Adenovirus-Derived Vectors for Prostate Cancer Gene Therapy

Abstract

Prostate cancer is a leading cause of death among men in Western countries. Whereas the survival rate approaches 100% for patients with localized cancer, the results of treatment in patients with metastasized prostate cancer at diagnosis are much less successful. The patients are usually presented with a variety of treatment options, but therapeutic interventions in prostate cancer are associated with frequent adverse side effects. Gene therapy and oncolytic virus therapy may constitute new strategies. Already a wide variety of preclinical studies has demonstrated the therapeutic potential of such approaches, with oncolytic prostate-specific adenoviruses as the most prominent vector. The state of the art and future prospects of gene therapy in prostate cancer are reviewed, with a focus on adenoviral vectors. We summarize advances in adenovirus technology for prostate cancer treatment and highlight areas where further developments are necessary.

Introduction

A lthough many viruses are being evaluated as oncolytic agents, human adenoviruses (HAdVs) are among the most popular to be developed. There are several good reasons for making HAdV vectors such a popular choice. Recombinant HAdV vectors have a good safety profile as a gene therapy vector, can be produced under GMP conditions, and various commercially operating manufacturing facilities are available, allowing research groups access to batches of HAdV vectors that meet the required quality standards. In addition, the wild-type HAdVs from which vectors are derived are only mildly pathogenic (Van der Vliet and Hoeben, 2006). Although HAdV genomes are stable enough to prevent the rapid development of heterogeneous populations of so-called quasispecies, they are easily amendable by genetic modification. Genetically modified HAdVs with altered host range (“targeted viruses”) have been generated by engineering new polypeptide ligands in the capsids of the particles, yielding viruses that preferentially infect specific cell or tissue types. In parallel, by engineering mutations at known receptor-binding sites in the capsid, “detargeted” HAdV vectors have been generated to reduce transduction of nontarget tissues. Also, HAdVs can be modified to carry therapeutic or reporter transgenes (Bachtarzi et al., 2008). Numerous and diverse transgenes have been inserted in E1-deleted HAdVs to be exploited as cytolytic agents. Such therapeutic transgenes include genes encoding prodrug-activating enzymes (e.g., herpes simplex virus thymidine kinase for activating ganciclovir, cytosine deaminase for activating 5-fluorocytosine, bacterial nitroreductase activating CB1954), genes encoding immune-stimulatory cytokines (interleukin [IL]-2, IL-12, granulocyte-macrophage colony-stimulating factor [GM-CSF], and IL-24), or genes encoding proteins inducing apoptosis (e.g., p53). More recently, conditionally replicating adenoviruses (CRAds) have been developed. Such viruses rely on the lytic replication cycle of HAdVs for tumor cell killing. Viral replication in the tumor increases the local vector concentration and may lead to spread of the virus within the tumor. In addition, HAdV may provide a danger signal that stimulates an antitumor immune response (Geutskens et al., 2000). Successful preclinical studies have led to various phase I clinical trials of replication-defective as well as conditionally replication-competent HAdV vectors in prostate cancer (Figueiredo et al., 2007; Freytag et al., 2007). So far these studies have confirmed the good safety profile of HAdV vectors. Taken together, these factors have made adenoviral vectors a prime candidate for developing viral oncolytic agents.

Getting the Virus to the Tumor

Intratumoral injection

An important lesson learned from preclinical and clinical research in cancer gene therapy is that efficient transduction of the cancer cells in the tumor is essential for efficacious treatment. Tumors are heterogeneous and contain a stromal compartment and extracellular matrix components that form physical barriers within the tumor (Fig. 1). Therefore even direct intratumoral administration of viral anticancer agents is often disappointingly inefficient. Expression of the receptor used by most HAdVs to enter the cell, that is, the coxsackievirus and adenovirus receptor (CAR), is often scanty. The high interstitial fluid pressure within most tumors causes a convective flow from the tumor. This inhibits the passive diffusion of viral particles into the tumor. The extracellular matrix may form physical barriers that prevent efficient spread of viral vectors (Kuppen et al., 2001; Li et al., 2004; Mok et al., 2009). In elegant studies, Jain and co-workers demonstrated that destruction of the matrix by collagenase treatment or overexpression of matrix metalloproteinases (MMP)-1 and -8 increases the volume distribution of oncolytic herpesviruses (McKee et al., 2006; Mok et al., 2007). The volume distribution can also be increased by multiple injections, or by convection-enhanced delivery procedures.

FIG. 1.

Schematic representation of the tumor structure and the hurdles to efficient adenoviral vector-mediated prostate cancer gene therapy. Several efficacy-lowering aspects are encountered on systemic or intratumoral delivery of adenoviral vectors to prostate cancer. Systemic delivery of vector particles to the primary tumor site and to metastasized tumor cells is hampered by innate and humoral immunity, sequestration from the blood stream through the binding of blood cells or plasma proteins, and limited permeability of blood vessels. The heterogeneous composition of the primary tumor mass results in inefficient penetration into the tumor and insufficient transduction of the neoplastic cells.

Vascular delivery

Theoretically, vascular delivery of vectors may lead to a larger distribution of viruses within the tumor. Also, it could provide an option for transducing (micro)metastatic tumors. However, vascular delivery, too, has been frustratingly inefficient so far. This is attributable to a wide variety of factors. Direct contact between malignant cells and the oncolytic HAdV may be difficult to obtain. Often blood vessels are confined to the tumor stroma, and therefore several layers of stromal cells must be passed before malignant cells are reached by vascularly applied therapeutic agents (Kuppen et al., 2001; Li et al., 2004).

HAdV vectors may become unavailable to the tumor by promiscuous association with nontarget tissues such as the liver. The primary receptor of many HAdVs is the CAR. After ligation of the adenoviral fiber with the CAR an integrin-binding RGD motif in the penton base binds α_vβ₃- or α_vβ₅-integrins. This promotes adenovirus internalization. Both CAR and the integrins are widely expressed on cells in the human body, resulting in transduction of nontarget tissues (Arnberg, 2009). Intriguingly, mutation of the CAR-binding site of the fiber and the RGD motif in the penton base was found not to reduce liver transduction, and on intravascular administration adenoviruses were still efficiently sequestered by Kupffer cells in the liver (Di Paolo et al., 2009). Subsequently it became evident that the interaction of the virus with host blood cells and plasma proteins is critical. Studies suggest that these interactions dictate the particle biodistribution of adenovirus in vivo. Various plasma proteins and in particular vitamin K-dependent coagulation factors IX and X can bind to hexon proteins in the capsid, and bridge the virus to receptors in the liver (Kalyuzhniy et al., 2008; Waddington et al., 2008). In this respect there is variability between human adenoviral serotypes. Whereas the serotypes commonly used as vector, that is, HAdV-5 and -2, strongly bind factor X, others such as serotype 26 and 46 do not (Waddington et al., 2007; Alba et al., 2009). Therefore (hexons of) different serotypes, or non-clotting factor-binding derivatives of the HAdV-5 hexon, can be used to decrease the loss of vector particles in the liver and to improve bioavailability to the tumors. Indeed, such mutations significantly decrease liver transduction of HAdV-5 vectors in mice (Waddington et al., 2007; Alba et al., 2009). The HAdV-5 fiber harbors a site with high affinity for heparan sulfate proteoglycans; however, mutation of the binding motif KKTK barely affects liver transduction in mice (Kritz et al., 2007; Di Paolo et al., 2009).

Not only these interactions with clotting factors thwart efficient tumor cell transduction, but so do neutralizing immunoglobulins. A majority of humans have preexisting humoral neutralizing activity against HAdV-5 and HAdV-2 as a result of prior exposure to these viruses. Intravascular administration of adenovirus to recipients with preexisting humoral immunity will strongly reduce gene transfer. The use of vectors derived from HAdV with a low seroprevalence in the general population, or from nonhuman adenoviruses, may reduce the magnitude of the problem (Abbink et al., 2007).

An unexpected finding came with the observation that human, in contrast to murine, erythrocytes bind HAdV-5. Human erythrocytes present CAR at their surface, which stably interacts with HAdV-5 particles (Carlisle et al., 2009; Seiradake et al., 2009). Also human, but not murine, erythrocytes present complement receptor-1 (CR1), which binds HAdV-5 in the presence of antibodies and complement (Carlisle et al., 2009). Transplantation of human erythrocytes into immune deficient mice extended the blood circulation time of HAdV-5, reduced liver transduction, and decreased extravasation of the virus into human xenograft tumors. Similarly, HAdV-5 showed extended circulation and decreased liver transduction in transgenic mice presenting either CAR or CR1 on their erythrocytes. Erythrocytes may therefore restrict HAdV-5 infection in humans, independent of antibody status, presenting another challenge to HAdV-5-based anticancer viruses (Carlisle et al., 2009; Seiradake et al., 2009). Although much insight has been acquired on the interaction of adenoviruses with blood cells and plasma proteins, many other areas of virus–host interactions remain underexplored and in general we understand little of it.

Shielding Vector Particles from Neutralizing Immunity

Although formidable, the challenges of vascular delivery summarized previously may not be insurmountable. One approach potentially leading to improvements in delivery of adenoviral vectors for cancer gene therapy involves chemical coating of the vector particles. This approach is based on established and clinically applicable technology of packaging drugs or therapeutic biologicals in synthetic polymers. It is hoped that this can lead to improved pharmacological parameters, such as improved solubility and stability, reduced dosing frequency, potentially reduced toxicity, and extended circulation time. Amongst others, poly[N-(2-hydroxypropyl)-methacrylamide] and polyethylene glycol (PEG) are often used to covalently coat therapeutics (Kreppel and Kochanek, 2008).

Coating with multivalent polymers based on poly[N-(2-hydroxypropyl)-methacrylamide] abrogated normal HAdV-5 infectious tropism (Fisher et al., 2001). In addition, neutralization by antibodies was decreased up to 50-fold, and the resulting polymer-coated adenoviral particles have a greatly extended plasma circulation time in mice (Fisher et al., 2001; Green et al., 2004). Although normal tropism was blocked, polymer-coated adenovirus accumulated within a solid subcutaneous tumor 40 times more efficiently than unmodified virus, and mediated higher levels of transgene expression within tumors. This has been attributed to the enhanced permeability and retention effect, which leads to the nonspecific accumulation of circulating macromolecules within tumors (Matsumura and Maeda, 1986).

After blocking, infectivity can be restored by linking targeting peptides onto the surface of the polymer-coated viruses. Addition of a synthetic –SIKVAV– peptide, which binds α₆-integrin, can restore viral infectivity of PC-3 cells. Competition assays confirmed that entry of retargeted viruses was mediated via the incorporated ligand. Intravenous administration of retargeted viruses to tumor-bearing mice resulted in slower plasma clearance and greatly reduced liver tropism, and hence toxicity compared with unmodified virus, while maintaining reporter gene expression in the tumor (Stevenson et al., 2007). Similarly, polymer-coated HAdV could be targeted with cetuximab to target the epidermal growth factor (EGF) receptor (Morrison et al., 2009). The data demonstrate that the polymer-coating technology is compatible with peptide-based tumor targeting.

Other groups used coating of adenoviral particles with polyethylene glycol (PEG) molecules for particle shielding, and showed that this allows escape from neutralizing antibodies and to some extend allowed vector readministration. In this respect the size of the PEG molecules matters. With the use of large PEG molecules (e.g., 20-kDa PEG), vector particles were detargeted from muscle after local delivery and from liver after systemic delivery in mouse models. Surprisingly, fully detargeted PEGylated adenoviral vectors still induced strong cellular and humoral immune responses to vector-encoded transgene products. PEGylation does not affect the kinetics of transgene product-specific cytotoxic immune responses (Wortmann et al., 2008). These data have been corroborated by Barry and collaborators, who demonstrated that PEGylation with 20-kDa PEG was as efficient at detargeting adenovirus from Kupffer cells and hepatocytes as virus predosing and warfarinization (Hofherr et al., 2007, 2008; Weaver and Barry, 2008; Doronin et al., 2009). Bioluminescence imaging of viral distribution in a xenograft model in nude mice demonstrated that PEGylation with 20-kDa PEG reduced liver infection 19- to 90-fold. Tumor transduction levels were similar for 20-kDa PEGylated and un-PEGylated vectors. Anticancer efficacy after a single intravenous injection was retained at the level of unmodified vector in large established LNCaP prostate carcinoma xenografts, resulting in complete elimination of tumors in all animals and long-term tumor-free survival (Doronin et al., 2009). It should be noted that the protective effects of PEGylation could be more pronounced in the presence of human erythrocytes, as PEGylation will also reduce association with CAR and complement receptor I. Taken together, these data suggest that chemical shielding of HAdV particles is a powerful approach to prevent interaction of HAdV particles with blood proteins, erythrocytes, and nontarget tissues, and thereby may increase the bioavailability, and as a result the uptake of viruses into tumors, by enhanced permeability and retention.

Vascular Permeability

To further stimulate extravasation of vector particles, new physiological regulators of vascular permeability (i.e., vascular endothelial growth factor [VEGF]) may be used. This strategy allows enhanced transduction of striated muscle by combining intravenous AAV6-vector administration with infusion of VEGF (Gregorevic et al., 2004). Alternatively, strategies are being developed that employ the endothelial receptor-mediated transcytosis pathway. The transferrin receptor is an example of a receptor that binds transferrin and its associated iron, resulting in caveolar uptake of the complex. Via a series of vesicles these complexes are transported across the endothelium and released at the basolateral side. Curiel and co-workers provided evidence that this pathway can be used by adenoviruses. By using a bifunctional adaptor, for example, a soluble CAR–transferrin fusion protein, the particles could be taken up by Caco-2 cells and transported across a polarized monolayer (Zhu et al., 2004). These data suggest that adenoviruses can be redirected to the transcytosis pathway, although it remains to be established how efficiently this route can be recruited in the tumor endothelium.

Targeting Adenoviruses

Many strategies have been pursued to improve gene transfer into CAR-negative cells. In addition to the use of non-CAR-binding serotypes and fiber-swap vectors (Murakami et al., 2009; Sandberg et al., 2009), recombinant HAdV vectors with altered tropism have also been generated by engineering new ligands for cellular receptors into surface loops of capsid components. The favorite locations have been the C terminus and the HI loop of the knob domain of the fiber, the RGD loop of the penton base protein, and the L1 loop of the hexon. Although effective, the applicability of this approach was initially limited by the restricted tolerance for inserting new ligands at these positions (Arnberg, 2009). In addition, new ligands that are to be incorporated genetically into adenoviral vectors must be able to fold correctly in the reducing environment of the mammalian cell cytoplasm. This excludes most ligands dependent on disulfide bond formation for proper folding such as epidermal growth factor and most single-chain variable fragments (Lindholm et al., 2008). Promising candidate ligands are Affibody^® molecules (Affibody AB, Bromma, Sweden), which are affinity proteins based on a 58-amino acid three-helix bundle structure, termed “Z,” that is derived from the immunoglobulin-binding domain of staphylococcal protein A. Display libraries have been constructed on the basis of randomization of 13 surface accessible amino acids in the Z domain, from which novel Affibody molecules to desired targets have been selected. These Affibodies can be efficiently used for genetic retargeting of adenovirus (Henning et al., 2002; Lindholm et al., 2008). Although initial experiments were thwarted by structural constraints, Lindholm and collaborators managed to insert two Affibodies in tandem in the HI loop, by connecting them via small flexible linkers. This resulted in HAdV-5 vectors genetically retargeted with a HER2/neu-specific Affibody molecule inserted in the HI loop of the fiber knob of a CAR binding-ablated fiber (Magnusson et al., 2007). With this technology, vectors can be generated by incorporating two Affibody molecules with different specificities (Myhre et al., 2009). Camelid and human single-domain antibody fragments may be applicable in a similar manner (Harmsen and De Haard, 2007).

In another approach, the knob and shaft of the fiber have been replaced by an artificial trimerization domain, which was linked to an heterologous receptor-binding ligand (Krasnykh et al., 2001; Magnusson et al., 2001; Schagen et al., 2008). Via this strategy Willemsen and co-workers managed to retarget HAdV-5 to tumor cells by replacing the shaft and knob of HAdV-5 by a single-chain T cell receptor specific for HLA-A1 molecules that present a MAGE-A1 peptide (Sebestyen et al., 2007). Similar single-chain T cell receptors have been fused to the C terminus of the minor capsid protein IX (de Vrij et al., 2008). Immunoaffinity studies suggested that the C termini of protein IX molecules are positioned near the capsid surface (Akalu et al., 1999; Vellinga et al., 2005b). This has been confirmed by cryoelectron microscopy studies, which suggest that the C termini are located near the peripentonal hexons. Here the leucine zipper domain in the C-terminal part of protein IX interacts with the zipper of other molecules, forming a coiled coil (Scheres et al., 2005; Saban et al., 2006; Fabry et al., 2009). These protein IX–protein IX associations are not necessary for pIX–capsid incorporation and thermostability of the particles (Vellinga et al., 2005a).

If ligands are to be fused with pIX, a spacer may expose the ligands above the outer surface of hexon capsomers to ensure its accessibility to cellular receptors. Vellinga and co-workers have demonstrated that linkers up to a length of 75 Å can be added to the C terminus of protein IX without affecting the incorporation of protein IX into the capsid (Vellinga et al., 2004). Indeed, a wide variety of targeting polypeptides could be functionally incorporated by genetic fusion at the C terminus of protein IX. In this way small targeting peptides, a hyperstable single-chain Fv, and a single-chain T cell receptor could be functionally incorporated into the capsid (Vellinga et al., 2004, 2006, 2007; de Vrij et al., 2008). In addition, other functional proteins were incorporated in the adenoviral capsid through linkage to pIX, such as fluorescent proteins that allow particle tracing by fluorescence microscopy (Le et al., 2004; Meulenbroek et al., 2004).

An elegant combination of genetic modification and chemical modification has been developed by Kreppel and co-workers. HAdV-5 vectors were genetically modified to contain cysteines at solvent-exposed positions in the capsid (Kreppel et al., 2005). The introduced thiol groups are highly reactive, and procedures were established for their controlled covalent coupling to protein and nonprotein ligands. Depending on the chemistry used, ligands could be coupled by formation of thioether or disulfide bonds. The latter method yields viruses that release the coupled ligand in the endosome. In addition, thiol groups in the fiber knob were still accessible after amino PEGylation, allowing PEG shielding to be combined with targeting by ligand coupling to the thiol groups (Kreppel et al., 2005). Coupling of transferrin to engineered cysteine residues at the C terminus of protein IX allowed targeting of HAdV particles in mice in vivo (Corjon et al., 2008). This validates the applicability of the technique in procedures involving intravenous administrations of adenoviral vectors.

Taken together, these data show that HAdV vector technology has matured and constitutes a functional and robust platform for generating shielded, retargeted vectors that can be used for developing oncolytic HAdV vectors for cancer gene therapy.

Targetable Receptors in Prostate Cancer

With the targeting platform in place, a key question concerns which receptors could be targeted in prostate cancer. Extensive target exploration has yielded several cell surface receptors that are expressed preferentially or specifically in prostate cancer cells.

A prime candidate is prostate-specific membrane antigen (PSMA). It is expressed both on benign and malignant prostate cells. It is a type 2 membrane receptor, which can be efficiently internalized. The ligands that trigger internalization remain to be identified (Wang et al., 2007). Nearly all prostate tumors and prostate cancer cells express PSMA and increased expression correlates with aggressive tumors (Tasch et al., 2001). PSMA is also upregulated after androgen deprivation in model systems whereas other markers such as prostate-specific antigen (PSA) are decreased after androgen withdrawal (Israeli et al., 1994). PSMA can serve as a tissue-specific target for adenoviral vectors (Kraaij et al., 2005). Retargeting of viral particles to prostate cancer cell lines was obtained through the attachment of bispecific molecules, which consisted of conjugates between an anti-adenoviral fiber knob Fab′ fragment and anti-PSMA monoclonal antibodies (Kraaij et al., 2005).

Besides PSMA, various other cell surface molecules have been demonstrated to be upregulated in prostate cancer, such as prostate stem cell antigen (PSCA). Successful targeting to PSCA-expressing prostate cancer cells has been achieved for genetically engineered T cells that have been equipped with a chimeric T cell receptor recognizing PSCA (Morgenroth et al., 2007). These findings support the exploration of PSCA targeting in the context of prostate cancer-targeted oncolytic viruses.

The urokinase-type plasminogen activator receptor (uPAR) has also been exploited for targeting of oncolytic viruses to prostate cancer cells. uPAR is overexpressed in tumors as well as in stromal cells of multiple malignancies, including prostate cancer (Romer et al., 2004; Li and Cozzi, 2007). uPAR is involved in tumor angiogenesis. For example, tumor cell-conditioned media can upregulate endothelial uPAR expression (Seghezzi et al., 1996). The principle of targeting tumor endothelium by aiming at uPAR, rather than at the cancer cells themselves, is highly attractive, because such an approach may lead to improved viral trafficking from the bloodstream into the tumor tissue. HAdV targeting to uPAR is feasible (Drapkin et al., 2000). More recently, tumor and vascular targeting of an oncolytic measles virus has been achieved (Jing et al., 2009).

In addition, other cell surface molecules are upregulated in the tumor vasculature, including members of the vascular endothelial growth factor receptor (VEGFR) family. Both VEGFR1 (Flt-1) and VEGFR2 (Flk-1) are selectively expressed on endothelial cells and are highly upregulated in proliferating (angiogenic) capillary cells of numerous types of prostate tumor (Kollermann and Helpap, 2001). The potential of VEGFR2 as a target has been shown in studies on systemic targeting of drug-loaded microspheres to subcutaneous prostate tumors in mice, which demonstrated significant inhibition of tumor growth after conjugation of anti-VEGFR2 antibodies to the microspheres (Lu et al., 2008).

One class of potentially specific receptors is the group of tumor-specific cancer testis (CT) antigens (Costa et al., 2007). Peptides of these CT antigens are presented on the cell surface in complex with major histocompatibility class (MHC) I molecules. With the exception of their expression in the testis, an immune-privileged site due to the absence of MHC expression, the CT antigens are expressed exclusively in cancer cells. Proof-of-principle of targeting adenoviral vectors to CT antigens has been shown by genetically fusing viral capsid proteins with single-chain T cell receptors, which could specifically recognize the melanoma-specific CT antigen MAGE-A1 in complex with HLA-A1 (Sebestyen et al., 2007; de Vrij et al., 2008). Multiple cancer CT antigens have been found in patients with prostate cancer, including SSX-2 and MAD-CT-1 and -2 (Hoeppner et al., 2006; Dubovsky and McNeel, 2007). A peptide present in the majority of MAGE-A gene family members could serve as an ideal target as most tumors, both solid and blood-borne, express at least one member of this MAGE-A gene family (Scanlan et al., 2002). In addition to cell surface antigens, intracellular antigens, for example, PSA, have now become available for targeting and warrant further exploration as targets for gene therapy vectors (Hoeppner et al., 2006; Dubovsky and McNeel, 2007). Combining the selective power of phage display, which allows for the testing of tens of billions of individual clones, with high-throughput selection of Fabs with peptide–MHC complex-binding capacity will yield new human “T cell receptor (TCR)-like” Fab fragments that specifically target viruses to tumor cells expressing intracellular tumor antigens (Willemsen et al., 2008).

Prostate-Targeted Conditionally Replicative Adenoviruses

HAdVs have been generated that replicate specifically in certain cell types. In HAdV infection, the E1A gene acts as the master switch that activates the viral gene expression cascade. Therefore, by controlling E1A expression with a tumor- or tissue-specific promoter, viral replication can be restricted to certain cell types. Along these lines, Essand and collaborators developed a series of prostate-specific adenoviruses that replicate exclusively in normal and neoplastic prostate epithelial cells (Cheng et al., 2006; Dzojic et al., 2007; Danielsson et al., 2008). In these vectors expression of E1A is controlled by the recombinant prostate-specific PPT sequence. The PPT sequence is composed of a prostate-specific antigen (PSA) enhancer, a prostate-specific membrane antigen (PSMA) enhancer, and a T cell receptor γ-chain alternate reading frame protein (TARP) promoter. The PSMA enhancer, which upregulates PSMA expression in androgen-depleted prostate cancer cells, also ensures that the PPT sequence is active under androgen-deprived conditions. The mouse H19 insulator (I), with enhancer-blocking activity, was placed upstream of PPT to protect it from interfering signals from the adenoviral backbone (Cheng et al., 2006; Dzojic et al., 2007). The most advanced version, the so-called Ad[i/PPT-E1A, E3] virus, induced regression of aggressively growing LNCaP tumors, and yielded significantly prolonged survival for treated mice compared with the control groups (Danielsson et al., 2008).

Not only can E1 regulation be used to restrict replication to cancer cells, but the strategy has also been used to prevent expression of E1A, and thereby expression of other viral genes, in sensitive nontarget tissues. HAdV5 vectors can mediate significant hepatotoxicity. To prevent viral gene expression in hepatocytes, multiple binding sites for a hepatocyte-specific microRNA, miR-122, were placed in the 3′ untranslated region of the E1A gene (Ylosmaki et al., 2008; Cawood et al., 2009). miR-122 is highly and selectively expressed in hepatocytes and this modification might prevent expression of E1A within hepatocytes, and hepatotoxicity, while maintaining its replicative capacity in tumor cells. Animals receiving a lethal dose of wild-type Ad5 (5 × 10¹⁰ viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, whereas inclusion of miR-122 binding sites decreased replication 50-fold and virtually abrogated liver toxicity, demonstrating the efficiency of the approach (Cawood et al., 2009). These examples demonstrate that we can endow replicating HAdV vectors with tumor cell selectivity, while protecting sensitive nontarget tissues.

New Directions

A robust technology platform for cancer-targeted and oncolytic HAdV vector generation has been established. Many elegant tools and techniques have been developed with well-chosen experiments to show their proof-of-concept. However, it seems fair to state that most improvements have been incremental and only a few of the vectors that showed promise in preclinical studies have reached the stage of clinical evaluation. So, where do we go from here? In what fields are new developments necessary to fulfill the promise of efficacious prostate cancer-targeted HAdV vectors? It may be useful to step back, reflect, and place our activities in perspective.

Rational design or evolution of new oncolytic viruses?

So far, most vectorologists have followed a “rational design” or reverse-genetics approach for building new therapeutic vectors. In other words, on the basis of a priori knowledge of virus biology, tumor cell biology, and pharmaceutical parameters, we have built our new vectors to have the desired phenotype and to perform as anticipated. This approach has been most useful and has delivered most of the vectors that are in use in clinical gene therapy to date. Nevertheless, one should realize that this is not the classical approach in microbiology.

Classical virology studies viruses by employing selection strategies to isolate mutants with desired phenotypes and to study these to obtain insight into viral genetics and virus biology. There has been a revival of interest in the classical, more evolutionary approaches involving bioselection strategies for developing improved HAdV oncolytic vectors. Yan and co-workers have provided a fine example of the power of this approach (Yan et al., 2003). In vitro chemical mutagenesis of HAdV-5 was combined with a bioselection strategy. This yielded a mutant virus that replicated more efficient in the HT29 colorectal tumor cells that were used for selection. The mutation truncates an open reading frame in the late i-leader transcript. Not only in HT29, but also in several other tumor cell lines, replication was enhanced by the causative i-leader mutation at nucleotide 8350 of the HAdV-5 genome (Yan et al., 2003). Along similar lines, Gros and co-workers used in vivo bioselection and obtained an E3/19K mutant with enhanced antitumoral potency (Gros et al., 2008). By selecting for HAdVs with large-plaque phenotypes, Subramanian and co-workers isolated a series of mutants, including mutants in the i-leader and in the E3/19K gene (Subramanian et al., 2006). Taken together, these data suggest that the oncolytic activity of wild-type HAdV-5 can be enhanced by mutations.

Hermiston and collaborators took this approach a step further (Kuhn et al., 2008). On the basis of the notion that there is no evidence that HAdV-5 is the optimal start point for selecting more potent oncolytic HAdVs, they pooled an array of HAdV serotypes. These pools were passaged under conditions that invite recombination between serotypes. They isolated a mutant, designated coloAd1, which replicates more efficiently than any wild-type virus in human colon cancer cells. Characterization of coloAd1 revealed that it is a complex hybrid between HAdV-3 and -11p (Kuhn et al., 2008). It is evident that none of the bioselected viruses would have been created on the basis of preexisting knowledge. In fact, we have no clear understanding why these mutants replicate so much better in the cell systems that were used for their isolation. This underscores the potential of the classical strategy. It is to be expected that evolutionary approaches involving bioselections will become more widely applied. These approaches will yield new viruses, and the characterization of these viruses may provide new insights concerning critical aspects of virus and tumor cell biology. Uil and co-workers presented an approach that employs modified adenoviral polymerases with deficits in the polymerase proofreading function. This strategy will facilitate the isolation of more complex HAdV mutants for cancer gene therapy (Uil et al., 2009).

Taken together, it seems reasonable to anticipate that adenoviral mutations that have been isolated by bioselection procedures will soon be incorporated in clinically applicable oncolytic adenoviruses.

Cellular delivery of viruses

Many studies have demonstrated that the delivery of vector particles into tumors is inefficient. Both intratumoral and vascular delivery are thwarted by a variety of factors (see above), and therefore new delivery methods are essential. An attractive option is to use cells with the capacity to migrate to tumors as delivery vehicles for oncolytic viruses. In this strategy the tumor-targeting cells are loaded with viruses and administered to the patient. After migration the cells should hand off the cell-associated viruses or, if the virus replicates in the delivery cells, their progeny. In this way viruses should be delivered to tumor cells. Several cell types can migrate to tumors in vivo, including cytokine-induced killer cells, tumor antigen-specific T cells, macrophages, endothelial progenitor cells, and mesenchymal stem cells (Power and Bell, 2008). In addition, virus-loaded dendritic cells have been used to eradicate tumor cells from tumor-draining lymph nodes (Ilett et al., 2009).

Cellular delivery of viral anticancer agents may also circumvent the effects of neutralizing immunity (Power et al., 2007). This is evidenced in a study employing human reoviruses as the oncolytic agent. In reovirus-immune mice with B16tk lymph node melanoma metastases, in vivo delivery of free reovirus to the melanoma was ineffective, whereas effective antitumor responses and long-term tumor clearance were obtained if mature dendritic cells as well as T cells were used as carriers (Ilett et al., 2009). This and other studies suggest that cellular delivery is feasible and may be applicable for the delivery of viral oncolytic agents at tumor sites. It should be noted that cellular delivery adds a new level of complexity to clinical gene therapy studies and is logistically challenging.

Tumor stem cells as new targets?

With the aid of such new technologies, that is, the bioselection-based strategies for vector improvement, and cell-based methods for vector delivery, new cancer gene therapeutics and therapeutic strategies may be developed that ensure efficient vector delivery into the tumors. It is known that tumors are usually markedly heterogeneous, consisting of complex mixtures of cancer cells with various grades of differentiation. A cell type that has attracted much attention is the tumor-initiating cell, or cancer stem cell. The tumor-initiating cell is a cell with the capacity to self-renew and differentiate into any of the lineages of cancer cells that comprise a tumor (Lobo et al., 2007). Presumably the tumor-initiating cells are derived from organ stem cells (Collins and Maitland, 2009). The latter are long-lived and change over the course of time by accumulating (epi)genetic alterations. In this model, the characteristics of a cancer-initiating cell, and therefore of the resulting tumor, depend (in part) on these alterations. The cancer stem cell population in a tumor may govern crucial tumor processes such as progression, invasion, and metastasis. Therapy resistance may be the consequence if a particular treatment does not effectively eradicate the cancer stem cell population. It may therefore be important to ensure that new oncolytic agents have the capacity to transduce and kill cancer stem cells.

Nonviral delivery of viral genomes

Systemic approaches for cancer gene therapy have been focused on delivering viruses to the tumor. With advances in the field of nonviral gene delivery, an alternative approach became feasible (Carlisle et al., 2006). Rather than delivering intact viruses to the tumor cells, a strategy is followed in which viral genomes are delivered. If transferred into cells, HAdV DNA can yield replicating virus. Although not particularly efficient, infectious HAdV can be reproducibly recovered from cell cultures on transfer of naked DNA.

The field of nonviral gene transfer has seen impressive advances in increased tumor accumulation of transgenes (Wolff and Rozema, 2008), but so far the therapeutic consequences remain to be improved. Individual steps in gene delivery need further improvement, especially those relating to intracellular trafficking of the complexes. This includes the timely release of the DNA complex from the targeting ligand, efficient escape from the endosomal compartment, and proper delivery of the genes into the nucleus (Ogris et al., 2007; Russ et al., 2008; Schwerdt et al., 2008). Furthermore, innate immunity must be evaded. Integration of controlled-release technologies into targeted gene delivery systems will provide more effective gene delivery systems (Meyer and Wagner, 2006; Philipp et al., 2008; Schaffert and Wagner, 2008).

With the delivery of viral genomes we could benefit from the best of two worlds. Nonviral vectors may be easier to produce and formulate, and may deliver their payload more reproducibly at the tumor site than viral vectors. If used for the delivery of viral genomes, tumor-selective replicating viruses may be generated on site, which can spread in the tumor and exert their therapeutic action, without causing collateral damage to nontarget tissues. It remains doubtful, however, that HAdV is the best choice of virus for such strategies.

New therapeutic genes

New transgenes in the vectors may enhance therapeutic efficacy. An intriguing class of prodrug-activating genes is based on deoxyribonucleoside kinases (dNKs). These enzymes catalyze the phosphorylation of deoxyribonucleosides to deoxyribonucleoside monophosphates and thereby provide the cell with deoxyribonucleoside triphosphates. The dNKs catalyze the first, and often rate-limiting, step of nucleoside analog activation. These enzymes are therefore promising candidates to be used in combination with nucleotide analogs as prodrug–enzyme combinations. Bioselection yielded mutants of the Drosophila melanogaster-derived dNK with enhanced sensitivity to a range of clinically approved nucleotide analogs, such as 3′-azido-3′-deoxythymidine (AZT), arabinosylcytosine (AraC), ddA, and ddC (Knecht et al., 2000, 2007). These mutants efficiently sensitized glioblastoma, osteosarcoma, and breast cancer cells to clinically accepted drugs (Knecht et al., 2007). The use of approved nucleoside analogs may facilitate swift acceptance of the strategy.

Future prospects

We have witnessed an enormous expansion of the gene transfer technology required for cancer gene therapy. Initial clinical safety studies have demonstrated the validity of the concept, and the feasibility of current approaches (Schenk et al., 2009). The viral vectors used in the clinical studies reported so far have been well tolerated and safe. Robust technology platforms have been established and many of the factors currently thwarting prostate cancer gene therapy have been identified. New platforms for preclinical evaluation of new oncolytic vectors are available (Maitland et al., 2009). Combining the technologies and building on new insights from prostate cancer biology and virology will facilitate the generation of new vectors that will, it is hoped, be as safe as current vectors, but more efficacious. With these we may keep the promise of gene therapists to provide new and effective treatment for malignant neoplastic disease in general and prostate cancer in particular.

Footnotes

Acknowledgment

This work was supported by the European Union through the 6th Framework Program GIANT (contract no. 512087).

Author Disclosure statement

Jeroen de Vrij, Ralph A. Willemsen, and Rob C. Hoeben declare no competing financial interests. Leif Lindholm is a shareholder of Got-a-Gene, which has intellectual property on adenovirus targeting technology.

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