Gene 525 (2013) 208216

Contents lists available at SciVerse ScienceDirect

Gene
journal homepage: www.elsevier.com/locate/gene

Trojan horse at cellular level for tumor gene therapies

Guillaume Collet a, Catherine Grillon a,, Mahdi Nadim a, b, Claudine Kieda a
a
b

Centre de Biophysique Molculaire, UPR4301 CNRS, Rue Charles Sadron, 45071, Orlans, cedex 2, France
Libragen-Induchem Company, 3, rue des satellites, Bat. Canal Biotech, 31400, Toulouse, France

a r t i c l e

i n f o

Article history:
Accepted 7 March 2013
Available online 28 March 2013
Keywords:
Nucleic acid delivery
Exosome
Liposome
Virus
Progenitor cell
Tumor targeting

a b s t r a c t
Among innovative strategies developed for cancer treatments, gene therapies stand of great interest despite
their well-known limitations in targeting, delivery, toxicity or stability. The success of any given genetherapy is highly dependent on the carrier efciency. New approaches are often revisiting the mythic trojan
horse concept to carry therapeutic nucleic acid, i.e. DNAs, RNAs or small interfering RNAs, to pathologic
tumor site. Recent investigations are focusing on engineering carrying modalities to overtake the above
limitations bringing new promise to cancer patients.
This review describes recent advances and perspectives for gene therapies devoted to tumor treatment, taking
advantage of available knowledge in biotechnology and medicine.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Cancer remains a global health problem and a major cause of
death worldwide. Statistical analysis published by the International
Agency for Research on Cancer from the World Health Organization
reveals that if the estimated trends continue, the incidence of all cancer
cases will raise from 12.7 million new cases in 2008 to 21.2 million by
2030 (Bray et al., 2012).
Facing such an alarming disease progression, varieties of therapies
have been developed but expected efciency has still not been
reached. Thus the need to develop innovative strategies is crucial.
Progress in the knowledge about tumor biology and molecular
aspects of cancer has facilitated the design of new therapies aiming
to overtake the limitations faced by research during the past decades.

Abbreviations: AAV, adeno-associated viruses; Ad, adenovirus; CTGVT, cancer

targeting gene-viro-therapy; EGFR, epidermal growth factor receptor; EPCs, endothelial precursor cells; GAOVT, gene armed oncolytic virus therapy; GCV, ganciclovir; HRE,
hypoxia responsive element; HSV-TK, herpes simplex virus thymidine kinase; MDR1,
multi drug resistance protein; MSCs, mesenchymal stem cells; NK, natural killers;
OVs, oncolytic viruses; PAMAM, highly branched polyamidoamine; PEG, polyethylene
glycol; PEI, poly(ethylenimine); PLL, poly(L-lysine); PU-PEI, polyurethane-short
branch polyethylenimine; QD, quantum dots; SPION, superparamagnetic iron oxide
nanoparticles; TERT, telomerase reverse transcriptase; THL, Trojan Horse Liposome;
TK, thymidine kinase; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand;
VEGF, vascular endothelial growth factor.
Corresponding author at: Centre de Biophysique Molculaire, UPR4301 CNRS, Rue
Charles Sadron, 45071 Orlans cedex 2, France. Tel.: +33 2 38 25 78 04; fax: +33 2 38
25 54 59.
E-mail addresses: guillaume.collet@cnrs-orleans.fr (G. Collet),
catherine.grillon@cnrs-orleans.fr (C. Grillon), mahdi.nadim@cnrs-orleans.fr (M. Nadim),
claudine.kieda@cnrs-orleans.fr (C. Kieda).
0378-1119/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.gene.2013.03.057

The main goal in anti-cancer approaches is to maximize efcacy of

cancer treatments and to minimize systemic toxicity.
Remaining a challenge, new approaches are now developed focusing
on tumor microenvironment in addition to tumor cells themselves.
This had led for example to the development of new methods for delivery
of therapies by targeting the tumor-associated vasculature, providing
thus promising antitumor effects with minimal systemic toxicity.
Gene therapy was born 50 years ago thanks to Dr. W. Szybalski and
Dr. E. Szybalska's pioneer experiments (Szybalska and Szybalski, 1962)
reporting the rst gene transfer to mammalian cells. This eld has been
steadily developing and fast growing keeping its main focus on cancer.
Moreover, a large number of anti-tumor strategies have been described.
However the main obstacles to tissue and cell-specic gene delivery
still remains. Biotechnology allows new strategies that improve and
succeed to change the means of cancer treatments. Among elaborated
approaches of gene therapies, the image of the trojan horse is largely
used by strategies that combine, in the same engineered entity, a
targeting unit and a specic drug/gene delivery system. Inspired by
the Greek Mythology, the trojan horse was the source for the presented
approaches and revisited for numerous applications. The trojan horse
is an engineered specic tool in form of: a liposome, an exosome, a
specialized cell or a modied virus in order to specically reach the
tumor site (Fig. 1). Hidden Odysseus's army symbolized the various
therapeutic genes or interfering RNAs that are supposed to accurately
target the gene of interest.
In gene therapy approaches, the nal protein level modulation
is obtained by exogenous DNA or mRNA delivery on the one hand,
giving rise to specic protein expression. On the other hand, small
RNAs (siRNAs, miRNAs) can be used to inhibit or modulate endogenous
protein translation. To be efcient, these molecules have to resist the
degradation in the circulation and further reach either cytoplasm (for

G. Collet et al. / Gene 525 (2013) 208216

engineered
virus

Customized
particles
(i,e, polyplex, liposome,
exosomes)

vector armed
targeting cells

209

carrier cell-based
delivery of
virus

(i.e. EPCs)

healthy cells

physiological
endothelium

Blood vessel

pathological
endothelium

tumor cells

Fig. 1. An overview of various trojan horse strategies developed for cancer gene therapy. Schematic representation of approaches revisiting the trojan horse strategy for specic
gene delivery to cancer cells. This double side scheme presents, in the upper part, a blood vessel in a physiological context, harboring a continuous and well organized endothelium.
The lower part refers to a blood vessel in a pathological context of cancer where endothelium is disorganized, with tumor cells taking place in the vessel wall with endothelial cells.
Such leaky and chaotic tumor vessels are supposed to give an access for therapy to cancer cells which can be targeted as well as tumor endothelium. In this aim, various trojan
horses approaches have been developed. Numerous customized molecules such as polyplexes, liposomes or even exosomes (A) can be used and derivatized to reach preferentially
the tumor area when injected in the blood stream, carrying either a DNA or interfering RNA (siRNA, miRNA). Viruses (B) can also be good carriers and bring an additional oncolytic
activity. Homing cells like EPCs or MSCs (C) are used to target neoangiogenic sites. Following systemic application, engineered cells are recruited to the tumor environment where
they will express the transgene, then acting on tumor cells. Targeted cells can be used for oncolytic viral particles production (D) upon recruitment in the tumor. To address
therapies to cancer cells, these trojan horses combine regulatory safety locks to protect healthy cells. (Figure produced using Servier Medical Art).

RNA) or nucleus (for DNA) as well as escape endosomal degradation. As

these active molecules are not predisposed to overcome physiological
barriers to be delivered to the target cells, carriers are needed.
Indeed, the latter should be able to facilitate nucleic acid stability in the
circulation, intracellular delivery and, when required, import into the
nucleus (Wang et al., 2012).
This review describes various approaches elaborated for tumor
treatment addressed to the cell and sub-cellular levels and using the
trojan horse tricky concept.
2. When the horse becomes a molecule
Non-viral gene delivery systems among which are cationic polymers
such as poly(ethylenimine) (PEI)/poly(L-lysine) (PLL), dendrimers, carbon nanotubes, Superparamagnetic Iron Oxide Nanoparticles (SPION)
or Quantum Dots (QD) are usually positively charged allowing
compression of anionic nucleic acids in solution and interaction with
negatively charged cell membranes. Thus, they may act as molecular
trojan horses to allow gene delivery.
The most widely used are cationic polymers, PLL and PEI, that
combine with DNA into particulate complex, called polyplex which
enable the gene transfer into cells.
PEI allows polyplexes to efciently escape the degradation within endosomes (Kichler et al., 2001). Polyurethane-short branch
polyethylenimine (PU-PEI) or PEI are myristilated to help targeting
brain tumor sites. They are used as a therapeutic-delivery vehicle in
the treatment of glioblastoma either by delivering tumor necrosis
factor-related apoptosis-inducing ligand (TRAIL) in intracranial U87
glioblastoma-bearing mice (Li et al., 2011) or by delivering miRNAs
(miR145) both in vitro in human glioblastoma-associated cancer stem
like cells (CD 133+) and in vivo in an orthotopic model induced by
transplantation of the same cells in immunocompromised mice (Yang
et al., 2012). Such polyplexes can be substituted by polyethylene glycol

(PEG) to enhance their stability. PLL alone has poor transfection ability
(Pouton et al., 1998). However PEG coating can increase both transfection efcacy and circulation half-life (Lee et al., 2002). Liu et al.
in a recent study showed that PLL when associated with PEG and
poly(lactic-co-glycolic acid) gets highly efcient to deliver adriamycin
and siRNA into hepatic carcinoma cells (Liu et al., 2012a). Similarly,
poly(lactic-co-glycolic acid) substituted-nanoparticles, having encapsulated an anti-miRNA to inhibit miR-155 activity, were shown to
efciently slow down the growth of pre-B-cell tumors (Babar et al.,
2012). To improve the therapeutic efcacy of these tools, physical/
chemical technics are utilized to modify them. Chen et al. (2012)
linked PEI-PEG to superparamagnetic iron oxide nanoparticles and a
single-chain variable fragment CD44v6 (scFvCD44v6-PEG-g-PEI-SPION).
Such engineered vectors act as a cancer-targeting as well as magnetic
resonance detectable nanocarrier. Tested in siRNA delivery, these complexes provide both imaging and therapeutic modalities. PEI-based
complexes could also be combined with physical tools as sonoporation
to enhance the transfer of therapeutic molecules as shown for miRNAs
(Chen et al., 2011).
Besides these synthetic macromolecules, natural cationic polymers
can also deliver foreign genetic materials into cancer cells. Chitosan, a
biodegradable and biocompatible linear aminopolysaccharide is a
well-established vector for DNA delivery (Rudzinski and Aminabhavi,
2010). Chitosan and its derivatives are also adequate to deliver RNA as
shown in siRNA-mediated silencing of gene expression in breast cancer
cells (Pille et al., 2006).
Cationic lipids are also largely used as gene delivery systems
(Miele et al., 2012). Due to their positive charge, they naturally
make complexes with DNA to form lipoplexes, protecting nucleic
acid from degradation and facilitating their delivery inside cells.
Novel classes of cationic lipids are currently synthetized, such as
cyclen-based cationic lipids which allow genetic material transfer
into different tumor cell lines (Huang et al., 2011a). Interestingly,

210

G. Collet et al. / Gene 525 (2013) 208216

cationic lipids modied with dexamethasone to target glucocorticoid

receptor-bearing cells and carrying the tumor suppressor gene p53
induce apoptosis and regression of tumor growth (Mukherjee et al.,
2009).
Numerous nanoparticles are being developed depending on size,
shape, charge, and surfactant coating to facilitate their uptake by cells.
These functionalized nanoparticles are for example fullerenes and carbon nanotubes, polymeric micelles, polymeric nanospheres, dendrimers,
polymer-coated nanocrystals, nanoshells, SPION and QDs.
Single-walled carbon nanotubes carrying siRNA were shown
efcient in carrying, releasing and delivering siRNA in vitro into mammalian HeLa cells (Kam et al., 2005). A study performed by Zhang
et al. shows the silencing of telomerase reverse transcriptase (TERT) expression, a key enzyme for the stabilization of chromosomes. Released
from nanotube sidewalls, TERT siRNA inhibited the corresponding
gene expression in various tumor types both in vitro and in vivo
(Zhang et al., 2006).
Highly branched polyamidoamine (PAMAM) dendrimers are a new
class of polymers in spherical conformation and soluble in aqueous solution. Conjugated with Angiopep-2, which target low-density lipoprotein
receptor-related protein-1 expressed on brain capillary endothelial and
glial cells, these particles cross the blood-brain barrier, carrying the therapeutic DNA to glial tumor cells (Huang et al., 2011b). In addition,
Taratula et al. demonstrated the ability of SPION-complexed dendrimers
to deliver siRNA in cancer cells (Taratula et al., 2011).
Recently, quantum dots functionalized by -cyclodextrin coupled
to amino acids were designed to facilitate the delivery of siRNA.
Silencing the multi-drug resistance 1 gene, these siRNA-carring QDs
are promising vehicles for nucleic acid delivery as they were shown
to reverse the multidrug resistance in the case of HeLa cells (Li
et al., 2012).
Designed microbubbles can specically deliver nucleic acids to
tumor sites when exposed to ultrasound. Indeed, when loaded
with an active molecule such as nucleic acid, ultrasounds trigger
microbubbles destruction and release of their content. Using this
method, Carson et al. reported the efcacy of epidermal growth factor
receptor (EGFR)-directed siRNA in reducing squamous cell carcinoma
growth (Carson et al., 2012). Similarly, customized microbubbles
were used to reduce drug resistance of cancer cells by delivering
multi drug resistance protein 1 (MDR1) siRNA which led to the decrease of P-glycoprotein activity. As inhibition of MDR1 also increases
cancer cell sensitivity to drugs, a strategy combining gene silencing
and chemotherapy could be proposed for cancer treatments taking
advantage of this synergistic effect (He et al., 2011).
3. When the horse becomes a liposome
Selective delivery systems are still lacking. Ideally, they should
specically target the disease niche and allow an efcient transfer of
the therapeutic nucleic acid inside the targeted cells. Such strategies
are designed in the purpose of avoiding the toxicity due to the
systemic distribution of injected molecules.
In this respect liposomes as vectors brought promise (Bangham,
1995). In fact, liposomes, vesicles with an aqueous compartment
limited by a phospholipid bilayer, can protect nucleic acids from
enzymatic degradation. According to their lipid composition they
can allow delivery of nucleic acids into cells by interacting with the
negatively charged cell membrane.
Liposomes were rst used for protein and drug delivery
(Gregoriadis and Ryman, 1971) and more than ten years later for
gene transfer (Cline, 1985; Soriano et al., 1983). Neutral liposomes
the production of which is easy were studied in anticancer therapies
for in vitro and in vivo delivery of nucleic acids because of their low
toxicity and immunogenicity (Halder et al., 2006; Landen et al.,
2005). However, due to their lack of surface charges, they led to low
transfection efcacy. Therefore, cationic liposomes were developed

and optimized, improving entry into cells and protection of nucleic

acid against degradation (Spagnou et al., 2004). The Trojan Horse
Liposome (THL) Technology was largely developed for non-viral
gene transfer and is based on pegylated cationic liposomes (Boado,
2007). It is suitable for brain targeting as opposed to conventional delivery systems. Indeed, viruses or cells do not cross the blood-brain
barrier which is only permeable to lipophilic molecules smaller than
400 Da (Patel et al., 2009). Furthermore, encapsulated molecules
can be targeted to specic cells if the liposomes are substituted on
their outer membrane by either monoclonal antibodies (against insulin or transferrin receptors, for example) or other specic molecules,
leading to so-called immunoliposomes. A successful therapeutic
increase of the brain beta-glucuronidase activity was obtained by
administration of non-viral plasmid DNA encoding lysosomal enzyme
encapsulated in liposomes bearing monoclonal antibody to mouse
transferrin receptor (Zhang et al., 2008).
This approach has been further improved by limiting gene expression to specic regions of the brain by a specic promoter. Xia et al.
reported a study on rats with experimental Parkinson's disease
and treated with liposomes containing a plasmid DNA coding for the
glia-derived neurotrophic factor and targeted with a monoclonal antibody to the rat transferrin receptor. The expression of the transgene
was conned to catecholaminergic cells which was achieved by conditioning the gene expression to the rat tyrosine hydroxylase promoter
(Xia et al., 2008). This intravenous non-viral therapy displayed almost
no toxic side effect in rat (Zhang and Pardridge, 2009).
Such THLs can also be used for small RNA delivery (Boado, 2007;
Pardridge, 2010). Zhang et al. reported a 90% increase in survival time
of mice with intracranial brain tumors following weekly treatment
with monoclonal antibodies-targeted THLs that encapsulated an
expression plasmid encoding RNAi knocking down the EGFR (Zhang
et al., 2004). Recently, liposomes were shown to be efcient vectors
for sens/antisens miRNA able to regulate protein expression. This was
illustrated by the targeting of liposome-encapsulated anti-miR-296 to
endothelial cells, inhibiting v3 integrin expression and leading to
a decrease of angiogenesis both in vitro and in vivo (Liu et al., 2011).
An additional example is given by miR-7-containing cationic liposomes
which were shown to be effective in suppressing EGFR expression
in vitro and in reducing tumor volume in vivo in a mouse xenograft
model (Rai et al., 2011).
4. When the horse becomes an exosome
Exosomes can be described as naturally cell-produced liposomes.
These small-sized vesicles produced by various cells are composed by
a lipid bilayer membrane and contain proteins, nucleic acids, miRNAs
in a composition depending on the cell type (Fevrier and Raposo,
2004; Thery et al., 2002). They are natural mediators allowing specic
transfer of molecules between cells, independently of cell contact.
Exosomes are found in biological uids as serum, lymph, saliva but
also milk, allowing a trans-individual transfer of information (Gu
et al., 2012). Besides their role in physiology, they have been shown
to be involved in pathological conditions. Several natural infectious
processes use exosomes as natural trojan horses. In prion disease,
abnormally folded prion protein was found associated with exosomes,
suggesting their contribution in the spreading of prions throughout
the organism (Fevrier et al., 2004). More recently, they have been
shown to be largely involved in neurodegenerative disorders (Ghidoni
et al., 2008; Rajendran et al., 2006; Schneider and Simons, 2012).
Moreover, the trojan exosome hypothesis proposes that retroviruses
including HIV can take advantage of the intercellular vesicle trafc
and exosome exchange to move between cells in the absence of fusion
events in search of adequate target cells (Izquierdo-Useros et al., 2010).
Cancer cells also produce their own exosomes in excessive amount
as compared to normal cells. They were shown to mediate tumor
growth by suppressing immune response: inhibiting the functions of

G. Collet et al. / Gene 525 (2013) 208216

T cells and natural killers (NK), the differentiation of precursors to

mature antigen-presenting cells, increasing the number and/or activity
of immune suppressor cells (Zhang and Grizzle, 2011). The exchange
of exosomes between cancer cells and tumor stroma may also promote
the transfer of oncogenes and onco-miRNAs from cell to cell (Kharaziha
et al., 2012).
Exosomes are considered as carriers both in pathological as well
as physiological conditions. Indeed, exosomes were found to contain
endogenous miRNAs. Consequently, exosomes provide these fundamental regulators of various biological activities the mean to be
specically targeted to distant sites. Taking advantage of this natural
targeting process, exosomes were loaded with small RNAs to reach
specic sites/cells in order to modify desired protein expression
(Lee et al., 2012; O'Loughlin et al., 2012; Tan et al., 2012).
Using exosome properties, attempts to control the immune
system were developed. Plasma exosomes were used as vectors for
gene delivery in order to carry exogenous siRNA to human monocytes
and lymphocytes. This was shown to successfully lead to the selective
gene silencing in the case of mitogen-activated protein kinase 1
(Wahlgren et al., 2012). In addition, exosomes were shown to transfer
miRNAs from T cells to antigen-presenting cells, modulating gene
expression in recipient cells (Mittelbrunn et al., 2011). This effect was
also demonstrated in vivo. Dendritic cell-derived exosomes expressing
a neuron-specic peptide, loaded with siRNA, and injected intravenously
were shown to deliver siRNA specically to brain cells, leading to a
specic gene knockdown (Alvarez-Erviti et al., 2011). These results
show that exosomes can be efcient tools to make nucleic acid cross
the blood-brain barrier.
Cancer cells could also be directly targeted. For example, the delivery
of tumor-suppressive miRNA was achieved to desired sites using
exosomes and the gene-silencing effect on recipient cell was obtained,
resulting in the inhibition of cancer progression (Kosaka et al., 2012).
According to these recent results, the concept of utilizing exosomes
as natural vectors for gene delivery is promising. In fact, the nonimmunogenic property of autologous exosomes allow nucleic acid to
reach the brain through a natural targeting which can be modulated
by engineering the exosome producing cells.

5. When the horse becomes a cell: the cell-mediated gene therapy

Some cells are known to be able to target specically a welldened part from the organism. Endothelial precursor cells (EPCs)
are among these specic cells. They are devoted to reach selectively
neoangiogenesis areas as well as vascular remodeling regions.
From the 1990s, Asahara and colleagues reported the existence
of CD34 +expressing cells in the blood of adult mice, which could
differentiate in vitro into endothelial cells (Asahara et al., 1997).
They showed later on that this EPCs mobilization depends on
chemoattracting signals such as vascular endothelial growth factor
(VEGF) (Asahara et al., 1999b). Moreover EPCs contribute to postnatal
physiological and pathological neovascularization as well (Asahara
et al., 1999a), thus presenting a perfectly adapted tool for tumor
targeting (Varma et al., 2012).
Then the trojan horse was making sense once more, revisiting the
Asahara's pioneer works on EPCs and using gene transfer techniques
that provide the possibility to arm cells with therapeutic genes
prior systemic injection (Asahara et al., 2000; Debatin et al., 2008;
Dudek, 2010).
Alternatively to EPCs, mesenchymal stem cells (MSCs) were considered as potential candidates for gene transfer (Tang et al., 2010).
The homing of MSCs toward tumors has prompted extensive research
to test their use for cancer-specic gene delivery (Dwyer et al., 2010; Sun
et al., 2011). Depending on the site to target, such approach could be extended to neural stem cells (Zhao et al., 2012), macrophages (Burke et al.,
2002) or neutrophils (Tazzyman et al., 2009), and generally to all cells

211

that have homing properties to a targeted site (Dudek, 2010; Tabatabai

et al., 2011).
Various methods and vectors can be used to engineer cells making
them express therapeutic transgenes. The viral constructs are often
preferred also not excluding the possibilities offered by non-viral
methods. For such applications, vectors are mainly based on adenoviruses, retroviruses, adeno-associated viruses (Herrlinger et al., 2000;
Okada and Ozawa, 2008; Ozawa et al., 2000; Ramezani et al., 2003;
Stender et al., 2007), and more recently baculoviruses (Zhao et al.,
2012). The choice of the viral type of vectors depends on the expected
effect, like integration into the genome of the recipient cell, ability to
transduce, immunogenic potential, level of transgene expression and
durability of expression.
Once the cell vehicle is determined, the nucleic acid to transfer
(cDNA for protein expression or small RNAs, i.e. siRNA or miRNA,
for modulation of protein expression) is chosen as a function of the
desired type of tumor treatment. Various therapeutic genes have
been reported including the prodrug-activating enzymes (cytosine
deaminase, carboxylesterase, thymidine kinase), treatments applied
in combination with the proper pro-drug molecules (Aboody et al.,
2000, 2006a; Bak et al., 2010; Conrad et al., 2011; Zhao et al., 2012).
Various genes expressing interleukins (IL-2, IL-4, IL-12, IL-23) (Elzaouk
et al., 2006; Nakamura et al., 2004; Yuan et al., 2006), interferon-
(Dickson et al., 2007; Sims et al., 2008), apoptosis-promoting genes
such as TRAIL (Ehtesham et al., 2002; Shah et al., 2005), soluble VEGF
receptor (sFlk1) as VEGF-trap (Davidoff et al., 2001) and even expression
of fractalkine by cells (Xin et al., 2007) were successfully applied
and led to either tumor growth inhibition and/or in improvement of
animal survival. In addition to the tumor development delay brought
by 131Iodide-based therapy using natrium-iodide symporter delivered
by MSCs, such stategy has been shown to allow an imaging modality
when used with 123I for scintigraphy or 124I for PET (Dwyer et al.,
2011; Knoop et al., 2011).
Further improvements of such cell-mediated approaches were
reported by Zhao et al. describing the use of neural stem cells to target
a glioma tumor armed by baculoviral vector to introduce the herpes
simplex virus thymidine kinase (HSV-TK) suicide gene. Then, the TK
gene product in combination with the pro-drug ganciclovir (GCV)
produces a potent toxin which affects replicative cells and inhibits
tumor growth (Bak et al., 2010; Zhao et al., 2012). Baculoviral vector
advantages are rst due to their non-integration and transient transgene expression in human cells, both dividing and non-dividing cells
including human embryonic stem cells and MSCs (Bak et al., 2010;
Zeng et al., 2007). Baculoviral vectors are presented as a safe class
of gene delivery vectors because they do not replicate nor cause any
toxicity in mammalian cells (Hu, 2008; Wang and Balasundaram,
2010).
To improve the regulation specicity, Conrad et al. and Niess et al.
reported the possibility to engineer MSCs to express the therapeutic
gene (TK-GCV couple) under the selective control of Tie2 promoter/
enhancer (Conrad et al., 2011; Niess et al., 2011). Actively recruited
to growing tumor vasculature, the construction drives the therapeutic
gene expression only in the context of angiogenesis. This selective
expression restricted to a tumor-specic toxic environment confers
another degree of control to render the approach safer.
In a validation purpose, authors injected the engineered cells
inside or close to the tumor. Nevertheless, the best adapted strategy
for clinical application would be the systemic injections, taking
advantage of the natural targeting specic potential of engineered
cells to reach the tumor site (Aboody et al., 2006a, 2006b; Dickson
et al., 2007; Studeny et al., 2002).
6. When the horse becomes a virus
Viruses are known for their extremely high efciency to transfer
genetic material into cells. They represent for this reason a tempting

212

G. Collet et al. / Gene 525 (2013) 208216

delivery tool for gene therapy. Besides previous classical methods,

viruses used as trojan horse for tumor targeted approaches and
viral gene therapy appear as a potential new strategy in anti-cancer
treatments. Ability to carry and introduce a transgene does not
warrantee success since virus-based therapies efcacy is impaired
by poor control of targeting. Viral vectors for gene therapy should
infect only desired tissue to limit the toxicity towards surrounding
cells. To date, reality is still far from this. Nevertheless, research is
actively performed in this purpose.
Among viruses used for gene therapy, retroviruses have been withdrawn from clinical trials because of uncontrolled insertion mutagenesis
which led to adverse events such as disruption and transcriptional
activation of genes, including oncogenes and transmission via germ
cells (Kay et al., 2001). Adenovirus (Ad) is a widely used vector for cancer
gene therapy, while adeno-associated viruses (AAV) are emerging for
cancer gene therapy with promising results.
Ad are recognized as tools of choice for cancer gene therapy : they
can be produced at very high titers, have relatively high capacity for
transgene insertion and efciently transduce both quiescent and
actively dividing cells without incorporation of viral DNA into the host genome (Alemany et al., 2000; Majhen and Ambriovic-Ristov, 2006). Using
adenoviruses, various cancer gene therapy strategies were developed and
classied into ve categories of effects, including mutation compensation,
molecular chemotherapy, genetic immunopotentiation, oncolytic agents
and inhibition of angiogenesis (Majhen and Ambriovic-Ristov, 2006).
Mutation compensation aims to correct deregulated gene
expression of either tumor suppressor genes (i.e. p53, PTEN or BRCA1)
or oncogenes (ErbB2) (Ding et al., 2008, 2012). Thus, wild-type p53
gene encoding Ad vectors can be used to restore a proper cell control
and induce apoptosis (Pisters et al., 2004) while blocking oncogene
expression such as c-myc can be achieved by introducing antisense
oligonucleotides in Ad-vector (Chen et al., 2001). Both strategies can
be combined as demonstrated by Irie et al. using Ad vector restoring
p53 and reducing the overexpressed ErbB2 by expressing an antiErbB2 ribozyme within the same entity (Irie et al., 2006).
The molecular chemotherapy, also known as suicide gene therapy,
is based on the introduction into cells of pro-drug activating enzymes
such as cytosine deaminase, carboxylesterase or TK. These therapies
are used in combination with the proper pro-drugs (i.e. GCV for TK)
since the expressed gene products are able to generate the active form
of the toxic drug (Song, 2005). Moreover, this approach is reported to
induce a lateral diffusion of the activated pro-drug, leading to the so
called bystander effect which means that one treated cell causes the
death of nearby untreated cells.
Due to the weak immunogenicity induced by tumor cells, the
immunopotentiation approach aims to increase the anti-tumor cell
specic immune response. Dendritic cells can be transduced by Ad
coding for a tumor-specic antigen then activate cytotoxic
T-lymphocytes and induce a protective immunity against tumor
cells (Kaplan et al., 1999). Barajas et al. reported activation of NK
cells and inhibition of angiogenesis as antitumor mechanisms linked
to injection of adenovirus expressing IL-12 (Barajas et al., 2001), as
IL-12 was shown to inhibit angiogenesis by mediating NK recruitment
(Bielawska-Pohl et al., 2010).
More elaborated and used for clinical application, oncolytic viruses (OVs) are genetically modied viruses designed to kill cancer cells,
taking advantage of their abnormalities (i.e. p53 deciency) without
affecting normal tissues. Resulting from cell lysis, OVs provide the
additional benet of local amplication and highly immunogenic
response made from the tumor cell epitopes released (Hemminki,
2012). Designed to be intravenously administered (although intratumoral way is used too), this approach provides a real trojan
horse. Furthermore, an additional modality has been developed by
using OVs as carrier for various protein encoding genes, so called
armed viruses (Guo and Fang, 2009), such as interleukins, i.e.
IL-12 (Zhu et al., 2012) or IL-24 (Liu et al., 2012b), TRAIL to enhance

the anti-tumor activity, (Guo et al., 2006), or shRNA for targets (Kon
et al., 2012) such as IL-8 (Yoo et al., 2008), VEGF (Yoo et al., 2007), or
survivin (Shen et al., 2009).
Naturally oncolytic viruses called reoviruses are of great interest
for cancer therapy (Maitra et al., 2012). The basis of the ability of
reovirus to target and kill tumor cells without infecting normal
nonproliferating cells lies in its ability to usurp the highly activated
signaling pathway found in tumor cells (Lal et al., 2009; Wilcox
et al., 2001). This is most clearly established for Ras or elements
from its downstream pathways but ongoing evidences reveal the
involvement of other complex molecular mechanisms which remain
to be claried. Based on this oncolytic property, reovirus-based
therapies are currently in clinical trials for the treatment of various
cancers (Galanis et al., 2012; Morris et al., 2012).
To supply nutrients and oxygen for cell proliferation, tumor
initiates angiogenesis leading to neo-formation of blood vessels. Due
to its involvement in tumor growth, angiogenesis constitutes a target
of choice to inhibit tumor development. Thus, an adenoviral vector
was designed by Popkov et al. to deliver a recombinant single-chain
antibody fragment (pAd-2S03) able to inhibit Tie-2 receptor (Popkov
et al., 2005), leading to tumor growth inhibition by reducing tumor
vasculature.
One limitation of such adenovirus-based gene therapy resides in
the low level of selectivity of the treatment towards cancer cells.
Thus, other control must operate such as transductional targeting,
when the vector is directed toward desired cells or transcriptional
targeting which connes the transgene expression to certain tissues
(Majhen and Ambriovic-Ristov, 2006).
The attachment and entry of Ad are mediated by bers which
confer their tropisms to the viral particles. Addition of a RGD motif
allows RGD-Ad to recognize and bind v-integrins, a class of membrane protein overexpressed in various cancer types (Dmitriev
et al., 1998), leading to a better infectivity of cancer cells (Buskens
et al., 2003; Kanerva et al., 2002). To enhance the Ad particles infectivity, combination of different serotypes, chimerism and targeting
ligand(s) incorporation in ber molecules can be designed. To this
end, Shinozaki et al. engineered a hybrid Ad5/35 virus, where the
serotype 5 bers, that are poorly infective for endothelial cells,
have been replaced by the serotype 35 bers to allow targeting
tumor vasculature (Shinozaki et al., 2006). Only found in the
angiogenesis-rich border region of the tumor this virus was suggested as a useful vector in anticancer strategies for the targeting
of tumor endothelial cells.
At the transcriptional targeting level, the gene of interest is to be
under the control of a tumor-specic promoter (Robson and Hirst,
2003). Thus expression is restricted to tumor cells by hTERT (Song,
2005; Zhu et al., 2012) or to prostate cancer by DD3 promoters
(Ding et al., 2012). Okada et al. described an Ad-RGD mediated
HSV-TK/GCV system where HSV-TK gene was controlled either by
melanoma-specic tyrosinase promoter or by TERT (Okada et al.,
2005). Consequently, made to be safe, this vector system for suicide
gene therapy is efcient and ts perfectly with the trojan horse
approach. Moreover, gene expression could also be advantageously
dependent upon the conditions which are characteristic of the
tumor microenvironment such as hypoxia (Kwon et al., 2010; Xie
et al., 2009). A hypoxia/HIF-dependent replicative adenovirus displays
hypoxia-conditioned cytolytic activity towards hypoxic but not
normoxic cells (Cho et al., 2004; Post and Van Meir, 2003).
In this constant quest for a tight regulation and tumor specicity,
new approaches are proposed combining available mechanisms. It
gives rise to so-called Cancer Targeting Gene-Viro-Therapy (CTGVT)
or Gene Armed Oncolytic Virus Therapy (GAOVT). Both are based on
the insertion of an antitumor gene into an oncolytic virus. These
sophisticated combination strategies are addressed to the tumor
cells, which ultimately are attacked from different sides to be killed
(Cai et al., 2012; Liu et al., 2012b). They have a much higher

G. Collet et al. / Gene 525 (2013) 208216

antitumor activity than either gene therapy or oncolytic virotherapy

alone (Liu et al., 2012c).
Nevertheless, immunity reduces drastically virotherapy efcacy.
Liver and spleen take up the major fraction of injected viruses
from the circulation. In addition to this innate barrier, the adaptative
immunity barriers are built in reaction to systemic delivery by production of antiviral antibodies. Immunity compromises the possibility
of multiple virotherapeutic injections (Power and Bell, 2008).
Attemps were undertaken to limit the visibility of the viral antigens by the immune system. Two approaches were mainly reported:
the covalent binding of PEG to protect Ad from antibody neutralization (O'Riordan et al., 1999; Yao et al., 2009), the loading into a carrier
cell to be protected and hidden. Additional benets are provides by
the carrier cell targeting properties (Power and Bell, 2008).
Among the currently developed viral vectors, AAV is considered as
a promising vector because of its lack of pathogenicity and toxicity,
its ability to infect dividing and non-dividing cells of various tissue
origins, its very low immunogenicity and long-term expression
without integration into host chromosome (Ponnazhagan et al.,
2001). Basically, approaches can be divided similarly to adenoviral
approaches: antiangiogenesis, immunomodulation, suicide gene therapy and repair of damaged tumor cells (Li et al., 2005). Comparably to
adenovirus, specic tumor cell targeting is made possible by control
of transduction as well as transcription with tumor cell-specic
enhancers/promoters (Nicklin et al., 2003). Hypoxia and hypoxia
responsive element (HRE) triggering sequence is used to allow a
tumor hypoxia selective transcription of the transgene (Ruan et al.,
2001). Various therapeutic approaches can be reported such as expression of a soluble Flt1 to sequester the VEGF and inhibit angiogenesis
(Mahendra et al., 2005) but also angiostatin and endostatin
(Ponnazhagan et al., 2004), or TK for combination with GCV (Pan et al.,
2012).
The viral vectors reviewed here are not an exhaustive list, but
represent those which are used in current clinical trials or under
advanced preclinical development.

213

accomplishing their therapeutic effect (oncolytic killing for example).

Moreover, thanks to the absence of immune response, several injections can be performed to enhance therapeutic efcacy as opposed
to naked viruses (Power et al., 2007).
However, efcient delivery of viruses/vectors to tumor sites
remains a challenge that tightly depends on the carrier. Deng and
Jia hypothesized the use of EPCs to achieve oncolytic viruses delivery
(Deng and Jia, 2008). Asahara et al. described that, when systemically
administered, EPCs should migrate via peripheral blood to home to
the site of tumor neovasculature (primary and metastatic) to exert
therapy (Asahara et al., 1997). Currently, several technical problems
have to be solved before clinical application, such as EPC expansion
in keeping their progenitor cell characteristics and limitation of lytic
viral activity in EPCs before they reach the tumor site.
Alternatively to EPCs other cells have been used such as MSCs
(Komarova et al., 2006; Pereboeva et al., 2003; Stoff-Khalili et al.,
2007), activated T cells (Ong et al., 2007), monocytes, endothelial cells
(outgrowth endothelial cells) (Jevremovic et al., 2004), peripheral
blood mononuclear cells (Iankov et al., 2007) and even tumor cells
(Garcia-Castro et al., 2005; Raykov et al., 2004).
To restrict the therapy to cancer cells, a dual targeting of transgene
expression to cancer cells using both transcriptional and mRNA
translational control was developed (Stoff-Khalili et al., 2008). This
combines a cancer specic gene transcriptional control using the
CXCR4 gene promoter and cancer specic mRNA translational control
utilizing a 5-UTR element from the broblast growth factor-2 mRNA.
Replicative specicity demonstrated signicant improvement in tumor
selectivity.
This very promising combination of a carrier cell and a virus opens
great new avenues for therapies. Nevertheless, many hurdles remain
before such strategy can be clinically applied to patients. The choice of
the carrier cell is a prominent obstacle in order to achieve an efcient
targeting of the tumor site. Studies on stem cells and precursors cells
raise the prerequisite of phenotype stability in vivo as well as in vitro.
Moreover the lytic viral activity has to be controlled in the carrier
cells until they reach their tumor targets (Deng and Jia, 2008).

7. When the horse becomes a virus-producing cell

For their efciency to infect cells and to transfer genetic material,
viruses are tools of choice to introduce transgenes into cells. Nevertheless,
safe clinical applications remain the challenge.
The well-known homing of cells such as EPCs or MSCs is already
used in cancer therapy to reach the tumor site, being carried by the
blood to the site of interest (Asahara et al., 1997; Studeny et al.,
2002). This cell mediated targeting complies with host tolerance.
The idea of making both approaches fused in a unique strategy
could bring a breakthrough for more efcient therapy (Coukos et al.,
1999; Guo et al., 2008; Harrington et al., 2002; Power and Bell,
2008). The cell provides the vehicle to reach the tumor site and offers
the platform for locally virus release allowing viral particles to transduce surrounding tumor cells. In such situation, cells serve as trojan
horse vehicles to evade antiviral mechanisms encountered in the
bloodstream (i.e. antibodies, cells) and to avoid uptake by off-target
tissues. Such carrier cell-based delivery of virus combination is fully
included in the trojan horse approaches.
Involved in the development of such a strategy, Power and Bell
reported that its success is tightly dependent on three coordinated
sequential phases which are (1) ex vivo loading, (2) stealth delivery
and (3) virus production at the tumor site (Power and Bell, 2008).
Power et al. brought the proof-of-concept that viruses-infected carrier cells, when intravenously injected, escape from antiviral defenses
and reach the tumor site during the eclipse phase (prior to viral
protein synthesis and virion release) (Power et al., 2007). In the
tumor site, the protective vehicle role of carrier cells shifts to
virus factory able to produce up to hundreds of virions in the tumor
environment where they infect the surrounding tumor cells thus

8. Concluding remarks
The last ve decades have brought huge improvement in cancer
therapy and genetic engineering still opens new horizons through
better targeting, addition of safety locks to secure patient treatment
and to overcome reported limitations. In fact, among various trojan
horse types, oncolytic virus studies are the most completed and
several are already in phase II or III clinical trials (Schmidt, 2011).
Interdisciplinary research is needed to develop innovative therapeutic
approaches for an effective transfer system and a selective gene targeting.
According to new progresses and promising results, synergy effect should
be obtained by combining the scientic tool boxes. Success should
come with a judicious assembly of a controlled trafcking to a specic
targeting, and a tight regulation making therapeutic delivery highly
restricted to cancer cells, keeping in mind the investigations to nd
new angles from which to attack and defeat the tumor. Future will
show if these trojan horse like therapeutic approaches will bring
improvements in gene therapy efcacy to help cancer treatments.
Acknowledgments
The authors are grateful to Agata Matejuk for revising the
English text.
References
Aboody, K.S., et al., 2000. Neural stem cells display extensive tropism for pathology in
adult brain: evidence from intracranial gliomas. Proc. Natl. Acad. Sci. U. S. A. 97,
1284612851.

214

G. Collet et al. / Gene 525 (2013) 208216

Aboody, K.S., et al., 2006a. Development of a tumor-selective approach to treat metastatic

cancer. PLoS One 1, e23.
Aboody, K.S., et al., 2006b. Targeting of melanoma brain metastases using engineered
neural stem/progenitor cells. Neuro Oncol. 8, 119126.
Alemany, R., Balague, C., Curiel, D.T., 2000. Replicative adenoviruses for cancer therapy.
Nat. Biotechnol. 18, 723727.
Alvarez-Erviti, L., Seow, Y., Yin, H., Betts, C., Lakhal, S., Wood, M.J., 2011. Delivery
of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat.
Biotechnol. 29, 341345.
Asahara, T., et al., 1997. Isolation of putative progenitor endothelial cells for angiogenesis.
Science 275, 964967.
Asahara, T., et al., 1999a. Bone marrow origin of endothelial progenitor cells responsible
for postnatal vasculogenesis in physiological and pathological neovascularization.
Circ. Res. 85, 221228.
Asahara, T., et al., 1999b. VEGF contributes to postnatal neovascularization by mobilizing
bone marrow-derived endothelial progenitor cells. EMBO J. 18, 39643972.
Asahara, T., Kalka, C., Isner, J.M., 2000. Stem cell therapy and gene transfer for regeneration.
Gene Ther. 7, 451457.
Babar, I.A., et al., 2012. Nanoparticle-based therapy in an in vivo microRNA-155 (miR155)-dependent mouse model of lymphoma. Proc. Natl. Acad. Sci. U. S. A. 109,
E1695E1704.
Bak, X.Y., Yang, J., Wang, S., 2010. Baculovirus-transduced bone marrow mesenchymal
stem cells for systemic cancer therapy. Cancer Gene Ther. 17, 721729.
Bangham, A.D., 1995. Surrogate cells or Trojan horses. The discovery of liposomes.
Bioessays 17, 10811088.
Barajas, M., et al., 2001. Gene therapy of orthotopic hepatocellular carcinoma in rats
using adenovirus coding for interleukin 12. Hepatology 33, 5261.
Bielawska-Pohl, A., et al., 2010. The anti-angiogenic activity of IL-12 is increased in
iNOS-/-mice and involves NK cells. J. Mol. Med. (Berl) 88, 775784.
Boado, R.J., 2007. Blood-brain barrier transport of non-viral gene and RNAi therapeutics.
Pharm. Res. 24, 17721787.
Bray, F., Jemal, A., Grey, N., Ferlay, J., Forman, D., 2012. Global cancer transitions
according to the Human Development Index (20082030): a population-based
study. Lancet Oncol. 13 (8), 790801.
Burke, B., Sumner, S., Maitland, N., Lewis, C.E., 2002. Macrophages in gene therapy:
cellular delivery vehicles and in vivo targets. J. Leukoc. Biol. 72, 417428.
Buskens, C.J., et al., 2003. A genetically retargeted adenoviral vector enhances viral
transduction in esophageal carcinoma cell lines and primary cultured esophageal
resection specimens. Ann. Surg. 238, 815824 (discussion 825-6).
Cai, Y., Liu, X., Huang, W., Zhang, K., Liu, X.Y., 2012. Synergistic antitumor effect of TRAIL
and IL-24 with complete eradication of hepatoma in the CTGVT-DG strategy. Acta
Biochim. Biophys. Sin. (Shanghai) 44, 535543.
Carson, A.R., et al., 2012. Ultrasound-targeted microbubble destruction to deliver siRNA
cancer therapy. Cancer Res. 72 (23), 61916199.
Chen, J.P., et al., 2001. Molecular therapy with recombinant antisense c-myc adenovirus
for human gastric carcinoma cells in vitro and in vivo. J. Gastroenterol. Hepatol. 16,
2228.
Chen, Z.Y., Liang, K., Qiu, R.X., Luo, L.P., 2011. Enhancing microRNA transfection to
inhibit survivin gene expression and induce apoptosis: could it be mediated by
a novel combination of sonoporation and polyethylenimine? Chin. Med. J. (Engl)
124, 35923594.
Chen, Y., et al., 2012. Development of an MRI-visible nonviral vector for siRNA delivery
targeting gastric cancer. Int. J. Nanomedicine 7, 359368.
Cho, W.K., et al., 2004. Oncolytic effects of adenovirus mutant capable of replicating
in hypoxic and normoxic regions of solid tumor. Mol. Ther. 10, 938949.
Cline, M.J., 1985. Perspectives for gene therapy: inserting new genetic information into
mammalian cells by physical techniques and viral vectors. Pharmacol. Ther. 29,
6992.
Conrad, C., et al., 2011. Linking transgene expression of engineered mesenchymal stem
cells and angiopoietin-1-induced differentiation to target cancer angiogenesis.
Ann. Surg. 253, 566571.
Coukos, G., et al., 1999. Use of carrier cells to deliver a replication-selective herpes
simplex virus-1 mutant for the intraperitoneal therapy of epithelial ovarian cancer.
Clin. Cancer Res. 5, 15231537.
Davidoff, A.M., et al., 2001. Bone marrow-derived cells contribute to tumor
neovasculature and, when modied to express an angiogenesis inhibitor, can restrict
tumor growth in mice. Clin. Cancer Res. 7, 28702879.
Debatin, K.M., Wei, J., Beltinger, C., 2008. Endothelial progenitor cells for cancer gene
therapy. Gene Ther. 15, 780786.
Deng, W., Jia, J., 2008. Endothelial progenitor cells as cellular vehicles to deliver
oncolytic virus therapies to metastatic tumors: the Trojan horse approach.
Med. Hypotheses 70, 842844.
Dickson, P.V., et al., 2007. Intravascular administration of tumor tropic neural progenitor
cells permits targeted delivery of interferon-beta and restricts tumor growth in a
murine model of disseminated neuroblastoma. J. Pediatr. Surg. 42, 4853.
Ding, L., et al., 2008. Somatic mutations affect key pathways in lung adenocarcinoma.
Nature 455, 10691075.
Ding, M., et al., 2012. Prostate cancer-specic and potent antitumor effect of a DD3controlled oncolytic virus harboring the PTEN gene. PLoS One 7, e35153.
Dmitriev, I., et al., 1998. An adenovirus vector with genetically modied bers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptorindependent cell entry mechanism. J. Virol. 72, 97069713.
Dudek, A.Z., 2010. Endothelial lineage cell as a vehicle for systemic delivery of cancer
gene therapy. Transl. Res. 156, 136146.
Dwyer, R.M., Khan, S., Barry, F.P., O'Brien, T., Kerin, M.J., 2010. Advances in mesenchymal stem cell-mediated gene therapy for cancer. Stem Cell Res. Ther. 1, 25.

Dwyer, R.M., et al., 2011. Mesenchymal stem cell-mediated delivery of the sodium
iodide symporter supports radionuclide imaging and treatment of breast cancer.
Stem Cells 29, 11491157.
Ehtesham, M., et al., 2002. Induction of glioblastoma apoptosis using neural stem cellmediated delivery of tumor necrosis factor-related apoptosis-inducing ligand.
Cancer Res. 62, 71707174.
Elzaouk, L., Moelling, K., Pavlovic, J., 2006. Anti-tumor activity of mesenchymal stem
cells producing IL-12 in a mouse melanoma model. Exp. Dermatol. 15, 865874.
Fevrier, B., Raposo, G., 2004. Exosomes: endosomal-derived vesicles shipping extracellular
messages. Curr. Opin. Cell Biol. 16, 415421.
Fevrier, B., et al., 2004. Cells release prions in association with exosomes. Proc. Natl.
Acad. Sci. U. S. A. 101, 96839688.
Galanis, E., et al., 2012. Phase II trial of intravenous administration of Reolysin((R))
(Reovirus Serotype-3-dearing Strain) in patients with metastatic melanoma. Mol.
Ther. 20, 19982003.
Garcia-Castro, J., et al., 2005. Tumor cells as cellular vehicles to deliver gene therapies
to metastatic tumors. Cancer Gene Ther. 12, 341349.
Ghidoni, R., Benussi, L., Binetti, G., 2008. Exosomes: the Trojan horses of neurodegeneration.
Med. Hypotheses 70, 12261227.
Gregoriadis, G., Ryman, B.E., 1971. Liposomes as carriers of enzymes or drugs: a new
approach to the treatment of storage diseases. Biochem. J. 124, 58P.
Gu, Y., et al., 2012. Lactation-related microRNA expression proles of porcine breast
milk exosomes. PLoS One 7, e43691.
Guo, W., Fang, B., 2009. Potentiation of oncolytic virotherapy with armed shRNA.
Cancer Biol. Ther. 8, 9293.
Guo, W., et al., 2006. Combination effect of oncolytic adenovirotherapy and TRAIL
gene therapy in syngeneic murine breast cancer models. Cancer Gene Ther. 13,
8290.
Guo, Z.S., Thorne, S.H., Bartlett, D.L., 2008. Oncolytic virotherapy: molecular targets in
tumor-selective replication and carrier cell-mediated delivery of oncolytic viruses.
Biochim. Biophys. Acta 1785, 217231.
Halder, J., et al., 2006. Focal adhesion kinase targeting using in vivo short interfering
RNA delivery in neutral liposomes for ovarian carcinoma therapy. Clin. Cancer
Res. 12, 49164924.
Harrington, K., et al., 2002. Cells as vehicles for cancer gene therapy: the missing
link between targeted vectors and systemic delivery? Hum. Gene Ther. 13,
12631280.
He, Y., et al., 2011. Ultrasound microbubble-mediated delivery of the siRNAs targeting
MDR1 reduces drug resistance of yolk sac carcinoma L2 cells. J. Exp. Clin. Cancer
Res. 30, 104.
Hemminki, A., 2012. Portrait of a leader in immunotherapeutics: oncolytic viruses for
treatment of cancer. Hum. Vaccin. Immunother. 8.
Herrlinger, U., et al., 2000. Helper virus-free herpes simplex virus type 1 amplicon
vectors for granulocyte-macrophage colony-stimulating factor-enhanced vaccination
therapy for experimental glioma. Hum. Gene Ther. 11, 14291438.
Hu, Y.C., 2008. Baculoviral vectors for gene delivery: a review. Curr. Gene Ther. 8,
5465.
Huang, Q.D., et al., 2011a. Cyclen-based cationic lipids for highly efcient gene delivery
towards tumor cells. PLoS One 6, e23134.
Huang, S., et al., 2011b. Dual targeting effect of Angiopep-2-modied, DNA-loaded
nanoparticles for glioma. Biomaterials 32, 68326838.
Iankov, I.D., et al., 2007. Infected cell carriers: a new strategy for systemic delivery of
oncolytic measles viruses in cancer virotherapy. Mol. Ther. 15, 114122.
Irie, A., et al., 2006. Growth inhibition efcacy of an adenovirus expressing dual
therapeutic genes, wild-type p53, and anti-erbB2 ribozyme, against human bladder
cancer cells. Cancer Gene Ther. 13, 298305.
Izquierdo-Useros, N., et al., 2010. HIV and mature dendritic cells: Trojan exosomes
riding the Trojan horse? PLoS Pathog. 6, e1000740.
Jevremovic, D., et al., 2004. Use of blood outgrowth endothelial cells as virus-producing
vectors for gene delivery to tumors. Am. J. Physiol. Heart Circ. Physiol. 287,
H494H500.
Kam, N.W., Liu, Z., Dai, H., 2005. Functionalization of carbon nanotubes via cleavable
disulde bonds for efcient intracellular delivery of siRNA and potent gene silencing.
J. Am. Chem. Soc. 127, 1249212493.
Kanerva, A., et al., 2002. Gene transfer to ovarian cancer versus normal tissues with
ber-modied adenoviruses. Mol. Ther. 5, 695704.
Kaplan, J.M., et al., 1999. Induction of antitumor immunity with dendritic cells transduced with adenovirus vector-encoding endogenous tumor-associated antigens.
J. Immunol. 163, 699707.
Kay, M.A., Glorioso, J.C., Naldini, L., 2001. Viral vectors for gene therapy: the art of
turning infectious agents into vehicles of therapeutics. Nat. Med. 7, 3340.
Kharaziha, P., Ceder, S., Li, Q., Panaretakis, T., 2012. Tumor cell-derived exosomes:
a message in a bottle. Biochim. Biophys. Acta 1826, 103111.
Kichler, A., Leborgne, C., Coeytaux, E., Danos, O., 2001. Polyethylenimine-mediated
gene delivery: a mechanistic study. J. Gene Med. 3, 135144.
Knoop, K., et al., 2011. Image-guided, tumor stroma-targeted 131I therapy of hepatocellular cancer after systemic mesenchymal stem cell-mediated NIS gene delivery.
Mol. Ther. 19, 17041713.
Komarova, S., Kawakami, Y., Stoff-Khalili, M.A., Curiel, D.T., Pereboeva, L., 2006.
Mesenchymal progenitor cells as cellular vehicles for delivery of oncolytic adenoviruses.
Mol. Cancer Ther. 5, 755766.
Kon, T., et al., 2012. Oncolytic virus-mediated tumor radiosensitization in mice through
DNAPKcs-specic shRNA. Transl. Cancer Res. 1, 414.
Kosaka, N., et al., 2012. Exosomal tumor-suppressive microRNAs as novel cancer
therapy: exocure is another choice for cancer treatment. Adv. Drug Deliv. Rev.
65 (3), 376382.

G. Collet et al. / Gene 525 (2013) 208216

Kwon, O.J., Kim, P.H., Huyn, S., Wu, L., Kim, M., Yun, C.O., 2010. A hypoxia- and {alpha}fetoprotein-dependent oncolytic adenovirus exhibits specic killing of hepatocellular
carcinomas. Clin. Cancer Res. 16, 60716082.
Lal, R., Harris, D., Postel-Vinay, S., de Bono, J., 2009. Reovirus: rationale and clinical trial
update. Curr. Opin. Mol. Ther. 11, 532539.
Landen Jr., C.N., et al., 2005. Therapeutic EphA2 gene targeting in vivo using neutral
liposomal small interfering RNA delivery. Cancer Res. 65, 69106918.
Lee, H., Jeong, J.H., Park, T.G., 2002. PEG grafted polylysine with fusogenic peptide for
gene delivery: high transfection efciency with low cytotoxicity. J. Control. Release
79, 283291.
Lee, Y., El Andaloussi, S., Wood, M.J., 2012. Exosomes and microvesicles: extracellular
vesicles for genetic information transfer and gene therapy. Hum. Mol. Genet. 21,
R125R134.
Li, C., Bowles, D.E., van Dyke, T., Samulski, R.J., 2005. Adeno-associated virus vectors:
potential applications for cancer gene therapy. Cancer Gene Ther. 12, 913925.
Li, J., et al., 2011. The use of myristic acid as a ligand of polyethylenimine/DNA
nanoparticles for targeted gene therapy of glioblastoma. Nanotechnology 22, 435101.
Li, J.M., et al., 2012. Multifunctional QD-based co-delivery of siRNA and doxorubicin to
HeLa cells for reversal of multidrug resistance and real-time tracking. Biomaterials
33, 27802790.
Liu, X.Q., Song, W.J., Sun, T.M., Zhang, P.Z., Wang, J., 2011. Targeted delivery of antisense
inhibitor of miRNA for antiangiogenesis therapy using cRGD-functionalized nanoparticles. Mol. Pharm. 8, 250259.
Liu, P., Yu, H., Sun, Y., Zhu, M., Duan, Y., 2012a. A mPEG-PLGA-b-PLL copolymer carrier
for adriamycin and siRNA delivery. Biomaterials 33, 44034412.
Liu, X., et al., 2012b. Gene-viro-therapy targeting liver cancer by a dual-regulated
oncolytic adenoviral vector harboring IL-24 and TRAIL. Cancer Gene Ther. 19,
4957.
Liu, X.Y., Li, H.G., Zhang, K.J., Gu, J.F., 2012c. Strategy of cancer targeting gene-virotherapy (CTGVT) a trend in both cancer gene therapy and cancer virotherapy.
Curr. Pharm. Biotechnol. 13, 17611767.
Mahendra, G., et al., 2005. Antiangiogenic cancer gene therapy by adeno-associated
virus 2-mediated stable expression of the soluble FMS-like tyrosine kinase-1
receptor. Cancer Gene Ther. 12, 2634.
Maitra, R., Ghalib, M.H., Goel, S., 2012. Reovirus: a targeted therapeuticprogress and
potential. Mol. Cancer Res. 10, 15141525.
Majhen, D., Ambriovic-Ristov, A., 2006. Adenoviral vectorshow to use them in cancer
gene therapy? Virus Res. 119, 121133.
Miele, E., Spinelli, G.P., Di Fabrizio, E., Ferretti, E., Tomao, S., Gulino, A., 2012. Nanoparticlebased delivery of small interfering RNA: challenges for cancer therapy. Int. J.
Nanomedicine 7, 36373657.
Mittelbrunn, M., et al., 2011. Unidirectional transfer of microRNA-loaded exosomes
from T cells to antigen-presenting cells. Nat. Commun. 2, 282.
Morris, D.G., et al., 2012. REO-001: a phase I trial of percutaneous intralesional
administration of reovirus type 3 dearing (Reolysin(R)) in patients with advanced
solid tumors. Invest. New Drugs. http://dx.doi.org/10.1007/s10637-012-9865-z.
Mukherjee, A., et al., 2009. Selective cancer targeting via aberrant behavior of cancer
cell-associated glucocorticoid receptor. Mol. Ther. 17, 623631.
Nakamura, K., et al., 2004. Antitumor effect of genetically engineered mesenchymal
stem cells in a rat glioma model. Gene Ther. 11, 11551164.
Nicklin, S.A., et al., 2003. Transductional and transcriptional targeting of cancer cells
using genetically engineered viral vectors. Cancer Lett. 201, 165173.
Niess, H., et al., 2011. Selective targeting of genetically engineered mesenchymal stem
cells to tumor stroma microenvironments using tissue-specic suicide gene
expression suppresses growth of hepatocellular carcinoma. Ann. Surg. 254,
767774 (discussion 774-5).
Okada, T., Ozawa, K., 2008. Vector-producing tumor-tracking multipotent mesenchymal stromal cells for suicide cancer gene therapy. Front. Biosci. 13, 18871891.
Okada, Y., Okada, N., Mizuguchi, H., Hayakawa, T., Nakagawa, S., Mayumi, T., 2005.
Transcriptional targeting of RGD ber-mutant adenovirus vectors can improve
the safety of suicide gene therapy for murine melanoma. Cancer Gene Ther. 12,
608616.
O'Loughlin, A.J., Wofndale, C.A., Wood, M.J., 2012. Exosomes and the emerging eld of
exosome-based gene therapy. Curr. Gene Ther. 12, 262274.
Ong, H.T., Hasegawa, K., Dietz, A.B., Russell, S.J., Peng, K.W., 2007. Evaluation of T cells
as carriers for systemic measles virotherapy in the presence of antiviral antibodies.
Gene Ther. 14, 324333.
O'Riordan, C.R., et al., 1999. PEGylation of adenovirus with retention of infectivity
and protection from neutralizing antibody in vitro and in vivo. Hum. Gene Ther.
10, 13491358.
Ozawa, C.R., Springer, M.L., Blau, H.M., 2000. A novel means of drug delivery: myoblastmediated gene therapy and regulatable retroviral vectors. Annu. Rev. Pharmacol.
Toxicol. 40, 295317.
Pan, J.G., Zhou, X., Luo, R., Han, R.F., 2012. The adeno-associated virus-mediated HSVTK/GCV suicide system: a potential strategy for the treatment of bladder carcinoma. Med. Oncol. 29, 19381947.
Pardridge, W.M., 2010. Preparation of Trojan horse liposomes (THLs) for gene transfer
across the bloodbrain barrier. Cold Spring Harb. Protoc. http://dx.doi.org/10.1101/
pdb.prot5407.
Patel, M.M., Goyal, B.R., Bhadada, S.V., Bhatt, J.S., Amin, A.F., 2009. Getting into the
brain: approaches to enhance brain drug delivery. CNS Drugs 23, 3558.
Pereboeva, L., Komarova, S., Mikheeva, G., Krasnykh, V., Curiel, D.T., 2003. Approaches to
utilize mesenchymal progenitor cells as cellular vehicles. Stem Cells 21, 389404.
Pille, J.Y., et al., 2006. Intravenous delivery of anti-RhoA small interfering RNA loaded in
nanoparticles of chitosan in mice: safety and efcacy in xenografted aggressive
breast cancer. Hum. Gene Ther. 17, 10191026.

215

Pisters, L.L., et al., 2004. Evidence that transfer of functional p53 protein results in
increased apoptosis in prostate cancer. Clin. Cancer Res. 10, 25872593.
Ponnazhagan, S., Curiel, D.T., Shaw, D.R., Alvarez, R.D., Siegal, G.P., 2001. Adeno-associated
virus for cancer gene therapy. Cancer Res. 61, 63136321.
Ponnazhagan, S., et al., 2004. Adeno-associated virus 2-mediated antiangiogenic cancer
gene therapy: long-term efcacy of a vector encoding angiostatin and endostatin
over vectors encoding a single factor. Cancer Res. 64, 17811787.
Popkov, M., Jendreyko, N., McGavern, D.B., Rader, C., Barbas III, C.F., 2005. Targeting
tumor angiogenesis with adenovirus-delivered anti-Tie-2 intrabody. Cancer Res.
65, 972981.
Post, D.E., Van Meir, E.G., 2003. A novel hypoxia-inducible factor (HIF) activated
oncolytic adenovirus for cancer therapy. Oncogene 22, 20652072.
Pouton, C.W., Lucas, P., Thomas, B.J., Uduehi, A.N., Milroy, D.A., Moss, S.H., 1998. Polycation
DNA complexes for gene delivery: a comparison of the biopharmaceutical properties
of cationic polypeptides and cationic lipids. J. Control. Release 53, 289299.
Power, A.T., Bell, J.C., 2008. Taming the Trojan horse: optimizing dynamic carrier cell/
oncolytic virus systems for cancer biotherapy. Gene Ther. 15, 772779.
Power, A.T., et al., 2007. Carrier cell-based delivery of an oncolytic virus circumvents
antiviral immunity. Mol. Ther. 15, 123130.
Rai, K., et al., 2011. Liposomal delivery of MicroRNA-7-expressing plasmid overcomes
epidermal growth factor receptor tyrosine kinase inhibitor-resistance in lung
cancer cells. Mol. Cancer Ther. 10, 17201727.
Rajendran, L., et al., 2006. Alzheimer's disease beta-amyloid peptides are released in
association with exosomes. Proc. Natl. Acad. Sci. U. S. A. 103, 1117211177.
Ramezani, A., Hawley, T.S., Hawley, R.G., 2003. Performance- and safety-enhanced
lentiviral vectors containing the human interferon-beta scaffold attachment region
and the chicken beta-globin insulator. Blood 101, 47174724.
Raykov, Z., Balboni, G., Aprahamian, M., Rommelaere, J., 2004. Carrier cell-mediated
delivery of oncolytic parvoviruses for targeting metastases. Int. J. Cancer 109,
742749.
Robson, T., Hirst, D.G., 2003. Transcriptional targeting in cancer gene therapy. J. Biomed.
Biotechnol. 2003, 110137.
Ruan, H., Su, H., Hu, L., Lamborn, K.R., Kan, Y.W., Deen, D.F., 2001. A hypoxia-regulated
adeno-associated virus vector for cancer-specic gene therapy. Neoplasia 3,
255263.
Rudzinski, W.E., Aminabhavi, T.M., 2010. Chitosan as a carrier for targeted delivery of
small interfering RNA. Int. J. Pharm. 399, 111.
Schmidt, C., 2011. Amgen spikes interest in live virus vaccines for hard-to-treat
cancers. Nat. Biotechnol. 29, 295296.
Schneider, A., Simons, M., 2012. Exosomes: vesicular carriers for intercellular communication in neurodegenerative disorders. Cell Tissue Res.
Shah, K., Tung, C.H., Breakeeld, X.O., Weissleder, R., 2005. In vivo imaging of S-TRAILmediated tumor regression and apoptosis. Mol. Ther. 11, 926931.
Shen, W., Wang, C.Y., Wang, X.H., Fu, Z.X., 2009. Oncolytic adenovirus mediated
Survivin knockdown by RNA interference suppresses human colorectal carcinoma
growth in vitro and in vivo. J. Exp. Clin. Cancer Res. 28, 81.
Shinozaki, K., et al., 2006. Efcient infection of tumor endothelial cells by a capsidmodied adenovirus. Gene Ther. 13, 5259.
Sims Jr., T.L., et al., 2008. Neural progenitor cell-mediated delivery of interferon
beta improves neuroblastoma response to cyclophosphamide. Ann. Surg. Oncol.
15, 32593267.
Song, J.S., 2005. Adenovirus-mediated suicide SCLC gene therapy using the increased
activity of the hTERT promoter by the MMRE and SV40 enhancer. Biosci.
Biotechnol. Biochem. 69, 5662.
Soriano, P., et al., 1983. Targeted and nontargeted liposomes for in vivo transfer to
rat liver cells of a plasmid containing the preproinsulin I gene. Proc. Natl. Acad.
Sci. U. S. A. 80, 71287131.
Spagnou, S., Miller, A.D., Keller, M., 2004. Lipidic carriers of siRNA: differences in
the formulation, cellular uptake, and delivery with plasmid DNA. Biochemistry
43, 1334813356.
Stender, S., et al., 2007. Adeno-associated viral vector transduction of human mesenchymal stem cells. Eur. Cell. Mater. 13, 9399 (discussion 99).
Stoff-Khalili, M.A., et al., 2007. Mesenchymal stem cells as a vehicle for targeted delivery
of CRAds to lung metastases of breast carcinoma. Breast Cancer Res. Treat. 105,
157167.
Stoff-Khalili, M.A., et al., 2008. Cancer-specic targeting of a conditionally replicative
adenovirus using mRNA translational control. Breast Cancer Res. Treat. 108,
4355.
Studeny, M., Marini, F.C., Champlin, R.E., Zompetta, C., Fidler, I.J., Andreeff, M., 2002.
Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta
delivery into tumors. Cancer Res. 62, 36033608.
Sun, X.Y., Nong, J., Qin, K., Warnock, G.L., Dai, L.J., 2011. Mesenchymal stem cellmediated cancer therapy: a dual-targeted strategy of personalized medicine.
World J. Stem Cells 3, 96103.
Szybalska, E.H., Szybalski, W., 1962. Genetics of human cess line. IV. DNA-mediated
heritable transformation of a biochemical trait. Proc. Natl. Acad. Sci. U. S. A. 48,
20262034.
Tabatabai, G., Wick, W., Weller, M., 2011. Stem cell-mediated gene therapies for malignant
gliomas: a promising targeted therapeutic approach? Discov. Med. 11, 529536.
Tan, A., Rajadas, J., Seifalian, A.M., 2012. Exosomes as nano-theranostic delivery platforms for gene therapy. Adv. Drug Deliv. Rev. 65 (3), 357367.
Tang, C., Russell, P.J., Martiniello-Wilks, R., Rasko, J.E., Khatri, A., 2010. Concise review:
nanoparticles and cellular carriers-allies in cancer imaging and cellular gene therapy? Stem Cells 28, 16861702.
Taratula, O., Garbuzenko, O., Savla, R., Wang, Y.A., He, H., Minko, T., 2011.
Multifunctional nanomedicine platform for cancer specic delivery of siRNA by

216

G. Collet et al. / Gene 525 (2013) 208216

superparamagnetic iron oxide nanoparticlesdendrimer complexes. Curr. Drug

Deliv. 8, 5969.
Tazzyman, S., Lewis, C.E., Murdoch, C., 2009. Neutrophils: key mediators of tumour
angiogenesis. Int. J. Exp. Pathol. 90, 222231.
Thery, C., Zitvogel, L., Amigorena, S., 2002. Exosomes: composition, biogenesis and
function. Nat. Rev. Immunol. 2, 569579.
Varma, N.R., et al., 2012. Endothelial progenitor cells (EPCs) as gene carrier system for
rat model of human glioma. PLoS One 7, e30310.
Wahlgren, J., et al., 2012. Plasma exosomes can deliver exogenous short interfering
RNA to monocytes and lymphocytes. Nucleic Acids Res. 40, e130.
Wang, S., Balasundaram, G., 2010. Potential cancer gene therapy by baculoviral
transduction. Curr. Gene Ther. 10, 214225.
Wang, T., Upponi, J.R., Torchilin, V.P., 2012. Design of multifunctional non-viral
gene vectors to overcome physiological barriers: dilemmas and strategies. Int. J.
Pharm. 427, 320.
Wilcox, M.E., et al., 2001. Reovirus as an oncolytic agent against experimental human
malignant gliomas. J. Natl. Cancer Inst. 93, 903912.
Xia, C.F., Boado, R.J., Zhang, Y., Chu, C., Pardridge, W.M., 2008. Intravenous glialderived neurotrophic factor gene therapy of experimental Parkinson's disease
with Trojan horse liposomes and a tyrosine hydroxylase promoter. J. Gene
Med. 10, 306315.
Xie, M., et al., 2009. A novel triple-regulated oncolytic adenovirus carrying PDCD5 gene
exerts potent antitumor efcacy on common human leukemic cell lines. Apoptosis
14, 10861094.
Xin, H., et al., 2007. Targeted delivery of CX3CL1 to multiple lung tumors by mesenchymal
stem cells. Stem Cells 25, 16181626.
Yang, Y.P., et al., 2012. Inhibition of cancer stem cell-like properties and reduced
chemoradioresistance of glioblastoma using microRNA145 with cationic
polyurethane-short branch PEI. Biomaterials 33, 14621476.
Yao, X., et al., 2009. Systemic administration of a PEGylated adenovirus vector with a
cancer-specic promoter is effective in a mouse model of metastasis. Gene Ther.
16, 13951404.

Yoo, J.Y., et al., 2007. VEGF-specic short hairpin RNA-expressing oncolytic adenovirus elicits potent inhibition of angiogenesis and tumor growth. Mol. Ther. 15,
295302.
Yoo, J.Y., et al., 2008. Short hairpin RNA-expressing oncolytic adenovirus-mediated
inhibition of IL-8: effects on antiangiogenesis and tumor growth inhibition.
Gene Ther. 15, 635651.
Yuan, X., Hu, J., Belladonna, M.L., Black, K.L., Yu, J.S., 2006. Interleukin-23-expressing
bone marrow-derived neural stem-like cells exhibit antitumor activity against
intracranial glioma. Cancer Res. 66, 26302638.
Zeng, J., Du, J., Zhao, Y., Palanisamy, N., Wang, S., 2007. Baculoviral vector-mediated
transient and stable transgene expression in human embryonic stem cells.
Stem Cells 25, 10551061.
Zhang, H.G., Grizzle, W.E., 2011. Exosomes and cancer: a newly described pathway
of immune suppression. Clin. Cancer Res. 17, 959964.
Zhang, Y., Pardridge, W.M., 2009. Near complete rescue of experimental Parkinson's
disease with intravenous, non-viral GDNF gene therapy. Pharm. Res. 26,
10591063.
Zhang, Y., Zhang, Y.F., Bryant, J., Charles, A., Boado, R.J., Pardridge, W.M., 2004. Intravenous RNA interference gene therapy targeting the human epidermal growth factor
receptor prolongs survival in intracranial brain cancer. Clin. Cancer Res. 10,
36673677.
Zhang, Z., et al., 2006. Delivery of telomerase reverse transcriptase small interfering
RNA in complex with positively charged single-walled carbon nanotubes
suppresses tumor growth. Clin. Cancer Res. 12, 49334939.
Zhang, Y., Wang, Y., Boado, R.J., Pardridge, W.M., 2008. Lysosomal enzyme replacement of the brain with intravenous non-viral gene transfer. Pharm. Res. 25,
400406.
Zhao, Y., et al., 2012. Targeted suicide gene therapy for glioma using human embryonic
stem cell-derived neural stem cells genetically modied by baculoviral vectors.
Gene Ther. 19, 189200.
Zhu, W., Wei, L., Zhang, H., Chen, J., Qin, X., 2012. Oncolytic adenovirus armed with IL-24
Inhibits the growth of breast cancer in vitro and in vivo. J. Exp. Clin. Cancer Res. 31, 51.