Gene therapy of prostate cancer: current and future directions Endocrine-Related Cancer

Transcription

Endocrine-Related Cancer (2002) 9 115–139
Gene therapy of prostate cancer:
current and future directions
N J Mabjeesh, H Zhong and J W Simons
Winship Cancer Institute, Department of Hematology and Oncology, Emory University School of Medicine,
1365 Clifton Road, Suite B4100, Atlanta, Georgia 30322, USA
(Requests for offprints should be addressed to J W Simons; Email: jonathan—[email protected])
Abstract
Prostate cancer (PCA) is the second most common cause of death from malignancy in American
men. Developing new approaches for gene therapy for PCA is critical as there is no effective
treatment for patients in the advanced stages of this disease. Current PCA gene therapy research
strategies include cytoreductive approaches (immunotherapy and cytolytic/pro-apoptotic) and
corrective approaches (replacing deleted or mutated genes). The prostate is ideal for gene therapy. It
is an accessory organ, offers unique antigens (prostate-specific antigen, prostate-specific membrane
antigen, human glandular kallikrein 2 etc.) and is stereotactically accessible for in situ treatments.
Viral and non-viral means are being used to transfer the genetic material into tumor cells. The number
of clinical trials utilizing gene therapy methods for PCA is increasing. We review the multiple issues
involved in developing effective gene therapy strategies for human PCA and early clinical results.
Introduction
New therapeutics like gene therapy are needed urgently for
advanced prostate cancer (PCA). PCA remains the most
common solid tumor and the second leading cause of cancerrelated deaths among men in the USA. An estimated 180 400
new cases and 31 900 PCA-related deaths are expected for
2000 (Cookson 2001). The current standard therapies
employed for organ-confined PCA include radiation or surgery, in some circumstances incorporating neoadjuvant or
adjuvant hormonal therapy. While these therapies are relatively effective in the short-term, a significant proportion of
patients initially presenting with localized disease ultimately
relapse. Moreover, each of these therapies may incur
unwanted side-effects. Ultimately, the major risk faced by
patients with PCA is the development of metastatic disease.
For 60 years the mainstay of therapy for metastatic disease
has been androgen ablation. While this provides cytoreduction and palliation, progression to hormone-refractory
disease typically occurs within the order of 14–20 months
(Crawford et al. 1989). A great number of clinical research
studies have been reported in the field of chemotherapy for
advanced androgen-independent PCA during the last ten
years (Culine & Droz 2000). So far, no combination of
chemotherapy reported has improved overall survival of
patients. Docetaxel combined with estramustine, however, is
in randomized trials to assess its impact on survival. An
advance in medical oncology of the 1990s is the demon-
stration of the impact of chemotherapy (mitoxantrone+
prednisone) on quality of life as compared with prednisone
alone in advanced hormone-refractory PCA. At the present
time, chemotherapy should be considered as a palliative or
investigational treatment in patients with symptomatic androgen-independent disease (Morris & Scher 2000).
An improving understanding of the biology of PCA has
changed the outlook for patients with this disease. We now
have available approaches designed to modulate the immune
system, inhibit the metastatic cascade, block angiogenesis,
promote apoptosis and alter signal transduction.
Gene therapy has been much heralded since its inception
with the first patient treated in 1990, but in oncology has yet
to reach phase III trials or show clear cancer patient benefits
in large statistically powered phase II trials (Anderson 1998).
Nonetheless, in recent years there has been progress in the
design and execution of gene transfer clinical studies. Progress in this field provides a rational basis for optimism that
gene therapeutic strategies may significantly improve PCA
treatment outcome administered either alone or in combination with current therapies. In this report we review the multiple issues involved in developing effective gene therapy
strategies for human PCA.
Rationale for PCA gene therapy
The promise of gene therapy lies in its potential for selective
potency. To achieve this aim, cancer gene therapy strategies
1351-0088/02/009–000  2002 Society for Endocrinology Printed in Great Britain
Online version via http://www.endocrinology.org
N J Mabjeesh et al.: Prostate cancer gene therapy
attempt to exploit the biological uniqueness of each particular
tumor. The human prostate is an accessory organ and is not
required for potency or urinary continence. Thus unique targets showed by PCA and the normal prostate are candidates
for new treatment. The Prostate Expression Database is an
online resource designed to access and analyze gene expression information derived from the human prostate (Nelson et
al. 2000). It has revealed more than 55 000 expressed
sequence tags (ESTs) from 43 cDNA libraries; of these
approximately 500 are prostate-unique (Nelson et al. 2000).
The presence of this large number of prostate-unique promoters and candidate antigens promises to facilitate the design
of prostate-specific gene therapy. Ideally, both the clinical
and genetic features of PCA should be taken into account in
the design of effective prostate gene therapeutic strategies
(Table 1).
Cancer gene therapy may be defined as the transfer of
recombinant DNA into human cells to achieve an antitumor
effect. Depending upon the strategy, DNA may be introduced
either into cells removed from the body – the ex vivo
approach – or introduced directly into cells in their normal
location – the in vivo approach (Anderson 1998).
Gene transfer systems
Efficient gene transfer requires the use of a vector. All vectors contain at a minimum the transgene of interest linked
to a promoter to drive its expression (Galanis et al. 2001).
Increasingly wider ranges of viral and synthetic vectors are
available, each with characteristic advantages and limitations
(Table 2). However, most vectors used for gene therapy contain promoters that are tissue-non-specific, allowing gene
expression to occur in unintended tissues and resulting in the
potential for systemic toxicity.
In choosing between vectors, attributes to consider
include maximal transgene size permissible, maximal titer of
viruses attainable, tendency to provoke inflammatory/
immune response, persistence of gene expression, ability to
transduce non-dividing cells, effect of vector genome on
desired therapy, target cell-specificity, and transduction effi-
Table 1 PCA-specific gene therapy strategies
Anatomy
PCA-specific features
Relevance to gene therapy
Localized tumor accessible for procedures
such as brachytherapy
Accessibility for localized gene therapy and
combined modality therapy in vivo by
transurethral/transrectal routes
Complete ablation of the normal organ in addition
to the tumor is clinically undeleterious
Cytoreductive transgene expression required only
for duration necessary for prostate tissue
ablation
Preventive gene therapy strategies may be
feasible
Definition of genetic/epigenetic events underlying
molecular progression provides rational basis for
preventive/corrective strategies
Targeting of systemic gene therapy for advanced
disease must effectively target bone and
lymphatics
Supports use of novel combined modality
treatments to improve local control
Pressing need for alternative systemic therapies
Effective vectors for in vivo gene delivery must
transduce both non-dividing as well as dividing
cells
Tumors are resistant to S-phase-dependent
cytotoxic strategies
Define gene therapy targets including tumor
suppressors, oncogenes etc., for prevention and
for treatment of disease at various stages
Accessory organ
Natural history
Long pre-clinical latency
Propensity for metastatic spread to lymphatics/
bone
Current treatment efficacy Failures even for localized disease
Biology
Prostate-specific genes
and antigens
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Ineffective for advanced disease
Low mitotic rate
Clinical/biological transitions:
쐌 Prostatic intraepithelial neoplasial
(PIN)씮carcinoma
쐌 Androgen-dependent씮androgenindependent tumor growth
앑500 or more prostate-specific ESTs in
addition to current known markers such as
PSA/PSMA
Facilitate prostate specific promoter/antigen
targeting and therapeutic monitoring
www.endocrinology.org
Table 2 Vectors for gene therapy
Vector
Gene therapy
applications
Advantages
Disadvantages
Insert
size
Immune
response
MMLV-based
used in majority
of current
clinical trials
Ex vivo
transduction for
stable gene
expression
Transmitted to progeny of
transduced cell
Potentially long term
transgene expression
6–8 kb
No
Lentiviruses
Preclinical
testing, stable
infection of
non-dividing
cells
Can infect non-dividing cells
Long-term transgene
expression
Not inactivated by human
serum
6–8 kb
No
HFVs
Preclinical
testing, stable
infection of
non-dividing
cells
Non-pathogenic
Infect non-dividing cells
Polycations not required for
efficient transduction
Not inactivated by human
serum
Infect actively dividing
cells only
Risk of insertional
mutagenesis
Relatively low titers
Low transduction
efficiency
Inactivated by
complement in vivo
Difficult to target
attachment
Safety: possible
recombination with
endogenous HIV/other
viruses
Risk of insertional
mutagenesis
Low titers
5–7 kb
No
Vaccine-based
gene therapy
Large insert size
Replicates in vivo
25 kb
Yes
HSV
Neural tropism
10–100 kb
Yes
Ad
In vivo use
Potentially large insert size
(amplicons)
Episomal no risk insertional
mutagenesis
Moderate efficiency
Wide target cell range
Concentrate to high titers
High transduction efficiency
Episomal, no risk of
insertional mutagenesis
High transgene expression
Low efficiency
Pre-existing immunity
from prior vaccinations
limits expression
duration to 앑1 month
Expression transient
Cytopathic
Difficult to produce
high titers
7.5 kb
AAV
In vivo use
Transient expression
without passage to
progeny
Ad infection provokes
cell-mediated immune
response limiting
duration of expression
Humoral response
makes reinfection less
feasible with rapid
clearance of vector
Local tissue
inflammation
Limited insert size
Propensity for
rearrangement during
integration
Risk of helper Ad and
wild-type AAV
contamination
Insertional
mutagenesis possible if
rep gene deleted
RNA virus RV vectors
RVs
(oncoviruses)
DNA virus vectors
Vaccinia virus
Non-pathogenic
Wide target cell range
High transduction efficiency
Site-specific integration
chr 19
2–4.5 kb
Yes
No
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Table 2 cont.
Vector
Synthetic vectors
Naked DNA
Gene therapy
applications
Advantages
Disadvantages
Insert
size
Immune
response
DNA vaccines
Low cost
Simple preparation
Non-toxic
Large insert size
Safe for repeated
administration
Non-toxic
Low cost
Suited for targeting
Large insert size
Safe for repeated
administration
Non-toxic
Low cost
Suited for targeting
Poor efficiency
Unlimited
No
Poor efficiency
Prone to degradation/
short expression
50 kb
No
Poor efficiency
Prone to degradation/
short expression
50 kb
No
Liposomes
In vivo/ex vivo
use
DNA–protein
complex
In vivo
ciency. Furthermore, it is important to consider the goals of
the overall strategy. Broad categories encompassing the current gene therapy strategies include cytoreductive (includes
immunotherapy and cytolytic/pro-apoptotic approaches) and
corrective approaches.
Viral vectors
Viral vectors are used in the vast majority of gene therapy
trials owing to their relatively higher efficiency at gene transfer compared with the synthetic vectors. Viral vectors may
be RNA virus-based, or DNA virus-based. RNA viruses
include the retroviruses (RVs) among which are the oncoviruses such as murine leukemia virus (MuLV) and mouse
mammary tumor virus (MMTV), the lentiviruses derived
from human immunodeficiency virus (HIV), and the spumaviruses such as human foamy virus (HFV). The DNA viruses
include adenovirus (Ad), adeno-associated viruses (AAVs),
vaccinia virus, and herpes simplex virus (HSV). To increase
their safety, viral vectors may be designed to be replicationdeficient, with no further virus particles generated following
infection of the target cells. Alternatively the viral vectors
may be replication-competent or replication-attenuated, in
which case viral replication can occur in permissive cells.
Ad vectors
Ad vectors are well suited to a variety of gene therapy strategies owing to their broad host range, low potential for
pathogenicity, and relatively high gene transfer efficiency.
Ad consists of a linear double-stranded DNA genome of 30–
40 kb. Binding and internalization of Ad to target cells are
dependent on the presence of the coxsackie Ad receptor
(CAR) and of the αvβ3 and αvβ5 integrins (Wickham et al.
1993, Roelvink et al. 1998). The interaction of Ad particles
with CAR is mediated by the Ad fiber protein knob domain
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(Roelvink et al. 1998). The fiber protein may be reengineered
to bind to a variety of cell-surface molecules (Miller et al.
1998). Fiber protein reengineering together with prostatespecific promoter/enhancers may allow construction of Ad
vectors targeted for binding to prostate-specific cell-surface
markers such as prostate-specific membrane antigen (PSMA)
and at the same time restricted for replication or transgene
expression to prostate cells (Lee et al. 1996, Rodriguez et
al. 1997, Gotoh et al. 1998). Following attachment, Ad is
internalized through coated pits into endosomes, then is disassembled and extruded into the cytosol prior to lysosomal
fusion, thereby avoiding lysosomal degradation (Silman &
Fooks 2000). The DNA-protein core then penetrates the
nuclear pores into the nucleus and gene expression commences. The first of two phases of Ad gene expression results
in expression of the early genes (E1–E4), which are essential
to DNA replication, and subversion of host antiviral defenses. If replication-competent, the Ad proceeds to replicate
episomally, culminating in host cell lysis and the release of
newly produced viral particles. The lack of proviral integration results in gene expression that is less sustained than
with the RVs and gradually lost with subsequent cell divisions. Ad vectors have high safety owing to their mild associated clinical pathogenicity and extrachromosomal life
cycle, which eliminates the risk of insertional mutagenesis.
The administration of an Ad vector triggers both cellular
and humoral immune responses, which clear remaining virus
and also function to eliminate transduced cells, thereby limiting the duration of transgene expression. The anti-Ad
immune response rapidly clears subsequent injections of Ad
vector, severely limiting the efficacy of readministered vector
(Yang et al. 1996). Intratumoral readministration of Ad elicits profound humoral and cellular immune responses to Ad
and their transgenes (Nagao et al. 2001). Following endocytosis, virions are released from endosomes and undergo
MHC-1 processing and presentation prior to any de novo Ad
protein synthesis.
It has been possible to increase the duration of Ad
transgene expression through the use of cytotoxic T-lymphocyte (CTL) blocking agents such as cytotoxic T-lymphocyte
antigen-4Ig (Kay et al. 1995) or immunosuppressive drugs
(Dai et al. 1995). In addition to specific anti-Ad/transgenedirected immune responses, non-specific inflammation may
also significantly reduce transgene expression (Otake et al.
1998). The ability to suppress immunogenicity towards Ad
vectors would be of enormous benefit to in vivo gene therapy
and would allow multiple reinfections with the same or different transgenes delivered.
The resulting improvements in gene localization from
targeted Ad vectors are likely to reduce immunogenicity and
toxicity, increase safety, and enable the systemic administration of these vectors for cancer. Two basic requirements are
necessary to create a targeted Ad vector: interaction of Ad
with its native receptors must be removed and novel, tissuespecific ligands must be added to the virus. Two general
approaches have been used to achieve these basic requirements. In the ‘two-component’ approach, a bispecific
molecule is complexed with the Ad. The bispecific component simultaneously blocks native receptor binding and
redirects virus binding to a tissue-specific receptor. In the
‘one-component’ approach the Ad is genetically modified to
ablate native receptor interactions and a novel ligand is genetically incorporated into one of the Ad coat proteins. Twocomponent systems offer great flexibility in rapidly validating the feasibility of targeting via a particular receptor.
One-component systems offer the best advantages in producing a manufacturable therapeutic agent and in more completely ablating all native Ad receptor interactions. The
coming challenges for targeted Ad vectors will be the demonstration that the technology performs in vivo. Ultimately,
or in parallel, ‘receptor-targeting’ technology can be combined with improved Ad backbones and with ‘transcriptionaltargeting’ approaches to create Ad which deliver genes selectively, safely, and with minimal immune response (Wickham
2000).
In addition to their immunogenicity, another limitation
of Ad vectors is their limited transgene capacity of approximately 8 kb. Increased insert capacity and decreased
immunogenicity has been observed with modified Ad vectors
known as ‘gutless’ Ad vectors, or as encapsidated Ad minichromosomes (EAMs). In EAMs, almost the entire Ad
genome has been deleted with the exception of the inverted
repeats necessary for replication, and the encapsidation/packaging signals (Kumar-Singh & Farber 1998). EAMs have a
theoretical cloning capacity of 36 kb and have facilitated the
transfer of multi-gene expression units as large as 28 kb.
These vectors offer exciting prospects for the transfer of
genes with large regulatory regions intact, or of multiple gene
cassettes. An even more radical Ad modification for gene
transfer is the ‘Ad dodecahedron’ which is composed of only
two of the 11 Ad structural proteins and contains no Ad
DNA. The vector consists of the penton base and fiber protein, which are sufficient to mediate efficient attachment, cell
entry, and escape from endosomal degradation (Fender et al.
1997). The reduction in virion proteins presented to the
immune system is intended to decrease the immunogenicity
of this vector, making it even more suitable than EAMs for
repeat application.
AAVs
The potential applications of the AAV as a gene transfer
system vector have expanded dramatically in the last decade.
AAVs are a non-enveloped DNA virus belonging to the
family Parvoviridae. AAVs have a linear 4.7 kb genome
encoding a group of regulatory genes – the rep genes – and
a group of structural genes – the cap genes. AAVs must be
coinfected with a helper virus such as Ad for productive lytic
infection, otherwise they remain a latent provirus in the host
genome. The Ad E1/E4 gene products are essential for AAV
transcription and the AAV lytic phase (Richardson &
Westphal 1981). Another unique feature of the AAVs is their
capacity for site-specific integration. The preferred site of
integration for wild-type AAV is chromosome 19q14.3
(Kotin et al. 1990). The capacity for site-specific integration
is rep gene-dependent and deletion of rep results in random
integration. AAVs are limited to a transgene size of approximately 4.5 kb, smaller than the capacity of the Ad vectors.
Unlike RVs, AAVs may infect non-dividing cells. In
addition, the AAVs elicit little or no immune/inflammatory
response, which together with their integration into the target
cell genome confers the potentially repeated administrations
and stable long-term transduction. The AAVs are not associated with any known pathogenicity or oncogenicity. Recent
advances in the production of high-titer purified rAAV vector
stocks have made the transition to human clinical trials a
reality (Monahan & Samulski 2000).
RV vectors
RVs of the oncovirus subfamily are single-stranded RNA
viruses with a 7–10 kb genome. The RV life cycle begins
with attachment to a cell-surface receptor via an envelope
(env) surface protein. Depending on their env glycoprotein,
RVs may be either amphotropic – able to infect all species,
or ecotropic – able to infect only mouse cells. The amphotropic variety of MuLV attaches to cells via the RAM-1
receptor (Kavanaugh et al. 1994), a widely expressed
inducible sodium phosphate symporter. Receptor-mediated
endocytosis occurs following attachment. Then, the outer env
protein coat is shed and the virus genome undergoes reverse
transcription by viral reverse transcriptase resulting in the
synthesis of a double-stranded DNA intermediate. This viral
nucleoprotein complex then enters the cell nucleus and integrates randomly via the viral long terminal repeat sequences
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(LTRs) into the host genome. The stably integrated viral
genome is termed a provirus. Because of their stable integration into the target cell genome and transmission to the
progeny of the transduced parent cell, RV vectors have the
potential for sustained duration of transgene expression, a
major advantage particularly for corrective gene therapy strategies (Lin 1998).
The capacity for random proviral integration into the host
genome raises the concern of potential insertional mutagenesis. Wild-type Moloney muzine leukemia virus
(MMLV) can induce leukemias and lymphomas in mice after
long latency. To limit this risk, the minimum total RV dose
necessary to achieve a therapeutic endpoint must be utilized.
The use of replication-deficient virus also minimizes the
amount of virus applied to the target tissue.
For the oncovirus subfamily of RVs which includes
MMLV- and MMTV-based RVs, the entry into the cell
nucleus to undergo proviral integration is cell-division
dependent (Miller et al. 1990). This is a significant limitation
on the usefulness of oncovirus family-based vectors for gene
therapy in vivo, particularly in PCA with its low mitotic rate.
Another feature of RV vectors which may limit their suitability for targeted gene therapy strategies in vivo is the relatively non-specific tropism of the amphotropic viruses to the
ubiquitous RAM-1 receptors. The tropism of RV vectors can
be modified through modification or replacement of the env
gene product. Modification of RV targeting through the display of single-chain antibodies to antigens such as carcinoembryonic antigen, transferrin receptor, Her2/neu, and CD34
has been recently reported, suggesting that this approach may
improve the suitability of RV vectors for use in vivo (Jiang
et al. 1998). Another major hindrance to the use of RV vectors in vivo is the rapid inactivation of RVs by human complement.
In view of the above limitations, RV vectors are typically
used for ex vivo gene therapy for which they are well suited,
as the issues of non-specific transduction and inactivation by
complement are avoided (Cornetta et al. 1990). The most
common use of the RV vectors for PCA gene therapy has
been for ex vivo cytoreductive immunotherapy.
Lentivirus and spumavirus vectors
The lentiviruses are a subfamily of the RVs, which includes
HIV, bovine leukemia virus, and human T-cell leukemia
virus (HTLV)-1 and HTLV-2. Unlike members of the oncoviridae subfamily such as MMLV and MMTV, the lentiviruses are able to integrate into non-dividing cells, an important advantage for gene therapy in vivo. For the lentivirus
HIV, this ability is a result of the viral proteins vpr and viral
matrix protein p17 (Bukrinsky et al. 1993, Gallay et al.
1995a). The phosphorylation of a terminal tyrosine residue
on p17 permits transport of the HIV pre-integration complex
into the nucleus even in the absence of mitosis (Gallay et al.
1995a,b). Another favorable feature of the lentiviral vectors
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is their ability to direct sustained transgene expression for
greater than 6 months’ duration (Miyoshi et al. 1997). The
target cell range for wild-type HIV is restricted mainly to
CD4+ cells. This limitation has been circumvented experimentally for HIV-based vectors by substituting env
sequences from other RVs for those of HIV to confer varying
and broader cell specificity (Naldini et al. 1996, Nascone &
Mercola 1997). Safety concerns have tempered enthusiasm
for the use of HIV-based recombinant vectors. While helperfree virus stocks of HIV-based vectors have been successfully produced, the small risk of pathogenic recombination in
vivo supports the development of non-HIV-based/non-human
lentiviral vectors for clinical application. Recently, Loimas
et al. (2001) found that lentiviral vectors carrying a fusion
gene of HSV-thymidine kinase (TK) (HSV-TK) were efficient vehicles for three human PCA cell lines DU-145,
LNCaP and PC-3. Despite sufficient gene transfer rates (25–
45%) in the ganciclovir (GCV)-sensitivity experiment, only
DU-145 cells were efficiently destroyed under clinically relevant GCV concentrations (Loimas et al. 2001).
The spumaviruses are another subfamily of the RVs and
include the HFVs. HFV is non-pathogenic and has the
capacity for integration into the genome of a wide range of
non-dividing cells (Russell & Miller 1996). These are not
inactivated by human serum as are the oncoviruses and may
represent a safer alternative for clinical gene therapy than
HIV.
Herpes virus vectors
The herpes viruses are enveloped DNA viruses of the family
Herpesviridae. The HSV-1 genome consists of a doublestranded linear DNA of 152 kb. Other members of the herpes
virus family include HSV-2, varicella zoster, cytomegalovirus (CMV), Epstein–Barr virus, human herpes virus
(HHV)-6 and HHV-7. The herpes viruses replicate in the
nucleus episomally. Following replication, viral particles
reach the cell surface via the endoplasmic reticulum. HSV-1based vectors have been used extensively in gene therapy for
neural tissue and tumors, given their inherent neural tropism.
Advantages of HSV-1-based vectors include their large insert
capacity of 35 kb, and their ability to infect dividing as well
as non-dividing cells. Disadvantages of the HSV-based vectors include their potential pathogenicity, poor transduction
efficiency and the short duration of gene expression
achieved.
Vaccinia virus vectors
Vaccinia virus-based vectors have been used extensively for
in vivo immunotherapy strategies. These DNA virus vectors
have a wide host range and may transfer large inserts of up
to 25 kb, facilitating multi-gene transfer (Peplinski et al.
1998). They have been used extensively for delivery of antigen and immune-stimulatory genes for vaccine-based gene
therapy (Hodge et al. 1994). These vectors may replicate in
vivo, further increasing the efficiency of host cell transduction. Their utility for in vivo gene therapy is limited by the
strong immune response they elicit, and pre-existing antivaccinia immunity is common as a result of childhood smallpox immunization. The immunological elimination of the
vaccinia-transduced cells limits gene expression to about 4
weeks. For the vaccine applications for which vaccinia vectors are typically used, this duration of expression is sufficient for priming an immune response. Booster vaccine
administrations may be more effective if given using a
heterologous vector, which is not immunologically crossreactive (Irvine et al. 1997).
Synthetic gene transfer systems
Several non-viral methods for gene delivery are under intense
investigation. These include chemical methods such as a variety of liposomes and physical methods such as microinjection electroporation and biobalistics (Prince 1998).
Plasmid DNA vectors
The most technically simple form of gene therapy is the use
of naked DNA. A plasmid containing the gene of interest
and appropriate promoter is injected directly into a desired
site. Administered in this way, the efficiency of transgene
uptake/expression is very low. The transforming DNA does
not integrate into the host genome and the duration of expression is short. Of all sites tested so far, the greatest transduction efficiency is observed in muscle (Wolff et al. 1990). In
this application, brief low-level expression may be sufficient
for eliciting a therapeutic immune response. Animal studies
have indicated that the use of syngeneic dendritic cells (DCs)
that have been transfected ex vivo with DNA for tumorspecific antigen results in tumor regression and decreased
number of metastases. Additional studies have also suggested
the possibility of modulating the DCs in vivo either by
‘naked’ DNA immunization or by injecting replicationdeficient viral vectors that carry the tumor-specific DNA.
Using PSMA as a target molecule, Mincheff et al. (2000)
have initiated a clinical trial for immunotherapy of PCA.
They have included the extracellular human PSMA DNA as
well as the human CD86 DNA into separate expression vectors (PSMA and CD86 plasmids), and into a combined
PSMA/CD86 plasmid. In addition, the expression cassette
from the PSMA plasmid was inserted into a replicationdeficient Ad expression vector. Twenty-six patients with
PCA were entered into a phase I/II toxicity–dose escalation
study. No immediate or long-term side-effects following
immunizations have been recorded. All patients who
received initial inoculation with the viral vector followed by
PSMA plasmid boosts showed signs of immunization as evidenced by the development of a delayed-type hypersensitivity reaction after the PSMA plasmid injection. In contrast, of
the patients who received a PSMA plasmid and CD86 plas-
mid, only 50% showed signs of successful immunization.
However, several responders, as evidenced by a change in
the local disease, distant metastases, and prostate-specific
antigen (PSA) levels, were reported (Mincheff et al. 2000).
A phase II clinical study to evaluate the effectiveness of the
therapy is currently underway.
Liposome vectors
Liposomes are the second most frequently used gene transfer
method in current clinical trials. Liposome vectors consist of
a DNA plasmid surrounded by a liposomal coat. The charge
characteristics of the liposomal coat play a major role in
determining the targeting capacity, intracellular stability and
transgene size capacity of the vector. Anionic liposomebased vectors do not display non-specific cell-surface binding
and thus may be targeted to specific cell-surface moieties
(Lee & Huang 1996). Anionic liposomes are endocytosed by
the target cell, targeted to endosomes, and subsequently
prone to degradation by lysosomal nucleases severely limiting efficiency of transgene expression. Cationic liposomes
bind avidly and non-specifically to the cell surface and are
taken up into cells by a non-receptor-mediated process. They
are less likely to undergo endosomal degradation than
anionic liposomes and can also package much larger
transgenes. A commonly used cationic liposomal vector in
clinical trials is Lipofectin.
Liposomes display preferential uptake in cells of the
reticuloendothelial system (RES), complicating attempts at
targeting. Modification to decrease this unwanted uptake
includes the addition of sialic acid residues to make ‘stealth’
liposomes (Lasic et al. 1991) which may evade RES uptake.
It has been suggested that the presence of leaky vessels in
solid tumors may favor the selective accumulation of sterically stabilized large vectors such as liposomes (Brown &
Giaccia 1998). Liposomal vectors are inexpensive and relatively easy to prepare in large quantities. Their large insert
capacity permits the transfer of multi-gene cassettes of
unlimited size. Since they lack proteins, they elicit weaker or
no immune/inflammatory responses. A disadvantage of liposomal gene transfer is its inefficiency; thousands of liposomes are necessary per cell for successful transduction
(Yotsuyanagi & Hazemoto 1998).
DNA–protein complex vectors and hybrid vectors
DNA–protein complexes have been developed for gene
transfer in which the transgene is complexed with a targeting
protein. This allows the possibility of high-specificity gene
transfer of large inserts with minimal immune response.
DNA–protein complexes, like anionic liposomes are, however, highly susceptible to endosomal targeting and lysosomal nuclease degradation, greatly reducing their transduction
efficiency.
The major limitation of viral vectors is maximum insert
size. The major limitation of the synthetic vectors is their
121
poor gene transfer efficiency, a result of poor uptake of
vector DNA into cell/nucleus and the lysosomal degradation
that occurs following internalization. As a means to circumvent some of the limitations of both synthetic and viral vectors, hybrid vectors combining viral and synthetic approaches
have been devised. DNA segments can be complexed to Ad
virions to which polylysine or DEAE-dextran has been conjugated (Wagner et al. 1992, Curiel 1994, Forsayeth &
Garcia 1994). These conjugates improve transfer efficiency
by orders of magnitude in comparison with naked DNA.
Similar improvements have been noted for an Ad–liposome
complex which resulted in a 1000-fold increase in gene transfer efficiency relative to naked plasmid (Raja-Walia et al.
1995). Transgenes of up to 48 kb have been successfully
transferred using this technique (Cotten et al. 1992). The Ad
to which the DNA is complexed may be replication-defective
or UV-inactivated. The presence of the virion proteins in the
DNA–virus complex are sufficient to improve vector attachment and uptake, and more importantly, allow escape of the
vector DNA from endosomes prior to fusion with the lysosomes, where they may be degraded (Cristiano et al. 1993,
1998).
Corrective PCA gene therapy
Corrective gene therapy involves the functional complementation of an abnormal or absent gene. Corrective strategies may be designed to prevent tumorigenesis in phenotypically normal cells. Alternatively, corrective gene therapy
may be administered to induce phenotypic reversion or
regression of transformed cells to a non-neoplastic or less
transformed phenotype. Numerous potential candidate genes
for corrective gene therapy approaches are suggested by the
wide variety of genetic and epigenetic changes known to
occur accompanying prostate tumorigenesis (Table 3) including allelic loss, alterations in DNA methylation patterns, activating mutations of oncogenes, and inactivating mutations of
tumor suppressors and cell-adhesion molecules (Isaacs et al.
1995, Konishi et al. 1997, Roylance et al. 1997).
The most commonly observed allelic loss in PCA
involves chromosomes 8, 10, 11 and 16 (Isaacs et al. 1994,
1995), suggesting the presence of tumor-suppressor genes on
these chromosomes. The potential for functional tumor suppression by genes encoded in these deleted chromosomal
regions has been demonstrated in microcell-mediated chro-
Table 3 Corrective gene therapy strategies for PCA
Corrective gene
therapy targets
Tumor suppressors
p53
Rb
pTEN
p16 (CDKN2)
KAI-1
Oncogenes
myc
bcl-2
Ras
c-met
Growth factors
IGFs/IGFBPs
TGF-β
Cell-adhesion molecules
E-cadherin
α-catenin
β-catenin
CD44
Detoxification
GSTP1
122
Normal function
Defect in PCA
Regulation of cell cycle G1/S checkpoint,
Mutated in 20–75% of PCAs
apoptosis, response to stress/DNA damage
Cell cycle regulation and differentiation
Allelic loss in 27–40% of PCAs
Regulation of PI3K cell survival pathway
Decreased expression in 50% of PCAs
Cyclin-dependent kinase inhibitor involved in cell Markedly reduced expression in 43% untreated
cycle regulation
primary PCAs; no alteration in BPH
Down regulation by androgen
Cell–cell and cell–matrix interactions
Decreased expression in metastatic PCAs
Transcription factor regulating proliferation/
differentiation
Inhibition of apoptosis
GTP-binding proteins involved in growth
regulation
Receptor tyrosine kinase for hepatocyte growth
factor
Growth factors/growth factor regulatory proteins
Amplified in 20% of advanced PCAs
Uniformly elevated in androgen-independent
PCAs
Mutated in 25% PCA in US men; in Japanese
cases PCA 앑25% mutated
Elevated in 100% bone metastases
Growth factor – growth-inhibitory in normal
prostate
Increased IGF-I levels associated with increased
risk of PCA
Tumor progression may involve dysregulation of
TGF-β growth inhibition
Signal transduction/intercellular adhesion
Intercellular adhesion/cell–matrix interactions
Deficient or absent expression in 앑50% of PCAs
Deficient expression in 42% of PCAs
Mutated in 5% of PCAs
Decreased expression with increasing stage
Detoxification of electrophilic carcinogens
Absent expression secondary to promoter
hypermethylation in PIN and 98% of clinical PCAs
mosome transfer studies. Transfer of portions of chromosomes 8, 10, 11, 17 and 19 into Dunning R-3327 rat PCA
cells results in decreased formation of metastases and
decreased tumorigenicity (Isaacs et al. 1995, Gao et al.
1999). Analysis of allelic loss involving chromosome 11 led
to the cloning of the KAI1 metastasis-suppressor gene (Dong
et al. 1995), a potential candidate for use in PCA gene therapy strategies aimed at prevention of metastases. Further analysis of allelic loss during tumor progression may identify
new candidates suitable for corrective gene therapy specific
to various stages of tumorigenesis.
Tumor-suppressor genes such as p53, retinoblastoma
(Rb), p16, and pTEN show evidence of mutation or aberrant
expression in some PCAs (Bookstein et al. 1993, Isaacs et
al. 1994, 1995, Pesche et al. 1998, Suzuki et al. 1998). The
tumor-suppressor gene p53 encodes a transcription factor
involved in regulation of the cell cycle and apoptosis.
Mutations in p53 are widespread in human cancer and are
observed in between 20 and 75% of PCAs, more commonly
in advanced metastatic tumors (Heidenberg et al. 1995). The
ability of overexpressed p53 to inhibit the growth of primary
cultures derived from radical prostatectomy specimens has
been demonstrated, surprisingly even when p53 status is
normal (Asgari et al. 1997).
Mutations of the tumor-suppressor pTEN have been frequently observed in PCA (Pesche et al. 1998, Suzuki et al.
1998, Whang et al. 1998). pTEN appears to be involved in
a regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt
pathway, a pathway involved in cell survival. Candidates for
effectors downstream in this signaling pathway include
GSK-3/β-catenin (Bullions & Levine 1998), and members of
the bcl-2 family. Another tumor-suppressor, p16 (CDKN2)
shows markedly reduced expression in 43% of untreated primary PCAs but is unaltered in benign prostatic hyperplasia
(BPH) (Chi et al. 1997). p16 is a cyclin-dependent kinase
inhibitor involved in cell cycle regulation. Down-regulation
of p16 by androgen, promoter methylation, or other inactivating mutations may increase progression through the cell
cycle (Jarrard et al. 1997, Lu et al. 1997).
Methylation changes affecting gene expression have
been widely observed in prostate and other tumors (Lee et al.
1994, Nelson et al. 1997). A frequent target for inactivating
methylation changes is the glutathione S-transferase P1 gene,
a gene involved in detoxification of electrophilic carcinogens
(Lee et al. 1994). The absence of this mutation in normal
prostate or BPH, and its presence in prostatic intraepithelial
neoplasia (PIN) and greater than 98% of clinical prostate
tumors suggests that it is an early step in prostate tumorigenesis, and thus may be an appropriate target for PCA preventive gene therapy.
Oncogenes that may be activated in PCA include c-Myc,
Ras, bcl-2 and c-met (Table 3) (Isaacs et al. 1995, Konishi
et al. 1997, Roylance et al. 1997). Gene therapy strategies
to correct oncogene activation include the use of dominant
negative gene products, antisense oligodeoxynucleotides,
hammerhead ribozymes, and single-chain antibodies. The
overexpression of the oncogene c-Myc is frequently observed
in advanced prostate tumors (Fleming et al. 1986, Buttyan et
al. 1987). Disruption of c-Myc overexpression using antisense c-Myc transduced by a replication-deficient MMTVderived RV (Steiner et al. 1998) resulted in a 94.5%
reduction in tumor size of DU145 PCA cell xenografts.
Another oncogene, which could be targeted in a corrective
gene therapy approach, is bcl-2, which is frequently overexpressed in androgen-independent PCAs (McDonnell et al.
1992, Furuya et al. 1996, Beham et al. 1998). A hammerhead
ribozyme designed to disrupt bcl-2 expression in LNCaP
PCA cells showed pro-apoptotic activity and resulted in
increased cell sensitivity to secondary pro-apoptotic agents
including phorbol ester (Dorai et al. 1997).
Cell-adhesion molecules such as the cadherins and catenins show frequent alterations accompanying tumorigenesis
and may constitute appropriate targets for corrective gene
strategies. The cadherins are a family of transmembrane celladhesion proteins involved in calcium-dependent cell–cell
adhesion, and in transduction of extracellular growth regulatory signals. Deficient or absent E-cadherin expression due
to allelic loss, or promoter hypermethylation, is observed in
approximately half of PCAs. The level of E-cadherin expression is prognostic for survival, metastases, tumor stage,
grade, and risk of post-prostatectomy recurrence (Umbas et
al. 1994, Richmond et al. 1997). E-cadherin-mediated cell
adhesion also requires the function of the cytoplasmic protein
α-catenin which couples E-cadherin to the cytoskeleton.
Deficient α-catenin expression has been observed in approximately 42% of PCAs and is prognostic for patient survival
(Richmond et al. 1997). The constitutive expression of
α-catenin in PC-3 PCA cells through microcell-mediated
transfer of chromosome 5 results in suppression of tumorigenicity in an E-cadherin-dependent fashion (Ewing et al.
1995). β-catenin, another catenin involved in E-cadherinmediated cell–cell adhesion as well as in signal transduction
through the Wnt/wingless gene signaling pathway, has
recently been shown to be mutated focally in some prostate
tumors, suggesting that it may play a role in tumor progression (Voeller et al. 1998).
Growth factors are another potential target for corrective
gene therapy. The insulin-like growth factors (IGFs) are
potent mitogens for prostate epithelial cells. The ability of
IGF-I to activate the androgen receptor (AR) even in the
absence of hormone suggests a potential role in PCA progression (Culig et al. 1995a,b). The IGF-binding proteins
(IGFBPs), produced in the prostate and elsewhere, are a
family of seven related proteins which can modulate epithelial cell growth, either via binding and modulation of
IGF-I and -II activity or through IGF-independent mechanisms (Oh 1997). The IGFBPs are induced by growth inhibitory molecules such as transforming growth factor (TGF)-β
123
Class II MHC +
Tumor Antigen
Secreted cytokine
DC
Th
CD-4
CTL
Class I MHC
+
PCA Tumor
Antigen
Cytokine-Vector
Autologous/allogeneic
tumor cell
!!
CTL
DC
ex vivo
Immunostimulatory
Gene Transfer
Th
!!
!!
Th
!!
CD-8
CTL
Vaccine Site
Distant
Metastases
Figure 1 Ex vivo cytokine-transduced tumor vaccine gene therapy. Tumor cells transduced ex vivo with cytokine such as
GM-CSF are irradiated then reintroduced into the patient. Transduced GM-CSF improves tumor cell antigen presentation and
fully activates DCs, the most potent APCs in the immune system. DCs present prostate tumor vaccine-associated antigens they
have processed to both CD4 (helper) and CD8 (cytolytic) T cells in the draining lymph node of the vaccination sites, promoting
systemic tumoricidal immunity.
and p53 (Hwa et al. 1997) and IGFBP-3 transgene expression
in PC-3 cells defective for p53 results in an IGF- and p53independent induction of apoptosis (Rajah et al. 1997).
It is estimated that approximately 9% of PCAs occurring
prior to the age of 85 are due to genetic predisposition
(Gronberg et al. 1997, Walsh & Partin 1997). The basis for
hereditary PCA has been mapped to several loci – the first
susceptibility locus identified, HPC-1, resides on chromosome 1q24–25 (Smith et al. 1996). A second susceptibility
locus, the recently identified HPC-X, resides on the X chromosome at Xq27–28 (Xu et al. 1998). Identification of the
involved gene(s) at these loci should provide targets for preventive/corrective gene therapy, particularly for high genetic
susceptibility risk probands.
A potential limitation of corrective gene therapy is the
requirement that not even a single neoplastic clonogen must
develop. To achieve this goal would require 100% efficiency
of gene transfer, as well as sustained transgene expression,
or tolerance for repeated vector application. Even if efficiency could be improved to the extent required, alternative
mechanisms of progression may circumvent whatever the
corrective strategy employed.
Cytoreductive PCA gene therapy
Immunotherapy strategies
PCA offers potentially unique antigens which may be
exploited for the genetic induction of autoimmune antitumor
124
immune responses. Immunotherapy strategies for cancer
gene therapy utilize gene transfer to facilitate a dormant host
immune response directed against the tumor. Evasion of
autologous host cellular immunity is a common feature of
tumor cell neoantigens. Tumor cells are poor antigenpresenting cells (APCs). In PCA in particular, defects in
MHC class I expression are observed in a striking 85% of
primary and 100% of metastatic tumors (Blades et al. 1995),
suggesting that evasion of MHC class I tumor-associated
antigen is important in prostate tumor development. ‘Cancer
vaccine’ strategies are based on optimization of the context
in which tumor antigens or tissue-specific antigens are
presented to the host immune system. When appropriately
primed, the activated host immune system can then act
against tumor cells systemically.
One means to utilize gene therapy to optimize tumorantigen presentation is through the targeted expression of
cytokines in tumor cells. Targeted paracrine expression eliminates the toxicities associated with systemic cytokine administration. The transduced cytokines result in a combination
of improved tumor cell vaccine antigen presentation, and
activation of APCs, both essential for effective priming of the
cellular immune response (Fig. 1). Granulocyte-macrophage
colony stimulating factor (GM-CSF) has emerged as a cytokine with significant efficacy in the induction of an antitumor
immune response (Dranoff et al. 1993). GM-CSF is the most
potent cytokine signal tested for activation of antigen processing and presentation by macrophages and DCs (Cella et
al. 1997a,b). GM-CSF may be transduced into autologous or
allogeneic tumor cells ex vivo using either an RV or other
vectors. The transduced tumor cells are then irradiated both
to minimize malignant potential and to improve immunogenicity (Simons & Mikhak 1998). The cells are then reintroduced by vaccination into the patient and tumor response
monitored. Tumor cell vaccine-expressed GM-CSF may activate quiescent DCs which then capture prostate tumor cell
vaccine antigens. DCs are the most potent APCs in the
immune system and are central to the success of these
genetically engineered tumor vaccine strategies. Activated
DCs can present prostate tumor vaccine-associated antigens
they have processed to both CD4 (helper) and CD8
(cytolytic) T cells in the draining lymph node of the vaccination sites (Fig. 1) (Banchereau & Steinman 1998,
Simons & Mikhak 1998), activating a systemic tumoricidal
immune response.
By presenting tumor antigen/MHC I in the context of
supraphysiological local levels of transduced cytokines such
as GM-CSF or interleukin (IL)-2, the CTL-based clinical
antitumor immune response has been enhanced (Simons &
Mikhak 1998). A potent antitumor CTL response has also
been attained through co-presentation of tumor antigen/MHC
II with a transduced costimulatory signal such as B7 (Baskar
et al. 1993). The duration of the transduced cytokine expression must be of sufficient duration to permit cell harvesting
ex vivo and for antigen processing/presentation in vivo. For
autologous vaccine, RV and Ad vectors have been favored
for ex vivo tumor vaccine trials. GM-CSF gene transfer has
also been accomplished using cationic immunoliposome
AAV vectors (Koppenhagen et al. 1998).
Ex vivo cancer vaccine strategies have also directly
employed APCs such as DCs to facilitate effective tumorantigen presentation. The rationale for these strategies is that
the APC, properly primed, will express the most appropriate
combination of factors for eliciting an effective immune
response. DC-based vaccines have been generated by RV
vector-mediated transduction of antigen (Specht et al. 1997),
pulsing with tumor-antigen RNA (Boczkowski et al. 1996),
and pulsing with tumor- or tissue-specific peptide (Murphy
et al. 1999).
As complete ablation of PSA-expressing tissue is an
ideal therapeutic endpoint, in vivo cytoreductive immunotherapy approaches have centered on prostate-specific recombinant viral vaccines. The vector-induced inflammatory/
immune response functions as an adjuvant to the transduced
antigen, resulting in local release of cytokines and influx of
APCs to the vaccine site. The direct transduction and expression of tumor-associated antigens in DCs can enhance the
efficacy of viral vaccines (Bronte et al. 1997). Cotransduction of cytokines together with the tumor-associated antigen
may further enhance viral vaccine efficacy (Bronte et al.
1995).
Another vaccine strategy is the DNA or polynucleotide
vaccine. In this approach, an expression cassette containing
the transgene against which an immune response is desired
is injected directly into host cells, typically i.m. (Benton &
Kennedy 1998). The expression of the transfected gene in
vivo may then engender humoral and cellular immune
responses. DNA vaccines are technically easier to prepare
than peptide- or viral-based vaccines and pose no risk of
pathogenicity as they are non-replicating. A modification of
the DNA vaccine technique which may improve its efficacy
is the fusion of the desired transgene with the sequence for
a pathogen-derived gene to engender a stronger immune
response. Fragment C of tetanus toxin has been used in a
fusion with single-chain Fv sequences from B-cell lymphoma, and resulted in striking enhancement of both humoral
and cellular immunity (Falo & Storkus 1998, King et al.
1998). Recently, the preclinical testing of a PSA-based DNA
vaccine in mice was reported (Kim et al. 1998). Injection
i.m. of the PSA expression cassette resulted in a strong CTL/
humoral response against PSA-positive tumor cells. Evaluation of the effects of vaccines incorporating various regions
of the PSA-coding sequence identified four dominant coding
sequences for Th epitopes of PSA. Future vaccine
approaches can be predicted to target other prostate-unique
antigens such as PSMA, mucin (MUC-2), human glandular
Kallikrein 2 (hK2), and other novel prostate-unique antigens
whose identification will be facilitated through the use of
genomics and proteomics techniques (Pang et al. 1997,
Nelson et al. 1998).
A major limitation of cytoreductive immunotherapy is
the limited tumor burden which the immune system can eliminate in experimental models. Thus, applicability is probably
limited to clinical settings of low bulk or micrometastatic
disease, or in combination with other debulking therapies.
Additionally, the harvesting and culture of autologous or
allogeneic tumor or immune cells for ex vivo gene therapy is
expensive and technically challenging. The observed safety
of cytokine gene-transduced vaccines is in no small measure
responsible for ongoing enthusiasm for clinical testing as an
outpatient adjuvant therapy.
Polyvalent vs single antigen PCA vaccines
Polyvalent cancer vaccines targeting the entire antigenic
spectrum on tumor cells may represent a superior therapeutic
strategy for cancer patients than vaccines solely directed
against single antigens (Heiser et al. 2001). Heiser et al.
(2001) showed that autologous DCs transfected with RNA
amplified from microdissected tumor cells are capable of stimulating CTL against a broad set of unidentified and critical
PSAs. In this strategy of gene therapy, the tumor’s candidate
antigens were transfected as a cDNA library. Although the
polyclonal CTL responses generated with amplified tumor
RNA-transfected DCs encompassed as a subcomponent a
response against PSA as well as against telomerase reverse
125
transcriptase, the tumor-specific CTLs were consistently
more effective than PSA or telomerase reverse transcriptase
CTLs in lysing tumor targets, suggesting the superiority of
the polyclonal response. Although tumor RNA-transfected
DCs stimulated CTLs which recognized not only tumor but
also self-antigens expressed by benign prostate tissue, these
cross-reactive CTLs were exclusively specific for the PSA,
indicating an immunodominant role of PSA in the PCAspecific immune response. These data suggest that tumor
RNA-transfected DCs may represent a broadly applicable,
potentially clinically effective vaccine strategy for PCA
patients, which is not limited by tumor tissue availability for
antigen preparation and may minimize the risk of clonal
tumor escape.
Cytolytic/pro-apoptotic strategies
Enzyme/prodrug gene therapy
Enzyme/prodrug gene therapy, also referred to as ‘suicide
gene therapy’, relies on the conversion of an inactive prodrug
into a toxic drug using an enzyme vectored only to the target
tumor cells. In this way, active drug is limited spatially to the
transduced cells and adjacent surrounding cells, facilitating
higher tumor drug concentrations without increased normal
tissue toxicity. As opposed to the corrective gene therapy
approach noted above, it is essential that the vector be targeted with great specificity to tumor cells to avoid increased
normal tissue injury. Prodrug-activating enzymes which have
been employed in this approach include cytosine deaminase
(CD) which catalyzes the conversion of the non-toxic
5-fluorocytosine (5-FC) to the cytotoxic 5-fluorouracil
(5-FU), and HSV-TK which together with cellular enzymes
facilitates the conversion of GCV into the toxic GCV triphosphate. Using this approach it has been shown that even
when only 2% of a tumor contains CD-transduced cells, significant tumor regression is observed, suggesting the presence of a significant bystander effect (Kim et al. 1998). The
cytotoxic efficacy of suicide gene therapy in vivo may benefit
from a systemic antitumor response precipitated by tumor
lysis and inflammation, which may be mediated by natural
killer (NK) cells (Hall et al. 1998). The capacity for prostatespecific expression of suicide genes has been demonstrated
using PSA, PSMA, and hKLK2 promoters (Harris et al.
1994).
Cytotoxins
Another cytoreductive strategy involves the targeted expression of a cytotoxin such as the diphtheria toxin A chain, or
pseudomonas exotoxin A. In a screen of cytotoxins using a
wide range of PCA cell lines, Rodriguez et al. (1998) demonstrated a cell cycle- and p53-independent cytotoxic activity of
the diphtheria toxin A chain in PCA cell lines through both
126
apoptotic and non-apoptotic pathways. Another cytoreductive
agent that has shown activity in PCA cells in vitro is expanded
polyglutamine. This is a pro-apoptotic molecule implicated in
eight inherited neurodegenerative disorders (Ikeda et al. 1996).
Its pro-apoptotic potential can be modulated by varying the
expression level or the length of the expanded polyglutamine.
This molecule has demonstrated pro-apoptotic activity in PCA
cell lines when expressed from a vector under control of a PSA
promoter (Segawa et al. 1998). A critical concern with the use
of potent cytotoxins for cytoreductive gene therapy is the target
cell specificity of the vectoring strategy. From a practical viewpoint, even low levels of expression of a cytotoxin may be
toxic to the packaging cell line if a recombinant viral strategy
is employed. With tumor/tissue-specific promoter or antigen
targeting, unwanted expression of the cytotoxin in non-target
cells either through non-specific antigen targeting or through a
‘leaky’ tissue-specific promoter could result in significant
adverse normal tissue effects.
Oncolytic viruses
Viral vectors may themselves be designed to target and kill
tumor cells without insertion of a foreign transgene. Proof of
this principle was demonstrated therapeutically as early as
the 1950s with tumor responses noted following injections of
wild-type Ad into patients with cervical cancer. The Ad life
cycle includes a lytic phase, which can result in host cell
death independently of entry into the cell cycle – an important advantage for PCA therapy given the low mitotic rate.
Ad has evolved a potent repertoire of gene products, which
may exert profound effects on the growth regulation of the
host cell, in order to facilitate viral replication. A replicationcompetent Ad designed to preferentially replicate in p53
mutant cells, the ONYX-015 vector, is currently in clinical
trials (Kirn et al. 1998). The design of this vector was based
on the findings that a consequence of Ad infection was the
induction of p53, whose pro-apoptotic activity would be detrimental to the viral life cycle and was therefore blocked
directly by the virally encoded E1B 55 kDa protein
(Debbas & White 1993). In addition, E1B 19 kDa protein
expression resulted in the production of a bcl-2 type
molecule with anti-apoptotic effects downstream of the p53
pathway (Chiou et al. 1994). It was reasoned therefore that
an E1B-deficient Ad would replicate preferentially in p53deficient tumors (Bischoff et al. 1996). Recent findings suggest, however, that control of viral replication is more complex than the initial model suggests, as the E1B-deficient
virus replicates in p53 wild-type tumor cells as well
(Rothmann et al. 1998). To evaluate the selectivity of
ONYX-015 replication and cytopathic effects for the first
time in humans, a phase II clinical testing of intratumoral
and peritumoral ONYX-015 injection in 37 patients with
recurrent head and neck carcinoma was carried out
(Nemunaitis et al. 2000). Post-treatment biopsies docu-
mented selective ONYX-015 presence and/or replication in
the tumor tissue of 7 of 11 patients biopsied. Tissue destruction was also highly selective; significant tumor regression
(>50%) occurred in 21% of evaluable patients, whereas no
toxicity to injected normal peritumoral tissues was demonstrated. p53 mutant tumors were significantly more likely to
undergo ONYX-015-induced necrosis (7 of 12) than were
p53 wild-type tumors (0 of 7; P = 0.017). High neutralizing
antibody titers did not prevent infection and/or replication
within tumors. ONYX-015 is the first genetically engineered
replication-competent virus to demonstrate selective intratumoral replication and necrosis in patients. This agent demonstrates the promise of replication-selective viruses as a
novel therapeutic platform against cancer (Nemunaitis et al.
2000).
Among the Ad gene products which may function to
induce cell death are E1A (Rao et al. 1992), the E3–11.6k
protein also known as the Ad death protein, which is required
for efficient cell lysis during productive viral infection
(Tollefson et al. 1996b), and the E4orf4 protein, which may
induce p53-independent apoptosis (Marcellus et al. 1998).
The E1A 243-residue protein is able to induce a p53dependent apoptosis in the absence of other viral proteins
using a caspase effector cascade which involves activation
of procaspase-8, followed by mitochondrial redistribution of
cytochrome c and activation of procaspase-3 (Fearnhead et
al. 1998, Nguyen et al. 1998). An Ad vector with the E1A
gene placed under the control of a PSA minimal promoter
enhancer, the CN706 vector (Fig. 2) showed potent PSAselective cytotoxic activity in preclinical testing and is currently being utilized in a phase I clinical trial (Rodriguez et
al. 1997) (see below; Principal Investigator J W Simons,
Johns Hopkins University).
Searching for additional approaches that would induce
therapeutic apoptosis of PCA cell lines, Li et al. (2001)
recently used a binary Ad system to overexpress the proapoptotic molecule Bax. Bax was dramatically overexpressed
and caused apoptosis of every cell line infected by engaging
the mitochondrial pathway, including proteolytic cleavage
and catalytic activation of the caspases, cleavage of caspase
substrates, release of cytochrome c from the mitochondria,
and DNA fragmentation. Furthermore, three injections of the
Bax-overexpression system into PC-3 cell tumors in nude
mice in vivo caused a 25% regression in tumor size corresponding to a 90% reduction relative to continued tumor
growth in animals that received injections with the control
binary system expressing Lac-Z. These experiments show
that Ad-mediated Bax overexpression is capable of inducing
therapeutic programmed cell death in vitro and in vivo by
activating the mitochondrial pathway of apoptosis. On the
basis of these studies, it can be concluded that manipulation
of Bax expression is an attractive new gene therapy approach
for the treatment of PCA (Li et al. 2001).
Prostate-specific targeting
Regardless of the vector or gene therapy strategy used,
restriction of cytoreductive effects to target tissue is an essential factor in determining overall therapeutic index. Strategies
to restrict cytoreductive effects of gene therapy may utilize
tissue- or tumor-specific antigens or promoters. The presence
of several well-characterized prostate-specific markers such
as PSA and PSMA, and 500 or more prostate-unique genes,
provides a biological foundation for prostate-localized gene
therapy treatment. The prostate-restricted expression of PSA
has stimulated a variety of PSA-dependent gene therapy strategies. Antigen-based strategies include the PSA vaccines in
clinical trial discussed below. Several groups have placed
therapeutic genes under the control of various PSA gene cisregulatory regions. Varying segments of the 5′-flanking
region of PSA have been evaluated for their suitability for
prostate-specific expression of a desired gene. A minimal
composite PSA promoter/enhancer element (PSE) was used
to drive expression of Ad E1A in the attenuated replicationcompetent vector CN706 (Fig. 2) (Schuur et al. 1996, Rodriguez et al. 1997). Within the PSA enhancer region employed
in the PSE, a functional androgen response element (ARE)
at position 4136 (Fig. 2) results in an up to 100-fold increase
in expression in the presence of testosterone or the steroid
analog R1881. The regulation of E1A expression by the PSE
element effectively limits cytolytic viral replication to PSAexpressing cells, with a resulting therapeutic ratio of between
20:1 and 3000:1 depending upon the cell line tested.
One issue with PSA-promoter/enhancer-based targeting
is whether sufficient levels of transgene expression are
obtained, particularly in the absence of androgen. For clinical
PCA gene therapy, efficient transgene expression in the
absence of androgen would be preferred. In a PSA promoterbased strategy in preclinical testing, the HSV-tk gene was
placed under control of a long 5.8 kb PSA promoter in an
Ad construct. This vector showed activity in both the presence and absence of androgen (Gotoh et al. 1998). Another
approach to amplify PSA-specific expression independently
of androgen is through a two-step transcriptional activation
system in which the PSA promoter drives the expression of
a potent transcriptional transactivator which in turn regulates
expression of the desired transgene (Segawa et al. 1998).
PSMA is a potential target in PCA patients because it is
very highly expressed and because it has been reported to be
upregulated by androgen deprivation. O’Keefe et al. (2000)
recently described analysis of the PSMA enhancer for the
most active region(s) using the enhancer in combination with
the E. coli CD gene for suicide-driven gene therapy that converts the non-toxic prodrug 5-FC into the cytotoxic drug
5-FU in PCA cells. Deletion constructs of the full-length
PSMA enhancer were subcloned into a luciferase reporter
vector containing either the PSMA or SV-40 promoter. The
127
Adenovirus type 5 genome
E3-deleted
Adenovirus
E1A E1B
ITR
E4
ITR
E4
ITR
pE1A
Insertion of PSE
E3 Deletion
(28133-30818)
E1A E1B
ITR
CN706
512
PSE
512
ARE
-5322
PSA enhancer
-3738
-541
+12
PSA promoter
Figure 2 Structure of CN706. CN706 was derived from an E3-deleted type 5 Ad into which a minimal human PSA gene composite promoter/enhancer element (PSE) was inserted at nucleotide 512 of the Ad type 5 genome just 3′ to the E1A promoter
(pE1A). The PSE-driven E1A permits restriction of CN706 replication to PSA-positive cells. The PSE was derived from a fusion
of PSA enhancer sequence from nucleotides −5322 to −3738 to the PSA promoter spanning nucleotides −541 to +12 of the
PSA gene. At position −4136 in the PSA enhancer is a functional ARE. Note presence of inverted terminal repeats (ITR), and
packaging sequence ψ in the vector.
most active portion of the enhancer was then determined via
luciferase activity in the C4–2 cell line. The luciferase gene
was then replaced with the E. coli CD gene in the subclone
that showed the most luciferase activity. The specificity of
this technique was examined in vitro, using the PCA cell
line LNCaP, its androgen-independent derivative C4-2, and
a number of non-prostatic cell lines. Deletion constructs
revealed that at least two distinct regions seem to contribute
to expression of the gene in PCA cells, and therefore the best
construct for prostate-specific expression was determined to
be 1648 bp long. Transfection with the 1648 nucleotide
PSMA enhancer and the PSMA promoter to drive the CD
gene enhanced toxicity in a dose-dependent manner more
than 50-fold, while cells that did not express the PSMA gene
were not significantly sensitized by transfection.
Tissue-specific transcriptional regulatory elements can
increase the safety of gene therapy vectors. Unlike PSA/
hK3, whose expression displays an inverse correlation with
PCA grade and stage, hK2 is upregulated in higher grade
and stage disease. Therefore, Xie et al. (2001) developed
a prostate-specific hK2-based promoter for targeted gene
128
therapy. They identified the minimum ‘full-strength’ hK2
enhancer and built transcriptional regulatory elements composed of multiple tandem copies of this 1.2 kb enhancer,
fused to the hK2 minimal promoter. Relative to the weak
induction of the minimal hK2 promoter by androgen analog
(R1881) in AR-positive LNCaP cells, transcriptional
activity was increased by 25-, 44-, 81- and 114-fold when
one to four enhancers were spliced to the hK2 promoter
respectively. In contrast, the PSEs were inactive in the
AR-negative PCA line PC-3 and in a panel of non-prostate
lines, including 293, U87, MCF-7, HuH-7 and HeLa cells.
These results suggest that the hk2 multi-enhancer/promoter
should be a powerful reagent for targeted gene therapy of
PCA.
The search for additional prostate-specific genes for
use in PCA gene therapy is still going on. Recently, Steiner
et al. (2000) reported on a novel gene pHyde that was
cloned from Dunning rat PCA cell lines. A replicationdeficient recombinant Ad containing pHyde cDNA gene
under the control of a truncated RSV promoter
(AdRSVpHyde) was generated. AdRSVpHyde inhibited
growth of prostate cell lines (DU145 and LNCaP) by about
80% as well as DU145 xenograft tumors by 75% (Steiner
et al. 2000).
Combined modality therapy: integration
with radiotherapy
Even if optimal gene delivery is achieved, the success of
gene therapy, like conventional therapy, may be impeded by
tumor cell resistance and intratumoral cell heterogeneity. The
use of combined treatment modalities provides a rational
paradigm to improve upon the clinical efficacy of cancer
gene therapy. Within the modality of gene therapy itself,
multiple therapies may be combined in an attempt to benefit
from additive or synergistic efficacy. Multi-gene therapy
approaches already under evaluation include the transduction
of dual immunostimulatory molecules for immunotherapy
(Albertini et al. 1996), and dual suicide genes for enzyme/
prodrug strategies (Uckert et al. 1998).
The use of radiation-inducible promoters has been proposed as another means of achieving spatial localization with
combined modality treatment (Advani et al. 1997). This type
of promoter has been used in a construct directing expression
of tumor necrosis factor-α in a spatially and temporally
restricted fashion (Hallahan & Weichselbaum 1998).
Improvements in conformal radiation treatment permit the
precise delivery of anatomically restricted radiation doses,
improving the potential anatomical precision of this
approach. Conceivably, single or multiple cytotoxic or radiosensitizing genes could be delivered in a spatially and temporally restricted fashion with either therapeutic or regulatory
radiation doses. The therapeutic gain achievable by this
approach has been limited by the ‘leakiness’ of the radiationresponsive promoters.
One combined modality approach particularly attractive
in PCA is the combination of radiation and gene therapy.
There is strong evidence that synergy exists between these
modalities. Recent work reveals greater than expected tumor
regression of U-87MG glioma cell xenografts following
combined radiation and viral treatment with the HSV mutant
R3616 (Advani et al. 1998). In irradiated xenografts, viral
replication was increased 2- to 5-fold per cell. In unirradiated
tumors, virally infected cells were restricted to regions of the
xenograft immediately adjacent to the infecting needle track.
In irradiated tumors on the other hand, virally infected cells
were more widely distributed throughout the xenograft and
away from the needle track than in unirradiated tumors.
Increased doses of virus to unirradiated cells resulted in no
increase in viral spread from the vicinity of the needle track,
supporting the idea that radiation was facilitating not only
viral replication but viral spread as well. Thus radiation may
potentially be used to improve the efficacy of corrective/cytoreductive gene therapy approaches by increasing the capacity
to transduce cells spatially distributed throughout a tumor.
Observations in our own laboratory reveal evidence for
synergy between the oncolytic CN706 Ad (see above and
Fig. 2) and radiation in the LNCaP and LAPC-4 PCA cell
lines. An obvious combined modality strategy for PCA is the
combined implantation of brachytherapy seed sources with a
gene therapy vector. The capacity of Ad proteins such as
E1A and E4orf4 to engage cellular pro-apoptotic machinery
suggests a mechanistic basis for the observed therapeutic
synergy (Marcellus et al. 1998, Nguyen et al. 1998). Supporting this idea is the observation that E1A expression
enhances apoptotic cell killing by a variety of chemotherapeutic agents in ovarian cell lines (Brader et al. 1997).
Further investigations into the interactions between radiation,
cellular and oncolytic virus gene products may clarify the
mechanisms by which radiation might potentiate oncolytic
gene therapy.
Gene therapy may also be used for radiosensitization
strategies in which the activation of a prodrug, such as GCV
(Nishihara et al. 1997) or 5-FC (Hanna et al. 1997, Gabel et
al. 1998), can function to sensitize cells to the effects of radiation. While chemotherapy has thus far been ineffective for
PCA, novel combinations of gene therapy and chemotherapy
may display synergistic improvements in efficacy. The combination of paclitaxel and gene therapy utilizing an Ad
expressing p53 under the control of the CMV promoter
revealed synergy for the combined modality treatment for a
wide variety of tumor cell lines in vitro and in xenografts
including the DU-145 PCA cell line (Nielsen et al. 1998).
Combinations of three modalities have also been demonstrated – radiotherapy, viral-cytopathic (E1B-deleted Ad)
and radiosensitizing double-suicide gene therapy – with
marked enhancement in efficacy in vitro with DU-145 cells
(Freytag et al. 1998).
HSV-tk gene therapy may be effective in combination with
radiation therapy due to complementary mechanisms and distinct toxicity profiles. In a study (Chhikara et al. 2001) mouse
prostate tumors transplanted s.c. were treated by either gene
therapy involving intratumoral injection of Ad-tk followed by
systemic GCV or local radiation therapy or the combination of
gene and radiation therapy. Both single-therapy modalities
showed a 38% decrease in tumor growth compared with controls. The combined treatment resulted in a decrease of 61%.
The combination led to an additional 50% reduction in lung
colonization. Primary tumors that received the combination
therapy had a marked increase in CD4 T-cell infiltration. This
is the first report showing a dramatic systemic effect following
the local combination treatment of radiation and Ad-tk. A clinical study using this strategy has been initiated and patient
accrual is ongoing (Chhikara et al. 2001).
Translation of concepts to human gene
therapy clinical trials
At present there are 40 approved clinical gene therapy trials
for PCA in progress in the USA (ORDA 010/2001)
129
(HUMAN GENE THERAPY PROTOCOLS. http://
www4.od.nih.gov/oba/rdna.htm Updated 10–01–2001 edn
(National Institutes of Health (NIH) Office of Recombinant
DNA Activities 2001). Of these, 22 are for cytoreductive
immunotherapy, 14 are for cytoreductive cytolytic/proapoptotic strategies, and four are corrective strategies
(Table 4).
The preliminary results of only part of these trials have
only recently been published. The results of the phase I clinical trial of irradiated GM-CSF-secreting autologous prostate
tumor vaccine therapy in eight patients (Principal Investigator J W Simons, Johns Hopkins University) indicated that
such treatment was safe and induced both B-cell and T-cell
immune responses against PCA cell-associated antigens
(Simons et al. 1999). GM-CSF gene-transduced PCA vaccines increased antibody titers against prostate tumor cell
line-associated antigens. Increasing titers of antibodies to
PCA antigens were detected among three of the men treated
with irradiated GM-CSF-secreting autologous PCA cell vaccines. This is the first report of induction of new antibody
responses to PCA antigens in patients using cytokine genemodified tumor vaccines, peptide-pulsed DCs, or any other
strategy of immunotherapy.
Phase II studies have commenced with GM-CSF genetransduced allogeneic vaccines. Preliminary results of the
initial trial approved to use direct transrectal prostatic gene
therapy injection were reported by Steiner & Gingrich
(2000). A total of 21 men with PCA in whom standard therapy failed underwent ultrasound-guided injection of the RV
LXSN containing BRCA-1 under the control of the viral promoter LTR. No viral symptoms or evidence of viremia developed. Serum PSA in these cases of metastatic disease
remained unchanged. The study indicated the safety of PCA
gene replacement therapy by direct injection.
Herman et al. (1999) reported results of a phase I
clinical trial of a replication-deficient Ad containing the
HSV-tk injected directly into the prostate, followed by i.v.
administration of the prodrug GCV. Only 1 of 18 patients
at the highest dose level developed spontaneously reversible grade 4 thrombocytopenia and grade 3 hepatotoxicity.
Most of the patients at the highest dose levels achieved
objective response (fall in serum PSA levels by 50% or
more).
Of the clinical trials ongoing at the University of California at Los Angeles preliminary results have been
reported. In a phase II trial, intratumoral injection of a
plasmid coding for IL-2, formulated in a liposomal, cationic lipid mixture vehicle, revealed decreases in serum
PSA levels at 2 weeks post-injection in 80% of the patients
treated (Pantuck et al. 2000). IL-2 gene therapy was well
tolerated, with no grade 3 or 4 toxicity occurring. In
another immunotherapy phase I trial in MUC-1-positive
patients using vaccinia virus MUC-1-IL2 revealed an
immunological response associated with a clinical PSA
130
decline. In addition, an upregulation of cytokine expression,
augmented T-cell activation signals and augmented MUC1-targeted cytotoxicity were noted (Pantuck et al. 2000).
Integration of the following concepts: (i) PSA-based
targeting, (ii) virus-directed cytolytic therapy, (iii) stereotactically-guided vector administration for improved target
tissue distribution, and (iv) use of replication-competent
virus for improved tissue penetration, formed the basis
for a recently initiated clinical trial for patients with a
post-radiation therapy local recurrence of PCA (Principal
Investigator J W Simons, Johns Hopkins University).
A major limitation in the use of replication-deficient
gene therapy in solid tumors in vivo is the diffusion-limited
tissue penetration into the target tissue. The ability of viral
replication to increase tissue penetration has been observed
in vitro and in vivo (Advani et al. 1998, Han et al. 1998).
Replication-competent Ad spread in vivo may be facilitated
not only through transcellular movement and vector amplification, but also through greater tissue exposure resulting
from virus-dependent cell lysis. Thus, a replicationcompetent Ad can be expected to achieve greater tissue
penetration relative to a replication-deficient Ad or other
non-lytic vector. CN706 (Fig. 2) is attenuated for replication competence only in PSA-expressing cells, in which
it may be expected to amplify titer and achieve greater
tissue penetration. In addition, the local immune response
against the Ad-infected cells may aid in killing of the
target tumor cells.
The concept of dose uniformity for cytotoxic PCA therapy
has been well developed in radiation therapy. Local underdosing is thought to increase the risk of treatment failure – a concept supported by the improved local control observed with
conformal radiation dose-escalation trials (Hanks et al. 1998,
Zelefsky et al. 1998). It follows that similar principles may
apply to the tissue distribution and dosing of viral cytolytics
such as CN706. By analogy to radiation dosimetry for prostate
brachytherapy, the tissue surrounding a seed implant is
exposed to varying concentrations of virus. The extent to
which a replication-competent virus spreads depends on initial
viral titer, infectivity, lytic ability, and host immune response.
Empirical determinations of viral spread from tumor models
allows an estimation of the effective radius of virus activity.
This can then be used iteratively in a computer 3D model to
designate viral injection patterns for optimal tissue distribution
(Fig. 3). Existing brachytherapy treatment planning systems
may be adapted for 3D viral vector spread modeling. Validation of predicted viral spread through biopsy analysis of Ad
hexon protein immunohistochemistry will permit increasing
accuracy in viral spread predictions. Spatial evaluation of
oncolysis relative to viral spread will help define potentially
new parameters involved in oncolytic efficacy and augment
comparisons of alternative vectors. Clinical direct injection
trials utilizing this precision in vector delivery benefit from the
increased uniformity of gene delivery between patients.
Table 4 PCA gene therapy: current clinical trials summary
NIH No.
Phase
Principal
investigator
Institution
Vector
Gene
Modality
9408-082
I–II
Simons
Johns Hopkins University
RV
GM-CSF
9509-123
I
I
Antisense myc
RNA
PSA cDNA
9510-132
I
9601-144
I
Lyerly &
Paulson
Scardino
9609-160
9702-176
I
I–II
Eder & Kufe
Sanda
Vanderbilt University School
of Medicine
National Naval Medical
Center
Duke University Medical
Center
Memorial Sloan-Kettering
Cancer Center
Dana-Farber Cancer Institute
The University of Michigan
RV
9509-126
Holt &
Steiner
Chen
Ex vivo autologous
PCA vaccine
In vivo intraprostatic
injection
In vivo i.d. injection
9703-184
I
Belldegrun
University of California, Los
Angeles
9705-187
I
Hall & Woo
9706-192
I
Belldegrun
9708-205
9710-217
I–II
I–II
Simons
Logothetis
9801-229
I
Kadmon
Mount Sinai School of
Medicine
Angeles
The University of Texas MD
Anderson Cancer Center
Baylor College of Medicine
9802-236
I
Simons
9805-251
I–II
Figlin
9812-276
I
9901-282
II
Gardner &
Chang
Eder
9901-283
I–II
Small
9902-293
II
9904-306
I
Kaufman &
DiPaola
Vieweg
9905-312
II
Belldegrun
9905-315
II
9906-321
I
Smith &
Small
Freytag &
Kim
Vaccinia virus
Angeles
University of Virginia Health Ad serotype 5
Science System
Dana-Farber Cancer Institute Vaccinia virus/
fowlpox virus
University of California, San RV
Francisco
Eastern Cooperative
Vaccinia virus/
Oncology Group
fowlpox virus
RNA
Center
Cationic liposome
Angeles
complex/
DMRIE-DOPE
University of California, San RV
Francisco
Henry Ford Health System
Ad
9906-324
I–II
9909-338
I
AguilarCordova
& Butler
Gingrich
9910-344
I–II
Terris
Vaccinia virus
Cationic liposome
complex
Ad serotype 5
IL-2 cDNA
HSV-TK cDNA
Vaccinia virus
Vaccinia virus
PSA cDNA
PSA cDNA
Cationic liposome
complex/
DMRIE-DOPE
Ad serotype 5
IL-2 cDNA
HSV-TK cDNA
Ad serotype 5
p53 cDNA
RV
Ad serotype 5
GM-CSF
p53 cDNA
Ad serotype 5
HSV-TK cDNA
Ad serotype 5/
replicationcompetent virus
Promoter and
enhancer
elements of the
PSA
MUC-1/IL-2
HSV-TK cDNA
Ex vivo autologous
PCA vaccine
In vivo intraprostatic
injection
In vivo i.d. injection
In vivo i.d. injection,
autologous
Intratumoral direct
injection
Intratumoral direct
injection
Intratumoral direct
injection
s.c. injection
Intratumoral direct
injection
Intratumoral direct
injection
Intratumoral direct
injection
i.m. injection
PSA
Intratumoral direct
injection
i.m. injection
GM-CSF
s.c. injection
PSA
i.m. or i.d. injection
PSA
i.v. injection
IL-2 cDNA
Intratumoral direct
injection
GM-CSF
s.c. injection
Intratumoral direct
injection
Ad
E. coli CD
cDNA/HSV-TK
DNA
HSV-TK cDNA
University of Tennessee,
Coleman College of Medicine
Stanford University Palo Alto
Veterans Administration
Medical Center
Ad serotype 5
p16 cDNA
Ad serotype 5/
Promoter and
enhancer
elements of the
PSA
Intratumoral direct
injection
Intratumoral
injection
Intratumoral
injection
DMRIE, 1,2-Dimyristyloxpropyl-3-dimethyl-hydroyethyl ammonium bromide; DOPE, dioleoyl-phosphatidyl-ethanolamine.
131
Table 4 cont.
NIH No.
Phase
Principal
investigator
Institution
Vector
Gene
Modality
9910-345
I–II
Wilding
University of Wisconsin
Comprehensive Cancer
Center
Ad serotype 5/
i.v. injection
9910-352
I–II
Belldegrun
Angeles
9912-368
II
NCI
0001-373
II
Gulley &
Dahut
Arlen
0008-410
I
Vieweg
Cationic liposome
complex/
DMRIE-DOPE
Vaccinia virus/
fowlpox virus
Vaccinia virus/
fowlpox virus
RNA
Promoter and
enhancer
elements of the
PSA
IL-2 cDNA
0010-418
II
Pollack
0010-426
I
Gardner
0010-428
I
Freytag &
Kim
0101-447
0101-449
I
I
0101-450
NCI
Center
The University of Texas MD
Anderson Cancer Center
Ad serotype 5
Indiana University Medical
Center
Ad
Lubaroff
Miles
University of Iowa
Ad serotype 5
Ad serotype 5
II
DeWeese
Ad serotype 5
0101-451
II
Small
University of California, San
Francisco
Ad serotype 5
0103-459
0104-464
I
I
Dula
Freytag &
Kim
West Coast Clinical Research AAV
Ad serotype 5
Twenty men with locally recurrent PCA following radiation
therapy were treated with CV706 between September 1998
and May 2000 (Principal Investigator J W Simons, Johns Hopkins University). CV706 was found to be safe and was not
associated with irreversible grade 3 or any grade 4 toxicity.
Post-treatment prostatic biopsy and detection of a delayed
‘peak’ of circulating virus provided evidence of intraprostatic
replication of CV706. This study revealed that all patients
treated with the highest doses of CV706 achieved a 50% or
more reduction in serum PSA levels (DeWeese et al. 2001).
Future directions
The challenges facing the implementation of successful gene
therapeutic strategies will be better understood as the early
clinical trials for PCA gene therapy begin to return more
results. Vector development with increased transgene size
capacity, optimized immunogenic properties, and improved
132
Ad serotype 5
PSA/B7.1
(CD80)
PSA/B7.1
(CD80)
Total tumor
RNA
p53 cDNA
Osteocalcin
promoter
E. coli CD
cDNA/HSV-TK
cDNA
PSA cDNA
IL-2 cDNA
Promoter and
enhancer
elements of
PSA
Promoter and
enhancer
elements of
PSA
GM-CSF cDNA
E. coli CD
cDNA/HSV-TK
cDNA
Intratumoral
injection
i.v. injection
Percutaneous/
intraprostatic
injection
Intratumoral
injection
Intratumoral
injection
s.c. injection
Intratumoral
injection
Intratumoral
injection
i.v. injection
s.c. injection
Intratumoral
injection
transduction efficiency and targeting will facilitate the next
generation of gene therapy strategies. The burgeoning field
of genomics provides an exciting new resource for the design
of prostate-specific gene therapy strategies. The design of
oncolytic viruses should be aided by new insights into the
mechanism of killing by lytic viruses. Already, the potential
of Ad genes such as E4orf4, E3–11k and E1A to function in
virus-induced cell death has been observed (Tollefson et al.
1996a, Nevels et al. 1997, Marcellus et al. 1998, Nemunaitis
et al. 2000, Li et al. 2001). Harnessing these viral gene products and others for use as oncolytic drugs offers exciting
prospects for a whole new class of cytoreductive gene therapy strategies.
As the diversity of molecular lesions underlying prostate
tumorigenesis is better characterized, new targets for corrective and cytoreductive approaches will emerge. Effective
anticancer gene therapy may ultimately require individualized molecular profiles. Manipulation of the cellular
A.
B.
C.
D.
Figure 3 Injection of CN706 oncolytic Ad into the prostate under stereotactic guidance. (A) Intraoperative ultrasound prostate
imaging with superimposed brachytherapy template. (B) Image (A) imported into brachytherapy treatment planning system
(Radionics). Yellow and green circles indicate injection needle tracks, filled circles correspond to actual injection sites in the
plane of the imaged section. Urethra and prostate outlines contoured in green and red respectively. (C) 3-D reconstruction of
prostate volume and injection positions. Urethra imaged in red. (D) Predicted virus spread from injection sites in yellow.
apoptotic machinery for cytoreductive PCA gene therapy
may permit development of more generalized antineoplastic
treatment, which bypasses upstream signal cascade lesions.
Components of the apoptotic machinery such as the death
receptors, caspases, and the bcl-XL/BH3 family may be
utilized in strategies which result in efficient oncolysis even
in the presence of diverse upstream lesions. Cell death pathways involving the death receptors such as CD95, DR3, 4
and 5, may provide potent therapeutic targets. A recent preclinical study shows that Ad- or RV-transduced fas ligand
(FasL) or FADD can efficiently induce apoptosis in human
glioma cells (Shinoura et al. 1998).
Dong et al. (1995) showed that intratumoral delivery of
FasL using an Ad vector could force PCA cells into apoptosis.
They placed the tetracycline transactivator gene under the control of a prostate-specific ARR2PB promoter, and a proapoptotic FasL-GFP gene under the control of the tetracycline
responsive element. The latter expression cassette was inserted
into Ad 5. High levels of expression were exclusively observed
in LNCaP cells but not in other cell lines of other origins (Hyer
et al. 2000). This may hold promise in the future.
Solid tumors meet their demands for nascent blood vessels and increased glycolysis, to combat hypoxia, by activating multiple genes involved in angiogenesis and glucose
metabolism. Hypoxia inducible factor-1 (HIF-1) is a constitutively expressed basic helix-loop-helix transcription factor,
formed by the assembly of HIF-1alpha and HIF-1beta (Arnt),
that is stabilized in response to hypoxia, and rapidly degraded
under normoxic conditions. It activates the transcription of
genes important for maintaining oxygen homeostasis
(Dachs & Tozer 2000, Semenza et al. 2000). Sun et al.
(2001) recently demonstrated that engineered downregulation of HIF-1alpha by intratumoral gene transfer of an
antisense HIF-1alpha plasmid leads to the down-regulation
of vascular endothelial growth factor (VEGF), and decreased
tumor microvessel density. Antisense HIF-1alpha monotherapy resulted in the complete and permanent rejection of
small EL-4 tumors. It induced NK-dependent rejection
133
of tumors, but failed to stimulate systemic T-cell-mediated
antitumor immunity, and synergized with B7-1-mediated
immunotherapy (Sun et al. 2001). This approach holds promise to form the foundation for the transition between the traditional anticancer therapies of the past four decades and the
molecular antineoplastic pharmacology of the future.
Other approaches are to develop new gene therapy vectors whose expression is selectively activated by hypoxia. As
VEGF is upregulated by hypoxia, such regulatory mechanisms would enable us to achieve hypoxia-inducible expression of therapeutic genes. Constructs with multiple copies of
hypoxia-responsive elements (HREs) derived from the 5′untranslated region of the human VEGF showed excellent
transcriptional activation at low oxygen tension relevant to
tumor hypoxia (Shibata et al. 2000). It was found that the
combination of 5-HRE and a CMV minimal promoter
exhibited hypoxia responsiveness (over 500-fold) to a level
similar to the intact CMV promoter. Thus it can be proposed
that this vector would be useful for tumor-selective gene
therapy in the future. Of course many other replicationdeficient Ad could be designed to contain multiple copies of
HREs and the HSV-tk, which would be injected directly into
the prostate and followed by i.v. administration of the prodrug GCV. In this case only hypoxic tumor cells, which are
expressing high levels of HIF-1, will be killed.
Acknowledgements
This work was supported by NIH PCA SPORE Grant
(J W S) CA-58236. N J M is a recipient of a fellowship
from The American Physicians Fellowship for Medicine in
Israel. H Z is an Avon Scholar supported by the Avon Products Foundation.
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Gene therapy of prostate cancer: current and future directions Endocrine-Related Cancer

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