Current Gene Therapy - Volume 6, Issue 1, 2006

Herpesviruses possess a number of characteristics which make them promising gene delivery vectors. These include their capacity to package large amounts of heterologous DNA and an ability to establish persistent, lifelong infections, where the viral genome remains as a circular non-integrated episome. Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus and is currently being developed as a potential gene delivery vector. In addition to the above properties, HVS-based vectors have the ability to infect a wide range of human cell lines and primary cultures with high efficiencies. Moreover, upon infection the viral genome persists as high copy number, circular, non-integrated episomes which segregate to progeny cells upon division. This allows the HVS-based vector to stably transduce a dividing cell population and provide sustained heterologous gene expression. As such, it offers the characteristics of an artificial chromosome combined with a highly efficient delivery system. This review aims to describe the assessment of HVS-based vectors in both in vitro and in vivo studies, highlighting new developments and possible applications for the treatment of genetic diseases.

A high proportion of oncogenes and tumor suppressor genes encode transcription factors. Deregulated expression or activation and inactivation of transcription factors as well as mutations and translocations play critical roles in tumorigenesis. Furthermore, the majority of oncogenic signaling pathways converge on sets of transcription factors that ultimately control gene expression patterns resulting in tumor formation and progression as well as metastasis. Under normal physiological conditions whole sets of genes with similar functions are regulated by highly specific, tightly regulated upstream transcriptional regulators, whereas in cancer aberrant activation of these transcription factors leads to deregulated expression of multiple gene sets associated with tumor development and progression. The activity of these transcription factors can be modulated by multiple mechanisms including posttranslational modifications. Activation or inactivation of transcription factors promote cancer development, cell survival and proliferation and induce tumor angiogenesis. Since many of these transcription factors are inactive under normal physiological conditions and their expression and activities are tightly regulated, these transcription factors represent highly desirable and logical points of therapeutical interference in cancer development and progression. Three major families of transcription factors have emerged as important players in human cancer and are validated targets in drug discovery for cancer therapy: 1) the NF-kB and AP-1 families of transcription factors, 2) the STAT family members and 3) the steroids receptors. This review aims to elucidate the divergent molecular mechanisms involved in the deregulated activation of transcription factor signaling in malignant transformation, although additional transcription factor families such as the Ets factors, ATF family members, basic helix-loophelix transcription factors etc. are additional critical transcriptional regulators in human cancer. We explore new approaches to specifically inhibit these transcription factors in cancer in order to validate them as a drug targets. Efforts to develop novel viral vectors for therapeutic applications are also discussed.

The attachment of viruses to the host cell surface is a critical stage that will largely condition cell permissivity and productive infection. The understanding of such mechanisms is therefore essential for gene therapy applications involving viruses, as this step will influence both targeting and delivery efficiency of the gene of interest. Viral attachment depends upon the recognition and binding of viral envelope/capsid proteins to specific cellular receptors that can be from very diverse origins. Amongst them are heparan sulphate proteoglycans (HSPGs), a family of glycoproteins which, through the large binding properties of their heparan sulphate (HS) polysaccharide chains, serve as attachment receptor for a great number of viruses. The aim of this review is to provide an update on the multiple roles of HSPGs during viral infection, with a special focus on viruses used as gene delivery vectors. Consequences of HS binding for gene therapy applications will be assessed, as well as the various strategies that have been developed to potentiate the advantages or to overcome the drawbacks resulting from viral vector interaction with HS.

Autoimmune diseases such as type 1 diabetes and multiple sclerosis pose a significant health burden on our society. As a whole, autoimmune diseases affect approximately 6% of the population and are the third largest disease burden after heart disease and cancer. Such pathologic manifestations arise by way of damaging reactions of B-cell derived antibodies and/or T-cells to self-antigens and are triggered by genetic and environmental factors. Currently there is no known cure, with treatment restricted to toxic, long-term immunosuppressive regimes, replacement therapy and in intractable cases, transplantation of autologous or allogeneic haematopoietic stem cells. In experimental models of autoimmunity, gene therapeutic approaches have demonstrated promise in treating the autoimmune diseases. These include delivery of anti-inflammatory cytokines and exploitation of regulatory T cells. However, none of these approaches provide lasting, long-term benefit. We hypothesise that therapeutically transduced haematopoietic stem cells followed by transplantation is an alternative strategy to establish permanent immune tolerance that can not only prevent autoimmunity but also cure these diseases. Our approach is focused on directing autoimmune disease-specific autoantigen expression in the thymus by genetic manipulation of haematopoietic stem cells to establish molecular chimeras. Our hypothesis originates from experimental studies with a mouse model of experimental autoimmune gastritis (EAG) and more recently with the nonobese diabetic (NOD) mouse model for type 1 diabetes (T1D).

Currently, the major drawback of gene therapy is the gene transfection rate. The two main types of vectors that are used in gene therapy are based on viral or non-viral gene delivery systems. There are several non-viral systems that can be used to transfer foreign genetic material into the human body. In order to do so, the DNA to be transferred must escape the processes that affect the disposition of macromolecules. These processes include the interaction with blood components, vascular endothelial cells and uptake by the reticuloendothelial system. Furthermore, the degradation of therapeutic DNA by serum nucleases is also a potential obstacle for functional delivery to the target cell. Cationic polymers have a great potential for DNA complexation and may be useful as non-viral vectors for gene therapy applications. The objective of this review was to address the state of the art in gene therapy using synthetic and natural polycations and the latest strategies to improve the efficiency of gene transfer into the cell.

Despite recent advances in treatment strategies, the overall 5-year survival rate for patients with common epithelial cancers is poor largely because of the difficulty in treating metastatic cancers. Therefore, therapeutic agents are urgently needed that can effectively inhibit both primary epithelial tumors and their metastases. One such agent that has shown promise in preclinical studies is the tumor suppressor/cytokine, melanoma differentiation associated gene-7 also known as interleukin-24 (mda-7/IL-24). Preclinical studies from our and other laboratories have shown that overexpression of MDA-7/IL-24 causes a strong tumor- suppressive effect in many human cancer cells but spares normal cells. This gene therapy also enhances the tumorsuppressive activity of radiotherapy and chemotherapy. Secreted MDA-7 protein that is glycosylated also has been shown to have potent antiangiogenic activity both in vitro and in vivo. Studies examining the immune properties of mda-7 have shown that MDA-7/IL-24 unlike the related IL-10, functions as a Th1 cytokine. Recently, an MDA-7 protein-mediated "bystander effect" on tumor cells has been documented. Building on these findings we successfully completed a Phase I clinical trial of adenovirus-based mda-7 cancer therapy that confirmed the safety of this gene therapy. Phase II trials evaluating the efficacy of mda-7-based gene therapy are warranted. The outcome of such ongoing mda-7-based gene therapy trials will allow us to better understand this therapy's clinical utility.

There is now conclusive evidence that gene therapy can lead to real clinical benefit. Initial enthusiasm has been muted by set-backs related to viral vectors including retroviral oncogenesis and adenoviral inflammatory response. Plasmid- mediated muscle-targeted gene transfer offers the potential of a cost-effective pharmaceutical grade therapy delivered by simple intramuscular injection without the need for anaesthetic, cell culture, transplantation or immunosuppression. This approach is particularly appropriate for long-term circulating therapeutic protein replacement currently requiring repeated injection therapy. Wide-ranging clinical applications include haemophilia, chronic anaemia, growth hormone deficiency and diabetes. Inadequate transgene expression, unregulated protein delivery and immune response have been major limiting factors. Recent innovations including in situ electroporation enabling sustained systemic protein delivery within the therapeutic range are reviewed. Pharmacological and physiological approaches to regulation are discussed in addition to the role of innate and humoral immunity. Translation of advances in all of these areas to clinical success will enable muscle-targeted gene therapy to capitalise on its inherent strengths and realise its long-standing promise.

Enhanced DNA repair in many cancer cells can be correlated to the resistance to cancer treatment, and thus contributes to a poor prognosis. Ionizing radiation and many anti-cancer drugs induce DNA double-strand breaks (DSBs), which are usually regarded as the most toxic types of DNA damages. Repair of DNA DSBs is vital for maintaining genomic stability and hence crucial for survival and propagation of all cellular organisms. Therefore, reducing the capacity of cancer cells to repair DSBs could sensitize tumors to radio/chemotherapy. Many investigators have used gene therapy strategies to down-regulate or inactivate proteins involved in the repair of DSBs in order to reduce the survival of cancer cells. Herein, are reviewed several protein candidates that have been targeted by different gene therapy approaches. Results obtained from in vitro and in vivo experiments are presented and discussed in the perspective of potential gene therapy clinical trials.

The implementation of experimental gene therapy in animal models of neurological diseases is an area of growing interest. Although the neuroendocrine system offers unique advantages for the assessment of in vivo gene therapy, little work has been done in this model. Here we review the core of documented studies in which in vivo gene therapy has been implemented in the neuroendocrine system of rodent models. In the hypothalamus, restorative gene therapy has been successfully implemented in Brattleboro rats, an arginine vasopressin (AVP) mutant which suffers from diabetes insipidus, in Koletsky (faκ/faκ) and in Zucker (fa/fa) rats which have leptin receptor mutations that render them obese, hyperphagic and hyperinsulinemic. In the above models, viral vectors expressing AVP, leptin receptor b and proopiomelanocortin, respectively were stereotaxically injected in the relevant hypothalamic regions. In rats, aging brings about a progressive degeneration and loss of hypothalamic tuberoinfundibular dopaminergic neurons, which are involved in the tonic inhibitory control of prolactin secretion and lactotrophic cell proliferation. Stereotaxic injection of an adenoviral vector expressing Insulin-like Growth Factor-I (IGF-I) was able to correct their chronic hyperprolactinemia and restore tuberoinfundibular dopaminergic (TIDA) neuron numbers. In young and old F-344 male rats, Glial Cell Line-derived Neurotrophic Factor (GDNF) gene delivery in the hypothalamus induced body weight loss. These results suggest that further implementation of gene therapy strategies in neuroendocrine models may be highly rewarding.

There are numerous examples in the literature of gene therapy applications for recessive disorders. There are precious few instances, however, of studies conducted to treat dominantly inherited pathologies. The reasons are simple: there are fewer cases of dominantly inherited diseases on one hand, but mostly it is far easier to correct recessive mutations than dominant ones. Typically recessive mutations cause a loss of (or reduced) gene function which can be compensated for by introduction of a replacement allele into the cell. In contrast, dominant negative mutations not only display impaired function, but also exhibit a novel one that is pathologic to the cell. Treating these conditions by gene therapy implies silencing the dominant allele without altering the expression of the wild-type gene. We describe here different strategies aimed at silencing dominant mutations through mRNA destruction and provide examples of their application to known autosomal dominant diseases. An overview of the most common molecular tools (antisense DNA and RNA, ribozymes and RNA interference) suitable to utilize these strategies is also presented and we discuss the relevant aspects involved in the choice of a particular approach in a gene therapy experiment.

Ciliary neurotrophic factor (CNTF) is a cytokine with neurotrophic activity across a broad spectrum of peripheral and central nervous system (CNS) cells. While its therapeutic potential for CNS diseases has been clear for sometime, the blood brain barrier (BBB) hinders the systemic delivery of CNTF and direct bolus injections are not suitable due to the short half-life of CNTF. One means of overcoming the BBB while providing continuous delivery of CNTF is with immunoisolated cellular implants that produce and deliver CNTF directly to the region of interest. Cells can be protected from host rejection by encapsulating, or surrounding, them within an immunoisolatory, semipermeable membrane that admits oxygen and required nutrients and releases bioactive cell secretions, but restricts passage of larger cytotoxic agents from the host immune defense system. The selective membrane eliminates the need for chronic immunosuppression of the host and allows the implanted cells to be obtained from nonhuman sources. In this review we discuss cell immunoisolation for Huntington's disease and retinitis pigmentosa. These two indications are highlighted because of extensive pre-clinical data supporting the general concept and recent clinical data that both strengthens the theoretical role of CNTF for treating neurodegeneration and justifies additional clinical evaluation in these and other diseases.