Download gene therapy for prostate cancer: where are we now?

0022-5347/00/1644-1121/0
THE JOURNAL OF UROLOGY®
Copyright © 2000 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 164, 1121–1136, October 2000
Printed in U.S.A.
Review Article
GENE THERAPY FOR PROSTATE CANCER: WHERE ARE WE NOW?
MITCHELL S. STEINER
AND
JEFFREY R. GINGRICH
From the Department of Urology, University of Tennessee, Memphis, Tennessee
ABSTRACT
Purpose: The ability to recombine specifically and alter DNA sequences followed by techniques to
transfer these sequences or even whole genes into normal and diseased cells has revolutionized
medical research and ushered the clinicians of today into the age of gene therapy. We provide
urologists a review of relevant background information, outline current treatment strategies and
clinical trials, and delineate current challenges facing the field of gene therapy for advanced prostate
cancer.
Materials and Methods: We comprehensively reviewed the literature, including PubMed and
recent abstract proceedings from national meetings, relevant to gene therapy and advanced
prostate cancer. We selected for review literature representative of the principal scientific
background for current gene therapy strategies and National Institutes of Health Recombinant
DNA Advisory Committee approved clinical trials.
Results: Current prostate cancer gene therapy strategies include correcting aberrant gene expression, exploiting programmed cell death pathways, targeting critical cell biological functions, introducing toxic or cell lytic suicide genes, enhancing the immune system antitumor response and
combining treatment with conventional cytotoxic chemotherapy or radiation therapy.
Conclusions: Many challenges lie ahead for gene therapy, including improving DNA transfer
efficiency to cells locally and at distant sites, enhancing levels of gene expression and overcoming
immune responses that limit the time that genes are expressed. Nevertheless, despite these
current challenges it is almost certain that gene therapy will be part of the urological armamentarium against prostate cancer in this century.
KEY WORDS: prostatic neoplasms, prostate, immunotherapy, gene therapy, DNA
Prostate cancer is the most frequently diagnosed malignancy
and the second leading cause of cancer death in American men
today. An estimated 179,300 new cases of prostate cancer and
37,000 deaths were predicted for 1999.1 The risk of prostate
cancer increases steeply with age and continues to increase by
a projected 3% to 4% yearly in older men as fewer die of cardiovascular disease.2 Despite concerns that increasing the early
detection of prostate cancer by widespread serum prostate specific antigen (PSA) testing may lead to many more cases of
small indolent cancers treated unnecessarily, no change was
noted in the proportion of such cases at many large medical
centers from 1983 to 1996.3 The majority of patients carefully
selected for treating clinically localized disease with radical
prostatectomy continue to harbor pathologically more advanced
disease. Badalament et al evaluated 4 large prostatectomy series with a total of 5,661 patients and reported that 55% had
extracapsular disease at surgery.4 The finding of locally advanced prostate cancer, defined as extracapsular extension,
increases the likelihood of positive surgical margins at radical
prostatectomy and portends a poor prognosis. Repeat examination of the role of radical prostatectomy as monotherapy for
extracapsular disease indicates that it is really not curative
since 10 and 15-year disease-free survival after radical prostatectomy is only 12% to 60% and 20% to 28%, respectively.
Particularly local recurrence rates are as high as 41% 5 years
after radical prostatectomy.5 Hence, radical prostatectomy only
is not curative in the majority of patients with significant extracapsular disease.
Although it was well established that testosterone depriva-
tion only rarely cures prostate cancer,6 neoadjuvant hormone
deprivation was recently administered in an attempt to improve the probability of local cancer control by downsizing or
down staging locally advanced prostate cancer before radical
prostatectomy. Early studies indicated a decrease in the positive surgical margin rate7 but no changes were noted in the rate
of PSA recurrence in a retrospective analysis8 or in prospective
randomized studies with 2 years of followup.9, 10 Thus, neoadjuvant hormone deprivation has not been shown to change the
prostate cancer tumor progression or survival rate.9 –12
External beam radiation treatment only also has a high
local failure rate in advanced prostate cancer. Zagars et al
reported increasing serum PSA after external beam radiation in 17% and in as many as 60% of patients with PSA less
and greater than 40 ng./ml., respectively.13 Overall Holzman
et al observed a 53% local recurrence rate 8 years after
external beam radiation.14 The reported disease-free survival rate for stage T3 disease is 64% at 5, 10% to 35% at 10
and 15% to 18% at 15 years.15, 16 When a serum PSA criterion
is used, the biochemical failure rate exceeds 90% at 10 years
for stage T3 prostate cancer.16 Similarly in a series from
Stanford University greater than 60% of patients had increasing serum PSA, indicating cancer progression, at a
mean followup of 17 months after definitive external beam
radiation treatment,17 including more than 90% with a biopsy positive for prostate cancer.18 In addition, although the
true clinical significance remains to be determined, the grade
of recurrent prostate cancer after radiation therapy has been
shown to be higher than that of the original cancer.19, 20
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GENE THERAPY FOR PROSTATE CANCER
Thus, surgical, radiation or hormonal therapy as monotherapy or in combination does not appear to be adequate to
control locally clinical or pathological stage T3 prostate cancer, which ultimately leads to morbidity, distant metastasis
and decreased survival.16 Because androgen independent
prostate cancer cells eventually lead to death,21 successful
strategies to modify the biological behavior of these cells may
potentially have the most significant clinical impact. Clearly
other novel treatment approaches to advanced or recurrent
disease are desperately needed to achieve long-term local
control and particularly to develop effective systemic therapy
for metastatic prostate cancer.
The concept that cancer is a genetic disease is based on
observations that normal gene expression is frequently altered. Aberrant gene expression may develop due to various
reasons, including inherited or acquired gene gain, loss or
mutation as cells become malignant. Furthermore, gene expression may be affected at the RNA and protein levels. This
altered gene expression may affect normal cellular functions,
leading to the phenotypic and characteristic changes associated with malignancy, such as increased proliferation, invasion, the achievement of androgen independence and the
ability to metastasize. Therefore, it is conceivable that resuming normal gene expression at 1 or more of these points
in the pathway to fatal disease may reverse, arrest or delay
this progression. Recombinant DNA technology involving the
ability to recombine specifically and alter DNA sequences
followed by techniques to transfer these specific sequences or
even whole genes into normal and diseased cells has revolutionized medical research and ushered clinicians today into
an age of medicine directed at the molecular level. Modifying
endogenous gene expression (genes originally in the cell chromosomes) and introducing exogenous genes (genes inserted
into the cell that are then expressed in addition to the endogenous genes) into living humans, particularly by viruses,
are now possible and currently under way in several clinical
trials for prostate cancer. This new type of medicine is called
gene therapy. We provide practicing urologists with a review
of relevant background information, outline current treatment strategies and clinical trials available for patients, and
delineate current challenges facing the field of gene therapy
for advanced prostate cancer.
GENE TRANSFER IN HUMANS FOR THE TREATMENT OF
DISEASE
Gene therapy is a new frontier in the treatment of human
disease that represents the pinnacle of applied genetic research.
The feasibility of using genes as pharmacological agents for
targeting inherited and acquired diseases was only realized
within the last 5 to 10 years. It is only 36 years since the genetic
code was deciphered, and only within the last 2 decades have
scientists manipulated and transferred foreign genetic material
into a cell, subsequently altering its phenotypic and functional
characteristics. Broadly, genes may be transferred for gene
therapy by ex vivo or in vivo techniques (fig. 1). Ex vivo
gene therapy introduces foreign genes in the laboratory into
cells harvested from the body. The genetically modified cells are
then administered back into the patient. For in vivo gene therapy foreign genes are introduced directly into cells while the
cells remain within patients. The initial human gene transfer
trial in 1989 involved ex vivo gene therapy techniques to replace
the defective hemoglobin gene with a normal gene in a patient
with thalassemia.22 The initial in vivo gene therapy trial was
done in 1992 and by 1994 more in vivo than ex vivo gene
transfer trials were approved by the Food and Drug Administration, and the National Institutes of Health (NIH) Recombinant DNA Advisory Committee.22, 23
Gene therapy begins with the identification and characterization of human genes. Genes consist of specific DNA sequences whose copying or transcription into messenger (m)
RNA followed by conversion or translation into a protein is
controlled by adjacent sequences of DNA called promoters.
These genes must be isolated or cloned from their surrounding DNA into smaller, workable lengths. Cloning is generally
performed by creating complementary (c) DNA from the gene
mRNA and inserting it into another fragment of DNA, usually a piece of circular bacterial DNA also known as a plasmid. The plasmid containing the inserted DNA is then commonly referred to as a plasmid vector. The vector may then be
replicated in greater quantities by bacteria to provide a large
enough quantity of cDNA for sequencing. It also serves as a
vehicle for transferring the gene into a target cell. Since each
cell in the body contains the complete genetic information
necessary to construct the whole body, the genes transcribed
into RNA and translated into protein for a particular cell are
highly regulated by combined factors within the cell, including the gene promoters. In addition, regions known as enhancers that markedly increase the level of gene expression
may function only in the presence of other cell specific proteins. These factors provide the basis of how certain cells
make specific proteins characteristic of those cells, such as
PSA in the prostate, although the cells contain all of the
information needed to make any protein in the body. For
example, highly prostate specific promoters are only actively
expressed in prostate cells and not in other cell types, such as
those of the lung or breast. These tissue specific promoters or
FIG. 1. Prostate cancer gene therapy delivery is broadly categorized as using ex vivo or in vivo techniques to transfer therapeutic genes.
In vivo techniques treat tumor cells in situ. Ex vivo treated tumor cells may be used for subcutaneous (s. c.) vaccination or treated effector
cells may be infused intravenously (i. v.) as adoptive immunotherapy. GM-CSF, granulocyte macrophage colony stimulating factor.
GENE THERAPY FOR PROSTATE CANCER
enhancers may be exploited by designing gene therapy strategies, so that the transfer and expression of genes may be
directed to specific cell types, avoiding potential toxicity due
to undesirable therapeutic gene expression in adjacent or
distant tissue.
A VECTOR IS THE VEHICLE THAT MOVES GENES INTO CELLS
Gene therapy for human disease may be likened to classic
pharmacology, in which the administered drug is the gene
product encoded by the transferred DNA sequence and the
syringe the vector containing its gene with its promoter.
Vector technology for delivering genes is also rapidly evolving. The original methods of introducing genes into cells were
inefficient and crude. Practically speaking only ex vivo gene
therapy was previously considered. However, newer methods
have been developed that enable in vivo gene therapy to
become a reality. Generally the 2 vector categories used to
transfer genes into cells are the nonviral and viral based
vector systems. The Appendix shows the features and limitations of some common vector systems. Generally nonviral
vectors may carry large pieces of DNA and are easily mass
produced but tend to have a low gene transfer rate and
uncertain toxicity. Viral based vector systems take advantage of the genetic composition of viruses, which already have
the necessary tools to shuttle foreign DNA into cells and in
some cases facilitate the incorporation of viral DNA into host
cell DNA for optimal gene expression. Viruses produce disease by introducing their genes into a cell and then using the
cell machinery to produce more viruses. Viruses for gene
therapy are usually genetically altered, so that they do not
independently reproduce and, therefore, are not pathogenic.
Through DNA recombination techniques viral genes that encode for virus replication are replaced with the cDNA of the
therapeutic gene (fig. 2, A). The resulting replication deficient,
recombinant virus acts essentially as a molecular syringe for
injecting therapeutic genes into target cells (fig. 2, B).22–24
Each virus type has different properties that may be therapeutically exploited. For example, retroviruses have the
ability to integrate the therapeutic gene into the genome of
dividing target cells. Therefore, the transferred gene becomes
an inherited component of cells and may result in a permanent change in cell characteristics. This approach may be
ideal for corrective gene therapy when the intent is to correct
an underlying gene mutation of each cell. However, a limitation of retroviral vectors is that genes may only be introduced
into dividing cells, which may be problematic since prostate
cancer tends to have a low proliferative index, meaning that
prostate cancer cells do not generally grow and divide rapidly. In contrast, adenoviral vectors may transfer genes into
FIG. 2. Adenoviral vector development. A, therapeutic gene cDNA
under regulatory control of promoter sequence is substituted into
viral DNA, replacing genes controlling virus replication. B, using
appropriate helper cells replication deficient adenoviruses carrying
therapeutic gene may be produced for gene therapy.
1123
dividing and nondividing cells but therapeutic gene expression is only transient. Thus, this vector may not be satisfactory when the intent is to express permanently the therapeutic gene as part of the target cell genome. On the other hand,
transient gene expression of a toxic gene product may provide
a built-in safety feature.22 In addition, viral and generally
nonviral based vector systems cannot target specific cell
types. This limitation is being addressed by a number of
strategies, including incorporating tissue specific expression
elements such as promoters, or through tumor cell targeting
using surface protein antigens or antibodies that localize the
vector to more specific cell types. Although some features of
each vector system may be exploited for gene therapy, to our
knowledge no vector system is currently ideal. However,
waiting for the perfect vector before performing preliminary
gene therapy trials would be similar to waiting for the perfect
car engine before designing a car. The ultimate goal of gene
therapy is to develop the ability to administer vectors systemically, providing highly efficient tissue specific gene replacement or expression. Vector technology is rapidly evolving and the vectors of the future are expected to be different
from those used today. Nonetheless, in the meantime it is
paramount that our understanding of the basic science of
gene therapy and vector technology continue in parallel.
GENE THERAPY AND PROSTATE CANCER
Prostate cancer is a particularly suitable malignancy to
study and use in gene therapy trials because of several features. Prostate cancer is common and there is no cure in the
majority of patients diagnosed with advanced disease. The
prostate gland produces several known gene products, such
as PSA, prostate secretory protein, prostatic acid phosphatase and human glandular kallikrein, and hundreds of others
that remain uncharacterized to date and are relatively
unique to the prostate, of which each protein or antigen may
potentially be exploited for vector targeting or gene vaccine
immunization.25, 26 Moreover, prostate specific promoters
and other enhancers that direct the transcription of these
prostate unique proteins may also be incorporated into vectors to direct the prostate specific expression of therapeutic
genes.23 The prostate gland does not serve any critical life
sustaining function, which obviates the need to distinguish
normal from cancerous prostate tissue for targeted gene therapy. The prostate gland is also easily accessible by transurethral, transperineal and transrectal approaches for the intratumoral administration of gene therapy. After gene
therapy the prostate may be easily evaluated by transrectal
ultrasound, digital rectal examination and other standard
radiological imaging, such as magnetic resonance imaging
(MRI) and computerized tomography. The pattern of prostate
cancer spread is also predictable, since it commonly metastasizes to the pelvic lymph nodes and then to the axial skeleton. Therefore, this pattern of metastasis warrants the development of gene therapy strategies to purge bone marrow
of malignant cells or develop vectors that have tropism (attraction or affinity) for bone.24, 27, 28 Although prostate cancer
gene therapy is controversial, it may readily be followed by
serum PSA testing. It may not be a single absolute end point
by which to make treatment decisions but it may serve as a
surrogate marker of prostate cancer progression.29, 30
As mentioned, prostate cancer also poses some unique and
interesting challenges for current gene therapy technology.
Because less than 5% of cells are actively dividing at any time
within a tumor, the mean doubling time of prostate cancer is
usually estimated as greater than 150 days.31 Due to this low
proliferative index vectors capable of providing high gene
transfer rates independent of cell division are required for
treating prostate cancer. Another challenge is that prostate
cancer is heterogeneous, not only among but also within
individuals. For example, the underlying genetic mutations
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GENE THERAPY FOR PROSTATE CANCER
that dictate the phenotype of the primary tumor may not be
identical to those responsible for metastasis.21, 32 Thus, appropriate corrective gene therapy in an individual may not
necessarily be effective for another patient or even for another lesion in the same individual.
PROSTATE TARGETED DELIVERY OF GENE THERAPY
Current technology cannot achieve the ultimate goal of in
vivo systemic administration of a vector that results in adequate tissue specific expression of the therapeutic gene in
100% of target cells without other specific or systemic toxicity. Innovative gene therapy approaches being sought to circumvent the limitations of current vectors include more effective delivery routes, tumor targeting, the incorporation of
tissue specific promoters and/or enhancers into vectors and
increasing cell death by enhancing the phenomenon known
as the bystander effect.
Delivery approaches. Theoretically the treatment of metastatic disease may require the systemic delivery of gene vectors intravenously. However, in locally advanced, stages T3
and T4 prostate cancer various delivery strategies may help
to target gene therapy vectors to the prostate. Lu et al evaluated the effectiveness of an adenoviral vector containing a
␤-galactosidase reporter gene (a gene whose expression may
be detected but does not have significant physiological effects) delivered to the canine prostate by intravenous, intraarterial and intraprostatic administration routes.33 The
intra-arterial approach was performed by cannulating the internal iliac artery and advancing the catheter into the inferior
vesicle and prostatic arteries. The transduction efficiency of
adenovirus ␤-galactosidase was assessed by X-galactosidase
staining of prostate tissue frozen sections, colorimetric
␤-galactosidase assay and polymerase chain reaction. Intraprostatic administration of the adenoviral vector resulted in the
highest gene transfer rate by far with the least systemic adenoviral dissemination.33 This study provided direct support for
clinical intratumoral prostatic injections of gene therapy vectors under ultrasound guidance via the transrectal or transperineal approach in prostate cancer gene therapy trials.34 Further
optimization of local gene therapy delivery and gene expression
may ultimately be achieved by using various matrixes for the
vector, such as a gelatin sponge matrix as recently reported by
Siemens et al.35
Tumor targeting. The identification of cellular pathways or
cell surface antigens that are unique or may be exploited for
developing drugs or other tumor targeting strategies has
been a major pursuit of oncological research for several decades. Using large computerized databases of gene expression36, 37 it is now more feasible to develop intentionally
monoclonal antibodies toward known specific antigens that
are more likely to be selective for particular tumor cell types
and may act as a means of localizing immunotoxins or other
conjugates.38, 39 Refining this concept to yet another level,
immunotoxins such as Pseudomonas exotoxin or cytotoxic
agents such as doxorubicin, may now be targeted by peptide
conjugates, which bind to specific antigens or cell receptors
such as those for fibroblast growth factors or luteinizing
hormone releasing hormone.40, 41 Via such tumor targeting
mechanisms systemic toxicity may be minimized while maximizing delivery to the tumor cell targets.
Tissue specific promoters. Viral vectors have the ability to
bind to and transfer their genes to any mammalian cells
expressing the appropriate receptor for the specific virus.
Dannull et al demonstrated that a high dose of vectors that
are not tissue specific may have systemic toxicity.42 A strategy for viral and other vectors to target theoretically only
prostate cells is the incorporation of a prostate tissue specific
promoter and/or enhancer. Therefore, although potentially
incorporated into other cell types, expression of the therapeutic gene would be limited to prostate cells since only they
would contain the appropriate complement of transcription
factors to activate the prostate specific promoter. However,
this result depends on tight regulation of the prostate tissue
specific promoter-gene vector expression system, so that it
would not be expressed at a low level in some tissue, or under
changing or leaky conditions that would result in the inadvertent expression of the therapeutic gene in unintended
tissue. Unfortunately the prostate promoters currently used
in gene therapy are leaky to some degree. What remains to be
determined is whether this low level of expression in nonprostate tissue is clinically relevant.
Prostate epithelial cell promoters used for gene therapy
include PSA,33, 43– 46 probasin,47 mouse mammary tumor virus48 –50 and prostate specific membrane antigen.51 The PSA
promoter is most commonly used in vector constructs.43, 44 A
theoretical concern is that the PSA promoter requires that
each androgen receptor and circulating androgen be active.
However, the majority of patients with advanced and hormone refractory prostate cancer also undergo androgen deprivation therapy, which may compromise activation of the
PSA promoter. To address this potential obstacle Gotoh et al
characterized the 5,837 bp PSA promoter as more active than
the short 631 bp PSA promoter in an androgen depleted
environment and in androgen insensitive cells in vitro.52
Another strategy was modifying a prostate tissue specific
promoter, so that activation by another hormone occurred
while the tissue specificity of a promoter such as probasin
was preserved. Zhang et al developed a retroviral construct
containing a glucocorticoid responsive element upstream of
the androgen responsive element, which enabled activation
of the probasin promoter with dexamethasone.47 Similarly
Rodriguez et al demonstrated that the androgen sensitive
probasin promoter may also be activated by phenylbutyrate
in the absence of androgen.53 Thus, gene therapy using constructs containing modified prostate specific promoters may
be administered in advanced prostate cancer that is also
managed by androgen deprivation therapy.
A limitation of prostate tissue specific promoters is that the
level of expression or activity tends to be less than that of
other nonspecific or viral promoters.33 There tends to be an
inverse correlation of promoter activity with tissue specificity. In a canine prostate model PSA, probasin and mouse
mammary tumor virus promoters were prostate specific but
had 10 to 100-fold less activity than the Rous sarcoma virus
promoter in vivo.33 To help circumvent this problem Pang et
al cloned the mutated PSA promoter PCPSA from a patient
with prostate cancer and elevated serum PSA.46 The PCPSA
promoter had 50-fold greater activity than the standard
PSA promoter. Similarly upstream regulatory sequences of
the PSA promoter called prostate specific enhancer sequences were cloned from normal and cancerous prostate
tissue. In vitro prostate specific enhancer sequences increased PSA promoter activity 72-fold when isolated from
normal prostate compared with 1,000-fold when cloned from
prostate cancer tissue.44 Recently Dannull et al incorporated
the 822 bp prostate specific enhancer and 611 bp PSA promoter into an E1 deleted adenoviral vector.43 Unfortunately
intratumoral injection of this vector into various subcutaneous human prostate SCID mouse xenografts resulted in less
robust PSA promoter activity than that in vitro. Activity
remained markedly less than that of the cytomegalovirus
(CMV) viral promoter in the same system.
An alternate tactic involves gene therapy to treat the supporting bone stromal cells in an effort to eradicate prostate
epithelial cells metastatic to the bone. Using the osteocalcin
promoter, which is active in bone stroma, including osteoblasts,27 prostate cancer gene therapy may be directed toward bony metastatic sites. Ko et al reported that osteosarcoma tumors are inhibited after intratumoral injection of an
adenoviral vector composed of the osteocalcin promoter controlling thymidine kinase followed by systemic ganciclovir admin-
GENE THERAPY FOR PROSTATE CANCER
27
istration. Moreover, the osteocalcin promoter is active in
spontaneous canine prostate cancer bone metastasis, implying
that this strategy may be useful for targeting bone metastasis
in humans.54 Combined PSA enhancers, and other prostate
specific epithelial and stromal promoters are currently under
intense investigation. Whether any of these promoter combinations are ultimately effective for the systemic treatment of prostate cancer with the required level of promoter activity remains
to be determined.55
Bystander effect. A phenomenon after the application of
gene therapy in preclinical models is called the bystander
effect, which denotes that more cells are destroyed or biologically altered after gene therapy than those predicted by gene
transfer (transduction) only. This result is fortuitous in that
no single vector system is currently available that may be
applied clinically to transfer therapeutic genes into 100% of
target cells. Thus, the ability to affect more cells than just
those transfected makes gene therapy more clinically feasible. Several hypotheses attempt to explain the bystander
effect mechanism, such as intratumoral cell-to-cell transfer
of the therapeutic gene, gene product or gene activated toxic
prodrug through cellular vesicles, endocytosis or diffusion
through gap junctions and cell channels. Alternatively the
therapeutic gene or vector antigens may induce an intense
immune response that contributes to cell kill. This immune
response appears to be natural killer cell mediated.56 In
addition, the bystander effect may be associated with tissue
ischemia resulting from endovascular injury secondary to
toxic gene product or a nonspecific immunological response.57 Although to our knowledge the exact mechanism is
not known, the bystander effect is a clinical entity that may
help to compensate partially for the inefficient in vivo gene
transfer limitations of currently available vectors.
GENE THERAPY STRATEGIES TO COMBAT PROSTATE CANCER
Gene therapy strategies are rapidly evolving as new gene
targets, better vectors and improved gene expression systems
become available. The initial approach involving the transfer
of genes into cells growing outside of the body in tissue
culture is classified as ex vivo gene therapy (fig. 1). Primarily
because of new technological advances, including viral based
vectors, in vivo gene therapy is also possible. Innovative gene
therapy strategies currently used for treating prostate cancer
include enhancing the immune system antitumor response,
correcting specific aberrant gene expression, exploiting programmed cell death pathways, introducing toxic or cell lytic
suicide genes and combined strategies in conjunction with
conventional cytotoxic chemotherapy or radiation therapy.
Immunotherapy. Class I major histocompatibility complex
protein expression by tumor cells is critical for achieving an
effective antitumor immunological response. Alterations in
or the loss of class I major histocompatibility complex expression represents a common means by which prostate cancer
cells may evade the host immune system.58, 59 Several approaches have been used to stimulate or augment the body
antitumor immune response to circumvent essentially the
loss of the critical class I major histocompatibility complex
proteins (fig. 1). The general immunological approaches developed for prostate cancer involve autologous or nonautologous gene vaccine therapy using ex vivo gene transfer techniques, direct in vivo intratumoral injection of gene therapy
vectors containing cytokine genes, adoptive immunotherapy
for treating effector immune cells, such as dendritic cells,
tumor infiltrating lymphocytes and cytotoxic T lymphocytes,
by ex vivo gene transfer techniques, and cytokine immunotherapy. However, the latter is not gene therapy in the purest
sense since patients are treated systemically with purified
cytokines (immune system signaling proteins), such as interleukin (IL)-2 or granulocyte macrophage colony stimulating
factor.44
1125
Gene vaccine therapy (fig. 1). Tumor vaccines have been
generated using autologous tumor or fibroblast cells harvested from patients by biopsy or surgery, while alternatively
nonautologous cells such as tumor cell lines are modified by
ex vivo gene therapy with genes that encode for cytokines.60
In addition, before inoculation the genetically altered cells
are irradiated to destroy their replication capacity. Cytotoxic
T lymphocytes not only recognize tumor specific antigens
present on the surface of these inoculated, irradiated cells,
but also are induced by local secretion of the transferred
stimulatory cytokines. Activated cytotoxic T lymphocytes expand in number and then target and destroy tumor cells
throughout the body that share these antigens on the cell
surface.61 Gene therapy with cytokine genes, IL-2 and granulocyte macrophage colony stimulating factor would theoretically stimulate an antitumor response independent of the
class I major histocompatibility complex level by using class II
major histocompatibility complex expression and natural killer
cell mediated tumor lysis.62, 63 In contrast, to mediate their
immune effects tumor necrosis factor-␣ and interferon-␥ depend
on class I major histocompatibility complex proteins, which as
mentioned are often altered in prostate cancer.64, 65 Preclinical
studies clearly showed that gene vaccines are not efficacious for
a large tumor burden but theoretically may be more useful
against micrometastasis after primary tumor debulking.23, 66, 67
Other clinical obstacles of autologous prostate cancer gene vaccines include the fact that sufficient prostate cancer tissue may
not be obtainable, tumor tissue may not be easily cultured,
cytokine gene transfer efficiency may be poor and large scale
production yields of genetically modified tumor cells may be
inadequate.68
Gene vaccines with some efficacy against prostate cancer
in animal models include the gene transfer of cytokine genes
granulocyte macrophage colony stimulating factor,69
IL-2,70, 71 interferon-␥, tumor necrosis factor-␣ and B7.71, 72
Using the transgenic adenocarcinoma of mouse prostate
transgenic model of prostate cancer and derived cell
lines73–75 Kwon et al demonstrated that increased expression
of B7 by normally tumorigenic cell lines resulted in the
complete rejection of cancer cells injected subcutaneously
into immune and intact but not into nude immunocompromised mice.72 MAT-LyLu cells grown in Copenhagen rats
that produced an increased level of the cytokines IL-2, granulocyte macrophage colony stimulating factor and
interferon-␥ were also evaluated.58, 70, 76 –78 The retroviral
delivery of IL-2 or granulocyte macrophage colony stimulating factor inhibited prostate cancer tumors and increased
animal survival with up to 30% cured after treatment with
granulocyte macrophage colony stimulating factor gene vaccine.70, 77, 79, 80 Similar results were reported for ex vivo IL-2
liposomal gene vaccine therapy.81 In contrast, Kawakita et al
used canary pox virus for genes IL-2, interferon-␥, tumor
necrosis factor-␣ and B7, and observed that only tumor necrosis factor-␣ and IL-2 delayed RM1 tumors in C57BL/6
mice.71
Another approach alters prostate cancer cells or immune
cells by the ex vivo gene transfer of genes encoding tumor
specific antigens. When inoculated, these modified prostate
cells recruit immune effector cells capable of eliciting a wide
array of immunological antitumor responses, sensitizing the
host immune system against these newly introduced prostate
tumor specific antigens.44, 82 Tumor specific antigens that
appear to have relative prostate cancer specificity include
PSA, prostate specific membrane antigen,51 GAGE-7, PAGE,
TAG-72,83 and the prostate mucin antigens mucin-184, 85 and
mucin-2.44, 82, 86, 87 Lubaroff et al demonstrated that a PSA
producing adenoviral vector induced potent antitumor immunity in vivo mediated by cytotoxic T lymphocytes and humoral immune responses.88 Overall by producing cytokines
or presenting tumor specific antigens cancer gene vaccines
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GENE THERAPY FOR PROSTATE CANCER
aim to enhance the induction of T cell immunity for eradicating prostate cancer micrometastasis.
Direct in vivo intratumoral injection of gene therapy vectors
containing cytokine genes (fig. 1). This immunotherapy approach treats tumor cells more directly by injecting vectors
containing cytokine genes intratumorally. In animal models
of prostate cancer Naitoh et al noted that the intratumoral
injection of liposome and adenoviral vectors containing IL-2
gene expression systems resulted in the activation of specific
T cell antitumor responses.82 Similarly Sanford et al intratumorally injected adenoviral vectors containing IL-12 for
treating primary prostate cancer tumors in mice, which significantly decreased the number of lung metastases.89 The
molecular mechanism may include the stimulation of T and
natural killer cells, induction of interferon-␥ and upregulation of fas expression.89, 90 Phase I clinical trials of IL-2
gene transfer vectors for the intratumoral injection of prostate cancer are nearing completion.91
Adoptive immunotherapy (fig. 1). Effector immune cells,
including dendritic cells, tumor infiltrating lymphocytes and
cytotoxic T lymphocytes, are genetically modified by ex vivo
gene transfer techniques. This form of therapy for prostate
cancer is still in its infancy. The difficulty of this approach in
prostate cancer is in selectively obtaining specific effector cell
types from patients, ex vivo amplification of the effector cells
and ex vivo gene transfer of specific biological modifiers, such
as cytokine genes, back into patients.
Thus, immunotherapy may ultimately be effective against
micrometastatic disease but greater metastatic or primary
tumor volume would most likely require some other nonimmunological therapeutic intervention. Another concern is the
theoretical possibility that the autologous or nonautologous
cell production of a low level of tumor associated antigens
would paradoxically induce immune tolerance, which would
suppress the host immunity against prostate cancer. In addition, generally tumor antigens are also weak inducers of
the immune system. Therefore, only well designed clinical
trials of gene immunotherapy may answer these critical questions.
Corrective gene therapy (fig. 3, A). Corrective gene therapy
seeks to replace inherited or acquired defective genes that
are important for the normal growth regulation of the cell
cycle. The molecular components of the cell cycle include
proto-oncogenes, tumor suppressor genes and growth factors
with their respective receptors. Similar to most tumor cell
types, individual prostate cancer cells represent an accumulation of 1 or more gene mutations or other genetic events.
Therefore, it is unlikely that correcting a single gene alteration would have a significant biological consequence on the
cancer cell phenotype. Furthermore, this problem is confounded by the fact that current vector technology cannot
typically achieve stable in vivo expression of target genes in
100% of prostate cancer tumor cells, as mentioned. Nevertheless, replacing or correcting single gene alterations with
various vectors and genes altered the malignant phenotype
in several preclinical studies and in some cases eradicated
prostate tumors.34, 92–95 These observations are consistent
with models in which some genetic mutations were more
critical for cellular control than others.34, 49, 96 They imply
that additional phenomena, such as the bystander effect in
suicide gene therapy,56, 97 may also have a necessary or complementary role. In the more likely scenario that an association of several genetic events responsible for clinically significant prostatic adenocarcinoma is identified delivery
techniques for replacing 1 or more genes simultaneously or
sequentially may ultimately be required for effective gene
therapy.
Most corrective gene therapy strategies involve retroviral
or adenoviral vectors administered by intratumoral injection.
Prostate cancer preclinical studies were reported in which an
assortment of tumor suppressor genes were replaced, includ-
ing AdCMVp53,96, 98 –100 retroviral LXSN BRCA-1,34
AdCMVp2196, 99 and AdCMV CAM1.101 Another critical cell
cycle component, cell cycle dependent kinase inhibitor p16, is
commonly changed in prostate cancer.102, 103 Inactivation of
p16 is common in the majority of human prostate cancer cell
lines104, 105 and alterations of p16 were also reported in prostate cancer.103 Initially p16 mutations in primary human
prostate cancer were thought to be rare. Subsequently controversy arose about whether p16 inactivation was critical
only in rapidly dividing cancer cells in tissue culture rather
than in primary human prostate cancer because homozygous
deletions or intragenic mutations of p16 were apparently
rare.106 However, this controversy was laid to rest by the
recent discovery of frequent microdeletions within the p16
gene.
These microdeletions were difficult to confirm by standard
molecular techniques because normal cells were present
within the tumor specimen.106 Microsatellite analysis using
markers close to the p16 gene revealed that a wide range of
tumor types, including prostate cancer, had less than 200 kb
deletions of each p16 allele.106 Unlike other tumor suppressor genes that are primarily inactivated by point mutation,
small homozygous deletions represent a major mechanism of
p16 inactivation in cancer.106 In fact, using this technique
Cairns103 and Jarrard104 et al identified p16 homozygous
deletions in 40% of human primary prostate cancers. Moreover, with progression 46% of prostate cancer metastatic
lesions involve the loss of p16 heterozygosity.104 Even more
interesting is the fact that when androgen deprivation therapy fails, there is a 71% allelic loss of 9p in the region of
p16.21 Using the adenoviral vector GTx-001 containing p16
Steiner et al noted that p16 replacement suppresses cell
growth and induces cell senescence in various prostate cancer cell lines.94 A single in vivo intratumoral injection of
GTx-001 resulted in a 70% decrease in PPC-1 human prostate xenografts in nude mice and prolonged animal survival.94, 107, 108 In another animal prostate cancer model Gotoh
et al had similar results using an AdCMVp16 vector.96
Oncogene over expression is another way that cancer cells
commonly lose control of the cell cycle.109 An approach to
decrease the growth response directs the expression of a
reverse or antisense strand of mRNA, which is complementary to the normal or sense strand for that gene. Antisense
mRNA binds or anneals to the sense strand and effectively
prevents translation of the protein from that mRNA, suppressing its translation into protein. Since prostate cancer
commonly involves c-myc over expression, Steiner et al constructed a retroviral LXSN vector containing a prostate specific mouse mammary tumor virus promoter driving antisense to the c-myc gene.49 A single intratumoral injection of
retroviral mouse mammary tumor virus antisense c-myc
markedly suppressed and even eradicated some DU145 prostate cancer xenografts growing in nude mice. They determined that the molecular mechanism was the down regulation of c-myc mRNA and protein expression as well as the
induction of apoptosis by the down regulation of bcl-2 protein.49 Using a similar approach Kim et al observed that an
adenoviral vector containing an antisense erb-B-2 gene (Ad
anti-erb-B-2) inhibited the over expression of growth factor
erb-B-2 in prostate cancer cells, resulting in their destruction.28 This tactic was used in vitro to purge selectively
metastatic prostate cancer cells from bone marrow.28
In vivo animal model systems have also been used to identify possible alterations in peptide growth factor and growth
factor receptor expression during prostate cancer development and progression as potential targets for gene corrective
therapy. Interestingly peptide growth factor receptor fibroblast growth factor receptor 2 IIIb is altered with prostate
cancer progression.110 Matsubara et al reported that in the
Dunning rat adenocarcinoma cell line AT3 fibroblast growth
factor receptor 2 IIIb restored the suppression of the prostate
GENE THERAPY FOR PROSTATE CANCER
1127
FIG. 3. Mechanistic approaches to gene therapy. A, replace missing or correct mutated genes. B, exploit programmed cell death. C, target
critical cell machinery. D, express suicide genes. tk, thymidine kinase. GCV, ganciclovir. E, viral oncolysis of tumor cells.
cancer growth rate after transfection with fibroblast growth
factor receptor 2 kinase.111 Thus, restoring a single underlying mutation may change the malignant phenotype. Similarly when investigating changes in the insulin-like growth
factor family axis, Kaplan et al recently observed that
prostate specific insulin-like growth factor-I mRNA expression increased and insulin-like growth factor-2 mRNA expression decreased during prostate cancer progression.112
However, the expression of types 1 and 2 insulin-like growth
factor receptor mRNA was not altered during primary prostate cancer progression in intact transgenic adenocarcinoma
of mouse prostate mice but dramatically decreased in metastatic lesions and in androgen independent disease. As in
reports of clinical disease, serum insulin-like growth factor-1
levels increased early as prostate disease developed. These
results imply that the insulin-like growth factor growth factor axis may also serve as a therapeutic target for treating
prostate cancer.
Generally corrective gene therapy holds the promise
that restoring the expression of 1 or more genes may revert
the malignant phenotype of the cancer cell to or toward
normal. Other corrective gene therapy approaches indicate
that gene replacement induces cell death in a proportion of
tumor cells. Still others may incite host responses, such as
the bystander effect and other immunological responses,
which result in additional tumor cell death via complementary
or synergistic mechanisms.
Preliminary studies of corrective gene therapy also raise important clinical concerns unique to gene therapy. It has always
been a dictum in cancer therapy that each cancer cell must be
1128
GENE THERAPY FOR PROSTATE CANCER
eradicated to effect a long-term cure. Corrective gene therapy
with its goal of correcting or repairing alterations in the cell
cycle or other growth regulatory components challenges this
concept. It is possible that the clinical end point of corrective
gene therapy strategy would simply be that the cell behaves
more normally and no longer threatens the life of the patient.
To this end leaving cancer cells in an individual that have
become more phenotypically normal due to gene transfer may
be an acceptable clinical end point. As the human genome
project progresses and new prostate cancer genes are identified,
corrective gene therapy will have an increasing role in prostate
cancer prevention and management.
Exploitation of programmed cell death gene therapy (fig. 3,
B). Gene therapy strategies for activating apoptotic pathways, also known as programmed cell death, are also being
developed. In this approach the ultimate goal is to force the
cancer cell to commit irreversibly to programmed cell death.
Segawa et al used the elaborate PSA based promoter system
GAL-4-VP16 to activate GAL-4 responsive elements.113
A GAL-4 responsive element is placed upstream of the polyglutamine gene. Polyglutamine is a potent apoptotic protein
that in this study was produced selectively in PSA producing
cells. Similarly Hyer et al observed that adenovirus mediated
transduction of the fas ligand, a component of cell death
pathways, induced apoptosis in LNCaP, PC3 and DU145
prostate cancer cell lines in vitro.90 Marcelli et al reported
that transducing prostate cancer LNCaP cells with an adenoviral vector containing caspase-7, a potent and critical modulator of apoptosis, also induced programmed cell death.114
Another molecular approach targeted bcl-2, an oncogene with
anti-apoptotic activity, with an adenoviral vector containing
a hammerhead ribozyme directed against bcl-2.115, 116 The
bcl-2 ribozyme is an RNA molecule that specifically catalyzes
or disrupts bcl-2 mRNA, making the cell more susceptible to
apoptosis. Interestingly adenoviral hammerhead bcl-2 ribozyme treatment induced apoptosis in androgen sensitive
but not androgen insensitive prostate cancer cells. Although
no preclinical in vivo studies have yet been reported, exploiting programmed cell death gene therapy approaches is attractive and may potentially be effective.
Gene therapy for targeting critical cell machinery (fig. 3, C).
As in classic pharmacology, the basis of rational anticancer
gene therapy design may be to target critical cellular processes.
Lee et al designed liposomal vectors containing a PSA promoter
upstream of antisense topoisomerase II or antisense DNA polymerase ␣.117 Topoisomerase II and DNA polymerase ␣ are
critical molecular components of DNA replication. The treatment combination of PSA-antisense topoisomerase II and PSAantisense DNA polymerase ␣ liposomes had the greatest inhibitory effects on the prostate cancer cell lines LNCaP, DU145
and PC3 in vitro. Similarly Williams et al used a retroviral
vector that incorporated the antisense eIF4E gene for treating
prostate cancer cells.118 Prostate cancer cells were previously
shown to have over expression of eIF4E, which is a rate limiting
factor in the translation initiation of growth controlling genes
such as cyclin D1, c-fos, c-myc, v-epidermal growth factor and
b-fibroblast growth factor. A single intratumoral injection of
retroviral antisense eIF4E suppressed prostate cancer xenograft growth for up to 65 days.118 Thus, the rational design of
gene therapy vectors for disrupting critical molecular events
required for cellular function is an enticing strategy against
prostate cancer.
Suicide gene therapy (fig. 3, D). A suicide type of gene
therapy strategy may have the most promising clinical potential. In this strategy vectors are used to introduce the
therapeutic gene into cancer cells. After the gene product is
expressed the cell is destroyed without regard to the underlying genetic mutations responsible for the malignant phenotype. The types of suicide gene therapy strategies that
have emerged are gene directed enzyme prodrug treatment
and gene directed production of a cellular toxin.
Gene Directed Enzyme Prodrug Treatment: The gene directed enzyme prodrug treatment approach involves prodrug
enzyme gene therapy followed by the systemic administration of its specific prodrug. After gene transfer of the prodrug
enzyme gene cells producing the prodrug enzyme render the
cell capable of converting a nontoxic prodrug into an activated lethal drug. This activated drug not only kills the cell
that produced the toxic drug, but also its neighboring cancer
cells. This bystander effect may be impressive and kill 100 to
1,000-fold more cells than predicted by the gene transfer rate
only. Thus, low gene transfer efficiency may be compensated
by a high bystander effect. Local activation of the cancer killing
drug maximizes the toxic metabolite concentration at the tumor
site and minimizes systemic toxicity as the drug becomes diluted in the total blood stream volume of distribution.
The most widely used gene directed enzyme prodrug treatment system against prostate cancer is the Herpes simplex
virus (HSV)-thymidine kinase and ganciclovir system.97, 119
The nucleoside analogue ganciclovir is converted by HSVthymidine kinase, an enzyme not normally expressed in
mammalian cells, into a phosphorylated compound that is then
incorporated into DNA during DNA replication. This incorporation causes DNA chain termination and the selective killing
of dividing cells. Eastham et al used an adenoviral vector containing h. simplex virus-thymidine kinase to sensitize human
and murine prostate cancer cells to the toxic effects of ganciclovir in in vitro and in vivo models.119 Ad HSV-thymidine kinase
gene therapy followed by ganciclovir suppressed prostate cancer growth and prolonged survival in mice with prostate tumors.119 Hall et al also reported that Ad HSV-thymidine kinase
injection of orthotopic mouse prostate reconstitution model cell
line tumors followed by ganciclovir administration suppressed
tumor growth and decreased the rate of spontaneous lung metastasis.56, 97 An immune basis for these effects was demonstrated by challenging mice with an injection of prostate cancer
cells into the tail vein followed by excising subcutaneous prostate cancer tumors. In the animals with previously treated
primary tumors lung metastasis decreased 40%. This effect
appeared to be partially mediated by natural killer cells.56
Other gene directed enzyme prodrug treatment strategies
investigated in prostate cancer include the cytosine deaminaseflucytosine system, in which cytosine deaminase converts flucytosine to the chemotherapeutic agent 5-fluorouracil.120, 121 Kim
et al transferred the cytosine deaminase or HSV-thymidine
kinase gene into the murine bone marrow stroma cell line
D1.121 Co-cultures of D1 cells and human prostate cancer cell
lines followed by the appropriate prodrug destroyed prostate
cancer cells when as little as 20% of the D1 cells in the coculture produced the prodrug enzyme. Blackburn et al used an
adenoviral vector incorporating the heat shock protein (HSP) 70
promoter and the cytosine deaminase or HSV-thymidine kinase
gene for treating PC3 cells.120 In this system hyperthermia to
41C activated the HSP-70 promoter, which then directed prodrug enzyme expression. Thus, systemic administration of the
prodrug and local heat enabled selective expression of the prodrug enzyme in the intended tissue.120 Martiniello-Wilks et al
used another system with an E1a deleted adenovirus containing the Escherichia coli DeoD gene purine nucleoside phosphorylase as a prodrug enzyme under the control of the PSA promoter.122 The prodrug used was 6-methyl-9 (2 deoxy-␤-D
erythro-pentofuranosyl) purine (6 MPDR), which converts
6 MPDR into a toxic nonphosphorylated purine capable of killing quiescent and proliferating cells when incorporated in
mRNA or DNA during synthesis.122 The purine nucleoside
phosphorylase-6 MPDR system was efficacious against the human prostate cancer cell line PC3.122 Other gene directed enzyme prodrug treatment systems using assorted prostate specific promoters and vector types are currently under intense
investigation.
Gene Directed Production of Cell Toxin: With this strategy,
which is similar to gene directed enzyme prodrug treatment,
GENE THERAPY FOR PROSTATE CANCER
the transferred gene kills the cell independent of the underlying cancer gene mutations. Unlike gene directed enzyme
prodrug treatment this approach does not require a prodrug.
Rodriguez et al screened numerous direct biological toxins
known to kill mammalian cells by cell cycle independent
mechanisms to determine which would be best against human prostate cancer.123 Diptheria toxin was the most toxic
substance used. This toxin kills rapidly independent of p53 or
androgen sensitivity status, and kills dividing and nondividing cells alike.123 However, this approach has limitations. This
toxic gene must be incorporated into vectors that contain highly
prostate specific promoters that are under tight regulatory control since diptheria toxin is so biologically toxic that even a
small amount of leaky promoter activity in nonprostatic tissue
may be lethal. Also, mass production of adenoviral vector containing the diptheria toxin gene is difficult because of the toxic
effects of the diptheria toxin gene product on the packaging cell
line, resulting in low production titers.23
Oncolytic virus gene therapy (fig. 3, E). Because of safety
reasons, almost all current vectors are engineered to be replication incompetent. This term means that after the virus
with the gene is transported into a cell, the virus cannot
express its viral genes that commandeer the cell to produce more virus and enter the lytic cycle, which would release more virus particles to infect additional cells. Consequently viral vector effectiveness directly correlates with
transduction efficiency and the ability to be administered in
repeat doses. Recently 2 types of replication competent viral
vectors were developed. A conditionally competent adenoviral vector was mutated, such that the virus does not express
the viral protein E1b.124 The wild-type adenovirus uses the
E1b protein to block p53 from performing its normal function, which is to prevent the replication of cells with damaged
DNA. Theoretically mutant E1b virus may infect, replicate and
lyse p53 deficient cells but it does not affect normal cells with
functional p53.124 Thus, these mutant viruses are oncolytic to
cancer cells harboring p53 mutations. However, in prostate
cancer there is generally a lower rate of p53 mutations than in
other cancer types. Only 10% to 20% of prostate cancers have
mutated p53 and most such mutations tend to develop in higher
grades and stages of prostate cancer.119, 125, 126
Other replication competent oncolytic viruses were designed
based on an attenuated cytotoxic adenovirus type 5 vector incorporating a prostate specific enhancer and promoter coupled
to the E1a gene.127, 128 The E1a viral product enables the virus
to reproduce and enter the lytic cycle. The PSA promoter theoretically limits E1a production to PSA producing cells.127 The
level of E1a production was higher in PSA producing cells, such
as LNCaP, than in those producing little or no PSA.23
In vivo CN706 viral vector caused the regression of LNCaP
tumors and decreased PSA production after a single intratumoral injection.23, 127 Although intratumoral injection of
CN706 in patients with prostate cancer is the basis of a
clinical phase I trial, CN706 raises several clinical concerns.
Due to a lack of adequate large animal models to our knowledge there is no experimental support that systemically injecting CN706 lyses prostate cells exclusively. In some systems wide variation in the level of E1a expression has little
effect on inducing viral replication, implying that the level of
E1a does not correlate with viral replication and subsequent
cell lysis. This implication is especially worrisome since the
PSA promoter has been shown to be leaky because other
types of cells produce PSA in addition to prostate cells. For
example, cells that line the urethra produce abundant PSA.
Theoretically CN706 only ceases replication and lysis when
all PSA producing cells are eradicated. Furthermore, there is
some question of the usefulness of any PSA promoter vector
for treating cells that do not produce PSA and in patients on
androgen deprivation therapy. Nevertheless, studies of the
CN706 or any other vector containing the PSA promoter that
involve intratumoral injection would be critical for increasing
1129
our understanding of this field until newer, tissue specific
vector technology becomes a reality.
Combined gene therapy approaches and other treatment
modalities. As discussed, it is clear that neither surgery,
radiation therapy, hormonal therapy nor chemotherapy is
currently adequate alone or in combination for treating advanced prostate carcinoma. Gene therapy as monotherapy
against prostate cancer currently remains in its infancy.
Although preventive strategies are being entertained, the
ultimate clinical use of gene therapy for improving cancer
treatment would most likely be in combination with surgery,
radiation or chemotherapy in men with pathologically advanced disease or those at high risk for locally advanced
disease. Moreover, clinical trials using this multimodal approach are more justified until the science of gene therapy
becomes better understood. The most commonly used strategies involve gene therapy combined with DNA damaging
agents, although effective regimens combining gene therapy
with antimetabolites were also recently reported.129 Improved prostate cancer tumor suppression and apoptotic induction were described for Ad5CMVp53 combined with paclitaxel130 and Ad5CMVp53,131 Adp16 or Ad.Egr tumor
necrosis factor-␣132 combined with ionizing radiation. Taxol
appears to up-regulate Coxsackie adenovirus receptors on
the cell surface, enhancing transgene delivery and, therefore,
better gene expression.133 Other combined approaches to
prostate cancer include gene directed enzyme prodrug treatment Ad cytosine deaminase and flucytosine with radiation,134 and gene directed enzyme prodrug treatment. Ad
HSV-thymidine kinase with ganciclovir and AdIL-12 cytokine therapy were studied in prostate cancer.135 These types
of multimodal therapy for improving conventional prostate
cancer treatment represent the most promising immediate
clinical applications for prostate gene therapy.
PROSTATE CANCER GENE THERAPY CLINICAL TRIALS
Challenges of gene therapy clinical trials. In many ways
identifying a promising gene therapy target and generating
preclinical supporting data in the laboratory are the easy
initial steps on a long pathway to clinical trials. As with any
new drug, before performing a clinical trial basic in vivo
pharmacology and toxicology studies are mandatory. However, gene therapy trials are currently complicated by the
obligation to establish not only the pharmacology and toxicology of the therapeutic gene, but also the toxicity of the
gene vector, for example adenovirus, retrovirus, liposomes
and so forth. Successful efforts to improve the production of
increasingly higher viral titers have resulted in the ability to
deliver efficiently trillions of viral particles directly to a site.
The local, regional and systemic host distribution of and
response to these vectors must be established and monitored.
When a replication deficient viral vector is used to limit
potential transmission and expression toxicity, assays must
be developed and performed to detect replication competent
viruses in addition to screening for contaminating viruses.
Optimizing vector delivery by intravenous, intra-arterial or
direct injection may require additional investigation. Furthermore, viral shedding from a patient to the surrounding
equipment, personnel and family must be considered. As
standard gene delivery vectors and techniques are developed
and experience with them accumulates, these additional requirements may decrease with time
The toxicity of gene expression would largely be determined by the particular therapeutic strategy of a trial. For
example, gene replacement therapy that attempts to restore
physiological levels of gene expression may have limited toxic
side effects since most normal cells already express the target
gene. In other cases to limit toxicity expression of the therapeutic gene may be restricted to a specific tissue or cell type
by tissue specific gene regulatory elements such as PSA
1130
GENE THERAPY FOR PROSTATE CANCER
Current prostate cancer gene therapy trials136
NIH No.
Principal
Phase
Investigators
Institution
9408-082
I-II
Simons
9503-102
I-II
9509-123
I
9509-126
I
Gansbacher Memorial Sloan
Kettering
Steiner
University of
TennesseeVanderbilt
Chen
Bethesda Naval
9510-132
I
Paulson
9601-144
I
Scardino
9609-160
I
9702-176
Sponsor
Johns Hopkins
Pt. Population
Vector
Gene
Metastasis
Retrovirus
Prostate Ca
Retrovirus
Advanced disease
Retrovirus
Prostate Ca
Vaccinia virus PSA cDNA
Duke University
Baylor College
Locally advanced disease ⫹ metastasis
Radiation recurrence
AAV ⫹ liposome
Adenovirus
Dana-Farber
University
University of
Michigan
Prostate Ca
I-II
Kufe and
Eder
Sanda
9703-184
I
Belldegrun
9705-187
I
Hall
University of
Vical, Inc.
California,
Los Angeles
Mt. Sinai Medical Center
9706-192
I
Belldegrun
MFG-granulocyte colony Ex vivo autologous
stimulating factor
prostate Ca vaccine
Ex vivo allogeneic
IL-2 ⫹ interferon-␥
prostate Ca vaccine
Antisense myc
In vivo intraprostatic
injection
9708-205
I-II
Simons
9710-217
I-II
Introgen
9801-229
I-II
Logothethis M.D. Anderson
and
Cancer CenSteiner
ter University of Tennessee
Kadmon
Baylor
9802-236
I
Simons
Johns Hopkins
9805-251
I-II
Figlin
9812-276
I
9909-338
I-II
Gardner
and
Chung
Gingrich
IL-2
Rous sarcoma virus
HSV-thymidine kinase ⫹ ganciclovir
Vaccinia virus PSA cDNA
In vivo intradermal
injection
Ex vivo
In vivo intraprostatic
injection
In vivo intradermal
injection
In vivo intradermal
injection, autologous
In vivo autologous,
intratumoral
PSA recurrence after
radical retropubic
prostatectomy
Locally advanced
disease
Vaccinia virus PSA cDNA
Liposome
IL-2
Stages T1c, T2b ⫹ c
Adenovirus
Rous sarcoma virus
HSV-thymidine kinase ⫹ ganciclovir
Locally advanced disease ⫹ recurrence
Adenovirus
p53 Wild-type cDNA
Prostate Ca
Retrovirus
No treatment, high
risk
Adenovirus
Granulocyte colony
stimulating factor
p53 Wild-type cDNA
Ex vivo allogeneic
prostate Ca vaccine
In vivo intraprostatic
injection, radical
retropubic prostatectomy
No treatment, high
risk
Adenovirus
Rous sarcoma virus
HSV-thymidine kinase ⫹ ganciclovir
Calydon
Radiation recurrence
Adenovirus
University of
California,
Los Angeles
University of
Virginia
Transgene, SA
1-Mucin-1 pos.
PSA-E1a replication
competent
Vaccinia virus 1-mucin-1 IL2
In vivo intraprostatic
injection, radical
retropubic prostatectomy
In vivo intraprostatic
injection
Immunotherapy
Metastasis
Adenovirus
Osteocalcin HSV-kinase
⫹ valacyclovir
In vivo intratumoral
injection
University of
Tennessee
Genotherapeutics
Locally advanced
disease
Adenovirus
p16
In vivo intraprostatic
injection, radical
retropubic prostatectomy
University of
California,
Los Angeles
Johns Hopkins
Therion Biologics Corp.
Modality
Schering-Plough
Corp.
promoter. Therefore, restricted expression must be demonstrated in vitro and subsequently confirmed in an appropriate in vivo model, including toxicity studies before initiating
clinical trials. When the therapeutic approach involves conversion of a prodrug to an active metabolite in a suicide gene
therapy strategy, the simultaneous efficacy and toxicity of
the vector, prodrug and active metabolites must be investigated and differentiated.
These studies must be completed and then presented in
various formats, including a detailed clinical protocol and
investigator brochure to obtain approval from 1 or more
institutional review boards, biosafety committees, the NIH
Recombinant DNA Advisory Committee, and the Food and
Drug Administration. Because producing only the clinical
grade product for a gene therapy trial exceeds $300,000,
private foundation or corporate sponsorship is almost universally required. Laboratory research and clinical trials are
currently sponsored by a number of companies that are partially or completely devoted to urology related gene therapy
research (see table).136
In addition to identifying genetic targets and developing
adequate vector delivery systems with acceptable toxicity, a
major challenge in the field of gene therapy is establishing
In vivo intraprostatic
injection, radical
retropubic prostatectomy
In vivo intratumoral
injection
appropriate clinical end points by which to assess efficacy.
Traditional radiological methods, such as computerized tomography and MRI estimation of tumor volume before, during and after therapy, are unlikely to reflect adequately
changes in the molecular milieu within or surrounding cancer cells that ultimately determine the success or failure of
any given gene therapy. Although the PSA response has been
used as a surrogate marker of prostate tumor burden, changing PSA levels have not gained complete acceptance as an
accurate reflection of prostate cancer progression or response
to therapy. Therefore, appropriate direct or indirect biochemical end points or the expression of specific molecular markers involved in the initiation and progression of disease must
be identified and developed. It is likely that the molecular
response, stabilization or progression of disease would ultimately be monitored by combined gene expression in biopsy
samples, the detection of novel serum markers and more
dynamic imaging modalities, including the antibody detection of protein expression such as that of the ProstaScint*
111
indium capromab pendetide scan kit, or by more sensitive
* Cytogen Corp., Princeton, New Jersey.
GENE THERAPY FOR PROSTATE CANCER
direct functional assays using technologies such as positron
emission tomography.
Current gene therapy protocols for the prostate. At this time
18 gene therapy trials for prostate cancer have been approved by the NIH (see table).136 To date the approved trials
have had a phase I or I-II design. The preliminary results of
these trials have only recently been forthcoming as meeting
abstracts and peer reviewed publications. The initial approved gene therapy trial in prostate cancer of Simons et al
involved patients with metastatic prostate cancer in the
lymph nodes at radical prostatectomy (see table).67, 137 As in
previous studies of metastatic renal cell carcinoma treatment,138 the ex vivo transduction of autologous, irradiated
prostate tumor cells with retroviral MFG-granulocyte macrophage colony stimulating factor was done to generate vaccines that were administered subcutaneously every 2 weeks
until available cells were exhausted. Vaccination site biopsy
revealed infiltrating macrophages, dendritic cells, eosinophils and T cells. No dose limiting toxicity was observed. At
an average followup of 20 weeks ultrasensitive PSA criteria
revealed progression in 7 of 8 patients. This study demonstrated the feasibility of autologous granulocyte macrophage
colony stimulating factor transduced prostate cancer vaccines, limited only by the in vitro expansion of vaccine cells.
To circumvent this limitation a followup trial by Simons et
al was approved and is currently in progress that is powered
to estimate the efficacy of using the ex vivo, granulocyte
macrophage colony stimulating factor transduced, allogeneic
prostate cancer cell lines PC3139 and LNCaP140 as a vaccine
(see table). Patients are vaccinated weekly for 8 weeks with
irradiated, granulocyte macrophage colony stimulating factor secreting PC3 and LNCaP prostate cancer cells. Of the 21
patients treated to date 1 has a partial PSA response of
greater than 7 months in duration, 14 stable disease and 6
progression.141 By 3 months PSA velocity or slope decreased
in 71% of cases. No dose limiting toxicity was identified.
Although the dose and schedule are still being optimized to
determine potential therapeutic efficacy, numerous new
post-vaccination IgG1 antibodies were identified, indicating
that immune tolerance to prostate cancer associated antigens
may be broken. Therefore, this approach appears to be a
clinically feasible means of treating prostate cancer.
Preliminary results of the initial trial approved to use direct
transrectal prostatic gene therapy injection were recently reported by Steiner et al.34 To our knowledge this study was the
first to evaluate gene replacement strategy. A total of 21 men
with prostate cancer in whom standard therapy failed underwent ultrasound guided injection of the retrovirus LXSN containing BRCA-1 under the control of the viral promotor LTR.
No viral symptoms or evidence of viremia developed. Viral DNA
was detected by polymerase chain reaction at least 2 years after
injection (unpublished data). Although average prostate volume
plus or minus standard deviation was not decreased 1 month
after injection (30.9 ⫾ 6.4 versus 36.5 ⫾ 6.5 cc), serum PSA in
these cases of metastatic disease remained unchanged. This
study indicated the safety of prostate cancer gene replacement
therapy by direct injection.
Logothetis et al recently reported initial clinical gene therapy
results using a similar gene replacement strategy of replication
defective adenovirus containing wild-type p53 (AdCMVp53)
driven by the CMV promoter before radical prostatectomy.142
To date 17 patients with locally advanced prostate cancer received at least 1 course of AdCMVp53, involving 3 doses of 3 ml.
in 4 to 6 divided transperineal injections 14 days apart under
transrectal ultrasound guidance. In this phase I-II study the
number of viral particles delivered was escalated from 3 ⫻ 1010
per treatment per patient to 3 ⫻ 1012. Three patients who
completed a repeat course of therapy had a greater than 25%
decrease in tumor size, as measured by endorectal coil MRI
after the initial treatment course. There was no grade 3 or 4
toxicity in the 14 evaluable patients. This study confirmed the
1131
relative feasibility and safety of intraprostatic gene therapy
injection. Further results on efficacy due to the apparent radiological responses, correlations with gene expression, pathological findings at prostatectomy and surgical outcomes are pending at this time.
The initial trial of intraprostatic injection of a locally cytotoxic
or suicide type of gene therapy was recently completed by Herman et al.42 For locally recurrent prostate cancer after definitive radiation therapy 18 men received a single 1.1 cc injection
of replication deficient adenovirus containing the HSVthymidine kinase gene under the RSV promoter, followed by 14
days of intravenous ganciclovir. The dose of Ad HSV-thymidine
kinase was escalated from 1 ⫻ 108 to 1 ⫻ 1011 IU. After treatment 3 and 1 patients had a greater than 50% decrease in PSA
and negative biopsy, respectively. Local and systemic toxicity
was mild except for the final patient, in whom severe thrombocytopenia developed with abnormal liver function test results.
Another study to investigate multiple sites of injection before
radical prostatectomy is currently in progress. These followup
studies may provide important histological confirmation of the
tumor response to intraprostatic cytotoxic gene therapy. In addition, assessing the long-term patient outcomes of beneficial
local or possible systemic immunological bystander effects
would be interesting.
An alternative approach to intraprostatic gene replacement
or cytotoxic therapy that may indirectly enhance a host immunological response against tumor cells is the administration of
gene therapy to stimulate intentionally an immune response.
Using a liposomal vector Patel et al treated 12 patients before
radical prostatectomy and 9 with recurrent prostate cancer
after radiation or cryotherapy with 2 injections of intraprostatic
IL-2.143 A total of 40 injections of 300 to 1,500 ␮g. IL-2 were
administered. In the men treated before radical prostatectomy
average PSA decreased by 5.3 ng./ml. preoperatively and in
75% undetectable PSA was maintained 24 to 56 weeks postoperatively. Average PSA decreased 3.6 and 1.3 ng./ml. after 1 or
2 injections, respectively, in patients with recurrent disease.
Although the significance of these PSA responses and differentiation of the effects of gene therapy from surgery only are not
yet clear, this liposomal delivery strategy appears to be safe. In
addition, it may be effective locally and promote a systemic
immune response.
Three trials were approved and initiated using Prostvac,†
the recombinant vaccinia virus expressing PSA, as a tumor
associated antigen immunotherapy strategy (see table). Chen
et al treated 30 men with hormone refractory prostate cancer
who were withdrawn from antiandrogen therapy with a vaccinia inoculation followed by Prostavac immunization.144 Patients received 2.65 ⫻ 105 or 2.65 ⫻ 106 plaque forming units
by dermal scarification, or subcutaneous administration of
2.65 ⫻ 106 or 2.65 ⫻ 108 plaque forming units once monthly
for 3 months, followed by repeat staging. Local erythema
developed at the vaccination site in all cases and all toxicity
was grade 0 to 1. Disease was stable in 4 of the 14 patients
who completed the treatment course, including 2 with subsequent progression at 5 and 6 months, while disease continued to progress in 10.
In a population with less advanced disease Eder et al treated
24 men with increasing PSA after radical prostatectomy and/or
radiation therapy with 2.65 ⫻ 106 – 8 plaque forming units as 3
consecutive monthly doses without significant toxicity.145 Patients were withdrawn from the protocol when there was clinical progression or 3 monthly increases in PSA greater than
50% of baseline. Of the 23 men 12 maintained stable disease
status for 10 months or greater. In another study Sanda et al
administered Prostvac once in 6 men with androgen modulated
prostate cancer recurrence after radical prostatectomy.146 In
addition to toxicity, they evaluated time to serum PSA increase
after the interruption of androgen deprivation therapy and per† Therion Biologics, Cambridge, Massachusetts.
1132
GENE THERAPY FOR PROSTATE CANCER
formed Western blot analysis to determine anti-PSA antibody
production. Again no dose limiting toxicity was observed. Undetectable serum PSA was maintained in 1 case more than 8
months after antiandrogen therapy was withdrawn and immunization done. This study emphasized the variable interval to
the return of serum testosterone levels after antiandrogen therapy is withdrawn, as observed by Oefelein.147 An IgG antibody
against PSA developed after immunization in 1 case. Notably 2
of 6 patients had anti-PSA antibodies before immunization. The
significance of this finding and the implications of vaccination
against tumor associated antigens is unclear at this time. However, it was demonstrated that an immune response may be
solicited by this gene therapy strategy. Additional trials of variations of these types of gene therapy strategies were approved
for locally advanced and metastatic prostate cancer (see table).
The NIH Office of Recombinant DNA Activities regularly updates its comprehensive list of gene therapy trials.136
CONCLUSIONS
This decade marks the beginning of history when human
gene therapy has become a biomedical commodity. Since the
initial gene therapy trial in 1990, there have been more than
125 phase I, 25 phase II and 1 phase III human clinical trials
in the United States. Worldwide more than 363 clinical gene
therapy trials have been approved with more than 4,000
patients enrolled. Of the various diseases treated malignancy
is first at 68% of cases, followed by AIDS at 18% and cystic
fibrosis at 8%. Widespread clinical acceptance of this new
field and progress in identifying new potential target genes
aided by the Human Genome Project have further fueled the
rapid expansion of this new technology.
Gene therapy for advanced localized prostate cancer is
safe and feasible. However, it is clear that gene therapy for
prostate cancer remains in its infancy since to our knowledge no clinical data yet demonstrate even short-term gene
therapy activity with a significant clinical response. The
challenges that lie ahead for the widespread use of this
technology in this century include finding the appropriate
target or therapeutic genes for gene therapy, determining
the safety of gene therapy in humans, identifying strong
tissue specific promoters and other ways to target prostate
cancer exclusively, systemically delivering gene therapy to
distant prostate cancer cell targets, and ultimately developing appropriate short-term and mid-term end points to
assess the efficacy of this new treatment option for human
prostate therapy.
APPENDIX: FEATURES AND LIMITATIONS OF VECTORS THAT MAY BE USED FOR PROSTATE CANCER GENE THERAPY
Vector
Nonviral:
Cationic liposomeDNA complex
Features
Synthetic DNA complex
No size limitations of gene insert
Low immunogenicity
Protein/DNA
complex
Potential for cell-specific targeting
No size limitations of gene insert
Colloidal gold DNA
complex
Gene gun
No size limitation of DNA
Viral:
Retrovirus
Adenovirus
Adeno-associated
virus (Parvovirus)
Vaccinia virus
Limitations
No integration
Low efficiency gene transfer
Systemic administration unlikely
Toxicity (?)
No integration
Immunogenicity
Low efficiency gene transfer
Toxicity (?)
Local damage to normal cells
Low efficiency gene transfer
Toxicity (?)
May require surgical procedure
Systemic administration unlikely
Ex vivo use only (?)
Commonly employed in clinical trials
Transduces many cell types
Integrates into cell’s genome
Safety
Commonly employed in clinical trials
Transduces many cell types
Transduces both dividing and nondividing cells
High viral titer production
High gene transfer efficiency
Transient gene expression
Integrates into cell’s genome
Transduces many cell types
Transduces dividing and nondividing cells
High viral titer production
High gene transfer efficiency
Nonpathogenic
Immunogenicity
May carry large gene
HSV
Neuronal tropism
Latency expression
Avianpox virus
Transduces both dividing and nondividing cells
Immunogenicity
Transduces only dividing cells
Low efficiency gene transfer
Systemic administration unlikely
Low viral titer production
Does not integrate
Systemic administration unlikely
Immunogenicity
Transient gene expression
Immunogenicity
Difficult to mass produce for clinical trials
Systemic administration uncertain
Immunogenicity
Low efficiency gene transfer
Transient gene expression
Toxicity (?)
Toxicity (?)
Latency expression
Mass production (?)
Low efficiency gene transfer
Toxicity (?)
GENE THERAPY FOR PROSTATE CANCER
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