Chimeric adeno-associated virus and bacteriophage: a potential targeted gene therapy vector for malignant glioma.

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Therapeutic Delivery

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Chimeric adeno-associated virus and bacteriophage: a potential targeted gene therapy vector for malignant glioma

The incipient development of gene therapy for cancer has fuelled its progression from bench to bedside in mere decades. Of all malignancies that exist, gliomas are the largest class of brain tumors, and are renowned for their aggressiveness and resistance to therapy. In order for gene therapy to achieve clinical success, a multitude of barriers ranging from glioma tumor physiology to vector biology must be overcome. Many viral gene delivery systems have been subjected to clinical investigation; however, with highly limited success. In this review, the current progress and challenges of gene therapy for malignant glioma are discussed. Moreover, we highlight the hybrid adenoassociated virus and bacteriophage vector as a potential candidate for targeted gene delivery to brain tumors.

Background Cancer is an enigmatic disease that is at the forefront of disease-related mortality in both the developing and developed world. Despite its historic past, the amount of interest given to cancer research has lost none of its prominence. Of all cancers that have been reported, central nervous system (CNS) malignancies, particularly gliomas, are renowned for their aggressive phenotypes and poor prognoses. They represent a form of resistant cancers, for which its successful treatment may pave new paths to treat other cancers and related diseases. The available treatment for gliomas and other cancers has developed progressively through time, experiencing several paradigm shifts from cytotoxic agents to personalized targeted therapeutics. Alongside other frameworks that have become available through bioinformatics, gene therapy is now considered a useful treatment approach for cancer and other genetic diseases with varying degrees of feasibility [1] . Relative to other next-generation treatment approaches, such as cell-based therapies, gene therapy has a high potential to provide effective treatment with lower toxicity and higher safety than other currently available agents. The large

10.4155/TDE.14.58 © 2014 Future Science Ltd

number of clinical trials that are, or have yet to be completed reflects the global demand, and to an extent, the success of gene therapy as a curative treatment approach. In the past few decades, an intense amount of research has been devoted to developing gene delivery vehicles that treat malignant glioma and other cancers by delivering transgenes. A large number of these vectors are recombinant viruses, such as adenovirus, herpes-simplex virus or lentiviruses, which lack pathogenicity to humans [2] . Although clinical benefits have not been consistently reported, these studies all indicate optimistic responses and identify areas of improvement that is paramount to successful future clinical translation [3–5] . Currently, new generations of gene delivery vectors are constantly being developed. An exemplary candidate is the chimeric adeno-associated virus/phage (AAVP), which is able to deliver transgenes for therapy and molecular imaging in a targeted fashion, to mammalian cells in vitro and in vivo. Its safety profile and production methods are a few amongst many other attributes that make this chimeric virus an attractive vector for clinical gene therapy in the future.

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Paladd Asavarut1, Kevin O’Neill2, Nelofer Syed2 & Amin Hajitou*,1,3 Phage Therapy Group, Department of Medicine, Imperial College London, Burlington Danes Building, Hammersmith Hospital, Du Cane Road, London, UK 2 The John Fulcher Molecular NeuroOncology Laboratory, Division of Brain Sciences, Faculty of Medicine, Imperial College London, Burlington Danes Building, Hammersmith Hospital, Du Cane Road, London, UK 3 Qatar Biomedical Research Institute (QBRI), Qatar Foundation, Education City, Doha, Qatar *Author for correspondence: Tel.: +44 207 594 6546 [email protected] 1

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Key terms Malignant glioma: A class of cancers arising from glial cells that account for over 80% of all brain tumors. Particularly aggressive forms include high-grade astrocytomas, often called glioblastoma multiforme. Adeno-associated virus/phage: A hybrid targeted gene delivery vector constructed by inserting recombinant adeno-associated virus into the genome of filamentous bacteriophage. Molecular imaging: A non-invasive method for visualizing cells, tissues or organs by exploiting their specific biochemical or molecular features. Blood–brain barrier: A biochemical and electrical barrier formed by specialized endothelial cells that exceptionally restrict trafficking of molecules into the central nervous system. Angiogenesis: The cellular process of generating new blood vessels from existing vessels.

This review aims to summarize the current contributions that viral vector-mediated gene therapy has made to cancer treatment in the context of malignant gliomas. Herein, we provide an overview of glioma and CNS physiology, critically evaluate adeno-associated virus (AAV) vectors for brain tumor gene delivery and suggest future strategies to enhance research and clinical translation. In particular, we highlight ligand-targeted AAVP vectors as promising candidates for gene delivery to brain tumors and other cancers Gene delivery vectors & the CNS Gene delivery across the blood–brain barrier

Effective gene delivery and expression relies on three key factors. First, the type of therapeutic transgene must be suitable, including its promoter and expression in tumor cells. Second, the vector must be able to bind and become endocytosed by tumor cells with specificity. Last, the vectors and transgenes must have an acceptable level of safety to achieve a high therapeutic index. Creating efficacious gene therapy vectors is a challenge that has yet to be overcome. However, designing those for the CNS adds further layers of complexity. The physiology of the CNS and the brain tumor microenvironment are both physical and chemical barriers to the delivery of therapeutic transgenes. The blood–brain barrier (BBB) is the first key obstacle that impedes non-invasive therapy of glioma through systemically administered agents. Unlike peripheral vasculature, the BBB endothelial cells express tight junction proteins that highly restrict paracellular permeability and create electrical resistance [6,7] . At deeper levels, the neurovascular unit, which includes multiple cell types, such as pericytes, astrocytes and neurons, help maintain the integrity of the

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BBB through intercellular communication with the endothelial layer. Movement of solutes and macromolecules from blood to brain can be achieved through passive and facilitated diffusion, and receptor-mediated and adsorptive transcytosis (RMT). Although it may seem rather simple, transcytosis is tightly restricted to a small number of molecules, and even the diffusion of small polar and apolar drugs is not easily achievable. Thus, the brain is designated as a privileged site as a result of this restriction of paracellular and transcellular permeability. Despite being protective, the BBB prevents systemic therapeutic agents from being delivered to the tumor site. For example, unlike other cancers, the use of conventional chemotherapeutic agents, such as alkylating agents, is limited by their inability to cross the BBB. A key feature that has been attributed to poor drug penetrance is the p-glycoprotein efflux pump, which is enriched in the BBB and even further upregulated in the glioma neovasculature [8] . Furthermore, molecular immunotherapy or immunomodulation is of highly limited use due to immunological protection. Efforts have been made to circumvent this problem by using cell-based therapies, which exploit homing and trafficking of modified immunological cells that easily extravasate and cross through the BBB [9] . Because facilitated exchange of substances across the BBB is virtually impossible without the help of highly selective endothelial receptors or transport proteins, delivery strategies of therapeutic agents is very limited. Several approaches that have been employed in an attempt to infiltrate therapeutic substances in to the CNS rely on RMT. In the context of gene therapy, import of macromolecules and gene delivery vectors across the BBB is virtually only achievable through RMT. Few circulating ligands that are transported across the BBB through RMT have been reported, such as glucose, insulin, transferrin and leptins. Most, if not all, of techniques currently used to overcome the BBB rely on molecular mimicry to deliver a ‘Trojan horse’ [10] . Earlier examples have achieved success through the use of conjugation chemistry with peptidomimetic monoclonal antibodies [11–13] . Recently, new peptides and biomaterials enable RMT to occur based on overexpression of receptors or ligands in gliomas that are permissive of transcytosis. These include the receptor-binding domain of Apolipoprotein E, Angiopep-2 or glucose-analogue coated nanoparticles that have emerged and warrant further clinical investigation [14–16] . In malignant glioma, the BBB becomes disrupted at varying degrees depending on tumor phenotype and genotype. The ability to create and maintain an intact BBB is characteristic of normal cells in the CNS. How-

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Chimeric adeno-associated virus & bacteriophage: a potential targeted gene therapy vector for malignant glioma

ever, deregulation of the BBB worsens with increasing glioma grade. High-grade gliomas directly degrade tight junctions, promote endothelial dysfunction and alter astrocyte polarity through the secretion of soluble factors [17,18] . As a result, the physical and electrical integrity of the BBB are compromised. Intriguing data from multiple studies have revealed that the BBB is heterogeneously disrupted, with the core of the tumor being more disrupted and ‘leaky’ relative to the tumor periphery [19–21] . This indeed contributes to subsequent relapse and high morbidity of malignant glioma, and poses great challenges to successful treatment, even after aggressive chemo- and/or radiotherapy. The effect of glioma tumor physiology on resistance to genetic & other therapies

Glioblastoma represents the largest and most aggressive class of gliomas, accounting for poorest prognoses and survival rates [22] . High-grade gliomas possess genetic and epigenetic abnormalities that provide enabling physical and biochemical characteristics. As a result, they are aggressive, invasive, and highly resilient to multiple therapeutic strategies, including gene therapy, which may otherwise be effective against other cancers. Angiogenesis and metastasis are characteristics that have become synonymous with cancer and highgrade gliomas [23,24] . These characteristics present a substantial obstacle for systemically delivered agents to be able to reach and treat malignant cells [25,26] . One key feature that underlies invasiveness is the glioma extracellular matrix (ECM), which in itself is a physical barrier to gene delivery vectors. More importantly, it cooperates with malignant cells to produce a selfperpetuating invasive phenotype [27–29] . In particular, the role of glioma ECM in facilitating intercellular signalling between various classes of tumor, ependymal, endothelial and glial cells has been heavily implicated in tumor outgrowth and morphology [26] . The sheer size and mass of gliomas is a major barrier to successful therapy. This poses a major challenge to gene delivery vectors, as even small molecules are unable to completely penetrate through the tumor. Due to their fast-growing nature, the unreachable hypoxic and often necrotic brain tumor core is an additional force that drives tumor survival and metastatic behavior [30] . At the hypoxic core, hypoxia-inducible factor and vascular and endothelial growth factor signalling pathways provide intracellular signalling cascades that promote growth and survival through chemotaxis and morphological changes [31] . It has been demonstrated by many studies that glioblastoma cells elicit mesenchymal-like properties, which are signified by poor differentiation and tumor grade. Clearly, the physical


Review

features of gliomas account for a substantial portion of their aggressiveness and impunity to therapy. Metabolic changes play an equally important role in providing glioma with enabling characteristics and resistance to therapy. Even after successful delivery of therapeutic agents across the BBB, genetic and epigenetic changes confer resistance through the upregulation of compensatory signalling pathways to counter tumor killing [32–34] . Survival and proliferation pathways, such as those of epidermal and tumor growth factors, have been targeted in brain tumor therapy [35,36] . However, their inhibition has been proven ineffective, as gliomas are able to counter these pathways by both epigenetic enzyme and normal enzyme regulation [33,37,38] . Often, genetic and epigenetic deregulation also affects other pathways that contribute to glioma phenotype. As previously mentioned, mesechymal-like properties of gliomas permit physical invasiveness, but more importantly, genetic and developmental studies suggest that its poor differentiation may signify glioma stem cell niches [39–42] . Popular and controversial in equal respect, the tumor stem cell hypothesis is a valuable perspective in accounting for glioma phenotype and its reputation for relapse. It is also important to consider the indirect consequences that gliomas have on the tumor microenvironment and the immune system. For example, gliomas are known to impose control over inflammation and immunomodulation through attracting adult haematopoietic stem cells [43,44] . Furthermore, evidence suggests that gliomas also downregulate tumor immunosurveillance that is an important supporter of tumor killing [45,46] . Despite the large body of studies investigating glioma cell biology and its molecular characteristics, much more needs to be known if current therapeutic barriers are to be overcome. The current progress of gene therapy for glioma Therapeutic approaches to glioma gene therapy in clinical trials

Over recent years, numerous clinical gene therapy trials have flourished, offering the possibility to treat a large number of diseases that would otherwise be incurable. The number of clinical trials however, is only the tip of the iceberg considering preclinical studies that have yet to reach translatable stages. In regards to gene therapy for brain tumors, adenovirus, retrovirus and several alternative viral vectors have been investigated in clinical trials. Due to limitations posed by the BBB, all current virotherapy trials rely on intracerebral injections or direct injection/inoculation of gene therapy vectors after surgical debulking as adjuvant treatment [3] . The

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Review Asavarut, O’Neill, Syed & Hajitou transgenes that are carried by these vectors exert antitumor effects through several key approaches, such as suicide, oncolytic viral vectors, immunomodulatory and others types of gene therapy. Several combinations of vectors and therapeutic approaches have provided variable results in tumor control or regression. Suicide gene therapy

The treatment approach that has been used in glioma gene therapy with high popularity is the suicide gene and prodrug system. In such systems, HSV-tk is transduced to tumor cells, allowing conversion of ganciclovir (GCV) to GCV-triphosphate, a nucleoside analogue that is a suicide inhibitor of DNA synthesis [3] . Such systems induce a strong anti-tumor response due to a ‘bystander effect’, or the killing of neighbouring tumor cells that do not express HSV-tk. The precise mechanism of this effect has been attributed to intercellular communication via gap junctions, which allow GCVtriphosphate or other phosphorylated deoxyguanosine analogues to enter nearby cells [47] . The termination of DNA synthesis through this mechanism induces tumor control or regression through apoptosis [48] . In practice, adenovirus and retrovirus-mediated HSV-tk suicide gene system is perhaps the most widely used system in clinical trials. Phase I/II trials using adenoviral and retroviral vectors to deliver HSV-tk have shown limited success, achieving short-term tumor regression or control in less than half of patients in a trial with the most optimistic results, although most studies report rates lower than 33% [49–60] . In a Phase III trial conducted by Rainov et al., the results suggested that retroviral suicide gene therapy did not provide additional benefits when compared with standard-of-care therapies (radiotherapy with or without adjuvant chemotherapy) [61] . More recently, another completed Phase III trial (ASPECT) by Westphal et al. reported that adenoviral HSV-tk/GCV gene therapy could increase time to death, but not improve survival of glioblastoma multiforme patients [62] . A problem with assessing these clinical trials together arises due a variety of factors, such as ambiguity of study measures, that is, patient survival or tumor size regression, or viral vector load and administration frequency. Although adenoviral and retroviral vectors used in such suicide gene systems are replication-incompetent, the administration of retroviral vectors in many HSVtk trials are indirect, as virus-producing cells (GLI 328) are injected adjacent to the tumor site to promote in situ virus production and tumor transduction [54,63] . It is known that adenoviral vectors enter the target cell through the coxsackie and adenovirus receptor, while retroviral vectors possess varying degrees of affinity to target receptors, such as sulphatide or galactocerebro-

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side, which are expressed in neurons and glial cells [64,65] . The poor account of viral capsid modification or strain in glioma gene therapy trials in general make relative evaluation of success nearly impossible. We can however speculate that at least for adenoviral gene therapy, coxsackie and adenovirus receptor is progressively downregulated with malignant glioma grade, and this may probably account for disappointing clinical trial results [66] . The difficulty in comparing clinical trial data from HSV-tk suicide gene therapy confounds the explanations of why these clinical trials produced disappointing results. Despite vector-related issues, we can speculate several key factors that are highly likely to be involved in glioma resistance to suicide gene therapy. The effectiveness of HSV-tk suicide gene therapy depends largely on the bystander effect, as gliomas are large solid tumors that are not permissive of vector penetration. It is often assumed that gliomas express gap junctions, which renders them susceptible to the bystander effect. However, the expression of certain gap junction connexins, such as Cx43 and Cx26, are essential for bystander killing [48,67–69] . Less-differentiated glioma cell types, such as glioma stem cells, express lower levels of connexins compared with their more-differentiated daughter cells [70] . Consequently, they are able to resist HSV-tk gene therapy and relapse after an initially observable tumor regression. The expression of connexins has indeed been implicated in a variety of roles that further affects glioma phenotype. It has been shown that Cx43 plays a crucial role in inhibiting gliomagenesis, whereby their expression is associated with epithelial morphology and increased cellular aggregation [71] . Additionally, they enable intercellular communication between gliomas and the astrocytic network, further suggesting involvement with invasiveness. Recently, the expression of connexins have also been linked with electro-chemical physiology, resistance to apoptosis and proliferation of gliomas [68,70,72] . As a result, it is rational to hypothesize that the control of connexins under genetic and epigenetic means may hold the key for not only sensitizing gliomas to suicide gene therapy, but also phenotype modulation to a lower grade. It is also worth mentioning that even though HSV-tk/GCV suicide gene therapy systems are popular, alternatives exist. One that uses the same antitumor mechanisms is the cytosine deaminase (CD) and 5-fluorocytosine (5-FC) system, which await clinical trials [73] . Suicide gene therapy that exclusively kill tumors directly through p53 transduction have also been implemented in Phase I trials, but only shortterm tumor control in a third of subjects was achieved [74] . Clearly, suicide gene therapy may potentially be an



effective approach to treating gliomas. Further research and development in tumor physiology is essential to ensure maximal effectiveness and realistic clinical trial results. Oncolytic viral gene therapy

Unlike the suicide gene approach, oncolytic viral gene therapy relies on a replication-competent virus that conditionally infects and kills tumor cells. Although this particular approach to therapy raises safety concerns when compared with replication incompetent viruses, it has shown highly variable success. Recombinant herpes simplex viruses (HSV) have been used in this treatment approach, such as the G207 or 1716 strains, which have been genetically modified to ablate their pathogenicity [75,76] . HSVs enter the cell through heparin sulphate proteoglycans, herpes virus entry mediator, or Nectin-1, however the status of these receptors on malignant glioma is not well characterized [77,78] . As recombinant HSVs are able to infect dividing cells but not neurons, they serve as favorable candidates for oncolytic glioma therapy. To date, five oncolytic HSV trials have been completed. Overall, less than half of subjects enrolled in these studies benefitted from oncolytic viral therapy [79–83] . In addition to oncolytic recombinant HSV, several other oncolytic viruses have also been investigated. Newcastle disease virus exhibits pathogenicity specifically to tumor cells via the Ras pathway, and is also able to stimulate the immune system [84] . Several single subject or small subject group clinical studies have shown optimistic results in tumor control, whereas its Phase I clinical trials reported long-term response in less than 30% of subjects [85–87] . Another vector that has been given interest is Reovirus, which also conditionally infects cells with over-expressive Ras, although both recent Phase I trials have reported disappointing anti-tumor response in over 90% of subjects [88,89] . The possibility of using oncolytic adenovirus against malignant glioma has also been explored to a significant extent. Recombinant adenoviruses can be made oncolytic through transcriptional targeting of tumor cells, such as those that are p53-decificent; however, no significant benefit was reported in a Phase I trial [90] . Other strategies have also been employed to enhance tumor/tissue specificity of oncolytic adenoviruses, including metabolic targeting or the use of microRNAs to drive specificity [91,92] . This has indeed contributed to current thinking, which suggests that oncolytic adenoviral systems can be used to simultaneously sensitize glioma cells to the chemotherapeutic agent temozolomide [93] . This notion of combinatorial viroand chemotherapy has also been explored in other viral


Review

systems and is likely to provide fruitful alternatives to traditional stand-alone virotherapy [94] . Immunomodulatory gene therapy

Cell and cytokine-based immunomodulatory gene therapy has been investigated in numerous preclinical studies. By stimulating the immune system, stronger tumor immunosurveillance or apoptosis and inhibition of tumor proliferation can be achieved. Gliomas and indeed other malignant tumors have immunological profiles that differ from the surrounding tissue, such as the expression of tumor-specific antigens. The use of cytokines that facilitate cell-mediated immunity and subsequent tumor degradation, such as interleukins (e.g., IL-2, IL-4) or interferons (e.g., IFNϒ, IFNβ), is attractive due to their self-sustaining ability (via positive feedback mechanisms), which may partially circumvent the issue of vector penetrance. Clinical trial and preclinical data have shown the feasibility of such systems to date, although its therapeutic efficacy may not be impressive. In a Phase I trial that used cationic liposomes to deliver IFNβ plasmids to tumor cells by stereotactic injection, two out of five subjects showed partial and complete tumor responses in the first 10 weeks of therapy [95,96] . However, the use of adenoviral vectors to deliver IFNβ in a recent Phase I trial proved to be less effective, as disease progression occurred in all 11 patients within 4 months after treatment [97] . Combinatorial therapies of immunomodulation and suicide gene therapies also exist. In 2011, a Phase I trial for combinatorial gene therapy was recently launched; the approach is to use signalling pathways from damage-associated molecular patterns (such as high-mobility group box 1 protein) and HSV-tk/GCV to enhance tumor killing via dendritic cell activation [98–100] . Gene silencing & interference through RNA technology

Another form of gene therapy that has attracted much attention in preclinical research is gene silencing or interference. The control of gene expression by RNA, such as siRNA or microRNA, plays a crucial role in controlling glioma growth and invasiveness [101] . This has led to the use of small siRNAs to post-transcriptionally deactivate genes that are implicated in disease, and indeed gliomas. Silencing of intracellular proteins expressed or overexpressed in glioma cells, such as Notch proteins, EGFR and matrix metalloproteases using siRNAs have been fruitful in inhibiting glioma growth, invasiveness and angiogenesis in vitro and in vivo [102–104] . The delivery of these small nucleic acid molecules can be achieved using synthetic biomaterials, such as nanoparticles that contain or are coated by siR-


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Key term Phage display: A technology used for identifying novel protein–protein interactions through the insertion of random peptide libraries on filamentous bacteriophage coat proteins.

NAs or microRNAs [105,106] . However, the approach of gene interference has also been incorporated into viral vectors in the form of antisense therapy, which similarly inhibits post-transcriptional expression by binding to and forming double-stranded mRNA complexes [107,108] . The feasibility of antisense therapy in glioma treatment has been frequently reported, including a Phase I clinical trial that reported complete tumor response in two and partial response in four subjects out of 12 that were enrolled [109] . Although preclinical data can be highly promising, targeting synthetic molecules, let alone viral vectors for systemic therapy or stereotactic injections, remain a major challenge to glioma gene therapy, inhibiting their progress to clinical translation. Characterisation of hybrid adeno-associated virus/phage vectors & their contribution to gene therapy Adeno-associated viral vectors: progress & challenges

Over recent years, AAV vectors have become widely popular and show great promise for successful clinical translation. Although delivery of therapeutic agents to the CNS and glioma are challenging as previously described, attempts have been made to enhance the capabilities of the AAV toolkit through multiple approaches. A prominent principle by which specificity and targeting is achieved is through viral capsid selection or modification. AAV are non-enveloped eukaryotic viruses, and its reproduction and evolution is closely tied to its relative, the adenovirus. The relatively small (4.7Kb) AAV genome, containing replicative (rep) and capsid (cap) elements are flanked by inverted terminal repeat sequences (ITRs), which form hairpin-like secondary structures [110] . While providing a protective function, ITRs confer the ability of (concatameric) AAV episomes to persist in mammalian cells. In contrast to wild-type AAV, recombinant AAV used for gene therapy lacks rep and cap elements, which are replaced by therapeutic and/or reporter genes [2] . The life cycle of AAV after infection is lysogenic, unless adeno-helper proteins are present, whereby a productive, lytic cycle occurs. It is worth considering that alternative viruses, such as HSV or baculovirus, are superior alternatives for providing adeno-helper genes in large-scale AAV production systems [111,112] .

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In gene therapy, the lysogenic AAV life cycle is extremely useful in providing stable expression of transgenes. Moreover, AAV has the ability to infect both dividing and non-dividing cells, making it ideal for CNS application [110] . Although AAV may integrate with the host genome, albeit with very low probability, the occurrence is not likely to generate any safety concerns [113–115] . Despite their advantageous genetic properties, a major drawback of AAV vectors is low transduction efficacy in the target tissue. The transduction efficacy of AAV vectors is significantly stunted by the broad tropism that AAV has to multiple tissue types. Several strategies have been employed to minimize the safety risks associated with non-specific transduction, such as using tissue-specific promoters [116–119] . However, this does not solve the core of the problem, which concerns the AAV capsid [116] . Indeed, the tissue tropism of AAV depends on the capsid serotype, such as AAV serotype 2 (AAV2). Currently, nine capsid serotypes have made regular appearances in research literature, with AAV2 being most heavily investigated [120] . As AAV are viruses that have native tropism to mammalian tissue, the host innate, humoral and cell-mediated immune response is another obstacle for efficient gene transduction [121] . Earlier reported attempts to overcome CNS and brain tumor gene delivery involved using recombinant AAV of different capsid serotypes. The AAV capsid is known to interact specifically with cell surface receptors that confer tissue tropism. Pseudotyping, or the alteration of tissue tropism of viruses through capsid and coat protein modifications has been extensively used to alter recombinant AAV vectors. By constructing a separate vector containing rep genes from AAV2 and cap genes from multiple serotypes, ‘gutted’ AAV2 genomes with ITR-flanked reporter/therapeutic genes can be assembled in to tissue-targeted virions given the presence of adenohelper functions [122] . However, modification of tissue tropism is not easily solvable. In addition to ablating broad tropisms that certain AAV serotypes have, the route of administration also determines transduction efficacy in the target tissue in vivo [120] . This presents a problem when comparing tropism between studies for future vector design. Using new technologies, generations of recombinant AAV (rAAV) vectors that exhibit higher tissue specificity and transduction efficacy have been identified. DNA family shuffling represents a next-generation technique of pseudotyping that has allowed complex chimeric AAV capsids to be constructed from up to eight serotypes [123] . Not only do these hybrid serotypes (e.g., AAV-D/J and AAV-DJ/8) provide high



efficacy and specificity in liver transduction compared with AAV2, they also provide a novel platform for peptide display and directed evolution of AAV vectors. Another technique derived from peptide display that has found its way in to AAV gene therapy is direct insertion of identified targeting peptides that have been fruitful in providing improved targeted gene delivery to venous endothelia, lungs and the brain in vitro and in vivo [124,125] . In light of glioma gene therapy, AAV vectors have been given increasing interest. With respect to the possibility of systemic gene therapy, it has recently been demonstrated that AAV serotype 9 (AAV9) is able to cross the BBB in mice after systemic injection [126] . Interestingly, transduction targets were different between injections to neonatal (widespread CNS transduction) and adult mice (predominantly astrocytes), indicating that the BBB remains an obstacle for AAV gene therapy. On the contrary, gliomas are known to have a leaky BBB, as they secrete soluble factors that disrupt tight junctions and astrocyte polarity, which may work to the advantage of molecular and gene therapy [17,18] . Unfortunately, the physical and behavioral characteristics of gliomas overshadow this therapeutic window for therapy. The feasibility of AAV for glioma gene therapy as currently demonstrated should warrant further preclinical and clinical studies in the future, as only few have been previously conducted [127,128] . Nonetheless, AAV vectors show great promise for gene delivery in brain tumors. To achieve such goals, better targeting approaches or administration routes must be developed. Regardless of its less favorable features, the genetic properties of AAV surpass most other vectors, especially in safety and expression patterns.

as mere discovery tools for other therapies. Phage display exploits the display of peptides on the pIII or pVIII coat proteins of filamentous bacteriophage for screening positive protein–protein interactions on an immobilized phase [129] . Their simplistic genome– phenome relationship allowed DNA-sequencing to easily identify molecular targets with high accuracy. Not only small, but also large proteins, such as light chain antibody fragments could be displayed on phagemid particles, yielding valuable information for antibody production and antibody-based immunotherapy [131] . Eventually, phage display technology expanded from in vitro to in vivo use, where it became a fundamental research tool for identifying tissue or organ-specific targets in cancer and cardiovascular proteomics [132–138] . The first demonstration of ligand-targeted bacteriophage-mediated gene delivery to mammalian cells was reported in 1998 [139] . Filamentous bacteriophages and phagemid particles displaying complete FGF2 or EGF ligands on the pIII coat protein were able to transduce a reporter gene to mammalian cells displaying FGFR, EGFR/HER2 receptors [139–144] . In addition to gene therapy, these vectors provide a platform for improved phage display library screening, as only targets that are bound and internalized will be detected [142] . The use of these vectors in the clinic would be even more attractive in terms of safety when compared with viral vectors because they do not possess inherent tropism for mammalian cells. Despite their numerous advantages, interest in other viral vectors outcompeted phage-mediated gene therapy as they showed superior performance. Consequently, the research in phage-mediated gene delivery vehicles came to a halt as of 2002.

Filamentous bacteriophage: a new vector for gene delivery to mammalian cells

AAVP: a chimera between display technology & gene therapy

Bacteriophages are prokaryotic viruses that have become instrumental in biotechnology. Due to their simplistic life cycle, amenability to genetic modification and physical integrity, phages have been extensively used in basic research, in the food industry and clinical antibiotic therapy. Most importantly, phage display technology has made paramount contributions to the identification of therapeutic targets in a vast range of contexts [129,130] . The use of bacteriophage for gene delivery to mammalian cells has only emerged in the end of the 20th century, where it became intermittently halted given the rise of alternative viral and nonviral vectors that have overtaken them by performance and popularity. The possibility of using bacteriophages for gene delivery began in phage display, which formerly served

Safety and effectiveness of gene delivery vehicles has long been an issue of sacrifice. Although phagebased vectors provide a safe alternative to mammalian viruses, their specificity comes at the cost of efficiency. The development of phage display technology and recent advances in vectorology has provided scientists the necessary tools to overcome the shortcomings of individual vectors. The hybrid AAVP vector is an amalgamation of two well-characterized singlestranded DNA vector entities that show great promise for future clinical use [145] . Since its characterisation in 2006, research using AAVP vector has made significant progress in demonstrating its fidelity in gene transduction in vitro and in vivo [145,146] . Future studies are warranted to further explore its application in clinical gene therapy and research.



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Review Asavarut, O’Neill, Syed & Hajitou AAVP vectors are constructed using genetic engineering to incorporate cis genetic elements from rAAV into an intergenomic region of the filamentous bacteriophage genome (Figure 1) . Because the major and minor capsid proteins (pVIII and pIII, respectively) can be modified by gene insertion to display fusion peptides (i.e., phage display), ligand-directed targeting can be conferred to AAVP vectors. At the same time, the nature of transgene expression from the vector is stable and persistent due to protection by ITRs from AAV-2 under general or tumor specific promoters, such as cytomegalovirus or the glucose-regulated protein 78 (Grp78) respectively [147,148] . To ascertain whether after transduction, the ITR-flanked rAAV cassette drives gene expression, an AAV-rescue experiment confirmed that after internalisation by the target cell, the AAVP persists as an AAV [146] . Taken together, its physical and genetic properties make it an attractive vector for gene therapy and for a wide range of research use, where ligand-directed targeting is required. A number of studies have used AAVPs containing different transgene cassettes to demonstrate its ability to target and kill tumor cells. In AAVP vectors, ligandA

directed targeting of tumors was achieved through insertion and display of the double cyclic CDCRGDCFC (RGD4C) peptide ligand for ανβ3 integrins, which is expressed on tumor cells and angiogenic blood vessels of solid tumors [135,149] . Indeed, this particular integrin has long been implicated in adhesion and invasiveness of high-grade gliomas and has served as a target for therapy and molecular imaging [150–153] . Targeting tumor endothelia as opposed to tumor cells is an attractive approach in many aspects. This is principally due to the fact that the tumor endothelium is genetically stable and less likely to resist therapy, in addition to being a suitable primary target for systemic therapy [154] . Furthermore, because the tumor vasculature is vital for growth, successful targeting of a single tumor endothelial cell may be effective in exerting strong indirect anti-tumor effects. Transgenes that have been used in the AAVP system include HSVtk and TNF-α, which are able to kill tumor cells of various types in vitro, including human and rat glioblastoma (U87 and 9L respectively), as well as the human embryonic kidney (HEK293), human Kaposi’s sarcoma (KS1767) and human prostate carcinoma

B

pIII minor coat protein i

ii Recombinant AAV cassette

C i

RGD

pIII GOI

ii

ITR

GFP/Luc/HSV-tk/TNF-α

ITR

Figure 1. Schematic diagram of the hybrid adeno-associated virus/phage vector. (A) Schematic cross-section of the AAVP vector, showing the genetic construct and modified pIII minor coat protein displaying a fusion peptide for tumor targeting (black). (B) Simplified genetic construct of the AAVP. (C) i. The modified pIII minor coat protein gene of AAVP containing the cyclic CDCRGDCFC (RGD4C) peptide sequence for targeting ανβ3 integrins displayed on angiogenic blood vessels and tumor cells of solid tumors. ii. Recombinant AAV cassette inserted into an intergenomic region of a filamentous bacteriophage genome, which is adaptable for expression of a GOI, flanked by ITR, such as GFP, Luc, HSV-tk and TNF-α. AAV: Adeno-associated virus; AAVP: Adeno-associated virus/phage; GOI: Gene of interest; ITR: Inverted terminal repeats.

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(DU145) cell lines [48,155–157] . AAVP vectors also serve as a platform for combinatorial drug and gene therapy. It has been demonstrated that administration of drugs, such as proteasome inhibitors or epigenetic modulators, enhance AAVP-mediated gene transduction and tumor killing in a variety of cell lines in vitro [156,158] . In a similar manner, in vivo administration of AAVP in both murine and canine cancer models demonstrate its fidelity for clinical use. Transgenes such as luc, or HSV-tk in combination with radiolabelled 2-Deoxy2-trifluoromethanesulfonyl-1,3,5-tri-O-benzoyl-alphaD-ribofuranose ([18F]FEAU) can be used for live molecular imaging of cancer in vivo in mice [145,146,159] . Moreover, these molecular imaging techniques can also be used to predict systemic cytotoxic drug response of soft-tissue sarcomas through PET [160] . In moving AAVP vector applications closer to human clinical trials, the Comparative Oncology Trials Consortium was established by the National Cancer Institute, USA to translate findings in murine tumor xenograft models to natural mesenchymal tumors of larger animals [161] . The application of tumor endothelium-targeted RGD4C-AAVP that delivers TNF-α in pet dogs has been shown to be variably effective in killing sarcomas of various types that were identified in the study [161] . However, this depends on a variety of factors including drug response or resistance that have been established a priori, and specific tumor pheno/genotypes. Remarkably, one of two dog patients with previously defined partial response (Response Evaluation Criteria in Solid Tumors) showed over 85% reduction in soft tissue sarcoma size after eight weekly doses of treatment. The compelling evidence from in vivo studies showing the therapeutic efficacy and flexibility of AAVP vectors demonstrates its importance and promise in clinical and laboratory applications. The AAVP represents a new generation of viral vectors that defy the conventions of gene delivery vehicles. Its growing evidence base from in vitro and in vivo studies clearly show its emergence in the field of gene delivery. Although much remains to be investigated, AAVP vectors show no less potential compared with other viral vectors, particularly rAAV, which are popularly used for clinical gene therapy at present. Future perspective: targeted vectors & newer strategies Malignant gliomas remain one of the most elusive cancers due to their aggressiveness, invasiveness and resistance to therapy. Designing treatment strategies that effectively target glioma cells is an extremely difficult task, as considerations must be made on both the vector and the intricate physiology of these brain tumors. Future development of gene delivery vectors


Review

Key term Integrins: Trans-membrane receptors that play multiple key roles in intercellular and intracellular cell signalling, cellcycle regulation and cell adhesion and motility.

requires more innovative approaches and a deeper understanding of glioma cancer biology. Viral vectors that are currently in use have shown immense potential in vitro and in vivo preclinical studies; however, this is not necessarily reflected by data from clinical trials. In order to achieve a high therapeutic index, vectors benefits must outweigh their harm. As most viral vectors have the issue of their native tropism for mammalian cells, the lack of transduction specificity means that administration of vectors or vector-producing cells has to be done invasively, through stereotactic injections. If vectors were able to cross the BBB with specificity, relatively harmless systemic administration would be possible. Although synthetic non-viral vectors is a paradigm that has become popular, chimeric vectors with high potential, such as the AAVP, may provide a novel viral platform that avoids the physical limitations posed by recombinant mammalian viruses. By combining molecular targets from phage display with economic advantages in vector production, AAVPs have potential translatability that other vectors do not. Thus, innovating previous approaches and adapting them to modern techniques is equally as important as generating new technologies in the context of vector development. To ensure that in vitro and in vivo preclinical studies offer realistic and accurate findings, a better understanding of glioma tumor physiology is required. Currently, in vitro and in vivo brain tumor models are of limited validity, as they do not accurately reflect the natural brain tumor physiology. In particular, the vast number of cell types that are involved in gliomagenesis and the tumor ECM must be carefully considered. Consequently, it is very likely that these limitations account for clinical trial data that are often disappointing. Indeed, due to the complicated nature of malignant glioma, gene therapy alone may not be sufficient, and clinical trials for combinatorial therapy between tumor-modulating drugs and gene therapy is highly demanded. Thus, if better brain tumor models could be generated and translated to even larger animals such as primates, treatment approaches that are brought into clinical trials may provide more optimistic results. In conclusion, gene therapy is a highly attractive treatment approach for malignant glioma. However, a deeper understanding of the complex tumor microenvironment and of different viral vectors is required. Future success of clinical gene therapy trials for brain tumors requires clinical investigation of new-generation vectors, such as AAVP, to be put forth.


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Executive summary Background • Gliomas are brain malignancies renowned for their aggressiveness, invasiveness and resistance to therapy when compared with all other cancers. • Over the past few decades, gene therapy has been explored as a curative therapeutic approach, however with very limited success.

Gene delivery to the CNS • Gene delivery across the blood–brain barrier –– The CNS and its tumors possess features like the blood–brain barrier that impede the administration of gene delivery vehicles and other therapeutic agents. • The effect of glioma tumor physiology on resistance to genetic and other therapies –– The physical barriers to delivery of therapeutic agents include the blood–brain barrier, tumor extracellular matrix and physical size, while intricate networks of cell-signalling, genetic and epigenetic regulation provide biochemical resistance.

The current progress of gene therapy for malignant glioma • Therapeutic approaches to glioma gene therapy in clinical trials –– Current gene therapy approaches consist of the use of viral vectors that deliver suicide genes and modulate the immune system, are oncolytic or inhibit gene expression through epigenetic silencing. –– The HSV-tk and ganciclovir suicide gene system is the most popular approach, and has been put to test in clinical trials. The results however, seem to be disappointing due to a number of factors concerning tumor cell biology. –– In order to improve the performance of gene therapy in a clinical context, better in vivo models are required to accurately mimic the complex cellular physiology of brain tumors.

Characterization of the hybrid adeno-associated virus/phage vector for gene therapy • Adeno-associated viral vectors: progress & challenges –– In delivering stably expressed transgenes with high safety, adeno-associated viral vectors are ideal for CNS gene therapy, however they have not yet been clinically applied to treat human malignant glioma. • Filamentous bacteriophage: a new vector for gene delivery to mammalian cells –– Bacteriophage vectors are highly suitable for eukaryotic ligand-directed targeting as they have a strong track record in display technology and possess no inherent tropism for mammalian cells. • Adeno-associated virus/phage: a chimera between display technology and gene therapy –– Being both single-stranded DNA viruses, combining the adeno-associated virus and mammalian celltargeted bacteriophage offers a safe and effective gene delivery vector that has a potential for treating malignant glioma.

Conclusion • By further developing accurate in vivo brain tumor models and better-targeted vectors, such as the adenoassociated virus/phage, safe and effective gene therapy for malignant glioma may be achieved.

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment,

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Review

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The findings of this study report the potential of using adeno-associated virus/phage vectors for translational and diagnostic applications.

•

This study reports the first preclinical trial results and safety of adeno-associated virus/phage vector-mediated gene therapy of cancer in large animals (pet dogs with spontaneous tumors).


Gene therapy for malignant glioma.

human bocavirus 1 parvovirus vector efficiently transduces human airway epithelia".

Cornea-targeted gene therapy using adenovirus vector.

Bacteriophage-derived vectors for targeted cancer gene therapy.

Adeno-Associated Virus (AAV) as a Vector for Gene Therapy.

Potential application of temozolomide in mesenchymal stem cell-based TRAIL gene therapy against malignant glioma.

Tomato thymidine kinase-based suicide gene therapy for malignant glioma--an alternative for Herpes Simplex virus-1 thymidine kinase.

Human menstrual blood-derived mesenchymal stem cells as a cellular vehicle for malignant glioma gene therapy.

Split vector systems for ultra-targeted gene delivery: a contrivance to achieve ethical assurance of somatic gene therapy in vivo.

Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma.

Oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer.

Retrovirus-mediated gene transfer targeted to malignant glioma cells in murine brain.

Targeted therapy in pediatric low-grade glioma.

Epigenetic Targeted Therapy for Diffuse Intrinsic Pontine Glioma.

Immunotherapy for malignant glioma.

Gene therapy of malignant brain tumors: a rat glioma line bearing the herpes simplex virus type 1-thymidine kinase gene and wild type retrovirus kills other tumor cells.

The GIST of targeted therapy for malignant melanoma.

Potential vector for West Nile virus prevalent in Kent.

Adeno-associated virus vector-based gene therapy for monogenetic metabolic diseases of the liver.

A potential approach for gene therapy targeting hepatoma using a liver-specific promoter on a retroviral vector.

Gene therapy using a liver-targeted AAV vector restores nucleoside and nucleotide homeostasis in a murine model of MNGIE.

Nanoparticle-loaded macrophage-mediated photothermal therapy: potential for glioma treatment.

Interstitial radiotherapy for malignant glioma.

Mesoporous silica nanoparticles: a potential targeted delivery vector for reproductive biology?