Current Gene Therapy - Volume 5, Issue 6, 2005

Novel treatments for cancer will require the development of approaches, which encompass several mechanisms of action, including different modes of cell death and immune stimulation. These novel therapies would work synergistically with the best treatment options currently available. Gene-based therapies constitute a very attractive approach for cancer. There are several approaches currently being pursued; which include oncolytic viruses, which will selectively replicate in cancer cells, causing their destruction; anti-angiogenic targets which aim at depriving the growing tumor of new blood vessels needed for it to spread; targeted toxins as oncolytic molecules; immune-stimulatory targets which aim at eliciting an anti-tumor immune response which should inhibit tumor growth and also metastatic disease. Also, there are efforts directed at inhibiting signal transduction pathways, which are activated in certain cancers and which have been shown to mediate tumor progression. Although many of these approaches have shown excellent efficacy with low or no toxicity in preclinical animal models, their success has not been reproduced in human clinical trials. The reason for this can be several, such as the need to enhance the specificity and efficacy of the gene transfer vectors and the therapies. This can be achieved by the use of targeted vectors, which have been engineered so that they can only infect tumor cells, or the use of cancer cells' specific promoters which will drive the expression of therapeutic molecules exclusively in the tumor. Another important hurdle, especially with the use of oncolytic viruses, it to be able to limit the viral replication to only cancerous cells, this will also prevent untoward systemic toxicity and spread of the vectors onto other tissues throughout the body. The abiltity to generate a systemic long lived antitumor immune response is also a critical advancement which would prove very powerful for the treatment of cancers which can metastasize throughout the body, and also in the case of recurrences. Immune stimulatory approaches mediated through the delivery of genes which induce immune cell recruitment and/or activation is an approach which is being actively pursued. For the clinical implementation of these therapies, it is imperative to be able to monitor disease progression and persistence of the genetic-based therapy in vivo, using non-invasive imaging techniques. This is an exciting area of cancer gene therapy research, which is currently being actively pursued, and will enable the monitoring of the persistence of the therapeutic vectors and also their putative biodistribution. Perhaps the biggest challenge before these gene-based therapies can be sussessfully and safely implemented to treat human cancers, is the issue of the toxicity and biodistribution of the gene transfer vectors used. Also, the pre-existing immune response to the vectors can have very serious deleterious effects, not only by down-modulating the effects of the therapy by inhibiting therapeutic gene expression, but also by causing severe immune-related systemic adverse side effects. Again, this is an area which is the focus of many investigations and has led to the development of better and safer vectors. In this exciting issue, I have aimed at including reviews which span all the areas which I highlighted above, from novel therapeutic targets, to the use of safer and more efficacious gene transfer vectors, to novel non-invasive in vivo imaging approaches. I hope it will be useful to steer renewed enthusiasm to continue working in this exciting field and bring it closer to clinical success. I would like to thank Dr. Ignacio Anegon for giving me the opportunity to put together this exciting Special Issue of the journal, Dr. Pedro Lowenstein for his unfailing support for this project and all our gene therapy efforts aimed at bringing cancer therapeutics closer to the bedside. Very importantly, I wish to convey my infinite gratitude to all the authors, who with zest and enthusiasm, in spite of increasingly mounting academic pressures, took the time to prepare exciting and timely reviews in their areas of expertise. Thank you all for all your support and enjoy!

The most common primary brain tumor in adults is glioblastoma. These tumors are highly invasive and aggressive with a mean survival time of nine to twelve months from diagnosis to death. Current treatment modalities are unable to significantly prolong survival in patients diagnosed with glioblastoma. As such, glioma is an attractive target for developing novel therapeutic approaches utilizing gene therapy. This review will examine the available preclinical models for glioma including xenographs, syngeneic and genetic models. Several promising therapeutic targets are currently being pursued in pre-clinical investigations. These targets will be reviewed by mechanism of action, i.e., conditional cytotoxic, targeted toxins, oncolytic viruses, tumor suppressors/oncogenes, and immune stimulatory approaches. Preclinical gene therapy paradigms aim to determine which strategies will provide rapid tumor regression and long-term protection from recurrence. While a wide range of potential targets are being investigated preclinically, only the most efficacious are further transitioned into clinical trial paradigms. Clinical trials reported to date are summarized including results from conditionally cytotoxic, targeted toxins, oncolytic viruses and oncogene targeting approaches. Clinical trial results have not been as robust as preclinical models predicted, this could be due to the limitations of the GBM models employed. Once this is addressed, and we develop effective gene therapies in models that better replicate the clinical scenario, gene therapy will provide a powerful approach to treat and manage brain tumors.

Pituitary tumors are the most common primary intracranial neoplasms. Although most pituitary tumors are considered typically benign, others can cause severe and progressive disease. The principal aims of pituitary tumor treatment are the elimination or reduction of the tumor mass, normalization of hormone secretion and preservation of remaining pituitary function. In spite of major advances in the therapy of pituitary tumors, for some of the most difficult tumors, current therapies that include medical, surgical and radiotherapeutic methods are often unsatisfactory and there is a need to develop new treatment strategies. Gene therapy, which uses nucleic acids as drugs, has emerged as an attractive therapeutic option for the treatment of pituitary tumors that do not respond to classical treatment strategies if the patients become intolerant to the therapy. The development of animal models for pituitary tumors and hormone hypersecretion has proven to be critical for the implementation of novel treatment strategies and gene therapy approaches. Preclinical trials using several gene therapy approaches for the treatment of anterior pituitary diseases have been successfully implemented. Several issues need to be addressed before clinical implementation becomes a reality, including the development of more effective and safer viral vectors, uncovering novel therapeutic targets and development of targeted expression of therapeutic transgenes. With the development of efficient gene delivery vectors allowing long-term transgene expression with minimal toxicity, gene therapy will become one of the most promising approaches for treating pituitary adenomas.

Tumor formation and growth depends mainly on the inability of the organism to elicit a potent immune response, and on the formation of new blood vessels that enable tumor nutrition. Interleukin-12 (IL-12) therapy can target both processes. And IL-12-based gene therapy may restrict IL-12 production to the relevant site in order to obtain enhanced antitumor activity and reduced toxicity. In the clinical setting, IL-12 gene transfer can be used either to improve the pharmacokinetic/pharmacodynamic profile of the cytokine, to transduce dendritic cells or to enhance the efficiency of antitumor vaccination. It can also synergize with other procedures involving the simultaneous transfer of other transgenes or non-gene based strategies. The strong anti-tumoral power shown in many different animal models has not been found in early clinical trials in which cancer patients were treated by peritumoral injections of autologous fibroblasts producing IL-12, intratumoral injections of an adenoviral vector encoding human IL-12 genes, or intratumoral injection of autologous dendritic cells transduced ex vivo with this same adenoviral vector. However, these trials have set the proof-ofconcept that local production of IL-12 inside a tumor can stimulate tumor infiltration by effector immune cells and that in some cases it is followed by tumor regression. From the many questions that arise after these disappointing results the most relevant concerns the duration and intensity of transgene expression and the capability to monitor this topics in vivo. New vectors that might achieve regulated, long-term production of this cytokine might have better results and merit clinical testing.

Adoptive transfer of antigen-specific T cells is an attractive means to provide cancer patients with immune cells of a desired specificity and the efficacy of such adoptive transfers has been demonstrated in several clinical trials. Because the T cell receptor is the single specificity-determining molecule in T cell function, adoptive transfer of TCR genes into patient T cells may be used as an alternative approach for the transfer of tumor-specific T cell immunity. On theoretical grounds, TCR gene therapy has two substantial advantages over conventional cellular transfer, as it can circumvent the demanding process of in vitro generation of large numbers of specific immune cells and it allows the use of a set of particularly effective TCR genes in large patient groups. Conversely, TCR gene therapy may be associated with a number of specific problems that are not confronted during classical cellular therapy. Here we review our current understanding of the potential and possible problems of TCR gene therapy, as based on in vitro experiments and mouse model systems. Furthermore, we discuss the prospects of clinical application of this gene therapy approach, and the possible barriers on the route towards clinical use.

Over the past decade, adenovirus (Ad)-based vectors have been used extensively in the context of cancer gene therapy. Two basic strategies have been pursued for the use of Ad vectors in cancer gene therapy: 1) approaches aimed at direct tumor cell killing through delivery of replicating oncolytic viruses or non-replicating vectors encoding tumor suppressor genes, suicide genes or anti-angiogenic genes, and 2) immunotherapeutic approaches aimed at inducing host antitumor immune responses that can destroy tumor cells at both primary and metastatic locations. Both strategies offer the potential of selective tumor cell destruction without damage to normal tissues. Extensive pre-clinical and clinical studies have been conducted based on these strategies. Encouraging results have been obtained but robust clinical efficacy remains elusive. Several obstacles limiting the therapeutic activity of Ad vectors have been encountered, including efficiency of tumor cell transduction and inhibition of efficacy by anti-Ad host immune responses. However, expanding knowledge in the areas of Ad biology and tumor biology continues to lead to increasingly sophisticated approaches to address these issues. A review of various Ad-based cancer gene therapy approaches and recent progress in the area are presented herein.

Gene-based therapy is a promising and flexible therapeutic approach to manage diverse types of cancer. The lack of convincing therapeutic success of current gene therapy protocols in part, can be attributed to the inability to monitor gene expression at the targeted site in the living subject. Linking molecular imaging to gene therapy will enable real-time assessment of the therapeutic process and the refinement of treatment protocols. This review will cover two common imaging modalities, positron emission tomography (PET) and bioluminescence imaging (BLI), used in preclinical and clinical gene therapy applications. Strategies to develop more specific and robust cancer gene therapy and imaging approaches will be discussed. Coupling PET to gene therapy of cancer has already been implemented in several clinical studies. This approach would help to improve the efficacy and safety of future gene therapy clinical trials.

The ability to grow and differentiate dendritic cells (DC) ex vivo has allowed their genetic manipulation to enhance immune activation against tumor antigens. Gene engineering of DC can be achieved with a variety of physical methods and using different viral vectors. RNA or DNA transfection, either alone (naked), coated with liposomes or using electroporation or gene guns leads to T cell activation while transgene expression is frequently undetectable. Adenoviral and retroviral vectors have proven to be highly efficient in DC genetic modification, and have been widely used in preclinical models. Other vectors like lentivirus, poxvirus, herpes virus and adeno-associated virus (AAV) can also lead to foreign transgene expression in DC leading to immune cell activation. DC have been genetically engineered to provide constitutive and high level of tumor antigen expression or to introduce genes that further enhance their immune stimulatory ability. The promising results from preclinical animal models and from in vitro human immune cell culture systems have provided a strong rationale to initiate pilot clinical trials. Recently published or communicated clinical experiences and ongoing trials have used defined tumor antigen RNA transfection for prostate carcinoma and melanoma, liposomeencoated DNA transfection for breast or pancreatic cancer, adenoviral vector tumor antigen gene modification for melanoma and small cell lung cancer, and poxvirus-mediated expression of costimulatory molecules for colon carcinoma. These preliminary experiences suggest that genetically modified DC can safely induce T cell responses but few clinical responses.

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.

Despite advances in therapy, advanced ovarian cancer maintains a dismal overall survival of 15-30%. Thus, the need for novel therapeutic modalities exists. Gene therapy represents one such approach and the purpose of this review is to present a logical rationale for the investigation of gene therapy for the treatment of ovarian cancer. The different strategies of gene therapy (molecular chemotherapy (prodrugs), mutation compensation, immunotherapy approaches, altered drug sensitivity, and virotherapy) for cancer treatment are discussed separately with attention to investigations with clinical applicability. Furthermore, the different viral vectors utilized for improvements in targeted therapy are presented. The advancements, discovery, and shortcomings are reviewed which lend itself to future directions. These future directions involve coxsackie-adenovirus receptor (CAR) independent pathways to improve infectivity and specificity to ovarian tumor cells, the potential of utilizing gene therapy as an imaging modality in detecting cancer, and incorporating the recently described technique of RNA interference. Due to the advancements in detection and targeting of ovarian cancer, coupled with the containment to the intraperitoneal cavity, gene therapy remains a promising treatment modality for ovarian cancer.

As cancer gene therapy employing replication-defective vectors has met with limited clinical success, there is renewed interest in using replication-competent viruses for oncolytic virotherapy. In preclinical and clinical studies, various attenuated vaccine strains and engineered virus vectors are currently being tested for their ability to achieve tumorselective cell killing. However, significant improvements are still required in tumor selectivity, cytolytic potency, and modulating immune responses to achieve anti-tumor effects without prematurely terminating virus spread. Recently, we have developed murine leukemia virus (MLV)-based replication-competent retrovirus (RCR) vectors for highly efficient, selective, and persistent gene transfer to cancer cells, and found that such vectors may offer significant advantages as oncolytic agents. In a variety of preclinical models, RCR vectors can achieve efficient and persistent gene delivery as the virus replicates throughout an entire tumor mass after inoculation with initial multiplicities of infection as low as 0.001. When engineered to deliver suicide genes, RCR vectors achieve highly efficient and synchronized cell killing triggered by pro-drug administration, both in culture and in tumor models in vivo. Further strategies are being explored to enhance the packaging capacity, efficiency, and specificity of this vector system through the development of semi-replicative RCR vectors, adenovirus-RCR hybrids, and incorporation of tumor targeting mechanisms via modification of binding tropism and transcriptional regulation. In addition, the ability of these vectors to achieve stable transgene expression in infected tumor cells may allow therapeutic applications that move beyond oncolysis per se.

Treatment of a physiological disorder in the genetic level (gene therapy) and induction of a specific immunity by means of a genetic material (genetic vaccination), are considered two revolutionary approaches for clinical medicine. The implementation strategies for these basic concepts demand a vehicle for nucleic acid delivery. Viral delivery systems, although highly efficient, possess severe limitations in terms of life safety and thus non-viral synthetic systems have become increasingly desirable. Intensive efforts for the last 3 decades enabled the development of a lot of synthetic devices, most of which belong to cationic lipids, peptides and other polymers, but comparatively little attention was paid to inorganic materials. This is the first article aimed at reviewing the dramatic progress of non-viral gene delivery research focusing on the functional inorganic materials. Both biodegradable and non-biodegradable inorganic particles have been fabricated in the nano-scale with the attributes of binding DNA, internalizing across the plasma membrane and finally releasing it in the cytoplasm for final expression of a protein. Some in vivo trials also brought highly satisfactory results demonstrating their potential applications in the clinical medicine.

Despite the fact that the etiopathogenesis of systemic lupus erythematosus is largely unknown, key steps in the pathophysiology of the disease have been recognized and targeted using gene therapy techniques. In animal models of lupus, gene transfer has been used to block the action of pro-inflammatory cytokines and co-stimulatory molecules leading to clinical improvement. In humans, ex vivo experiments have shown the feasibility of gene transfer in live T cells and its potential for restoring normal phenotype in T cells from patients with lupus. Still in experimental phase, gene therapy in lupus promises to correct the aberrant immunological response without the numerous side-effects of the currently used immunosuppressant medications.

Recent progress in gene therapy has increased the need for non-invasive imaging methods that would allow early diagnosis of successful treatment. Magnetic resonance methods, magnetic resonance imaging (MRI) and spectroscopy (MRS), have shown great promise to achieve this goal. The current mini-review describes recent advances in experimental MRI and MRS to detect individual treatment steps in gene therapy of tumours from gene transfection to therapy response. Limitations of the current techniques are also discussed.