Recent Progress in Gene Therapy for Ovarian Cancer

Abstract

Ovarian cancer is the most lethal gynecological malignancy in developed countries. This is due to the lack of specific symptoms that hinder early diagnosis and to the high relapse rate after treatment with radical surgery and chemotherapy. Hence, novel therapeutic modalities to improve clinical outcomes in ovarian malignancy are needed. Progress in gene therapy has allowed the development of several strategies against ovarian cancer. Most are focused on the design of improved vectors to enhance gene delivery on the one hand, and, on the other hand, on the development of new therapeutic tools based on the restoration or destruction of a deregulated gene, the use of suicide genes, genetic immunopotentiation, the inhibition of tumour angiogenesis, the alteration of pharmacological resistance, and oncolytic virotherapy. In the present manuscript, we review the recent advances made in gene therapy for ovarian cancer, highlighting the latest clinical trials experience, the current challenges and future perspectives.

Full text

International Journal of
Molecular Sciences
Review
Recent Progress in Gene Therapy for Ovarian Cancer
Ángela Áyen 1, Yaiza Jiménez Martínez 2,3, Juan A. Marchal 1,2,3,4 ID and Houria Boulaiz 1,2,3,4,*ID
1Department of Human Anatomy and Embryology, University of Granada, 18016 Granada, Spain;
aayen@correo.ugr.es (A.A.); jmarchal@ugr.es (J.A.M.)
2
Biopathology and Medicine Regenerative Institute (IBIMER), University of Granada, 18016 Granada, Spain;
yaijmartinez@correo.ugr.es
3Biosanitary Institute of Granada (ibs.GRANADA), SAS-Universidad de Granada, 18016 Granada, Spain
4Excellence Research Unit “Modeling Nature” (MNat), University of Granada, 18016 Granada, Spain
*Correspondence: hboulaiz@ugr.es; Tel.: +34-958-241-271
Received: 8 June 2018; Accepted: 27 June 2018; Published: 30 June 2018


Abstract:
Ovarian cancer is the most lethal gynecological malignancy in developed countries. This is
due to the lack of specific symptoms that hinder early diagnosis and to the high relapse rate after
treatment with radical surgery and chemotherapy. Hence, novel therapeutic modalities to improve
clinical outcomes in ovarian malignancy are needed. Progress in gene therapy has allowed the
development of several strategies against ovarian cancer. Most are focused on the design of improved
vectors to enhance gene delivery on the one hand, and, on the other hand, on the development
of new therapeutic tools based on the restoration or destruction of a deregulated gene, the use of
suicide genes, genetic immunopotentiation, the inhibition of tumour angiogenesis, the alteration
of pharmacological resistance, and oncolytic virotherapy. In the present manuscript, we review
the recent advances made in gene therapy for ovarian cancer, highlighting the latest clinical trials
experience, the current challenges and future perspectives.
Keywords:
ovarian cancer; gene therapy; delivery systems; promoter; suicide genes; ovarian cancer
stem cells
1. Introduction
Ovarian cancer (OC) is the seventh most frequent cancer among women worldwide with an
incidence of 238.700 new cases and 151.900 annual deaths [
1
]. In developed countries, it is the second
most common gynaecological cancer (99.800 new cases) and the most lethal (65.000 deaths), since
the majority of OC are diagnosed at an advanced stage [
1
]. OC must be considered as a group of
different diseases with differences in epidemiologic and risk factors, premalignant lesions (serous tubal
intraepithelial neoplasia or endometriosis), molecular events, response to chemotherapy and prognosis.
Primary fallopian tube cancer and peritoneal serous carcinoma are considered rare malignancies,
however, many tumours that were classified as serous carcinomas of the ovary or peritoneal cancers
appear to have their origin in the fallopian tube [
2
]. These carcinomas have similarities in histology,
genetic and clinical behaviour with OC, so they should be considered collectively and are managed
similarly [
3
]. The International Federation of Gynecology and Obstetrics (FIGO) staging system
combined ovarian, fallopian tube, and peritoneal cancers into a single classification in 2014, based on
surgical stage [4].
Based on clinical, histologic and molecular factors, epithelial ovarian cancer (EOC) can be divided
into two subtypes: Type I and Type II. Type I tumors are slow growing, remain confined to the
ovary for long periods and have an indolent course. They include (i) endometriosis-related tumors
(endometrioid, clear cell, and seromucinous carcinomas); (ii) low-grade serous carcinomas; and (iii)
mucinous carcinomas and malignant Brenner tumors. In contrast, type II tumors are high-grade,
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Int. J. Mol. Sci. 2018,19, 1930 2 of 29
spread rapidly and are highly aggressive at their onset. They include high-grade serous carcinoma
(poorly and moderately differentiated), carcinosarcomas (malignant mixed mesodermal tumors) and
undifferentiated carcinoma [5].
1.1. Risk Factors and Prevention Strategies
The risk of OC is increased in women with a family history of OC, personal history of breast
cancer, mutation in BRCA1 or BRCA2, Lynch syndrome, increased age, infertility, nulliparity, hormonal
factors like early age at menarche or late age at menopause, inflammatory states such as endometriosis
or pelvic inflammatory disease, and obesity. In contrast, multiparity, history of breastfeeding, taking
oral contraceptives, hysterectomy and tubal ligation appear to have a protective role [
6
]. The risk
factors for the fallopian tube and peritoneal carcinoma are unclear [7].
1.2. Diagnostic and Early Detection
The clinical presentation of EOC is commonly insidious, making diagnosis at an early stage more
difficult. The majority of women have stage III or stage IV at diagnosis, and present abdominal pain
or discomfort, menstrual irregularities, dyspepsia and other gastrointestinal symptoms, and urinary
symptoms of frequency or retention. The advanced disease respiratory symptoms appear from ascites
or pleural effusion, and bowel obstruction [3].
Most cases of OC are diagnosed at later stages, with a high mortality. There is no adequate
screening test for ovarian cancer in asymptomatic women without high risk of developing this
pathology, since strategies based on measurement of serum CA-125 concentration, transvaginal
ultrasound, or both, are not sensitive enough to early stage detection of this disease. Moreover,
they generate a negative balance between the important harm derived from false positives and the
number of OCs detected [
8
]. In a recent study, a panel of four markers, CA-125, HE4, E-CAD and IL-6,
was selected and showed great potential in the detection of high-grade serous ovarian carcinoma at
earlier stages in samples collected from 172 patients. However, additional validation studies using the
combination of biomarkers in patients with OC are needed to confirm its effectiveness [
9
]. In addition,
woman with a suspected high-risk hereditary cancer syndrome (BRCA1 and BRCA2 mutation and
Lynch syndrome) should receive genetic counselling, and, if the mutation is confirmed, to consider
prophylactic surgery (risk-reducing bilateral salpingo-oophorectomy [3]).
As shown in Figure 1, the histologic diagnosis, stage and prognosis of epithelial ovarian, fallopian
tube or peritoneal cancer require surgical exploration. A clinical assessment and measurement of
serum CA-125 aids diagnosis. Human gonadotropin (hCG) and alpha-fetoprotein (AFP) allow us to
exclude the origin in the germ cell. Transvaginal ultrasonography is the ideal imaging investigation
in the visualisation of ovarian masses, allowing us to see characteristics suggestive of malignancy
(International Ovarian Tumor Analysis (IOTA) simple rules) [
10
]. Prior to surgery, radiographers
should take a chest X-Ray and a CT scan of the abdomen and pelvis to evaluate the presence of
metastatic disease. Cancer of the ovary, fallopian tube, or peritoneum is staged according to the 2017
FIGO staging system [3].
1.3. Current Treatments of Ovarian Cancer
The current standard of care for EOC is an optimal cytoreductive surgery followed by a
combination of chemotherapy with a platinum and taxane regimen (Figure 1). The most important
prognostic indicator in patients with advanced stage is the volume of residual disease after surgery,
so these patients should undergo a total hysterectomy, bilateral salpingo-oophorectomy, omentectomy,
and a maximal attempt at optimal cytoreduction. In addition, peritoneal washings, multiple peritoneal
biopsies, appendectomy in mucinous histology and removal of bulky para-aortic and pelvic nodes
are performed. In some patients with advanced stage (IIIC or IV) and unresectable tumours it is
necessary for 2–3 cycles of neoadjuvant chemotherapy initially, followed by surgical cytoreduction

Int. J. Mol. Sci. 2018,19, 1930 3 of 29
and additional chemotherapy. This approach may also be used in a patient with a primary suboptimal
cytoreduction [3,10].
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 29
Figure 1. Algorithm for management of ovarian cancer. Figo Staging ovarian images modified from
Cancer Research UK/Wikimedia Commons.
In relation to adjuvant chemotherapy in an early stage, it is not indicated in stage IA and IB
grade 1–2, but it offers benefits in patients with grade 3 of differentiation, stage IC and II or clear-cell
histology, in which 3–6 cycles of carboplatin and paclitaxel (PTX) are administered. Chemotherapy
is recommended for all patients with stage II-IV, concretely 6 cycles of carboplatin and PTX (or
docetaxel if PTX is not tolerated) [3,10].
Despite an adequate approach, the majority of women with advanced-stage OC will relapse due
to platinum-resistant or refractory cancer, with a median time to recurrence of 16 months [3]. Thus,
there has been significant interest in developing innovative strategies more targeted at treating this
Figure 1.
Algorithm for management of ovarian cancer. Figo Staging ovarian images modified from
Cancer Research UK/Wikimedia Commons.
In relation to adjuvant chemotherapy in an early stage, it is not indicated in stage IA and IB
grade 1–2, but it offers benefits in patients with grade 3 of differentiation, stage IC and II or clear-cell
histology, in which 3–6 cycles of carboplatin and paclitaxel (PTX) are administered. Chemotherapy is
recommended for all patients with stage II-IV, concretely 6 cycles of carboplatin and PTX (or docetaxel
if PTX is not tolerated) [3,10].
Despite an adequate approach, the majority of women with advanced-stage OC will relapse due
to platinum-resistant or refractory cancer, with a median time to recurrence of 16 months [
3
]. Thus,

Int. J. Mol. Sci. 2018,19, 1930 4 of 29
there has been significant interest in developing innovative strategies more targeted at treating this
pathology and gene therapy represents a good therapeutic option. In this review, we will summarize
the latest advances in the application of gene therapy in OC, providing a basic understanding of
current vector technology, possible relevant genetic targets and a summary of clinical trials.
2. Gene Therapy in Ovarian Cancer
Although gene therapy for OC is in continuous progress and innovation, it is far from reaching the
patient. Hence, several strategies have been developed to improve the performance of these systems.
2.1. Improved Vectors for Gene Delivery
One of the limitations of clinical success in gene therapy is still the lack of a safe and highly
efficient gene delivery system. An optimal vector for gene therapy should selectively target the tumour
cells allowing (i) an improvement in transfection efficiencies; (ii) minimization of off-target transfection;
and (iii) reduction of genotoxicity, which have long been recognized as the major obstacles of gene
therapy [
11
]. In this context, many efforts are being made and a wide range of viral and non-viral
vectors have been developed.
2.1.1. Viral Vectors
Multiple Viral Vectors Have Been Evaluated in OC
Lentiviruses are one of the most studied vectors for targeted gene therapy due to their ability to
transduce both dividing and non-dividing cells and allow long-term transgene expression
in vivo
and
in vitro
through integration into host cell genomes. Huhtala et al. used cetuximab (anti-epidermal
growth factor receptor (EGFR) antibody)-conjugated lentivirus vectors to improve the effectiveness of
the treatment in nude mice with orthotropic SKOV-3m human ovarian carcinoma xenografts. This
vector induced a significant antitumour immunity leading to tumour regression [12].
Adenoviral vectors (ADV) infect both dividing and non-dividing cells, are safe, have a large
cloning capacity and facilitate gene expression in 12 h after infection. However, their capacity to infect
cells is dependent upon the presence of Coxsackievirus and adenovirus receptors (CAR) that have a
low concentration in target tumours including OC, meaning a restricted transfection efficacy. To avoid
this limitation, Rawlinson et al. incorporated an Arg-Gly-Asp peptide (RGD) that allows the virus to
use an alternative receptor during transduction and increased its efficiency [
13
]. Another problem of
the recombinant adenovirus is that repeated administration will develop an immune response, with
neutralization of the adenovirus by antibodies. The use of protective polymer-coating on the virus
particles can avoid this problem. Yoshihara et al. coated adenoviruses by layer-on-layer deposition of
ionic polymers (polyethyleneimine (PEI) and hyaluronic acid) onto adenovirus particles to produce
multilayer-coated virus vectors. They reported that the infectivity of the virus in the presence of an
anti-adenovirus antibody increased with the number of layers, showing relatively high infectivity
efficiency on cultured cells and in intraperitoneally metastatic OC [
14
]. Their high tropism to the liver
and the induction of strong innate immune responses by macrophages and dendritic cells are another
important hurdle that might limit its use.
Adeno-associated virus (AAV) can also infect a broad range of cells, like adenoviruses. AAVs
have been used in treating OC delivering bevacizumab [
15
], Kringle 5 [
16
] or endostatin [
17
]. In a
recent study, AAV9 was used in a single intraperitoneal (IP) injection to deliver albumin leader Q425R
MIS (LRMIS), showing elevated and sustained serum levels of a Mullerian-inhibiting substance (MIS),
which inhibited the growth of xenografts from ascites (PDXa) from patients with highly resistant
recurrent OC, without overt toxicity [18].
Human papillomavirus (HPV) pseudovirions can efficiently deliver DNA into multiple cell
lines, protecting it from nucleases, but pseudovirus infection of human tumours may be HPV type
and tumour specific. In a study, IP injection of human papillomavirus 16 (HPV 16) pseudovirion

Int. J. Mol. Sci. 2018,19, 1930 5 of 29
to deliver the herpes simplex virus thymidine kinase (HSV-TK) gene to ovarian tumour cells was
able to preferentially infect murine and human ovarian tumour cells in tumour-bearing nude mice.
Subsequent administration of ganciclovir led to significant therapeutic anti-tumour effects
in vitro
and
in vivo. This system could be used to deliver other candidate genes [19].
2.1.2. Non-Viral Vectors
Viral vectors have been reported to have several problems, such as inflammatory and immune
responses, insertional mutagenesis, limited loading capacity and difficult production. Non-viral
vectors have a higher gene-loading capacity, lower immune response and are safer. Non-viral delivery
gene systems include injection of naked DNA, transfection using liposomes, polyplexes, lipopoliplexes
and nanoparticles, as well as ultrasound (US)/microbubble (MB)-mediated gene delivery [20].
Cationic lipid–DNA complexes (lipoplexes) have relatively high transfection efficiency
in vitro
when locally delivered at low doses. One example of this is PEI, one of the most effective non-viral
gene carriers [
21
]. However, the application of PEI is restricted by its non-biodegradable nature
and relatively high cytotoxicity. These limitations are overcome with biodegradable cationic heparin
polyethileneimine (HPEI) nanogels, used to deliver several genes like survivin-T34A [
22
], FILIP1L [
23
]
or gelonin toxin [
24
], with low cytotoxicity and high transfection efficiency and stability. Another
lipoplexe is chitosan. The tumour-targeted polyethylene glycol-chitosan lactate nanoparticles with
folic acid (FA) as the targeting ligand (FA-PEG-COL nanoparticles) have been effective for delivery of
siRNA in OC gene therapy, thanks to its encapsulating efficiency and good protection of siRNA from
serum degradation [
25
]. Moreover, a bioreducible disulfide-based cationic dextran system was efficient
for prolonged gene delivery targeting SKOV-3 cells
in vitro
and in a mouse model by intravenous
(iv) injection [
26
]. The transport of siRNA is difficult due to its hydrophilic character and its negative
electric charge. The use of cationic cholesterol derivative-based liposomes is efficient as an interfering
RNA (siRNA) delivery system, showing low toxicity and excellent cellular uptake and gene silencing
efficiency [27].
Nanoparticles show great promise for gene delivery. N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimeth
ylammoniummethyl sulphate and monomethoxy poly(ethylene glycol)
−
poly(D,L-lactide) (DPP),
a particle with very low toxicity, was enhanced with low-dosage PTX, creating a PTX-encapsulated
DPP (P-DPP) nanoparticle, which increases the gene delivery and transfection efficiency of DPP
nanoparticles. Furthermore PTX exerts a synergistic effect a with vesicular stomatitis virus matrix
protein (VSVMP) gene that induces apoptosis, acting via multiple mechanisms in OC treatment. This
system can efficiently inhibit the OC in vitro and in vivo [28].
Gene transfer by ultrasound-targeted microbubble destruction (UTMD) is a safe and promising
technique for gene delivery, but has low gene transfection efficiency. This system produces transient
pores in cell membranes and stimulates cell membrane permeabilization. In several studies, this system
has been used for siRNA delivery, attempting to overcome the drawback of its low concentration at
the target site. In this context, Florinas et al. combined UTMD with an arginine-grafted bioreducible
polymer (ABP) and was able to synergize the advantages of each delivery systems own enhancing gene
silence efficiency and siRNA transfection efficacy and VEGF protein knockdown in OC cells
in vitro
and
in vivo
[
29
]. There are several strategies to overcome suboptimal gene transfection efficiency such
as the use of OC targeting microbubbles by conjugating LHRHa on the surface of the lipid microbubbles
since it is expressed in a high percentage of OC cell lines, improving p53 gene transfection efficiency
and inducing cells apoptosis by IP delivery in OC cell lines [
30
]; or the recombinant expression plasmid
of shRNA targeting the survivin gene (pshRNA survivin) with a higher cell apoptosis rate (by down
regulating caspase-3 and caspase-9 expression) and cell proliferation inhibitory rate [31].
2.1.3. Cell-Based Vectors
T-cell-based immunotherapy is a therapeutic strategy that is gaining strength more and more
in the treatment of cancer. A variant of this therapy that is receiving considerable attention in the

Int. J. Mol. Sci. 2018,19, 1930 6 of 29
investigation is chimeric antigen receptor–modified T (CAR-T)-cell therapy which was selected by
the American Society of Clinical Oncology (ASCO) as the “ASCO 2018 Advance of the Year” [
32
].
Immunotherapy with CAR-T cells involves reprogramming the T cells of patients to express Chimeric
Antigen Receptor (CAR) on their cell membrane using gene transfer technology. This receptor counts
with an external target-binding domain designed to recognize a specific tumor antigen and an internal
activation domain responsible for activating the T cell when the CAR-T binds its target. Second and
third generation CAR-Ts have additional costimulatory domains that further enhance the immune
response. In this way, the cytotoxic potential of lymphocytes T target cancer cells (Figure 2). CAR-T cells
combine both T-lymphocyte activation properties and antigen specificity in a single fusion molecule.
This system has been first used in patients with hematological tumours with excellent results [
33
],
but it still needs to be improved to avoid the side effects caused. However, its use to detect solid
tumours is a challenge, probably due to the characteristics of their histopathological structure and
the difficulty for the infiltration of T cells in tumour sites. Despite these several challenges, its use in
solid tumour, including OC, has been investigated and thoroughly reviewed by Zhu et al. [
34
] and
Zhang et al. [35]
. In this context, the most common antigens targeted by CARs in OC used in active
clinical trials, include MUC16, folate receptor-
α
(FR
α
) and mesothelin, with promising preliminary
results [34].
Another innovative therapeutic modality that is generating great expectations in cancer therapy
is cell therapy based on the use of stem cells. The use of mesenchymal stem cells (MSCs) that have the
ability to migrate to tumours as vehicles for drug delivery is an emerging strategy that could solve
many of the problems generated by vectors (Figure 2). This tumour tropism is due to the repair function
in which MSCs are recruited by sites of tissue injury and inflammation. Furthermore, they also have the
advantage of being able to be obtained from multiple sources such as the liver, bone marrow, placenta
or the umbilical cord, and could be stably amplified
in vitro
[
36
].
Zhang et al.
evaluated MSCs derived
from human umbilical cord for IL-21 delivery via lentiviral vector, with which they seek to obtain a
more lasting expression of IL-21, to develop a therapeutic effect on SKOV3 OC xenograft-bearing nude
mice. MSCs-LV-IL-21 showed an important therapeutic effect on inhibition of OC growth and safety as
they do not form gross or histological teratomas up to 60 days post-transplantation in murine lung, liver,
stomach and spleen [
37
]. In another work, MSCs derived from human bone marrow transfected with a
recombinant adenovirus encoding endostatin possessed significant migratory capacity and inhibited
the proliferation of SKOV3 cells by cell cycle arrest and promotion of apoptosis [
38
].
Dembinski et al.
found that microenvironments of OC recruit MSCs after its intraperitoneally administered to participate
in their stroma development, and gene-modified MSC to express IFN-
β
, achieved to control or eradicate
ovarian tumour in tumour xenograft models, resulting in reduction of tumour growth and prolonged
survival [39].
Moreover, targeting abilities of MSCs can be enhanced via the introduction of artificial receptors.
In this context, Komarova et al. [
40
], developed, by transduction with genetically modified adenoviral
vectors, a human MSC expressing an artificial receptor that binds to erbB2, a tumour cell marker
(MSC-AR). MSC-AR properties were tested in human ovarian carcinoma cell line SKOV3ip1 and
in vivo
using transient transgenic mice that express human erbB2 in the ovarian xenograft tumour
model. The binding of MSC-AR to erbB2-expressing cells was enhanced in both models suggesting
that the application of this strategy enhances the efficacy of cell-based therapy [
40
]. However, although
these preclinical studies clearly demonstrated the therapeutic benefits of using MSCs as vectors, very
few clinical trials have been approved for cancer treatments. Until now, a clinical phase 1 study
to determine the effects of MSCs secreting interferon beta in patients with advanced OC is being
developed. The aim is to treat women with recurrent OC. For that, MSCs, isolated from healthy male
donors will be genetically engineered and then be intraperitoneally administered into patients. This
study, enrolled in MD Anderson with the participation of up to 21 patients, will undoubtedly bring
promising results (NCT02530047) [
41
]. The fact that there are not many approved clinical trials can be
partly due to reports that MSCs not only show a potential for malignant transformation, but may also

Int. J. Mol. Sci. 2018,19, 1930 7 of 29
lead to the induction of metastasis [
42
]. To solve this problem, a novel delivery platform based on the
use of natural membrane-derived vesicles, termed nanoghosts (NGs), was developed from different
biological sources. NGs developed from MSCs membrane (MSC-NGs) retain MSC surface markers
and behave broadly as MSCs in relation to tumour identification capabilities
in vitro
and
in vivo
. MSC-
NGs utility was proven in several types of cancers although not yet in OC [
41
]. MSC-NG could be a
good alternative to MSCs, they are potentially safer, as they are not associated with the common risks
that arise from the administration of living proliferating cells.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 29
and in vivo. MSC- NGs utility was proven in several types of cancers although not yet in OC [41].
MSC-NG could be a good alternative to MSCs, they are potentially safer, as they are not associated
with the common risks that arise from the administration of living proliferating cells.
Figure 2. Summary of current therapies targeting ovarian cancer through the use of cell-based vectors.
(A) MSCs as vehicles for drug delivery; (B) reprogramming the T cells of patients to express CARs
targeting ovarian cancer.
Figure 2.
Summary of current therapies targeting ovarian cancer through the use of cell-based vectors.
(
A
) MSCs as vehicles for drug delivery; (
B
) reprogramming the T cells of patients to express CARs
targeting ovarian cancer.

Int. J. Mol. Sci. 2018,19, 1930 8 of 29
2.1.4. Targeted Vectors for Ovarian Cancer Gene Therapy
Not only the type of vector used is important, but also the design of the vector to achieve the
targeted organ. In this sense, there are different strategies, based on the use of gene regulatory elements
such as promoters, or proteins that have high affinity for specific cells such as receptors that selectively
eliminate cancer cells without harming healthy cells.
Based on the frequent overexpression of EGFR or its mutant in OC cells, carrying vectors guided
by synthetic nano-antibodies for EGFRvIII and EGFR were developed to deliver human recombinant
DNases (DNASE1, DNASE1L3, DNASE2, DFFB) into human OC cells from ascites and cultures,
achieving the degradation of their genomic DNA and, consequently, cancer cell death, without affecting
the healthy cells [
43
]. The human telomerase reverse transcriptase (hTERT) promoter can direct target
gene expression to OC cells, since it is expressed in this cell type and repressed in normal tissues.
In a study, hTERT was integrated in a systemic amplifier expression vector (VISA, VP16-Gal4-WPRE),
enhancing transgene expression with lower toxicity than a CMV promoter. E1A, an adenoviral type 5
transcription factor that possesses various anticancer activities, was used as a therapeutic gene with this
platform, being specifically targeted to OC cells and showing a significant reduction of OC cell growth
in a mouse model and significantly prolonged survival compared with a control [
44
]. This promoter
has also been used to achieve tumour-specific Thymosin
β
10 (T
β
10) gene expression, a protein that
regulates actin dynamics, affecting metastasis and proliferation in many cancer cells [
45
].
Huang et al.
demonstrated that IP administration of cationic biodegradable poly(
β
-amino ester) polymers may
efficiently deliver diphtheria toxin subunit-A (DT-A) DNA to mice bearing ovarian tumours, using
transcriptional regulation with the promoters of two genes, tumour-specific human epididymis protein
4 (HE4) and MSLN, whose activity is increased in OC cells [
46
]. HE4 promoter is overexpressed in a
high percentage of serous and endometrioid EOC, and was used to drive the HSV-TK gene, showing it
to be a possible treatment strategy for patients with high levels of serum HE4 [
13
]. Cocco et al. [
47
]
designed a dual-targeting approach to exploit the overexpression of claudin-3/-4, the receptors for
Clostridium perfringens enterotoxin (CPE), and the p16 promoter in OC cells, using an IP injection of
nanoparticles (NPs) modified with the carboxy-terminal–fragment of CPE (c-CPE-NP) for the delivery
of plasmid encoding for the DT-A. These particles showed themselves to be efficient in transfecting
OC cells
in vivo
, and DT-A was effective in inhibiting ovarian tumour growth [
47
]. Overexpression
of FR
α
is characteristic of OC, and could be used to direct target gene expression in human OC cells.
In a recent work, FR
α
-targeted folate modified lipoplexes with an hTERT promoter were successfully
used to drive the expression of a matrix protein (MP) of the vesicular stomatitis virus, F-LP/pMP [
48
].
He et al.
used a FR
α
-targeted lipoplex (folate modified liposome, F-P-LP) with CLDN3-short hairpin
RNA (shRNA) [
49
]. Finally, it should be noted that, given the metastasis of OC is generally confined to
the abdominal cavity, OC is a good candidate for local gene therapy via IP administration [50].
2.2. Gene Therapy for Ovarian Cancer Treatment
Different treatment approaches have been explored in relation to gene therapy in OC: (i) tumour
suppressor gene therapy that restores cell control through replacing tumour suppressor genes;
(ii) oncogene inhibition strategies inactivating dominant oncogenes; (iii) suicide gene therapy by
enzyme/prodrug system or activating expression of a toxin; (iv) genetic immunopotentiation by
strengthening the immune response to tumour cells, augmenting the expression of tumour antigens
or the production of cytokines, interleukins and growth factor; (v) antiangiogenic gene therapy
(alterations in tumour vascularity to drain blood supply; (vi) multi drug resistance (MDR) associated
genes strategies using genes such as PRP-4 and surviving; and (vii) oncolytic virotherapy. In Table 1,
we summarize the strengths and weaknesses of these gene therapy strategies.

Int. J. Mol. Sci. 2018,19, 1930 9 of 29
Table 1. Strengths and weaknesses of various gene therapy strategies for ovarian cancer treatment.
Therapeutic Strategy Gene/System Strong Points Weak Points
Tumor Suppressor
gene
p53
Altered gene in a high percentage of OC.
The use of Gendicine (a recombinant
human Ad-p53) has been approved in the
treatment of OC in China.
Several clinical trials are currently ongoing.
Not useful in cells with a normal
p53 gene.
No therapeutic benefit in the first
clinical trials due to the use of a
wrong delivery system.
WWOX
Promising results in vivo
Inhibition of proliferation and promotion of
apoptosis in OCSC.
It has not been evaluated in
clinical trials in OC.
Oncofactor inhibition
strategies
EGFR
Gene widely studied in cancer.
Its use increases the sensitivity
to chemotherapy
It has not been evaluated in vivo
or in clinical trials in OC.
CLDN3
Several strategies of silencing have been
studied in OC (siRNA and shRNA).
Promising results
in vivo
, with inhibition of
malignant ascites formation.
It has not been evaluated in
clinical trials in OC.
Suicide gene therapy
HVS-TK
Strategy widely studied in gene therapy
for cancer.
Several clinical trials are currently ongoing.
Promising results in vivo and in
clinical trials.
Bystander effect.
Not phase 2 or 3 clinical
trials published.
DT-A Promising results in vivo, with
minimal cytotoxicity.
It has not been evaluated in
clinical trials in OC.
Antiangiogenic gene
therapy
VEGFRs
Promising results
in vivo
, with inhibition of
ascities formation.
Synergist effect in combination
with chemotherapy.
It has not been evaluated in
clinical trials in OC.
The work showed increased
proliferation of tumour cells with
the use of VEGFR2 and Ti2 in
combination with PTX
and carboplatin.
Endostatin
Promising results in vivo.
Clinical trials have been carried out in other
types of cancer.
Transient expression due to
humoral immune response if
adenovirus is used as delivery
system.
It has not been evaluated in
clinical trials in OC.
Genetic
immunopotentiation
IL-12
Potent immune-modulatory properties and
ability to inhibit tumour angiogenesis.
Promising results in vivo.
Several clinical trials are currently ongoing
or have been completed.
It has showed to be safe and feasible in
phase I trials.
Poor clinical benefits in the
completed phase II trial.
CAR-T cell
Possibility of target any specific
tumour antigen.
Promising preliminary results in clinical
trials in OC.
Important side effects caused in
patients.
Multi-Drug Resistance
MDR1
Several strategies have been developed to
knockdown it.
Its silencing enhances sensitivity to
anticancer drugs.
Promising results in vivo.
It has not been evaluated in
clinical trials in OC.
Survivin
Several strategies targeting this gene have
been developed, including combined
therapy with anticancer drugs.
Promising results in vivo.
It has not been evaluated in
clinical trials in OC.
Oncolytic virotherapy VSV
Can be genetically designed to deliver
therapeutic genes.
A phase I trial is currently ongoing.
Neurotoxicity and Induction of
neutralizing antibodies.
IFN response intact in most OC
cells, blocking virus replication.

Int. J. Mol. Sci. 2018,19, 1930 10 of 29
2.2.1. Tumor Suppressor Gene Therapy
Several studies of replacement of an altered tumour suppressor gene have been developed and
shown antitumour efficacy (Figure 3). One of the most studied genes in cancer is p53, a protein with
a wide variety of anticancer functions, thus it is involved in response to DNA-damaging, apoptosis
and cell cycle and growth arrest. In a high percentage of OCs, there is a loss of p53 function. Many
gene therapy approaches have focused on the role of p53 mutation, with good results both
in vitro
and
in vivo
. The transduction of wild-type p53 allows tumour proliferation inhibition [
51
] and increased
sensitivity to cisplatin [
52
] and PTX [
53
] in preclinical research. Although, in cells with a normal
p53 gene, this therapy does not provide any additional benefit [
54
]. Furthermore, a recent
in vitro
study showed that Ad-p53 infection is an effective method to activate the apoptosis of cancer cells and
re-sensitize the resistant OC cells to taxol [
55
], mediated by p53 upregulated modulator of apoptosis
(PUMA), the direct downstream pro-apoptotic effector of p53.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 10 of 29
2.2.1. Tumor Suppressor Gene Therapy
Several studies of replacement of an altered tumour suppressor gene have been developed and
shown antitumour efficacy (Figure 3). One of the most studied genes in cancer is p53, a protein with
a wide variety of anticancer functions, thus it is involved in response to DNA-damaging, apoptosis
and cell cycle and growth arrest. In a high percentage of OCs, there is a loss of p53 function. Many
gene therapy approaches have focused on the role of p53 mutation, with good results both in vitro
and in vivo. The transduction of wild-type p53 allows tumour proliferation inhibition [51] and
increased sensitivity to cisplatin [52] and PTX [53] in preclinical research. Although, in cells with a
normal p53 gene, this therapy does not provide any additional benefit [54]. Furthermore, a recent in
vitro study showed that Ad-p53 infection is an effective method to activate the apoptosis of cancer
cells and re-sensitize the resistant OC cells to taxol [55], mediated by p53 upregulated modulator of
apoptosis (PUMA), the direct downstream pro-apoptotic effector of p53.
Figure 3. Tumour suppressor genes are involved in a wide variety of antitumour functions. (A) If
these functions are inhibited, the tumour appears; (B) diverse strategies in gene therapy have tumour
suppressor genes as a molecular target, enabling the recovery of gene function and tumour
destruction.
Figure 3.
Tumour suppressor genes are involved in a wide variety of antitumour functions. (
A
) If
these functions are inhibited, the tumour appears; (
B
) diverse strategies in gene therapy have tumour
suppressor genes as a molecular target, enabling the recovery of gene function and tumour destruction.
However, when this strategy was studied for the first time in phase II/III trials employing an
adenoviral transgene delivery system, there was no therapeutic benefit and there appeared to be

Int. J. Mol. Sci. 2018,19, 1930 11 of 29
complications in respect to targeting ovarian tumour cells with Ad vectors due to the lack of expression
of coxsackie-adenovirus receptors, or anti-Ad antibodies in ascites [
56
]. However, there is new, more
promising data from the use of Gendicine, a gene therapy product approved for clinical use in China
in 2003 based on the injection of recombinant human Ad-p53. Its use to treat OC was studied with a
response rate of 90%, and 100% resolution of the peritoneal effusion condition [57].
Moreover, overexpression of human PNAS-4, a pro-apoptotic gene participating in the early
response to DNA damage and one of the targets of p53 tumor suppressor protein, results in a great
decrease of proliferation and induces apoptosis of SKOV3 OC cells
in vitro
. Its iv administration
through a cationic liposome showed efficient inhibition of growth and prolongation of survival in an
OC mouse model, due to induction of apoptosis and inhibition of angiogenesis [58].
Furthermore, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumour
suppressor gene, frequently mutated in OC. Transfection of OC cells line with exogenous PTEN
plasmids showed to be a good approach in increasing the expression of PTEN gene, which achieved
significant growth suppression in OC cells by apoptosis and arrest at the G1 phase of the cell cycle.
This might be due to the suppression of the PI3K/AKT pathway, besides reduction of cell migration
and invasion by decreasing MMP-9 expression [59].
Another tumour suppressor gene is p16, whose upregulation in OC was demonstrated to reduce
the proliferation of ovarian tumour cells by downregulation of eukaryotic translation elongation factor
1
α
2 protein (eEF1A2) [
60
]. In a recent study, Lu et al. showed that EZH2, a histone methyltransferase,
negatively regulates the expression of p16, and its inhibition reduces OC cell proliferation and
migration in vitro and suppresses ovarian tumour formation in vivo [61].
Finally, WW domain containing oxidoreductase (WWOX) gene has been identified as a tumour
suppressor gene and its expression has been shown to be reduced or absent in ovarian tumours.
The expression of WWOX gene in OC induces apoptosis and inhibits cell proliferation both in
differentiated tumor cells [62] and in cancer stem cells (CSCs) [63].
2.2.2. Oncofactor Inhibition Strategies
Many genes associated with the regulation of proliferation and angiogenesis are mutated in
cancer, resulting in uncontrolled proliferation. These genes are a potential target for the gene silencing
therapy. There are several strategies to inactivate it such as using antisense oligonucleotides binding to
the target mRNA; blocking its transduction; or the use of RNA interferences (RNAi) mediated by short
hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) (Table 2).
Table 2. Most frequent oncofactor inhibition approaches used in ovarian cancer.
Gene Function in Ovarian Cancer Silencing Strategy Model Ref.
EGFR Cell migration, proliferation and differentiation siRNA carried in nanogels In vitro [64]
NOB1
Protein degradation through ubiquitin
proteasome pathway (maturation of the
20S proteasome)
shRNA carried in a
lentiviral system In vitro [65]
MACC1
Regulation of MET, which is involved in cellular
growth and migration, angiogenesis, invasion
and metastasis
shRNA plasmid In vitro [66]
MTA1
Component of histone deacetylase 1 involved in
transcriptional regulation. May enhance cell
invasion, migration, adhesion and
anoikis-resistance.
siRNA plasmid In vitro [67]
COX2
Prostaglandin synthesis, involved in stimulation
of proliferation and angiogenesis in cancer siRNA and shRNA plasmids In vitro and
in vivo [68,69]
WT1 Proliferation and differentiation of the
urogenital system ASODN carried in liposomes In vitro [70]
STAT3
Regulation of multiple oncogenes and
suppressor gene expressions involved in cell
proliferation and apoptosis and angiogenesis
shRNA carried in
DOTAP-cholesterol liposomes
In vitro and
in vivo [71]

Int. J. Mol. Sci. 2018,19, 1930 12 of 29
Table 2. Cont.
Gene Function in Ovarian Cancer Silencing Strategy Model Ref.
H1F-1α
Transcriptional regulator of the adaptive
response to hypoxia by activation of genes
involved in cell proliferation and migration,
angiogenesis, apoptosis and glucose metabolism
siRNA through
FA-PEG-COL nanoparticles In vitro [25]
CLDN3
Component of tight junction (TJ) of epithelial
cells and cancer cells, so is involved in invasion
and metastasis
siRNA carried in lipidoid
molecules, shRNA carried in
PLGA-NPs, shRNA carried
in F-P-LP
In vitro and
in vivo [72,73]
NOTCH1
Cell development, proliferation, differentiation
and apoptosis.
siRNA carried in cationic
cholesterol
derivative-based liposomes
In vitro [27]
CD59 Inhibition of cytolytic activity of complement shRNA carried by a
recombinant retrovirus
In vitro and
in vivo [74]
gDNMT1
DNA methylation, involved in tumorigenesis,
relapse and resistance of ovarian cancer. CRISPR-Cas9 delivered by F-LP In vitro and
in vivo [75]
One of the most studied oncogenes is EGFR, related to cell migration, proliferation and
differentiation, and is highly expressed in OC. In a preclinical study, siRNAs targeting EGFR were
transferred to erythropoietin-producing hepatocellular A2 (EphA2) receptor positive OC cells by a
core/shell hydrogel nanoparticle (nanogels) targeted to the EphA2 receptor, showing decreased EGFR
expression levels and an increase in the sensitivity of these cells to docetaxel [64].
Moreover, the expression of Nin one binding protein (NOB1p), a protein overexpressed in OC cells
and involved in protein degradation through the ubiquitin proteasome pathway (UPP), was knocked
down by a lentiviral shRNA delivery system, which led to a marked reduction of the proliferation and
colony formation of OC cells [65].
Metastasis associated in colon cancer 1 (MACC1) is upregulated in several types of cancer.
Its downregulation by MACC1-specific shRNA showed inhibition of proliferation, migration capability
and invasive potential of ovarian carcinoma cells. In addition, enhancement of apoptosis that was
observed might be as a consequence of inhibition of HGF/Met and MEK/ERK pathways, which
are widely implicated in carcinogenesis (reduced expression of Met, p-MEK1/2, p-ERK1/2, cyclin
D1 and matrix metalloproteinase (MMP) protein, and an increased level of cleaved caspase 3) [
66
].
Metastasis-associated gene 1 (MTA1) also plays an important role in the invasion and metastasis in OC.
Its inhibition by siRNA transfection reduced the cell invasion potential, migration and intercellular
adhesion, and induced cell anoikis, a form of apoptosis in cells detached from the surrounding
extracellular matrix (OC cells must acquire anoikis resistance to survive in ascites), maybe through a
change of PTEN/AKT and beta 1 integrin/AKT pathway functions (since Beta 1 integrin, MMP-9 and
phosphor-AKT protein levels were significantly down-regulated and PTEN upregulated) in A2780
cancer cells [67].
Cyclooxigenase-2 (COX-2) is an inducible enzyme highly expressed in OC tissues, and has an
important role in the proliferation, growth, invasion and metastasis of OC cells, acting as an oncogene
by stimulating proliferation and angiogenesis. Effective COX-2 silencing in human OC cells by a
COX-2 specific siRNA plasmid vector [
68
] or a COX-2 shRNA sequence [
69
] inhibits cell proliferation
by blocking the cell cycle in G1 phase, attenuating invasion and migration ability by a decrease in
vascular growth factor, MMP-2 and MMP-9 protein expression, and suppressing the growth of OC
in vitro and in vivo models.
Another gene overexpressed in ovarian epithelial carcinoma but not in normal ovarian tissue
is Wilms tumor gene (WT1). Its expression was inhibited by WT1 antisense oligodeoxynucleotide
(ASODN) that significantly inhibited cell proliferation, arrested cell cycle at G0–G1 phase and increased
apoptosis in SKOV3 ovarian carcinoma cells [70].

Int. J. Mol. Sci. 2018,19, 1930 13 of 29
Signal transducer and activator of transcription 3 (STAT3) is frequently activated in OC, being
associated with the tumour formation and chemoresistance of OC. Jiang et al. used a shRNA to silence
it, showing apoptosis and inhibition of cell proliferation
in vitro
and
in vivo
. Reduced tumour weight
and angiogenesis were also observed. These effects could be related to STAT3 being shown to induce
the expression of cleaved caspase-3 and to reduce the expression of survivin, Bcl-2, cyclin D1 and
vascular endothelial growth factor [
71
]. In addition, the silencing of inducible factor 1-
α
(H1F-1
α
),
often overexpressed in OC and associated with multiple tumour characteristics, achieved effective
inhibition of cell proliferation in human OC in vitro [25].
Claudin3 (CLDN3) is a tight junction protein that is upregulated in a high percentage of ovarian
tumours but not in normal ovarian tissue. Its overexpression is associated with proliferation, invasion
and metastasis of ovarian tumours. Several studies have shown that downregulation of CLDN3 inhibits
tumour growth
in vivo
and
in vitro
by promoting tumour cell apoptosis, inhibiting cell proliferation
and reducing angiogenesis. Moreover, malignant ascites formation was inhibited in treated mice [
72
,
73
].
Huang et al. used an intratumoural injection of siRNA-lipidoid formulation [
72
] and Sun et al. used
IP administration of shRNA-nanoparticle formulation, based on poly(lactic-coglycolic acid (PLGA)
nanoparticle, a system with good biodegradability, biocompatibility and low toxicity [73].
The suppression of Notch1 activation in SKOV3 human OC cells, a protein that acts as an oncogene
in OC, showed low toxicity and excellent cellular uptake and gene silencing efficiency. This study also
demonstrated that this system could inhibit the growth of SKOV3 cells and promote apoptosis [27].
Downregulation of CD59, a membrane complement regulatory protein (mCRPs) that inhibits the
cytolytic activity of complement, by a recombinant retrovirus encoding shRNA targeted human CD59,
enhanced complement mediated cell damage, increasing apoptosis
in vitro
and inhibiting tumour
growth in nude mice [74].
Finally, a promising tool that can be used for the inactivation of oncogenes is the Clustered
Regularly Interspersed Short Palindromic Repeats (CRISPR)-caspase 9 (Cas9) genome editing
technology, an editing tool that can generate deletions, insertions and replacements in the mammalian
genome. He et al. targeted the OC-related DNA methyltransferase 1 gene (gDNMT1), an enzyme of
DNA methylation whose overexpression inactivates tumour suppressor genes and which is related to
tumorigenesis and resistance in OC. They found that cationic liposomal vectors (in this case a folate
receptor-targeted cationic liposome, F-LP) are effective delivery systems for CRISPR-Cas9 technology,
and F-LP/gDNMT1 inhibited growth of ovarian tumours
in vivo
, with few side effects other than a
high-dose PTX injection [75].
2.2.3. Suicide Gene Therapy
Suicide gene therapy is based on delivery of a gene encoding a toxin, or enzymes that convert
nontoxic prodrug into toxic drugs following the administration of the inactive prodrug. The cytotoxic
action is localized only in the tumour or, if applicable, in neighbouring tumour cells that have not been
transduced but undergo oncolysis due to the so called “bystander effect”. This strategy is turning into
tumour-targeted chemotherapy and can be categorized into two groups, direct strategies which use a
gene that encodes a toxin, or indirect strategies using a prodrug [76].
The most commonly used suicide gene approach is based in herpes simplex virus-thymidine
kinase (HSV-TK) followed by treatment with an antiviral drug such as ganciclovir (GCV), which is
transformed into toxic metabolites by the action of thymidine kinase (TK) and other cellular enzymes,
causing a failure in DNA replication and, as a consequence, cell death by apoptosis. Rawlinson et al.
developed a replication-deficient adenovirus bearing the HSV-TK gene driven by the tumour-specific
HE4 promoter. Its administration, followed by GCV treatment, increased the killing of cells by
up to ten-fold in cisplatin-sensitive and resistant A2780 OC cell lines [
13
]. In other recent studies,
the ultrasound was combined with an HSV-TK system to transfect an OC model in mice showing an
enhanced tumour inhibitory effect due to apoptosis and reducing microvessel density compared with
control groups [77,78].

Int. J. Mol. Sci. 2018,19, 1930 14 of 29
Escherichia coli cytosine deaminase (CD) gene is another system with the ability to convert
5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). Sher et al. [
79
] developed a strategy based on the
survivin promoter (protein downregulated by wild type p53 but not by mutant p53) plus a transgene
amplification vector (VISA) to deliver an endostatin cytosine deaminase fusion protein (hEndoyCD)
composed of an endostatin domain with antiangiogenic capacity and a CD domain that converts
the 5-FC into 5-FU. This system has high tumour-specific targeting effects and induced OC cell
death
in vitro
and
in vivo
without affecting normal tissues, inhibiting tumour growth and prolonging
survival in mouse xenograft models. An almost synergistic cytotoxic effect in combination with
cisplatin was shown [
79
]. Another example of suicide genes used in OC models is Escherichia
coli nitroreductase (NTR), which has been shown to activate the alkylating agent CB1954 and
increase survival in ovarian-infected murine models [
80
]. The same effect was observed using purine
nucleoside phosphorylase (PNP), whose use in combination with conventional chemotherapy in
multidrug-resistant OC cells showed a significant enhancement of apoptosis in OC cell lines [81].
Focusing on toxins, there are many examples that have been used in preclinical studies on OC
cells. Bai et al. evaluated the antitumour activity of a recombinant plasmid DNA expressing gelonin,
a toxin that causes cell death by inactivating the 60s ribosomal subunit. This plasmid was delivered
by HPEI nanogels, reducing cancer cell growth and inducing apoptosis in SKOV3 cells and in mice
with intraperitoneal ovarian carcinomatosis, without significant side effects [
24
]. In addition, DT-A
is a potent inhibitor of protein synthesis. Its delivery to chemotherapy-resistant OC cells using an
IP injection of nanoparticles showed an effective inhibition of OC tumour growth. This toxin was
more effective than treatment with cisplatin and PTX and with minimal nonspecific cytotoxicity,
and a prolonged life span compared to control mice [
46
,
47
]. Matrix protein (MP) of the vesicular
stomatitis virus (VSV) inhibited the growth of tumours and enhanced the survival of mice, mediated
by the induction of cancer cell apoptosis, inhibition of tumour cell proliferation and suppression of
tumour angiogenesis, with a good safety profile [
48
]. Moreover, carbonyl reductase 1 (CBR1) reduces
microvessel density and induces apoptosis. Delivering CBR1 DNA to OC cells via a polyamidoamine
(PAMAM) dendrimer increased survival in mice with peritoneal carcinomatosis of OC by inhibition of
dissemination and proliferation of malignant cells in mice, without significant adverse reactions [82].
2.2.4. Antiangiogenic Gene Therapy
In cancer, the balance between pro- and antiangiogenic growth factors is modified in favour of
angiogenesis, or new blood vessel formation from the pre-existing vasculature. A key aspect of the
success of antiangiogenic treatment is maintaining optimal levels for a prolonged period with the
inhibitor in circulation; gene therapy is a good strategy to achieve it.
Angiogenesis is regulated by the vascular endothelial growth factors (VEGFs) and their tyrosine
kinase receptors (VEGFR-1/Ftl-1, VEGFR-2, VEGFR-3/Ftl-4), and angiopoietin growth factor and their
receptors (Tie1 and Tie2). This pathway has been targeted in multiple studies, both in monotherapy
and in combination therapy. Soluble Flt-1 (fms-like tyrosine kinase receptor, a potent VEGF antagonist)
have shown efficacy inhibiting tumour growth and ascities formation, and increasing survival
in vivo
model [
83
]. Soluble VEGF decoy receptor (VEGF Trap) combined with PTX has enhanced the
survival by complete inhibition of ascities formation and tumour metastasis, and reduced the tumour
burden [
84
]. Several reports have studied the combined antiangiogenic gene therapy with soluble
decoy VEGFRs (-1, -2 and -3, without tyrosine kinase part) delivered with adenovirus, and combination
therapy has a more powerful antitumor effect than single gene therapy, suppressing tumour growth in
a mouse model of ovarian carcinoma [
85
]. The survival is prolonged if PTX is added to combination
gene therapy [
86
], being safe in healthy rats [
87
]. Furthermore, adenovirus gene therapy with soluble
VEGFR2 and Ti2 reducing tumour growth and formation of ascities, enhanced reduction of ascities
when gene therapy was combined with PTX and carboplatin, although in this last case, it increased the
proliferation of tumour cells [88].

Int. J. Mol. Sci. 2018,19, 1930 15 of 29
Other angiogenesis inhibitors that have been widely studied are angiostatin and endostatin.
Adenovirus-mediated gene transfer of angiostatin or endostatin effectively inhibited malignant ascites
and blood vessel formation and repressed tumour growth in OC but with transient expression [
89
].
To escape from the neutralization caused by humoral immune response, recombinant human endostatin
adenovirus (Ad-hEndo) was encapsulated into PEG-PE cationic liposome, enhancing transfection
efficiency on CAR-negative OC. Systemic administration reduced tumour growth in an established
OC model, by decreasing the number of micro-vessels and increasing apoptosis of tumour cells [
17
].
Moreover, AAV vectors provide stable gene expression and have an excellent safety profile with
persistent, long term expression. Recombinant adeno-associated virus (rAAV)-mediated delivery of a
mutant endostatin (P125A-endostatin) has been shown to inhibit blood vessel formation and ovarian
carcinoma growth [
90
]. A single intramuscular injection of rAVV-mediated delivery of K5, a potent
angiogenic inhibitor, inhibited VEGF and tumour cell-induced angiogenesis in subcutaneous and
intraperitoneal human OC cells in mouse models, conferring survival advantage without toxicity.
Furthermore, they affect the nascent vessels more than the mature ones. This system achieved sustained
levels of K5 in circulation [
16
]. In a more recent study, a single administration of AAVrh10.BevMab,
a rhesus serotype 10 adeno-associated viral vector coding for bevacizumab, achieved persistent and
high levels of bevacizumab in the peritoneal cavity with low systemic concentration, with significant
reduction of OC growth through inhibition of angiogenesis, and increased survival in an OC murine
model. Furthermore, combination with chemotherapeutic agents such as PTX or topotecan shows an
additive effect, being more effective than monotherapy [15].
2.2.5. Immunopotentiation
Strategies are based upon tumour-associated antigens and the ability of the immune system
to recognize these molecules, and as such, construct encoding-known tumour antigens to elicit an
immune response against it.
IL-21 has been extensively applied to significantly augment antitumour immunity in multiple
murine ovarian tumour models enhancing NK cytotoxicity [
91
]. Hu et al. used a recombinant
pIRES2-IL-21-EGFP and transfected it into CD34+ human umbilical cord blood stem cells (UCBSCs)
for treatment of OC xenograft mice, with an increase in the therapeutic effect by reducing the tumour
sizes and extending survival rate, markedly increasing levels of IFN-
γ
and TNF-
α
in the mouse serum,
which may increase the NK cytotoxicity by upregulation of the expression of NKG2D and MIC A
molecules in the tumour tissues. However, IL-21 expression was gradually decreased in the mouse
tumour sites [
92
]. Furthermore Zhang et al. obtained a more lasting expression of IL-21 on SKOV3 OC
xenograft-bearing nude mice triggering a reduction of tumour sizes, with inhibition of OC growth
by downregulation expression of
β
-catenin and cyclin-D1, and the elevation of the aforementioned
cytokines [
37
]. Fewell et al. developed a synthetic polymeric delivery vehicle (PPC) incorporating
the anticancer cytokine IL-12 gene (pmIL-12) and studied its IP administration in a mouse model
of disseminated OC. IP administration of pmIL-12/PPC led to elevated murine IL-12 (mIL-12) and
IFN-γlevels in ascites fluid, with a significant decrease in VEGF protein that resulted in inhibition of
ascites accumulation and improved survival, with no significant evidence of systemic toxicity due to
IP administration (IL-12 iv administered show significant toxicities). Animal survival was improved
by adding iv taxol and paraplatin chemotherapy treatment with no augmented side effects over that
associated with chemotherapy alone [93].
Dendritic cells (DCs) could be loaded by self-tumour antigen and induce specific anti-tumour
immunity against tumour cells carrying the target antigen. In this case, Her-2/neu was used as an
antigen and was transduced into DCs by rAAV vector inducing a strong and rapid stimulation of
cytotoxic T-lymphocyte (CTL) directed against OC cells [94].

Int. J. Mol. Sci. 2018,19, 1930 16 of 29
2.2.6. Multi-Drug Resistance (MDR)
The development of acquired drug resistance is the primary cause of chemotherapy failure
in the treatment of OC, posing a major impediment to the clinical treatments. Knockdown of drug
resistance-associated genes is a strategy used in OC cell lines. Inactivation of p53 by negative regulators
such as murine double minute 2 (MDM2) can contribute to resistance to p53 gene therapy. Gu et al.
used a dual expression plasmid with MDM2-specific siRNA and wild-type p53, which was effective in
increasing the sensitivity of cisplatin-resistant OC cells in vitro and in vivo [95].
Multidrug resistance gene 1 (MDR1) is overexpressed in chemoresistant OC cells. This gene
encodes a membrane-bound P-glycoprotein (P-gp) that works as a drug pump, and several strategies
have been developed to knock it down. Yang et al. developed hyaluronic acid-based nanoparticles
that can target CD44 receptors, overexpressed on MDR OC, to deliver MDR1 siRNA, and efficiently
downregulate the expression of MDR1, increasing sensitivity to PTX in MDR OC mouse models [
96
].
Another option is a combined therapy such as the system developed by Zhang et al., containing two
anticancer drugs (doxorubicin DOX and cisplatin CIS), and two antisense oligonucleotides targeted to
MDR1 an BCL2 mRNA (suppressors of cellular resistance) [
97
], or the use of an oncolytic adenovirus
(Ad5/3) under the control of the MDR1 promoter with PTX [98].
Survivin (SVV), a member of the inhibitor of apoptosis protein (IAP) family, is associated with
chemotherapy and radiotherapy resistance in OC and apoptosis inhibition of cells. This protein is
upregulated in various cancers but not expressed in normal adult tissues, and silencing SVV expression
induces the apoptosis of OC cells. Many therapeutic strategies targeting the SVV gene have been
developed for OC treatment. Vivas-Mejia et al. have used siRNA to target SVV for OC and to
suppress its expression [
99
]. Jiang et al. used adenovirus-mediated knockdown of SVV by shRNA
(ad5-SVV) in cisplatin-resistant OC cells, inhibiting proliferation and invasion and inducing apoptosis,
via downregulation of PCNA and MMP-2 expression and upregulation of caspase-3 expression [
100
].
Other studies have demonstrated a promising result in the development of vehicles such as polymeric
micelles [
101
] and degradable HPEI nanoparticles [
22
] for the combined therapy of SVV siRNA and
chemotherapy drugs in treatment of chemoresistant tumours.
The EGFR/extracellular signal-regulated kinase (ERK) pathway leads to tumour cell proliferation,
survival and chemotherapy resistance, and it is induced by PTX, and inhibited by MicroRNA-7 (miR-7)
by downregulating EGFR expression. Cui et al. have developed a dual-drug-delivery system based
on biodegradable polymer nanoparticles that do not have these problems, to simultaneously deliver
PTX and miR-7. Despite the results of products of this combination, this therapy has some limitations,
because it was not able to completely eradicate the tumours [102].
Another way to act on chemoresistance is through the clock gene, an important regulator of the
inherent circadian rhythm in mammals. Formations of a heterodimer transcription factor complex
with another protein activate the expression of many genes regulating metabolism, eating, physiology
and behaviours. Sun et al. found that suppressing the Clock gene expression can induce autophagy
and can upregulate the cisplatin-induced apoptosis in OC cells
in vitro
. Furthermore, enhancing
cisplatin chemosensitivity by the inhibition of the expression of resistant genes like MRP2 or P-gp,
by combined therapy cisplatin and interfering Clock expression, have superior cytotoxicity to cisplatin
alone [
103
]. In addition, glucose regulated protein 78 (GRP78) is involved in cell survival during
endoplasmic reticulum stress, and contributes to development of chemoresistance. Its silencing by
siRNA transfection increases the sensitivity to PTX in OC cell line (HO-8910) by induction of cancer
cell apoptosis [
104
]. Moreover, the TNF-related apoptosis-inducing ligand (TRAIL) is a protein with
the ability to induce apoptosis in a broad range of cancer cells, without affecting normal cells, but
has instability
in vivo
and resistance to cancer cells. A study showed that the use of retrovirus
encoding TRAIL gene inhibited growth of drug-resistant A2780/DDP ovarian carcinoma cells
in vitro
via a caspase-activated apoptotic mechanism, and in combination with cisplatin-enhanced anticancer
activity
in vitro
and in a xenograft nude mouse model. This may be an efficient approach to treat
drug-resistant OC [105].

Int. J. Mol. Sci. 2018,19, 1930 17 of 29
2.2.7. Oncolytic Virotherapy
Cancer virotherapy is a strategy in which viruses are modified to preferentially replicate in tumour
cells and lead to cell death, through targeted alterations in the cancer cells, such as p53 mutation, viral
deletion, tissue-specific transcriptional control, or tumour-specific receptors. Furthermore, oncolytic
viruses (OVs) can be genetically designed to deliver therapeutic genes as suicide genes.
The viral glycoproteins hemagglutinin (H) of measles virus (MV), responsible for receptor
attachment and rigger in cell-entry, can be genetically engineered to use any cell surface receptor of
choice for cell entry. Designed Ankyrin repeat proteins (DARPin) domains allow the generation of
oncolytic viruses with double specificity, simultaneously targeting HER2 and EpCAM54 (a cancer stem
cell marker), handling intratumoural variation of antigen expression and targeting simultaneously
CSCs and the tumour mass, showing oncolytic potential in a disseminated OC xenograft model in
mice, furthermore, showing the superior efficacy of bispecific over monospecific viruses [106].
Reoviruses kill ovarian-cancer cells
in vitro
, this effect is reduced by ascites due to the presence
of neutralizing antibodies (NAb). However, cytotoxicity can be restored using a combination of
lymphokine-activated killer and dendritic cells (LAKDC) as carriers, which protect the virus from
NAb in the ascites [107].
The combination of oncolytic herpes simplex viruses (HSV) with immunostimulatory cytokines
has recently been studied in attempts to increase its efficacy. Genetically engineered HSV were
transformed to express the cytokines IL-12, having a cytotoxic effect in an OC cell line, and its IP
administration in mice models had a longer survival and lower rates of peritoneal metastasis, with
an increased CD8+ T-cell immune response [
108
]. HF10 intraperitoneal injection, a highly attenuated
variant of the HSV type 1 (HSV-1), decreased tumour size in a murine OC model, and its combination
with a Granulocyte–macrophage colony-stimulating factor (GM-CSF) can elicit immune response, with
higher antitumoural effects [109].
The VSV is one of the most potent oncolytic viruses, but presents neurotoxicity and induction
of Nab; limitations are overcome by pseudotyping VSV with the glycoprotein of the lymphocytic
choriomeningitis virus (LCMV)—resulting in virus VSV-GP. Selectivity replication in cancer cells
by this virus is determined by reduced antiviral defense due to aberrations in the type I interferon
(IFN) system, which is very common in tumour cells. However, in OC cell lines, most cells have the
IFN response intact, and VSV-GP oncolysis can be enhanced by combination with an inhibitor of the
interferon response, such as Ruxolitinib, inhibitor of Jak1 and Jak2. Dold et al. showed that IP treatment
with VSP-GP was able to infect, replicate in and kill most OC cell lines tested, but tumour remission
in mice was only temporary. However, the combined therapy with the Jak1/2 inhibitor ruxolitinib
enhanced efficacies compared with monotherapy, and there was no increase in virus toxicity [110].
The combination of Myxoma virus (MYXV) and cisplatin produced a significant improvement
in overall survival in a mouse model of disseminated OC, and reduced secretion of cytokines
immunosuppressives by CD14+ myeloid cells [
111
]. The recombinant oncolytic adenovirus
ZD55-MnSOD (an antioxidant enzyme with tumour suppressor activity) enhances cisplatin-mediated
growth suppression and apoptosis in OC cells,
in vitro
and
in vivo
, so the combination therapy of
cisplatin and ZD55-MnSOD results in an improved survival rate, compared to monotherapy [112].
Different studies describe the use of suicide gene therapy in combination with virotherapy
for ovarian carcinoma treatment. Hartkopf et al. developed a combined strategy based on a
recombinant MeV armed with a bifunctional suicide fusion gene that encodes for CD and uracil
phosphoribosyltransferase (MeV-SCD), that enhance the sensitivity of chemoresistant cancer to 5-FU
by its conversion into the toxic metabolite 5-fluorouridine monophosphate (5-FUMP). This combined
therapy showed an effective infection and lysis of human OC cell lines and primary tumour cells
derived from malignant ascites of OC patients, and 5-FC significantly enhanced the antineoplasic
activity of MeV-SCD [113].

Int. J. Mol. Sci. 2018,19, 1930 18 of 29
3. Clinical Trials
According to The Journal of Gene Medicine Clinical Trial site (http://www.abedia.com/wiley/
indications.php), there are a total of 1688 clinical trials for cancer gene therapy, representing 65% of
the total clinical trials based on gene therapy compiled in this registry. Clinical trials have explored
the feasibility and effectiveness of several gene therapy treatment strategies for OC listed before,
including tumour suppressors, suicide genes and oncolytic virotherapy (Table 3). To date, the clinical
trial registry contains 2176 trials about OC (Source: clinicaltrials.gov), and 31 clinical trials involving
gene therapy have been registered.
Table 3.
Current ovarian cancer gene therapy clinical trials available in [
114
] “clinicaltrials.gov” until
May 2018 using the terms “ovarian cancer” and “gene therapy” as key words.
Therapeutic
Strategy Intervention Clinical Trial
Reference Phase Year (First–Last
Posted)
Suicide gene
therapy
HSV-TK + GCV
Vector: Adenovirus (Ad5.SSTR/TK.RGD) NCT00964756 Phase 1 2009–2013
HSV-TK + GCV
Vector: Vector producer cells (VPC) NCT00005025 Phase 2 2003–2013
CD + 5-FC
Vector: Toca 511, a purified retroviral replicating
vector encoding a modified yeast CD gene
NCT02576665 Phase 1 2015–2018
Tumour suppressor
gene
Inserting the p53 gene
Vector: Adenovirus (Ad5CMV-p53) NCT00003450 Phase 1 2003–2009
Inserting the p53 gene
Vector: Adenovirus (Ad5CMV-p53) NCT00003588 Phase 1 2004–2013
Inserting the p53 gene + chemotherapy (PTX and
carboplatin)
Vector: Adenovirus (SCH-58500)
NCT00003880 Phase 2
Phase 3 2004–2015
Inserting the p53 gene + chemotherapy (cisplatin
and PTX) NCT02435186 Phase 2 2015–2015
Oncolytic
virotherapy
LOAd703 (an oncolytic adenovirus serotype
5/35 encoding immunostimulatory transgenes:
TMZ-CD40L and 41BBL) + chemotherapy
or gemcitabine
NCT03225989 Phase 1
Phase 2 2017–2018
Recombinant carcinoembryonic antigen
(CEA)-expressing measles virus (MV-CEA) and
oncolytic measles virus encoding thyroidal
sodium iodide symporter (MV-NIS)
NCT00408590 Phase 1 2006–2018
Vesicular Stomatitis Virus expressing Human
Interferon Beta and Sodium-Iodide Symporter
(VSV-hIFNbeta-NIS)
NCT03120624 Phase 1 2017–2018
Immunopotentiation
EGEN-001 (IL-12 Plasmid Formulated With
PEG-PEI-Cholesterol Lipopolymer) +
chemotherapy
NCT00473954 Phase 1 2007–2013
EGEN-001 (IL-12 Plasmid Formulated With
PEG-PEI-Cholesterol Lipopolymer) NCT00137865 Phase 1 2005–2013
GEN-1 (IL-12 Plasmid Formulated With
PEG-PEI-Cholesterol Lipopolymer) +
chemotherapy (PTX and carboplatin)
NCT02480374 Phase 1 2015–2018
NYESO-1(C259) transduced autologous T cells NCT01567891 Phase 1
Phase 2 2012–2018
TBI-1301 (Autologous T cells engineered to
express a T cell receptor (TCR) targeting
NY-ESO-1) + cyclophosphamide
NCT02869217 Phase 1 2016–2017
TBI-1301 (Autologous T cells engineered to
express a T cell receptor (TCR) targeting
NY-ESO-1) + cyclophosphamide ±fludarabine
NCT02366546 Phase 1 2015–2017
Autologous T cells engineered to express a T cell
receptor (TCR) targeting NY-ESO-1 +
cyclophosphamide + fludarabine
NCT02457650 Phase 1 2015–2016

Int. J. Mol. Sci. 2018,19, 1930 19 of 29
Table 3. Cont.
Therapeutic
Strategy Intervention Clinical Trial
Reference Phase Year (First–Last
Posted)
Immunopotentiation
TBI-1201 (MAGE-A4-specific TCR gene
transduced T lymphocytes) + cyclophosphamide
±fludarabine
NCT02096614 Phase 1 2014–2017
Gen modified lymphocytes with MOv-gamma
chimeric receptor gene (MOv-PBL) + IL-2 NCT00019136 Phase 1 2003–2015
TCR-Transduced PBL (T-Cells Genetically
Engineered to Express T-Cell Receptors Reactive
Against Mutated Neoantigens)
NCT03412877 Phase 2 2018–2018
Anti-mesothelin CAR transduced PBL (retroviral
vector that contains a chimeric T cell receptor
(CAR) that recognizes mesothelin) +
cyclophosphamide, fludarabine and aldesleukin
NCT01583686 Phase 1
Phase 2 2012–2018
Anti-hCD70 CAR PBL (Transducing PBL with a
chimeric antigen receptor (CAR) that engages
CD70) + cyclophosphamide, fludarabine
and aldesleukin
NCT02830724 Phase 1
Phase 2 2016–2018
ZYC300 (vaccine which encodes the cytochrome
P450 family member, CYP1B1, a known human
tumor-associated antigen) + cyclophosphamide
Vector: PGL-encapsulated plasmid DNA
NCT00381173 Phase 1 2006–2013
Vigil (vaccine composed of autologous tumor
cells which are transfected extracorporeally with
a plasmid encoding for the gene for GM-CSF, an
immune-stimulatory cytokine, and a bifunctional
short hairpin RNA that targets furin, convertase
responsible for activation of both TG
β
1 and
β
2) +
Atezolizumab
NCT03073525 Phase 2 2017–2018
ALVAC(2)-NY-ESO-1 (M)/TRICOM vaccine +
IDO1 inhibitor NCT01982487 Phase 1
Phase 2 2013–2013
ALVAC(2)-NY-ESO-1 (M)/TRICOM vaccine +
sirolimus + GM-CSF NCT01536054 Phase 1 2012–2018
ALVAC(2)-NY-ESO-1 (M)/TRICOM
vaccine + sargramostim NCT00803569 Phase 1 2008–2011
atezolizumab ±guadecitabine ±CDX-1401
vaccine (a vaccine composed of a human mAb
specific for DEC-205 fused to the full-length
tumor antigen NY-ESO-1)
NCT03206047 Phase 1
Phase 2 2017–2018
p53MVA vaccine (modified vaccinia virus ankara
vaccine expressing tumor protein p53) +
gemcitabine hydrochloride
NCT02275039 Phase 1 2014–2018
p53MVA vaccine + Pembrolizumab NCT03113487 Phase 2 2017–2018
p53 peptide vaccine + ISA-51 + IL-2 ±GM-CSF NCT00001827 Phase 2 1999–2017
Immunopotentiation is the strategy with the most clinical trials in gene therapy of OC
made so far. As we can see in Table 2, delivery of cytokines to the tumour (NCT00473954,
NCT00137865, NCT02480374), administration of tumour vaccines based on tumour-associated antigens
(TAAs) (NCT00381173, NCT03073525) or T-Cells genetically engineered to express T-cell receptors
reactive against mutated neoantigens (NCT01567891, NCT02869217, NCT02366546, NCT02457650,
NCT01583686) were used. IL-12 is one of the most widely studied cytokines in this sense, due to its
potent immune-modulatory properties and its ability to inhibit tumour angiogenesis. The latest studies
have been directed at the evaluation of EGEN-001, a system composed of a human IL-12 plasmid and
a delivery system polyethyleneglycol-polyethyleneimine-cholesterol (PCC) that facilities its delivery
in vivo
. Research groups have brought about two phase I [
115
,
116
] and one phase II trials [
117
] in
patients with recurrent OC. In the phase I trials, EGEN-001 was administered IP alone or in combination
with chemotherapy, showing to be safe and feasible with low grade and manageable side effects and
disease response in some patients. The phase II trial evaluated the toxicity and antitumour activity

Int. J. Mol. Sci. 2018,19, 1930 20 of 29
of EGEN-001 administered IP at the 24 mg/m
2
dose, in patients with platinum-resistant recurrent
ovarian, fallopian tube or primary peritoneal cancer. Specific toxicity was similar to that found in phase
I studies. More frequent adverse events presented in the 20 treated patients were grade 1/2, including
nausea, vomiting, pain, fatigue and anemia, but EGEN-001 was less tolerated than in the previous
studies. Of the 16 patients evaluable for response, seven had stable disease and nine had progressive
disease, with no partial or complete response, showing that EGEN-001 in monotherapy had limited
activity in platinum-resistant EOC patients. Another more recent study evaluated the administration
of EGEN-001 at a higher dose (36 mg/m
2
) in combination with liposomal doxorubicin, which presents
the ability to modulate the immune system in various ways, was initiated [
118
]. This phase I trial
showed promising results, with a clinical benefit (partial responses and stable disease) of 57.1% in the
14 patients with measurable disease, achieving 28.6% of partial responses and 57.1% of stable disease
at dose level 3.
Reintroduction of tumour suppressor genes has been widely studied in preclinical studies, and
p53 is one of the most extensively studied. In the clinical trial registry we can find that several studies
about the insertion of the p53 gene are being carried out (NCT00003450, NCT00003588, NCT00003880,
NCT02435186), alone or in combination with chemotherapy.
Numerous groups have evaluated suicide-based gene therapy in OC (NCT00964756,
NCT00005025, NCT02576665). The suicide gene most commonly investigated has been HSV/TK
with several clinical trials completed. Kim et al. studied a new strategy to enhance the infectivity
of a RGD-modified adenovirus (Ad5-SSTR/TK-R-GD) expressing HSV-TK gene and a somatostatin
receptor (SSTR) used as a non-invasive strategy to assess gene transfer through nuclear imaging [
119
].
This phase I study was conducted in a cohort of 12 patients, showing the safety of using this vector
intraperitoneally, with limited clinical toxicities (manageable constitutional or pain symptoms), related
to the dose. In relation to effectiveness, no partial or complete responses were observed, although five
patients had stable disease 1 month after treatment and Ca-125 levels decreased in three. Furthermore,
a patient had a delayed and durable complete response, being clinically free of disease 25 months
after treatment.
Over the last two decades, several clinical trials based on oncolytic virotherapy have been
developed. One of the main approaches has been the use of serotype-5 conditionally-replicative
adenoviruses (CRAds), which has shown problems in relation to the non-expression of its receptor,
the CAR, on the surface of OC cells. This has led to the development of novel strategies to enhance
the infectivity of adenoviruses cells [
120
–
122
]. In a phase I study in 10 recurrent OC patients, Kim
et al. used Ad5/3-
∆
24, a CRAd which incorporates a modification that alters the tropism towards
serotype-3 adenoviral receptors, and a deletion for selective replication in Rb-p16-deficient tumour
cells [
120
]. Its IP administration daily for three consecutive days at dosages up to 1
×
10
12
vp, showed
it to be safe, with mild and manageable vector-related toxicities. Clinical efficacy analyses showed that
75% of patients had RECIST-defined stable disease and 37.5% had a decrease in CA-125 levels, but a
marked anti-adenoviral antibody response was also noted. These results are similar to those of other
trials evaluating other strategies of increased infectivity of CRAds in OC, such as the study carried out
by Koski et al. in 21 patients with a variety of solid tumours including four patients with OC [
121
].
A serotype 5/3 chimeric oncolytic adenovirus expressing GMCSF was administrated and one of the
OC patients had RECIST-defined stable disease and a slight decrease in CA-125 levels.
4. Future Directions
Despite all the efforts made to treat and cure OC, approximately 80% of patients diagnosed with
ovarian epithelial cancer will relapse after standard first-line treatment, which includes platinum-based
and taxane-based chemotherapy [
123
]. These suggest that CSCs may play an important role in OC
and cannot be ignored. It is believed that CSCs are responsible for tumour initiation and contribute
to chemo and radioresistance, which explains relapse and resistance in OC. Ovarian cancer stem
cells (OCSCs) are a subpopulation of OC cells with self-renewing ability, and can differentiate into

Int. J. Mol. Sci. 2018,19, 1930 21 of 29
heterogeneous tumour cell types. Some markers have been found in OCSCs, including CD44, CD47,
CD133, ALDH1, CD24, CD117 (c-kit), epithelial cell adhesion molecule (EpCAM), and SOX2, with
a heterogenic expression [
124
]. Moreover, ALDH plays an important role in the resistance of OC to
chemotherapeutics, especially in OC stem cells [
125
]. In a recent study, Shaomin and Guang correlated
ALDH2 mutation to the low incidence and mortality of OC in East Asian women [
126
]. Furthermore,
an overexpression of several OCSCs markers have been found in residual ovarian tumours after
treatment with chemotherapy [
124
]. Thus, OCSCs markers have been considered as useful therapeutic
targets to minimize drug resistance and the tumour relapse of OC. Targeting some of these markers
has been studied in several works [
127
]. In addition, certain substances have been shown to be
effective in treating OCSCs, such as metformin, niclosamide (commonly used against parasites) and
salinomycin (antibiotic isolated from Streptococcus albus bacteria and has been tested in humans too);
hence, these drugs could be used in combination with gene therapy to improve the response to
treatment. Some studies support the ability of OCSCs to adhere and to spread on mesothelial layers,
showing characteristics of mesenchymal cells [127].
Another approach is the use of anti-angiogenic drugs such as Bevacizumab or Trebananib, since
there is a link between CSCs and angiogenesis [
128
]. Many inmunotherapeutic strategies can be used
to target OCSCs, and some of them have already been researched in some works: NK cell, cancer
therapeutic vaccine (a CD177/CD44 vaccine), monoclonal antibody immunotherapy (catumaxomab,
which can bind to CD133 and EpCAM) and the blockade of immune checkpoints.
In the field of gene therapy applied on OCSCs, Chimeric antigen receptor (CAR)-T cells, genetically
modified T cells, could be used to target these specific antigens on OCSCs, and have been used to
target CD133 in OC cell lines [
129
]. Moreover, Long et al. studied its elimination by truncated Bid
(tBid), a potent inducer of cell apoptosis, delivery by the Cre/LoxP system, targeting CD133, a cell
surface marker of OCSCs. They constructed two recombinant adenoviruses, the first facilitates the
expression of the Cre in CD133+ CSCs, which cut-off LoxP sequences from the second, thus allowing
the overexpression of tBid, driven by the CMV promoter, in CD133+ CSCs. This system induced the
apoptosis and inhibited growth of OCSCs in vitro and in vivo, in addition to increasing the cytotoxic
effect of cisplatin [
130
]. Furthermore, it has been shown that OCSCs expressed several genes related to
primordial germ cells, germinal lineage, and pluripotency, such as Nanog, Oct4 and Sox2; therefore,
their involvement in the manifestation of OC is not excluded [
131
]. The gene therapy with shRNA or
siRNA targeting Nanog has already shown some promising therapeutic potential [132].
Furthermore, the low transduction efficiency of the vector within the tissue, and the creation of
neutralizing antibodies to viral vectors by the immune system is a great limitation of the actual gene
therapy systems. New non-viral vectors for OC are increasingly investigated. The use of nanoghosts,
vesicles derived from the natural membrane as a vehicle for gene delivery, represents an innovative and
promising approach for targeted OC. Although there are several types of vesicles, such as exosomes
and red blood cells, that are being manipulated for the delivery of genes and/or drugs, MCSs are
undoubtedly the best option for ovarian cancer since they have inherent targeting capabilities and can
be produced using a technologically scalable and pharmaceutically applicable process [42].
5. Conclusions
No effective therapies exist for patients with advanced-stage OC. This malignancy is confined
to the abdominal cavity, offering the option of a localized treatment with protection of the organs
out of the peritoneal cavity and gene therapy could be a promising strategy. This review showed
the development of multiple systems for OC treatment based on gene therapy with encouraging
preclinical results. However, the translation to humans has not yet shown a significant clinical
benefit due to, among others, the lack of efficient vectors. This fact has led to the development of
numerous new vectors, many of them from non-viral origin. To improve tumour targeting, specific
tumour promoters, tumour gene targets and markers have been identified. They were used to achieve
effective penetration within advanced tumour masses and to minimize toxicity in normal tissues.

Int. J. Mol. Sci. 2018,19, 1930 22 of 29
These improved vectors are beginning to be analysed in phase I trials, and constitute one of the main
fields of research for future works. On the other hand, the use of gene therapy as monotherapy
has not been as successful as had been expected. Combining gene therapy with chemotherapy and
radiotherapy has been shown to improve the effectiveness and safety of the treatment by reducing the
dosages. This could also be applicable to the combination with other emerging therapeutic strategies,
such as targeting molecular pathways with angiogenesis inhibitors (bevacizumab) or Poly ADP
Ribose Polymerase (PARP) inhibitors (olaparib), and immunotherapy (ipilimumab, an anti-CTLA-4,
or nivolumab, an anti-PD-1 agent). Finally, OCSCs have been shown to contribute to cancer progression,
metastasis, chemoresistance and recurrence, and their discovery has opened a new area of research in
OC. Several OCSCs markers have been suggested with a high variability of their phenotypical features
in the different studies. Even so, there is no doubt that these markers will open the way to the targeting
of OCSCs to minimize the drug resistance and tumour relapse.
In conclusion, there is still much work to be done in order to reach the full potential that gene
therapy can offer for the treatment of OC. More phase I and II clinical trials are needed to investigate
current preclinical strategies so as to translate this powerful strategy to the clinic.
Author Contributions:
Á.Á.: drafting the manuscript; Y.M.J.; performs figures; J.A.M.: substantively article
review; H.B.: design, drafting and substantively manuscript revision.
Funding:
This research was supported by the Fundación Mutua Madrileña by the proyect FMM-AP16683-2017,
Consejería de Salud Junta de Andalucía (PI-0089-2017) and from the Chair “Doctors Galera-Requena in cancer
stem cell research”.
Conflicts of Interest: The authors declare no conflict of interest.
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