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Current Challenges of Gene Therapy for Prostate Cancer

Current Challenges of Gene Therapy for Prostate Cancer

ABSTRACT: Gene therapy for prostate cancer faces hurdles similar to those being encountered for other cancers and nonmalignant processes. The greatest obstacle is the identification of efficient delivery systems, since numerous animal models and cell culture systems have shown potential efficacy when most cells express the introduced genetic material. Early prostate cancers are easily accessible to gene vector introduction, and the predictable metastatic patterns of this cancer may offer additional advantages for gene therapy. This article reviews gene vectors and gene products, as well as ongoing trials of gene therapy that have recently begun in prostate cancer. [ONCOLOGY 11(6):845-856, 1997]

Introduction

Prostate cancer is the most rapidly increasing cancer diagnosis in the
United States, owing both to its prevalence in an aging "baby boom"
population and to increased use of screening with improved technologies,
such as the prostate-specific antigen (PSA) test.[1,2] Although early detection
offers the hope of improved survival, evidence for this outcome is lacking.
Furthermore, current therapeutic strategies for prostate cancer, even when
localized, are controversial because of unwanted side effects and disagreement
over their efficacy.[3,4] Neoadjuvant hormone therapy has shown some promise
in reducing the incidence of positive surgical resection margins and biochemical
relapse after radiation treatment, but as yet no data indicate improved
overall survival.[5-9]

Although advanced prostate cancer is well-palliated for 2 to 4 years
by hormonal ablation of testosterone, it inevitably progresses. Further
treatment with cytotoxic chemotherapy, radiopharmaceuticals, or external-beam
radiation to areas of bone pain achieve transient responses but have no
definite impact on survival. Thus, increasing attention has been given
to the development of alternative treatments for prostate cancer of all
stages, including gene therapy.

Definition of Gene Therapy

The term "gene therapy" is broadly applied to encompass all
approaches involving the introduction of genetic material into patients'
cells. This may involve replacement of an absent or defective gene, such
as adenosine deaminase (ADA) in ADA deficiency, the low-density lipoprotein
in familial hypercholesterolemia, and the chloride-transporter gene in
cystic fibrosis.

Alternatively, gene therapy may involve the introduction of anticancer
or antiviral genes, such as those encoding tumor-suppressor proteins (leading
to tumor regression), ribozymes (catalytic RNAs that cleave targeted mRNAs),
antisense RNA (interfering with normal RNA processing), and dominant mutations
that knock out functions essential for viral activity or oncogenesis.

Finally, many of the ongoing gene therapy clinical trials involve a
vaccination approach with gene-modified tumor cells. In this paradigm,
tumor cells from the patient are removed and genetically engineered to
secrete a cytokine or growth factor, such as interleukin-2 (IL-2) or granulocyte-macrophage
colony-stimulating factor (GM-CSF), or to express a foreign protein, such
as HLA-B7. Upon reinfusion of the cells, expression of the introduced gene
should stimulate the patient's own immune system to reject the tumor. A
modification of this approach involves the injection of a DNA vector directly
into tumor deposits, with the intention of triggering local and systemic
immune responses.

Gene Therapy
Challenges Common to All Diseases

The challenges of gene therapy for prostate cancer include those which
are common to gene therapy approaches for all diseases and those which
are unique for this disease. The common issues are grouped into three broad
categories: gene transfer, gene regulation, and safety. These have been
extensively reviewed recently.[10-12] Thus, we will provide only a brief
summary here, with the emphasis on recent clinical outcomes.

Gene Transfer

Efficient delivery of foreign genes into the appropriate target cells
is the major hurdle facing all gene therapy approaches today. Delivery
methods can be divided into viral (eg, retrovirus, adenovirus, and adeno-associated
virus [AAV]) and nonviral (plasmid DNA delivered either as naked DNA or
by physical means, such as cationic liposomes and particle-mediated bombardment).

Two major considerations in determining the optimal delivery method
are: (1) the target cell and its accessibility to the vector used to deliver
the DNA, and (2) whether or not the treatment requires long-term expression
of the gene. The first consideration also involves a decision to deliver
the DNA either in vivo or ex vivo, as well as a determination of the specific
route for in vivo delivery (ie, systemic administration or localized injection).
The length of expression required varies with the nature of the defect
being corrected, as well as the delivery system. For example, approaches
that involve physically mediated, localized, in vivo delivery of DNA can
be administered repeatedly. In contrast, methods that involve ex vivo delivery
of DNA into hematopoietic stem cells derived from bone marrow, followed
by reinfusion of these cells, are limited in terms of the number of repetitions
due to the invasiveness of the procedures.

For most approaches, long-term sustained expression of the therapeutic
gene is desirable. With current technologies, most nonviral methods of
gene delivery cannot produce sustained expression. Of the viral vectors,
two (retrovirus and AAV) are at least theoretically capable of producing
long-term expression; in fact, this has been demonstrated in ongoing clinical
trials using retroviral vectors.

Specific features of the different delivery systems have been reviewed
recently[12,13] and are summarized in Table
1.

Viral Vectors--All viral vectors share the feature of being disabled,
so that the viruses may deliver DNA into the target cell but cannot undergo
replication once inside the target cell (ie, they are replication-incompetent).
Since all of the viruses used are known to infect human cells, this is
one of the major safety concerns. (For further discussion of this concern,
see "Safety" below.) Features that distinguish the different
viral vectors include the size of the gene insert accepted, whether or
not the virus infects nondividing cells, the duration of expression, and
possible host immune response to viral proteins.

Of the approved clinical trials, over 85% have used viral vectors; the
majority of these have been retroviral vectors, which have the longest
usage history (5 years) in gene therapy studies. Bone marrow hematopoietic
cells that were "marked" with retroviral vectors could still
be detected in patients for up to 3 years following autologous transplantation;
in these vectors, the polymerase chain reaction (PCR) technique was used
to amplify retroviral sequences.[14] Retrovirally marked tumor cells were
detected in leukemia and neuroblastoma patients who relapsed following
autologous transplantation with unpurged bone marrow cells.[15] This unequivocally
demonstrated that the graft had residual tumor cells, and pointed to the
need for purging.

In the first gene replacement study, conducted in patients with ADA
deficiency, ADA sequences retrovirally introduced into patients' T-cells
could still be detected 2 years following infusion.[16,17] The ADA gene
has also been introduced into hematopoietic cells derived from cord blood,
and ADA sequences have been detected 18 months following infusion.[18]

Together, these studies point to the feasibility of using retroviral
vectors to deliver genes for long-term expression. However, with current
methodologies this method is still quite inefficient.

Adenoviral vectors offer the most efficient method for gene transfer,
and thus, have been the most frequently used vectors for localized in vivo
gene delivery, such as delivery of the CFTR gene for cystic fibrosis.[19]
These vectors can infect nondividing cells, which is advantageous for applications
that target such cells. However, in the case of tumors, retroviral vectors
(which do not infect nondividing cells) offer the possibility of selective
targeting to the dividing tumor cell, whereas adenoviral vectors target
dividing and nondividing cells indiscriminately.

Another disadvantage of adenoviral vectors is that they generally result
in short-term expression, since the viral DNA is not integrated into the
host genome. In the case of cystic fibrosis, the longest-lasting expression
was 4 to 9 days. Thus, this approach requires repeated administrations.

Adeno-associated virus vectors are being investigated as an alternative
to adenoviral vectors for delivery of CFTR into the lungs of patients with
cystic fibrosis. These vectors can infect nondividing cells, albeit at
a reduced efficiency compared with dividing cells. Development of producer
systems to reproducibly generate high yields of AAV has been problematic
and has slowed the clinical application of this vector system.[20]

Nonviral Vectors--The nonviral delivery methods offer several
advantages over viral vectors with respect to safety and ease of production.
Rigorous tests do not need to be applied to validate the absence of replication-competent
viruses, which saves both time and money. Another advantage is that nonviral
vectors can deliver larger pieces of DNA than can viral vectors.

Such an approach, utilizing liposomes to deliver HLA-B7 along with beta-2-microglobulin
genes directly into tumor cells in vivo, has been applied in the treatment
of metastatic melanoma, hepatic metastases of colon cancer, and other tumors.[21,22]
This approach falls into the immunotherapy category, in which the foreign
antigen is recognized and serves to boost the local immune response. In
some patients treated with this approach, tumor shrinkage has been noted.

Although gene transfer with nonviral vectors is promising, its clinical
application has lagged behind viral vectors. This is due primarily to the
relatively short-term expression of nonviral vectors. However, short-term
expression may be acceptable or even preferable for some applications,
such as expression of a potent toxin or conditionally lethal gene in a
localized cancer. Thus, this strategy for gene delivery has some potential
applications for prostate cancer, as discussed below.

Gene Regulation

Diseases that have been targeted for initial gene therapy studies do
not require stringent regulation of the transferred gene. There are two
related sides to this issue: On the one hand, expression in a nontargeted
cell should not be toxic; on the other hand, even a low level of expression
in the target cell should be effective, since the expression of foreign
genes tends to be low and decreases with time. The ADA gene met these requirements,
and thus, ADA deficiency was an ideal candidate for gene replacement even
though it is a rare disease.

A related consideration is whether to use constitutive regulatory sequences
(promoters) that are active in all cell types, or to use a tissue-specific
promoter in order to target expression to a particular cell type. Again,
this depends on the gene and the disease. For example, delivery of a gene
encoding a lethal product that induces the death of tumor cells would ideally
require a tumor-specific promoter so that its expression would be limited
to tumor cells. Unfortunately, such "magic bullets" that direct
expression of the lethal gene exclusively to tumor cells have not yet been
identified.

Promoters that are much more active in the target cell type than in
other cell types have been used in preclinical studies, however. In reality,
these promoters tend to be weaker than the constitutive promoters, and
consequently, their in vivo use is hampered by the problem of low expression.
Of course, many gene therapy approaches skirt the issue of obtaining tissue-specific
expression by using delivery methods that preferentially target the appropriate
cells. This is accomplished either by localized injection or by taking
advantage of vectors that preferentially target dividing cells.

Safety

Currently, the major safety concerns about gene therapy relate to: (1)
generation of replication-competent virus with altered phenotype and/or
host range; (2) activation of an oncogene or inactivation of a tumor-suppressor
gene due to insertional mutagenesis; and (3) potential immunogenicity resulting
from expression of foreign proteins. The first concern relates exclusively
to viral vectors; the second also relates mainly to viral vectors since
most nonviral vectors are not integrated into the host genome.

Generation of Replication-Competent Viruses--All viruses in current
use for clinical studies are replication-incompetent. Viral proteins are
provided in "trans," either in a stable packaging cell line or
a transient expression system. "Trans" means that the virus that
is packaged in these cells contains all the necessary viral proteins (supplied
by engineered packaging cells) but lacks the genes to produce more viral
proteins. Instead, the viral genome encodes the therapeutic gene under
appropriate regulatory sequences. The resulting virus can thus undergo
one round of infection, but, once inside the target cell, it is unable
to produce more virus, since no viral proteins are made.

Concern about the generation of replication-competent virus stems from
one study in which a retroviral stock known to contain replication-competent
virus induced T-cell lymphoma in primates.[23] Thus, rigorous testing of
viral stocks is required, as well as monitoring of patients at specified
periods following infusion to ensure the absence of virus. Replication-competent
virus has not been detected in any of the more than 500 patients treated
with viral vectors to date.

Insertional Mutagenesis--Theoretically, there is always the possibility
that insertion of material into the genome may occur at a site, which would
have a deleterious effect. However, this has not occurred in any of the
clinical trials to date.

Immunogenicity--In contrast, immunogenicity has proven to be
a problem in clinical studies involving adenoviral vectors. In early cystic
fibrosis trials, an immunogenic response to adenoviral proteins encoded
in the vector genome led to decreased dosages and attempts to engineer
the vector to make it less immunogenic. It will be important to resolve
this problem, since the immunogenic response can lead not only to localized
inflammation at the site of delivery but also to rapid elimination of transduced
cells.

There have been no deleterious, long-lasting effects attributed to genetic
intervention in any of the patients treated in over 60 clinical protocols
to date.

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