Biomedicines 2014, 2, 149-162; doi:10.

3390/biomedicines2020149
OPEN ACCESS

biomedicines
ISSN 2227-9059
www.mdpi.com/journal/biomedicines/
Review

Gene Therapy Used in Cancer Treatment

Thomas Wirth 1,* and Seppo Ylä-Herttuala 1,2
1
A.I Virtanen Institute, Department of Biotechnology and Molecular Medicine,
University of Eastern Finland; Neulaniementie 2, 70211 Kuopio, Finland;
E-Mail: seppo.ylaherttuala@uef.fi
2
Research Unit and Gene Therapy Unit, Kuopio University Hospital, 70210 Kuopio, Finland

* Author to whom correspondence should be addressed; E-Mail: thomas.wirth@uef.fi;

Tel.: +358-40-3553561.

Received: 15 January 2014; in revised form: 12 March 2014 / Accepted: 18 March 2014 /
Published: 8 April 2014

Abstract: Cancer has been, from the beginning, a target of intense research for gene
therapy approaches. Currently, more than 60% of all on-going clinical gene therapy trials
worldwide are targeting cancer. Indeed, there is a clear unmet medical need for novel
therapies. This is further urged by the fact that current conventional cancer therapies are
frequently troubled by their toxicities. Different gene therapy strategies have been employed
for cancer, such as pro-drug activating suicide gene therapy, anti-angiogenic gene therapy,
oncolytic virotherapy, gene therapy-based immune modulation, correction/compensation of
gene defects, genetic manipulation of apoptotic and tumor invasion pathways, antisense,
and RNAi strategies. Cancer types, which have been targeted with gene therapy, include
brain, lung, breast, pancreatic, liver, colorectal, prostate, bladder, head and neck, skin,
ovarian, and renal cancer. Currently, two cancer gene therapy products have received
market approval, both of which are in China. In addition, the stimulation of the host’s
immune system, using gene therapeutic approaches, has gained vast interest. The intention
of this review is to point out the most commonly viral and non-viral vectors and methods
used in cancer gene therapy, as well as highlight some key results achieved in clinical trials.

Keywords: cancer; glioma; gene therapy; gene transfer; viral vectors; non-viral vectors;
safety; clinical trials
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1. Introduction

Cancer is a major global health problem accounting, annually, for more than eight million deaths
globally. It is a complex, multifactorial disease involving changes in the genome, which is orchestrated
by host and environmental interactions [1]. The hallmarks of cancer are self-sufficiency in growth
signals, insensitivity to anti-growth signals, ability for tissue invasion and metastasis, limitless
replicative potential, sustained angiogenesis, and evasion of apoptosis [1]. The tumor microenvironment,
which is composed of various non-malignant cells expressing various regulatory proteins, as well as
the extracellular matrix, plays a pivotal role in the initiation and progression of cancers [2]. Gene
therapy aims at delivering genetic material into target cells or tissue and to express it with the intention
to gain a therapeutic effect. It has the advantage over conventional therapies due to the fact that it can
be administered locally, thereby delivering, locally, a high therapeutic dose without risking systemic
adverse effects. Furthermore, since most gene therapies are single time applications, they can be cost
effective in the long run.

2. Gene Therapy for Cancer: An Overview

Rogers et al. was one of the first to demonstrate an initial proof-of-concept of virus mediated gene
transfer. What he showed was that foreign genetic material can be transferred to cells of interest by
utilizing viruses [3]. Motivated by the results he went even further and tested it in humans. With this
experiment, Rogers became the first to perform a human gene therapy trial. In that study, Rogers used
a wild-type Shope papilloma virus with the intention to introduce the gene for arginase into two girls
suffering from a urea cycle disorder (i.e., hyperargininemias) [4,5]. He hypothesized that the Shope
papilloma virus would naturally encode the gene for arginase activity and that this gene could be
transferred by introducing the virus to the patients. Unfortunately, the outcome of the trial was
negative. There was no change in the arginine levels, nor was there a change in the clinical course of
the disease in these patients. Even though Rogers “out of the box” thinking was intriguing, it was
doomed to fail as it later turned out that the Shope papilloma virus genome does not encode the
arginase gene.
The US Food and Drug Administration (FDA) approved the first gene therapy protocol, which was
carried out in 1989. Therein, tumor infiltrating lymphocytes collected from advanced melanoma
patients were ex vivo transduced with a marker gene (i.e., not a therapeutic gene), expanded in vitro,
and re-infused to the patients [6]. The first clinical trial on cancer with an therapeutic intend was
started in the following year, wherein patients with advanced melanoma were treated with tumor
infiltrating lymphocytes genetically modified ex vivo to express tumor necrosis factor [6].
Another important milestone in the history of gene therapy was the study conducted by Cline et al.
Cline treated thalassaemia patients, wherein he extracted bone marrow cells from these patients and
transfected ex vivo with plasmids containing the human globulin gene. After cells were transfected
they were administered back to the patients [7,8]. The reason why this study presents a milestone in the
history of gene therapy is not because of the failure of the study itself, but because the study was done
without the consent to perform these studies from the University of California, Los Angeles (UCLA)
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Institutional Review Board. This case demonstrated that knowledge was very limited and that human
gene therapy would be technically, as well as ethically much more complex than expected.

3. Gene Transfer Methods and Vectors Used for Gene Therapy

The challenge in gene therapy is to deliver an adequate amount of genetic material into target cells
or tissues and to maintain gene expression for a desired period of time. Genetic material can be
introduced to their target cells or tissues via different methods of delivery. In principle, we can group
them into: (1) physical; (2) viral; (3) non-viral methods; and (4) bacterial or yeast.
Electroporation, ultrasound, and gene gun deliveries are examples of physical methods that have
been used. As the name already implies, with viral vectors a biological (i.e., virus) vector is used as a
vehicle to deliver the genetic material into the cells, whereas with non-viral gene transfer methods a
synthetic carrier (liposomes or nanoparticles) is used. Different vectors have different properties in
relation to their transduction efficiency and their efficacy to express the introduced genes. In addition,
they differ in respect of the duration of expression of the transgene, as well as their safety profile.
Depending on the requirements, different vectors can be used for different therapeutic purposes.
Currently, viral vectors are considered as the most effective of all gene delivery methods for in vivo
gene transfer. Ideally, the gene transfer vector should be able to target a specific tissue with high
transduction efficiency and sustain a stable, regulated gene expression without any side effects or
immunogenic responses. Unfortunately, none of the currently used gene delivery vectors fulfil all these
criteria. Local injection of a vector typically results in a limited, but accurate effect area. On the
contrary, systemic administration of a vector can result in a system wide expression. Consequently,
vectors and their administration methods have been modified in order to achieve targeted delivery, as
well as to increase transduction efficiency [9]. Most viral vectors have, however, already natural
tropism to certain cell types or tissues, which can be utilized for therapeutic approaches [10].

3.1. Viral Vectors

The most commonly used viral vectors used for gene transfer are adenoviruses, lenti- and
retroviruses (including the human immunodeficiency virus (HIV)), vaccinia viruses, adeno associated
viruses (AAV), and baculoviruses. These vectors differ from each other regarding their cell
tropisms, expression profiles, transgene capacities, immunogenicity, as well as different duration of
transgene expression.
In addition to their origin, viral vectors can be divided into integrating and non-integrating vectors.
Adenoviruses and baculoviruses are examples of non-integrating vectors. They lack the ability to
integrate their genome (and, hence, with it also the transgene) into the host genome. Lenti- and
retroviruses, as well as AAVs, on the contrary, are examples of vectors that do integrate into the host
genome. While the expression of the transgene is transient in case of non-integrating viral vectors
(diminishing in a few weeks), integrating vectors commonly results in long-term expression (months,
up to years). This integration of the transgene into the host genome has raised concerns about the
safety of these vectors. This is due to the fact that integration has been observed with retroviral vectors
to occur occasionally in actively expressed sites (i.e., insertional mutagenesis) [11–13].
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Genetic material can be delivered also by ex vivo gene transfer approach. Therein, the genetic
material is introduced to the cell outside the patient (i.e., ex vivo), into previously isolated autologous
cells, which then are re-introduced back to the patient.
Currently, adenoviruses are the most dominant gene delivery vectors used in gene therapy. More
than 50 different serotypes have been identified for adenoviruses, which can be divided further into six
subgroups (A–F) [14]. Of those, the serotypes 2 and 5 are the most commonly used ones in gene
therapy. A limiting factor with adenoviruses is the fact that detectable levels of pre-existing antibodies
can be found in 97% of individuals, which potentially may influence transduction efficiency and
therapeutic outcome.

3.2. Non-Viral Vectors

Viral vectors have been shown to be efficient gene transfer tools. Nevertheless, drawbacks such as
rapid clearance of viral vectors from the bloodstream (when injected systemically), their immunogenic
and inflammatory potential, has urged the development of new synthetic gene delivery vectors. In fact,
non-viral gene delivery systems are a topic that is currently being studied extensively as alternatives
for viral delivery systems. The simplest form of a non-viral system is naked plasmid DNA. The
advantage of naked plasmid is that it poses the lowest form of toxicity or other unwanted reactions. In
addition, it is easy to formulate and inexpensive to produce. However, its disadvantage is the low
transfection efficiency compared to viral-mediated gene transfer [15]. As a result, to improve transfection
efficiency, cationic polymers, or lipids formulations have been developed to condense plasmid DNA to
protect the degradation of DNA and to enhance uptake and transfection of plasmids [15]. The
advantage with those formulations is that polymers or lipids can comparatively easily be designed to
attain certain properties. For example, non-viral vectors can easily be targeted to a target tissue or cell
by coupling of cell- or tissue-specific targeting moieties on the carrier. Furthermore, by determining
the size of the micro- or nanoparticle the biodistribution, cellular internalization, and intracellular
trafficking of the micro- or nanoparticle can be influenced [16]. Unfortunately, the success of non-viral
delivery systems in clinical applications in gene therapy has been limited. Compared to viral vectors,
non-viral vectors have not gone through the evolutionary process of time that viruses have, which
typically can be seen as low transduction efficiencies in vivo.
The success of the non-viral gene therapy is dependent on the various extra- and intracellular
barriers that affect the efficacy of all gene delivery systems, including cellular uptake, endosomal
escape, nuclear uptake, and gene expression [16–18].

4. Clinical Efficacy of Gene Therapy

Different gene therapy approaches using different gene transfer vectors have been studied for
cancer gene therapy. These include induction of apoptosis, oncolytic virotherapy, immune modulation,
anti-angiogenic gene therapy, correction of gene defects, inhibition of tumor invasion, gene therapy to
enhance chemo- and radiotherapy, myeloprotective gene therapy, antisense and RNA interference
(RNAi) based strategies, and pro-drug activation/suicide gene therapy. Unfortunately, only few of
these strategies have made it actually to the clinic. One commonly used strategy in cancer gene therapy
has been the use of a commonly occurring mutation in the p53 protein. In 2003, Lang et al. described a
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phase I clinical trial using an adenoviral vector encoding for the tumor suppressor gene TP53 to treat
patients with recurrent malignant gliomas, wherein 15 patients ought to undergo intratumoral
stereotactic injection of the adenoviral vector via an implanted catheter, followed by en bloc resection
of the tumor and treatment of the post-resection cavity [19]. Due to the design of the study, tumor
response could not be assessed, but the study demonstrated minimal toxicity. No systemic viral
dissemination was observed and a maximum tolerated dose was not reached in this study. Furthermore,
analysis of tumor specimens demonstrated restricted transgene expression close to the injection site.
Another study, and similar to the virus used by Lang et al. is Gendicine™. Gendicine™ is a
replication-incompetent adenovirus encoding for the TP53 gene (in place of the viral E1 gene) used for
the treatment of a variety of cancers. What makes Gendicine™ interesting is the fact that it became the
first gene therapy product that has been approved for clinical use [20]. In a phase I clinical trial, with
12 laryngeal cancer patients, Gendicine™ demonstrated therapeutic potential, as none of the patients
treated with Gendicine™ had tumor relapse during the five-year follow-up after the treatment [21]
Additionally, Gendicine™ demonstrated good safety profile, exemplified by a phase II/III trials with
132 head and neck squamous cell carcinoma patients. Therein, 32% showed fever as the only
side-effect of the treatment [22]. When Gendicine™ was used in combination with radiotherapy, 64%
of the patients responded with a complete regression and 29% with a partial regression while with
radiotherapy alone, 19% showed a complete regression and 60% a partial regression, suggesting
synergistic effect of the combination treatment [22].
A second gene therapy product that received market approval by the Chinese SFDA is Oncorine™,
developed by Chinese Shanghai Sunway Biotech. Oncorine™ is a conditionally replicative adenovirus,
which is produced by deleting the adenoviral E1B 55K gene. The deletion of this gene prevents the
virus to bind and inactivate the wild-type p53 protein, which is an essential self-defence mechanism of
the host against virus infection [23]. When E1B 55K activity is removed, the replication in normal
cells is blocked, allowing only replication in p53-deficient cells. In malignant cells the viral
proliferation leads to oncolysis, used as a cancer therapy to treat solid tumors. Interesting to this
product is the fact that ONYX-015 (developed by Onyx Pharmaceutical’s), which is analogous to
Oncorine™, never received market approval. Compared to Oncorine™ ONYX-015 was not able to
demonstrate therapeutic benefit in clinical settings. For example, in a phase I dose-escalation trial
published by Chiocca et al., 24 patients with recurrent malignant glioma were injected with the
oncolytic virus in a total of 10 injections into 10 different sites of the cavity of resected tumors [24].
Even though the study demonstrated that ONYX-015 was safe and that none of the patients
experienced serious adverse events that could be attributed to the virus, all patients showed tumor
progression. One patient with anaplastic astrocytoma had stable disease and two patients who underwent
a second resection had lymphocytic and plasmacytoid cell infiltration at the site of injection.
Oncorine™ and ONYX-015 have together provided a vast amount of safety data for various types
of cancer, including glioma, head and neck, pancreatic, and ovarian cancers, demonstrating an
acceptable safety profile [20]. Typical complications included fever, injection site pain, nausea,
alopecia, leucopenia, and flu-like symptoms [25].
In order to improve efficacy of oncolytic viruses, additional therapeutic proteins have been added to
the viruses. An example for this is Onco VEXGM-CSF, which is a second-generation oncolytic
herpes simplex virus (HSV), additionally coding for the therapeutic protein granulocyte macrophage
Biomedicines 2014, 2 154

colony-stimulating factor. A phase I safety study showed that Onco VEXGM-CSF was well tolerated
and safe when administered by intratumoral injection in patients with cutaneous or subcutaneous
deposits of breast, head and neck and gastrointestinal cancers, and malignant melanoma who had failed
prior therapy [26]. In addition, evidence of an antitumor effect was seen in that study, which was further
supported by a Phase I/II, in which Onco VEXGM-CSF was given in combination with radiotherapy
and cisplatin to patients with untreated stage III/IV squamous cell cancer of the head and neck [27].

4.1. Gene Therapeutic Approaches to Stimulate the Immune System

Immunotherapy is a topic that has gained much attention recently. Typically, in immunotherapy the
objective is to enhance either the recognition or presentation of tumor-associated antigens (TAA’s).
Unfortunately, there are common challenges that have been faced by immunotherapies, including the
natural tolerance towards TAAs and the strongly immunosuppressive tumor microenvironment.
Particularly, the genetic engineering of T cells has been of intense research [28]. An example for
genetic engineering of T cells is the introduction of a T cell receptor (TCR) against a known TAA.
An example of such an approach is the clinical report by Morgan et al., wherein they transduced
normal peripheral blood lymphocytes (PBLs) using retroviral vectors with an anti-MART1 TCR
transgene that was isolated from tumor infiltrating lymphocytes (TILs) of patients with
melanoma [29]. Therein, they demonstrated durable engraftment of the T cells in 15 patients at levels
exceeding 10% of peripheral blood lymphocytes for at least two months after cell infusion.
Furthermore, they observed high sustained levels of circulating, engineered PBLs at one year after
infusion in two patients who both demonstrated objective regression of metastatic melanoma lesions.
In another clinical trial T cells were transduced with a TCR against the antigen NY-ESO-1, a
cancer/testis (CT) antigen expressed in various cancers [30]. In addition, in this trial, an objective
clinical response in patients was observed, providing evidence that introduction of a TCR targeting a
TAA represents a feasible option for the treatment of cancer.
Similarly to introducing a TCR an artificial T cell receptor (typically referred to as a chimeric
antigen receptor; CAR) can be introduced to T cells. Utilizing CAR to target T cells to cancer cells has
resulted in impressive response rates in the clinic against haematological malignancies [31]. An example
is the study performed by Kochenderfer et al., who assessed in a clinical phase I trial the potential and
safety of adoptive transfer of genetically modified T cells expressing CAR against CD19 [32].
Another way to boost an anti-tumoral immune response was evaluated by Herman et al. in a
randomized phase III clinical trial among patients with locally advanced pancreatic cancer [33].
A second generation replication-deficient adenovirus of the serotype 5 containing the TNF-α cDNA
under the early growth response protein 1 (Egr-1) promoter was assessed for this purpose. The Egr-1 is
a promoter, which is induced by ionizing radiation, thus restricting the expression of the transgene to
the radiation field. In that study, 304 patients were randomly assigned 2:1 to standard of care plus gene
therapy (i.e., adenovirus encoding for TNF-α) versus standard of care alone. The results revealed that
even though standard of care plus gene therapy was safe it did not result in a survival benefit in
patients with locally advanced pancreatic cancer [33]. A more promising result, in contrast, was
presented in a study by Malmström et al., wherein they studied the immunostimulating effects of
gene therapy with adenoviral vectors expressing CD40 ligand [34]. CD40L belongs to the TNF gene
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superfamily and is known to be a potent immune stimulator of T helper 1 cells. This study recruited
eight patients with invasive bladder cancer for a phase I/IIa trial evaluating the safety, efficacy of gene
transfer, immune effects, and antitumor responses [34]. The results demonstrated that the presence of
IFN-γ was increased in the biopsies of tumors, whereas levels of circulating T regulatory cells were
reduced. Further histologic evaluation indicated that adenoviral CD40L gene therapy reduced the load
of malignant cells in the bladder.
In another study performed by Chiocca et al., 11 patients were injected with different doses of
interferon-β-expressing adenoviruses ranging from 2 × 1010 to 2 × 1011 viral particles stereotactically
into the tumor [35]. This was followed by surgical removal of the tumor four to eight days later with
additional injections of the adenovirus into the tumor bed. Unfortunately, all patients had disease
progression and/or recurrence within four months of the treatment. The median time to tumor
progression was 9.3 weeks and the median overall survival was 17.9 weeks.
In addition to the above mentioned strategies, the utilisation of a pro-drug activating suicide gene
therapy is an approach that has been extensively explored pre-clinically and in the clinic for cancer
therapy, which will be discussed in more detail below.

4.2. Pro-Drug Activating Suicide Gene Therapy

The principle of pro drug activating suicide gene therapy is to introduce a transgene encoding for an
enzyme that is either absent in mammalian cells or present in a very inactive form, into the tumor. The
enzyme produced by the transduced cells will convert the subsequently administered inactive pro drug
into its active form, evoking the death of cells expressing the therapeutic gene. Therein, the bystander
effect (a phenomenon wherein also the neighboring non-transduced cells are killed) is fundamental for
the therapeutic success [36]. In this concept, brain tumors bear several features that make them
particularly amenable to pro-drug activating gene therapy. First of all, brain tumors are typically
single, localized lesions of rapidly dividing cells in a background of non-dividing cells. Furthermore,
recurrence typically happens in the close vicinity of the original lesion. Unfortunately, the first results
were not very promising. Transduction efficiency was a major problem resulting in a poor therapeutic
efficacy. The use of retroviral vectors in those early studies was most likely a major reason for poor
transduction efficiency. In comparison to retroviral vectors, adenoviral vectors have shown to have
much higher transduction efficacy as well as transgene expression [37]. One of the reasons is that in
comparison to retroviruses, adenoviruses transduce both dividing and quiescent cells. This feature may
provide an important advantage, as not all cancer cells proliferate within the tumor at a given time
point. In 1996, Eck et al. published the first phase I clinical trial, where the pro-drug activating enzyme
Herpes simplex virus—thymidine kinase (HSV-tk) packed into an adenovirus was used with the
intention to treat patients with recurrent gliomas [38]. The first completed trial using adenovirus
HSV-tk in patients with malignant glioma, however, was published by Sandmair et al. in 2000 [37]. In
that study, Sandmair et al. compared the efficacy of both the retrovirus-packaging cells for HSV-tk and
the adenovirus mediated HSV-tk gene therapy for the treatment of primary or recurrent gliomas.
Twenty-one patients were enrolled in that study. The mean survival time in the adenovirus HSV-tk
group was 15 months and significantly longer when compared to a 7.4 months survival time in the
retrovirus-packaging cell group. The control group, which received adenovirus LacZ had a mean
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survival time of 8.3 months. Although the retrovirus-packaging cell approaches were found safe, no
efficacy was observed. The low gene transfer efficacy with retrovirus and the lack of the treatment
response indicated that retroviral HSV-tk gene therapy may not be efficient enough in human clinical
settings. The lack of efficacy was further confirmed in the first randomized, open-label, parallel group
phase III clinical trial of 248 patients, where HSV-tk was produced by retroviral producing cells. The
study did not show any improvement of survival [39]. The clinical efficacy of HSV-tk gene therapy
was first demonstrated in two separate phase II clinical trials; a phase IIa trial and a randomized and
controlled phase IIb trial [37,40]. Therein, 17 patients with operable or recurrent malignant gliomas
receiving HSV-tk gene therapy out of 36 patients implicated a survival advantage over control patients,
who did not receive HSV-tk gene therapy [40]. The mean survival of the patients treated with HSV-tk
gene therapy was significantly longer (p < 0.0095) when compared to the standard care group or a
historical control group (p < 0.0017). This study was also historically the first randomized, controlled
trial with an adenoviral vector using the HSV-tk pro-drug activating suicide gene, where survival
benefit could be demonstrated. Encouraged by these results, a multicenter, standard care controlled,
randomized clinical phase III trial was commenced. Therein, 250 patients were recruited and randomly
allocated, whereof 124 were allocated to the experimental group and 126 to the standard care group.
The median time to death or re-intervention was longer in the experimental group (308 days) than in
the control group (268 days). Interestingly, in a subgroup of patients with non-methylated status of the
DNA repair gene MGMT (O6-alkylguanine DNA alkyltransferase), the hazard ratio (HR) was 1.72
(p = 0·008). However, no statistical difference in the overall survival between the groups was
observed [41]. Although the study did not demonstrate improvement of overall survival, the findings
suggested that the use of HSV-tk gene therapy after tumor resection can increase time to death or
re-intervention in patients with newly diagnosed supratentorial glioblastoma multiforme. Furthermore,
this study demonstrates that locally delivered gene therapy for glioblastoma should be further
developed, especially for patients who are unlikely to respond to standard chemotherapy. This study is,
thus far, the only adenoviral vector study that has completed a phase III clinical trial, which is based on
the suicide gene therapy with HSV-tk.

5. Safety of Gene Therapy

Despite the tragic case of Jesse Gelsinger, who died as a result of gene therapy using adenoviral
vectors, the safety data collected from different human gene therapy trials have been uniformly
satisfactory. However, it should be pointed out that viral vectors used in gene therapy are typically
human pathogens, and hence, pre-existing antibodies against the viral vector may be present, which
might result in an unwanted immune response. For example, an injection of adenoviral vectors will
result in an initial non-specific immune response in the host, i.e., release of a variety of cytokines
followed by a specific antibody and cell-mediated immune response directed against transduced cells.
However, the response towards adenoviruses is serotype dependent. For example, a study by
Thoma et al. demonstrated that the immediate cytokine response of macrophages following adenovirus
stimulation differs between adenovirus serotypes, hence, is serotype-specific. Particularly, in a
long-term study, wherein either adenovirus of the serotype 11 (Ad11) or 5 (Ad5) was administered
intra peritoneally, Ad11 caused no/mild and Ad5 moderate/severe toxicity [42].
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Generally, there is still not much long-term safety data using viral vectors in humans. Nevertheless,
several meta-analysis already exist for adenoviruses demonstrating an adequate safety profile in
humans [41,43]. The tolerability towards adenoviral vectors has been acceptable and the side effects
have mostly been mild without any serious adverse events related to gene therapy.
Different means with the intention of improving the safety of gene therapy have been implemented.
One approach is to develop targeting strategies in order to enhance the delivery of gene transfer
vectors, and hence, to improve the duration and efficacy of gene expression. Generally, one of the
major shortcomings with gene therapy is their lack of specificity to their target cells and their low
transduction efficiency. Improving specificity and/or transduction efficacy ultimately would result also
in a better safety profile. Consequently, the improvement of transduction efficacy of gene transfer
vectors has come along with the development of vector technologies, including re-engineering of viral
vectors using epitope insertion, chemical modification, and molecular evolution [44]. An example for
this was demonstrated in a phase I clinical trial by Kim et al., where they modified the RGD fiber knob
on adenoviruses, thereby enhancing viral infectivity of cancer cells [45].
The role of innate immunity, as well as the activation of T and B cells in response to the vector and
its transgene product is a topic of intense research. Particularly, the possible effects of gene transfer
vectors and/or their expressed proteins on local lymph nodes are topics that require further evaluation.
The pre-existence of neutralizing antibodies (e.g., against several adenovirus serotypes or AAVs) has
been acknowledged already for quite some time and it is known that these pre-existing neutralizing
antibodies can substantially diminish transduction efficiency [46].
In order to improve specificity, as well as transduction efficiency, viral surface proteins have been
modified, removed or replaced. For example, lentiviral vectors have been generated, wherein a cell
type specific ligand or antibody has been fused to the viral envelope (i.e., pseudotyping) [47].
The downside of this has been that different modifications resulted in low vector titers during
lentivirus production [13]. Furthermore, it has been shown that targeting may also potentially
compromise the entry of the vector into the cell [13,47]. On the contrary to targeting viral vectors to
specific cells, pseudotyping can also be used to broaden tropism of the viral vector to other cells. For
example, retroviruses and lentiviruses are frequently pseudotyped with the Vesicular Stomatitis virus
G-protein (VSV-G) to widen their tropism and to increase their yield in production [48].
Another approach to increase specificity of viral vectors to their target cells is the use of
tissue-specific or conditional promoters. An example for conditional dependent gene expression is the
use of hypoxia-specific regulatory systems, where gene expression is aimed to be induced and
restricted to ischemic tissues [49]. Commonly, these hypoxia-specific regulatory systems have been
applied to various ischemic disease models, including ischemic myocardium, stroke, and injured spinal
cord, but could also be used in cancer gene therapy [50]. Gene expression can also be regulated based
on a genotypic feature (e.g., a mutated TP53 gene in cancer cells), which has been discussed already
above in case of Oncorine™.
The risk of insertional mutagenesis with integrating vectors is a safety risk. Retroviruses,
lentiviruses and AAVs are examples of viruses that integrate their genome into their host
chromosomes. By doing so, there is a chance that these vectors may integrate into gene regulatory
areas or into transcriptionally active areas, respectively, which potentially can adversely result in
insertional mutagenesis and oncogenesis. Several approaches have been developed to circumvent these
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problems. Therefore, targeted integration of transgenes to predetermined genomic sites has been one of
the most important topics in current vector development. One of the most efficient methods to achieve
targeted integration into human cells is based on DNA double-strand break-enhanced homologous
recombination [51]. In addition, lentivirus/transposon hybrids have been developed in order to reduce
the risk of insertional mutagenesis [52]. For example, the Sleeping Beauty transposon system is an
attractive approach allowing stable integration of the transgene through transposition into the target
cell genome [53,54]. The advantage of the Sleeping Beauty transposon system is that it does not
exhibit a preference for integration within active genes and the inverted repeats have only very low
residual promoter/enhancer activity. The risk of genotoxicity/mutagenesis caused by gene therapy has
been one of the main arguments against human gene therapy is. However, the fact, that conventional
cancer therapies (i.e., radiation therapy and chemotherapy) also can cause genetic alterations is often
disregarded. It is fact that many chemotherapeutic drugs, as well as radiation therapy, may cause
genetic alterations and oncogenesis in patients [55–57].
In addition, by developing the manufacturing of gene transfer vectors (i.e., development of
production cell lines, production methods, as well as the purification steps) the safety profile of gene
transfer vectors can be improved. For example, gutless adenoviral vectors are vectors, where all other
genes but those essential for virus production are removed and replaced with the gene of interest,
driven by a suitable promoter. As a result, gutless adenoviruses still exhibit high transduction
efficiency and similar tropism to previous vectors, but are less immunogenic than the first generation
adenoviral vectors. However, since gutless vectors are devoid of all viral genes, co-infection with a
helper adenovirus is required that provides proteins needed for its genome replication, packaging, and
capsid formation. As both helper and gutless vectors have the same viral capsid, separation must be
addressed before purification, which is laborious and has not been without challenges [58].

6. Conclusions

Gene therapy is an intriguing and potential approach to treat various diseases, including cancer.
Currently most gene therapy protocols are limited to the local administration of the gene transfer
vector, or to ex vivo gene transfer approaches. One of the challenges in gene therapy is still the low
transduction efficiency and its minimal distribution of the vector within the tissue. However, it should
be emphasized that focus should not only be directed towards vector development itself, but also
towards the manufacturing of these vectors. The high cost involved in viral vector manufacturing,
which is the result of tedious downstream purifications steps, has been challenging. In addition, the
concept of using gene therapy as a single agent therapy has not been as successful as being hoped.
Consequently, combination therapy with existing conventional modalities or other new therapies
should be considered and may offer additional benefit in cancer gene therapy.

Acknowledgments

The authors would like to acknowledge the European Union’s Seventh Framework Programme, the
Finnish Academy, the Kuopio University Hospital (EVO grant), the Spearhead Project-programme of
the University of Eastern Finland and the North-Savo Cancer Foundation.
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Conflicts of Interest

The authors declare no conflict of interest.

References

1. Hanahan, D.; Weinberg, R.A. The hallmarks of cancer. Cell 2000, 100, 57–70.
2. Bissell, M.J.; Hines, W.C. Why donʼt we get more cancer? A proposed role of the
microenvironment in restraining cancer progression. Nat. Med. 2011, 17, 320–329.
3. Rogers, S.; Pfuderer, P. Use of viruses as carriers of added genetic information. Nature 1968, 219,
749–751.
4. Rogers, S.; Lowenthal, A.; Terheggen, H.G.; Columbo, J.P. Induction of arginase activity with the
Shope papilloma virus in tissue culture cells from an argininemic patient. J. Exp. Med. 1973, 137,
1091–1096.
5. Terheggen, H.G.; Lowenthal, A.; Lavinha, F.; Colombo, J.P.; Rogers, S. Unsuccessful trial of
gene replacement in arginase deficiency. Z. Kinderheilkd. 1975, 119, 1–3.
6. Rosenberg, S.A.; Aebersold, P.; Cornetta, K.; Kasid, A.; Morgan, R.A.; Moen, R.; Karson, E.M.;
Lotze, M.T.; Yang, J.C.; Topalian, S.L. Gene transfer into humans—Immunotherapy of patients
with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene
transduction. N. Engl. J. Med. 1990, 323, 570–578.
7. MacMillan, P. The Cline affair. Nurs. Times 1982, 78, 383.
8. Beutler, E. The Cline affair. Mol. Ther. 2001, 4, 396–397.
9. Raty, J.K.; Lesch, H.P.; Wirth, T.; Yla-Herttuala, S. Improving safety of gene therapy.
Curr. Drug Saf. 2008, 3, 46–53.
10. Coughlan, L.; Alba, R.; Parker, A.L.; Bradshaw, A.C.; McNeish, I.A.; Nicklin, S.A.; Baker, A.H.
Tropism-modification strategies for targeted gene delivery using adenoviral vectors. Viruses 2010,
2, 2290–2355.
11. Montini, E. Quest for safety at AAValon. Blood 2011, 117, 3249–3250.
12. Biffi, A.; Bartolomae, C.C.; Cesana, D.; Cartier, N.; Aubourg, P.; Ranzani, M.; Cesani, M.;
Benedicenti, F.; Plati, T.; Rubagotti, E.; et al. Lentiviral vector common integration sites in
preclinical models and a clinical trial reflect a benign integration bias and not oncogenic selection.
Blood 2011, 117, 5332–5339.
13. Matrai, J.; Chuah, M.K.; VandenDriessche, T. Recent advances in lentiviral vector development
and applications. Mol. Ther. 2010, 18, 477–490.
14. Sharma, A.; Li, X.; Bangari, D.S.; Mittal, S.K. Adenovirus receptors and their implications in
gene delivery. Virus Res. 2009, 143, 184–194.
15. Heyde, M.; Partridge, K.A.; Oreffo, R.O.; Howdle, S.M.; Shakesheff, K.M.; Garnett, M.C.
Gene therapy used for tissue engineering applications. J. Pharm. Pharmacol. 2007, 59, 329–350.
16. Pathak, A.; Patnaik, S.; Gupta, K.C. Recent trends in non-viral vector-mediated gene delivery.
Biotechnol. J. 2009, 4, 1559–1572.
17. Mudhakir, D.; Harashima, H. Learning from the viral journey: How to enter cells and how to
overcome intracellular barriers to reach the nucleus. AAPS J. 2009, 11, 65–77.
Biomedicines 2014, 2 160

18. Escoffre, J.M.; Teissie, J.; Rols, M.P. Gene transfer: How can the biological barriers be
overcome? J. Membr. Biol. 2010, 236, 61–74.
19. Lang, F.F.; Bruner, J.M.; Fuller, G.N.; Aldape, K.; Prados, M.D.; Chang, S.; Berger, M.S.;
McDermott, M.W.; Kunwar, S.M.; Junck, L.R.; et al. Phase I trial of adenovirus-mediated p53
gene therapy for recurrent glioma: Biological and clinical results. J. Clin. Oncol. 2003, 21,
2508–2518.
20. Raty, J.K.; Pikkarainen, J.T.; Wirth, T.; Yla-Herttuala, S. Gene therapy: the first approved
gene-based medicines, molecular mechanisms and clinical indications. Curr. Mol. Pharmacol.
2008, 1, 13–23.
21. Han, D.M.; Huang, Z.G.; Zhang, W.; Yu, Z.K.; Wang, Q.; Ni, X.; Chen, X.H.; Pan, J.H.;
Wang, H. Effectiveness of recombinant adenovirus p53 injection on laryngeal cancer: Phase I
clinical trial and follow up. Zhonghua Yi Xue Za Zhi 2003, 83, 2029–2032.
22. Peng, Z. Current status of gendicine in China: Recombinant human Ad-p53 agent for treatment of
cancers. Hum. Gene Ther. 2005, 16, 1016–1027.
23. Bischoff, J.R.; Kirn, D.H.; Williams, A.; Heise, C.; Horn, S.; Muna, M.; Ng, L.; Nye, J.A.;
Sampson-Johannes, A.; Fattaey, A.; et al. An adenovirus mutant that replicates selectively in
p53-deficient human tumor cells. Science 1996, 274, 373–376.
24. Chiocca, E.A.; Abbed, K.M.; Tatter, S.; Louis, D.N.; Hochberg, F.H.; Barker, F.; Kracher, J.;
Grossman, S.A.; Fisher, J.D.; Carson, K.; et al. A phase I open-label, dose-escalation,
multi-institutional trial of injection with an E1B-Attenuated adenovirus, ONYX-015, into the
peritumoral region of recurrent malignant gliomas, in the adjuvant setting. Mol. Ther. 2004, 10,
958–966.
25. Yu, W.; Fang, H. Clinical trials with oncolytic adenovirus in China. Curr. Cancer. Drug Targets
2007, 7, 141–148.
26. Hu, J.C.; Coffin, R.S.; Davis, C.J.; Graham, N.J.; Groves, N.; Guest, P.J.; Harrington, K.J.;
James, N.D.; Love, C.A.; McNeish, I.; et al. A phase I study of OncoVEXGM-CSF, a
second-generation oncolytic herpes simplex virus expressing granulocyte macrophage
colony-stimulating factor. Clin. Cancer Res. 2006, 12, 6737–6747.
27. Harrington, K.J.; Hingorani, M.; Tanay, M.A.; Hickey, J.; Bhide, S.A.; Clarke, P.M.;
Renouf, L.C.; Thway, K.; Sibtain, A.; McNeish, I.A.; et al. Phase I/II study of oncolytic HSV
GM-CSF in combination with radiotherapy and cisplatin in untreated stage III/IV squamous cell
cancer of the head and neck. Clin. Cancer Res. 2010, 16, 4005–4015.
28. Kershaw, M.H.; Westwood, J.A.; Darcy, P.K. Gene-engineered T cells for cancer therapy.
Nat. Rev. Cancer 2013, 13, 525–541.
29. Morgan, R.A.; Dudley, M.E.; Wunderlich, J.R.; Hughes, M.S.; Yang, J.C.; Sherry, R.M.;
Royal, R.E.; Topalian, S.L.; Kammula, U.S.; Restifo, N.P.; et al. Cancer regression in patients
after transfer of genetically engineered lymphocytes. Science 2006, 314, 126–129.
30. Robbins, P.F.; Morgan, R.A.; Feldman, S.A.; Yang, J.C.; Sherry, R.M.; Dudley, M.E.;
Wunderlich, J.R.; Nahvi, A.V.; Helman, L.J.; Mackall, C.L.; et al. Tumor regression in patients
with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes
reactive with NY-ESO-1. J. Clin. Oncol. 2011, 29, 917–924.
Biomedicines 2014, 2 161

31. Kochenderfer, J.N.; Rosenberg, S.A. Treating B-cell cancer with T cells expressing anti-CD19
chimeric antigen receptors. Nat. Rev. Clin. Oncol. 2013, 10, 267–276.
32. Kochenderfer, J.N.; Dudley, M.E.; Feldman, S.A.; Wilson, W.H.; Spaner, D.E.; Maric, I.;
Stetler-Stevenson, M.; Phan, G.Q.; Hughes, M.S.; Sherry, R.M.; et al. B-cell depletion and
remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19
chimeric-antigen-receptor-transduced T cells. Blood 2012, 119, 2709–2720.
33. Herman, J.M.; Wild, A.T.; Wang, H.; Tran, P.T.; Chang, K.J.; Taylor, G.E.; Donehower, R.C.;
Pawlik, T.M.; Ziegler, M.A.; Cai, H.; et al. Randomized phase III multi-institutional study of
TNFerade biologic with fluorouracil and radiotherapy for locally advanced pancreatic cancer:
Final results. J. Clin. Oncol. 2013, 31, 886–894.
34. Malmstrom, P.U.; Loskog, A.S.; Lindqvist, C.A.; Mangsbo, S.M.; Fransson, M.; Wanders, A.;
Gardmark, T.; Totterman, T.H. AdCD40L immunogene therapy for bladder carcinoma—The first
phase I/IIa trial. Clin. Cancer Res. 2010, 16, 3279–3287.
35. Chiocca, E.A.; Smith, K.M.; McKinney, B.; Palmer, C.A.; Rosenfeld, S.; Lillehei, K.;
Hamilton, A.; DeMasters, B.K.; Judy, K.; Kirn, D. A phase I trial of Ad.hIFN-beta gene therapy
for glioma. Mol. Ther. 2008, 16, 618–626.
36. Freeman, S.M.; Abboud, C.N.; Whartenby, K.A.; Packman, C.H.; Koeplin, D.S.; Moolten, F.L.;
Abraham, G.N. The “bystander effect”: Tumor regression when a fraction of the tumor mass is
genetically modified. Cancer Res. 1993, 53, 5274–5283.
37. Sandmair, A.M.; Loimas, S.; Puranen, P.; Immonen, A.; Kossila, M.; Puranen, M.;
Hurskainen, H.; Tyynela, K.; Turunen, M.; Vanninen, R.; et al. Thymidine kinase gene
therapy for human malignant glioma, using replication-deficient retroviruses or adenoviruses.
Hum. Gene Ther. 2000, 11, 2197–2205.
38. Eck, S.L.; Alavi, J.B.; Alavi, A.; Davis, A.; Hackney, D.; Judy, K.; Mollman, J.; Phillips, P.C.;
Wheeldon, E.B.; Wilson, J.M. Treatment of advanced CNS malignancies with the recombinant
adenovirus H5.010RSVTK: a phase I trial. Hum. Gene Ther. 1996, 7, 1465–1482.
39. Rainov, N.G. A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and
ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with
previously untreated glioblastoma multiforme. Hum. Gene Ther. 2000, 11, 2389–2401.
40. Immonen, A.; Vapalahti, M.; Tyynela, K.; Hurskainen, H.; Sandmair, A.; Vanninen, R.; Langford, G.;
Murray, N.; Yla-Herttuala, S. AdvHSV-tk gene therapy with intravenous ganciclovir improves survival
in human malignant glioma: A randomised, controlled study. Mol. Ther. 2004, 10, 967–972.
41. Westphal, M.; Yla-Herttuala, S.; Martin, J.F.; Warnke, P.; Menei, P.; Eckland, D.; Kinley, J.;
Kay, R.; Ram, Z. Adenovirus-mediated gene therapy with stimagene ceradenovec followed by
intravenous ganciclovir for patients with operable high-grade glioma (ASPECT): A randomised,
open-label, phase 3 trial. Lancet Oncol. 2013, in press.
42. Thoma, C.; Bachy, V.; Seaton, P.; Green, N.K.; Greaves, D.R.; Klavinskis, L.; Seymour, L.W.;
Morrison, J. Adenovirus serotype 11 causes less long-term intraperitoneal inflammation than
serotype 5: Implications for ovarian cancer therapy. Virology 2013, 447, 74–83.
43. Wirth, T.; Hedman, M.; Makinen, K.; Manninen, H.; Immonen, A.; Vapalahti, M.;
Yla-Herttuala, S. Safety profile of plasmid/liposomes and virus vectors in clinical gene therapy.
Curr. Drug Saf. 2006, 1, 253–257.
Biomedicines 2014, 2 162

44. Wang, J.; Faust, S.M.; Rabinowitz, J.E. The next step in gene delivery: Molecular engineering of
adeno-associated virus serotypes. J. Mol. Cell. Cardiol. 2011, 50, 793–802.
45. Kim, K.H.; Ryan, M.J.; Estep, J.E.; Miniard, B.M.; Rudge, T.L.; Peggins, J.O.; Broadt, T.L.;
Wang, M.; Preuss, M.A.; Siegal, G.P.; et al. A new generation of serotype chimeric
infectivity-enhanced conditionally replicative adenovirals: The safety profile of ad5/3-Delta24 in
advance of a phase I clinical trial in ovarian cancer patients. Hum. Gene Ther. 2011, 22, 821–828.
46. Wang, L.; Calcedo, R.; Bell, P.; Lin, J.; Grant, R.L.; Siegel, D.L.; Wilson, J.M. Impact of
pre-existing immunity on gene transfer to nonhuman primate liver with adeno-associated virus 8
vectors. Hum. Gene Ther. 2011, 22, 1389–1401.
47. Barnett, B.G.; Crews, C.J.; Douglas, J.T. Targeted adenoviral vectors. Biochim. Biophys. Acta
2002, 1575, 1–14.
48. Cronin, J.; Zhang, X.Y.; Reiser, J. Altering the tropism of lentiviral vectors through pseudotyping.
Curr. Gene Ther. 2005, 5, 387–398.
49. Kim, H.A.; Mahato, R.I.; Lee, M. Hypoxia-specific gene expression for ischemic disease gene
therapy. Adv. Drug Deliv. Rev. 2009, 61, 614–622.
50. Harvey, T.J.; Hennig, I.M.; Shnyder, S.D.; Cooper, P.A.; Ingram, N.; Hall, G.D.; Selby, P.J.;
Chester, J.D. Adenovirus-mediated hypoxia-targeted gene therapy using HSV thymidine kinase
and bacterial nitroreductase prodrug-activating genes in vitro and in vivo. Cancer Gene Ther.
2011, 18, 773–784.
51. Urnov, F.D.; Rebar, E.J.; Holmes, M.C.; Zhang, H.S.; Gregory, P.D. Genome editing with
engineered zinc finger nucleases. Nat. Rev. Genet. 2010, 11, 636–646.
52. Staunstrup, N.H.; Moldt, B.; Mates, L.; Villesen, P.; Jakobsen, M.; Ivics, Z.; Izsvak, Z.;
Mikkelsen, J.G. Hybrid lentivirus-transposon vectors with a random integration profile in human
cells. Mol. Ther. 2009, 17, 1205–1214.
53. Mates, L.; Chuah, M.K.; Belay, E.; Jerchow, B.; Manoj, N.; Acosta-Sanchez, A.; Grzela, D.P.;
Schmitt, A.; Becker, K.; Matrai, J.; et al. Molecular evolution of a novel hyperactive Sleeping
Beauty transposase enables robust stable gene transfer in vertebrates. Nat. Genet. 2009, 41,
753–761.
54. VandenDriessche, T.; Ivics, Z.; Izsvak, Z.; Chuah, M.K. Emerging potential of transposons for
gene therapy and generation of induced pluripotent stem cells. Blood 2009, 114, 1461–1468.
55. Patel, S.R. Radiation-induced sarcoma. Curr. Treat. Options Oncol. 2000, 1, 258–261.
56. Harris, C.C. The carcinogenicity of anticancer drugs: A hazard in man. Cancer 1976, 37,
1014–1023.
57. Boffetta, P.; Kaldor, J.M. Secondary malignancies following cancer chemotherapy. Acta Oncol.
1994, 33, 591–598.
58. Alba, R.; Bosch, A.; Chillon, M. Gutless adenovirus: Last-generation adenovirus for gene therapy.
Gene Ther. 2005, 12, S18–S27.

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