Endocrine-Related (2002) 9 115–139

Gene therapy of prostate cancer: current and future directions

N J Mabjeesh, H Zhong and J W Simons

Winship Cancer Institute, Department of Hematologyand Oncology,EmoryUniversitySchool of Medicine, 1365 Clifton Road, Suite B4100, Atlanta, Georgia 30322, USA

(Requests for offprints should be addressed to J W Simons; Email: jonathan—[email protected]) Abstract Prostate cancer (PCA) is the second most common cause of death from malignancyin American men. Developing new approaches for therapyfor PCA is critical as there is no effective treatment for patients in the advanced stages of this . Current PCA gene therapyresearch strategies include cytoreductive approaches (immunotherapy and cytolytic/pro-apoptotic) and corrective approaches (replacing deleted or mutated ). The prostate is ideal for gene therapy. It is an accessoryorgan, offers unique (prostate-specific , prostate-specific membrane antigen, human glandular kallikrein 2 etc.) and is stereotacticallyaccessible for in situ treatments. Viral and non-viral means are being used to transfer the genetic material into tumor cells. The number of clinical trials utilizing gene therapymethods for PCA is increasing. We review the multiple issues involved in developing effective gene therapystrategies for human PCA and earlyclinical results. Endocrine-Related Cancer (2002) 9 115–139

Introduction stration of the impact of chemotherapy (mitoxantrone+ prednisone) on quality of life as compared with prednisone New therapeutics like gene therapy are needed urgently for alone in advanced hormone-refractory PCA. At the present advanced prostate cancer (PCA). PCA remains the most time, chemotherapy should be considered as a palliative or common solid tumor and the second leading cause of cancer- investigational treatment in patients with symptomatic andro- related deaths among men in the USA. An estimated 180 400 gen-independent disease (Morris & Scher 2000). new cases and 31900 PCA-related deaths are expected for An improving understanding of the biology of PCA has 2000 (Cookson 2001). The current standard therapies changed the outlook for patients with this disease. We now employed for organ-confined PCA include radiation or sur- have available approaches designed to modulate the immune gery, in some circumstances incorporating neoadjuvant or system, inhibit the metastatic cascade, block , adjuvant hormonal therapy. While these therapies are rela- promote and alter . tively effective in the short-term, a significant proportion of Gene therapy has been much heralded since its inception patients initially presenting with localized disease ultimately with the first patient treated in 1990, but in oncology has yet relapse. Moreover, each of these therapies may incur to reach phase III trials or show clear cancer patient benefits unwanted side-effects. Ultimately, the major risk faced by in large statistically powered phase II trials (Anderson 1998). patients with PCA is the development of metastatic disease. Nonetheless, in recent years there has been progress in the For 60 years the mainstay of therapy for metastatic disease design and execution of gene transfer clinical studies. Pro- has been androgen ablation. While this provides cytore- gress in this field provides a rational basis for optimism that duction and palliation, progression to hormone-refractory gene therapeutic strategies may significantly improve PCA disease typically occurs within the order of 14–20 months treatment outcome administered either alone or in combina- (Crawford et al. 1989). A great number of clinical research tion with current therapies. In this report we review the mul- studies have been reported in the field of chemotherapy for tiple issues involved in developing effective gene therapy advanced androgen-independent PCA during the last ten strategies for human PCA. years (Culine & Droz 2000). So far, no combination of chemotherapy reported has improved overall survival of patients. Docetaxel combined with estramustine, however, is Rationale for PCA gene therapy in randomized trials to assess its impact on survival. An The promise of gene therapy lies in its potential for selective advance in medical oncology of the 1990s is the demon- potency. To achieve this aim, cancer gene therapy strategies

Endocrine-Related Cancer (2002) 9 000–000 Online version via http://www.endocrinology.org 1351-0088/02/009–000  2002 Societyfor Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/28/2021 08:33:34AM via free access N J Mabjeesh et al.: Prostate cancer gene therapy attempt to exploit the biological uniqueness of each particular approach – or introduced directly into cells in their normal tumor. The human prostate is an accessory organ and is not location – the in vivo approach (Anderson 1998). required for potency or urinary continence. Thus unique tar- gets showed by PCA and the normal prostate are candidates Gene transfer systems for new treatment. The Prostate Expression Database is an online resource designed to access and analyze gene expres- Efficient gene transfer requires the use of a vector. All vec- sion information derived from the human prostate (Nelson et tors contain at a minimum the transgene of interest linked al. 2000). It has revealed more than 55 000 expressed to a promoter to drive its expression (Galanis et al. 2001). sequence tags (ESTs) from 43 cDNA libraries; of these Increasingly wider ranges of viral and synthetic vectors are approximately 500 are prostate-unique (Nelson et al. 2000). available, each with characteristic advantages and limitations The presence of this large number of prostate-unique promo- (Table 2). However, most vectors used for gene therapy con- ters and candidate antigens promises to facilitate the design tain promoters that are tissue-non-specific, allowing gene of prostate-specific gene therapy. Ideally, both the clinical expression to occur in unintended tissues and resulting in the and genetic features of PCA should be taken into account in potential for systemic toxicity. the design of effective prostate gene therapeutic strategies In choosing between vectors, attributes to consider (Table 1). include maximal transgene size permissible, maximal titer of Cancer gene therapy may be defined as the transfer of viruses attainable, tendency to provoke inflammatory/ recombinant DNA into human cells to achieve an antitumor immune response, persistence of , ability to effect. Depending upon the strategy, DNA may be introduced transduce non-dividing cells, effect of vector on either into cells removed from the body – the desired therapy, target -specificity, and transduction effi-

Table 1 PCA-specific gene therapystrategies PCA-specific features Relevance to gene therapy Anatomy Localized tumor accessible for procedures Accessibilityfor localized gene therapyand such as brachytherapy combined modality therapy in vivo by transurethral/transrectal routes Accessoryorgan Complete ablation of the normal organ in addition to the tumor is clinicallyundeleterious Cytoreductive transgene expression required only for duration necessaryfor prostate tissue ablation Natural history Long pre-clinical latency Preventive gene therapystrategies maybe feasible Definition of genetic/epigenetic events underlying molecular progression provides rational basis for preventive/corrective strategies Propensity for metastatic spread to lymphatics/ Targeting of systemic gene therapy for advanced bone disease must effectivelytarget bone and lymphatics Current treatment efficacyFailures even for localized disease Supports use of novel combined modality treatments to improve local control Ineffective for advanced disease Pressing need for alternative systemic therapies BiologyLow mitotic rate Effective vectors for in vivo gene deliverymust transduce both non-dividing as well as dividing cells Tumors are resistant to S-phase-dependent cytotoxic strategies Clinical/biological transitions: Define gene therapytargets including tumor ¼ Prostatic intraepithelial neoplasial suppressors, etc., for prevention and (PIN)Ǟcarcinoma for treatment of disease at various stages ¼ Androgen-dependentǞandrogen- independent tumor growth Prostate-specific genes ȁ500 or more prostate-specific ESTs in Facilitate prostate specific promoter/antigen and antigens addition to current known markers such as targeting and therapeutic monitoring PSA/PSMA

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Table 2 Vectors for gene therapy Vector Gene therapy Advantages Disadvantages Insert Immune applications size response RNA virus RV vectors RVs MMLV-based Transmitted to progenyof Infect activelydividing 6–8 kb No (oncoviruses) used in majoritytransduced cell cells only of current Potentiallylong term Risk of insertional clinical trials transgene expression mutagenesis Ex vivo Relativelylow titers transduction for Low transduction stable gene efficiency expression Inactivated by complement in vivo Difficult to target attachment Lentiviruses Preclinical Can infect non-dividing cells Safety: possible 6–8 kb No testing, stable Long-term transgene recombination with infection of expression endogenous HIV/other non-dividing Not inactivated byhuman viruses cells serum Risk of insertional mutagenesis HFVs Preclinical Non-pathogenic Low titers 5–7 kb No testing, stable Infect non-dividing cells infection of Polycations not required for non-dividing efficient transduction cells Not inactivated byhuman serum DNA virus vectors Vaccinia virus Vaccine-based Large insert size Low efficiency25 kb Yes gene therapyReplicates in vivo Pre-existing immunity from prior vaccinations limits expression duration to ȁ1 month HSV Neural tropism Potentiallylarge insert size Expression transient 10–100 kb Yes (amplicons) Cytopathic Episomal no risk insertional Difficult to produce mutagenesis high titers Infect non-dividing cells Moderate efficiency Ad In vivo use Infect non-dividing cells Transient expression 7.5 kb Yes Wide target cell range without passage to Concentrate to high titers progeny High transduction efficiencyAd infection provokes Episomal, no risk of cell-mediated immune insertional mutagenesis response limiting High transgene expression duration of expression Humoral response makes reinfection less feasible with rapid clearance of vector Local tissue inflammation AAV In vivo use Non-pathogenic Limited insert size 2–4.5 kb No Infect non-dividing cells Propensityfor Wide target cell range rearrangement during High transduction efficiencyintegration Site-specific integration Risk of helper Ad and chr 19 wild-type AAV contamination Insertional mutagenesis possible if rep gene deleted

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Table 2 cont. Vector Gene therapy Advantages Disadvantages Insert Immune applications size response Synthetic vectors Naked DNA DNA vaccines Low cost Poor efficiencyUnlimited No Simple preparation Non-toxic Liposomes In vivo/ex vivo Large insert size Poor efficiency50 kb No use Safe for repeated Prone to degradation/ administration short expression Non-toxic Low cost Suited for targeting DNA– In vivo Large insert size Poor efficiency50 kb No complex Safe for repeated Prone to degradation/ administration short expression Non-toxic Low cost Suited for targeting ciency. Furthermore, it is important to consider the goals of (Roelvink et al. 1998). The fiber protein may be reengineered the overall strategy. Broad categories encompassing the cur- to bind to a variety of cell-surface molecules (Miller et al. rent gene therapy strategies include cytoreductive (includes 1998). Fiber protein reengineering together with prostate- immunotherapy and cytolytic/pro-apoptotic approaches) and specific promoter/enhancers may allow construction of Ad corrective approaches. vectors targeted for binding to prostate-specific cell-surface markers such as prostate-specific membrane antigen (PSMA) Viral vectors and at the same time restricted for replication or transgene expression to prostate cells (Lee et al. 1996, Rodriguez et Viral vectors are used in the vast majority of gene therapy al. 1997, Gotoh et al. 1998). Following attachment, Ad is trials owing to their relatively higher efficiency at gene trans- internalized through coated pits into endosomes, then is dis- fer compared with the synthetic vectors. Viral vectors may assembled and extruded into the cytosol prior to lysosomal be RNA virus-based, or DNA virus-based. RNA viruses fusion, thereby avoiding lysosomal degradation (Silman & include the retroviruses (RVs) among which are the onco- Fooks 2000). The DNA-protein core then penetrates the viruses such as murine virus (MuLV) and mouse nuclear pores into the nucleus and gene expression com- mammary tumor virus (MMTV), the lentiviruses derived mences. The first of two phases of Ad gene expression results from human immunodeficiency virus (HIV), and the spuma- in expression of the early genes (E1–E4), which are essential viruses such as human foamy virus (HFV). The DNA viruses to DNA replication, and subversion of host antiviral defen- include adenovirus (Ad), adeno-associated viruses (AAVs), ses. If replication-competent, the Ad proceeds to replicate vaccinia virus, and herpes simplex virus (HSV). To increase episomally, culminating in host cell lysis and the release of their safety, viral vectors may be designed to be replication- newly produced viral particles. The lack of proviral inte- deficient, with no further virus particles generated following gration results in gene expression that is less sustained than infection of the target cells. Alternatively the viral vectors with the RVs and gradually lost with subsequent cell div- may be replication-competent or replication-attenuated, in isions. Ad vectors have high safety owing to their mild asso- which case viral replication can occur in permissive cells. ciated clinical pathogenicity and extrachromosomal life cycle, which eliminates the risk of insertional mutagenesis. Ad vectors The administration of an Ad vector triggers both cellular Ad vectors are well suited to a variety of gene therapy stra- and humoral immune responses, which clear remaining virus tegies owing to their broad host range, low potential for and also function to eliminate transduced cells, thereby limit- pathogenicity, and relatively high gene transfer efficiency. ing the duration of transgene expression. The anti-Ad Ad consists of a linear double-stranded DNA genome of 30– immune response rapidly clears subsequent injections of Ad 40 kb. Binding and internalization of Ad to target cells are vector, severely limiting the efficacy of readministered vector dependent on the presence of the coxsackie Ad receptor (Yang et al. 1996). Intratumoral readministration of Ad elic-

(CAR) and of the αvβ3 and αvβ5 integrins (Wickham et al. its profound humoral and cellular immune responses to Ad 1993, Roelvink et al. 1998). The interaction of Ad particles and their transgenes (Nagao et al. 2001). Following endo- with CAR is mediated by the Ad fiber protein knob domain cytosis, virions are released from endosomes and undergo

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MHC-1processing and presentation prior to any de novo Ad transfer is the ‘Ad dodecahedron’ which is composed of only protein synthesis. two of the 11 Ad structural and contains no Ad It has been possible to increase the duration of Ad DNA. The vector consists of the penton base and fiber pro- transgene expression through the use of cytotoxic T-lympho- tein, which are sufficient to mediate efficient attachment, cell cyte (CTL) blocking agents such as cytotoxic T-lymphocyte entry, and escape from endosomal degradation (Fender et al. antigen-4Ig (Kay et al. 1995) or immunosuppressive drugs 1997). The reduction in virion proteins presented to the (Dai et al. 1995). In addition to specific anti-Ad/transgene- immune system is intended to decrease the immunogenicity directed immune responses, non-specific inflammation may of this vector, making it even more suitable than EAMs for also significantly reduce transgene expression (Otake et al. repeat application. 1998). The ability to suppress immunogenicity towards Ad vectors would be of enormous benefit to in vivo gene therapy AAVs and would allow multiple reinfections with the same or dif- The potential applications of the AAV as a gene transfer ferent transgenes delivered. system vector have expanded dramatically in the last decade. The resulting improvements in gene localization from AAVs are a non-enveloped DNA virus belonging to the targeted Ad vectors are likely to reduce immunogenicity and family . AAVs have a linear 4.7 kb genome toxicity, increase safety, and enable the systemic administra- encoding a group of regulatory genes – the rep genes – and tion of these vectors for cancer. Two basic requirements are a group of structural genes – the cap genes. AAVs must be necessary to create a targeted Ad vector: interaction of Ad coinfected with a helper virus such as Ad for productive lytic with its native receptors must be removed and novel, tissue- infection, otherwise they remain a latent provirus in the host specific ligands must be added to the virus. Two general genome. The Ad E1/E4 gene products are essential for AAV approaches have been used to achieve these basic require- and the AAV lytic phase (Richardson & ments. In the ‘two-component’ approach, a bispecific Westphal 1981). Another unique feature of the AAVs is their molecule is complexed with the Ad. The bispecific compo- capacity for site-specific integration. The preferred site of nent simultaneously blocks native receptor binding and integration for wild-type AAV is chromosome 19q14.3 redirects virus binding to a tissue-specific receptor. In the (Kotin et al. 1990). The capacity for site-specific integration ‘one-component’ approach the Ad is genetically modified to is rep gene-dependent and deletion of rep results in random ablate native receptor interactions and a novel ligand is gen- integration. AAVs are limited to a transgene size of approxi- etically incorporated into one of the Ad coat proteins. Two- mately 4.5 kb, smaller than the capacity of the Ad vectors. component systems offer great flexibility in rapidly validat- Unlike RVs, AAVs may infect non-dividing cells. In ing the feasibility of targeting via a particular receptor. addition, the AAVs elicit little or no immune/inflammatory One-component systems offer the best advantages in produc- response, which together with their integration into the target ing a manufacturable therapeutic agent and in more com- cell genome confers the potentially repeated administrations pletely ablating all native Ad receptor interactions. The and stable long-term transduction. The AAVs are not associ- coming challenges for targeted Ad vectors will be the dem- ated with any known pathogenicity or oncogenicity. Recent onstration that the technology performs in vivo. Ultimately, advances in the production of high-titer purified rAAV vector or in parallel, ‘receptor-targeting’ technology can be com- stocks have made the transition to human clinical trials a bined with improved Ad backbones and with ‘transcriptional- reality (Monahan & Samulski 2000). targeting’ approaches to create Ad which deliver genes selec- tively, safely, and with minimal immune response (Wickham RV vectors 2000). RVs of the oncovirus subfamily are single-stranded RNA In addition to their immunogenicity, another limitation viruses with a 7–10 kb genome. The RV life cycle begins of Ad vectors is their limited transgene capacity of approxi- with attachment to a cell-surface receptor via an envelope mately 8 kb. Increased insert capacity and decreased (env) surface protein. Depending on their env glycoprotein, immunogenicity has been observed with modified Ad vectors RVs may be either amphotropic – able to infect all species, known as ‘gutless’ Ad vectors, or as encapsidated Ad mini- or ecotropic – able to infect only mouse cells. The ampho- chromosomes (EAMs). In EAMs, almost the entire Ad tropic variety of MuLV attaches to cells via the RAM-1 genome has been deleted with the exception of the inverted receptor (Kavanaugh et al. 1994), a widely expressed repeats necessary for replication, and the encapsidation/pack- inducible sodium phosphate symporter. Receptor-mediated aging signals (Kumar-Singh & Farber 1998). EAMs have a endocytosis occurs following attachment. Then, the outer env theoretical cloning capacity of 36 kb and have facilitated the protein coat is shed and the virus genome undergoes reverse transfer of multi-gene expression units as large as 28 kb. transcription by viral reverse transcriptase resulting in the These vectors offer exciting prospects for the transfer of synthesis of a double-stranded DNA intermediate. This viral genes with large regulatory regions intact, or of multiple gene nucleoprotein complex then enters the cell nucleus and inte- cassettes. An even more radical Ad modification for gene grates randomly via the viral long terminal repeat sequences www.endocrinology.org Downloaded from Bioscientifica.com at 09/28/2021119 08:33:34AM via free access N J Mabjeesh et al.: Prostate cancer gene therapy

(LTRs) into the host genome. The stably integrated viral is their ability to direct sustained transgene expression for genome is termed a provirus. Because of their stable inte- greater than 6 months’ duration (Miyoshi et al. 1997). The gration into the target cell genome and transmission to the target cell range for wild-type HIV is restricted mainly to progeny of the transduced parent cell, RV vectors have the CD4+ cells. This limitation has been circumvented experi- potential for sustained duration of transgene expression, a mentally for HIV-based vectors by substituting env major advantage particularly for corrective gene therapy stra- sequences from other RVs for those of HIV to confer varying tegies (Lin 1998). and broader cell specificity (Naldini et al. 1996, Nascone & The capacity for random proviral integration into the host Mercola 1997). Safety concerns have tempered enthusiasm genome raises the concern of potential insertional muta- for the use of HIV-based recombinant vectors. While helper- genesis. Wild-type Moloney muzine leukemia virus free virus stocks of HIV-based vectors have been success- (MMLV) can induce and lymphomas in mice after fully produced, the small risk of pathogenic recombination in long latency. To limit this risk, the minimum total RV dose vivo supports the development of non-HIV-based/non-human necessary to achieve a therapeutic endpoint must be utilized. lentiviral vectors for clinical application. Recently, Loimas The use of replication-deficient virus also minimizes the et al. (2001) found that lentiviral vectors carrying a fusion amount of virus applied to the target tissue. gene of HSV-thymidine kinase (TK) (HSV-TK) were effi- For the oncovirus subfamily of RVs which includes cient vehicles for three human PCA cell lines DU-145, MMLV- and MMTV-based RVs, the entry into the cell LNCaP and PC-3. Despite sufficient gene transfer rates (25– nucleus to undergo proviral integration is cell-division 45%) in the ganciclovir (GCV)-sensitivity experiment, only dependent (Miller et al. 1990). This is a significant limitation DU-145 cells were efficiently destroyed under clinically rel- on the usefulness of oncovirus family-based vectors for gene evant GCV concentrations (Loimas et al. 2001). therapy in vivo, particularly in PCA with its low mitotic rate. The spumaviruses are another subfamily of the RVs and Another feature of RV vectors which may limit their suit- include the HFVs. HFV is non-pathogenic and has the ability for targeted gene therapy strategies in vivo is the rela- capacity for integration into the genome of a wide range of tively non-specific tropism of the amphotropic viruses to the non-dividing cells (Russell & Miller 1996). These are not ubiquitous RAM-1receptors. The tropism of RV vectors can inactivated by human serum as are the oncoviruses and may be modified through modification or replacement of the env represent a safer alternative for clinical gene therapy than gene product. Modification of RV targeting through the dis- HIV. play of single-chain to antigens such as carcino- embryonic antigen, transferrin receptor, Her2/neu, and CD34 Herpes virus vectors has been recently reported, suggesting that this approach may The herpes viruses are enveloped DNA viruses of the family improve the suitability of RV vectors for use in vivo (Jiang Herpesviridae. The HSV-1genome consists of a double- et al. 1998). Another major hindrance to the use of RV vec- stranded linear DNA of 152 kb. Other members of the herpes tors in vivo is the rapid inactivation of RVs by human comp- virus family include HSV-2, varicella zoster, cytomegalo- lement. virus (CMV), Epstein–Barr virus, human herpes virus In view of the above limitations, RV vectors are typically (HHV)-6 and HHV-7. The herpes viruses replicate in the used for ex vivo gene therapy for which they are well suited, nucleus episomally. Following replication, viral particles as the issues of non-specific transduction and inactivation by reach the cell surface via the . HSV-1- complement are avoided (Cornetta et al. 1990). The most based vectors have been used extensively in gene therapy for common use of the RV vectors for PCA gene therapy has neural tissue and tumors, given their inherent neural tropism. been for ex vivo cytoreductive immunotherapy. Advantages of HSV-1-based vectors include their large insert capacity of 35 kb, and their ability to infect dividing as well Lentivirus and spumavirus vectors as non-dividing cells. Disadvantages of the HSV-based vec- The lentiviruses are a subfamily of the RVs, which includes tors include their potential pathogenicity, poor transduction HIV, bovine leukemia virus, and human T-cell leukemia efficiency and the short duration of gene expression virus (HTLV)-1and HTLV-2. Unlike members of the onco- achieved. viridae subfamily such as MMLV and MMTV, the lenti- viruses are able to integrate into non-dividing cells, an impor- Vaccinia virus vectors tant advantage for gene therapy in vivo. For the lentivirus Vaccinia virus-based vectors have been used extensively for HIV, this ability is a result of the viral proteins vpr and viral in vivo immunotherapy strategies. These DNA virus vectors matrix protein p17 (Bukrinsky et al. 1993, Gallay et al. have a wide host range and may transfer large inserts of up 1995a). The phosphorylation of a terminal tyrosine residue to 25 kb, facilitating multi-gene transfer (Peplinski et al. on p17 permits transport of the HIV pre-integration complex 1998). They have been used extensively for delivery of anti- into the nucleus even in the absence of mitosis (Gallay et al. gen and immune-stimulatory genes for vaccine-based gene 1995a,b). Another favorable feature of the lentiviral vectors therapy (Hodge et al. 1994). These vectors may replicate in

120 Downloaded from Bioscientifica.comwww.endocrinology.org at 09/28/2021 08:33:34AM via free access Endocrine-Related Cancer (2002) 9 115–139 vivo, further increasing the efficiency of host cell transduc- mid, only 50% showed signs of successful immunization. tion. Their utility for in vivo gene therapy is limited by the However, several responders, as evidenced by a change in strong immune response they elicit, and pre-existing anti- the local disease, distant metastases, and prostate-specific vaccinia immunity is common as a result of childhood small- antigen (PSA) levels, were reported (Mincheff et al. 2000). pox immunization. The immunological elimination of the A phase II clinical study to evaluate the effectiveness of the vaccinia-transduced cells limits gene expression to about 4 therapy is currently underway. weeks. For the vaccine applications for which vaccinia vec- tors are typically used, this duration of expression is suf- Liposome vectors ficient for priming an immune response. Booster vaccine Liposomes are the second most frequently used gene transfer administrations may be more effective if given using a method in current clinical trials. Liposome vectors consist of heterologous vector, which is not immunologically cross- a DNA plasmid surrounded by a liposomal coat. The charge reactive (Irvine et al. 1997). characteristics of the liposomal coat play a major role in determining the targeting capacity, intracellular stability and transgene size capacity of the vector. Anionic liposome- Synthetic gene transfer systems based vectors do not display non-specific cell-surface binding Several non-viral methods for gene delivery are under intense and thus may be targeted to specific cell-surface moieties investigation. These include chemical methods such as a var- (Lee & Huang 1996). Anionic liposomes are endocytosed by iety of liposomes and physical methods such as microinjec- the target cell, targeted to endosomes, and subsequently tion electroporation and biobalistics (Prince 1998). prone to degradation by lysosomal nucleases severely limit- ing efficiency of transgene expression. Cationic liposomes Plasmid DNA vectors bind avidly and non-specifically to the cell surface and are The most technically simple form of gene therapy is the use taken up into cells by a non-receptor-mediated process. They of naked DNA. A plasmid containing the gene of interest are less likely to undergo endosomal degradation than and appropriate promoter is injected directly into a desired anionic liposomes and can also package much larger site. Administered in this way, the efficiency of transgene transgenes. A commonly used cationic liposomal vector in uptake/expression is very low. The transforming DNA does clinical trials is Lipofectin. not integrate into the host genome and the duration of expres- Liposomes display preferential uptake in cells of the sion is short. Of all sites tested so far, the greatest transduc- reticuloendothelial system (RES), complicating attempts at tion efficiency is observed in muscle (Wolff et al. 1990). In targeting. Modification to decrease this unwanted uptake this application, brief low-level expression may be sufficient includes the addition of sialic acid residues to make ‘stealth’ for eliciting a therapeutic immune response. Animal studies liposomes (Lasic et al. 1991) which may evade RES uptake. have indicated that the use of syngeneic dendritic cells (DCs) It has been suggested that the presence of leaky vessels in that have been transfected ex vivo with DNA for tumor- solid tumors may favor the selective accumulation of steric- specific antigen results in tumor regression and decreased ally stabilized large vectors such as liposomes (Brown & number of metastases. Additional studies have also suggested Giaccia 1998). Liposomal vectors are inexpensive and rela- the possibility of modulating the DCs in vivo either by tively easy to prepare in large quantities. Their large insert ‘naked’ DNA immunization or by injecting replication- capacity permits the transfer of multi-gene cassettes of deficient viral vectors that carry the tumor-specific DNA. unlimited size. Since they lack proteins, they elicit weaker or Using PSMA as a target molecule, Mincheff et al. (2000) no immune/inflammatory responses. A disadvantage of lipo- have initiated a clinical trial for immunotherapy of PCA. somal gene transfer is its inefficiency; thousands of lipo- They have included the extracellular human PSMA DNA as somes are necessary per cell for successful transduction well as the human CD86 DNA into separate expression vec- (Yotsuyanagi & Hazemoto 1998). tors (PSMA and CD86 plasmids), and into a combined PSMA/CD86 plasmid. In addition, the expression cassette DNA–protein complex vectors and hybrid vectors from the PSMA plasmid was inserted into a replication- DNA–protein complexes have been developed for gene deficient Ad expression vector. Twenty-six patients with transfer in which the transgene is complexed with a targeting PCA were entered into a phase I/II toxicity–dose escalation protein. This allows the possibility of high-specificity gene study. No immediate or long-term side-effects following transfer of large inserts with minimal immune response. immunizations have been recorded. All patients who DNA–protein complexes, like anionic liposomes are, how- received initial inoculation with the viral vector followed by ever, highly susceptible to endosomal targeting and lysoso- PSMA plasmid boosts showed signs of immunization as evi- mal nuclease degradation, greatly reducing their transduction denced by the development of a delayed-type hypersensitiv- efficiency. ity reaction after the PSMA plasmid injection. In contrast, of The major limitation of viral vectors is maximum insert the patients who received a PSMA plasmid and CD86 plas- size. The major limitation of the synthetic vectors is their www.endocrinology.org Downloaded from Bioscientifica.com at 09/28/2021121 08:33:34AM via free access N J Mabjeesh et al.: Prostate cancer gene therapy poor gene transfer efficiency, a result of poor uptake of Corrective PCA gene therapy vector DNA into cell/nucleus and the lysosomal degradation that occurs following internalization. As a means to circum- Corrective gene therapy involves the functional comp- vent some of the limitations of both synthetic and viral vec- lementation of an abnormal or absent gene. Corrective stra- tors, hybrid vectors combining viral and synthetic approaches tegies may be designed to prevent tumorigenesis in pheno- have been devised. DNA segments can be complexed to Ad typically normal cells. Alternatively, corrective gene therapy virions to which polylysine or DEAE-dextran has been con- may be administered to induce phenotypic reversion or jugated (Wagner et al. 1992, Curiel 1994, Forsayeth & regression of transformed cells to a non-neoplastic or less Garcia 1994). These conjugates improve transfer efficiency transformed phenotype. Numerous potential candidate genes by orders of magnitude in comparison with naked DNA. for corrective gene therapy approaches are suggested by the Similar improvements have been noted for an Ad–liposome wide variety of genetic and epigenetic changes known to complex which resulted in a 1000-fold increase in gene trans- occur accompanying prostate tumorigenesis (Table 3) includ- fer efficiency relative to naked plasmid (Raja-Walia et al. ing allelic loss, alterations in DNA methylation patterns, acti- 1995). Transgenes of up to 48 kb have been successfully vating mutations of oncogenes, and inactivating mutations of transferred using this technique (Cotten et al. 1992). The Ad tumor suppressors and cell-adhesion molecules (Isaacs et al. to which the DNA is complexed may be replication-defective 1995, Konishi et al. 1997, Roylance et al. 1997). or UV-inactivated. The presence of the virion proteins in the The most commonly observed allelic loss in PCA DNA–virus complex are sufficient to improve vector attach- involves chromosomes 8, 10, 11 and 16 (Isaacs et al. 1994, ment and uptake, and more importantly, allow escape of the 1995), suggesting the presence of tumor-suppressor genes on vector DNA from endosomes prior to fusion with the lyso- these chromosomes. The potential for functional tumor sup- somes, where they may be degraded (Cristiano et al. 1993, pression by genes encoded in these deleted chromosomal 1998). regions has been demonstrated in microcell-mediated chro-

Table 3 Corrective gene therapystrategies for PCA Corrective gene Normal function Defect in PCA therapy targets Tumor suppressors Regulation of cell cycle G1/S checkpoint, Mutated in 20–75% of PCAs apoptosis, response to stress/DNA damage Rb Cell cycle regulation and differentiation Allelic loss in 27–40% of PCAs pTEN Regulation of PI3K cell survival pathwayDecreased expression in 50% of PCAs p16 (CDKN2) Cyclin-dependent kinase inhibitor involved in cell Markedly reduced expression in 43% untreated cycle regulation primary PCAs; no alteration in BPH Down regulation byandrogen KAI-1 Cell–cell and cell–matrix interactions Decreased expression in metastatic PCAs Oncogenes myc Transcription factor regulating proliferation/ Amplified in 20% of advanced PCAs differentiation bcl-2 Inhibition of apoptosis Uniformlyelevated in androgen-independent PCAs Ras GTP-binding proteins involved in growth Mutated in 25% PCA in US men; in Japanese regulation cases PCA ȁ25% mutated c-met Receptor tyrosine kinase for hepatocyte growth Elevated in 100% bone metastases factor Growth factors IGFs/IGFBPs Growth factors/growth factor regulatoryproteins Increased IGF-I levels associated with increased risk of PCA TGF-β Growth factor – growth-inhibitoryin normal Tumor progression mayinvolve dysregulationof prostate TGF-β growth inhibition Cell-adhesion molecules E-cadherin Signal transduction/intercellular adhesion Deficient or absent expression in ȁ50% of PCAs α-catenin Signal transduction/intercellular adhesion Deficient expression in 42% of PCAs β-catenin Signal transduction/intercellular adhesion Mutated in 5% of PCAs CD44 Intercellular adhesion/cell–matrix interactions Decreased expression with increasing stage Detoxification GSTP1 Detoxification of electrophilic carcinogens Absent expression secondaryto promoter hypermethylation in PIN and 98% of clinical PCAs

122 Downloaded from Bioscientifica.comwww.endocrinology.org at 09/28/2021 08:33:34AM via free access Endocrine-Related Cancer (2002) 9 115–139 mosome transfer studies. Transfer of portions of chromo- negative gene products, antisense oligodeoxynucleotides, somes 8, 10, 11, 17 and 19 into Dunning R-3327 rat PCA hammerhead ribozymes, and single-chain antibodies. The cells results in decreased formation of metastases and overexpression of the c-Myc is frequently observed decreased tumorigenicity (Isaacs et al. 1995, Gao et al. in advanced prostate tumors (Fleming et al. 1986, Buttyan et 1999). Analysis of allelic loss involving chromosome 11 led al. 1987). Disruption of c-Myc overexpression using anti- to the cloning of the KAI1 -suppressor gene (Dong sense c-Myc transduced by a replication-deficient MMTV- et al. 1995), a potential candidate for use in PCA gene ther- derived RV (Steiner et al. 1998) resulted in a 94.5% apy strategies aimed at prevention of metastases. Further ana- reduction in tumor size of DU145 PCA cell xenografts. lysis of allelic loss during tumor progression may identify Another oncogene, which could be targeted in a corrective new candidates suitable for corrective gene therapy specific gene therapy approach, is bcl-2, which is frequently overex- to various stages of tumorigenesis. pressed in androgen-independent PCAs (McDonnell et al. Tumor-suppressor genes such as p53, retinoblastoma 1992, Furuya et al. 1996, Beham et al. 1998). A hammerhead (Rb), p16, and pTEN show evidence of mutation or aberrant ribozyme designed to disrupt bcl-2 expression in LNCaP expression in some PCAs (Bookstein et al. 1993, Isaacs et PCA cells showed pro-apoptotic activity and resulted in al. 1994, 1995, Pesche et al. 1998, Suzuki et al. 1998). The increased cell sensitivity to secondary pro-apoptotic agents tumor-suppressor gene p53 encodes a transcription factor including phorbol ester (Dorai et al. 1997). involved in regulation of the cell cycle and apoptosis. Cell-adhesion molecules such as the cadherins and caten- Mutations in p53 are widespread in human cancer and are ins show frequent alterations accompanying tumorigenesis observed in between 20 and 75% of PCAs, more commonly and may constitute appropriate targets for corrective gene in advanced metastatic tumors (Heidenberg et al. 1995). The strategies. The cadherins are a family of transmembrane cell- ability of overexpressed p53 to inhibit the growth of primary adhesion proteins involved in calcium-dependent cell–cell cultures derived from radical prostatectomy specimens has adhesion, and in transduction of extracellular growth regulat- been demonstrated, surprisingly even when p53 status is ory signals. Deficient or absent E-cadherin expression due normal (Asgari et al. 1997). to allelic loss, or promoter hypermethylation, is observed in Mutations of the tumor-suppressor pTEN have been fre- approximately half of PCAs. The level of E-cadherin expres- quently observed in PCA (Pesche et al. 1998, Suzuki et al. sion is prognostic for survival, metastases, tumor stage, 1998, Whang et al. 1998). pTEN appears to be involved in grade, and risk of post-prostatectomy recurrence (Umbas et a regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt al. 1994, Richmond et al. 1997). E-cadherin-mediated cell pathway, a pathway involved in cell survival. Candidates for adhesion also requires the function of the cytoplasmic protein effectors downstream in this signaling pathway include α-catenin which couples E-cadherin to the . GSK-3/β-catenin (Bullions & Levine 1998), and members of Deficient α-catenin expression has been observed in approxi- the bcl-2 family. Another tumor-suppressor, p16 (CDKN2) mately 42% of PCAs and is prognostic for patient survival shows markedly reduced expression in 43% of untreated pri- (Richmond et al. 1997). The constitutive expression of mary PCAs but is unaltered in benign prostatic hyperplasia α-catenin in PC-3 PCA cells through microcell-mediated (BPH) (Chi et al. 1997). p16 is a cyclin-dependent kinase transfer of chromosome 5 results in suppression of tumori- inhibitor involved in cell cycle regulation. Down-regulation genicity in an E-cadherin-dependent fashion (Ewing et al. of p16 by androgen, promoter methylation, or other inactivat- 1995). β-catenin, another catenin involved in E-cadherin- ing mutations may increase progression through the cell mediated cell–cell adhesion as well as in signal transduction cycle (Jarrard et al. 1997, Lu et al. 1997). through the Wnt/wingless gene signaling pathway, has Methylation changes affecting gene expression have recently been shown to be mutated focally in some prostate been widely observed in prostate and other tumors (Lee et al. tumors, suggesting that it may play a role in tumor pro- 1994, Nelson et al. 1997). A frequent target for inactivating gression (Voeller et al. 1998). methylation changes is the glutathione S-transferase P1gene, Growth factors are another potential target for corrective a gene involved in detoxification of electrophilic carcinogens gene therapy. The insulin-like growth factors (IGFs) are (Lee et al. 1994). The absence of this mutation in normal potent mitogens for prostate epithelial cells. The ability of prostate or BPH, and its presence in prostatic intraepithelial IGF-I to activate the androgen receptor (AR) even in the neoplasia (PIN) and greater than 98% of clinical prostate absence of hormone suggests a potential role in PCA pro- tumors suggests that it is an early step in prostate tumorigen- gression (Culig et al. 1995a,b). The IGF-binding proteins esis, and thus may be an appropriate target for PCA preven- (IGFBPs), produced in the prostate and elsewhere, are a tive gene therapy. family of seven related proteins which can modulate epi- Oncogenes that may be activated in PCA include c-Myc, thelial cell growth, either via binding and modulation of Ras, bcl-2 and c-met (Table 3) (Isaacs et al. 1995, Konishi IGF-I and -II activity or through IGF-independent mechan- et al. 1997, Roylance et al. 1997). Gene therapy strategies isms (Oh 1997). The IGFBPs are induced by growth inhibi- to correct oncogene activation include the use of dominant tory molecules such as transforming growth factor (TGF)-β www.endocrinology.org Downloaded from Bioscientifica.com at 09/28/2021123 08:33:34AM via free access N J Mabjeesh et al.: Prostate cancer gene therapy

Secreted Class II MHC + Tumor Antigen DC

Th CD-4 !! !! CTL Th

Class I MHC Cytokine-Vector + PCA Tumor Antigen !! !! CTL Th Autologous/allogeneic tumor cell DC CD-8 CTL ex vivo Immunostimulatory Distant Vaccine Site Gene Transfer Metastases

Figure 1 Ex vivo cytokine-transduced tumor vaccine gene therapy. Tumor cells transduced ex vivo with cytokine such as GM-CSF are irradiated then reintroduced into the patient. Transduced GM-CSF improves tumor cell antigen presentation and fullyactivates DCs, the most potent APCs in the immune system.DCs present prostate tumor vaccine-associated antigens they have processed to both CD4 (helper) and CD8 (cytolytic) T cells in the draining lymph node of the vaccination sites, promoting systemic tumoricidal immunity.

and p53 (Hwa et al. 1997) and IGFBP-3 transgene expression immune responses. Immunotherapy strategies for cancer in PC-3 cells defective for p53 results in an IGF- and p53- gene therapy utilize gene transfer to facilitate a dormant host independent induction of apoptosis (Rajah et al. 1997). immune response directed against the tumor. Evasion of It is estimated that approximately 9% of PCAs occurring autologous host cellular immunity is a common feature of prior to the age of 85 are due to genetic predisposition tumor cell neoantigens. Tumor cells are poor antigen- (Gronberg et al. 1997, Walsh & Partin 1997). The basis for presenting cells (APCs). In PCA in particular, defects in hereditary PCA has been mapped to several loci – the first MHC class I expression are observed in a striking 85% of susceptibility locus identified, HPC-1, resides on chromo- primary and 100% of metastatic tumors (Blades et al. 1995), some 1q24–25 (Smith et al. 1996). A second susceptibility suggesting that evasion of MHC class I tumor-associated locus, the recently identified HPC-X, resides on the X chro- antigen is important in prostate tumor development. ‘Cancer mosome at Xq27–28 (Xu et al. 1998). Identification of the vaccine’ strategies are based on optimization of the context involved gene(s) at these loci should provide targets for pre- in which tumor antigens or tissue-specific antigens are ventive/corrective gene therapy, particularly for high genetic presented to the host immune system. When appropriately susceptibility risk probands. primed, the activated host immune system can then act A potential limitation of corrective gene therapy is the against tumor cells systemically. requirement that not even a single neoplastic clonogen must One means to utilize gene therapy to optimize tumor- develop. To achieve this goal would require 100% efficiency antigen presentation is through the targeted expression of of gene transfer, as well as sustained transgene expression, in tumor cells. Targeted paracrine expression elim- or tolerance for repeated vector application. Even if effi- inates the toxicities associated with systemic cytokine admin- ciency could be improved to the extent required, alternative istration. The transduced cytokines result in a combination mechanisms of progression may circumvent whatever the of improved tumor cell vaccine antigen presentation, and corrective strategy employed. activation of APCs, both essential for effective priming of the cellular immune response (Fig. 1). Granulocyte-macrophage Cytoreductive PCA gene therapy colony stimulating factor (GM-CSF) has emerged as a cyto- kine with significant efficacy in the induction of an antitumor Immunotherapy strategies immune response (Dranoff et al. 1993). GM-CSF is the most PCA offers potentially unique antigens which may be potent cytokine signal tested for activation of antigen pro- exploited for the genetic induction of autoimmune antitumor cessing and presentation by macrophages and DCs (Cella et

124 Downloaded from Bioscientifica.comwww.endocrinology.org at 09/28/2021 08:33:34AM via free access Endocrine-Related Cancer (2002) 9 115–139 al. 1997a,b). GM-CSF may be transduced into autologous or Another vaccine strategy is the DNA or polynucleotide allogeneic tumor cells ex vivo using either an RV or other vaccine. In this approach, an expression cassette containing vectors. The transduced tumor cells are then irradiated both the transgene against which an immune response is desired to minimize malignant potential and to improve immuno- is injected directly into host cells, typically i.m. (Benton & genicity (Simons & Mikhak 1998). The cells are then reintro- Kennedy 1998). The expression of the transfected gene in duced by vaccination into the patient and tumor response vivo may then engender humoral and cellular immune monitored. Tumor cell vaccine-expressed GM-CSF may acti- responses. DNA vaccines are technically easier to prepare vate quiescent DCs which then capture prostate tumor cell than peptide- or viral-based vaccines and pose no risk of vaccine antigens. DCs are the most potent APCs in the pathogenicity as they are non-replicating. A modification of immune system and are central to the success of these the DNA vaccine technique which may improve its efficacy genetically engineered tumor vaccine strategies. Activated is the fusion of the desired transgene with the sequence for DCs can present prostate tumor vaccine-associated antigens a pathogen-derived gene to engender a stronger immune they have processed to both CD4 (helper) and CD8 response. Fragment C of tetanus toxin has been used in a (cytolytic) T cells in the draining lymph node of the vacci- fusion with single-chain Fv sequences from B-cell lym- nation sites (Fig. 1) (Banchereau & Steinman 1998, phoma, and resulted in striking enhancement of both humoral Simons & Mikhak 1998), activating a systemic tumoricidal and cellular immunity (Falo & Storkus 1998, King et al. immune response. 1998). Recently, the preclinical testing of a PSA-based DNA By presenting tumor antigen/MHC I in the context of vaccine in mice was reported (Kim et al. 1998). Injection supraphysiological local levels of transduced cytokines such i.m. of the PSA expression cassette resulted in a strong CTL/ as GM-CSF or interleukin (IL)-2, the CTL-based clinical humoral response against PSA-positive tumor cells. Evalu- antitumor immune response has been enhanced (Simons & ation of the effects of vaccines incorporating various regions Mikhak 1998). A potent antitumor CTL response has also of the PSA-coding sequence identified four dominant coding been attained through co-presentation of tumor antigen/MHC sequences for Th epitopes of PSA. Future vaccine approaches can be predicted to target other prostate-unique II with a transduced costimulatory signal such as B7 (Baskar antigens such as PSMA, mucin (MUC-2), human glandular et al. 1993). The duration of the transduced cytokine expres- Kallikrein 2 (hK2), and other novel prostate-unique antigens sion must be of sufficient duration to permit cell harvesting whose identification will be facilitated through the use of ex vivo and for antigen processing/presentation in vivo. For genomics and proteomics techniques (Pang et al. 1997, autologous vaccine, RV and Ad vectors have been favored Nelson et al. 1998). for ex vivo tumor vaccine trials. GM-CSF gene transfer has A major limitation of cytoreductive immunotherapy is also been accomplished using cationic immunoliposome the limited tumor burden which the immune system can elim- AAV vectors (Koppenhagen et al. 1998). inate in experimental models. Thus, applicability is probably Ex vivo cancer vaccine strategies have also directly limited to clinical settings of low bulk or micrometastatic employed APCs such as DCs to facilitate effective tumor- disease, or in combination with other debulking therapies. antigen presentation. The rationale for these strategies is that Additionally, the harvesting and culture of autologous or the APC, properly primed, will express the most appropriate allogeneic tumor or immune cells for ex vivo gene therapy is combination of factors for eliciting an effective immune expensive and technically challenging. The observed safety response. DC-based vaccines have been generated by RV of cytokine gene-transduced vaccines is in no small measure vector-mediated transduction of antigen (Specht et al. 1997), responsible for ongoing enthusiasm for clinical testing as an pulsing with tumor-antigen RNA (Boczkowski et al. 1996), outpatient adjuvant therapy. and pulsing with tumor- or tissue-specific peptide (Murphy et al. 1999). Polyvalent vs single antigen PCA vaccines As complete ablation of PSA-expressing tissue is an Polyvalent cancer vaccines targeting the entire antigenic ideal therapeutic endpoint, in vivo cytoreductive immuno- spectrum on tumor cells may represent a superior therapeutic therapy approaches have centered on prostate-specific recom- strategy for cancer patients than vaccines solely directed binant viral vaccines. The vector-induced inflammatory/ against single antigens (Heiser et al. 2001). Heiser et al. immune response functions as an adjuvant to the transduced (2001) showed that autologous DCs transfected with RNA antigen, resulting in local release of cytokines and influx of amplified from microdissected tumor cells are capable of sti- APCs to the vaccine site. The direct transduction and expres- mulating CTL against a broad set of unidentified and critical sion of tumor-associated antigens in DCs can enhance the PSAs. In this strategy of gene therapy, the tumor’s candidate efficacy of viral vaccines (Bronte et al. 1997). Cotransduc- antigens were transfected as a cDNA library. Although the tion of cytokines together with the tumor-associated antigen polyclonal CTL responses generated with amplified tumor may further enhance viral vaccine efficacy (Bronte et al. RNA-transfected DCs encompassed as a subcomponent a 1995). response against PSA as well as against telomerase reverse www.endocrinology.org Downloaded from Bioscientifica.com at 09/28/2021125 08:33:34AM via free access N J Mabjeesh et al.: Prostate cancer gene therapy transcriptase, the tumor-specific CTLs were consistently apoptotic and non-apoptotic pathways. Another cytoreductive more effective than PSA or telomerase reverse transcriptase agent that has shown activity in PCA cells is expanded CTLs in lysing tumor targets, suggesting the superiority of polyglutamine. This is a pro-apoptotic molecule implicated in the polyclonal response. Although tumor RNA-transfected eight inherited neurodegenerative disorders (Ikeda et al. 1996). DCs stimulated CTLs which recognized not only tumor but Its pro-apoptotic potential can be modulated by varying the also self-antigens expressed by benign prostate tissue, these expression level or the length of the expanded polyglutamine. cross-reactive CTLs were exclusively specific for the PSA, This molecule has demonstrated pro-apoptotic activity in PCA indicating an immunodominant role of PSA in the PCA- cell lines when expressed from a vector under control of a PSA specific immune response. These data suggest that tumor promoter (Segawa et al. 1998). A critical concern with the use RNA-transfected DCs may represent a broadly applicable, of potent cytotoxins for cytoreductive gene therapy is the target potentially clinically effective vaccine strategy for PCA cell specificity of the vectoring strategy. From a practical view- patients, which is not limited by tumor tissue availability for point, even low levels of expression of a cytotoxin may be antigen preparation and may minimize the risk of clonal toxic to the packaging cell line if a recombinant viral strategy tumor escape. is employed. With tumor/tissue-specific promoter or antigen targeting, unwanted expression of the cytotoxin in non-target cells either through non-specific antigen targeting or through a Cytolytic/pro-apoptotic strategies ‘leaky’ tissue-specific promoter could result in significant adverse normal tissue effects. Enzyme/prodrug gene therapy Enzyme/prodrug gene therapy, also referred to as ‘suicide Oncolytic viruses gene therapy’, relies on the conversion of an inactive prodrug Viral vectors may themselves be designed to target and kill into a toxic drug using an enzyme vectored only to the target tumor cells without insertion of a foreign transgene. Proof of tumor cells. In this way, active drug is limited spatially to the this principle was demonstrated therapeutically as early as transduced cells and adjacent surrounding cells, facilitating the 1950s with tumor responses noted following injections of higher tumor drug concentrations without increased normal wild-type Ad into patients with cervical cancer. The Ad life tissue toxicity. As opposed to the corrective gene therapy cycle includes a lytic phase, which can result in host cell approach noted above, it is essential that the vector be tar- death independently of entry into the cell cycle – an impor- geted with great specificity to tumor cells to avoid increased tant advantage for PCA therapy given the low mitotic rate. normal tissue injury. Prodrug-activating enzymes which have Ad has evolved a potent repertoire of gene products, which been employed in this approach include cytosine deaminase may exert profound effects on the growth regulation of the (CD) which catalyzes the conversion of the non-toxic host cell, in order to facilitate viral replication. A replication- 5-fluorocytosine (5-FC) to the cytotoxic 5-fluorouracil competent Ad designed to preferentially replicate in p53 (5-FU), and HSV-TK which together with cellular enzymes mutant cells, the ONYX-015 vector, is currently in clinical facilitates the conversion of GCV into the toxic GCV tri- trials (Kirn et al. 1998). The design of this vector was based phosphate. Using this approach it has been shown that even on the findings that a consequence of Ad infection was the when only 2% of a tumor contains CD-transduced cells, sig- induction of p53, whose pro-apoptotic activity would be det- nificant tumor regression is observed, suggesting the pres- rimental to the viral life cycle and was therefore blocked ence of a significant bystander effect (Kim et al. 1998). The directly by the virally encoded E1B 55 kDa protein cytotoxic efficacy of suicide gene therapy in vivo may benefit (Debbas & White 1993). In addition, E1B 19 kDa protein from a systemic antitumor response precipitated by tumor expression resulted in the production of a bcl-2 type lysis and inflammation, which may be mediated by natural molecule with anti-apoptotic effects downstream of the p53 killer (NK) cells (Hall et al. 1998). The capacity for prostate- pathway (Chiou et al. 1994). It was reasoned therefore that specific expression of suicide genes has been demonstrated an E1B-deficient Ad would replicate preferentially in p53- using PSA, PSMA, and hKLK2 promoters (Harris et al. deficient tumors (Bischoff et al. 1996). Recent findings sug- 1994). gest, however, that control of viral replication is more com- plex than the initial model suggests, as the E1B-deficient Cytotoxins virus replicates in p53 wild-type tumor cells as well Another cytoreductive strategy involves the targeted expres- (Rothmann et al. 1998). To evaluate the selectivity of sion of a cytotoxin such as the diphtheria toxin A chain, or ONYX-015 replication and cytopathic effects for the first pseudomonas exotoxin A. In a screen of cytotoxins using a time in humans, a phase II clinical testing of intratumoral wide range of PCA cell lines, Rodriguez et al. (1998) demon- and peritumoral ONYX-015 injection in 37 patients with strated a cell cycle- and p53-independent cytotoxic activity of recurrent head and neck carcinoma was carried out the diphtheria toxin A chain in PCA cell lines through both (Nemunaitis et al. 2000). Post-treatment biopsies docu-

126 Downloaded from Bioscientifica.comwww.endocrinology.org at 09/28/2021 08:33:34AM via free access Endocrine-Related Cancer (2002) 9 115–139 mented selective ONYX-015 presence and/or replication in Prostate-specific targeting the tumor tissue of 7 of 11 patients biopsied. Tissue destruc- Regardless of the vector or gene therapy strategy used, tion was also highly selective; significant tumor regression restriction of cytoreductive effects to target tissue is an essen- (>50%) occurred in 21% of evaluable patients, whereas no tial factor in determining overall therapeutic index. Strategies toxicity to injected normal peritumoral tissues was demon- to restrict cytoreductive effects of gene therapy may utilize strated. p53 mutant tumors were significantly more likely to tissue- or tumor-specific antigens or promoters. The presence undergo ONYX-015-induced (7 of 12) than were of several well-characterized prostate-specific markers such p53 wild-type tumors (0 of 7; P = 0.017). High neutralizing titers did not prevent infection and/or replication as PSA and PSMA, and 500 or more prostate-unique genes, within tumors. ONYX-015 is the first genetically engineered provides a biological foundation for prostate-localized gene replication-competent virus to demonstrate selective intra- therapy treatment. The prostate-restricted expression of PSA tumoral replication and necrosis in patients. This agent dem- has stimulated a variety of PSA-dependent gene therapy stra- onstrates the promise of replication-selective viruses as a tegies. Antigen-based strategies include the PSA vaccines in novel therapeutic platform against cancer (Nemunaitis et al. clinical trial discussed below. Several groups have placed 2000). therapeutic genes under the control of various PSA gene cis- ′ Among the Ad gene products which may function to regulatory regions. Varying segments of the 5 -flanking induce cell death are E1A (Rao et al. 1992), the E3–11.6k region of PSA have been evaluated for their suitability for protein also known as the Ad death protein, which is required prostate-specific expression of a desired gene. A minimal for efficient cell lysis during productive viral infection composite PSA promoter/enhancer element (PSE) was used (Tollefson et al. 1996b), and the E4orf4 protein, which may to drive expression of Ad E1A in the attenuated replication- induce p53-independent apoptosis (Marcellus et al. 1998). competent vector CN706 (Fig. 2) (Schuur et al. 1996, Rodri- The E1A 243-residue protein is able to induce a p53- guez et al. 1997). Within the PSA enhancer region employed dependent apoptosis in the absence of other viral proteins in the PSE, a functional androgen response element (ARE) using a effector cascade which involves activation at position 4136 (Fig. 2) results in an up to 100-fold increase of procaspase-8, followed by mitochondrial redistribution of in expression in the presence of testosterone or the steroid cytochrome c and activation of procaspase-3 (Fearnhead et analog R1881. The regulation of E1A expression by the PSE al. 1998, Nguyen et al. 1998). An Ad vector with the E1A element effectively limits cytolytic viral replication to PSA- gene placed under the control of a PSA minimal promoter expressing cells, with a resulting therapeutic ratio of between enhancer, the CN706 vector (Fig. 2) showed potent PSA- 20:1and 3000:1depending upon the cell line tested. selective cytotoxic activity in preclinical testing and is cur- One issue with PSA-promoter/enhancer-based targeting rently being utilized in a phase I clinical trial (Rodriguez et is whether sufficient levels of transgene expression are al. 1997) (see below; Principal Investigator J W Simons, obtained, particularly in the absence of androgen. For clinical Johns Hopkins University). PCA gene therapy, efficient transgene expression in the Searching for additional approaches that would induce absence of androgen would be preferred. In a PSA promoter- therapeutic apoptosis of PCA cell lines, Li et al. (2001) based strategy in preclinical testing, the HSV-tk gene was recently used a binary Ad system to overexpress the pro- placed under control of a long 5.8 kb PSA promoter in an apoptotic molecule Bax. Bax was dramatically overexpressed Ad construct. This vector showed activity in both the pres- and caused apoptosis of every cell line infected by engaging ence and absence of androgen (Gotoh et al. 1998). Another the mitochondrial pathway, including proteolytic cleavage approach to amplify PSA-specific expression independently and catalytic activation of the , cleavage of caspase of androgen is through a two-step transcriptional activation substrates, release of cytochrome c from the mitochondria, system in which the PSA promoter drives the expression of and DNA fragmentation. Furthermore, three injections of the a potent transcriptional transactivator which in turn regulates Bax-overexpression system into PC-3 cell tumors in nude expression of the desired transgene (Segawa et al. 1998). mice in vivo caused a 25% regression in tumor size corre- PSMA is a potential target in PCA patients because it is sponding to a 90% reduction relative to continued tumor very highly expressed and because it has been reported to be growth in animals that received injections with the control upregulated by androgen deprivation. O’Keefe et al. (2000) binary system expressing Lac-Z. These experiments show recently described analysis of the PSMA enhancer for the that Ad-mediated Bax overexpression is capable of inducing most active region(s) using the enhancer in combination with therapeutic programmed cell death in vitro and in vivo by the E. coli CD gene for suicide-driven gene therapy that con- activating the mitochondrial pathway of apoptosis. On the verts the non-toxic prodrug 5-FC into the cytotoxic drug basis of these studies, it can be concluded that manipulation 5-FU in PCA cells. Deletion constructs of the full-length of Bax expression is an attractive new gene therapy approach PSMA enhancer were subcloned into a luciferase reporter for the treatment of PCA (Li et al. 2001). vector containing either the PSMA or SV-40 promoter. The

www.endocrinology.org Downloaded from Bioscientifica.com at 09/28/2021127 08:33:34AM via free access N J Mabjeesh et al.: Prostate cancer gene therapy

Adenovirus type 5 genome E3-deleted

Adenovirus ITR E1A E1B E4 ITR pE1A

Insertion of PSE E3 Deletion (28133-30818)

CN706 ITR E1A E1B E4 ITR 512 512 PSE

ARE

-3738 +12 -5322 -541 PSA enhancer PSA promoter

Figure 2 Structure of CN706. CN706 was derived from an E3-deleted type 5 Ad into which a minimal human PSA gene com- posite promoter/enhancer element (PSE) was inserted at 512 of the Ad type 5 genome just 3′ to the E1A promoter (pE1A). The PSE-driven E1A permits restriction of CN706 replication to PSA-positive cells. The PSE was derived from a fusion of PSA enhancer sequence from −5322 to −3738 to the PSA promoter spanning nucleotides −541 to +12 of the PSA gene. At position −4136 in the PSA enhancer is a functional ARE. Note presence of inverted terminal repeats (ITR), and packaging sequence ψ in the vector. most active portion of the enhancer was then determined via therapy. They identified the minimum ‘full-strength’ hK2 luciferase activity in the C4–2 cell line. The luciferase gene enhancer and built transcriptional regulatory elements com- was then replaced with the E. coli CD gene in the subclone posed of multiple tandem copies of this 1.2 kb enhancer, that showed the most luciferase activity. The specificity of fused to the hK2 minimal promoter. Relative to the weak this technique was examined in vitro, using the PCA cell induction of the minimal hK2 promoter by androgen analog line LNCaP, its androgen-independent derivative C4-2, and (R1881) in AR-positive LNCaP cells, transcriptional a number of non-prostatic cell lines. Deletion constructs activity was increased by 25-, 44-, 81- and 114-fold when revealed that at least two distinct regions seem to contribute one to four enhancers were spliced to the hK2 promoter to expression of the gene in PCA cells, and therefore the best respectively. In contrast, the PSEs were inactive in the construct for prostate-specific expression was determined to AR-negative PCA line PC-3 and in a panel of non-prostate be 1648 bp long. with the 1648 nucleotide lines, including 293, U87, MCF-7, HuH-7 and HeLa cells. PSMA enhancer and the PSMA promoter to drive the CD These results suggest that the hk2 multi-enhancer/promoter gene enhanced toxicity in a dose-dependent manner more should be a powerful reagent for targeted gene therapy of than 50-fold, while cells that did not express the PSMA gene PCA. were not significantly sensitized by transfection. The search for additional prostate-specific genes for Tissue-specific transcriptional regulatory elements can use in PCA gene therapy is still going on. Recently, Steiner increase the safety of gene therapy vectors. Unlike PSA/ et al. (2000) reported on a novel gene pHyde that was hK3, whose expression displays an inverse correlation with cloned from Dunning rat PCA cell lines. A replication- PCA grade and stage, hK2 is upregulated in higher grade deficient recombinant Ad containing pHyde cDNA gene and stage disease. Therefore, Xie et al. (2001) developed under the control of a truncated RSV promoter a prostate-specific hK2-based promoter for targeted gene (AdRSVpHyde) was generated. AdRSVpHyde inhibited

128 Downloaded from Bioscientifica.comwww.endocrinology.org at 09/28/2021 08:33:34AM via free access Endocrine-Related Cancer (2002) 9 115–139 growth of prostate cell lines (DU145 and LNCaP) by about Observations in our own laboratory reveal evidence for 80% as well as DU145 xenograft tumors by 75% (Steiner synergy between the oncolytic CN706 Ad (see above and et al. 2000). Fig. 2) and radiation in the LNCaP and LAPC-4 PCA cell lines. An obvious combined modality strategy for PCA is the Combined modality therapy: integration combined implantation of brachytherapy seed sources with a gene therapy vector. The capacity of Ad proteins such as with radiotherapy E1A and E4orf4 to engage cellular pro-apoptotic machinery Even if optimal gene delivery is achieved, the success of suggests a mechanistic basis for the observed therapeutic gene therapy, like conventional therapy, may be impeded by synergy (Marcellus et al. 1998, Nguyen et al. 1998). Sup- tumor cell resistance and intratumoral cell heterogeneity. The porting this idea is the observation that E1A expression use of combined treatment modalities provides a rational enhances apoptotic cell killing by a variety of chemothera- paradigm to improve upon the clinical efficacy of cancer peutic agents in ovarian cell lines (Brader et al. 1997). gene therapy. Within the modality of gene therapy itself, Further investigations into the interactions between radiation, multiple therapies may be combined in an attempt to benefit cellular and oncolytic virus gene products may clarify the from additive or synergistic efficacy. Multi-gene therapy mechanisms by which radiation might potentiate oncolytic approaches already under evaluation include the transduction gene therapy. of dual immunostimulatory molecules for immunotherapy Gene therapy may also be used for radiosensitization (Albertini et al. 1996), and dual suicide genes for enzyme/ strategies in which the activation of a prodrug, such as GCV prodrug strategies (Uckert et al. 1998). (Nishihara et al. 1997) or 5-FC (Hanna et al. 1997, Gabel et The use of radiation-inducible promoters has been pro- al. 1998), can function to sensitize cells to the effects of radi- posed as another means of achieving spatial localization with ation. While chemotherapy has thus far been ineffective for combined modality treatment (Advani et al. 1997). This type PCA, novel combinations of gene therapy and chemotherapy of promoter has been used in a construct directing expression may display synergistic improvements in efficacy. The com- bination of paclitaxel and gene therapy utilizing an Ad of -α in a spatially and temporally expressing p53 under the control of the CMV promoter restricted fashion (Hallahan & Weichselbaum 1998). revealed synergy for the combined modality treatment for a Improvements in conformal radiation treatment permit the wide variety of tumor cell lines in vitro and in xenografts precise delivery of anatomically restricted radiation doses, including the DU-145 PCA cell line (Nielsen et al. 1998). improving the potential anatomical precision of this Combinations of three modalities have also been demon- approach. Conceivably, single or multiple cytotoxic or radio- strated – radiotherapy, viral-cytopathic (E1B-deleted Ad) sensitizing genes could be delivered in a spatially and tem- and radiosensitizing double-suicide gene therapy – with porally restricted fashion with either therapeutic or regulatory marked enhancement in efficacy in vitro with DU-145 cells radiation doses. The therapeutic gain achievable by this (Freytag et al. 1998). approach has been limited by the ‘leakiness’ of the radiation- HSV-tk gene therapy may be effective in combination with responsive promoters. radiation therapy due to complementary mechanisms and dis- One combined modality approach particularly attractive tinct toxicity profiles. In a study (Chhikara et al. 2001) mouse in PCA is the combination of radiation and gene therapy. prostate tumors transplanted s.c. were treated by either gene There is strong evidence that synergy exists between these therapy involving intratumoral injection of Ad-tk followed by modalities. Recent work reveals greater than expected tumor systemic GCV or local radiation therapy or the combination of regression of U-87MG glioma cell xenografts following gene and radiation therapy. Both single-therapy modalities combined radiation and viral treatment with the HSV mutant showed a 38% decrease in tumor growth compared with con- R3616 (Advani et al. 1998). In irradiated xenografts, viral trols. The combined treatment resulted in a decrease of 61%. replication was increased 2- to 5-fold per cell. In unirradiated The combination led to an additional 50% reduction in lung tumors, virally infected cells were restricted to regions of the colonization. Primary tumors that received the combination xenograft immediately adjacent to the infecting needle track. therapy had a marked increase in CD4 T-cell infiltration. This In irradiated tumors on the other hand, virally infected cells is the first report showing a dramatic systemic effect following were more widely distributed throughout the xenograft and the local combination treatment of radiation and Ad-tk. A clini- away from the needle track than in unirradiated tumors. cal study using this strategy has been initiated and patient Increased doses of virus to unirradiated cells resulted in no accrual is ongoing (Chhikara et al. 2001). increase in viral spread from the vicinity of the needle track, supporting the idea that radiation was facilitating not only Translation of concepts to human gene viral replication but viral spread as well. Thus radiation may therapy clinical trials potentially be used to improve the efficacy of corrective/cyto- reductive gene therapy approaches by increasing the capacity At present there are 40 approved clinical gene therapy trials to transduce cells spatially distributed throughout a tumor. for PCA in progress in the USA (ORDA 010/2001) www.endocrinology.org Downloaded from Bioscientifica.com at 09/28/2021129 08:33:34AM via free access N J Mabjeesh et al.: Prostate cancer gene therapy

(HUMAN GENE THERAPY PROTOCOLS. http:// decline. In addition, an upregulation of cytokine expression, www4.od.nih.gov/oba/rdna.htm Updated 10–01–2001 edn augmented T-cell activation signals and augmented MUC- (National Institutes of Health (NIH) Office of Recombinant 1-targeted cytotoxicity were noted (Pantuck et al. 2000). DNA Activities 2001). Of these, 22 are for cytoreductive Integration of the following concepts: (i) PSA-based immunotherapy, 14 are for cytoreductive cytolytic/pro- targeting, (ii) virus-directed cytolytic therapy, (iii) stereo- apoptotic strategies, and four are corrective strategies tactically-guided vector administration for improved target (Table 4). tissue distribution, and (iv) use of replication-competent The preliminary results of only part of these trials have virus for improved tissue penetration, formed the basis only recently been published. The results of the phase I clini- for a recently initiated clinical trial for patients with a cal trial of irradiated GM-CSF-secreting autologous prostate post-radiation therapy local recurrence of PCA (Principal tumor vaccine therapy in eight patients (Principal Investi- Investigator J W Simons, Johns Hopkins University). gator J W Simons, Johns Hopkins University) indicated that A major limitation in the use of replication-deficient such treatment was safe and induced both B-cell and T-cell gene therapy in solid tumors in vivo is the diffusion-limited immune responses against PCA cell-associated antigens tissue penetration into the target tissue. The ability of viral (Simons et al. 1999). GM-CSF gene-transduced PCA vac- replication to increase tissue penetration has been observed cines increased antibody titers against prostate tumor cell in vitro and in vivo (Advani et al. 1998, Han et al. 1998). line-associated antigens. Increasing titers of antibodies to Replication-competent Ad spread in vivo may be facilitated PCA antigens were detected among three of the men treated not only through transcellular movement and vector ampli- with irradiated GM-CSF-secreting autologous PCA cell vac- fication, but also through greater tissue exposure resulting cines. This is the first report of induction of new antibody from virus-dependent cell lysis. Thus, a replication- responses to PCA antigens in patients using cytokine gene- competent Ad can be expected to achieve greater tissue modified tumor vaccines, peptide-pulsed DCs, or any other penetration relative to a replication-deficient Ad or other strategy of immunotherapy. non-lytic vector. CN706 (Fig. 2) is attenuated for repli- Phase II studies have commenced with GM-CSF gene- cation competence only in PSA-expressing cells, in which transduced allogeneic vaccines. Preliminary results of the it may be expected to amplify titer and achieve greater initial trial approved to use direct transrectal prostatic gene tissue penetration. In addition, the local immune response therapy injection were reported by Steiner & Gingrich against the Ad-infected cells may aid in killing of the (2000). A total of 21men with PCA in whom standard ther- target tumor cells. apy failed underwent ultrasound-guided injection of the RV The concept of dose uniformity for cytotoxic PCA therapy LXSN containing BRCA-1under the control of the viral pro- has been well developed in radiation therapy. Local underdos- moter LTR. No viral symptoms or evidence of viremia devel- ing is thought to increase the risk of treatment failure – a con- oped. Serum PSA in these cases of metastatic disease cept supported by the improved local control observed with remained unchanged. The study indicated the safety of PCA conformal radiation dose-escalation trials (Hanks et al. 1998, gene replacement therapy by direct injection. Zelefsky et al. 1998). It follows that similar principles may Herman et al. (1999) reported results of a phase I apply to the tissue distribution and dosing of viral cytolytics clinical trial of a replication-deficient Ad containing the such as CN706. By analogy to radiation dosimetry for prostate HSV-tk injected directly into the prostate, followed by i.v. brachytherapy, the tissue surrounding a seed implant is administration of the prodrug GCV. Only 1of 18patients exposed to varying concentrations of virus. The extent to at the highest dose level developed spontaneously revers- which a replication-competent virus spreads depends on initial ible grade 4 thrombocytopenia and grade 3 hepatotoxicity. viral titer, infectivity, lytic ability, and host immune response. Most of the patients at the highest dose levels achieved Empirical determinations of viral spread from tumor models objective response (fall in serum PSA levels by 50% or allows an estimation of the effective radius of virus activity. more). This can then be used iteratively in a computer 3D model to Of the clinical trials ongoing at the University of Cali- designate viral injection patterns for optimal tissue distribution fornia at Los Angeles preliminary results have been (Fig. 3). Existing brachytherapy treatment planning systems reported. In a phase II trial, intratumoral injection of a may be adapted for 3D viral vector spread modeling. Vali- plasmid coding for IL-2, formulated in a liposomal, cat- dation of predicted viral spread through biopsy analysis of Ad ionic lipid mixture vehicle, revealed decreases in serum hexon protein immunohistochemistry will permit increasing PSA levels at 2 weeks post-injection in 80% of the patients accuracy in viral spread predictions. Spatial evaluation of treated (Pantuck et al. 2000). IL-2 gene therapy was well oncolysis relative to viral spread will help define potentially tolerated, with no grade 3 or 4 toxicity occurring. In new parameters involved in oncolytic efficacy and augment another immunotherapy phase I trial in MUC-1-positive comparisons of alternative vectors. Clinical direct injection patients using vaccinia virus MUC-1-IL2 revealed an trials utilizing this precision in vector delivery benefit from the immunological response associated with a clinical PSA increased uniformity of gene delivery between patients.

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Table 4 PCA gene therapy: current clinical trials summary NIH No. Phase Principal Institution Vector Gene Modality investigator 9408-082 I–II Simons Johns Hopkins UniversityRV GM-CSF Ex vivo autologous PCA vaccine 9509-123 I Holt & Vanderbilt UniversitySchool RV Antisense myc In vivo intraprostatic Steiner of Medicine RNA injection 9509-126 I Chen National Naval Medical Vaccinia virus PSA cDNA In vivo i.d. injection Center 9510-132 I Lyerly& Duke UniversityMedical Cationic liposome IL-2 cDNA Ex vivo autologous Paulson Center complex PCA vaccine 9601-144 I Scardino Memorial Sloan-Kettering Ad serotype 5 HSV-TK cDNA In vivo intraprostatic Cancer Center injection 9609-160 I Eder & Kufe Dana-Farber Cancer Institute Vaccinia virus PSA cDNA In vivo i.d. injection 9702-176 I–II Sanda The Universityof Michigan Vaccinia virus PSA cDNA In vivo i.d. injection, autologous 9703-184 I Belldegrun Universityof California, Los Cationic liposome IL-2 cDNA Intratumoral direct Angeles complex/ injection DMRIE-DOPE 9705-187 I Hall & Woo Mount Sinai School of Ad serotype 5 HSV-TK cDNA Intratumoral direct Medicine injection 9706-192 I Belldegrun Universityof California, Los Ad serotype5 p53 cDNA Intratumoral direct Angeles injection 9708-205 I–II Simons Johns Hopkins UniversityRV GM-CSF s.c. injection 9710-217 I–II Logothetis The Universityof Texas MD Ad serotype5 p53 cDNA Intratumoral direct Anderson Cancer Center injection 9801-229 I Kadmon Baylor College of Medicine Ad serotype 5 HSV-TK cDNA Intratumoral direct injection 9802-236 I Simons Johns Hopkins UniversityAd serotype5/ Promoter and Intratumoral direct replication- enhancer injection competent virus elements of the PSA 9805-251 I–II Figlin Universityof California, Los Vaccinia virus MUC-1/IL-2 i.m. injection Angeles 9812-276 I Gardner & Universityof Virginia Health Ad serotype5 HSV-TK cDNA Intratumoral direct Chang Science System injection 9901-282 II Eder Dana-Farber Cancer Institute Vaccinia virus/ PSA i.m. injection fowlpox virus 9901-283 I–II Small Universityof California, San RV GM-CSF s.c. injection Francisco 9902-293 II Kaufman & Eastern Cooperative Vaccinia virus/ PSA i.m. or i.d. injection DiPaola OncologyGroup fowlpox virus 9904-306 I Vieweg Duke UniversityMedical RNA PSA i.v. injection Center 9905-312 II Belldegrun Universityof California, Los Cationic liposome IL-2 cDNA Intratumoral direct Angeles complex/ injection DMRIE-DOPE 9905-315 II Smith & Universityof California, San RV GM-CSF s.c. injection Small Francisco 9906-321 I Freytag & Henry Ford Health System Ad E. coli CD Intratumoral direct Kim cDNA/HSV-TK injection DNA 9906-324 I–II Aguilar- Baylor College of Medicine Ad HSV-TK cDNA Intratumoral direct Cordova injection & Butler 9909-338 I Gingrich Universityof Tennessee, Ad serotype5 p16 cDNA Intratumoral Coleman College of Medicine injection 9910-344 I–II Terris Stanford UniversityPalo Alto Ad serotype5/ Promoter and Intratumoral Veterans Administration replication- enhancer injection Medical Center competent virus elements of the PSA DMRIE, 1,2-Dimyristyloxpropyl-3-dimethyl-hydroyethyl ammonium bromide; DOPE, dioleoyl-phosphatidyl-ethanolamine. www.endocrinology.org Downloaded from Bioscientifica.com at 09/28/2021131 08:33:34AM via free access N J Mabjeesh et al.: Prostate cancer gene therapy

Table 4 cont. NIH No. Phase Principal Institution Vector Gene Modality investigator 9910-345 I–II Wilding Universityof Wisconsin Ad serotype5/ Promoter and i.v. injection Comprehensive Cancer replication- enhancer Center competent virus elements of the PSA 9910-352 I–II Belldegrun Universityof California, Los Cationic liposome IL-2 cDNA Intratumoral Angeles complex/ injection DMRIE-DOPE 9912-368 II Gulley& NCI Vaccinia virus/ PSA/B7.1 i.m. or i.d. injection Dahut fowlpox virus (CD80) 0001-373 II Arlen NCI Vaccinia virus/ PSA/B7.1 i.m. or i.d. injection fowlpox virus (CD80) 0008-410 I Vieweg Duke UniversityMedical RNA Total tumor i.v. injection Center RNA 0010-418 II Pollack The Universityof Texas MD Ad serotype5 p53 cDNA Percutaneous/ Anderson Cancer Center intraprostatic injection 0010-426 I Gardner Indiana UniversityMedical Ad serotype5 Osteocalcin Intratumoral Center promoter injection 0010-428 I Freytag & Henry Ford Health System Ad E. coli CD Intratumoral Kim cDNA/HSV-TK injection cDNA 0101-447 I Lubaroff Universityof Iowa Ad serotype5 PSA cDNA s.c. injection 0101-449 I Miles Baylor College of Medicine Ad serotype 5 IL-2 cDNA Intratumoral injection 0101-450 II DeWeese Johns Hopkins UniversityAd serotype5 Promoter and Intratumoral enhancer injection elements of PSA 0101-451 II Small Universityof California, San Ad serotype5 Promoter and i.v. injection Francisco enhancer elements of PSA 0103-459 I Dula West Coast Clinical Research AAV GM-CSF cDNA s.c. injection 0104-464 I Freytag & Henry Ford Health System Ad serotype 5 E. coli CD Intratumoral Kim cDNA/HSV-TK injection cDNA

Twenty men with locally recurrent PCA following radiation transduction efficiency and targeting will facilitate the next therapy were treated with CV706 between September 1998 generation of gene therapy strategies. The burgeoning field and May 2000 (Principal Investigator J W Simons, Johns Hop- of genomics provides an exciting new resource for the design kins University). CV706 was found to be safe and was not of prostate-specific gene therapy strategies. The design of associated with irreversible grade 3 or any grade 4 toxicity. oncolytic viruses should be aided by new insights into the Post-treatment prostatic biopsy and detection of a delayed mechanism of killing by lytic viruses. Already, the potential ‘peak’ of circulating virus provided evidence of intraprostatic of Ad genes such as E4orf4, E3–11k and E1A to function in replication of CV706. This study revealed that all patients virus-induced cell death has been observed (Tollefson et al. treated with the highest doses of CV706 achieved a 50% or 1996a, Nevels et al. 1997, Marcellus et al. 1998, Nemunaitis more reduction in serum PSA levels (DeWeese et al. 2001). et al. 2000, Li et al. 2001). Harnessing these viral gene prod- ucts and others for use as oncolytic drugs offers exciting Future directions prospects for a whole new class of cytoreductive gene ther- apy strategies. The challenges facing the implementation of successful gene As the diversity of molecular lesions underlying prostate therapeutic strategies will be better understood as the early tumorigenesis is better characterized, new targets for correc- clinical trials for PCA gene therapy begin to return more tive and cytoreductive approaches will emerge. Effective results. Vector development with increased transgene size anticancer gene therapy may ultimately require indi- capacity, optimized immunogenic properties, and improved vidualized molecular profiles. Manipulation of the cellular

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A. B.

C. D.

Figure 3 Injection of CN706 oncolytic Ad into the prostate under stereotactic guidance. (A) Intraoperative ultrasound prostate imaging with superimposed brachytherapy template. (B) Image (A) imported into brachytherapy treatment planning system (Radionics). Yellow and green circles indicate injection needle tracks, filled circles correspond to actual injection sites in the plane of the imaged section. Urethra and prostate outlines contoured in green and red respectively. (C) 3-D reconstruction of prostate volume and injection positions. Urethra imaged in red. (D) Predicted virus spread from injection sites in yellow. apoptotic machinery for cytoreductive PCA gene therapy in LNCaP cells but not in other cell lines of other origins (Hyer may permit development of more generalized antineoplastic et al. 2000). This may hold promise in the future. treatment, which bypasses upstream signal cascade lesions. Solid tumors meet their demands for nascent blood ves- Components of the apoptotic machinery such as the death sels and increased glycolysis, to combat hypoxia, by activat- receptors, caspases, and the bcl-XL/BH3 family may be ing multiple genes involved in angiogenesis and glucose utilized in strategies which result in efficient oncolysis even metabolism. Hypoxia inducible factor-1(HIF-1)is a consti- in the presence of diverse upstream lesions. Cell death path- tutively expressed basic helix-loop-helix transcription factor, ways involving the death receptors such as CD95, DR3, 4 formed by the assembly of HIF-1alpha and HIF-1beta (Arnt), and 5, may provide potent therapeutic targets. A recent pre- that is stabilized in response to hypoxia, and rapidly degraded clinical study shows that Ad- or RV-transduced under normoxic conditions. It activates the transcription of (FasL) or FADD can efficiently induce apoptosis in human genes important for maintaining oxygen homeostasis glioma cells (Shinoura et al. 1998). (Dachs & Tozer 2000, Semenza et al. 2000). Sun et al. Dong et al. (1995) showed that intratumoral delivery of (2001) recently demonstrated that engineered down- FasL using an Ad vector could force PCA cells into apoptosis. regulation of HIF-1alpha by intratumoral gene transfer of an They placed the tetracycline transactivator gene under the con- antisense HIF-1alpha plasmid leads to the down-regulation trol of a prostate-specific ARR2PB promoter, and a pro- of vascular endothelial growth factor (VEGF), and decreased apoptotic FasL-GFP gene under the control of the tetracycline tumor microvessel density. Antisense HIF-1alpha mono- responsive element. The latter expression cassette was inserted therapy resulted in the complete and permanent rejection of into Ad 5. High levels of expression were exclusively observed small EL-4 tumors. It induced NK-dependent rejection

www.endocrinology.org Downloaded from Bioscientifica.com at 09/28/2021133 08:33:34AM via free access N J Mabjeesh et al.: Prostate cancer gene therapy of tumors, but failed to stimulate systemic T-cell-mediated Banchereau J & Steinman RM 1998 Dendritic cells and the control antitumor immunity, and synergized with B7-1-mediated of immunity. Nature 392 245–252. immunotherapy (Sun et al. 2001). This approach holds prom- Baskar S, Ostrand-Rosenberg S, Nabavi N, Nadler LM, Freeman GJ & Glimcher LH 1993 Constitutive expression of B7 restores ise to form the foundation for the transition between the tra- immunogenicity of tumor cells expressing truncated major ditional anticancer therapies of the past four decades and the histocompatibility complex class II molecules. PNAS 90 5687– molecular antineoplastic pharmacology of the future. 5690. 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