Suicide gene therapy mediated by the Herpes Simplex virus thymidine kinase gene/Ganciclovir system: fifteen years of application

Gene-directed enzyme prodrug therapy (GDEPT) is a two step therapeutic approach for cancer gene therapy. In the first step, the transgene is delivered into the tumor and expressed. In the second step a prodrug is administered and is selectively activated by the expressed enzyme. The first GDEPT system described was the thymidine kinase gene of the Herpes Simplex virus (HSVtk) in combination with the prodrug Ganciclovir (GCV). A large number of experiments have been performed with this system, in different types of tumors and initial studies in animal models were very promising. This encouraged investigators to move into clinical trials although poor results have been obtained so far. A large effort has been made with numerous different strategies to enhance HSVtk/GCV efficacy in cellular and in vivo models and very strong cytotoxic effects have been obtained. The present review describes the current state of preclinical research and summarizes the results of the clinical trials undertaken.     

Viral based gene therapy for prostate cancer

In the last few years, significant advances in gene therapy have been made as a result of advances in many areas of molecular and cell biology, including the improvement of both viral and nonviral gene delivery systems, discovery of new therapeutic genes, better understanding of mechanism of disease progression, exploration of tissue specific promoter, receptor- and antibody-mediated targeting delivery, and development of better prodrug enzyme/prodrug systems. In this article, viral based gene therapy for prostate cancer will be reviewed and discussed. The areas of emphasis in this review are: choice of viral vectors, comparison of delivery routes, development of prostate-targeted viruses, choice of therapeutic genes and strategies including corrective gene therapy (tumor suppressor gene and anti-oncogene gene approaches), suicide gene therapy, programmed cell death therapy, immunomodulation therapy, and conditional oncolytic virus approach. Among them, several examples will be discussed in detail for the scientific basis and therapeutic applications. In addition, prostate cancer gene therapy clinical trials, unresolved problems and future directions in this field will also be described.     

Gene Therapy for Brain Tumors

Gene therapy has opened new doors for treatment of neoplastic diseases. This new approach seems very attractive, especially for glioblastomas, since treatment of these brain tumors has failed using conventional therapy regimens. Many different modes of gene therapy for brain tumors have been tested in culture and in vivo. Many of these approaches are based on previously established anti-neoplastic principles, like prodrug activating enzymes, inhibition of tumor neovascularization, and enhancement of the normally weak anti-tumor immune response. Delivery of genes to tumor cells has been mediated by a number of viral and synthetic vectors. The most widely used paradigm is based on the activation of ganciclovir to a cytotoxic compound by a viral enzyme, thymidine kinase, which is expressed by tumor cells, after the gene has been introduced by a retroviral vector. This paradigm has proven to be a potent therapy with minimal side effects in several rodent brain tumor models, and has proceeded to phase 1 clinical trials. In this review, current gene therapy strategies and vector systems for treatment of brain tumors will be described and discussed in light of further developments needed to make this new treatment modality clinically efficacious.     

Viral vectors in cancer immunotherapy: which vector for which strategy?

Gene therapy involves the transfer of genetic information to a target cell to facilitate the production of therapeutic proteins and is now a realistic prospect as a cancer treatment. Gene transfer may be achieved through the use of both viral and non-viral delivery methods and the role of this method in the gene therapy of cancer has been demonstrated. Viruses represent an attractive vehicle for cancer gene therapy due to their high efficiency of gene delivery. Many viruses can mediate long term gene expression, while some are also capable of infecting both dividing and non-dividing cells. Given the broadly differing capabilities of various viral vectors, it is imperative that the functionality of the virus meets the requirements of the specific treatment. A number of immunogene therapy strategies have been undertaken, utilising a range of viral vectors, and studies carried out in animal models and patients have demonstrated the therapeutic potential of viral vectors to carry genes to cancer cells and induce anti-tumour immune responses. This review critically discusses the advances in the viral vector mediated delivery of immunostimulatory molecules directly to tumour cells, the use of viral vectors to modify tumour cells, the creation of whole cell vaccines and the direct delivery of tumour antigens in animal models and clinical trials, specifically in the context of the suitability of vector types for specific strategies.     

A novel technique to monitor carboxypeptidase G2 expression in suicide gene therapy using 19F magnetic resonance spectroscopy

Development and evaluation of new anticancer drugs are expedited when minimally invasive biomarkers of pharmacokinetic and pharmacodynamic behaviour are available. Gene‐directed enzyme prodrug therapy (GDEPT) is a suicide gene therapy in which the anticancer drug is activated in the tumor by an exogenous enzyme previously targeted by a vector carrying the gene. GDEPT has been evaluated in various clinical trials using several enzyme/prodrug combinations. The key processes to be monitored in GDEPT are gene delivery and expression, as well as prodrug delivery and activation. {4‐[bis(2‐chloroethyl)amino]‐3,5‐difluorobenzoyl}‐L‐glutamic acid, a prodrug for the GDEPT enzyme carboxypeptidase‐G2 (CPG2; K_m = 1.71 µM; k_cat = 732 s^?1), was measured with ¹⁹F magnetic resonance spectroscopy (MRS). The 1 ppm chemical shift separation found between the signals of prodrug and activated drug (4‐[bis(2‐chloroethyl)amino]‐3,5‐difluorobenzoic acid) is sufficient for the detection of prodrug activation in vivo. However, these compounds hydrolyze rapidly, and protein binding broadens the MR signals. A new CPG2 substrate was designed with hydroxyethyl instead of chloroethyl groups (K_m = 3.5 µM, k_cat = 747 s^?1). This substrate is nontoxic and stable in solution, has a narrow MRS resonance in the presence of bovine and foetal bovine albumin, and exhibits a 1.1 ppm change in chemical shift upon cleavage by CPG2. In cells transfected to express CPG2 in the cytoplasm (MDA MB 361 breast carcinoma cells and WiDr colon cancer cells), well‐resolved ¹⁹F MRS signals were observed from clinically relevant concentrations of the new substrate and its nontoxic product. The MRS conversion half‐life (470 min) agreed with that measured by HPLC (500 min). This substrate is, therefore, suitable for evaluating gene delivery and expression prior to administration of the therapeutic agent. Copyright © 2009 John Wiley & Sons, Ltd.     

Developments with targeted enzymes in cancer therapy.

Cancer therapy based on the delivery of enzymes to tumour sites has advanced in several directions since antibody-directed enzyme/prodrug therapy was first described. It has been shown that methoxypolyethylene glycol (MPEG) can be used to deliver enzyme to a variety of solid tumours. MPEG-enzyme conjugates show reduced immunogenicity and may allow repeated treatment with enzymes of bacterial origin. Enzyme delivery to tumours by polymers can be used to convert a low toxicity prodrug to a potent cytotoxic agent. An example of such a prodrug is CB1954, which can be activated by a human enzyme in the presence of a cosubstrate. Tumour-located enzymes can also be used in conjunction with a combination of antimetabolites and rescue agents. The rescue agent protects normal tissue but is degraded at cancer sites by the enzyme, thus deprotecting the tumour and allowing prolonged antimetabolite action.     

Viral gene therapy strategies: from basic science to clinical application

A major impediment to the successful application of gene therapy for the treatment of a range of diseases is not a paucity of therapeutic genes, but the lack of an efficient non-toxic gene delivery system. Having evolved to deliver their genes to target cells, viruses are currently the most effective means of gene delivery and can be manipulated to express therapeutic genes or to replicate specifically in certain cells. Gene therapy is being developed for a range of diseases including inherited monogenic disorders and cardiovascular disease, but it is in the treatment of cancer that this approach has been most evident, resulting in the recent licensing of a gene therapy for the routine treatment of head and neck cancer in China. A variety of virus vectors have been employed to deliver genes to cells to provide either transient (eg adenovirus, vaccinia virus) or permanent (eg retrovirus, adeno-associated virus) transgene expression and each approach has its own advantages and disadvantages. Paramount is the safety of these virus vectors and a greater understanding of the virus-host interaction is key to optimizing the use of these vectors for routine clinical use. Recent developments in the modification of the virus coat allow more targeted approaches and herald the advent of systemic delivery of therapeutic viruses. In the context of cancer, the ability of attenuated viruses to replicate specifically in tumour cells has already yielded some impressive results in clinical trials and bodes well for the future of this approach, particularly when combined with more traditional anti-cancer therapies.     

Selective replicating viral vectors : potential for use in cancer gene therapy

Treatment of cancer is limited by toxicity to normal tissue with standard approaches (chemotherapy, surgery and radiotherapy). The use of selective replicating viral vectors may enable the targeting of gene-modified viruses to malignant tissue without toxic effect. Studies of these vectors have demonstrated tumour-selective replication and minimal evidence of replication in normal tissue. The most advanced clinical results reported involve gene-modified adenoviral vectors. Several completed, histologically confirmed responses to local/regional injection have been induced, particularly in recurrent squamous cell carcinoma involving the head and neck region. Dose limiting toxicity above 10(13) viral particles per injection has been observed. Anti-tumour effect is demonstrable in animal models without evidence of significant toxicity when these vectors are used alone or in combination with chemotherapy, radiation therapy or as gene delivery vehicles. Preliminary clinical trials, particularly with E1B-deleted adenoviruses, report evidence of clinical activity in comparison with expected historical responses. Enhancement in replication selectivity to malignant tissue is also demonstrated preclinically and clinically with an E1B-deleted adenovirus utilising a prostate-specific antigen promoter. Other selective replicating viral vectors such as herpes simplex virus and vaccinia virus have also been explored clinically and suggest evidence of activity in patients with cancer. Modifications may one day enable more aggressive use of these new and exciting therapeutics as systemic gene delivery vehicles.     

Improvements in Gene Therapy

Gene therapy is an interesting approach for the correction of defective genes, the treatment of cancer and the introduction of immunomodulatory genes. Various techniques for gene transfer into cells or tissues have been developed within the last decade; these can be divided generally into viral and nonviral gene transfer systems. Nonviral techniques include the liposome- or gene gun-mediated introduction of therapeutic genes; however, the efficiency of gene transfer by these applications is still very low. In contrast, viruses have optimised their strategies for efficient infection of virtually any cell type in a mammalian organism. The genetic modification of genomes from different virus families (Adenoviridae, Retroviridae, Herpesviridae) led to the development of gene therapy vectors with a similar capacity to infect cells or tissues as that of wild type viruses. In contrast to wild type viruses, gene therapy vectors are engineered to transfer therapeutic genes into the target cells or tissues. In addition, they have lost their capacity for replication in target cells, because of the removal of essential genes, which allows replication only in specialised packaging cell lines engineered for the production of recombinant viruses. Despite considerable progress over the past decade in the generation of gene transfer systems with reduced immunogenic properties, the remaining immunogenicity of many gene therapy vectors is still the major hurdle, preventing their frequent application in clinical trials. Recombinant adenoviruses have been shown to be promising vectors for gene therapy, since they are able to transduce both quiescent and proliferating cells very efficiently. However, a major disadvantage of adenoviral vectors lies in the activation of both the innate and adaptive parts of the recipient's immune system when applied in vivo. The inflammatory responses induced by adenovirus particles can be very strong and can be fatal in patients treated with these adenoviral constructs. Therefore, many experiments have been performed in the effort to prevent these inflammatory responses mediated by adenoviral particles. The depletion of cell populations responsible for these inflammatory responses as well as the application of immunosuppressive drugs have been investigated. Moreover, the generation of less immunogenic adenoviral vectors by further genetic modification within the adenoviral genome has led to vectors with reduced immunogenic properties. Both strategies to reduce inflammatory responses against adenoviral particles are discussed in this review.     

Immune responses to adenovirus and adeno-associated vectors used for gene therapy of brain diseases: the role of immunological synapses in understanding the cell biology of neuroimmune interactions

Researchers have conducted numerous pre-clinical and clinical gene transfer studies using recombinant viral vectors derived from a wide range of pathogenic viruses such as adenovirus, adeno-associated virus, and lentivirus. As viral vectors are derived from pathogenic viruses, they have an inherent ability to induce a vector specific immune response when used in vivo. The role of the immune response against the viral vector has been implicated in the inconsistent and unpredictable translation of pre-clinical success into therapeutic efficacy in human clinical trials using gene therapy to treat neurological disorders. Herein we thoroughly examine the effects of the innate and adaptive immune responses on therapeutic gene expression mediated by adenoviral, AAV, and lentiviral vectors systems in both pre-clinical and clinical experiments. Furthermore, the immune responses against gene therapy vectors and the resulting loss of therapeutic gene expression are examined in the context of the architecture and neuroanatomy of the brain immune system. The chapter closes with a discussion of the relationship between the elimination of transgene expression and the in vivo immunological synapses between immune cells and target virally infected brain cells. Importantly, although systemic immune responses against viral vectors injected systemically has thought to be deleterious in a number of trials, results from brain gene therapy clinical trials do not support this general conclusion suggesting brain gene therapy may be safer from an immunological standpoint.     

Clinical applications of phage-derived sFvs and sFv fusion proteins

Single chain Fv antibodies (sFvs) have been produced from filamentous bacteriophage libraries obtained from immunised mice. MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA), a glycoprotein that is highly expressed in colorectal adenocarcinomas. MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery. Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer. We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2) for use in the ADEPT (antibody directed enzyme prodrug therapy) system and MFE::TNF alpha aims to reduce sequestration and increase tumor concentrations of systemically administered TNF alpha.     

Viruses in therapy--royal road or dead end?

The idea of using viruses as gene vehicles to combat disease is tantalizing for the simplicity of its principle, and for the unlimited perspectives that it raises. Yet the initial enthusiasm gave way to deep skepticism, when the complex challenges became apparent. Issues that hampered clinical successes include the specificity and efficiency of gene delivery; the immune response to viral vectors and targeted cells; standardized and affordable production of vectors; and safety for patients and environment. More recently, some obstacles could be mastered through a better understanding of vector-cell-interactions, vector-induced pathogenesis and principles of vector engineering technologies. First clinical successes became apparent, giving raise to a second waive of effort to exploit viruses in gene therapy. Future challenges include the targeting of stem cells, through receptor tropism and the regulation of gene expression; controlled evasion of host defense; combining the beneficial features of several virus vectors; realistic animal models; and clinical protocols for standardized evaluation of safety and efficacy. Monogenetic disorders were initially regarded as principal targets for gene therapy. However, most clinical trials are now addressing cancer or HIV infection. Cancer gene therapy is aiming at the destruction of malignant cells, whereas 'conventional' gene therapy frequently establishes or restores a long-term function in target cells. Therefore, the requirements for viruses to be used against cancer are fundamentally different from conventional vectors. Host cell death, immune response, and spread of replicating viruses can all contribute to oncolytic efficacy. However, limiting these deleterious effects to tumor cells is mandatory for clinical safety. A number of approaches have been taken to improve the specificity and/or efficacy of cancer virotherapy. Recent studies concerning oncolytic adenoviruses exemplify these strategies.     

Enzymes to die for: exploiting nucleotide metabolizing enzymes for cancer gene therapy

Suicide gene therapy is an attractive strategy to selectively destroy cancer cells while minimizing unnecessary toxicity to normal cells. Since this idea was first introduced more than two decades ago, numerous studies have been conducted and significant developments have been made to further its application for mainstream cancer therapy. Major limitations of the suicide gene therapy strategy that have hindered its clinical application include inefficient directed delivery to cancer cells and the poor prodrug activation capacity of suicide enzymes. This review is focused on efforts that have been and are currently being pursued to improve the activity of individual suicide enzymes towards their respective prodrugs with particular attention to the application of nucleotide metabolizing enzymes in suicide cancer gene therapy. A number of protein engineering strategies have been employed and our discussion here will center on the use of mutagenesis approaches to create and evaluate nucleotide metabolizing enzymes with enhanced prodrug activation capacity and increased thermostability. Several of these studies have yielded clinically important enzyme variants that are relevant for cancer gene therapy applications because their utilization can serve to maximize cancer cell killing while minimizing the prodrug dose, thereby limiting undesirable side effects.     

细胞色素P450与肿瘤的基因治疗

利用细胞色素P450治疗肿瘤是一种新的基因治疗方法,对提高肿瘤化疗的安全性和有效性有非常大的潜力。这种方法的首要目标就是选择性地使细胞色素P450在肿瘤细胞内超量表达。P450酶系降解的前药大部分在肿瘤内活化,提高了肿瘤细胞内药物的相对浓度,减小药物对其它组织的细胞毒性。本文概述了可被细胞色素P450降解的用于肿瘤治疗的前药以及重组细胞色素P450在基因治疗中的应用。     

Biological Approaches to Treatment of Head and Neck Cancer

There are few options for the treatment of advanced squamous cell carcinoma of the head and neck (SCCHN). Chemotherapy in such patients is not associated with survival improvement. However, recent reports suggest that approaches involving biological therapy may provide some benefits. The most promising therapeutics, currently in phase III investigation, involve p53 gene replacement with adenoviral vectors and tumour lysis with tumour-specific oncolytic viruses (ONYX-015). Improved understanding of cancer immunology appears to be opening doors through targeting tumour antigens and upregulation of co-stimulatory molecules with the use of gene therapy. Cellular therapy trials and other approaches involving antiangiogenic factors, superoxide dismutase and the thymidine kinase gene in SCCHN remain preliminary.     

Replicative oncolytic herpes simplex viruses in combination cancer therapies

Viruses that kill the host cell during their replication cycle have attracted much interest for the specific killing of tumor cells and this oncolytic virotherapy is being evaluated in clinical trials. The rationale for using replicative oncolytic viruses is that viral replication in infected tumor cells will permit in situ viral multiplication and spread of viral infection throughout the tumor mass thus overcoming the delivery problems of gene therapy. Improved understanding of the life cycle of viruses has evidenced multiple interactions between viral and cellular gene products, which have evolved to maximize the ability of viruses to infect and multiply within cells. Differences in viral-cell interactions between normal and tumor cells have emerged that have led to the design of a number of genetically engineered viral vectors that selectively kill tumor cells while sparing normal cells. These viruses have undergone further modifications to carry adjunct therapy genes to increase their anti-cancer abilities. Since these viruses kill cells by oncolytic mechanisms differing from standard anticancer therapies, there is an opportunity that synergistic interactions with other therapies might be found with the use of combination therapy. In this review, we focus on the oncolytic Herpes Simplex Virus-1 (HSV-1) vectors that have been examined in preclinical and clinical cancer models and their use in combination with chemo-, radio-, and gene therapies.     

Alphaviral cytotoxicity and its implication in vector development

A great variety of viruses have been engineered to serve as expression vectors. Among them, the alphaviruses Semliki Forest virus and Sindbis virus represent promising tools for heterologous gene expression in a wide variety of host cells. Several applications have already been described in neurobiological studies, in gene therapy, for vaccine development and in cancer therapy. Both viruses trigger stress pathways in the cells they infect, sometimes culminating in the death of the host. This inherent property is either an advantage or a drawback, depending on the type of application.This review covers the development and applications of alphavirus vectors and, as our work has been mainly with Semliki Forest virus, we have focused on this virus with special emphasis on how the understanding of Semliki Forest virus cytotoxicity enables it to be manipulated and used.     

Nonviral vectors for cancer gene therapy: prospects for integrating vectors and combination therapies

Gene therapy has the potential to improve the clinical outcome of many cancers by transferring therapeutic genes into tumor cells or normal host tissue. Gene transfer into tumor cells or tumor-associated stroma is being employed to induce tumor cell death, stimulate anti-tumor immune response, inhibit angiogenesis, and control tumor cell growth. Viral vectors have been used to achieve this proof of principle in animal models and, in select cases, in human clinical trials. Nevertheless, there has been considerable interest in developing nonviral vectors for cancer gene therapy. Nonviral vectors are simpler, more amenable to large-scale manufacture, and potentially safer for clinical use. Nonviral vectors were once limited by low gene transfer efficiency and transient or steadily declining gene expression. However, recent improvements in plasmid-based vectors and delivery methods are showing promise in circumventing these obstacles. This article reviews the current status of nonviral cancer gene therapy, with an emphasis on combination strategies, long-term gene transfer using transposons and bacteriophage integrases, and future directions.     

Adenoviral vectors--how to use them in cancer gene therapy?

Gene therapy is most often described as a technique for introducing the foreign genetic material into cells with a correction of a dysfunctional gene as its final goal. Today, it is well known that cancer is one of the leading causes of mortality in the world. Besides classical methods for cancer treatment new strategies against cancer are needed. Although originally being designed as a treatment for monogenetic illness, soon after, gene therapy appeared as a potential new strategy in cancer therapy. One of the widely used vectors for cancer gene therapy is adenovirus. In this review we have described molecular biology of adenoviruses and basis for construction of adenoviral vectors. We have also described concepts for cancer gene therapy including their in vitro and in vivo application. Special attention is drawn toward retargeting of adenovirus as a new approach in vector design for cancer gene therapy, in order to restrict transgene expression in tumor tissue. This approach uses biophysical as well as genetic characteristics of tumor itself and its supporting tissue, allowing new "bypass" in cancer gene therapy.