Key Words: gene transfer, gene therapy, ovarian cancer, cervical cancer, gynecological disease Contents Introduction.. Targeting Vectors to Ovarian Cancer Cells The majority of gene ther
Trang 1Gene Transfer Approaches for Gynecological Diseases
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 gene transfer 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 gene transfer 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 for gene transfer include both nonviral
and viral vectors, such as adenovirus, retrovirus,
adeno-associated virus (AAV), and herpes simplex virus (HSV)
Nonviral gene transfer is most commonly 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] Most cases are diagnosed at an advanced state, and long-term survival of patients with metastatic disease is rare Although
Trang 2chemo-therapy approaches featuring taxanes and platinums, when
given following optimal cytoreductive surgery, can increase
the survival of patients, treatment of metastatic disease
eventually results in drug resistance and disseminated
disease cannot be cured Therefore, novel treatment
approaches are needed Gene therapy, even at its current
rather adolescent stage, is an attractive modality for
ovarian cancer as this cancer frequently presents with
metastases confined to the peritoneal cavity, creating a
rationale for locoregional delivery
Targeting Vectors to Ovarian Cancer Cells
The majority of gene therapy approaches for ovarian
cancer (Table 1) are based on adenovirus 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 gene transfer 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 clinpreclin-ical efficacy
One possible reason is that there might be a
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 domain of Ad5 with 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 even fatal immune reactions [11] Therefore, it has 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], ovarian-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 approaches for 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 gene transfer 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] Gene transfer 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 with this approach,
Trang 3trans-TABLE 1: Overview of gene therapy approaches for gynecological cancers
Ovarian cancer
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]
clinical trial
No dose-limiting toxicity, no responses [33]
liposome
Inhibition of erbB2 Phase I
clinical trial
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
(CRAd type 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]
mda-7/IL-24
In vitro Inhibition of ovarian cancer cell growth,
induction of apoptosis, targeting to CD40
or EGFR
[102,103]
truncated EGFR (erbB1)
In vivo Inhibition of cancer cell growth, enhanced
sensitivity to cisplatin
[104]
gene therapy
In vitro Gene transfer to ovarian cancer cells [105]
gene therapy
In vivo Inhibition of ovarian cancer cell growth,
inhibition of angiogenesis
[48]
Ad5-D24RGD Adenovirus
(CRAd type I)
in vivo
Killing of ovarian cancer cells, infectivity enhancement
[52–54]
(CRAd type I)
in vivo
Killing of ovarian cancer cells, infectivity enhancement
[61]
Dearing reovirus
serotype 3
in vivo
Inhibition of ovarian cancer cell growth [106]
in vivo
Killing of ovarian cancer cells, expression of soluble marker peptide
[107]
MV-CEA
MV-Moraten
MuV-JL
Measles and mumps viruses
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]
gene therapy
In vitro Cell killing of HPV-positive cells [66]
Ad5-D24RGD Adenovirus
(CRAd type I)
in vivo
Inhibition of cervical cancer cell growth [67]
Ad-MN/Ca9-E1a Adenovirus
(CRAd type II)
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]
gene therapy
In vitro and
in vivo
Inhibition of endometrial cancer cell growth [72]
Trang 4duction 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 evaluated 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 Following 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 intraperitoneal AdHSV-TK with intravenous acyclovir and topotecan [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 gene transfer 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 intraperitoneal injection of an AAV expressing sFlt-1
[47] Also other antiangiogenic genes such as mutant
Teratocarcinoma
Ad5-flt-1luc Adenovirus Transcriptional
targeting via flt-1 promoter
In vitro High transgene expression in teratocarcinoma
cells
[112]
TABLE 1 (continued)
Trang 5endostatin 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 reduction in microvessel
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 replicatransduc-tion-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 inhibisequestra-tion of
neovascularization (D) Virotherapy Viral infection
of cancer cells results in replication, oncolysis, and
release of virions to surrounding cells.
Trang 6To improve tumor penetration, various naturally
occurring, inherently tumor-selective or engineered
oncolytic viruses have been utilized, including
adeno-virus, HSV, Newcastle disease adeno-virus, vaccinia, reoadeno-virus,
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
1011viral 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, cervgynecolog-ical 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] In a 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 observation 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
Trang 7as abnormal bleeding [73] Further, they can cause
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 endometrial
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-triosis is pronounced vascularization, and therefore
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 infected 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 morbidity, 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 placental growth 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 gene transfer 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 approaches for noncancer gynecological diseases
Leiomyomas
Endometriosis
Placental disorders
Ad-LacZ, LacZ plasmid Adenovirus, Liposome/plasmid Angiographically guided utero-placental
transfer of marker gene
Embryo implantation disorder
Trang 8with intrauterine administration of HOXA10 plasmid/
liposome complex to mice[83]
In general, nonmalignant gynecological diseases 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 suggests 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 gene transfer 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, gene transfer 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] For further
potentiation, replication-competent viruses 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 gene transfer in combinautiliza-tion with convenutiliza-tional
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
ACKNOWLEDGMENTS This work was supported by HUCH Research Funds (EVO), 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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