Download The Current Status of Gene Therapy for Prostate Cancer

The Current Status of Gene Therapy
for Prostate Cancer
Mohammad R. Nowroozi, MD, and Louis L. Pisters, MD
As a new treatment approach, gene therapy may affect both local and systemic control of prostate cancer.
Background: Due to limitations of local and systemic therapies for prostate cancer, interest has continued in the development of new treatment modalities. Gene therapy has emerged as a new approach that may prevent or treat disease by using the therapeutic information encoded in DNA sequences. Several institutions are actively experimenting with this approach.
Methods: The authors review the most common genetic alterations in prostate cancer, the principles of gene therapy, and gene delivery including both viral and nonviral vectors. Treatment strategies for both cytoreductive gene therapy as well as corrective gene therapy are described, and the available protocols to date with gene therapy for the treatment of prostate cancer are presented.
Results and Conclusions: More than 150 active protocols are ongoing to evaluate gene therapy in the treatment of cancer, with 13 of these open for patients with prostate cancer. The future of gene therapy as applicable to prostate cancer depends on additional development of vector systems and a better understanding of the genes involved in tumor induction and proliferation. Although gene therapy is clearly in its infancy, it is in an explosive growth phase and holds tremendous promise as a treatment modality for prostate cancer.
Introduction to Gene Therapy
Although the incidence rate and mortality rate of prostate cancer have decreased slightly in the past few years, prostate cancer remains a major health threat to American men. This disease remains the most commonly diagnosed internal malignancy in men and the second leading cause of cancer death among men in the United
States.1 In 1998, an estimated 185,500 men will be diagnosed with prostate cancer, and 39,200 will die of the disease.2 Biologically, prostate cancer represents a
heterogeneous disease entity that exhibits varying degrees of aggressiveness, patterns of metastasis, and response to therapy.
Universal agreement has not been reached as to the best treatment for prostate cancer at any stage. Radical prostatectomy, external-beam radiation therapy,
brachytherapy, and cryotherapy can affect local tumor control and are potentially curative in patients with clinically localized disease. In spite of the widespread use of
prostate-specific antigen (PSA) in early detection and screening, many cases are not diagnosed until the disease has advanced or metastasized beyond the reach of
these local treatment modalities. Hormonal therapy and chemotherapy are the only systemic treatments available at the present time. Unfortunately, progressive disease
develops in many patients who undergo these treatments, thus proving them to be noncurative.
Because of the significant limitations of currently used local and systemic therapies for prostate cancer, interest has continued in the development of new treatment modalities. Gene therapy has emerged as an exciting new treatment that may affect both local and systemic control of prostate cancer.
The term gene therapy broadly refers to the transfer of genetic material into human cells and the expression of that material in these cells for a therapeutic purpose.3
With respect to cancer, the goal of gene therapy is to prevent or treat disease by using the therapeutic information encoded in the DNA sequences.
Evidence suggests that tumor formation is caused by the overexpression of oncogenes or by mutations in suppressor genes in the presence or absence of cancercausing environmental events. In the first human experiment of gene therapy, Blaese and coworkers4 successfully transferred the gene for adenosine deaminase into the
cells of a 4-year-old girl with severe combined immunodeficiency caused by adenosine deaminase deficiency. The gene therapy dramatically improved her immune
system function. Presently, more than 150 active protocols are evaluating gene therapy in the treatment of cancer, with at least 13 of these protocols open for patients
with prostate cancer5 (Table 1).6
Table 1. -- Current Prostate Cancer Gene Therapy Protocols in the United States
Principal
Institution Investigator Gene
(s)
Vector
Treatment
Approach
Johns
Hopkins
Retrovirus
Immunotherapy
Subcutaneous Metastatic PC
injection
Simons
GM-CSF
Delivery
Target
Population
Vanderbilt
University
Steiner
Medical
Center
Antisense
Retrovirus
c-myc
Oncogene
inhibition
Intraprostatic
Advanced PC
injection
National
Naval
Medical
Center
PSA gene
Vaccinia
virus
Immunotherapy
Intradermal
injection
--
Cationic
liposome
complex
Immunotherapy
Intradermal
injection
Locally
advanced
Medical or
metastatic PC
Chen
Duke
University Paulson
Center
IL-2
Baylor
College of Scardino
Medicine
HSV-tk
Adenovirus
Suicide gene
(prodrug)
Local
Intraprostatic
recurrence of
injection
PC
DanaFarber
Cancer
Institute,
Boston
PSA gene
Vaccinia
virus
Immunotherapy
Intradermal
injection
Kufe
University
of
Sanda
Michigan
Vaccinia
PSA gene
virus
University
of
Belldegrun
California
IL-2
Cationic
liposome
complex
HSV-tk
Suicide gene
Adenovirus
(prodrug)
Mount
Sinai
School of
Medicine
Hall
Intradermal
Immunotherapy
injection
Immunotherapy
--
Serologic
recurrence of
PC following
radical
prostatectomy
Intratumoral
injection
--
Intratumoral
injection
Neoadjuvant
treatment for
patients with
clinically
localized PC
UCLA
School Belldegrun p53
Medicine
Adenovirus
Suppressor
gene
Intratumoral
injection
Locally
advanced of or
recurrent
adenocarcinoma
Johns
Hopkins
Retrovirus
Immunotherapy
Subcutaneous
injection
--
Simons
GM-CSF
MD
Anderson
Cancer
Logothetis,
Center and
p53
Steiner
University
of
Tennessee
Tumor
Adenovirus suppressor
gene
Baylor
Adenovirus
Kadmon
HSV-tk
Suicide gene
(prodrug)
Intraprostatic Locally
injection
advanced PC
Intratumoral
injection
Johns
Simons
Hopkins*
PSA gene Adenovirus
Vector-directed Intratumoral
cell lysis
injection
Vanderbilt
University
and
Smith,
University Steiner
of
Tennessee
BRCA1
Oncogene
Retrovirus
Neoadjuvant
prior to
prostatectomy
Recurrent
locally
advanced PC
Intraprostatic Advanced PC
inhibition
injection
*Protocol under review, not open yet.
GM-CSF = granulocyte macrophage-colony stimulating factor
PSA = prostate-specific antigen
IL-2 = interleukin-2
HSV-tk = herpes simplex virus-thymidine kinase
PC = prostate cancer
Genetic Alterations in Prostate Cancer
Tumorigenesis is a multistep process that involves initiation, proliferation, loss of contact inhibition, invasion, and metastasis of the cancer cell. It is likely that the pathway leading to malignancy undoubtedly involves many complex genetic and epigenetic influences, such as cell-cycle regulation, angiogenesis, immunoreactivity, and
cell adhesion. In fact, no single gene defect has been consistently implicated in the development of any type of cancer; rather, this process has been shown to involve
many different mutations.7
A number of genetic changes have been documented in prostate cancer, including allelic loss, point mutations, and changes in DNA methylation pattern. The most consistent changes are allelic loss events, with the majority of tumors examined showing loss of alleles from at least one chromosomal arm. The short arm of
chromosome 8 and the long arm of chromosome 16 appear to be the most frequent regions of loss, suggesting the presence of novel tumor suppressor genes.8 A
summary of the genetic alterations occurring in prostate cancer is presented in Table 2.
Table 2. -- Common Genetic Alterations in Prostate Cancer
Gene
Effect on
Tumorigenesis
Normal Gene
Function
Comments
ras
Oncogene
Regulates DNA
replication through
cell cycle
Mutated in only 2%-5% of
prostate cancers in US men
bcl-2
Oncogene
Interfaces with the
p53 pathway and
inhibits apoptosis
Elevated in most androgenindependent and advanced
prostate cancers
c-myc
Oncogene
DNA regulatory
protein involved in
DNA repair and cell
proliferation
May be involved in prostate
cancer progression
GST-P1
Suppressor gene
Detoxifies potential
carcinogens
Most common genomic
alteration in prostate cancer;
found in over 98% of cases
p53
G1 checkpoint in
response to DNA
Suppressor gene damage; also
involved in
apoptosis
Mutation associated with
androgen-independent
progression
p21
Cell-cycle control
Suppressor gene and apoptosis
pathway
Suppresses prostate cancer
cell lines in vitro
p16
Binds to and inhibits
Inhibits growth of prostate
Suppressor gene cdk4, cell-cycle
cancer cell lines
regulation
Retinoblastoma Suppressor gene Cell-cycle regulation
E-cadherin
Cell adhesion
Suppressor gene
molecule
C-CAM1
Suppressor gene
Cell adhesion
molecule
Allelic loss in ~27% of
prostate cancers
Reduced or absent in about
half of prostate cancers;
normal expression is a marker
of locally confined disease
Downregulated in some
prostate cancers; inhibits
prostate cancer growth in
animals
CAM1 = cell adhesion molecules 1
GST = glutathione-s-transferase gene P1
Alterations in gene function and expression also are involved in the emergence of androgen-independent disease. Androgen deprivation causes the loss of prostate
cancer cells by means of the cell-death process referred to as apoptosis. Some 70% to 80% of patients experience at least partial remission after hormonal therapy, but
treatment failure and tumor recurrence are almost inevitable.9 Several months or years after treatment, prostate cancer eventually progresses to hormone-refractory
status. Several potential mechanisms for the development of androgen-independent disease have been described, including activation of epidermal, fibroblast, or other
growth-factor pathways; alteration of genes such as ras; mxi1 (a negative regulator of the c-myc proto-oncogene); mutations; overexpression of the bcl-2 oncoprotein
(suppressor of apoptosis); amplification of c-myc; loss or mutation of tumor suppressor genes Rb and p53; and loss of metastasis-suppressor genes such as E-cad
(cadherin) and kai1 in prostate cancer cell lines.10-13 After failing hormonal therapy, most prostate cancers remain incurable with conventional chemotherapy.
Principles of Gene Therapy
When considering gene therapy as a treatment approach for prostate cancer, the treatment team must decide what genes to insert, how to deliver the genes, and how to express the therapeutic genes at the site of cancer. Two major categories of gene therapy are cytoreductive gene therapy and corrective gene therapy. Cytoreductive
gene therapy includes treatment strategies designed to selectively destroy malignant cells either directly (eg, toxic genes) or indirectly (eg, genes that stimulate immune
responses). Corrective gene therapy involves replacing or inactivating defective genes in preneoplastic or neoplastic cells with genes that can slow or reverse the loss of
growth-control mechanisms (eg, tumor suppressor genes). These therapeutic genetic modifications can be performed either ex vivo or in vivo, depending on the
strategy.
Gene Delivery
One of the rate-limiting factors affecting gene therapy is the development of a safe, reliable vector that can insert the desired gene into the target. Vectors are
engineered DNA or RNA sequences into which a therapeutic gene can be inserted. The therapeutic gene generally is positioned adjacent to a promoter sequence for
RNA polymerase. From this position, messenger RNA (mRNA) of the therapeutic gene is expressed within the cell. Promoter sequences regulate gene expression
downstream and thus can serve as critical pharmacologic targets to modulate gene function. Most viral promoter sequences are, in fact, the endogenous long-terminalrepeat sequences. The ability of a vector to successfully deliver the gene of interest into a large number of target cells is defined as gene transfer efficiency.14 For
instance, if 1 in 3 treated cells takes up the vector successfully, then the gene transfer efficiency is 33%. The overall efficacy of the vector is also determined by the
degree of gene transduction achieved. Delivery methods are categorized as viral (eg, retrovirus, adenovirus, and adeno-associated virus [AAV]) and nonviral (plasmid
DNA, liposome DNA). The advantages and disadvantages of specific types of vectors are summarized in Table 3.
Table 3. -- Vectors for Gene Therapy
Vector
Advantage
Disadvantage
Viral
retrovirus
Easy to produce, efficient transfer,
small genome, biology well
understood, nontoxic to host cells,
high-efficiency genomic integration,
stable expression
Targets only dividing cells, risk of
replication, carries small DNA
sequences only, low transduction
efficiency, integration with potential
oncogenesis, poor in vivo delivery
Adenovirus
Highly efficient transfer, targets
nondividing cells, nontoxic to host
cell, high transduction efficiency,
immunogenicity
Possible host immune reaction risk
of replication, carries small DNA
sequences only, low potential
oncogenesis, no integration,
transient expression
Adenoassociated
virus
Less likely to produce immune
reactions, targets nondividing cells,
Small capacity, immunogenic, not
nonpathogenic in humans, efficient
well studied, risk of replication
transfer, good in vivo delivery,
integrates into genome
Vaccinia
virus
High titer, large insert size
Herpes
Large insert size
simplex virus
Antivector immunity, toxicity
Toxicity
Nonviral:
Plasmid DNA No size limitation
Easy to produce, safety features,
Low efficiency
Liposome
less likely to produce immune
response, no limitation on size and
type of nucleic acid
Low efficiency
Nonviral Vectors
Nonviral vectors offer several advantages over viral vectors with respect to safety and ease of production. Rigorous tests are not required 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 viral vectors.15
However, there are some limitations to traditional methods of plasmid transfection. Several limitations are specific to the use of plasmid transfection in prostate cancer.
Researchers have encountered some difficulties when attempting to passage prostate cancer cells in vitro, and these difficulties make the practical application of
plasmid transfection virtually impossible to reproduce.16 Furthermore, when plasmid transfection is carried out, overall transfection efficiency is very low. With systemic
administration, naked DNA plasmids are rapidly cleared from the bloodstream, and DNA degradation occurs within 5 minutes.7 The stability and transformation
efficiency of naked DNA is enhanced by coating plasmids with lipids (liposome-DNA). In this form of nonviral vector delivery, a liposome shell surrounds the plasmid,
protecting the DNA from degradation after systemic administration to the host. In addition, the lipid envelope can fuse with tumor-cell membranes, resulting in direct
delivery of the therapeutic gene to the cytoplasm of the cell. Plasmid transfection also is not likely to produce immune response and decrease in efficiency because of
the presence of blocking antibodies. Because the majority of liposome gene complexes are rapidly cleared from the circulating bloodstream by the liver, systemic
therapy cannot be efficiently administered.17 It is possible that intratumoral injection of liposomes may be able to bypass this intrahepatic clearance of the gene and
therefore improve transfection efficiency.
Liposome vectors show significant promise; however, more studies are needed to determine their safety and efficacy in vivo before widespread use.
Viral Vectors
Retroviruses and AAVs have an advantage in that they facilitate stable integration and a steady level of expression once the therapeutic gene is transfected and incorporated into the host genome. As the target DNA is replicated, so too is the inserted therapeutic gene embedded in the transferred chromosomal DNA. Thus,
transduction via these vectors can produce durable gene expression. In corrective gene therapy, the durability of the replication is essential for maintaining the corrected
cell phenotype over the patient’s lifetime.18 Furthermore, this can be advantageous in tumor vaccine strategies in which a steady level of gene expression may enhance
efficacy. In contrast, adenovirus, vaccinia, and liposomal vector transfer are episomal methods: the transferred gene is expressed without actual integration of the gene
into the target cell genome. These vectors are not well suited to clinical strategies requiring durable expression of transferred therapeutic genes.
Retroviral gene expression and dissemination depend on the presence of a rapidly proliferating tumor-cell population. In some malignancies, such as prostate cancer,
in which the tumor is inherently slow growing, the retroviral approaches may not be effective.19 The possibility of pathogenic mutagenesis during chromosomal insertion
of the vector and difficulty in isolating high titers of retrovirus for clinical use further limit retroviral transfer.20
In contrast to retroviruses, adenoviral vectors can facilitate highly efficient transfection of therapeutic genes into cell culture. The adenovirus has several characteristics that make it ideal for use in prostate cancer gene therapy. The adenovirus enters target cells by receptor-mediated endocytosis after binding to integrins or fibronectins,
providing significant tropism to cells of epithelial origin.9 Unlike the retrovirus, the adenovirus does not depend on cell replication for transfer of expression of its genetic
material.21 The safety of adenoviral gene therapy vectors already has been shown in human trials involving
cystic fibrosis, ornithine transcarbamylase deficiency and factor IX deficiency.21,22 Because the adenovirus can be efficiently delivered into replicating or nonreplicating
cells of epithelial origin, it may be ideal for in vivo systemic therapy, provided an effective avenue of delivery to sites of metastatic cancer can be devised.
Genomic integration rates are low; consequently, there is little risk of long-term sequelae with the administration of adenovirus.21 Short-term expression has obvious
limitations for chronic gene replacement, but it may be considered advantageous for the treatment of neoplasias. For example, transient overexpression of p53 in
prostate cancer may be sufficient to activate apoptosis in neoplastic cells without the concerns associated with integration of genetic material into normal cells. Induction
of antiviral antibodies and high levels of hepatic deposition of circulating virus would make these vectors inefficient for readministration and systemic administration,
although it has been reported that the coadministration of adenovirus and immunosuppressive drugs such as cyclosporin A can drastically increase the duration of
transgene expression in hepatocytes and hence suppress cellular immunity against adenovirus.9,16
Some AAV vectors appear to allow durable genetic transduction and are stable for potential direct in vivo gene transfer.20 However, integration of AAV genomes
into target cell DNA appears to be less efficient and has been associated with tendency toward inactivations via deletions or rearrangement during gene transfer.
Cytoreductive Gene Therapy
According to the immune surveillance theory of Burnet, the immune system is responsible for eliminating newly transformed cells; therefore, the emergence of a tumor signals the failure of the immune system.5 One gene therapy approach involves the activation of antitumor immune responses via T-cell killing of cancer cells.
The most thoroughly evaluated form of cytoreductive gene therapy for cancer so far involves stimulation of an antitumor immune response against a malignancy by vaccinating affected patients with genetically modified tumor cells.18 In the ex vivo vaccine approach, tumor cells are removed at surgery from the patient, grown in cell
culture, and transfected with cytokine genes that stimulate an immune response to antigens present on the tumor cell vaccine. The gene-modified tumor vaccine is then
irradiated to prevent subsequent tumor growth and reinjected into the patient in an attempt to generate either a local or systemic immune response against the remaining
tumor burden in the patient. These immune effector cells activated at the vaccination site may include T cells, B cells, natural killer cells, and antigen-presenting cells
such as dendritic cells and macrophages.23 In theory, the B-cell or T-cell arm activated by vaccination can then circulate systemically and eradicate or slow the growth
of distant micrometastatic cells that share antigens with the genetically engineered vaccine cell.
Several therapeutic cytokine genes have been studied for use in gene therapy. Sanda et al24 and Blades et al25 showed a defect in cell-surface expression of class I
major histocompatibility complex (MHC) in prostate cancer. Cytokines that are dependent on MHC class I processing for immunostimulatory effects, eg, interferon
(IFN)-8 , interleukin (IL)-4, and IL-6, are not prime candidates for cytokine gene-therapy approaches. On the other hand, cytokines not dependent on MHC class I
antigen processing (IL-2 and granulocyte macrophage-colony stimulating factor [GM-CSF]) may be more suitable for prostate cancer gene therapy. Sanda and
coworkers26 found that therapy using gene-modified, irradiated vaccine cells genetically transduced to secrete GM-CSF prolonged survival in animals with prostate
cancer; thus, this approach is feasible and has potential for wide application as a treatment strategy for human prostate cancer.
There are, however, limitations to the ex vivo approach. The efficacy of this tumor vaccine approach depends on establishing reliable, high levels of cytokine
expression within the tumor. Transfection of tumor-cell vaccine often requires surgical removal of the primary tumor. Furthermore, the harvesting, in vitro culture, and
transfection of autologous cells makes the ex vivo vaccine approach labor intensive, time consuming, and expensive. Another cytokine therapy approach involves the
introduction of cytokine genes directly into viruses or packaged segments of DNA that can deliver the cytokine gene directly into the tumor cells. Transfection and
genomic incorporation result in the chronic local production of cytokines from within the tumor itself. Both viral vectors and liposomecytokine gene complexes have
been shown to be effective in animal and human studies.7
A second form of cytoreductive gene therapy under clinical development is the transfer of drug-susceptible or "suicide" genes. In this strategy, a gene is transfected
into tumor cells that encodes the active site of an enzyme, which converts a nontoxic prodrug form of an antineoplastic drug into a cytotoxic one in transfected tumor
cells. The prodrug agent is given intravenously after gene transfer. Herpes simplex virus thymidine kinase (HSV-tk) is a classic suicide gene. Others, such as varicella
zoster and E-coli cytosine deaminase, have been used in other models.5
HSV-tk converts nontoxic nucleoside analogues such as ganciclovir (GCV) into phosphorylated compounds that act as chain terminators of DNA synthesis.9 GCV
is safe when given systemically as an chemotherapeutic antivirus and is a high-affinity substrate for HSV-tk. The introduction of the HSV-tk gene has been achieved via
adenoviral or retroviral vectors. One of the potential advantages of HSV-tk therapy is that it demonstrates the "bystander effect," which is the percentage of cells killed
exceeds the percentage of cells originally transduced. There are several explanations for this phenomenon, including the possibility that toxic metabolites are transferred
between juxtaposed cells and the possibility that systemic immune response is induced.27 To date, close cell–cell contact appears necessary for the "bystander" effect
to work. Hall and colleagues28 demonstrated that adenovirus-mediated HSV-tk/GCV therapy leads to systemic activity against spontaneous and induced metastasis in
an orthotopic mouse model of prostate cancer. Eastham et al29 showed that HSV-tk/GCV cytotoxic gene therapy can inhibit the growth of mouse and human prostate
cancer cells in vitro and can interrupt tumor growth of an aggressive mouse prostate cancer cell line in vivo.
A modification of cytoreductive gene therapy involves tissue-specific expression of drug-susceptible genes. Cell toxin vectors are being constructed that contain
tissue-specific promoters to restrict expression of the transferred cytotoxic gene. In prostate cancer, this approach has additional promise for the creation of tissuespecific gene therapy because of the recent discovery of a bipartite, PSA enhancer-promoter sequence. This DNA sequence results in high levels of androgensensitive, prostate tissue-specific gene expression. In more than 95% of the cases of metastatic prostate cancers, PSA promoter is used to express detectable levels of
PSA protein. When the PSA enhancer-promoter sequence is combined with the HSV-tk, there is potential for prostate cell-specific expression of the transfected gene,
with cytotoxic effects limited only to the target tissue.7
An entirely new class of cytoreductive gene therapy vectors can be generated as a consequence of the identification of specific transcription elements: oncolytic, replication-restricted viruses. Minimal enhancer-promoter DNA sequences for PSA have been put into the adenovirus genome to drive the control of viral replication
genes.18 The human adenovirus E1B gene encodes a 55-kd protein (E1B 55K) that binds and inactivates p53. Bischoff et al30 showed that a mutant adenovirus that
does not produce this viral protein can replicate in and lyse p53-deficient human tumor cells but not cells with functional p53.
An alternative strategy for cytoreductive gene therapy focuses on bone metastasis of prostate cancer. Osteoblastic response to prostate cancer is the hallmark of progression at this metastatic site. Osteocalcin (OC), a noncollagenous bone matrix protein, is expressed in high levels by osteoblasts. Ko et al31 constructed a
recombinant adenovirus, AD-OC-tk, which contains the OC promoter that drives the expression of HSV-tk as suicide gene. They showed that the OC promoter
mediated high levels of expression in osteoblast cell lines. Treatment with the AD-OC-tk plus prodrug has potential to eradicate osteoblastic cells that may be required
to maintain the survival of osseous metastatic tumors in prostate cancer.
Corrective Gene Therapy
Many different genetic alterations have been identified in prostate cancer (Table 2). Most of the lesions represent either overexpression of an oncogene or inactivation of a tumor-suppressor gene. Tumor-suppressor genes and antisense oncogenes are used chiefly as reagents for corrective gene therapy.
In metabolic diseases in which a single gene defect has been identified as the cause of the disease state, such as in cystic fibrosis, replacing the defective gene product is a promising treatment approach. However, the main problem in cancer is that there is no single oncogene or tumor-suppressor gene defect.7
Tumor-suppressor genes are a diverse group of genes present in the normal genome. Their inactivation may result in the initiation or the progression of a cancer. The
p53 gene is the most commonly mutated gene in human cancer. The p53 gene replacement is a particularly attractive therapeutic strategy because in vitro restoration
of wild-type p53 in many tumor cell lines causes growth arrest or apoptosis. The most common genomic alteration in prostate cancer is inactivation of the glutathioneS-transferase P1 gene (GST-P1).32 This inactivation may occur as early as prostatic intraepithelial neoplasia (PIN), and it is identified in over 98% of cases of clinically
detected prostate cancer. GST-P1 is an attractive target gene for corrective gene therapy research. Many other suppressor genes also have been identified as potential
targets for prostatic cancer corrective gene therapy, including p21,33 CAMS, and kai1. The University of Texas M.D. Anderson Cancer Center with the University of
Tennessee and the University of California at Los Angeles have begun phase I clinical trials of p53 gene replacement therapy for prostate cancer patients (Table 1).6
Antisense oligodeoxynucleotides are short synthetic nucleotide sequences formulated to be complementary to specific DNA or RNA sequences.3 They may be
delivered to target cells with annealing of the strands and thus can potentially disrupt transcription or translation of target oncogenes.5 The bcl-2 oncoprotein suppresses
apoptosis, and when overexpressed in prostate cancer cells, bcl-2 makes these cells resistant to a variety of therapeutic agents, including hormonal ablation drugs.
Dorai et al11 have synthesized a hammerhead ribozyme against bcl-2 mRNA and demonstrated efficient cleavage in vitro. Antisense bcl-2 is only one example of many
antisense oligodeoxynucleotides that could be used in corrective gene therapy.
The p53 Gene -- An Appropriate Gene for Prostate Cancer Gene Therapy
The p53 tumor suppressor gene is a 393-amino acid nuclear phosphoprotein that acts as a transcription factor to control expression of proteins involved in the cell
cycle.34 The p53 gene maps to the P arm of chromosome 17 with loss of heterozygosity resulting in expression of a mutant allele.35 It is continuously synthesized and
degraded; its levels are low under physiological conditions. When p53 levels rise, the result is G1 arrest of cell cycle or apoptosis. The levels of p53 increase after
irradiation and other types of cell damage. These increased levels of p53 in the nucleus arise from an increase in its half-life. Increased levels of p53 downregulate bcl-2
(which inhibits apoptosis) and upregulate the expression of specific genes including p21 and Bax.36 Bax is a potent promoter of the apoptotic cell death pathway, and
p21 inhibits cyclin-dependent kinases. Because of its functions, p53 has been called the "guardian of the genome," and its loss has been implicated in tumor
progression.34
There is some evidence that p53 might exert some of its antitumor activity through inhibition of angiogenesis as a consequence of an altered production of
thrombospondin (bystander effect).37 In addition, the presence of the wild-type p53 gene is speculated to be useful in accelerating induction of apoptosis caused by
cytotoxic drugs such as cis-platinum.18 Thus, corrective gene therapy with p53 is viewed by some as having the potential to improve the therapeutic index of available
chemotherapeutic drugs.
The p53 gene is the most commonly mutated gene in human cancer.18 Early studies have shown that approximately 60% of prostate cancer cell lines have mutations
in the p53 gene.7 Although primary prostate tumors have few mutations in p53 gene, specimens from advanced stages of the disease and metastases as well as their cell
lines frequently display mutations or deletions at both alleles of the p53 gene.36 Transfection of wild-type p53 into prostate cancer cell lines expressing mutant alleles
results in loss of tumorigenic capability.38 Gotoh et al39 demonstrated that p53 was able to suppress tumorigenicity regardless of the background p53 status of the
tumor cells.40
Ad-p53 - Intraprostatic Gene Therapy (INGN 201)
Gene therapy strategies that attack the underlying genetic mechanism of this disease offer promise in improving outcomes. In the protocol described here, investigators at M.D. Anderson Cancer Center and the University of Tennessee propose to study the effect of intraprostatic AD-p53 (INGN 201) injection (Table 1).
INGN 201 is a replication-defective adenoviral vector that encodes a wild-type p53 gene driven by a cytomegalovirus (CMV) promoter.
Eligible patients will have clinical stage T1c or T2a with high-grade disease (Gleason grade 8-10) on initial biopsy or clinical stage T2b-T2c with Gleason grade 7
and PSA >10, or clinical stage T3.
Patients with locally advanced prostate cancer who are enrolled in the study undergo baseline magnetic resonance imaging (MRI) and ultrasound of the prostate. One course of p53 therapy is defined as three separate injection procedures separated by two weeks, with reevaluation using transrectal ultrasound and MRI. If these
show reduction in the size of measurable lesion, then a second course of p53 gene therapy is performed. If there is no change after the first course from the baseline
MRI and transrectal studies, then patients are treated with radical prostatectomy. Those patients completing a second course of p53 gene therapy are again reevaluated
and proceed to radical prostatectomy.
The Technique of AD-p53 Injection
Using a specially designed needle guide that fits to the transrectal ultrasound probe and positions a needle at a measurable distance above the ultrasound transducer head, 17-gauge needles with stylettes are inserted transperineally into the prostate at the positions shown in Fig 1. The needles are advanced to the apex of the prostate
and then the stylettes are withdrawn, which allows an 18-gauge core biopsy to be obtained longitudinally along the axis of the prostate. The stylette is then advanced
back through the needle, and the needle is advanced along the tract of the biopsy longitudinally from the apex to the base of the prostate. This allows a longitudinal core
biopsy to be obtained at the site of injection. The p53 gene in the adenoviral vector is preloaded in 1-mL plastic "TB" syringes that are then hooked onto the 17-gauge
needles and injected into the prostate as shown in Fig 2.
A total of 30 patients will be enrolled in this study. Because the study is ongoing and the results are preliminary, it is not possible to comment on the efficacy of the treatment at this time.
The Future of Gene Therapy
The future of gene therapy approaches to prostate cancer or other cancers will depend on further development of vector systems at the basic science level as well as a better understanding of the genes involved in tumor induction and proliferation. In the long term, we will look at the construction of artificial chromosomes that can
carry whole clusters of genes with their natural control elements into cells. With this new technology also comes new ethical responsibilities to ensure that these
strategies are safe for patients and staff.3
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From the Department of Urology, The University of Texas M.D. Anderson Cancer Center, Houston, Tex. Dr Nowroozi is currently at the Department of Urology, Imam Khomeini Hospital, Tehran University of Medical Sciences, Keshavarz Blvd, Tehran, Iran.
Address reprint requests to Louis L. Pisters, MD, at the Department of Urology, Box 110, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX
77030.
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