B,
Biological sciences. 364, 99-115
50
Carthew, R. W. (2006) Gene regulation by microRNAs. Current opinion
in genetics & development. 16, 203-208
51
Lund, E. and Dahlberg, J. E. (2006) Substrate selectivity of exportin 5
and Dicer in the biogenesis of microRNAs. Cold Spring Harbor symposia on
quantitative biology. 71, 59-66
52
Macfarlane, L. A. and Murphy, P. R. (2010) MicroRNA: Biogenesis,
Function and Role in Cancer. Current genomics. 11, 537-561
53
Esquela-Kerscher, A. and Slack, F. J. (2006) Oncomirs - microRNAs
with a role in cancer. Nature reviews. Cancer. 6, 259-269
54
Latterich, M., ed. (2008) RNAi. Taylor & Francis Group, New York
55
Boehm, J. S., Zhao, J. J., Yao, J., Kim, S. Y., Firestein, R., Dunn, I. F.,
Sjostrom, S. K., Garraway, L. A., Weremowicz, S., Richardson, A. L.,
Greulich, H., Stewart, C. J., Mulvey, L. A., Shen, R. R., Ambrogio, L.,
Hirozane-Kishikawa, T., Hill, D. E., Vidal, M., Meyerson, M., Grenier, J. K.,
Hinkle, G., Root, D. E., Roberts, T. M., Lander, E. S., Polyak, K. and Hahn, W.
C. (2007) Integrative genomic approaches identify IKBKE as a breast cancer
oncogene. Cell. 129, 1065-1079
56
Wohlbold, L., van der Kuip, H., Miething, C., Vornlocher, H. P.,
Knabbe, C., Duyster, J. and Aulitzky, W. E. (2003) Inhibition of bcr-abl gene
expression by small interfering RNA sensitizes for imatinib mesylate (STI571).
Blood. 102, 2236-2239
57
Vasconcelos, M. H., Lima, R. T. and Guimarães, J. E. (2007)
Overcoming K562Dox resistance to STI571 (Gleevec) by downregulation of
P-gp expression using siRNAs. Cancer Therapy. 5, 67-76
58
Lee, J. M., Yoon, T. J. and Cho, Y. S. (2013) Recent developments in
nanoparticle-based siRNA delivery for cancer therapy. Biomed Res Int. 2013,
782041
22
59
Castanotto, D. and Rossi, J. J. (2009) The promises and pitfalls of
RNA-interference-based therapeutics. Nature. 457, 426-433
60
Heilbronn, R. and Weger, S. (2010) Viral vectors for gene transfer:
current status of gene therapeutics. Handb Exp Pharmacol, 143-170
61
Campos, S. K. and Barry, M. A. (2007) Current advances and future
challenges in Adenoviral vector biology and targeting. Curr Gene Ther. 7,
189-204
62
Shen, C., Buck, A. K., Liu, X., Winkler, M. and Reske, S. N. (2003)
Gene silencing by adenovirus-delivered siRNA. FEBS Lett. 539, 111-114
63
Liu, Q. and Muruve, D. A. (2003) Molecular basis of the inflammatory
response to adenovirus vectors. Gene Ther. 10, 935-940
64
Hartman, Z. C., Appledorn, D. M. and Amalfitano, A. (2008) Adenovirus
vector induced innate immune responses: impact upon efficacy and toxicity in
gene therapy and vaccine applications. Virus Res. 132, 1-14
65
Marshall, E. (1999) Gene therapy death prompts review of adenovirus
vector. Science. 286, 2244-2245
66
Yang, Y., Greenough, K. and Wilson, J. M. (1996) Transient immune
blockade prevents formation of neutralizing antibody to recombinant
adenovirus and allows repeated gene transfer to mouse liver. Gene Ther. 3,
412-420
67
Wonganan, P. and Croyle, M. A. (2010) PEGylated Adenoviruses:
From Mice to Monkeys. Viruses. 2, 468-502
68
Wan, Y., Han, J., Fan, G., Zhang, Z., Gong, T. and Sun, X. (2013)
Enzyme-responsive liposomes modified adenoviral vectors for enhanced
tumor cell transduction and reduced immunogenicity. Biomaterials. 34, 30203030
69
Sakuma, T., Barry, M. A. and Ikeda, Y. (2012) Lentiviral vectors: basic
to translational. The Biochemical journal. 443, 603-618
70
Burnett, J. C., Rossi, J. J. and Tiemann, K. (2011) Current progress of
siRNA/shRNA therapeutics in clinical trials. Biotechnol J. 6, 1130-1146
71
DiGiusto, D. L., Krishnan, A., Li, L., Li, H., Li, S., Rao, A., Mi, S., Yam,
P., Stinson, S., Kalos, M., Alvarnas, J., Lacey, S. F., Yee, J. K., Li, M.,
Couture, L., Hsu, D., Forman, S. J., Rossi, J. J. and Zaia, J. A. (2010) RNAbased gene therapy for HIV with lentiviral vector-modified CD34(+) cells in
patients undergoing transplantation for AIDS-related lymphoma. Sci Transl
Med. 2, 36ra43
72
Yi, Y., Hahm, S. H. and Lee, K. H. (2005) Retroviral gene therapy:
safety issues and possible solutions. Curr Gene Ther. 5, 25-35
73
Hong, Y., Macnab, S., Lambert, L. A., Turner, A. J., Whitehouse, A.
and Usmani, B. A. (2011) Herpesvirus saimiri-based endothelin-converting
enzyme-1 shRNA expression decreases prostate cancer cell invasion and
migration. Int J Cancer. 129, 586-598
74
Smollich, M., Gotte, M., Yip, G. W., Yong, E. S., Kersting, C.,
Fischgrabe, J., Radke, I., Kiesel, L. and Wulfing, P. (2007) On the role of
23
endothelin-converting enzyme-1 (ECE-1) and neprilysin in human breast
cancer. Breast Cancer Research and Treatment. 106, 361-369
75
Wood, M. J. A., Byrnes, A. P., Pfaff, D. W., Rabkin, S. D. and Charlton,
H. M. (1994) Inflammatory Effects of Gene-Transfer into the Cns with
Defective Hsv-1 Vectors. Gene Therapy. 1, 283-291
76
McMenamin, M. M., Byrnes, A. P., Charlton, H. M., Coffin, R. S.,
Latchman, D. S. and Wood, M. J. (1998) A gamma34.5 mutant of herpes
simplex 1 causes severe inflammation in the brain. Neuroscience. 83, 12251237
77
Cheng, Z., Al Zaki, A., Hui, J. Z., Muzykantov, V. R. and Tsourkas, A.
(2012) Multifunctional nanoparticles: cost versus benefit of adding targeting
and imaging capabilities. Science. 338, 903-910
78
Choi, H. S., Liu, W., Misra, P., Tanaka, E., Zimmer, J. P., Itty Ipe, B.,
Bawendi, M. G. and Frangioni, J. V. (2007) Renal clearance of quantum dots.
Nature biotechnology. 25, 1165-1170
79
Matsumura, Y. and Maeda, H. (1986) A New Concept for
Macromolecular Therapeutics in Cancer Chemotherapy: Mechanism of
Tumoritropic Accumulation of Proteins and the Antitumor Agent Smancs1.
Cancer Research. 46, 6387-6392
80
Tabata, T., Murakami, Y. and Ikada, Y. (1998) Tumor accumulation of
poly(vinyl alcohol) of different sizes after intravenous injection. Journal of
Controlled Release. 50, 123-133
81
Zhang, Y., Yang, M., Portney, N. G., Cui, D., Budak, G., Ozbay, E.,
Ozkan, M. and Ozkan, C. S. (2008) Zeta potential: a surface electrical
characteristic to probe the interaction of nanoparticles with normal and cancer
human breast epithelial cells. Biomed Microdevices. 10, 321-328
82
Bondar, O. V., Saifullina, D. V., Shakhmaeva, I. I., Mavlyutova, I. I. and
Abdullin, T. I. (2012) Monitoring of the Zeta Potential of Human Cells upon
Reduction in Their Viability and Interaction with Polymers. Acta Naturae. 4,
78-81
83
Zahr, A. S., Davis, C. A. and Pishko, M. V. (2006) Macrophage uptake
of coreshell nanoparticles surface modified with poly(ethylene glycol).
Langmuir. 22, 8178-8185
84
Knop, K., Hoogenboom, R., Fischer, D. and Schubert, U. S. (2010)
Poly(ethylene glycol) in Drug Delivery: Pros and Cons as Well as Potential
Alternatives. Angewandte Chemie-International Edition. 49, 6288-6308
85
Bunka, D. H. J. and Stockley, P. G. (2012) CHAPTER 6 Therapeutic
Applications of Nucleic Acid Aptamer Conjugates. In DNA Conjugates and
Sensors. pp. 140-165, The Royal Society of Chemistry
86
Bareford, L. M. and Swaan, P. W. (2007) Endocytic mechanisms for
targeted drug delivery. Advanced drug delivery reviews. 59, 748-758
87
Behr, J. P. (1997) The Proton Sponge - A Trick to Enter Cells the
Viruses Did Not Exploit. Chimia. 51, 34-36
24
88
Mastico, R. A., Talbot, S. J. and Stockley, P. G. (1993) Multiple
presentation of foreign peptides on the surface of an RNA-free spherical
bacteriophage capsid. J Gen Virol. 74 ( Pt 4), 541-548
89
Wu, M., Brown, W. L. and Stockley, P. G. (1995) Cell-specific delivery
of bacteriophage-encapsidated ricin A chain. Bioconjug Chem. 6, 587-595
90
Galaway, F. A. and Stockley, P. G. (2013) MS2 viruslike particles: a
robust, semisynthetic targeted drug delivery platform. Mol Pharm. 10, 59-68
91
Ashley, C. E., Carnes, E. C., Phillips, G. K., Durfee, P. N., Buley, M. D.,
Lino, C. A., Padilla, D. P., Phillips, B., Carter, M. B., Willman, C. L., Brinker, C.
J., Caldeira Jdo, C., Chackerian, B., Wharton, W. and Peabody, D. S. (2011)
Cell-specific delivery of diverse cargos by bacteriophage MS2 virus-like
particles. ACS Nano. 5, 5729-5745
92
Steinmetz, N. F. and Manchester, M. (2009) PEGylated viral
nanoparticles for biomedicine: the impact of PEG chain length on VNP cell
interactions in vitro and ex vivo. Biomacromolecules. 10, 784-792
93
Strauss, J. H., Jr. and Sinsheimer, R. L. (1963) Purification and
properties of bacteriophage MS2 and of its ribonucleic acid. Journal of
molecular biology. 7, 43-54
94
Pang, L., Nowostawska, U., Ryan, J. N., Williamson, W. M., Walshe, G.
and Hunter, K. A. (2009) Modifying the surface charge of pathogen-sized
microspheres for studying pathogen transport in groundwater. J Environ Qual.
38, 2210-2217
95
Masaki, T., Shiratori, Y., Rengifo, W., Igarashi, K., Yamagata, M.,
Kurokohchi, K., Uchida, N., Miyauchi, Y., Yoshiji, H., Watanabe, S., Omata,
M. and Kuriyama, S. (2003) Cyclins and cyclin-dependent kinases:
comparative study of hepatocellular carcinoma versus cirrhosis. Hepatology.
37, 534-543
96
Lo, A., Lin, C. T. and Wu, H. C. (2008) Hepatocellular carcinoma cellspecific peptide ligand for targeted drug delivery. Molecular cancer
therapeutics. 7, 579-589
97
Greaves, M. (2013) Return of the malingering mutants. Br J Cancer.
109, 1391-1393
98
Pan, Y., Jia, T., Zhang, Y., Zhang, K., Zhang, R., Li, J. and Wang, L.
(2012) MS2 VLP-based delivery of microRNA-146a inhibits autoantibody
production in lupus-prone mice. Int J Nanomedicine. 7, 5957-5967
99
Pan, Y., Zhang, Y., Jia, T., Zhang, K., Li, J. and Wang, L. (2012)
Development of a microRNA delivery system based on bacteriophage MS2
virus-like particles. FEBS J. 279, 1198-1208
100 Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz,
M., Northrop, J. P., Ringold, G. M. and Danielsen, M. (1987) Lipofection: a
highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad.
Sci. U. S. A. 84, 7413-7417
101 Yitbarek, E. (2011) Characterization and Analytical Applications of DyeEncapsulated Zwitterionic Liposomes. BiblioBazaar
25
102 Kim, H. K., Davaa, E., Myung, C. S. and Park, J. S. (2010) Enhanced
siRNA delivery using cationic liposomes with new polyarginine-conjugated
PEG-lipid. International Journal of Pharmaceutics. 392, 141-147
103 Gao, X. and Huang, L. (1995) Cationic liposome-mediated gene
transfer. Gene Ther. 2, 710-722
104 Kim, H. R., Kim, I. K., Bae, K. H., Lee, S. H., Lee, Y. and Park, T. G.
(2008) Cationic solid lipid nanoparticles reconstituted from low density
lipoprotein components for delivery of siRNA. Mol Pharm. 5, 622-631
105 Gabizon, A. and Papahadjopoulos, D. (1988) Liposome formulations
with prolonged circulation time in blood and enhanced uptake by tumors.
Proc. Natl. Acad. Sci. U. S. A. 85, 6949-6953
106 Senior, J., Delgado, C., Fisher, D., Tilcock, C. and Gregoriadis, G.
(1991) Influence of surface hydrophilicity of liposomes on their interaction with
plasma protein and clearance from the circulation: studies with poly(ethylene
glycol)-coated vesicles. Biochim Biophys Acta. 1062, 77-82
107 Maier, M. A., Jayaraman, M., Matsuda, S., Liu, J., Barros, S., Querbes,
W., Tam, Y. K., Ansell, S. M., Kumar, V., Qin, J., Zhang, X., Wang, Q.,
Panesar, S., Hutabarat, R., Carioto, M., Hettinger, J., Kandasamy, P., Butler,
D., Rajeev, K. G., Pang, B., Charisse, K., Fitzgerald, K., Mui, B. L., Du, X.,
Cullis, P., Madden, T. D., Hope, M. J., Manoharan, M. and Akinc, A. (2013)
Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for
systemic delivery of RNAi therapeutics. Mol Ther. 21, 1570-1578
108 Laouini, A., Jaafar-Maalej, C., Limayem-Blouza, I., Sfar, S.,
Charcosset, C. and Fessi, H. (2012) Preparation, Characterization and
Applications of Liposomes: State of the Art. Journal of Colloid Science and
Biotechnology. 1, 147-168
109 Dalby, B., Cates, S., Harris, A., Ohki, E. C., Tilkins, M. L., Price, P. J.
and Ciccarone, V. C. (2004) Advanced transfection with Lipofectamine 2000
reagent: primary neurons, siRNA, and high-throughput applications. Methods.
33, 95-103
110 Baker, B. E., Kestler, D. P. and Ichiki, A. T. (2006) Effects of siRNAs in
combination with Gleevec on K-562 cell proliferation and Bcr-Abl expression.
J Biomed Sci. 13, 499-507
111 Bjorge, J. D., Pang, A. S., Funnell, M., Chen, K. Y., Diaz, R.,
Magliocco, A. M. and Fujita, D. J. (2011) Simultaneous siRNA targeting of Src
and downstream signaling molecules inhibit tumor formation and metastasis
of a human model breast cancer cell line. PloS one. 6, e19309
112 Bouchie, A. (2012) Companies in footrace to deliver RNAi. Nature
biotechnology. 30, 1154-1157
113 Tabernero, J., Shapiro, G. I., LoRusso, P. M., Cervantes, A., Schwartz,
G. K., Weiss, G. J., Paz-Ares, L., Cho, D. C., Infante, J. R., Alsina, M.,
Gounder, M. M., Falzone, R., Harrop, J., White, A. C., Toudjarska, I.,
Bumcrot, D., Meyers, R. E., Hinkle, G., Svrzikapa, N., Hutabarat, R. M.,
Clausen, V. A., Cehelsky, J., Nochur, S. V., Gamba-Vitalo, C., Vaishnaw, A.
K., Sah, D. W., Gollob, J. A. and Burris, H. A., 3rd. (2013) First-in-humans trial
26
of an RNA interference therapeutic targeting VEGF and KSP in cancer
patients with liver involvement. Cancer Discov. 3, 406-417
114 Chang, H. I. and Yeh, M. K. (2012) Clinical development of liposomebased drugs: formulation, characterization, and therapeutic efficacy. Int J
Nanomedicine. 7, 49-60
115 Davis, M. E. and Brewster, M. E. (2004) Cyclodextrin-based
pharmaceutics: past, present and future. Nature reviews. Drug discovery. 3,
1023-1035
116 Heidel, J. D. and Schluep, T. (2012) Cyclodextrin-containing polymers:
versatile platforms of drug delivery materials. Journal of drug delivery. 2012,
262731
117 Bellocq, N. C., Davis, M. E., Engler, H., Jensen, G. S., Liu, A.,
Machemer, T., Maneval, D. C., Quijano, E., Pun, S. H., Schluep, T. and Wen,
S. (2003) Transferrin-targeted, cyclodextrin polycation-based gene vector for
systemic delivery. Mol. Ther. 7, S290
118 Heidel, J. D., Yu, Z., Liu, J. Y., Rele, S. M., Liang, Y., Zeidan, R. K.,
Kornbrust, D. J. and Davis, M. E. (2007) Administration in non-human
primates of escalating intravenous doses of targeted nanoparticles containing
ribonucleotide reductase subunit M2 siRNA. Proc. Natl. Acad. Sci. U. S. A.
104, 5715-5721
119 Pun, S. H. and Davis, M. E. (2002) Development of a Non-Viral Gene
Delivery Vehicle for Systemic Application. Bioconjugate Chem. 13, 630-639
120 Bartlett, D. W. and Davis, M. E. (2007) Physicochemical and biological
characterization of targeted, nucleic acid-containing nanoparticles. Bioconjug
Chem. 18, 456-468
121 Davis, M. E. (2009) The first targeted delivery of siRNA in humans via
a self-assembling, cyclodextrin polymer-based nanoparticle: from concept to
clinic. Mol Pharm. 6, 659-668
122 Hu-Lieskovan, S., Heidel, J. D., Bartlett, D. W., Davis, M. E. and
Triche, T. J. (2005) Sequence-specific knockdown of EWS-FLI1 by targeted,
nonviral delivery of small interfering RNA inhibits tumor growth in a murine
model of metastatic Ewing's sarcoma. Cancer Res. 65, 8984-8992
123 Cerqueira, N. M., Fernandes, P. A. and Ramos, M. J. (2007)
Ribonucleotide reductase: a critical enzyme for cancer chemotherapy and
antiviral agents. Recent Pat Anticancer Drug Discov. 2, 11-29
124 Davis, M. E., Zuckerman, J. E., Choi, C. H., Seligson, D., Tolcher, A.,
Alabi, C. A., Yen, Y., Heidel, J. D. and Ribas, A. (2010) Evidence of RNAi in
humans from systemically administered siRNA via targeted nanoparticles.
Nature. 464, 1067-1070
125 Czauderna, F., Fechtner, M., Dames, S., Aygun, H., Klippel, A., Pronk,
G. J., Giese, K. and Kaufmann, J. (2003) Structural variations and stabilising
modifications of synthetic siRNAs in mammalian cells. Nucleic Acids
Research. 31, 2705-2716
27
126 Chiu, Y. L. and Rana, T. M. (2003) siRNA function in RNAi: a chemical
modification analysis. Rna. 9, 1034-1048
127 Soutschek, J., Akinc, A., Bramlage, B., Charisse, K., Constien, R.,
Donoghue, M., Elbashir, S., Geick, A., Hadwiger, P., Harborth, J., John, M.,
Kesavan, V., Lavine, G., Pandey, R. K., Racie, T., Rajeev, K. G., Rohl, I.,
Toudjarska, I., Wang, G., Wuschko, S., Bumcrot, D., Koteliansky, V., Limmer,
S., Manoharan, M. and Vornlocher, H. P. (2004) Therapeutic silencing of an
endogenous gene by systemic administration of modified siRNAs. Nature.
432, 173-178
128 Kenski, D. M., Butora, G., Willingham, A. T., Cooper, A. J., Fu, W., Qi,
N., Soriano, F., Davies, I. W. and Flanagan, W. M. (2012) siRNA-optimized
Modifications for Enhanced In Vivo Activity. Mol Ther Nucleic Acids. 1, e5
129 Robbins, M., Judge, A., Liang, L., McClintock, K., Yaworski, E. and
MacLachlan, I. (2007) 2'-O-Methyl-modified RNAs act as TLR7 antagonists.
Mol. Ther. 15, 1663–1669
130 Jackson, A. L., Burchard, J., Leake, D., Reynolds, A., Schelter, J., Guo,
J., Johnson, J. M., Lim, L., Karpilow, J., Nichols, K., Marshall, W., Khvorova,
A. and Linsley, P. S. (2006) Position-specific chemical modification of siRNAs
reduces "off-target" transcript silencing. Rna. 12, 1197-1205
131 Bramsen, J. B. and Kjems, J. (2012) Development of TherapeuticGrade Small Interfering RNAs by Chemical Engineering. Frontiers in genetics.
3, 154
132 Bramsen, J. B., Laursen, M. B., Nielsen, A. F., Hansen, T. B., Bus, C.,
Langkjaer, N., Babu, B. R., Hojland, T., Abramov, M., Van Aerschot, A.,
Odadzic, D., Smicius, R., Haas, J., Andree, C., Barman, J., Wenska, M.,
Srivastava, P., Zhou, C., Honcharenko, D., Hess, S., Muller, E., Bobkov, G.
V., Mikhailov, S. N., Fava, E., Meyer, T. F., Chattopadhyaya, J., Zerial, M.,
Engels, J. W., Herdewijn, P., Wengel, J. and Kjems, J. (2009) A large-scale
chemical modification screen identifies design rules to generate siRNAs with
high activity, high stability and low toxicity. Nucleic Acids Res. 37, 2867-2881
133 Glud, S. Z., Bramsen, J. B., Dagnaes-Hansen, F., Wengel, J., Howard,
K. A., Nyengaard, J. R. and Kjems, J. (2009) Naked siLNA-mediated gene
silencing of lung bronchoepithelium EGFP expression after intravenous
administration. Oligonucleotides. 19, 163-168
134 Hornung, V., Guenthner-Biller, M., Bourquin, C., Ablasser, A., Schlee,
M., Uematsu, S., Noronha, A., Manoharan, M., Akira, S., de Fougerolles, A.,
Endres, S. and Hartmann, G. (2005) Sequence-specific potent induction of
IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through
TLR7. Nature medicine. 11, 263-270
135 Bramsen, J. B., Laursen, M. B., Damgaard, C. K., Lena, S. W., Babu,
B. R., Wengel, J. and Kjems, J. (2007) Improved silencing properties using
small internally segmented interfering RNAs. Nucleic Acids Res. 35, 58865897
136 Langkjaer, N., Pasternak, A. and Wengel, J. (2009) UNA (unlocked
nucleic acid): a flexible RNA mimic that allows engineering of nucleic acid
duplex stability. Bioorganic & medicinal chemistry. 17, 5420-5425
28
137 Bramsen, J. B., Pakula, M. M., Hansen, T. B., Bus, C., Langkjaer, N.,
Odadzic, D., Smicius, R., Wengel, S. L., Chattopadhyaya, J., Engels, J. W.,
Herdewijn, P., Wengel, J. and Kjems, J. (2010) A screen of chemical
modifications identifies position-specific modification by UNA to most potently
reduce siRNA off-target effects. Nucleic Acids Res. 38, 5761-5773
138 Laursen, M. B., Pakula, M. M., Gao, S., Fluiter, K., Mook, O. R., Baas,
F., Langklaer, N., Wengel, S. L., Wengel, J., Kjems, J. and Bramsen, J. B.
(2010) Utilization of unlocked nucleic acid (UNA) to enhance siRNA
performance in vitro and in vivo. Molecular bioSystems. 6, 862-870
139 Ellington, A. D., Szostak, J. W. (1990) In vitro selection of RNA
molecules that bind specific ligands. Nature. 346, 818 - 822
140 Dey, A. K., Griffiths, C., Lea, S. M. and James, W. (2005) Structural
characterization of an anti-gp120 RNA aptamer that neutralizes R5 strains of
HIV-1. Rna. 11, 873-884
141 McNamara, J. O., 2nd, Andrechek, E. R., Wang, Y., Viles, K. D.,
Rempel, R. E., Gilboa, E., Sullenger, B. A. and Giangrande, P. H. (2006) Cell
type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nature
biotechnology. 24, 1005-1015
142 Dassie, J. P., Liu, X. Y., Thomas, G. S., Whitaker, R. M., Thiel, K. W.,
Stockdale, K. R., Meyerholz, D. K., McCaffrey, A. P., McNamara, J. O., 2nd
and Giangrande, P. H. (2009) Systemic administration of optimized aptamersiRNA chimeras promotes regression of PSMA-expressing tumors. Nature
biotechnology. 27, 839-849
143 Birmingham, A., Anderson, E. M., Reynolds, A., Ilsley-Tyree, D.,
Leake, D., Fedorov, Y., Baskerville, S., Maksimova, E., Robinson, K.,
Karpilow, J., Marshall, W. S. and Khvorova, A. (2006) 3' UTR seed matches,
but not overall identity, are associated with RNAi off-targets. Nature Methods
3, 199–204
144 Schwarz, D. S., Hutvagner, G., Du, T., Xu, Z., Aronin, N. and Zamore,
P. D. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell.
115, 199-208
145 Bartlett, D. W. and Davis, M. E. (2006) Insights into the kinetics of
siRNA-mediated gene silencing from live-cell and live-animal bioluminescent
imaging. Nucleic Acids Research. 34, 322-333
146 Lorenz, C., Hadwiger, P., John, M., Vornlocher, H. P. and Unverzagt,
C. (2004) Steroid and lipid conjugates of siRNAs to enhance cellular uptake
and gene silencing in liver cells. Bioorg Med Chem Lett. 14, 4975-4977
147 Zhang, J., Zheng, J., Lu, C., Du, Q., Liang, Z. and Xi, Z. (2012)
Modification of the siRNA passenger strand by 5-nitroindole dramatically
reduces its off-target effects. Chembiochem : a European journal of chemical
biology. 13, 1940-1945
148 Grimm, D., Streetz, K. L., Jopling, C. L., Storm, T. A., Pandey, K.,
Davis, C. R., Marion, P., Salazar, F. and Kay, M. A. (2006) Fatality in mice
due to oversaturation of cellular microRNA/short hairpin RNA pathways.
Nature. 441, 537-541
29
149 Ahn, M., Witting, S. R., Ruiz, R., Saxena, R. and Morral, N. (2011)
Constitutive expression of short hairpin RNA in vivo triggers buildup of mature
hairpin molecules. Hum Gene Ther. 22, 1483-1497
150 Rolfsson, O., Toropova, K., Morton, V., Francese, S., Basnak, G.,
Thompson, G. S., Homans, S. W., Ashcroft, A. E., Stonehouse, N. J., Ranson,
N. A. and Stockley, P. G. (2008) RNA packing specificity and folding during
assembly of the bacteriophage MS2. Comput Math Method M. 9, 339-349
151 Borodavka, A., Tuma, R. and Stockley, P. G. (2012) Evidence that viral
RNAs have evolved for efficient, two-stage packaging. Proc. Natl. Acad. Sci.
U. S. A. 109, 15769-15774
152 Stockley, P. G., Rolfsson, O., Thompson, G. S., Basnak, G., Francese,
S., Stonehouse, N. J., Homans, S. W. and Ashcroft, A. E. (2007) A simple,
RNA-mediated allosteric switch controls the pathway to formation of a T=3
viral capsid. Journal of molecular biology. 369, 541-552
153 Valegard, K., Murray, J. B., Stockley, P. G., Stonehouse, N. J. and
Liljas, L. (1994) Crystal structure of an RNA bacteriophage coat proteinoperator complex. Nature. 371, 623-626
154 Weinberg, R. A. (2007) The Biology of Cancer. Garland Science, New
York
155 Arteaga, C. L. (2002) Epidermal growth factor receptor dependence in
human tumors: More than just expression? Oncologist. 7, 31-39
156 Chen, Y. F., Wang, J. Y., Wu, C. H., Chen, F. M., Cheng, T. L. and Lin,
S. R. (2005) Detection of circulating cancer cells with K-ras oncogene using
membrane array. Cancer Lett. 229, 115-122
157 Guan, H. Y., Zhang, H., Cai, J. C., Wu, J. H., Yuan, J., Li, J., Huang, Z.
S. and Li, M. F. (2011) IKBKE is over-expressed in glioma and contributes to
resistance of glioma cells to apoptosis via activating NF-kappa B. J. Pathol.
223, 436-445
158 Lutterbach, B., Zeng, Q. W., Davis, L. J., Hatch, H., Hang, G. Z., Kohl,
N. E., Gibbs, J. B. and Pan, B. S. (2007) Lung cancer cell lines harboring
MET gene amplification are dependent on met for growth and survival.
Cancer Research. 67, 2081-2088
159 Yoshida, T., Okamoto, I., Okamoto, W., Hatashita, E., Yamada, Y.,
Kuwata, K., Nishio, K., Fukuoka, M., Janne, P. A. and Nakagawa, K. (2010)
Effects of Src inhibitors on cell growth and epidermal growth factor receptor
and MET signaling in gefitinib-resistant non-small cell lung cancer cells with
acquired MET amplification. Cancer Sci. 101, 167-172
160 Wilda, M., Fuchs, U., Wossmann, W. and Borkhardt, A. (2002) Killing
of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi).
Oncogene. 21, 5716-5724
161 Sharma, S. V. and Settleman, J. (2007) Oncogene addiction: setting
the stage for molecularly targeted cancer therapy. Genes & Development. 21,
3214-3231
30
162 Iida, S., Rao, P. H., Butler, M., Corradini, P., Boccadoro, M., Klein, B.,
Chaganti, R. S. K. and DallaFavera, R. (1997) Deregulation of MUM1/IRF4 by
chromosomal translocation in multiple myeloma. Nature Genet. 17, 226-230
163 Ashwell, S. and Zabludoff, S. (2008) DNA damage detection and repair
pathways - Recent advances with inhibitors of checkpoint kinases in cancer
therapy. Clin. Cancer Res. 14, 4032-4037
164 Muller, A. J. and Scherle, P. A. (2006) Targeting the mechanisms of
tumoral immune tolerance with small-molecule inhibitors. Nat. Rev. Cancer. 6,
613-625
31
Figure 1. Construction of an MS2 VLP-based siRNA delivery system.
MS2 coat proteins associate as non-covalent dimers (magenta, PDB: 1MSC).
Capsid assembly from these dimers can be triggered at neutral pH by addition
of the MS2 translational operator (TR), a 19-nt RNA stem-loop [150, 151]. TRbinding induces a conformation change in the coat protein dimer (green, PDB:
1ZDI), which can then act as an assembly initiation complex [152, 153]. Any
siRNA conjugated to TR will be packaged inside assembled capsids [90]. The
capsid surface can then be chemically modified to display ligands for
immunomasking, endosomal escape or tumour targeting. Examples
presented include PEG, a nucleic acid aptamer, transferrin (Tf) and an
antibody. These are not drawn to scale.
Figure 2. The RONDEL siRNA delivery platform.
(A) Chemical structure of a cyclodextrin-containing polymer (CDP) composed
of repeating units of β–cyclodextrin, positively charged amidine centres and
terminal imidazoles. (B) Schematic illustrating RONDEL assembly. CDPs form
high affinity inclusion complexes with adamantane (AD) coupled to PEG or
PEG-Tf. Addition of siRNAs triggers compaction of the components via
electrostatic interactions between amidines within CDPs and the phosphate
backbone of siRNA. The resulting nanoparticle, approximately 70 nm in
diameter, contains condensed siRNAs in its core and multiple PEG and PEGTf ligands on its exterior. Adapted from [121].
Table 1. Summary of (non-)oncogene dependencies and their incidence
in cancers.
Abbreviations: NSCLC, non-small cell lung cancer; CML, chronic myeloid
leukaemia, AML, acute myeloid leukaemia; ALL, acute lymphoblastic
leukaemia. Dash indicates not applicable.
(Table 1 is presented on p 33.)
32
CANCERS IN
WHICH MUTATED
CANCERS IN WHICH
OVEREXPRESSED
REFERENCE
EGFR
Cell surface receptor
tyrosine kinase; induction of
various proliferative
pathways including the
MAP kinase and PI3K
pathways
NSCLC
Various epithelial cancers
including 40%
glioblastomas,
10-20% squamous cell
carcinomas, NSCLC,
breast, head and neck,
gastric, colorectal,
prostate, bladder, renal,
pancreatic, ovarian
[27, 154, 155]
K-RAS
Signal transduction
intermediate for mitogenic
and anti-apoptotic stimuli
>50% colorectal;
75% pancreatic;
48% NSCLC; 44%
adrenocortical
tumours
5-10% lung, ovarian and
bladder carcinomas
[154, 156]
IKBKE
Serine/ threonine protein
kinase; anti-apoptotic
-
30% breast, ovarian,
prostate, glioma
[157]
MET
Cell surface receptor
tyrosine kinase; induction of
various proliferative
pathways
-
20% gastric carcinomas,
NSCLC, 4% oesophageal
& lung
[154, 158,
159]
BCR-ABL
Tyrosine kinase; ABL
normally a regulator of the
cell cycle
95% CML, 30%
ALL, 2% AML
-
[34, 160]
c-KIT
Receptor tyrosine kinase
Gastrointestinal
stromal tumours
Gastrointestinal stromal
tumours
[16]
HER2/
ERBB2
Cell surface receptor
tyrosine kinase related to
EGFR
-
30% breast, ovarian,
NSCLC
[161]
VEGF
(extrinsic)
Signal protein; promote
growth of blood vessels
-
-
[16]
IRF4
(intrinsic)
Transcription factor; B-cell
differentiation and
proliferation
Multiple myeloma
-
[162]
CHK1
(intrinsic)
Cell cycle checkpoint
kinase; DNA damage
response and cell cycle
arrest
-
-
[163]
COX2
(extrinsic)
Roles in immune
suppression, angiogenesis,
invasiveness
-
Colorectal, breast,
pancreatic, NSCLC
[164]
PROTEIN
FUNCTION
ONCOGENE
DEPENDENT
NONONCOGENE
DEPENDENT
33