Current Gene Therapy - Volume 11, Issue 6, 2011

Gene therapy is a promising approach to achieve long-term expression of targeted genes, and is expected to be a useful tool for the treatment of currently untreatable diseases. Although there are various methods to transfer genes, naked plasmid DNA has attracted researchers because of its ease of handling and safety. However, the design of gene cloning vectors and the choice of gene delivery vectors are important to achieve effective transgene expression. In this special series, these points are well discussed. For example, Prof. Oleg E. Tolmachov provided the principles and technical information for the effective design of plasmid gene vectors. His review also showed how to control transgene expression and how to avoid an immune response induced by the CpG motif in plasmid DNA, for highly efficient transfection efficiency. Prof. Yasufumi Kaneda introduced the concept of liposomes, fusion-mediated gene delivery systems using hemagglutinating virus of Japan (HVJ), and HVJ-envelope vector. This review discussed the usefulness of these delivery vectors for tissue targeting, using conjugation with biocompatible polymers or modification of fusion proteins with targeting of molecules by genetic engineering. Prof. Keiji Itaka and collaborators reviewed the progress and future prospects in the development of polyplex nanomicelles. They summarized the history of the development of cationic polymers, such as polyethylenimine, and described their problems in clinical use. As solutions to these problems, several types of polyplex nanomicelles have been mentioned. The review also indicates the potential of nanomicelles for systemic gene delivery and promising strategies for controlling the profile of transgene expression as well as targeting transgene expression. In addition to these techniques, enhancement of transgene expression could be achieved using special devices. Prof. Tatsufumi Murakami and collaborators reviewed the usefulness of electroporation to improve transfection efficiency in muscle and skin and its application in DNA vaccines against cancer and infectious diseases. Prof. Yoshiaki Taniyama introduced the feasibility of ultrasound with microbubbles. This review provided information on the mechanisms of enhancing transgene expression and showed examples of its usefulness in experimental diseases. Technical review of the parameters of ultrasound and the kind of microbubbles are informative. The clinical and preclinical applications of naked plasmid DNA are summarized in three reviews. Prof. Cevayir Coban and collaborators provided a review on the molecular and immunological mechanisms by which DNA vaccines work and strategies to improve their efficacy. This information will help researchers in the field of DNA vaccines develop better ways to increase immunogenicity of DNA vaccines. Prof. Tetsuya Matoba and collaborators described gene therapy using plasmid DNA for the treatment of atherosclerotic cardiovascular disease, focusing on gene therapy targeting monocyte-mediated inflammation, and a novel nanoparticle-mediated gene delivery system. Prof. Munehisa Shimamura and collaborators introduced the application of plasmid DNA for central nervous system diseases, such as stroke, Parkinson disease, Alzheimer disease, and multiple sclerosis. Overall, these reviews described recent developments, prospects, and problems in the field of naked plasmid DNA-based medicine. To date, its clinical use has not been widely accepted, but several clinical trials are underway. Further improvement of transfection efficiency and safety will shed light on the development of new therapies for untreatable diseases.

For efficient gene delivery, chimeric vectors combining non-viral vectors with viral components have been developed. In particular, increasing attention has been paid to viral fusion activity. HVJ (hemagglutinating virus of Japan; Sendai virus) fuses with the cell membrane at neutral pH, and HN and F, fusion proteins of the virus, contribute to the cell fusion. For fusion-mediated gene transfer, DNA-loaded liposomes were fused with UV-inactivated HVJ to form the fusion liposome, HVJ-liposome. Fusion-mediated delivery protects the molecules incorporated in the liposome from degradation in endosomes and lysosomes before reaching the cytoplasm. Reconstituted pseudovirions of fusion-competent viruses such as HVJ and influenza virus have been also developed by a detergent-lysis and-removal method. A more direct and practical approach is the conversion of fusion-competent virions to non-viral gene delivery particles. Based on this concept, the HVJ envelope vector was developed using inactivated particles of HVJ and has been utilized for gene therapy experiments and functional screening for therapeutic genes. A tissue-targeting HVJ envelope vector was also constructed.

Inflammation in the vascular wall is an essential hallmark during the development of atherosclerosis, for which major leukocytes infiltrated in the lesions are monocytes/macrophages. Therefore, monocyte chemoattractant protein-1 (MCP-1) and its primary receptor CC chemokine receptor 2 (CCR2) are feasible molecular targets for gene therapy to inhibit monocyte/macrophage-mediated inflammation in atherogenesis. A mutant MCP-1 that lacks N-terminal 7 amino acids (7ND) has been shown to heterodimerize with native MCP-1, bind to CCR2 and block MCP-1-mediated monocyte chemotaxis by a dominant-negative manner. Gene therapy using intramuscular transfection with plasmid DNA encoding 7ND showed inhibitory effects on atherosclerosis in hypercholesterolemic mice, and neointima formation after vascular injury in animal models. Bare metal stents for coronary intervention were coated with multiple thin layers of biocompatible polymer with 7ND plasmid. The 7ND gene-eluting stent inhibited macrophage infiltration surrounding stent struts and in-stent neointima formation in rabbit femoral arteries and cynomolgus monkey iliac arteries. Finally, the authors describe new application of 7ND plasmid encapsulated in polymer nanoparticle (NP) that functions as gene delivery system with unique in vivo kinetics. NP-mediated 7ND gene delivery inhibited MCP-1-induced chemotaxis of mouse peritoneal macrophage ex vivo, which may be applicable for the treatment of atherosclerotic cardiovascular disease. In conclusion, anti-inflammatory gene therapy targeting MCP-1/CCR2 signal, with a novel NP-mediated gene delivery system, is a potent therapeutic strategy for the treatment of cardiovascular diseases.

Simple plasmid DNA injection is a safe and feasible gene transfer method, but it confers low transfection efficiency and transgene expression. This non-viral gene transfer method is enhanced by physical delivery methods, such as electroporation and the use of a gene gun. In vivo electroporation has been rapidly developed over the last two decades to deliver DNA to various tissues or organs. It is generally considered that membrane permeabilization and DNA electrophoresis play important roles in electro-gene transfer. Skeletal muscle is a well characterized target tissue for electroporation, because it is accessible and allows for long-lasting gene expression (> one year). Skin is also a target tissue because of its accessibility and immunogenicity. Numerous studies have been performed using in vivo electroporation in animal models of disease. Clinical trials of DNA vaccines and immunotherapy for cancer treatment using in vivo electroporation have been initiated in patients with melanoma and prostate cancer. Furthermore, electroporation has been applied to DNA vaccines for infectious diseases to enhance immunogenicity, and the relevant clinical trials have been initiated. The gene gun approach is also being applied for the delivery of DNA vaccines against infectious diseases to the skin. Here, we review recent advances in the mechanism of in vivo electroporation, and summarize the findings of recent preclinical and clinical studies using this technology.

Plasmid DNA (pDNA)-based gene therapy is a promising strategy for treating many chronic diseases and pathological states. Using pDNA has several advantages such as the sustained synthesis of proteins and peptides in their natives form, ease of the combined use of two or more bioactive factors, and cost-effectiveness. For effective pDNA delivery, cationic polymers are good candidates providing high pDNAstability and functionality.In this article, the development of polyplex nanomicelles composed of poly(ethyleneglycol) (PEG)-polycation block copolymers and pDNA as well as their future prospects is reviewed. The issues of safety and the transfection efficiency are highlighted for considerations of in vivo pDNA delivery.

Plasmids are circular or linear DNA molecules propagated extra-chromosomally in bacteria. Evolution shaped plasmids are inherently mosaic structures with individual functional units represented by distinct segments in the plasmid genome. The patchwork of plasmid genetic modules is a convenient template and a model for the generation of artificial plasmids used as vehicles for gene delivery into human cells. Plasmid gene vectors are an important tool in gene therapy and in basic biomedical research, where these vectors offer efficient transgene expression in many settings in vitro and in vivo. Plasmid vectors can be attached to nuclear directing ligands or transferred by electroporation as naked DNA to deliver the payload genes to the nuclei of the target cells. Transgene expression silencing by plasmid sequences of bacterial origin and immune stimulation by bacterial unmethylated CpG motifs can be avoided by the generation of plasmid-based minimized DNA vectors, such as minicircles. Systems of efficient site-specific integration into human chromosomes and stable episomal maintenance in human cells are being developed for further reduction of the chances for transgene silencing. The successful generation of plasmid vectors is governed by a number of vector design rules, some of which are common to all gene vectors, while others are specific to plasmid vectors. This review is focused both on the guiding principles and on the technical know-how of plasmid gene vector design.

DNA vaccines can induce both humoral and cellular immune responses in animals. Some DNA vaccines are already licensed for infectious diseases such as West Nile virus encephalitis in horses. When used in humans, however, DNA vaccines suffer from lower immunogenicity profiles. Although the reasons for this are poorly understood, various hypotheses have been proposed. This review aims to provide better understanding of the molecular and immunological mechanisms by which DNA vaccines work and how such knowledge can be used to bring about improvements in their efficacy. Recent studies have provided evidence that the ‘adjuvant effect’ of plasmid DNA is mediated by its doublestranded structure. This structure activates stimulator of interferon genes/TANK-binding kinase 1 (STING/TBK1)- dependent innate immune signaling pathways in the absence of Toll-like receptors. Indeed, type-I interferons (IFNs), induced in vivo via the STING/TBK1 pathway, were found to be crucial for both direct- and indirect-antigen presentation via distinct cell types (i.e. dendritic cells (DC) and muscle cells, respectively). Importantly, incorporation of TBK1 into a DNA vaccine was found to enhance the antigen-specific humoral immune responses targeting the Plasmodium falciparum serine repeat antigen (SERA), a candidate vaccine antigen expressed in the blood-stages of human malaria parasites. Thus, the results of these studies may offer new ways to develop DNA vaccines, as well as delivering novel vaccine adjuvants against infectious diseases.

Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common option in the real world because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid DNA-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a Phase III clinical trial did not show sufficient efficiency. Recently, a Phase III clinical trial of hepatocyte growth factor gene therapy in peripheral artery disease (PAD) showed improvement of ischemic ulcers, but it could not salvage limbs from amputation. In addition, a Phase I/II clinical study of fibroblast growth factor gene therapy in PAD extended amputation-free survival, but it seemed to fail in Phase III. In this situation, we and others have developed plasmid DNA-based gene transfer using ultrasound with microbubbles to enhance its efficiency while maintaining safety. Ultrasound-mediated gene transfer has been reported to augment the gene transfer efficiency and select the target organ using cationic microbubble phospholipids which bind negatively charged DNA. Ultrasound with microbubblesis likely to create new therapeutic options inavariety of diseases.

Novel therapeutic strategies utilizing plasmid DNA (pDNA) have been sought for non-treatable neurological disorders, such as ischemic stroke, Parkinson disease (PD), Alzheimer disease (AD), and multiple sclerosis (MS). One strategy is to induce overexpression of growth factors, such as vascular endothelial growth factor (VEGF), glial cell-line derived neurotrophic factor (GDNF), and hepatocyte growth factor (HGF), in the brain. Since ischemic stroke, PD, and AD show damage of neurons, the transfer of pDNA encoding these genes has been examined and shown to protect neurons from damage, associated with a better behavioral outcome. These growth factors have also been shown to accelerate angiogenesis, neurite outgrowth, and neurogenesis in the brain, and overexpression of these factors showed therapeutic effects in cerebral ischemia in rodents. Another application of pDNA is as a “DNA vaccine” to induce immunity against amyloid Aβ in AD, which requires a predominantly Th2 response to avoid autoimmune encephalomyelitis evoked by a Th1 response. Since the combination of pDNA and special devices and/or modification of pDNA could induce a predominantly Th2 response to a targeted antigen, a pDNA-based vaccine would be ideal for AD. Interestingly, pDNA could also induce immune tolerance, and pDNA-based vaccines to induce immune tolerance to autoimmune antibodies have been extensively examined in an animal model of MS. Based on the results, a pDNA vaccine has already been tried in MS patients and reported to be safe and partly effective in phase I/II clinical studies. In this review, we discuss the potential and problems of pDNA-mediated medicine in neurological disorders.

The approaches now united under the term “gene therapy” can be divided into two broad strategies: (1) strategy using the ideology of molecular targeted therapy, but with genes in the role of agents targeted at certain molecular component(s) or pathways presumably crucial for cancer maintenance; (ii) strategy aimed at the destruction of tumors as a whole exploiting the features shared by all cancers, for example relatively fast mitotic cell division. While the first strategy is “true” gene therapy, the second one, as e.g. suicide gene therapy, is more like genetic surgery, when a surgeon just cuts off a tumor being not interested in subtle genetic mechanisms of cancer emergence and progression. This approach inherits the ideology of chemotherapy but escapes its severe toxic effects due to intracellular formation of toxic agents. Genetic surgery seems to be the most appropriate approach to combat cancer, and its simplicity is paradoxically adequate to the super-complexity of tumors. The review consists of three parts: (i) analysis of the reasons of tumor supercomplexity and fatally inevitable failure of molecular targeted therapy, (ii) general principles of the genetic surgery strategy, and (iii) examples of genetic surgery approaches with analysis of their drawbacks and the ways for their improvement.

Short interfering RNAs (siRNAs) are the most commonly used RNA interference (RNAi) triggers. They hold promise as potent therapeutic tools, as demonstrated by recent successful in vivo experiments. However, in addition to triggering intended sequence-specific silencing effects, the reagents of RNAi technology can often cause side effects, including immunological off-target effects. The cellular sensors of foreign RNA, such as RIG-I or Toll-like receptors, involved in innate immune antiviral responses, are activated by RNAi reagents. Stimulation of these pathways results in changes in the cellular transcriptome and proteome that can lead to the inhibition of cell division and growth and eventually apoptosis. An additional undesired effect in the context of research applications may be the misinterpretation of experimental results. To date, a number of the specific features of siRNA structure, sequence and delivery mode that are responsible for these effects have been identified. This knowledge may be helpful in designing safer gene-silencing reagents. In this article we discuss the recent developments in the field of non-specific toxic effects caused by RNAi triggers and their delivery vehicles. These data are critically discussed and evaluated, taking advantage of relevant information compiled in the recently launched RNAimmuno database (http://rnaimmuno.ibch.poznan.pl).

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease characterized by an excessive production of auto-antibodies against double-stranded DNA, nucleosomes, ribonucleoproteins and other nuclear components. Accumulation of self-reactive antibodies leads to immune complex deposition in blood vessels, activation of macrophages and complement, inflammation and subsequent tissue damage in several organs, such as the heart, kidneys, lungs and central nervous system. Although significant progress has been made in the past 30 years of research, no effective specific treatments are currently available. The course of this disease remains unpredictable and patients diagnosed with SLE face long-term treatments with the subsequent economic, social and health burden. From the immunological perspective, SLE is a genetic- and environment-controlled disease that involves almost every constituent of the immune system, including both innate and adaptive immunity. Therefore, several immune cell types and molecules could be susceptible for intervention and modulation to develop more effective and specific treatments. More importantly, such therapies are likely not to induce complete immunosuppression and show reduced side effects on patients. In this article we discuss recent work in the field of SLE pathogenesis with a focus on data that provide clues for therapy design and new treatments.