GeneTransferApproachesforGynecological Diseases
Mari Raki,
1,2
Daniel T. Rein,
3
and Anna Kanerva
1,2,4
Akseli Hemminki
1,2,
*
1
Cancer Gene Therapy Group, Rational Drug Design Program, University Helsinki, 00014 Helsinki, Finland
2
Department of Oncology and
4
Department of Obstetrics and Gynecology, Helsinki University Central Hospital, 00029 Helsinki, Finland
3
Department of Obstetrics and Gynecology, University of Du
¨
sseldorf Medical Center, 40001 Du
¨
sseldorf, Germany
*To whom correspondence and reprint requests should be addressed at P.O. Box 63, University of Helsinki, 00014 Helsinki,
Finland. Fax: +358 9 1912 5465. E-mail: akseli.hemminki@helsinki.fi.
Available online 2 May 2006
Gene transfer presents a potentially useful approach for the treatment of diseases refractory to
conventional therapies. Various preclinical and clinical strategies have been explored for treatment
of gynecological diseases. Given the direst need for novel treatments, much of the work has been
performed with gynecological cancers and ovarian cancer in particular. Although the safety of many
approaches has been demonstrated in early phase clinical trials, efficacy has been mostly limited so
far. Major challenges include improving genetransfer vectors for enhanced and selective delivery
and achieving effective penetration and spread within advanced and complex tumor masses. This
review will focus on current and developmental genetransfer applications for gynecological
diseases.
Key Words: gene transfer, gene therapy, ovarian cancer, cervical cancer, gynecological disease
Contents
Introduction 154
Gene Therapy for Ovarian Cancer 154
Targeting Vectors to Ovarian Cancer Cells 155
Replacement of an Altered Tumor Suppressor Gene 155
Inhibition of Growth Factor Receptors 157
Molecular Chemotherapy 157
Antiangiogenic Gene Therapy 157
Virotherapy 158
Gene Therapy for Other Gynecological Cancers 159
Gene Therapy for Other Gynecological Disorders 159
Future Directions 161
Acknowledgments 161
References 161
INTRODUCTION
An increasing understanding of the molecular mecha-
nisms that cause human disease has rationalized gene
transfer as an approach for the treatment of diseases
resistant to more conventional therapies. Gene therapy
aims at transfer of genes for correction of either genetic
or somatic disease phenotypes or for expression of
molecules within or near target cells for therapeutic
effect. Vehicles forgenetransfer include both nonviral
and viral vectors, such as adenovirus, retrovirus, adeno-
associated virus (AAV), and herpes simplex virus (HSV).
Nonviral genetransfer is most co mmonly based on
plasmid DNA, particle bombardment, or cationic lip-
osomes. Viral gene delivery has already been optimized
by evolution and is therefore generally more effective,
while nonviral approaches are pharmacologically more
attractive.
GENE THERAPY FOR OVARIAN CANCER
Ovarian cancer is the leading cause of death from gyne-
cological malignancies in developed countries [1].Mostcases
are diagnosed at an advanced state, and long-term survival of
patients with metastatic disease is rare. Although chemo-
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1525-0016/$30.00
therapy approaches featuring taxanes and platinums, when
given following optimal cytoreductive surgery, can increase
the survival of patients, treat ment of metastatic disease
eventually results in drug resistance and disseminated
disease cannot be cured. Therefore, novel trea tment
approaches are n eeded. Gene therapy, even at its current
rather adolescent stage, is an attractive modality for
ovarian cancer a s t his c ancer frequently p resents w ith
metastases confined to the peritoneal cavity, creating a
rationale for locoregional delivery.
Targeting Vectors to Ovarian Cancer Cells
The majority of gene therapy approachesfor ovarian
cancer (Table 1) are based on adeno virus serotype 5
(Ad5), which binds to the coxsackie–adenovirus receptor
(CAR). A number of approaches have been tested in phase I
clinical trials with impressive safety data. Moreover,
successful genetransfer has been demonstrated in most
cases in which it has been analyzed. In contrast, only rare
examples of efficacy have been published. This is partly
influenced by trial design (phase I trials usually have safety
as the main endpoint), but nevertheless the lack of res-
ponse implies that there is a discrepancy between preclin-
ical and clinical efficacy.
Onepossiblereasonisthattheremightbea
tendency for researchers to use models that allow
effective transduction, and therefore variable CAR
expression has been recognized only upon analysis of
clinical substrates. Another reason might be the greater
complexity of advanced solid tumor masses in compar-
ison to relatively rapidly growing xenografts. By
extension, this implies that it is crucial to perform
extensive sampling and biopsies in phase I trials to
acquire material for correlative studies. Obviously, this
is hampered by compliance and cost issues and the fact
that traditionally phase I trials have mostly looked at
safety.
Heretofore, all published studies have been per-
formed with CAR-binding viruses. Unfortunately, con-
current studies have suggested that expression of CAR is
frequently dysregulated in many types of advanced
cancers, including ovarian cancer [2]. Various strategies
have been evaluated to modify adenovirus tropism to
circumvent CAR deficiency, for increased transduction
of tumor cells and reduced normal tissue tropism.
Transductional targeting can be achieved by utilizing
bispecific molecules that block the interaction with CAR
and redirect the virus to a novel receptor. Several
ligands, including basic fibroblast growth factor [3],
anti-TAG-72 [4], and anti-CD40 [5], have been physically
linked to an Ad5-fiber-binding moiety for enhanced
transduction.
Another strategy involves genetic modifications of
the viral capsid. Enhanced infectivity of ovarian cancer
cells has been demonstrated by incorporating an integ-
rin-binding RGD-4C motif in the HI loop of the fiber
knob [6]. Fiber pseudotyping has also been evaluated.
Substitution of the knob do main of Ad5 wi th the
corresponding domain of serotype 3 (Ad3) allows bind-
ing and entry through the Ad3 receptor, which is
expressed to a high degree on ovarian cancer cells [7,8].
High tolerability of adenoviruses in cancer trials has
allowed administration of large doses. In most trials, the
maximum tolerated dose has not been reached and the
maximum affordable dose has become limiting instead.
Nevertheless, some trials have reported abdominal pain
or liver enzyme elevations [9,10], suggesting that trans-
duction of normal tissue has the potential for toxicity.
Also, while very safe in comparison to, e.g., chemothe-
rapy, it is now well known that adenoviruses can cause
eve n fatal immune reactions [11].Therefore,ithas
become attractive to restrict expression of viral genes or
transgenes to tumor cells by using tumor-specific pro-
moters (TSPs) in a strategy called transcriptional target-
ing. Several TSPs have been evaluated for ovarian cancer
specificity, including L-plastin [12], midkine [13], cyclo-
oxygenase-2 (cox-2) [13], ovar ian-specific promoter-1
[14], secretory leukoprotease inhibitor promoter (SLPI)
[15,16], and mesothelin [17].
Although transcriptional targeting can reduce toxicity
associated with transgene expression in nontarget tissues,
it does not reduce immunological recognition of virus
particles and infected cells. An immune response toward
infected tumor cells can be useful for eradication of
metastases and protection against relapse. In contrast, an
acute immune reaction or clearance of infected nontarget
cells can be harmful. Specific transductional targeting of
viruses to target cells is a useful way to retain the
potentially beneficial aspects of a vector-targeted
immune response while reducing immunological toxic-
ity. Other approachesfor reducing immune responses
toward adenovirus are discussed in the last section.
Replacement of an Altered Tumor Suppressor Gene
Mutation of the p53
tumor suppressor gene is one of the
most frequent genetic changes in cancer and it has been
found in nearly 60% of advanced ovarian cancers [18].
Preclinical studies have demonstrated that adenovirus-
mediated delivery of wild-type p53 inhibits growth of
ovarian cancer cells both in vitro and in vivo [19,20] (Fig.
1A). p53 genetransfer to ovarian cancer cells using catio-
nic nonviral vector has also been reported [21]. Adp53
was evaluated in a phase I/II trial and the treatment was
well tolerated [9,22,23]. Genetransfer and biological
activity were also demonstrated [24].
These findings led to a randomized phase II/III trial
in which Adp53 was given intraperitoneally in combi-
nation with chemotherapy. Although complete results
have unfortunately not been published, the first interim
analysis suggested a lack of therapeutic effect but
increased toxicity and the study was closed [25].In
parallel with most trials wit h this approach, trans-
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TABLE 1: Overview of gene therapy approachesforgynecological cancers
Construct Vehicle Approach Study Main result Reference
Ovarian cancer
Adp53 Adenovirus Delivery of p53 Phase I/II/III
clinical trial
Phase I/II: No dose-limiting toxicity
Phase III: No advantage, toxicity seen
[9,23,25]
LXSN-BRCA1sv Retrovirus Delivery of BRCA-I Phase I/II
clinical trial
Phase I: Safe, tumor reduction in 25%
Phase II: No responses
[26,27]
Ad21 Adenovirus Inhibition of erbB2 Phase I
clinical trial
No dose-limiting toxicity, no responses [33]
DCC-E1A Cationic
liposome
Inhibition of erbB2 Phase I
clinical trial
Safe, no responses [35]
AdHSV-TK Adenovirus HSV-TK suicide
gene therapy
Phase I
clinical trial
No dose-limiting toxicity, no responses [39]
ADV-RSV-tk Adenovirus HSV-TK suicide
gene therapy
Phase I
clinical trial
No dose-limiting toxicity [10]
ONYX-015 Adenovirus
(CRAd type I)
Virotherapy Phase I
clinical trial
Dose-limiting toxicity in one patient,
no responses
[60]
Sense and
antisense
OPCML
Plasmid Delivery of OPCML In vitro and
in vivo
Inhibition of ovarian cancer cell growth [101]
Ad-mda-7 Adenovirus Delivery of
mda-7/IL-24
In vitro Inhibition of ovarian cancer cell growth,
induction of apoptosis, targeting to CD40
or EGFR
[102,103]
EGFR-DNR Retrovirus Delivery of
truncated
EGFR (erbB1)
In vivo Inhibition of cancer cell growth, enhanced
sensitivity to cisplatin
[104]
HSV-T3 HSV type 1 HSV-TK suicide
gene therapy
In vitro Genetransfer to ovarian cancer cells [105]
rAAV-P125Aendo AAV Antiangiogenic
gene therapy
In vivo Inhibition of ovarian cancer cell growth,
inhibition of angiogenesis
[48]
Ad5-D24RGD Adenovirus
(CRAd type I)
Virotherapy In vitro and
in vivo
Killing of ovarian cancer cells, infectivity
enhancement
[52–54]
Ad5/3-D24 Adenovirus
(CRAd type I)
Virotherapy In vitro and
in vivo
Killing of ovarian cancer cells, infectivity
enhancement
[61]
Dearing reovirus
serotype 3
Reovirus Virotherapy In vitro and
in vivo
Inhibition of ovarian cancer cell growth [106]
MV-CEA Measles virus Virotherapy In vitro and
in vivo
Killing of ovarian cancer cells, expression of
soluble marker peptide
[107]
MV-CEA
MV-Moraten
MuV-JL
Measles and
mumps viruses
Virotherapy In vitro and
in vivo
Intercellular fusion of ovarian cancer cells,
cell death
[108]
Cervical cancer
Ad-p73 Adenovirus Delivery of p73 In vitro Growth inhibition of E6-positive cells [69]
AAV-TK AAV HSV-TK suicide
gene therapy
In vitro Cell killing of HPV-positive cells [66]
Ad5-D24RGD Adenovirus
(CRAd type I)
Virotherapy In vitro and
in vivo
Inhibition of cervical cancer cell growth [67]
Ad-MN/Ca9-E1a Adenovirus
(CRAd type II)
Virotherapy In vitro and
in vivo
Inhibition of cervical cancer cell growth [109]
Tissue-specific
promoters
Adenovirus Transcriptional
targeting
In vitro High activity of MK and VEGF promoters in
cervical cancer cell lines and primaries
[110]
Endometrial carcinoma
Adp21, Adp53 Adenovirus Delivery of p21 or
p53
In vitro Inhibition of endometrial cancer cell growth,
induction of apoptosis
[71]
SFG-F/S-IRES-tk Retrovirus HSV-TK suicide
gene therapy
In vitro Inhibition of endometrial cancer cell growth [111]
pNF
nB-TK Plasmid HSV-TK suicide
gene therapy
In vitro and
in vivo
Inhibition of endometrial cancer cell growth [72]
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duction of advanced and bulky tumor masses may not
have been sufficient for significant therapeutic effect,
while transduction of normal tissues may have been the
reason for side effects.
Retrovirus has also been clinically studied for ovarian
cancer therapy, utilizing transfer of BRCA1 [26,27].A
phase I study using intraperitoneal delivery showed
partial response in 25% of patients, and the majority
had stable disease. However, a subsequent phase II
study showed no responses and vector stability was
poor. Other viral and nonviral approaches are listed in
Table 1.
Inhibition of Growth Factor Receptors
Growth factor receptors such as erbB1–erbB4 of the
epidermal growth factor receptor family can be targeted
for replacement or inactivation. Deshane et al. con-
structed a gene that encodes an intracellular single-
chain antibody (intrabody) against erbB2/HER-2/neu
[28]. This receptor is highly expressed in 10–15% of
ovarian cancers with correlation with poor prognosis
[29]. Adenovirus (Ad21)-mediated transfer of the intra-
body to ovarian tumors resulted in induction of
apoptosis and cytotoxicity in vitro and enhanced
efficacy and survival in animal models of ovarian
cancer [30–32]. The strategy was subsequently evaluated
in a phase I trial [33]. Intraperitoneal treatment was
well tolerated without dose-limiting toxicity, and gene
transfer was demonstrated but no responses were
detected.
Adenoviral E1A has been shown to downregulate
erbB2 expression with concomitant growth inhibition
[34]. Hortobagyi et al. evaluat ed cationic liposome-
mediated E1A transfer in a phase I trial with breast
and ovarian cancer patients [35]. Expression of E1A and
downregulation of erbB2 expression were demonstrated
in peritoneal samples. Fo llowing dose escalation,
abdominal pain eventually identified the maximum
tolerated dose, but stable disease was detected in only
17% of patients, a rather low figure perhaps reflecting
the effectiveness of plasmid-based transduction in the
context of advanced disease. A similar strategy was used
in another phase I trial [36].
Molecular Chemotherapy
Molecular chemotherapy (a.k.a. suicide gene therapy) is
a strategy based on delivery of genes encoding a prodrug-
activating enzyme (Fig. 1B). The most popular approach
in the context of ovarian cancer has been herpes simplex
virus thymidine kinase (HSV-TK), which converts the
prodrug ganciclovir (GCV) into a toxic metabolite. The
HSV-TK/GCV system is associated with a bbystander
effect,Q i.e., killing of uninfected neighboring cells. Based
on promising preclinical results [37,38] Alvarez et al.
utilized intraperitoneal delivery of a replication-deficient
adenovirus (AdHSV-TK) followed by intravenous GCV
[39]. No dose-limiting side effects were seen and 38% of
patients had stable disease for the duration of the study.
Transgene expression could be detected from ascites
samples of patients. Another phase I study combined
intrape ritoneal AdHSV-TK with intravenous acyclovir
and topo tecan [10]. Again, no dose-limiting adverse
effects were seen, and the most common side effect
was myelosuppression most likely related to chemo-
therapy. The median survival of these patients was 18.5
months [40]. As an example of bench-to-bedside-and-
back translational work, when clinical specimens
revealed variable expression of CAR, the efficacy of the
HSV-TK/GCV approach was subsequently enhanced in
vitro and
in vivo by incorporating an integrin-binding
RGD-4C motif into the adenoviral fiber [41,42], and a
trial is forthcoming.
Antiangiogenic Gene Therapy
Antiangiogenic genetransfer inhibits formation of neo-
vasculature required for tumor growth and may also act
by collapsing immature tumor-associated vascular struc-
tures (Fig. 1C). Ovarian cancer cells have been shown to
express proangiogenic growth factors such as vascular
endothelial growth factor (VEGF) [43]. Effects of VEGF are
mediated through the endothelium-specific VEGF recep-
tors such as Flt-1 [44]. Soluble FMS-like tyrosine kinase
receptor 1 (sFlt-1) is a splice variant of Flt-1 and binds to
VEGF, inhibiting its angiogenic actions and may also
prevent dimerization of wild-type Flt-1. Mahasreshti et al.
evaluated the effect of adenovirus-mediated sFlt-1 transfer
against ovarian carcinoma [45,46]. Intraperitoneal deliv-
ery of an integrin-targeted virus encoding sFlt-1 inhibited
ovarian tumor growth and increased the survival of mice.
However, intravenous delivery of the same construct
resulted in hepatotoxicity.
Inhibition of angiogenesis was demonstrated after
intraperitone al injection of an AAV expres sing sFlt-1
[47]. Also other antiangiogenic genes such as mutant
Construct Vehicle Approach Study Main result Reference
Teratocarcinoma
Ad5-flt-1luc Adenovirus Transcriptional
targeting via
flt-1 promoter
In vitro High transgene expression in teratocarcinoma
cells
[112]
TABLE 1 (continued)
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endostatin have been packaged into AAV for in vivo
efficacy [48]. Lentiviruses have not been widely used for
ovarian cancer therapy, but transfer of interferon-a has
been evaluated in a murine model [49]. Antitumor effects
were associated with a decrease in the formation of
hemorrhagic ascites and a reductioninmicrovessel
density.
Virotherapy
Utilization of the oncolytic potential of viruses for
killing of tumor cells predates the concept of gene
therapy by more than half a decade [50]. Nevertheless,
due to safety concerns, most modern gene therapy
approaches have been based on viruses that are unable
to replicate in infected cells. However, the main result
from a generation of clinical trials with these agents is
that the utility of replication-deficient viruses may be
limited when faced with advanced and bulky disease.
Thus, intratumoral diffusion of nanosize carriers such as
viruses may be a limiting step. While tumor targeting
and infectivity enhancement have improved transduc-
tion rates of replication-deficient viruses preclinically, to
our knowledge no trials have been initiated yet,
although a number are in preparation (Table 1). A
specific obstacle with regard to analysis of oncolytic
viruses on clinical specimens is the limited viability of
the latter in vitro. This can be partly overcome by
maintaining clinical samples as multicellular tumor
clusters or spheroids [51]. This technology has been
applied to analysis of transductionally targeted oncolytic
adenoviruses [52–55], but correlation to clinical respon-
siveness is not yet available.
FIG. 1. Gene therapy approaches. (A) Replacement
of a mutated tumor suppressor gene. Delivery and
expression of a wild-type gene results in apoptosis
and cancer cell death. (B) Molecular chemotherapy.
Delivery and expression of a suicide gene results in
conversion of a nontoxic prodrug into a cytotoxic
metabolite. (C) Antiangiogenic gene therapy. Deliv-
ery of a soluble VEGF receptor results in sequestra-
tion of VEGF and subsequent inhibition of
neovascularization. (D) Virotherapy. Viral infection
of cancer cells results in replication, oncolysis, and
release of virions to surrounding cells.
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To improve tumor penetration, various naturally
occurring, inherently tumor-selective or engineered
oncolytic viruses have been utilized, including adeno-
virus, HSV, Newcastle disease virus, vaccinia, reovirus,
measles virus, and vesicular stomatitis virus [56].
Conditionally replicating adenoviruses (CRAds) are the
most widely studied members of this group (Fig. 1D),
and more than 500 cancer patients have been treated
with CRAds [2,57].
In type I CRAds, tumor-specific replication is achieved
by engineering deletions in genes critical for efficient
viral replication in normal but not in tumor cells [58].
The most widely studied CRAd, ONYX-015 (dl1520),
carries deletions in E1B, exhibits reduced binding of
p53, and replicates selectively in tumor cells [59]. ONYX-
015 has been evaluated in a phase I ovarian cancer trial
[60]. Treatment resulted in grade 3 abdominal pain and
diarrhea in one patient but the maximum tolerated dose
was not reached, and the bmaximum affordable doseQ was
10
11
viral particles. However, there were no clinical or
radiological responses in any patients.
In addition to ONYX-015, many type I CRAds have
been evaluated preclinically. Integrin-targeted Ad5-
D24RGD and serotype 3 receptor-targeted Ad5/3-D24
contain a 24-bp deletion in the retinoblastoma (Rb)
binding site of E1A. Therefore, these viruses replicate
selectively in cancer cells deficient in the Rb/p16 pathway.
Recent studies have demonstrated that both agents deliver
a powerful antitumor effect to ovarian cancer cells in vitro,
to clinical ovarian cancers, and in orthotopic models of
ovarian cancer, and both viruses are now proceeding
toward clinical testing [52,53,61].
Type II CRAds are designed to achieve replicative
specificity based on heterologous promoters placed into
the adenovirus genome to control the expression of the
early genes such as E1A, which is essential for viral
replication. The utility of these agents is subservient to
the identification of promoters that induce the appro-
priate inductivity vs specificity profile [62]. Promoters
that have shown utility for ovarian cancer include IAI.3B,
cox-2, and SLPI [55,63,64].
GENE THERAPY FOR OTHER GYNECOLOGICAL
CANCERS
While ovarian cancer is the most problematic gynecolog-
ical cancer in developed societies, cervical cancer remains
the leading cause of mortality worldwide [1]. Unfortu-
nately, neither improvements in surgery nor radiotherapy
has significantly decreased mortality [65], and patients
with advanced, recurrent, or metastatic disease still have a
poor chance of being cured. The pathogenesis of cervical
cancer follows a natural history characterized by human
papillomavirus (HPV) infection, a long latency period,
and progression in a fraction of patients through dysplasia
and carcinoma in situ to invasive cancer and metastatic
disease. Only a few viral strains are specifically responsible
for cervical neoplasms, of which HPV16 accounts for
more than one-half of reported cases. Carson et al.
demonstrated a novel gene-based strategy to prevent virus
replication in HPV-infected cells through the conditional
expression of the HSV-TK gene [66]. Delivery of HSV-TK
with AAV followed by GCV treatment resulted in efficient
cell killing of HPV-positive cells.
CRAds represent another promising treatment alter-
native. In a recent study, Ad5-D24RGD demonstrated
effective oncolysis in cervical cancer cells [67]. Moreover,
therapeutic efficacy could be demonstrated in a mouse
model of cervical cancer with both intratumoral and
intravenous application. Importantly, no toxicity was
seen with human peripheral blood mononuclear cells.
Another interesting approach, which takes advantage of
similarities between gene products of DNA viruses, is
complementation of adenovirus mutants by HPV genes
[68].
An alternative approach to inhibiting the growth of
cervical cancer cells is based on the observation that
tumor suppressor p53 functions are downregulated in
most cervical cancer cells. The product of HPV oncogene
E6 binds to and inactivates p53 by promoting its
degradation. p73 is similar to p53 in structure and
function but not degraded by the HPV E6 gene product.
Das et al. demonstrated growth inhibition of E6-positive
cell lines in vitro following infection with Ad-p73 [69].
Endometrial carcinoma is the most common neoplasm
of the female reproductive tract and it accounts for nearly
one-half of all gynecologic malignancies. Although usu-
ally curable with surgery, sometimes aggressive tumors
such as uterine papillary serous carcinomas (UPSC) are
seen. Immunohistochemical studies suggest that p53 is
aberrant in 50–90% of UPSC tumors in comparison to 10–
30% in typical endometrioid adenocarcinomas [70].Ina
recent study, adenoviral delivery of p53 or p21 resulted in
growth suppression and induction of apoptosis in a UPSC
cell line [71].
Another interesting gene therapy approach for endo-
metrial cancer is based on the obs ervation that the
gonadotropin-releasing hormone receptor (GnRH-R) is
expressed by the majority of ovarian and endometrial
cancers. GnRH-R is a promising tumor-specific target due
to limited normal tissue expression. Grundker et al.
demonstrated the efficacy of HSV-TK/GCV controlled
by GnRH-R-specific elements in intraperitoneal and
subcutaneous mouse models of endometrial and ovarian
cancer [72].
GENE THERAPY FOR OTHER GYNECOLOGICAL
DISORDERS
Leiomyomas are benign, proliferating, estrogen-depend-
ent uterine tumors, which become clinically relevant
only when they enlarge enough to elicit symptoms such
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as abnormal bleeding [73].Further,theycancause
infertility and miscarriages. Current treatment is usually
hysterectomy or myomectomy. However, the disease is
localized to the uterus, which makes it an ideal target for
local gene therapy via ultrasound-guided injections,
laparoscopy, or hysteroscopy (Table 2). A plasmid-based
strategy with HSV-TK/GCV was assessed in vitro both in
human clinical samples and in a rat leiomyoma cell line.
A bystander effect was demonstrated, and interestingly, it
was increased with estradiol treatment [74]. In a murine
leiomyoma xenograft model adenovirus-mediated
expression of a dominant negative estrogen receptor
inhibited subcutaneous tumor growth and cell prolifer-
ation, while increased apoptosis was found [75].
Endometriosis, the growth of ectopic endo metrial
tissue, is an estrogen-dependent disease that causes pain
and infertility. Moreover, there is an association between
untreated endometriosis and development of ovarian
cancer. Typically, it is treated with surgical removal of
the lesions and medical therapy aiming at a hypoestro-
genic state [73]. An important feature of active endome-
triosisispronouncedvascularization,andtherefore
antiangiogenic gene therapy has been evaluated [76].In
a murine model, intraperitoneal delivery of an adenovi-
rus encoding the angiogenesis inhibitor angiostatin
caused a decrease in the number, size, and density of
blood vessels. More importantly, established endome-
triosis was eradicated in all treated mice within 18 days
[76]. Fortin et al. evaluated the efficacy of HSV-TK/GCV
for treatment of endometriosis. Human endometrial
fragments were inf ected ex vivo with an adenovirus
containing HSV-TK and injected subcutaneously into
nude mice. GCV treatment induced a significant regres-
sion in endometrial implants [77].
Placental disorders and dysfunction cause significant
fetal and maternal mo rbidity, including fetal growth
retardation, preeclampsia or eclampsia, and mortality.
Initially, there is defective development of the early
placenta and its maternal blood supply. The clinical
syndrome arises from subsequent generalized maternal
endothelial dysfunction [73]. Pathologically, a hypoxic
and dysfunctional placenta releases factors such as sFlt-1,
which binds VEGF and p lacental g rowth factor [78].
Increased understanding of these mechanisms facilitates
development of gene therapeutic strategies for treatment
of preeclampsia and prolonging the pregnancy. Senut et al.
delivered gene-modified placental cells to the rodent
placenta in vivo and demonstrated that gene products
were secreted throughout gestation without deleterious
effects [79]. Plasmid DNA and adenoviruses have been
guided with angiography to uterine arteries in rabbits for
transfection of trophoblast cells. Transfection efficiency
was as high as 34% with adenovirus, while plasmid
complexes led to much lower rates [80]. Insulin-like
growth factors (IGFs) I and II are critical in fetal growth
because of their role in placental development and
function, and reduced levels have been reported in intra-
uterine growth retardation. Adenoviruses encoding IGF-I
or IGF-II were utilized for in vitro genetransfer to fresh,
human primary placental fibroblasts. IGFs exerted both
autocrine and paracrine effects on cell proliferation,
migration, and survival [81].
Molecular defects have been implicated in embryo
implantation disorder, making it a possible target for
gene therapy. Homeobox (HOX) genes are transcription
factors necessary for embryonic development. Unlike in
most adult tissues, HOXA10 and HOXA11 expression
persists in the endometrium, and they are essential for
endometrial development and receptivity in response to
sex steroids. Interestingly, it has been shown that mice
with disruption of the HOXA10
gene are infertile because
of implantation failure [82]. More importantly, defects in
endometrial HOX gene expression in infertile women
have been demonstrated [82]. Thus, augmenting HOX
gene expression with gene therapy to improve implanta-
tion becomes attractive and has already been achieved
TABLE 2: Gene therapy approachesfor noncancer gynecological diseases
Construct Vehicle Rationale Study Reference
Leiomyomas
pNGVL1-tk Plasmid HSV-TK suicide gene therapy In vitro [74]
Ad-ER-DN Adenovirus Inhibition of estrogen receptor In vitro and in vivo [75]
Endometriosis
AdAngiostatin Adenovirus Antiangiogenic gene therapy In vitro and in vivo [76]
AdTK Adenovirus HSV-TK suicide gene therapy In vitro and in vivo [77]
Placental disorders
Ad-LacZ, LacZ plasmid Adenovirus, Liposome/plasmid Angiographically guided utero-placental
transfer of marker gene
In vivo [80]
Ad-IGF-I, Ad-IGF-II Adenovirus Delivery of IGF-1 or IGF-II In vitro [81]
Embryo implantation disorder
HOXA10 cDNA Liposome/plasmid Delivery of HOXA10 In vitro and in vivo [83]
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Copyright C The American Society of Gene Therapy
with intrauterine administration of HOXA10 plasmid/
liposome complex to mice [83].
In general, nonmalignant gynecologicaldiseases are
less severe and more treatable than gynecological cancers.
Therefore, clinical translation of gene therapy strategies
probably requires even more stringent safety information.
Moreover, given the immunogenic nature of adenovi-
ruses, other vectors such as lentiviruses and AAV may be
more attractive for this group of diseases.
FUTURE DIRECTIONS
Recent evidence su ggests that relatively conventional
gene therapy approaches, when applied following max-
imal cytoreduction, can increase the survival of cancer
patients [84]. Nevertheless, only a few pioneering studies
have managed to harness fully the power of correlative
analysis in phase I trials and these studies have implied
that traditional delivery systems usually result in insuffi-
cient genetransfer when faced with advanced tumor
masses [85]. To improve the quality and quantity of
correlative data in early phase trials, it is important to
increase our capacity for detection of the persistence and
magnitude of virus replication. Because obtaining serial
biopsies is difficult due to safety, cost, and compliance
issues, noninvasive strategies are most attractive. Some
promising approaches include functional imaging of
transgenes, incorporation of secretable marker proteins,
and detection of fluorescent proteins incorporated into
virus capsids [42,86,87].
Several strategies are currently being explored to
improve transduction of target cells and effective pene-
tration of solid tumors. For example, genetransfer by
viral vectors can be enhanced by using modified agents
that are retargeted to receptors highly expressed on
target cells [88]. Nonetheless, viral spread in the tumor
can be limited by physical barriers such as stromal cells
and matrix and necrotic, hypoxic, or hyperbaric regions.
For overcoming these obstacles, selectively oncolytic
viruses may be useful and targeting oncolytic viruses to
tumor cells is a logical sequel [52,61 ].Forfurther
potentiation, replication-competent v iruses can be
armed with therapeutic transgenes such as cytokines,
suicide genes, and fusogenic, proteolytic, or antiangio-
genic moieties [89].
A powerful approach for increasing efficacy is utiliza-
tion of genetransfer in combination with conventional
anticancer therapies in a multimodal antitumor approach
[90], which has recently been validated in randomized
trials [57,91,92]. Gene therapy differs from traditional
modalities with regard to mechanism and side effects,
providing a possibility for additive or synergistic inter-
actions [93,94].
The aforementioned intratumoral complexities hinder
also conventional antitumor approaches such as chemo-
therapy, and it is known that effective treatments usually
require multiple rounds of administration; solid tumors
can usually be reduced only layer by layer. Thus, clinical
gene transfer might benefit from readministration of
virus, whose efficacy may be inhibited by neutralizing
antibodies (NAb). Strategies for facilitating re-treatment
include alternating related viruses with different capsids
(sero-switch) [95], cotreatment with immunosuppressive
drugs for temporary abrogation of NAb induction [96],or
physical removal of NAbs by using immunopheresis or an
adenovirus capsid protein column [97].
Most importantly, it remains crucial to translate
preclinical advances quickly into clinical trials, because
only in patients can we find out which approaches work
and which do not. Comprehensive correlative analysis of
specimens obtained in these trials allows the translational
process to cycle rapidly back to the lab for development of
next generation agents. It may be that the biggest obstacle
cancer gene therapy faces is the continually increasing
difficulty in rapidly setting up phase I trials in an ever-
tightening regulatory environment. Other challenges
include improving gene delivery and potency to levels
compatible with clinical responses. Also, given the recent
success of monoclonal antibodies and small molecular
inhibitors as effective and relatively nontoxic antitumor
agents, gene therapy needs to deliver emphatic clinical
results to attract resources compatible with transforma-
tion of a promising approach to a clinically successful
strategy. Fortunately, recent watershed clinical trials
[57,84,92,98–100] have demonstrated that the theoretical
considerations behind gene delivery for therapeutic effect
are sound, and the technology remains a viable and potent
approach for treatment of diseases resistant to available
modalities.
A
CKNOWLEDGMENTS
This work was supported by HUCH Research Funds (EV O), the Academy of
Finland, the Emil Aaltonen Foundation, the Finnish Cancer Society, the
University of Helsinki, the Sigrid Juselius Foundation, the Sohlberg Foundation,
the Biocentrum Helsinki, the Instrumentarium Research Fund, the Finnish
Oncology Association, the Research and Science Foundation Farmos, and
Regional Funds of the Finnish Cultural Foundation.
RECEIVED FOR PUBLICATION AUGUST 31, 2005; REVISED DECEMBER 13,
2005; ACCEPTED FEBRUARY 6, 2006.
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REVIEW ARTICLE
doi:10.1016/j.ymthe.2006.02.019
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. developmental gene transfer applications for gynecological
diseases.
Key Words: gene transfer, gene therapy, ovarian cancer, cervical cancer, gynecological. rationalized gene
transfer as an approach for the treatment of diseases
resistant to more conventional therapies. Gene therapy
aims at transfer of genes for correction