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Gene Therapy for Ovarian Cancer
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Gene Therapy for Ovarian Cancer
Review Article [1] | September 01, 2001 | Ovarian Cancer [2]
By George Coukos, MD, PhD [3] and Stephen C. Rubin, MD [4]
Advances in molecular virology and biotechnology have led to the engineering of vectors that can
efficiently transfer genes to target cells. Gene therapy strategies were developed along two lines:
Cytotoxic approaches
he recent advances in cell and molecular biology of cancer have elucidated some of the
mechanisms underlying malignant transformation, tumor cell migration, metastasis, angiogenesis,
and cell response to chemotherapy. Advances in molecular virology and biotechnology have allowed
for the engineering of vectors that can efficiently transfer genes to target cells. As a result, gene
therapy strategies for the treatment of cancer were developed along two fundamental directions.
Cytotoxic or suicide gene therapy approaches entail the transfer of genes that encode enzymes able
to convert inactive prodrugs into cytotoxic drugs. Only transfected cancer cells expressing the
specific enzyme become susceptible to killing. Corrective gene therapy strategies aim at repairing
specific molecular alterations occurring in signal transduction mechanisms that control the cell cycle
or induce apoptosis.
T
Gene Therapy Vectors
The Recombinant DNA Advisory Committee has approved hundreds of gene therapy protocols for the
treatment of genetic diseases or cancer. Although gene therapy strategies have produced highly
promising results in preclinical in vitro and animal models, their application in clinical practice has
proven to be much more difficult. A fundamental problem concerns the efficiency of gene transfer.
Viral vectors remain the vectors of choice, mainly because of their high efficiency of transfection.
After all, viruses have evolved successful strategies for introducing their genome into eukaryotic
cells and using the host cell biochemical machinery.
The vast majority of phase I trials employ viral vectors, but some use DNA protein complexes, DNA
particles, ribozymes, or lipid-based vehicles. To minimize side effects, viral vectors are rendered
replication-incompetent. To date, several vectors have been developed that are associated with
significant limitations related to low efficacy, inability to penetrate deeply in tumor nodules,
inactivation by the immune system, and undesired side effects (Table 1). To enhance efficacy,
inserted genes (transgenes) are positioned under the control of a strong exogenous promoter. A
reporter gene, such as the Escherichia coli LacZ gene encoding beta-galactosidase, is also inserted
to assess the efficacy of gene transduction.
Adenovirus
Adenoviral vectors are currently the vectors of choice in gene therapy, including cancer gene
therapy.[1] Adenoviral vectors are remarkably efficient, but yield only transient expression of
therapeutic genes and are generally administered repeatedly to lengthen the duration of gene
expression. Adenoviruses infect dividing and nondividing cells, and can be produced relatively easily
on a large scale. They are stable, may be manufactured without contamination by
replication-competent adenovirus (RCA),[2] and can accommodate up to 7.5-kb transcripts.
Backbones have been derived from Ad2 and Ad5 serotypes, both of which belong to the wild-type-C
adenovirus subgroup.
The vitronectin receptor alpha(v) beta(3) integrin and the coxsackie/adenovirus receptor mediate
virus entry into human cells through chlathrin-coated vesicles.[3] The viral genome remains
extrachromosomal in the nucleus. Recombinant adenovirus is produced on appropriate packaging
cell lines complementing the missing genes. The early genes—specifically, E1—were first targeted
because they control viral replication and regulate the expression of late genes. Substitution of E1A
and E1B genes by a designated transgene led to the creation of the first generation of adenoviral
vectors.
Due to the high likelihood of recombination events occurring during manufacturing in vitro,
engineering of strains lacking only one gene were characterized by a prohibitive degree of
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replication-competent adenovirus (RCA) contamination. This translated to high toxicity, particularly
hepatotoxicity, considering the elevated liver tropism of the wild-type virus. A second-generation
virus was then produced by adding an additional mutation in the E2A or E3 regions.[4] This improved
the toxicity profile of the vectors by dramatically decreasing RCA contamination.
Administration of adenovirus is followed by an intense inflammatory and immune response. An early
innate response consists of the release of inflammatory cytokines such as interferon (IFN)-gamma,
interleukin (IL)-1, and IL-6 as well as the recruitment of an acute inflammatory infiltrate followed by a
specific neutralizing antibody and T-cell response.[5] Both the transgene and viral genes expressed
by transfected cells are cross-presented on major histocompatibility complex (MHC) class I or II sites,
triggering specific CD8- and CD4-positive T cells. An intense inflammatory reaction in proximity to
the tumor may enhance tumor immune recognition. However, immune-mediated vector
neutralization may pose marked limitations on the transduction efficacy of the vector.
We and others have recently demonstrated the presence of neutralizing antibodies in the serum and
peritoneal fluid of patients with epithelial ovarian cancer that significantly decrease the efficacy of
adenoviral vectors in vitro. However, the presence of antiadenoviral antibody titers did not appear to
compromise the efficiency of gene transfer in a phase I trial of adenoviral-based gene therapy for
pleural mesothelioma conducted at our institution.[6] Nevertheless, there is still considerable
concern about the limitations posed by the immune system, particularly if repeated administrations
of vectors are planned. A third-generation adenoviral vector with somewhat decreased
immunogenicity was recently produced by the deletion of the E4 region (in addition to E1 region)
and preservation of the E3 region. The protein product of E3 inhibits MHC I transport to the cell
surface, thereby preventing immune recognition of adenovirus-infected cells.
To address the concerns of RCA contamination and immune-mediated response, "gutted" or
"gutless" adenovirus vectors that contain no viral genes have been generated.[7] These vectors
accept up to 38 kb of foreign DNA. Gutless adenovirus vectors are replication-deficient and require
helper viruses for propagation. One advantage of gutless vectors is that they are less immunogenic
and, by using helper viruses derived from different adenovirus serotypes, production of host
antibodies can be directed away from the therapeutic gutless vector. Gutless adenovirus vectors are
showing promise in gene therapy. In mice, sustained expression of transgene was seen after a single
injection, with no serious virus-associated toxicity.[8]
Epithelial ovarian cancer cells are susceptible to infection by adenoviral vectors.[9] We recently
demonstrated that an E1/E4-deleted adenovirus was similarly efficacious to an E1/E3-deleted one in
several epithelial ovarian cancer cell lines (unpublished observation). Current investigation is
focusing on improving the tissue selectivity of adenoviral vectors. Crosslinking of adenoviral particles
to basic fibroblast growth factor has proven to significantly increase the binding of adenovirus on
epithelial ovarian cancer cells (SKOV3.ip), resulting in a 10-fold enhancement of its efficacy in
vitro.[10] Furthermore, the incorporation of an Arg-Gly-Asp (RGD)-containing peptide to the HI loop
of the adenovirus fiber knob through genetic engineering directs the virus away from
coxsackie/adenovirus receptor binding sites and enhances its binding to the ovarian cancer cell
surface.[11]
Adeno-Associated Virus
Adeno-associated virus has a 4.7-kb single-stranded DNA genome surrounded by a protein coat.
Adeno-associated virus is not autonomous; in the absence of a helper virus, adeno-associated virus
enters a latent state of infection, while coinfection by an adenovirus or herpesvirus enables
adeno-associated virus to replicate.[12] Its genome has at least six transcripts with three internal
promoters and two inverted terminal repeats. Construction of recombinant adeno-associated virus
vectors can be accomplished by stripping off the gene sequences encoding viral structural proteins
and generating a backbone with the two inverted-terminal repeats surrounding the inserted
transgene. A packaging cell line is necessary for viral replication and production.
Wild-type virus integrates at a specific site on human chromosome 19. Adeno-associated virus
vectors also integrate into host DNA for sustained gene expression. Adeno-associated virus proteins
are not toxic to cells. Although 80% of the population is seropositive, adeno-associated virus is not
associated with any known disease in humans, and an existing immune response does not impair the
efficiency of transfection. Moreover, adeno-associated virus infection is not accompanied by
inflammation or generation of a strong recall immune response.[13]
Adeno-associated virus shows promise in the setting of gene therapy because it infects a wide
variety of cells. Its ability to infect both dividing and nondividing cells may be a major advantage in
targeting tumors with low S phase. Sustained expression from adeno-associated virus vectors has
been observed in several tissue types. Epithelial ovarian cancer cells harvested directly from
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patients are susceptible to infection by adeno-associated virus, making adeno-associated virus a
promising vector for further testing in cancer gene and immune gene therapy of this disease in
particular.
Herpes Simplex Virus
Herpes simplex virus (HSV) is an enveloped double-stranded DNA virus of approximately 150 kb
capable of infecting a great variety of human tissues. The HSV enters the cells through at least three
identified receptors. Manipulation of the HSV genome has generated a great variety of vectors,
including replication-incompetent virus vectors and amplicons.[14] Amplicon vectors are derived
from plasmids that carry HSV genes, including the HSV origin of DNA replication and the packaging
signals, together with bacterial genes. Amplicon vectors require the presence of a helper virus to
carry out transgene expression. Recombinant vectors contain full-length HSV genomes, in which
various viral genes have been deleted and substituted by transgenes.
Different strategies have been followed for the generation of replication-incompetent HSV vectors,
but they invariably affect key genes controlling viral replication, such as the ribonucleotide
reductase, thymidine kinase (tk), the UL5, or the ICP34.5.[15] In addition, because the HSV genome
contains at least 30 nonessential genes (ie, not essential for viral propagation in culture), multiple
genes may be deleted without compromising vector production. Herpes simplex virus vectors,
therefore, offer unique opportunities for multiple transgene insertion.
Ovarian cancer cells are particularly susceptible to infection by HSV vectors.[16,17] The advantages
of HSV vectors include the feasibility of large-scale production, versatility of the vector system, the
ability to carry large or multiple transgenes, and the ability to induce direct toxicity even in the
absence of replication. The application of HSV vectors may be hindered by preexisting immunity
against HSV-1, which may be highly prevalent in the adult population. It is possible that
HSV-neutralizing antibodies present in the serum or peritoneal fluid may decrease the efficacy of
HSV-mediated gene therapy.
Retroviruses
Retroviruses are diploid positive-strand RNA viruses that replicate in the host through an
intermediate step of DNA reverse transcription. The DNA transcribed on the viral RNA template is
subsequently integrated into the host genome in a pseudorandom fashion, and the host nuclear
machinery is used to produce RNA copies of the virus.[18] The murine leukemia virus was first used
to construct vectors, but lentiviruses such as recombinant human immunodeficiency virus (HIV)
strains are also currently being tested. Because of viral genomic integration, this strategy offers the
advantage of long-term gene expression. Almost 20 years of experience with retroviral vectors has
confirmed their feasibility and safety of administration.
The major concern about retroviral vectors relates to insertional muta genesis.[19] The
pseudorandom insertion of genes into the host genome might disrupt a tumor-suppressor gene or
modify (amplify) a growth-promoting gene, resulting in tumorigenesis (as exemplified by the action
of the murine leukemia virus in mice). A second potential concern is related to incidental insertion of
transgenes into the germ line. Although this phenomenon has been described in mice, no further
experiments in animals have substantiated this concern.
A potentially attractive characteristic of retroviruses is related to the lack of immune response
following their administration, as retroviruses result in the production of a single transgene but no
significant viral proteins. In addition, since some of these viruses are of murine extraction, no
preexisting immunity has been encountered in humans. This would facilitate repeated administration
of the virus without loss of its potency.
Liposomes
Direct gene delivery of plasmid DNA by relatively inert vehicles may represent a feasible alternative
to the use of viral vectors. Liposomes are amphipathic lipids containing a hydrophobic domain and a
hydrophilic domain composed of hydrocarbon chains such as fatty acids. Anionic liposomes do not
bind DNA directly and offer limited packaging ability for gene therapy. Cationic lipids are mixed with
a fusogenic lipid (such as dioleylphosphatidylethanolamine, or DOPE) to form liposomes that are
positively charged and bind DNA with great affinity.[20] Nonspecific ionic interactions often facilitate
liposome binding to the cell surface; the complexes cross the cell membrane possibly through a
fluid-phase endocytosis mechanism, but fusion mechanisms may also be important for some
compounds.
Liposomes are not associated with the toxicity of viral vectors and do not generate as potent an
immune response. They are easy to produce on a large scale and offer the advantage of carrying
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transgenes up to 50 kb in size. However, their transduction efficiency is significantly lower than viral
vectors, and expression of liposome-delivered transgenes is transient.
Cationic liposomes have proven efficient in delivering transgenes to a large variety of malignant cells
in vitro. In vivo, liposomes have been used intraperitoneally and intravenously and have proven to
be safe. Intratumoral injections have also been described. The uptake of liposomes by epithelial
ovarian cancer cells is efficient,[21] thus supporting their use in intraperitoneal or systemic gene
therapy for that disease. One potential problem with cationic liposomes is that they are avidly taken
up by the reticuloendothelial cell system.[22] Stealth technologies may need to be implemented to
increase the tumor selectivity of these agents. Moreover, significant work still needs to be done in
order to improve the transfer efficiency and tumor selectivity of liposomes.
Cancer Gene Therapy Strategies
Cytotoxic Gene Therapy
Cytotoxic, or suicide, gene therapy entails the introduction into tumor cells of a specific gene that
encodes an enzyme capable of converting a prodrug into a highly toxic drug. This conversion only
takes place within the cells that express the transgene. The most frequently used mechanism
involves the tk of HSV, which allows for the phosphorylation of ganciclovir into ganciclovir
monophosphate in cells expressing the transgene.[23] This product is further phosphorylated twice
into ganciclovir triphosphate by mammalian kinases, to become a potent inhibitor of DNA and RNA
synthesis. Multiple other systems of enzymes and prodrugs with different advantages and limitations
have been tested.
Incorporating ganciclovir triphosphate into DNA inhibits DNA polymerase and ultimately leads to DNA
fragmentation followed by apoptosis. Ganciclovir induces cell death in a cell-cycle-dependent
manner because incorporation of the triphosphate metabolite into DNA requires the cell to enter S
phase. This provides a mechanism for tumor selectivity. Great enthusiasm was generated by studies
indicating the existence of "bystander" amplification mechanisms that cause cell death in
nontransfected nearby cells.[24] Initial studies, in fact, indicated that it was enough to transfect 5%
to 15% of the cells in vitro in order to achieve 100% killing with the HSV-tk/ganciclovir system.
Diffusion of ganciclovir triphosphate toxic metabolite occurs through gap junctions into neighboring
cells.[25] Cytotoxic cytokines released from dying cells may also induce apoptosis in neighboring
cells. Moreover, the Fas/Fas ligand (FasL) system may be involved in apoptosis induced by HSV-tk,
because Fas, FasL, and two downstream caspases were found to be increased in
tk/ganciclovir-treated tumor cells undergoing cell cycle arrest and apoptosis.[26] HSV-tk cytotoxic
therapy may result in the generation of an antitumor immune response (Table 2), which could
amplify the effect of cytotoxic gene therapy. In fact, tk/ganciclovir cytotoxic therapy has proven
more efficacious in immunocompetent than in immunodeficient mice.[27]
The immunogenicity of tumors treated with HSV-tk and ganciclovir appears to be related to the
modality of cell death and up-regulation of heat-shock proteins. A specific CD4- and CD8-positive
T-cell infiltrate was seen in tumors undergoing necrosis following tk/ganciclovir treatment.[28] The
cytokine response suggested a T-helper cell type 1 (TH1) lymphocyte response, including increased
expression of IL-2, IL-12, IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), and
granulocyte-macrophage colony-stimulating factor (GM-CSF). Furthermore, stimulatory molecules
such as B7 and ICAM were up-regulated together with MHC class I molecules in treated cells. Other
suicide gene therapy systems have been tested experimentally (Table 3). Many of them use similar
bystander effects and generate antitumor immune responses.
Ovarian Cancer Studies—Ovarian cancer cells are sensitive to the tk/ganciclovir cytotoxic system
delivered through adenoviral or retroviral vectors as well as liposomes.[29] Multiple studies in vitro
and in vivo in the immunodeficient mouse model have documented the ability of the tk/ganciclovir
system to induce cytotoxicity, reduce tumor burden, and increase survival. We demonstrated that
epithelial ovarian cancer cell lines SKOV3, CaOV3, OVCAR3, and A2780 are susceptible to killing by
tk/ganciclovir in a dose-dependent manner. A single intraperitoneal dose of 1 million × 10³ particles
of an E1/E3-deleted adenoviral vector carrying tk under a Rous sarcoma virus (RSV) promoter
resulted in a significant reduction of tumor burden. This effect was even more pronounced after
repeated intraperitoneal administrations (personal observations).
The use of HSV-tk/ganciclovir to enhance the antitumor immune response in ovarian cancer has
been explored by Scott Freeman.[30] This author engineered the PA-1 human ovarian
teratocarcinoma cell line to express HSV-tk through retroviral transfection. Intraperitoneal
inoculation of these cells in mice followed by ganciclovir treatment led to regression of established
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intraperitoneal adenocarcinoma. Tumor response was probably mediated by bystander and/or
immune mechanisms, as established tumors are not directly transduced with HSV-tk. It is possible
that inflammation in proximity to tumor may have contributed to unmasking of tumor antigens and
the generation of the immune response. In fact, histologic analysis of tumors revealed that PA-1 cells
were adherent on tumor nodules, and an intense inflammatory reaction had developed within tumors
where PA-1 cells had adhered.
Based on promising preclinical studies, clinical trials using HSV-tk/ganciclovir cytotoxic gene therapy
have been initiated in patients with advanced or recurrent epithelial ovarian cancer.[31]
Dose-escalation studies using intraperitoneal administration of an adenoviral vector carrying HSV-tk
have indicated that this strategy is safe, but no significant tumor responses have been seen thus far.
Further studies with larger volumes of patients are required to assess the therapeutic efficacy of this
strategy.
Corrective Gene Therapy
Corrective gene therapy strategies are being tested clinically in ovarian cancer (Table 4). These
therapies may be divided in two main groups: replacement therapies and neutralization therapies. In
tumors with documented loss of a gene function, wild-type genes are delivered to achieve normal
levels of expression or overexpression of the wild-type gene within tumor cells, resulting in apoptosis
or cell-cycle arrest. Examples of this process include the administration of p16, p21, p53, and BRCA1
genes.[32] In tumors with documented overexpression of oncogenes, ablative gene therapies may
be carried out to neutralize oncogene function.
BRCA1/2—The molecular aspects of hereditary ovarian cancer have been partially elucidated with
the identification and cloning of BRCA1 and BRCA2 genes, which appear to be mutated in some
patients with hereditary ovarian/breast cancer. BRCA1 appears to be involved in tumor-suppressor
pathways. The observation that BRCA1 overexpression blocks cell-cycle progression via p21 and
causes tumor-growth inhibition mediated by the retinoblastoma gene (even in cells without BRCA1
mutation) led to preclinical gene therapy studies with a splice variant of wild-type BRCA1 gene
(BRCA1sv) delivered via a retroviral vector.[33] In vitro and animal studies indicated that
overexpression of BRCA1 induced arrest of tumor growth in epithelial ovarian cancer.
A phase I clinical trial was conducted at Vanderbilt University Cancer Center in patients with sporadic
ovarian cancer, none of whom harbored germ-line BRCA1 mutations.[34] Patients received one to
three intraperitoneal injections of retroviral vector carrying the BRCA1sv gene at a dose of 108 to
1010 vector particles. Toxicity included self-limiting clinical peritonitis, which was observed in 25% of
patients. An antiretroviral antibody response was seen with higher doses, and gene transfer was
documented by Southern blot in approximately 5% to 10% of tumor cells in laparoscopic biopsy
material. Of 12 initially reported patients, 4 progressed, 7 displayed stable disease, and 1 achieved a
partial response. Notably, the authors saw no correlation between antibody response and vector
inactivation. However, they did observe a trend for higher gene expression in patients with low total
complement activity (CH50), although there was no statistical correlation.
p53—Gene therapy with tumor-suppressor genes and genes involved in cell-cycle control have also
been tested in epithelial ovarian cancer and other solid malignancies. Approximately 50% to 75% of
epithelial ovarian cancer specimens harbor alterations in the p53 tumor-suppressor gene. Loss of
p53 may be a late event in ovarian carcinogenesis but may contribute to chemotherapy resistance
and represents an independent prognostic factor for a poor outcome.[32] In vitro and in vivo
experiments showed that Bax (a transcriptional target of p53) and p16 (a cyclin D inhibitor involved
in regulation of the cell cycle) might be appropriate targets for corrective gene therapy. However,
most experience has been with the p53 tumor-suppressor gene.
Preclinical evidence suggests that p53 corrective gene therapy via adenoviral or retroviral vectors or
liposome-plasmid DNA complexes results in an increased amount of apoptosis or cell-cycle arrest in
epithelial ovarian cancer cells in vitro, while tumor regression was seen in vivo in mouse models.[29]
Epithelial ovarian cancer cells transfected with wild-type p53 gene become more sensitive to
DNA-damaging agents such as platinum.[35] Adenoviral p53 gene therapy was recently initiated in
patients with epithelial ovarian cancer. Intraperitoneal inoculation with p53 was reported in 37
women at a single dose of 7.5 × 1010 to 7.5 × 1013 particle-forming units. No significant
complications occurred, and a reduction in CA-125 levels was seen in more than half of the
patients.[36]
HER2/neu—HER2/neu (c-erbB2) is an oncogene encoding a 185-kd epidermal growth factor
receptor-related protein with tyrosine kinase activity overexpressed in 30% of epithelial ovarian
cancers and other solid tumors.[32] HER2/neu overexpression is correlated with malignant
transformation and metastasis. Deshane et al engineered a gene encoding a monoclonal
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single-chain antibody (sFv)—expressed in an intracytoplasmic location—against HER2/neu.[37]
Measurable levels of intracytoplasmic antibody were obtained following transfection of epithelial
ovarian cancer cells with the gene. This was associated with decreased cell surface expression of
HER2/neu, most probably due to entrapment of the newly synthesized thymidine kinase receptor
within the endoplasmic reticulum. Preclinical data in mice showed regression of intraperitoneal
tumors and led to the initiation of a phase I trial at the University of Alabama.[38]
Adenoviral E1A gene product specifically suppresses the HER2/neu promoter and functions as a
tumor-suppressor gene in tumors overexpressing HER2/neu.[39] Stable transfection of SKOV3.ip1
cells with the E1A gene led to a dramatic reduction of malignant phenotype in vitro and in vivo. A
replication-incompetent adenovirus lacking E1B and E3 but with intact E1A gene (Ad.E1A-positive),
administered intraperitoneally to immunosuppressed mice bearing SKOV3.ip1 epithelial ovarian
cancer tumors, resulted in suppression of HER2/neu expression and prolonged survival.[40]
A phase I clinical trial of adenoviral E1A delivered through liposomes was initiated at the M. D.
Anderson Cancer Center in patients with metastatic breast or ovarian cancer with documented
overexpression of HER2/neu. Cationic liposomes carrying E1A were delivered weekly starting at an
intraperitoneal dose of 1.8 mg/m². The study reached a maximal tolerated dose of 3.6 mg/m², and
several patients achieved disease stabilization. Down-regulation of HER2/neu was observed in two
patients, while E1A gene expression was documented in tumors and normal organs such as the
kidney, lungs, and liver.[32]
Oncolytic Agents
The direct treatment of tumors with replication-competent viruses is not a novel idea. A number of
studies undertaken in the 1950s and 1960s with direct intratumoral injections of wild-type viruses
had limited success. The viral approaches were abandoned, however, because of the inability to
attenuate viruses. With the advent of molecular biology and recombinant technologies, mutant
viruses have been generated, renewing interest in virus-based tumor therapies.
HSV-1 Mutants
Replication-restricted HSV-1 mutants have been generated in several laboratories by altering genes
that control viral replication, such as thymidine kinase (UL23) or the ICP6 gene (UL39) encoding the
large subunit of HSV ribonucleotide reductase.[15,41] HSV oncolytic agents have also been
generated by altering both copies of the RL1 gene. Its product, the ICP34.5 protein, is critical for
neurovirulence and plays an important role in viral replication, viral exit from infected cells, and
prevention of the premature shutoff of protein synthesis in the infected host.[15]
Initially designed for the treatment of central nervous system tumors, recombinant
replication-competent HSV-1 mutants are emerging as potent oncolytic agents. Clinical trials are
currently investigating intracerebral tumor administration of such ICP34.5-negative mutants for the
treatment of malignant gliomas.[42] However, a continuously expanding list of tumors are proving
sensitive to HSV oncolysis, including ovarian cancer, breast cancer, prostate cancer, mesothelioma,
malignant melanoma, metastatic colon carcinoma, and head and neck squamous carcinomas.[15] In
animal studies, we could not document any spread of the virus beyond the intraperitoneal tumors by
immunohistochemistry or polymerase chain reaction following intraperitoneal administration of the
virus, and no toxicity was seen.[17,43] A growing bulk of evidence suggests that HSV-based
oncolytic therapy may represent a safe tool for the treatment of solid tumors (Table 5).
Epithelial ovarian cancer cells are highly susceptible to infection and killing by recombinant
HSV-1.[17,43] Notably, primary cultures were significantly more susceptible to HSV killing than
established lines. Furthermore, HSV-1 lacking ICP34.5 is equally efficient in chemotherapy-sensitive
and -resistant epithelial ovarian cancer cells in vitro and in vivo.[16] Epithelial ovarian cancer cells
were found to undergo varying degrees of apoptosis, depending on the cell line. Apoptosis was not
dependent on the p53 status of the cells, which may partially account for the fact that chemotherapy
resistance does not affect the sensitivity of epithelial ovarian cancer cells to HSV oncolysis.
We further investigated the tumor specificity of HSV-G207, a doubly deleted HSV-1 lacking ICP34.5
and ribonucleotide reductase that regulates viral proliferation. HSV-G207 induced a dose-dependent
lysis in epithelial ovarian cancer cells in vitro but spared normal human mesothelial cells.[17]
Oncolytic HSV-1 appears promising and warrants testing in epithelial ovarian cancer.
ONYX-15
Another oncolytic agent of potential interest is the E1B-defective adenovirus ONYX-015.[44]
Adenoviral E1B gene encodes a 55-kd protein that inactivates the p53 tumor-suppressor gene. The
initial speculation that p53 inhibits adenoviral replication prompted the engineering of a recombinant
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adenovirus lacking E1B. It was anticipated that this virus would replicate in p53-deficient tumor cells
but not in cells possessing wild-type p53, and great enthusiasm was generated by the observation
that this was, in fact, true. Intratumoral injection of ONYX-015 into human cervical carcinoma (where
p53 is rapidly degraded due to the presence of human papillomavirus E6 protein product) grown in
nude mice led to complete regression in 60% of the tumors.[44]
Further reports confirmed that intratumoral or intravenous administration of ONYX-015 to nude mice
bearing human xenografts from a variety of tumors had antitumoral efficacy and that the virus was
not toxic to normal human cells. Moreover, a synergism with platinum-based chemotherapy was
seen. However, conflicting results were obtained by different authors who reported that intact p53
function and p53-dependent apoptosis were necessary for adenoviral replication.[45]
Clinical trials of ONYX-015 in head and neck cancer and lung cancer are ongoing. In spite of the
controversy surrounding the mechanism of action and tumor selectivity of ONYX-015, preliminary
results from a phase II study of ONYX-015 plus fluorouracil/cisplatin chemotherapy in 30 head and
neck cancer patients indicated extremely promising results. There were substantial and persistent
objective responses, including a high proportion of complete responses.[46] A randomized phase III
trial currently under preparation in head and neck cancer will further clarify the efficacy of
ONYX-015. Because epithelial ovarian cancer patients often display mutations in p53, ONYX-015 may
be useful in the treatment of recurrent/persistent epithelial ovarian cancer.
Conclusions
Gene and oncolytic therapy of ovarian cancer is an intense area of investigation. Significant focus is
being placed on the development of vectors that yield improved gene transfer with low toxicity. As
our understanding of tumor cell biology, tumor immunology, and tumor angiogenesis increases, it is
likely that new molecular targets will be incorporated into cancer gene therapy strategies in an
attempt to enhance tumor cell death, generate potent antitumor immune responses, and suppress
angiogenesis. The existing clinical evidence suggests that these techniques are probably best suited
for patients with a minimal volume of residual disease. It is likely that multimodality approaches with
conventional strategies (eg, cytoreductive surgery, chemotherapy, radiation therapy) and novel
therapeutic tools including oncolytic, gene, angiostatic, and immune therapy, in various
combinations will prove advantageous, compared to single-modality treatments. Multicenter,
prospective double-blinded studies need to be designed to test these hypotheses.
References:
1. Ragot T, Opolon P, Perricaudet M: Adenoviral gene delivery. Methods Cell Biol 52:229-260, 1997.
2. Gao GP, Yang Y, Wilson JM: Biology of adenovirus vectors with E1 and E4 deletions for
liver-directed gene therapy. J Virol 70:8934-8943, 1996.
3. McDonald D, Stockwin L, Matzow T, et al: Coxsackie/adenovirus receptor (CAR)-dependent and
major histocompatibility complex (MHC) class I-independent uptake of recombinant adenoviruses
into human tumour cells (see comments). Gene Ther 6:1512-1519, 1999.
4. Bett AJ, Haddara W, Prevec L, et al: An efficient and flexible system for construction of adenovirus
vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci U S A 91:8802-8806,
1994.
5. Dai Y, Schwarz EM, Gu D, et al: Cellular and humoral immune responses to adenoviral vectors
containing factor IX gene: Tolerization of factor IX and vector antigens allows for long-term
expression. Proc Natl Acad Sci U S A 92:1401-1405, 1995.
6. Molnar-Kimber KL, Sterman DH, Chang M, et al: Impact of preexisting and induced humoral and
cellular immune responses in an adenovirus-based gene therapy phase I clinical trial for localized
mesothelioma. Hum Gene Ther 9:2121-2133, 1998.
7. Hammerschmidt DE: Development of a gutless vector. J Lab Clin Med 134:C3, 1999.
8. Balague C, Zhou J, Dai Y, et al: Sustained high-level expression of full-length human factor VIII and
Page 7 of 10
Gene Therapy for Ovarian Cancer
Published on Cancer Network (http://www.cancernetwork.com)
restoration of clotting activity in hemophilic mice using a minimal adenovirus vector. Blood
95:820-828, 2000.
9. Behbakht K, Benjamin I, Chiu HC, et al: Adenovirus-mediated gene therapy of ovarian cancer in a
mouse model. Am J Obstet Gynecol 175:1260-1265, 1996.
10. Rancourt C, Rogers BE, Sosnowski BA, et al: Basic fibroblast growth factor enhancement of
adenovirus-mediated delivery of the herpes simplex virus thymidine kinase gene results in
augmented therapeutic benefit in a murine model of ovarian cancer. Clin Cancer Res 4:2455-2461,
1998.
11. Vanderkwaak TJ, Wang M, Gomez-Navarro J, et al: An advanced generation of adenoviral vectors
selectively enhances gene transfer for ovarian cancer gene therapy approaches. Gynecol Oncol
74:227-234, 1999.
12. Lebkowski JS, McNally MM, Okarma TB, et al: Adeno-associated virus: A vector system for
efficient introduction and integration of DNA into a variety of mammalian cell types. Mol Cell Biol
8:3988-3996, 1988.
13. Fisher KJ, Jooss K, Alston J, et al: Recombinant adeno-associated virus for muscle directed gene
therapy. Nat Med 3:306-312, 1997.
14. Glorioso J, Bender MA, Fink D, et al: Herpes simplex virus vectors. Mol Cell Biol Hum Dis Ser
5:33-63, 1995.
15. Coukos G, Rubin SC, Molnar-Kimber KL: Application of recombinant herpes simplex virus-1
(HSV-1) for the treatment of malignancies outside the central nervous system. Gene Ther Mol Biol
3:78-89, 1999.
16. Coukos G, Makrigiannakis A, Kang EH, et al: Oncolytic herpes simplex virus-1 lacking ICP34.5
induces p53-independent death and is efficacious against chemotherapy-resistant ovarian cancer.
Clin Cancer Res 6:3342-3353, 2000.
17. Coukos G, Makrigiannakis A, Montas S, et al: Multi-attenuated herpes simplex virus-1 mutant
G207 exerts cytotoxicity against epithelial ovarian cancer but not normal mesothelium and is
suitable for intraperitoneal oncolytic therapy. Cancer Gene Ther 7:275-283, 2000.
18. Karavanas G, Marin M, Salmons B, et al: Cell targeting by murine retroviral vectors. Crit Rev
Oncol Hematol 28:7-30, 1998.
19. Boris-Lawrie K, Temin HM: The retroviral vector. Replication cycle and safety considerations for
retrovirus-mediated gene therapy. Ann N Y Acad Sci 716:59-70 (incl discussion 71), 1994.
20. Gao X, Huang L: Cationic liposome-mediated gene transfer. Gene Ther 2:710-722, 1995.
21. Xing X, Zhang S, Chang JY, et al: Safety study and characterization of E1A-liposome complex
gene-delivery protocol in an ovarian cancer model. Gene Ther 5:1538-1544, 1998.
22. Qi XR, Maitani Y, Nagai T: Rates of systemic degradation and reticuloendothelial system uptake
of calcein in the dipalmitoylphosphatidylcholine liposomes with soybean-derived sterols in mice.
Pharm Res 12:49-52, 1995.
23. Moolten FL: Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes:
Paradigm for a prospective cancer control strategy. Cancer Res 46:5276-81, 1986.
24. Freeman SM, Abboud CN, Whartenby KA, et al: The "bystander effect": Tumor regression when a
fraction of the tumor mass is genetically modified. Cancer Res 53:5274-5283, 1993.
25. Mesnil M, Piccoli C, Tiraby G, et al: Bystander killing of cancer cells by herpes simplex virus
Page 8 of 10
Gene Therapy for Ovarian Cancer
Published on Cancer Network (http://www.cancernetwork.com)
thymidine kinase gene is mediated by connexins. Proc Natl Acad Sci U S A 93:1831-1835, 1996.
26. Wei SJ, Chao Y, Shih YL, et al: Involvement of Fas (CD95/APO-1) and Fas ligand in apoptosis
induced by ganciclovir treatment of tumor cells transduced with herpes simplex virus thymidine
kinase. Gene Ther 6:420-431, 1999.
27. Vile RG, Nelson JA, Castleden S, et al: Systemic gene therapy of murine melanoma using tissue
specific expression of the HSV-tk gene involves an immune component. Cancer Res 54:6228-6234,
1994.
28. Yamamoto S, Suzuki S, Hoshino A, et al: Herpes simplex virus thymidine
kinase/ganciclovir-mediated killing of tumor cell induces tumor-specific cytotoxic T cells in mice.
Cancer Gene Ther 4:91-96, 1997.
29. Robertson MW III, Barnes MN, Rancourt C, et al: Gene therapy for ovarian carcinoma. Semin
Oncol 25:397-406, 1998.
30. Freeman SM, McCune C, Robinson W, et al: The treatment of ovarian cancer with a gene modified
cancer vaccine: A phase I study. Hum Gene Ther 6:927-939, 1995.
31. Alvarez RD, Curiel DT: A phase I study of recombinant adenovirus vector-mediated
intraperitoneal delivery of herpes simplex virus thymidine kinase (HSV-TK) gene and intravenous
ganciclovir for previously treated ovarian and extraovarian cancer patients. Hum Gene Ther
8:597-613, 1997.
32. Tong XW, Kieback DG, Ramesh R, et al: Molecular aspects of ovarian cancer. Is gene therapy the
solution? Hematol Oncol Clin North Am 13:109-33, viii, 1999.
33. Holt JT, Thompson ME, Szabo C, et al: Growth retardation and tumour inhibition by BRCA1. Nat
Genet 12:298-302, 1996.
34. Tait DL, Obermiller PS, Redlin-Frazier S, et al: A phase I trial of retroviral BRCA1sv gene therapy
in ovarian cancer. Clin Cancer Res 3:1959-1968, 1997.
35. Song K, Li Z, Seth P, et al: Sensitization of cisplatinum by a recombinant adenovirus vector
expressing wild-type p53 gene in human ovarian carcinomas. Oncol Res 9:603-609, 1997.
36. Alvarez RD, Gomez-Navarro J, Wang M, et al: Adenoviral-mediated suicide gene therapy for
ovarian cancer. Mol Ther 2:524-530, 2000.
37. Deshane J, Loechel F, Conry RM, et al: Intracellular single-chain antibody directed against erbB2
down-regulates cell surface erbB2 and exhibits a selective anti-proliferative effect in erbB2
overexpressing cancer cell lines. Gene Ther 1:332-337, 1994.
38. Alvarez RD, Barnes MN, Gomez-Navarro J, et al: A cancer gene therapy approach utilizing an
anti-erbB-2 single-chain antibody-encoding adenovirus (AD21): A phase I trial. Clin Cancer Res
6:3081-3087, 2000.
39.Yu DH, Scorsone K, Hung MC: Adenovirus type 5 E1A gene products act as transformation
suppressors of the neu oncogene. Mol Cell Biol 11:1745-1750, 1991.
40. Zhang Y, Yu D, Xia W, et al: HER2/neu-targeting cancer therapy via adenovirus-mediated E1A
delivery in an animal model. Oncogene 10:1947-1954, 1995.
41. Martuza RL: Conditionally replicating herpes vectors for cancer therapy. J Clin Invest 105:841846, 2000.
42. Markert JM, Medlock MD, Rabkin SD, et al: Conditionally replicating herpes simplex virus mutant,
G207 for the treatment of malignant glioma: Results of a phase I trial. Gene Ther 7:867-874, 2000.
Page 9 of 10
Gene Therapy for Ovarian Cancer
Published on Cancer Network (http://www.cancernetwork.com)
43. Coukos G, Makrigiannakis A, Kang EH, et al: Use of carrier cells to deliver a replication-selective
herpes simplex virus-1 mutant for the intraperitoneal therapy of epithelial ovarian cancer. Clin
Cancer Res 5:1523-37, 1999.
44. Heise C, Sampson-Johannes A, Williams A, et al: ONYX-015, an E1B gene-attenuated adenovirus,
causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard
chemotherapeutic agents. Nat Med 3:639-645, 1997.
45. Hall AR, Dix BR, O’Carroll SJ, et al: p53-dependent cell death/apoptosis is required for a
productive adenovirus infection. Nat Med 4:1068-1072, 1998.
46. Khuri FR, Nemunaitis J, Ganly I, et al: a controlled trial of intratumoral ONYX-015, a
selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with
recurrent head and neck cancer. Nat Med 6:879-885, 2000.
Source URL: http://www.cancernetwork.com/printpdf/gene-therapy-ovarian-cancer-0/page/0/1
Links:
[1] http://www.cancernetwork.com/review-article
[2] http://www.cancernetwork.com/ovarian-cancer
[3] http://www.cancernetwork.com/authors/george-coukos-md-phd
[4] http://www.cancernetwork.com/authors/stephen-c-rubin-md
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