- Email: [email protected]
Prostate cancer gene therapy
Surg Oncol Clin N Am 11 (2002) 607–620
Prostate cancer gene therapy Mitchell S. Steiner, MD*, Jeffrey R. Gingrich, MD, Ravi D. Chauhan, MD Department of Urology, University of Tennessee Memphis, 1211 Union Avenue, Suite 340, Memphis, TN 38104, USA
Gene therapy involves the transfer of DNA into cells to replace or to affect the expression of the cell’s native (endogenous) genes. This transfer may occur while the cell is outside the body (ex vivo) and then returned to the body, or the gene may be introduced into the cell while it remains in the body (in vivo) in its natural microenvironment. To transfer DNA into a cell with sufficient efficiency, a DNA transporter or ‘‘vector’’ is generally required. Methods used to transfer genes into cells are broadly categorized as either nonviral or viral based and have recently been nicely summarized [1–3]. The hope of identifying genes whose expression level can be modulated to affect the development and progression of prostate cancer has led to efforts to develop gene therapy as a viable new treatment strategy to treat or potentially even to prevent prostate cancer. In this article, current strategies for prostate cancer gene therapy and initial results of preliminary clinical trials are reviewed. Prostate cancer and gene therapy Prostate cancer is particularly suited for gene therapy for a number of reasons: (1) prostate cancer is quite common, and for the patients diagnosed with locally advanced disease there is no cure currently available; (2) the prostate gland does not serve any life-sustaining function and therefore complete ablation of benign, premalignant, and cancerous prostate tissue could be desirable; (3) the prostate is accessible by transurethral, transperineal, and transrectal approaches for administration of gene therapy; (4) the prostate may be monitored by digital rectal examination, transrectal This work was supported by the Assisi Foundation, Inc. and J. R. Hyde Family Foundation. * Corresponding author. E-mail address: [email protected] (M.S. Steiner). 1055-3207/02/$ - see front matter Ó 2002, Elsevier Science (USA). All rights reserved. PII: S 1 0 5 5 - 3 2 0 7 ( 0 2 ) 0 0 0 2 6 - 1
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ultrasound, endorectal coil magnetic resonance imaging, or by serum prostate specific antigen (PSA); (5) the prostate gland produces high levels of several characterized proteins, including prostate specific antigen, whose promoters and other enhancers may be incorporated into vectors to direct prostate-specific expression of therapeutic genes [4]. Current strategies of gene therapy for prostate cancer Numerous gene therapy strategies currently being employed for the treatment of prostate cancer have recently been reviewed [4]. Thirty-two gene therapy trials have been approved by the National Institutes of Health for the treatment of prostate cancer. The most recent trials (approved during the last three years) are listed in Table 1. To date, the approved trials have all been either phase I or phase I/II in design. They can be broadly categorized as: (1) immunotherapy—to enhance the host immune system anti-tumor response, (2) corrective gene therapy—to increase or abrogate specific aberrant gene expression, (3) ablative or ‘‘suicide’’ gene therapy—to introduce toxic or cell lytic genes, and (4) combination therapy—one or more of these strategies with conventional cytotoxic chemotherapy or radiation therapy. Immunotherapy Alterations of class I MHC expression have been shown to be a common way that prostate cancer cells may evade the host’s immune system [5,6]. The approaches that have been used to stimulate the host antitumor immune response are gene vaccine therapy using ex vivo gene transfer techniques, and in vivo intratumoral injection of gene therapy vectors containing cytokine genes. Tumor vaccines have been generated using autologous tumor or nonautologous cells, such as tumor cell lines. Before subcutaneous inoculation, the cells are modified by ex vivo gene therapy to express cytokine genes, and radiated to eliminate their replication capacity. Cytotoxic T lymphocytes (CTL) not only recognize tumor-specific antigens present on the surface of these inoculated cells, but are also induced by local secretion of the transferred stimulatory cytokines. The activated CTL expand in number and then destroy tumor cells that share these same antigens on their cell surfaces throughout the body. Preclinical studies have clearly shown that gene vaccines are not efficacious in the presence of a large tumor burden, but theoretically they may be more useful against micrometastasis following removal or debulking of the primary tumor for locally advanced disease, or in the setting of minimally recurrent disease. The first approved gene therapy trial for prostate cancer was by Simons [7]. It was designed for patients found to have metastatic prostate cancer in the lymph nodes at the time of radical prostatectomy. Similar to their previous renal cell carcinoma studies, ex vivo transduction of autologous, irradiated prostate tumor cells with a retroviral granulocyte-macrophage
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colony-stimulating factor (GM-CSF) construct was performed to generate vaccines. The vaccines were administered subcutaneously every two weeks until available cells were exhausted. Vaccination site biopsies revealed infiltrating macrophages, dendritic cells, eosinophils, and T-cells. No dose limiting toxicities were observed and all patients ultimately demonstrated progressive disease. Interestingly, sera from three of eight patients were shown to contain new antibodies recognizing similarly sized polypeptides present in the prostate cancer cell lines LNCaP and PC-3. This study demonstrated the feasibility of autologous GM-CSF transduced prostate cancer vaccines, and showed that both T-cell and B-cell immune responses to human prostate cancer can be generated and that treatment was restricted only by the limitations of in vitro cell expansion. To circumvent this limitation, a follow-up trial powered to estimate efficacy using ex vivo GM-CSF transduced allogeneic prostate cancer cell lines (PC-3 and LNCaP) was initiated. Patients were vaccinated weekly for eight weeks with irradiated, GMCSF secreting PC-3 and LNCaP prostate cancer cells. One of 21 patients had a partial PSA response of >7 months duration, 14 had stable disease, and 6 progressed [8]. At three months after treatment, PSA velocity or PSA slope decreased in 71% of patients. No dose-limiting toxicities were identified. Although the dosing and schedule to conclusively demonstrate therapeutic efficacy are still undergoing optimization, numerous post-vaccination IgG1 antibodies were identified, demonstrating that immune tolerance to prostate cancer-associated antigens may be broken, and that this therefore appears to be a clinically feasible strategy for prostate cancer treatment. Three trials using a different vaccine strategy have been approved using recombinant vaccinia virus expressing PSA (rV-PSA, PROSTVAC) as a tumor-associated antigen. Chen treated 30 patients with hormone refractory prostate cancer who had undergone withdrawal of anti-androgen therapy with a vaccinia inoculation followed by PROSTAVAC immunization [9]. Patients received 2.65105 or 2.65106 plaque forming units (PFU) once a month for 3 months. All patients developed local erythema at the vaccination site. Four of 14 patients had stable disease (2 progressed at 5 and 6 months) and 10 had continued disease progression. In a patient population with less advanced disease, Eder et al treated 33 men with rising PSA after radical prostatectomy, radiation therapy, both, or metastatic disease, with 2.6510 [6–8] PFU of rV-PSA as three consecutive monthly doses without significant toxicity [10]. Ten patients who received the highest dose also received 250 lg/m2 as an immunostimulatory adjunct without significant additional toxicity. Fourteen out of 33 men maintained a stable disease status for at least 6 months, and 6 remain progression free as of 21 months. This trial demonstrated that rV-PSA is safe and can elicit immune responses, arresting disease progression in some patients for up to 21 months or longer. In the third study, Sanda administered PROSTVAC once to 6 patients with androgen-modulated recurrence of prostate cancer after radical prostatectomy [11]. In addition to evaluation for toxicity, time until
9801-229
9802-236
9805-251 9812-276
9901-282
9901-283
9902-293
9904-306
9905-312
1
2
3 4
5
6
7
8
9
NIH#
II
I
II
I/II
II
I/II I
I
I/II
Phase
Belldegrun/ Corman/Klein
Vieweg
Kaufman
Small
Eder
Figlin Gardner/Chung
Simons
Kadmon
PI
Table 1 Prostate cancer gene therapy trials
UCLA/Puget Sound Health Care System/ Cleveland Clinic
Duke University
UCSF/Mt. Zion Cancer Center Albert Einstein
Dana-Farber
UCLA U of Virginia
Johns Hopkins University
Baylor College of Medicine
Institution
Vical, Inc.
Cell Genesys, Inc. Eastern Cooperative Oncology Group
NCI-CTEP
Transgene, SA
Calydon, Inc.
Sponsor
Hormone Refractory Prostate Cancer
Advanced
Prostate Cancer
Prostate Cancer
MUC-1 Positive Metastatic
High risk for advanced disease RadioRecurrence
Patient population
Liposome
RNA
Vaccinia/ Fowlpox Virus
Vaccinia/ Fowlpox Virus Retrovirus
Vaccinia virus Adenovirus
Adenovirus
Adenovirus
Vector
IL-2 cDNA
PSA
PSA
GM-CSF cDNA
PSA-Ela (replication competent) MUC-1/IL2 Osteocalcin HSV-tk/ valacyclovir PSA
RSV-HSV-tk/ ganciclovir
Gene
610 M.S. Steiner et al / Surg Oncol Clin N Am 11 (2002) 607–620
9906-321
9906-324
9909-338
9910-344
9910-345
9910-352
9911-356
9912-368
0001-373
0008-410
11
12
13
14
15
16
17
18
19
20
I
II
II
I
I/II
I/II
I/II
I/II
I/II
I
II
Vieweg
Arlen
Belldegrun/ Figlin Dahut/Gulley
Belldegrun/ Klein
Wilding
Terris
AguilarCordova/ Butler Gingrich/Steiner
Freytag/Kim
Small/Smith
Duke University
NIH/NCI
NIH/NCI
UCLA/ Cleveland Clinic UCLA
University of Wisconsin
University of Tennessee Stanford University
Texas Children’s Hospital
UCSF-Mt. Zion/VA San Diego Henry Ford Health Sys.
From http://www4.od.nih.gov/oba/rac/clinicaltrial.htm
9905-315
10
Transgene, SA
Vical, Inc.
Calydon, Inc.
Calydon, Inc.
GTx, Inc.
Cell Genesys, Inc.
Hormone Refractory Metastatic
Combined with Radiotherapy
Advanced
Hormone Refractory Metastatic RadioRecurrent
Locally Advanced RadioRecurrent
Combined with Radiotherapy
RadioRecurrent
Hormone Refractory
Vaccinia/ Fowlpox Virus Vaccinia/ FowlpoxVirus RNA
Vaccinia Virus
Liposome
Adenovirus
Adenovirus
Adenovirus
Adenovirus
Adenovirus
Retrovirus
Tumor RNA
PSA/B7.1
PSA
MUC-1/IL2
PSA Directed Cytolytic Adenovirus IL-2 cDNA
PSA
p16
Cytosine deaminase/ HSV - tk cDNA HSV - tk cDNA
GM-CSF cDNA
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rise in serum PSA after interruption of androgen deprivation therapy and Western blot analysis for anti-PSA antibody production were determined. One patient maintained an undetectable serum PSA for over 8 months after withdrawal of anti-androgen therapy and immunization. One patient developed an IgG antibody against PSA after immunization. It is interesting to note that one third of patients had anti-PSA antibodies before immunization. Sanda concluded that although immune responses against PSA may be present among some patients with prostate cancer at baseline, the immune response may be induced in others through vaccinia-PSA immunization. The significance of this finding and its implications regarding the therapeutic efficacy of vaccination against tumor-associated antigens is unclear. Direct in vivo intratumoral injection of gene therapy vectors containing cytokine genes attempts to treat tumor cells in situ in the hope of generating a beneficial systemic effect for metastatic disease. Naitoh et al, using animal models of prostate cancer, have shown that intratumoral injection of liposome and adenoviral vectors containing IL-2 gene expression systems resulted in the activation of specific T-cell antitumor responses [12]. Using a liposomal vector, clinical trials are under way as a neoadjuvant before radical prostatectomy and for local recurrence after radiation therapy (see Table 1, Belldegrun) [13]. Belldegrun et al treated 12 patients prior to radical prostatectomy and 9 patients with recurrent prostate cancer after radiation or cryotherapy with two injections of intraprostatic IL-2 [14]. A total of 40 injections of 300 to 1500 lg of IL-2 were administered. For patients treated before radical prostatectomy, the average PSA declined by 5.3 ng/ml before surgery and 75% maintained undetectable PSA levels at 24 to 56 weeks after surgery. Average PSA declines in patients with recurrent disease were 3.6 ng/ ml and 1.3 ng/ml after one or two injections respectively. Although the significance of these PSA responses and differentiation of the effects of the gene therapy from surgery alone are not yet clear, this strategy using liposomal delivery appears to be safe; it may also be effective locally and may promote a systemic immune response. Similarly, Sanford employed intratumorally injected adenoviral vectors containing IL-12 to treat primary prostate cancer tumors in mice, significantly reducing the number of lung metastases [15]. The molecular mechanism may include stimulation of T-cell and NK cells, induction of IFN-c, and upregulation of fas expression [15,16]. In summary, preclinical experience now suggests that immunotherapy may ultimately be effective against minimal residual, recurrent, or micrometastatic disease. Patients with greater metastatic or primary tumor volumes will most likely require some other nonimmunological therapeutic intervention. Corrective gene therapy Corrective gene therapy seeks to replace inherited or acquired defective genes that are important for normal growth regulation. Most corrective gene therapy strategies have used either retroviral or adenoviral vectors
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administered by intratumoral injection. Prostate cancer preclinical studies have been reported for the replacement of an assortment of tumor suppressor genes, including AdCMVp53 [17–19], retroviral LXSN BRCA-1 [17], AdCMVp21 [17,19], AdCMVp16 [18,20], and AdCMV CAM1 [21]. Antisense strategies (directing the expression of a reverse strand of mRNA complimentary to the normal or ‘‘sense’’ strand of oncogenes) effectively preventing the translation of the mRNA into protein have also been evaluated. A single intratumoral injection of retroviral MMTV antisense c-myc markedly suppressed and even eradicated DU145 prostate cancer xenografts growing in nude mice, through downregulation of c-myc mRNA and protein expression and the induction of apoptosis through downregulation of bcl-2 protein [22]. Using a similar approach, Kim et al have shown that an adenoviral vector containing an antisense erb-B-2 gene inhibited the overexpression of growth factor erb-B-2 in prostate cancer cells in vitro, thereby selectively purging metastatic prostate cancer cells from the bone marrow [23]. Preliminary results of the first approved trial using direct transrectal prostatic gene therapy injection have been reported by Steiner [19]. This was also the first study designed using a gene replacement strategy. Twenty-one prostate cancer patients who had failed standard therapy underwent ultrasoundguided injection of retrovirus LXSN containing BRCA1 under the control of the LTR viral promoter. No patients developed viral symptoms or evidence of viremia. Viral DNA was detected by PCR at least two years after injection (unpublished results). Average prostate volume was not decreased one month after injection and serum PSA in these patients with metastatic disease remained unchanged. This study demonstrated the feasibility and safety of direct, transrectal injection of prostate cancer gene replacement therapy. Initial clinical gene therapy results using a similar gene replacement strategy of replication-defective adenovirus containing wild-type p53 (AdCMVp53) driven by the CMV promoter before radical prostatectomy have recently been reported [24]. Seventeen patients with locally advanced prostate cancer received at least one course of ultrasound guided AdCMVp53 treatment consisting of three administrations, 14 days apart, of 3 ml in four to six divided injections. In this phase I/II study, the number of viral particles (vp) delivered was escalated from 31010 vp per treatment per patient to 31012 vp. Three patients completed a second course of therapy for >25% reduction in tumor size, as measured by ultrasound or endorectal coil MRI after the first course of treatment. This study confirmed the relative feasibility and safety of repeated adenoviral intraprostatic gene therapy injection. Further results regarding efficacy after the apparent radiologic responses, correlations with gene expression, pathologic findings at prostatectomy, and surgical outcomes are pending. In general, corrective gene therapy holds the promise of reversing the malignant phenotype. These approaches have shown that gene replacement may also indirectly lead to activation of cell death pathways. The ‘‘bystander effect’’ may then also result in additional tumor cell death through
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complementary or synergistic mechanisms. Contrary to historical surgical, chemotherapeutic, and radiation therapy goals to eradicate every cancer cell to effect a long-term cure, corrective gene therapy that restores a normal phenotype through a durable cytostatic effect or cytotoxic effect with induction of an adequate immune response may be an acceptable clinical endpoint. Ablative, or suicide gene, therapy ‘‘Suicide’’ gene therapy strategies may offer the most promising clinical potential. In this strategy, therapeutic gene expression results in cell destruction without regard to the underlying genetic mutations responsible for the malignant phenotype. Two types of suicide gene therapy strategies have emerged: gene-directed enzyme prodrug treatment, and gene-directed production of a cellular toxin or induction of apoptosis. Enzyme prodrug strategies use transfer of an enzyme expression vector. Cells producing the enzyme then convert a nontoxic, systemically administered prodrug into an activated, lethal product that is cytotoxic to the cell and thus may subsequently directly or indirectly kill neighboring cancer cells. This bystander effect can be quite dramatic, resulting in the death of 100 to 1000 fold more cells than would be predicted by gene transfer rates alone. Local activation of the cancer-killing drug theoretically maximizes the toxic metabolite concentration at the tumor site and minimizes systemic toxicity as the drug becomes diluted in the total blood stream volume of distribution. The most widely studied system for prostate cancer has been the Herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir (GCV) system [25,26]. The nucleoside analogue GCV is converted by the viral HSV-tk enzyme into a phosphorylated compound that is then incorporated into replicating DNA, causing DNA chain termination and selective killing of dividing cells. The toxic effects of GCV in both in vitro and in vivo models have resulted in suppressed prostate cancer growth, prolonged survival rates, and have decreased the rate of spontaneous metastases to the lung [25,27]. These effects appear to be mediated in part by natural killer cells. The first trial using intraprostatic injection of this type of gene therapy has recently been completed [28]. Eighteen patients with locally recurrent prostate cancer after definitive radiation therapy received a single 1.1 cc injection of replication-deficient adenovirus containing the HSV-tk gene (AdHSV-tk) under the RSV promoter, followed by 14 days of intravenous ganciclovir. The dose of AdHSV-tk was escalated from 1108 to 11011 IU. Three patients had >50% reduction in PSA and 1 had a negative biopsy after treatment. Local and systemic toxicity was mild except for the final patient, who developed severe thrombocytopenia and abnormal liver function tests. In a second trial, the toxicity of subsequent in situ gene therapy for patients with localized prostate cancer was also investigated [29]. A total of 52 patients received a total of s76 gene therapy cycles. Toxic events were observed in 16 of 29 men (55%) who were given multiple injections into the
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prostate. All toxic events were mild, however, (primarily grade 1 or 2 fever), and completely resolved once the therapy course was completed. No additive toxicity was noted in patients receiving multiple courses. A phase I/II neoadjuvant trial in patients predicted to have a greater than 50% probability of failure rate when treated by radical prostatectomy alone has been started [30]. Histopathology from the first 4 patients demonstrated varying degrees of necrosis, spanning the spectrum of Gleason grading and loss of nuclear detail, as a common finding in targeted cancer cells. Minimal necrotic change in normal prostate tissue was seen, although there was frequently a mononuclear infiltrate. Assessment of long-term patient outcomes due to beneficial local or possible systemic immunologic ‘‘bystander’’ effects will be important in determining the future utility of this type of therapy. Using the HSV-tk gene under the osteocalcin promoter, which directs gene expression to osteoblasts of numerous cancer types, including prostate cancer, Koeneman et al have initiated a phase I trial targeting androgenindependent metastatic prostate cancer [31]. In this ongoing dose escalation trial, an index metastatic lesion is injected with the adenovirus on day 1 and day 8 in combination with oral Valacyclovir, followed by a repeat biopsy on day 30. Preliminary results have shown no clinically significant toxicity. Other prodrug strategies under investigation in prostate cancer include the cytosine deaminase-flucytosine system, in which cytosine deaminase converts flucytosine to the chemotherapeutic agent 5-fluorouracil [32,33] and the E. coli DeoD gene purine nucleoside phosphorylase (PNP), which converts the prodrug 6-methyl-9 (2 deoxy-b-D erythro-pentofuranosyl) purine into a toxic nonphosphorylated purine capable of killing both quiescent and proliferating cells when incorporated in mRNA or DNA during synthesis [34]. Gene therapy strategies to activate ‘‘apoptotic’’ (programmed cell death) pathways, thereby committing the cell to death, are also under investigation. Segawa [35] has used a PSA-based promoter system to activate GAL-4 responsive elements upstream of the polyglutamine gene, which is a potent promoter of apoptosis. Similarly, Hyer has shown in vitro that adenovirus transduction of the fas ligand induced apoptosis in LNCaP, PC3, and DU145 cell lines [16]. Marcelli has reported that transduction of prostate cancer LNCaP cells with an adenoviral vector containing caspase-7 also induced programmed cell death [36]. Using another molecular approach, Dorai has demonstrated the ability of a bcl-2 hammerhead ribozyme to specifically catalyze or disrupt bcl-2 mRNA, making the cell more susceptible to apoptosis [37]. Therefore, several approaches to exploit programmed cell death gene therapy pathways appear attractive now. Because of safety concerns, in the past practically all early vectors were engineered to be replication incompetent. Recently, several types of replication-competent viral vectors have been developed. One conditionally competent adenoviral vector has been mutated so that the virus cannot express viral protein E1b [38]. The wild-type adenovirus uses its E1b protein
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to block p53 from performing its normal function, which is to prevent replication of cells that have damaged DNA. Theoretically, the mutant E1b-virus can infect, replicate, and lyse p53 deficient cells, but does not affect normal cells that have functional p53. Thus, these mutant viruses are oncolytic to cancer cells harboring p53 mutations. Although in prostate cancer there is generally a lower rate of p53 mutations compared with other types of cancers [39], this is a very promising strategy once the most appropriate gene targets become identified. Other oncolytic viruses that are replication competent have been designed based on an attenuated cytotoxic adenovirus type 5 vector incorporating a prostate-specific enhancer and promoter coupled to the E1a gene [40,41]. The E1a viral product allows the virus to reproduce and enter the lytic cycle. The level of E1a production was found to be higher in PSA-producing cells such as LNCaP than in cells that produce little or no PSA [42]. In vivo, CN706 viral vector produced tumor regression of LNCaP tumors and decreased PSA production following a single intratumoral injection [41,42]. This strategy is currently the basis of a clinical phase I trial that will provide important information, including whether sufficient E1a expression can be achieved for subsequent cell lysis, and information regarding the specificity of systemic injection of CN706, which relies on the PSA promoter to exclusively lyse prostate cells. Another oncolytic virus is the vesicular stomatitis virus (VSV), which is a nonsegmented, negative-strand (NNS) RNA virus, and is the prototype for the Rhabdoviridae family. The glycoprotein of VSV (VSV-G) has gained much attention in the gene therapy community because it is readily incorporated into retrovirus vectors and increases the tropism of these vectors. The incorporation of VSV-G also allows the concentration of retrovirus vectors by centrifugation without any loss of infectivity. The recent development of ‘‘reverse genetic’’ systems for VSV and other NNS viruses has made it possible to engineer the genome of these viruses for the construction of novel vectors that have potential uses as gene delivery vehicles. Several properties of VSV make it an ideal gene delivery vehicle. First, VSV replicates in all mammalian cells examined to date, including nondividing cells. Second, the replication of VSV occurs exclusively in the cytoplasm and does not require any nuclear functions. Third, VSV replication does not involve a DNA intermediate, eliminating the concern of chromosome insertional mutagenesis. The major limitation of VSV as a gene delivery vector, however, is that VSV is normally cytopathogenic. Cells infected with VSV begin exhibiting cytopathic effects as soon as 4 hours postinfection and are usually killed within 18 to 24 hours through the action of the matrix (M) protein and the induction of apoptosis [43,44]. Using wild type VSV, Stojdl et al [5] have shown that VSV is a replication-competent oncolytic virus in a variety of tumors including ovarian, lung, melanoma, colon, and prostate. Melanoma xenografts growing in nude mice demonstrated regression of tumor volume in 12 of 12 tumors, and 3 of these tumors were completely eradicated [45].
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These studies together suggest that targeted VSV could be a potentially useful strategy against prostate cancer. Combination of gene therapy approaches and other treatment modalities It is clear that neither surgery, radiation therapy, hormonal therapy, chemotherapy, nor gene therapy is currently adequate alone for the treatment of advanced prostate carcinoma. Although preventive strategies are being entertained, the ultimate clinical use of gene therapy to improve cancer treatment may be in combination with surgery, radiation, or chemotherapy for those patients who either have pathologically advanced disease or who are at high risk for locally advanced disease. Clinical trials using this multimodal approach are warranted until the science of gene therapy becomes better understood. Strategies using gene therapy in combination with chemotherapeutic agents and with androgen ablation have been reported [46,47]. Other combined approaches that have been employed in prostate cancer include suicide prodrug gene therapy with radiation [48–50] and with cytokine therapy [51]. Freytag et al examined the use of double suicide gene therapy using both the cytosine deaminase and HSV-tk genes encoded on a single, replication-competent adenovirus in a murine model. When used in the neoadjuvant setting, Ad5CD/Tkrep-mediated double-suicide gene therapy dramatically potentiated the effectiveness of radiation therapy and produced significant tumor regression [52]. These types of multimodal therapies to improve conventional prostate cancer treatment for patients with advanced disease at diagnosis or recurrence after definitive therapy represent some of the most promising immediate clinical applications for prostate gene therapy. Summary Numerous gene therapy trials in the United States and throughout the world using various strategies are in progress for the treatment of locally advanced and metastatic prostate cancer. Although vector technology advances at a rapid pace, progress in elucidating the molecular pathways critical for the development and progression of prostate cancer has been slower and more deliberate. Thus far, prostate gene therapy appears to be safe and well tolerated. Through these early clinical trials, the safety and efficacy of gene therapy alone or in combination with more conventional therapy as a basis for the treatment of prostate cancer will ultimately be determined. References [1] Huang L, Li S. Nonviral gene therapy: promises and challenges. Gene Ther 2000;7:31–4. [2] Lemoine NR, Russell SJ, Vile RG. Cancer gene therapy: hard lessons and new courses. Gene Ther 2000;7:2–8.
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[3] Monahan PE, Samulski RJ. AAV vectors: is clinical success on the horizon? Gene Ther 2000;7:24–30. [4] Gingrich JR, Steiner MS. Gene therapy for prostate cancer: where are we now? J Urol 2000;164:1121–36. [5] Blades RA, Keating PJ, McWilliam LJ, et al. Loss of HLA class I expression in prostate cancer: implications for immunotherapy. Urology 1995;46:681–686 [discussion: 686–687]. [6] Restifo N, Sanda M, Walsh J, et al. Molecular characterization of defective antigen processing in human prostate cancer. J Natl Cancer Inst 1995;87:280–85. [7] Chang JF, Mikhak B, Simons JW, et al. Induction of immunity to prostate cancer antigens: results of a clinical trial of vaccination with irradiated autologous prostate tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer. Cancer Res 1999;59:5160–68. [8] Simons JW, Mikhak B, Chang JF, et al. Clinical activity and broken immunologic tolerance from ex vivo GM-CSF gene transduced prostate cancer vaccines. J Urol 1999; 161(4):Suppl 51. [9] Bastian A, Chen AP, Dahut W, et al. A phase I study of recombinant vaccinia virus that expresses prostate specific antigen (PSA) in adult patients with adenocarcinoma of the prostate. J Clin Onc 1998;16:314. [10] Eder JP, Kantoff PW, Roper K, et al. A phase I trial of a recombinant vaccinia virus expressing prostate-specific antigen in advanced prostate cancer. Clin Cancer Res 2000; 6:1632–38. [11] Charles LG, Sanda MG, Smith DC, et al. Recombinant vaccinia-PSA (PROSTVAC) can induce a prostate-specific immune response in androgen-modulated human prostate cancer. Urology 1999;53:260–66. [12] Gotoh A, Ko S-C, Thalmann GN, et al. Molecular therapy with recombinant p53 adenovirus in an androgen-independent, metastatic human prostate cancer model. Human Gene Ther 1996;7:1683–91. [13] Belldegrun AS, Pantuck AJ, Zisman A. Gene therapy for prostate cancer at the University of California, Los Angeles: preliminary results and future directions. World J Urol 2000; 18:143–47. [14] Patel B, Naitoh J, Stiles A, et al. Effect of interleukin-2 (IL-2) gene therapy on prostate specific antigen in patients undergoing neoadjuvant or adjuvant gene therapy for prostate cancer. J Urol 1999;161(4):Suppl 337. [15] Sanford MA, Hassen W, Atkinson G, et al. Interleukin-12 gene therapy induced cell death through induction of FAS/FAS ligand-mediated death in metastatic mouse prostate cancer. J Urol 1999;161(4):Suppl 57. [16] Dong J-Y, Hyer M, Rubinchik S, et al. Gene therapy of prostate cancer with adenovirusmediated fas ligand (FasL) expression. In: Proceedings of the AACR 90th Annual Meeting. Philadelphia: 1999. p. 173. [17] Eastham JA, Hall SJ, Sehgal I, et al. In vivo gene therapy with p53 or p21 adenovirus for prostate cancer. Cancer Res 1995;55:5151–55. [18] Gotoh A, Kao C, Ko SC, et al. Cytotoxic effects of recombinant adenovirus p53 and cell cycle regulator genes (p21 WAF1/CIP1 and p16CDKN4) in human prostate cancers. J Urol 1997;158:636–41. [19] Steiner MS, Lerner J, Greenberger M, et al. Clinical phase I gene therapy trial using BRCA1 retrovirus is safe. J Urol 1998;159(5):Suppl 132. [20] Allay JA, Steiner MS, Zhang Y, et al. Adenovirus p16 gene therapy for prostate cancer. World J Urol 2000;18:111–20. [21] Hsieh JT, Luo W, Song W, et al. Tumor suppressive role of an androgen-regulated epithelial cell adhesion molecule (C-CAM) in prostate carcinoma cell revealed by sense and antisense approaches. Cancer Res 1995;55:190–97. [22] Anthony CT, Lu Y, Steiner MS, et al. Antisense c-myc retroviral vector suppresses established human prostate cancer. Hum Gene Ther 1998;9:747–55.
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[23] Deshane J, Kim M, Wright M, et al. A novel gene therapy strategy for elimination of prostate carcinoma cells from human bone marrow. Hum Gene Ther 1997;8:157–70. [24] Logothetis CJ, Hossan E, Pettaway CA, et al. AD-p53 intraprostatic gene therapy preceding radical prostatectomy (RP): an in vivo model for targeted therapy development. J Urol 1999;161(4):Suppl 297. [25] Chen SH, Eastham JA, Sehgal I, et al. Prostate cancer gene therapy: herpes simplex virus thymidine kinase gene transduction followed by ganciclovir in mouse and human prostate cancer models. Hum Gene Ther 1996;7:515–23. [26] Chen SH, Hall SJ, Mutchnik SE, et al. Adenovirus-mediated herpes simplex virus thymidine kinase gene and ganciclovir therapy leads to systemic activity against spontaneous and induced metastasis in an orthotopic mouse model of prostate cancer. Int J Cancer 1997; 70:183–87. [27] Atkinson G, Hall SJ, Sanford MA, et al. Induction of potent antitumor natural killer cell activity by herpes simplex virus-thymidine kinase and ganciclovir therapy in an orthotopic mouse model of prostate cancer. Cancer Res 1998;58:3221–25. [28] Adler HL, Aguilar-Cordova E, Herman JR, et al. In situ gene therapy for adenocarcinoma of the prostate: a phase I clinical trial. Hum Gene Ther 1999;10:1239–49. [29] Kadmon D, Shalev M, Teh BS, et al. Suicide gene therapy toxicity after multiple and repeat injections in patients with localized prostate cancer. J Urol 2000;163:1747–50. [30] Ayala G, Shalev M, Wheeler TM, et al. Cytopathic effect of in situ gene therapy in prostate cancer. Hum Pathol 2000;31:866–70. [31] Kao C, Ko SC, Koeneman KS, et al. Osteocalcin-directed gene therapy for prostate-cancer bone metastasis. World J Urol 2000;18:102–10. [32] Blackburn RV, Corry PM, Galoforo SS, et al. Adenoviral-mediated transfer of a heat-inducible double suicide gene into prostate carcinoma cells. Cancer Res 1998;58: 1358–62. [33] Kim SJ, Gardner TA, Ko S-C, et al. Enhancement of bystander prostate cancer cell killing by the utilization of genetically engineered bone stromal cells that confer either cytosine deaminase or thymidine kinase gene. J Urol 1999;161(4):Suppl 58. [34] Daja MM, Garcia-Aragon J, Martiniello-Wilks R, et al. In vivo gene therapy for prostate cancer: preclinical evaluation of two different enzyme-directed prodrug therapy systems delivered by identical adenovirus vectors. Hum Gene Ther 1998;9: 1617–26. [35] Kakehi Y, Segawa T, Takebayashi H, et al. Prostate-specific amplification of expanded polyglutamine expression: a novel approach for cancer gene therapy. Cancer Res 1998; 58:2282–87. [36] Cunningham GR, Marcelli M, Walkup M, et al. Signaling pathway activated during apoptosis of the prostate cancer cell line LNCaP: overexpression of caspase-7 as a new gene therapy strategy for prostate cancer. Cancer Res 1999;59:382–90. [37] Dorai T, Perlman H, Walsh K, et al. Enhanced apoptosis of prostate cancer cells by adenoviral hammerhead ribozyme mediated disruption of bcl-2. In: Proceedings of the AACR 90th Annual Meeting, Philadelphia: 1999. p. 482. [38] Bischoff JR, Kirn DH, Williams A, et al. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science 1996;274:373–76. [39] Bova GS, Brooks JD, Ewing CM, et al. An uncertain role for p53 gene alterations in human prostate cancers. Cancer Res 1996;56:3814–22. [40] Henderson DR, Sakamoto GT, Yu DC. Identification of the transcriptional regulatory sequences of human kallikrein 2 and their use in the construction of calydon virus 764, an attenuated replication competent adenovirus for prostate cancer therapy. Cancer Res 1999;59:1498–504. [41] Lim HY, Rodriguez R, Schuur ER, et al. Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells. Cancer Res 1997;57:2559–63.
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[42] Baccala AA, Lim HY, Simons JW. Ex vivo and in vivo gene therapy strategies for prostate cancer: emerging clinical pharmacology. In: Lattime E, Gerson S, editors. Gene therapy of cancer. San Diego: Academic Press; 1999. p. 333–345. [43] Whitt MA, Jayakar H, Zeng Y, et al. Development of recombinant vesicular stomatitis virus as a novel targeting gene delivery system. In: Proceedings of the International Society of Gene Therapy and Molecular Biology Conference. Corfu, Greece: 16–17, 2002. [44] Whitt MA. Genetic manipulation of rhabdoviruses: new insights to virus replication, transcription and assembly. J Microbiol 1998;36:1–8. [45] Atkins H, Bell JC, Knowles S, et al. Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. Nat Med 2000;6:821–5. [46] Dell J, Lipari P, Nielsen LL, et al. Adenovirus-mediated p53 gene therapy and paclitaxel have synergistic efficacy in models of human head and neck, ovarian, prostate, and breast cancer. Clin Cancer Res 1998;4:835–46. [47] Hall SJ, Mutchnik SE, Yang G, et al. Cooperative therapeutic effects of androgen ablation and adenovirus-mediated herpes simplex virus thymidine kinase gene and ganciclovir therapy in experimental prostate cancer. Cancer Gene Ther 1999;6:54–63. [48] Anello R, Atkinson G, Cohen S, et al. Adenovirus mediated cytosine deaminase gene transduction and 5- FLUOROCYTOSINE therapy sensitizes mouse prostate cancer cells to irradiation. J Urol 2000;164:2173–7. [49] Atkinson G, Hall SJ. Prodrug activation gene therapy and external beam irradiation in the treatment of prostate cancer. Urology 1999;54:1098–104. [50] Yin L, Fu S, Peng X, et al. Cytosine deaminase (CDA) adenoviral vector (ADV) and administration of the non toxic prodrug 5-fluorocytosine (5FC) used for therapy sensitization (TS) of prostate cancer cells. J Clin Onc 1998;16:348. [51] Hassen W, Sanford M, Atkinson G, et al. Natural killer cells induced by herpes simplex virus thymidine kinase gene transduction and ganciclovir therapy are enhanced by interleukin-12 gene therapy in an othotopic model of mouse prostate cancer. J Urol 1999; 161(4):Suppl 53. [52] Paielli DL, Rogulski KR, Wing MS, et al. Double suicide gene therapy augments the antitumor activity of a replication-competent lytic adenovirus through enhanced cytotoxicity and radiosensitization. Hum Gene Ther 2000;11:67–76.
Prostate cancer gene therapy Mitchell S. Steiner, MD*, Jeffrey R. Gingrich, MD, Ravi D. Chauhan, MD Department of Urology, University of Tennessee Memphis, 1211 Union Avenue, Suite 340, Memphis, TN 38104, USA
Gene therapy involves the transfer of DNA into cells to replace or to affect the expression of the cell’s native (endogenous) genes. This transfer may occur while the cell is outside the body (ex vivo) and then returned to the body, or the gene may be introduced into the cell while it remains in the body (in vivo) in its natural microenvironment. To transfer DNA into a cell with sufficient efficiency, a DNA transporter or ‘‘vector’’ is generally required. Methods used to transfer genes into cells are broadly categorized as either nonviral or viral based and have recently been nicely summarized [1–3]. The hope of identifying genes whose expression level can be modulated to affect the development and progression of prostate cancer has led to efforts to develop gene therapy as a viable new treatment strategy to treat or potentially even to prevent prostate cancer. In this article, current strategies for prostate cancer gene therapy and initial results of preliminary clinical trials are reviewed. Prostate cancer and gene therapy Prostate cancer is particularly suited for gene therapy for a number of reasons: (1) prostate cancer is quite common, and for the patients diagnosed with locally advanced disease there is no cure currently available; (2) the prostate gland does not serve any life-sustaining function and therefore complete ablation of benign, premalignant, and cancerous prostate tissue could be desirable; (3) the prostate is accessible by transurethral, transperineal, and transrectal approaches for administration of gene therapy; (4) the prostate may be monitored by digital rectal examination, transrectal This work was supported by the Assisi Foundation, Inc. and J. R. Hyde Family Foundation. * Corresponding author. E-mail address: [email protected] (M.S. Steiner). 1055-3207/02/$ - see front matter Ó 2002, Elsevier Science (USA). All rights reserved. PII: S 1 0 5 5 - 3 2 0 7 ( 0 2 ) 0 0 0 2 6 - 1
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ultrasound, endorectal coil magnetic resonance imaging, or by serum prostate specific antigen (PSA); (5) the prostate gland produces high levels of several characterized proteins, including prostate specific antigen, whose promoters and other enhancers may be incorporated into vectors to direct prostate-specific expression of therapeutic genes [4]. Current strategies of gene therapy for prostate cancer Numerous gene therapy strategies currently being employed for the treatment of prostate cancer have recently been reviewed [4]. Thirty-two gene therapy trials have been approved by the National Institutes of Health for the treatment of prostate cancer. The most recent trials (approved during the last three years) are listed in Table 1. To date, the approved trials have all been either phase I or phase I/II in design. They can be broadly categorized as: (1) immunotherapy—to enhance the host immune system anti-tumor response, (2) corrective gene therapy—to increase or abrogate specific aberrant gene expression, (3) ablative or ‘‘suicide’’ gene therapy—to introduce toxic or cell lytic genes, and (4) combination therapy—one or more of these strategies with conventional cytotoxic chemotherapy or radiation therapy. Immunotherapy Alterations of class I MHC expression have been shown to be a common way that prostate cancer cells may evade the host’s immune system [5,6]. The approaches that have been used to stimulate the host antitumor immune response are gene vaccine therapy using ex vivo gene transfer techniques, and in vivo intratumoral injection of gene therapy vectors containing cytokine genes. Tumor vaccines have been generated using autologous tumor or nonautologous cells, such as tumor cell lines. Before subcutaneous inoculation, the cells are modified by ex vivo gene therapy to express cytokine genes, and radiated to eliminate their replication capacity. Cytotoxic T lymphocytes (CTL) not only recognize tumor-specific antigens present on the surface of these inoculated cells, but are also induced by local secretion of the transferred stimulatory cytokines. The activated CTL expand in number and then destroy tumor cells that share these same antigens on their cell surfaces throughout the body. Preclinical studies have clearly shown that gene vaccines are not efficacious in the presence of a large tumor burden, but theoretically they may be more useful against micrometastasis following removal or debulking of the primary tumor for locally advanced disease, or in the setting of minimally recurrent disease. The first approved gene therapy trial for prostate cancer was by Simons [7]. It was designed for patients found to have metastatic prostate cancer in the lymph nodes at the time of radical prostatectomy. Similar to their previous renal cell carcinoma studies, ex vivo transduction of autologous, irradiated prostate tumor cells with a retroviral granulocyte-macrophage
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colony-stimulating factor (GM-CSF) construct was performed to generate vaccines. The vaccines were administered subcutaneously every two weeks until available cells were exhausted. Vaccination site biopsies revealed infiltrating macrophages, dendritic cells, eosinophils, and T-cells. No dose limiting toxicities were observed and all patients ultimately demonstrated progressive disease. Interestingly, sera from three of eight patients were shown to contain new antibodies recognizing similarly sized polypeptides present in the prostate cancer cell lines LNCaP and PC-3. This study demonstrated the feasibility of autologous GM-CSF transduced prostate cancer vaccines, and showed that both T-cell and B-cell immune responses to human prostate cancer can be generated and that treatment was restricted only by the limitations of in vitro cell expansion. To circumvent this limitation, a follow-up trial powered to estimate efficacy using ex vivo GM-CSF transduced allogeneic prostate cancer cell lines (PC-3 and LNCaP) was initiated. Patients were vaccinated weekly for eight weeks with irradiated, GMCSF secreting PC-3 and LNCaP prostate cancer cells. One of 21 patients had a partial PSA response of >7 months duration, 14 had stable disease, and 6 progressed [8]. At three months after treatment, PSA velocity or PSA slope decreased in 71% of patients. No dose-limiting toxicities were identified. Although the dosing and schedule to conclusively demonstrate therapeutic efficacy are still undergoing optimization, numerous post-vaccination IgG1 antibodies were identified, demonstrating that immune tolerance to prostate cancer-associated antigens may be broken, and that this therefore appears to be a clinically feasible strategy for prostate cancer treatment. Three trials using a different vaccine strategy have been approved using recombinant vaccinia virus expressing PSA (rV-PSA, PROSTVAC) as a tumor-associated antigen. Chen treated 30 patients with hormone refractory prostate cancer who had undergone withdrawal of anti-androgen therapy with a vaccinia inoculation followed by PROSTAVAC immunization [9]. Patients received 2.65105 or 2.65106 plaque forming units (PFU) once a month for 3 months. All patients developed local erythema at the vaccination site. Four of 14 patients had stable disease (2 progressed at 5 and 6 months) and 10 had continued disease progression. In a patient population with less advanced disease, Eder et al treated 33 men with rising PSA after radical prostatectomy, radiation therapy, both, or metastatic disease, with 2.6510 [6–8] PFU of rV-PSA as three consecutive monthly doses without significant toxicity [10]. Ten patients who received the highest dose also received 250 lg/m2 as an immunostimulatory adjunct without significant additional toxicity. Fourteen out of 33 men maintained a stable disease status for at least 6 months, and 6 remain progression free as of 21 months. This trial demonstrated that rV-PSA is safe and can elicit immune responses, arresting disease progression in some patients for up to 21 months or longer. In the third study, Sanda administered PROSTVAC once to 6 patients with androgen-modulated recurrence of prostate cancer after radical prostatectomy [11]. In addition to evaluation for toxicity, time until
9801-229
9802-236
9805-251 9812-276
9901-282
9901-283
9902-293
9904-306
9905-312
1
2
3 4
5
6
7
8
9
NIH#
II
I
II
I/II
II
I/II I
I
I/II
Phase
Belldegrun/ Corman/Klein
Vieweg
Kaufman
Small
Eder
Figlin Gardner/Chung
Simons
Kadmon
PI
Table 1 Prostate cancer gene therapy trials
UCLA/Puget Sound Health Care System/ Cleveland Clinic
Duke University
UCSF/Mt. Zion Cancer Center Albert Einstein
Dana-Farber
UCLA U of Virginia
Johns Hopkins University
Baylor College of Medicine
Institution
Vical, Inc.
Cell Genesys, Inc. Eastern Cooperative Oncology Group
NCI-CTEP
Transgene, SA
Calydon, Inc.
Sponsor
Hormone Refractory Prostate Cancer
Advanced
Prostate Cancer
Prostate Cancer
MUC-1 Positive Metastatic
High risk for advanced disease RadioRecurrence
Patient population
Liposome
RNA
Vaccinia/ Fowlpox Virus
Vaccinia/ Fowlpox Virus Retrovirus
Vaccinia virus Adenovirus
Adenovirus
Adenovirus
Vector
IL-2 cDNA
PSA
PSA
GM-CSF cDNA
PSA-Ela (replication competent) MUC-1/IL2 Osteocalcin HSV-tk/ valacyclovir PSA
RSV-HSV-tk/ ganciclovir
Gene
610 M.S. Steiner et al / Surg Oncol Clin N Am 11 (2002) 607–620
9906-321
9906-324
9909-338
9910-344
9910-345
9910-352
9911-356
9912-368
0001-373
0008-410
11
12
13
14
15
16
17
18
19
20
I
II
II
I
I/II
I/II
I/II
I/II
I/II
I
II
Vieweg
Arlen
Belldegrun/ Figlin Dahut/Gulley
Belldegrun/ Klein
Wilding
Terris
AguilarCordova/ Butler Gingrich/Steiner
Freytag/Kim
Small/Smith
Duke University
NIH/NCI
NIH/NCI
UCLA/ Cleveland Clinic UCLA
University of Wisconsin
University of Tennessee Stanford University
Texas Children’s Hospital
UCSF-Mt. Zion/VA San Diego Henry Ford Health Sys.
From http://www4.od.nih.gov/oba/rac/clinicaltrial.htm
9905-315
10
Transgene, SA
Vical, Inc.
Calydon, Inc.
Calydon, Inc.
GTx, Inc.
Cell Genesys, Inc.
Hormone Refractory Metastatic
Combined with Radiotherapy
Advanced
Hormone Refractory Metastatic RadioRecurrent
Locally Advanced RadioRecurrent
Combined with Radiotherapy
RadioRecurrent
Hormone Refractory
Vaccinia/ Fowlpox Virus Vaccinia/ FowlpoxVirus RNA
Vaccinia Virus
Liposome
Adenovirus
Adenovirus
Adenovirus
Adenovirus
Adenovirus
Retrovirus
Tumor RNA
PSA/B7.1
PSA
MUC-1/IL2
PSA Directed Cytolytic Adenovirus IL-2 cDNA
PSA
p16
Cytosine deaminase/ HSV - tk cDNA HSV - tk cDNA
GM-CSF cDNA
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rise in serum PSA after interruption of androgen deprivation therapy and Western blot analysis for anti-PSA antibody production were determined. One patient maintained an undetectable serum PSA for over 8 months after withdrawal of anti-androgen therapy and immunization. One patient developed an IgG antibody against PSA after immunization. It is interesting to note that one third of patients had anti-PSA antibodies before immunization. Sanda concluded that although immune responses against PSA may be present among some patients with prostate cancer at baseline, the immune response may be induced in others through vaccinia-PSA immunization. The significance of this finding and its implications regarding the therapeutic efficacy of vaccination against tumor-associated antigens is unclear. Direct in vivo intratumoral injection of gene therapy vectors containing cytokine genes attempts to treat tumor cells in situ in the hope of generating a beneficial systemic effect for metastatic disease. Naitoh et al, using animal models of prostate cancer, have shown that intratumoral injection of liposome and adenoviral vectors containing IL-2 gene expression systems resulted in the activation of specific T-cell antitumor responses [12]. Using a liposomal vector, clinical trials are under way as a neoadjuvant before radical prostatectomy and for local recurrence after radiation therapy (see Table 1, Belldegrun) [13]. Belldegrun et al treated 12 patients prior to radical prostatectomy and 9 patients with recurrent prostate cancer after radiation or cryotherapy with two injections of intraprostatic IL-2 [14]. A total of 40 injections of 300 to 1500 lg of IL-2 were administered. For patients treated before radical prostatectomy, the average PSA declined by 5.3 ng/ml before surgery and 75% maintained undetectable PSA levels at 24 to 56 weeks after surgery. Average PSA declines in patients with recurrent disease were 3.6 ng/ ml and 1.3 ng/ml after one or two injections respectively. Although the significance of these PSA responses and differentiation of the effects of the gene therapy from surgery alone are not yet clear, this strategy using liposomal delivery appears to be safe; it may also be effective locally and may promote a systemic immune response. Similarly, Sanford employed intratumorally injected adenoviral vectors containing IL-12 to treat primary prostate cancer tumors in mice, significantly reducing the number of lung metastases [15]. The molecular mechanism may include stimulation of T-cell and NK cells, induction of IFN-c, and upregulation of fas expression [15,16]. In summary, preclinical experience now suggests that immunotherapy may ultimately be effective against minimal residual, recurrent, or micrometastatic disease. Patients with greater metastatic or primary tumor volumes will most likely require some other nonimmunological therapeutic intervention. Corrective gene therapy Corrective gene therapy seeks to replace inherited or acquired defective genes that are important for normal growth regulation. Most corrective gene therapy strategies have used either retroviral or adenoviral vectors
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administered by intratumoral injection. Prostate cancer preclinical studies have been reported for the replacement of an assortment of tumor suppressor genes, including AdCMVp53 [17–19], retroviral LXSN BRCA-1 [17], AdCMVp21 [17,19], AdCMVp16 [18,20], and AdCMV CAM1 [21]. Antisense strategies (directing the expression of a reverse strand of mRNA complimentary to the normal or ‘‘sense’’ strand of oncogenes) effectively preventing the translation of the mRNA into protein have also been evaluated. A single intratumoral injection of retroviral MMTV antisense c-myc markedly suppressed and even eradicated DU145 prostate cancer xenografts growing in nude mice, through downregulation of c-myc mRNA and protein expression and the induction of apoptosis through downregulation of bcl-2 protein [22]. Using a similar approach, Kim et al have shown that an adenoviral vector containing an antisense erb-B-2 gene inhibited the overexpression of growth factor erb-B-2 in prostate cancer cells in vitro, thereby selectively purging metastatic prostate cancer cells from the bone marrow [23]. Preliminary results of the first approved trial using direct transrectal prostatic gene therapy injection have been reported by Steiner [19]. This was also the first study designed using a gene replacement strategy. Twenty-one prostate cancer patients who had failed standard therapy underwent ultrasoundguided injection of retrovirus LXSN containing BRCA1 under the control of the LTR viral promoter. No patients developed viral symptoms or evidence of viremia. Viral DNA was detected by PCR at least two years after injection (unpublished results). Average prostate volume was not decreased one month after injection and serum PSA in these patients with metastatic disease remained unchanged. This study demonstrated the feasibility and safety of direct, transrectal injection of prostate cancer gene replacement therapy. Initial clinical gene therapy results using a similar gene replacement strategy of replication-defective adenovirus containing wild-type p53 (AdCMVp53) driven by the CMV promoter before radical prostatectomy have recently been reported [24]. Seventeen patients with locally advanced prostate cancer received at least one course of ultrasound guided AdCMVp53 treatment consisting of three administrations, 14 days apart, of 3 ml in four to six divided injections. In this phase I/II study, the number of viral particles (vp) delivered was escalated from 31010 vp per treatment per patient to 31012 vp. Three patients completed a second course of therapy for >25% reduction in tumor size, as measured by ultrasound or endorectal coil MRI after the first course of treatment. This study confirmed the relative feasibility and safety of repeated adenoviral intraprostatic gene therapy injection. Further results regarding efficacy after the apparent radiologic responses, correlations with gene expression, pathologic findings at prostatectomy, and surgical outcomes are pending. In general, corrective gene therapy holds the promise of reversing the malignant phenotype. These approaches have shown that gene replacement may also indirectly lead to activation of cell death pathways. The ‘‘bystander effect’’ may then also result in additional tumor cell death through
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complementary or synergistic mechanisms. Contrary to historical surgical, chemotherapeutic, and radiation therapy goals to eradicate every cancer cell to effect a long-term cure, corrective gene therapy that restores a normal phenotype through a durable cytostatic effect or cytotoxic effect with induction of an adequate immune response may be an acceptable clinical endpoint. Ablative, or suicide gene, therapy ‘‘Suicide’’ gene therapy strategies may offer the most promising clinical potential. In this strategy, therapeutic gene expression results in cell destruction without regard to the underlying genetic mutations responsible for the malignant phenotype. Two types of suicide gene therapy strategies have emerged: gene-directed enzyme prodrug treatment, and gene-directed production of a cellular toxin or induction of apoptosis. Enzyme prodrug strategies use transfer of an enzyme expression vector. Cells producing the enzyme then convert a nontoxic, systemically administered prodrug into an activated, lethal product that is cytotoxic to the cell and thus may subsequently directly or indirectly kill neighboring cancer cells. This bystander effect can be quite dramatic, resulting in the death of 100 to 1000 fold more cells than would be predicted by gene transfer rates alone. Local activation of the cancer-killing drug theoretically maximizes the toxic metabolite concentration at the tumor site and minimizes systemic toxicity as the drug becomes diluted in the total blood stream volume of distribution. The most widely studied system for prostate cancer has been the Herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir (GCV) system [25,26]. The nucleoside analogue GCV is converted by the viral HSV-tk enzyme into a phosphorylated compound that is then incorporated into replicating DNA, causing DNA chain termination and selective killing of dividing cells. The toxic effects of GCV in both in vitro and in vivo models have resulted in suppressed prostate cancer growth, prolonged survival rates, and have decreased the rate of spontaneous metastases to the lung [25,27]. These effects appear to be mediated in part by natural killer cells. The first trial using intraprostatic injection of this type of gene therapy has recently been completed [28]. Eighteen patients with locally recurrent prostate cancer after definitive radiation therapy received a single 1.1 cc injection of replication-deficient adenovirus containing the HSV-tk gene (AdHSV-tk) under the RSV promoter, followed by 14 days of intravenous ganciclovir. The dose of AdHSV-tk was escalated from 1108 to 11011 IU. Three patients had >50% reduction in PSA and 1 had a negative biopsy after treatment. Local and systemic toxicity was mild except for the final patient, who developed severe thrombocytopenia and abnormal liver function tests. In a second trial, the toxicity of subsequent in situ gene therapy for patients with localized prostate cancer was also investigated [29]. A total of 52 patients received a total of s76 gene therapy cycles. Toxic events were observed in 16 of 29 men (55%) who were given multiple injections into the
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prostate. All toxic events were mild, however, (primarily grade 1 or 2 fever), and completely resolved once the therapy course was completed. No additive toxicity was noted in patients receiving multiple courses. A phase I/II neoadjuvant trial in patients predicted to have a greater than 50% probability of failure rate when treated by radical prostatectomy alone has been started [30]. Histopathology from the first 4 patients demonstrated varying degrees of necrosis, spanning the spectrum of Gleason grading and loss of nuclear detail, as a common finding in targeted cancer cells. Minimal necrotic change in normal prostate tissue was seen, although there was frequently a mononuclear infiltrate. Assessment of long-term patient outcomes due to beneficial local or possible systemic immunologic ‘‘bystander’’ effects will be important in determining the future utility of this type of therapy. Using the HSV-tk gene under the osteocalcin promoter, which directs gene expression to osteoblasts of numerous cancer types, including prostate cancer, Koeneman et al have initiated a phase I trial targeting androgenindependent metastatic prostate cancer [31]. In this ongoing dose escalation trial, an index metastatic lesion is injected with the adenovirus on day 1 and day 8 in combination with oral Valacyclovir, followed by a repeat biopsy on day 30. Preliminary results have shown no clinically significant toxicity. Other prodrug strategies under investigation in prostate cancer include the cytosine deaminase-flucytosine system, in which cytosine deaminase converts flucytosine to the chemotherapeutic agent 5-fluorouracil [32,33] and the E. coli DeoD gene purine nucleoside phosphorylase (PNP), which converts the prodrug 6-methyl-9 (2 deoxy-b-D erythro-pentofuranosyl) purine into a toxic nonphosphorylated purine capable of killing both quiescent and proliferating cells when incorporated in mRNA or DNA during synthesis [34]. Gene therapy strategies to activate ‘‘apoptotic’’ (programmed cell death) pathways, thereby committing the cell to death, are also under investigation. Segawa [35] has used a PSA-based promoter system to activate GAL-4 responsive elements upstream of the polyglutamine gene, which is a potent promoter of apoptosis. Similarly, Hyer has shown in vitro that adenovirus transduction of the fas ligand induced apoptosis in LNCaP, PC3, and DU145 cell lines [16]. Marcelli has reported that transduction of prostate cancer LNCaP cells with an adenoviral vector containing caspase-7 also induced programmed cell death [36]. Using another molecular approach, Dorai has demonstrated the ability of a bcl-2 hammerhead ribozyme to specifically catalyze or disrupt bcl-2 mRNA, making the cell more susceptible to apoptosis [37]. Therefore, several approaches to exploit programmed cell death gene therapy pathways appear attractive now. Because of safety concerns, in the past practically all early vectors were engineered to be replication incompetent. Recently, several types of replication-competent viral vectors have been developed. One conditionally competent adenoviral vector has been mutated so that the virus cannot express viral protein E1b [38]. The wild-type adenovirus uses its E1b protein
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to block p53 from performing its normal function, which is to prevent replication of cells that have damaged DNA. Theoretically, the mutant E1b-virus can infect, replicate, and lyse p53 deficient cells, but does not affect normal cells that have functional p53. Thus, these mutant viruses are oncolytic to cancer cells harboring p53 mutations. Although in prostate cancer there is generally a lower rate of p53 mutations compared with other types of cancers [39], this is a very promising strategy once the most appropriate gene targets become identified. Other oncolytic viruses that are replication competent have been designed based on an attenuated cytotoxic adenovirus type 5 vector incorporating a prostate-specific enhancer and promoter coupled to the E1a gene [40,41]. The E1a viral product allows the virus to reproduce and enter the lytic cycle. The level of E1a production was found to be higher in PSA-producing cells such as LNCaP than in cells that produce little or no PSA [42]. In vivo, CN706 viral vector produced tumor regression of LNCaP tumors and decreased PSA production following a single intratumoral injection [41,42]. This strategy is currently the basis of a clinical phase I trial that will provide important information, including whether sufficient E1a expression can be achieved for subsequent cell lysis, and information regarding the specificity of systemic injection of CN706, which relies on the PSA promoter to exclusively lyse prostate cells. Another oncolytic virus is the vesicular stomatitis virus (VSV), which is a nonsegmented, negative-strand (NNS) RNA virus, and is the prototype for the Rhabdoviridae family. The glycoprotein of VSV (VSV-G) has gained much attention in the gene therapy community because it is readily incorporated into retrovirus vectors and increases the tropism of these vectors. The incorporation of VSV-G also allows the concentration of retrovirus vectors by centrifugation without any loss of infectivity. The recent development of ‘‘reverse genetic’’ systems for VSV and other NNS viruses has made it possible to engineer the genome of these viruses for the construction of novel vectors that have potential uses as gene delivery vehicles. Several properties of VSV make it an ideal gene delivery vehicle. First, VSV replicates in all mammalian cells examined to date, including nondividing cells. Second, the replication of VSV occurs exclusively in the cytoplasm and does not require any nuclear functions. Third, VSV replication does not involve a DNA intermediate, eliminating the concern of chromosome insertional mutagenesis. The major limitation of VSV as a gene delivery vector, however, is that VSV is normally cytopathogenic. Cells infected with VSV begin exhibiting cytopathic effects as soon as 4 hours postinfection and are usually killed within 18 to 24 hours through the action of the matrix (M) protein and the induction of apoptosis [43,44]. Using wild type VSV, Stojdl et al [5] have shown that VSV is a replication-competent oncolytic virus in a variety of tumors including ovarian, lung, melanoma, colon, and prostate. Melanoma xenografts growing in nude mice demonstrated regression of tumor volume in 12 of 12 tumors, and 3 of these tumors were completely eradicated [45].
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These studies together suggest that targeted VSV could be a potentially useful strategy against prostate cancer. Combination of gene therapy approaches and other treatment modalities It is clear that neither surgery, radiation therapy, hormonal therapy, chemotherapy, nor gene therapy is currently adequate alone for the treatment of advanced prostate carcinoma. Although preventive strategies are being entertained, the ultimate clinical use of gene therapy to improve cancer treatment may be in combination with surgery, radiation, or chemotherapy for those patients who either have pathologically advanced disease or who are at high risk for locally advanced disease. Clinical trials using this multimodal approach are warranted until the science of gene therapy becomes better understood. Strategies using gene therapy in combination with chemotherapeutic agents and with androgen ablation have been reported [46,47]. Other combined approaches that have been employed in prostate cancer include suicide prodrug gene therapy with radiation [48–50] and with cytokine therapy [51]. Freytag et al examined the use of double suicide gene therapy using both the cytosine deaminase and HSV-tk genes encoded on a single, replication-competent adenovirus in a murine model. When used in the neoadjuvant setting, Ad5CD/Tkrep-mediated double-suicide gene therapy dramatically potentiated the effectiveness of radiation therapy and produced significant tumor regression [52]. These types of multimodal therapies to improve conventional prostate cancer treatment for patients with advanced disease at diagnosis or recurrence after definitive therapy represent some of the most promising immediate clinical applications for prostate gene therapy. Summary Numerous gene therapy trials in the United States and throughout the world using various strategies are in progress for the treatment of locally advanced and metastatic prostate cancer. Although vector technology advances at a rapid pace, progress in elucidating the molecular pathways critical for the development and progression of prostate cancer has been slower and more deliberate. Thus far, prostate gene therapy appears to be safe and well tolerated. Through these early clinical trials, the safety and efficacy of gene therapy alone or in combination with more conventional therapy as a basis for the treatment of prostate cancer will ultimately be determined. References [1] Huang L, Li S. Nonviral gene therapy: promises and challenges. Gene Ther 2000;7:31–4. [2] Lemoine NR, Russell SJ, Vile RG. Cancer gene therapy: hard lessons and new courses. Gene Ther 2000;7:2–8.
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