Polysaccharide gene transfection agents

Gene delivery is a promising technique that involves in vitro or in vivo introduction of exogenous genes into cells for experimental and therapeutic purposes. Successful gene delivery depends on the development of effective and safe delivery vectors. Two main delivery systems, viral and non-viral gene carriers, are currently deployed for gene therapy. While most current gene therapy clinical trials are based on viral approaches, non-viral gene medicines have also emerged as potentially safe and effective for the treatment of a wide variety of genetic and acquired diseases. Non-viral technologies consist of plasmid-based expression systems containing a gene associated with the synthetic gene delivery vector. Polysaccharides compile a large family of heterogenic sequences of monomers with various applications and several advantages as gene delivery agents. This chapter, compiles the recent progress in polysaccharide based gene delivery, it also provides an overview and recent developments of polysaccharide employed for in vitro and in vivo delivery of therapeutically important nucleotides, e.g. plasmid DNA and small interfering RNA.     

超声波介导的基因输送技术

近年来超声波介导的基因输送技术由于其相对安全性和操作上的简单得到关注。本文对超声波导致的声致穿孔的机制,空化核的作用——增强基因输送的效率,以及细胞和在体基因输送效率作了综述,讨论了超声波介导的基因输送效率的影响因素。此方法充满希望并且载药的空泡可以作为一种新型药物载体在超声作用下实现药物的靶向输送     

Baculovirus as an expression and/or delivery vehicle for vaccine antigens

The baculovirus/insect cell-expression system has proven to be a valuable tool for rapid expression of abundant recombinant proteins for research purposes and has been increasingly exploited for the production of vaccine candidates for commercial use. Furthermore, baculovirus has been discovered to be capable of efficiently transducing a wide variety of mammalian cells, thus leading to the emergence of baculovirus as a novel vector for in vivo and in vitro gene delivery. By incorporating a mammalian expression cassette into the viral genome and/or genetically modifying the baculovirus envelope for immunogen display, baculovirus has also been exploited recently as a vaccine expression/delivery vehicle. This review will focus primarily on past progress and recent advances with regards to employing baculovirus as an in vitro or in vivo expression/delivery vehicle for vaccine immunogens.     

Electroporation gene therapy: new developments in vivo and in vitro

Electroporation-based gene therapy has become a "hot field" for non-viral gene delivery. This review summarizes the progress made in intramuscular and intratumoral electrogenetransfer, which include new applications and modifications. The progress in dendritic cell (DC) and stem cell transfection by use of electroporation has also been discussed. Rapid progress during the past two years clearly demonstrates the great potential of this technology, but there are challenges faced by both in vitro and in vivo applications, which include how to enhance the transfection efficiency for intratumoral delivery, how to extend the duration of gene expression for intramuscular injection, and how to increase the survival rate for in vitro cell transfection. Resolving these issues will shed new light on this field.     

Target delivery of a gene into the brain using the RVG29-oligoarginine peptide

The development of non-viral delivery systems that are capable of mediating an efficient, exclusive, and non-invasive transfer of DNA across the blood-brain barrier into the brain is challenging, but essential for the clinical application of gene therapy to brain diseases. Compared with other non-viral DNA carriers (e.g., lipids or polymers), peptide-based DNA delivery systems have many advantages including the ease of synthesis, low immunogenicity, biocompatibility, and biodegradability in vivo. However, all of the existing peptide-based vehicles for DNA delivery lack selectivity toward cells or tissues, which largely limited their applications in vivo. In this study, we demonstrated that an RVG29-9rR peptide-based DNA delivery system was able to transfect Neuro 2a cells in vitro more efficiently and specifically than Lipofectamine LTX & Plus, one of the most efficient commercially available transfection reagents. More significantly, the peptide mediated efficient and brain-targeting reporter gene expression after intravenous injection into mice. Thus, the results herein suggest a new strategy for brain-targeting DNA delivery in vivo.     

Sonoporation: Mechanical DNA Delivery by Ultrasonic Cavitation

Development of nonviral gene transfer methods would be a valuable addition to the gene-therapy armamentarium, particularly for localized targeting of specific tissue volumes. Ultrasound can produce a variety of nonthermal bioeffects via acoustic cavitation including DNA delivery. Cavitation bubbles may induce cell death or transient membrane permeabilization (sonoporation) on a single cell level, as well as microvascular hemorrhage and disruption of tissue structure. Application of sonoporation for gene delivery to cells requires control of cavitation activity. Many studies have been performed using in vitro exposure systems, for which cavitation is virtually ubiquitous. In vivo, cavitation initiation and control is more difficult, but can be enhanced by cavitation nucleation agents, such as an ultrasound contrast agent. Sonoporation and ultrasonically enhanced gene delivery has been reported for a wide range of conditions including low frequency sonication (kilohertz frequencies), lithotripter shockwaves, HIFU, and evendiagnostic ultrasound (megahertz frequencies). In vitro, a variety of cell lines has been successfully transfected, with concomitant cell killing. In vivo, initial applications have been to cancer gene therapy, for which cell killing can be a useful simultaneous treatment, and to cardiovascular disease. The use of ultrasound for nonviral gene delivery has been demonstrated for a robust array of in vitro and mammalian systems, which provides a fundamental basis and strong promise for development of new gene therapy methods for clinical medicine.     

Gene delivery to tumor cells by cationic polymeric nanovectors coupled to folic acid and the cell-penetrating peptide octaarginine

Target ligand folic acid (FA) and cell-penetrating peptide octaarginine (R8) were coupled with the gene vectors (PEI(600)-CyD, PC) composed of β-cyclodextrin (β-CyD) and low-molecular-weight polyethylenimine (PEI, Mw 600) to form nanovectors for highly efficient gene delivery to tumor cells. The resultant ternary nanocomplexes of FA-PC/R8-PC/pDNA produced excellent gene transfaction abilities in the folate receptor (FR)-positive tumor cells in vitro and in vivo. The FR-mediated endocytosis and the R8-mediated transmembrane functionality together contributed to the high transfection levels. This study provides a promising means to produce gene nanovectors for in vivo applications.     

Nanosphere-mediated delivery of vascular endothelial growth factor gene for therapeutic angiogenesis in mouse ischemic limbs

Polymeric nanosphere-mediated gene delivery may sustain the duration of plasmid DNA (pDNA) administration. In this study, poly(lactic-co-glycolic acid) (PLGA) nanospheres were evaluated as a gene carrier. The pDNA-loaded PLGA nanospheres were formulated with high encapsulation efficiency (87%). The nanospheres sustained release of pDNA for 11 days. The released pDNA maintained its structural and functional integrity. Furthermore, the PLGA nanospheres showed lower cytotoxicity than polyethylenimine (PEI) in vitro and in vivo. The nanospheres with vascular endothelial growth factor (VEGF) gene were injected into skeletal muscle of ischemic limb model, and gene expression mediated by the PLGA nanospheres with VEGF gene was compared to that of PEI/pDNA or naked pDNA in vivo. PLGA nanosphere/pDNA had significantly higher VEGF expression levels in comparison to PEI/pDNA and naked pDNA at 12 days after administration. In addition, gene therapy using PLGA nanospheres resulted in more extensive neovascularization at ischemic sites than both naked pDNA and PEI/pDNA. These results indicated that PLGA nanosphere might be useful as a potential carrier for skeletal muscle gene delivery applications.     

Electrotransfer as a non viral method of gene delivery

Over the last few decades, various vectors have been developed in the field of gene therapy. There still exist a number of important unresolved problems associated with the use of viral as well as non viral vectors. These techniques can suffer from secondary toxicity or low gene transfer efficiency. Therefore an efficient and safe method of DNA delivery still needs to be found for medical applications. DNA electrotransfer is a physical method that consists of the local application of electric pulses after the introduction of DNA into the extra cellular medium. As electrotransfer has proven to be one of the most efficient and simple non viral methods of delivery, it may provide an important alternative technique in the field of gene therapy. The present review focuses on questions related to the mechanism of DNA electrotransfer, i.e. the basic physical processes responsible for the electropermeabilisation of lipid membranes. It also addresses the current limitations of the method as applied to DNA transfer, in particular its efficiency in achieving in vitro gene expression in cells and also its potential use for in vivo gene delivery.     

Progress in cationic lipid-mediated gene transfection: a series of bio-inspired lipids as an example

Over the last several years, various gene delivery systems have been developed for gene therapy applications. Although viral vector-based gene therapy has led to the greatest achievements in animal and human studies, synthetic non-viral vectors have also been developed as they offer several advantages over viral systems, including lower immunogenicity and greater nucleic acid packaging capacity. Nevertheless, the transfection efficiency of the current non-viral gene carriers still needs to be improved, especially as regards direct in vivo transfection. In particular, cationic lipid/nucleic acid complexes (termed lipoplexes) have been the subject of intensive investigation with a view to optimize their performance and to better understand their mechanisms of action, and consequently to design new approaches to overcome the critical barriers of cationic liposome-mediated gene delivery. A possible strategy may rely on considering the membrane constituents and properties of the vast variety of living organisms as a source of inspiration for the design of biocompatible, non-toxic and effective novel artificial liposomal systems. Thus, the present forward-looking review provides an overview of the progress already made during the last years in the field of cationic lipid-mediated gene transfection and also focuses on a series of novel bio-inspired lipids for both in vitro and in vivo gene transfection.     

Cell membrane modification for rapid display of proteins as a novel means of immunomodulation: FasL-decorated cells prevent islet graft rejection

Long-term display of exogenous proteins on the cell surface may have important research and therapeutic implications. We report a novel method for the cell-surface display of proteins that involves generation of a chimeric protein with core streptavidin, biotinylation of cells, and "decoration" with the protein. A chimeric protein with the extracellular portions of FasL (SA-FasL) was efficiently displayed on the cell surface within 2 hr without detectable cellular toxicity. Biotin and SA-FasL persisted on the cell surface for weeks in vitro and in vivo. Immunomodulation with SA-FasL-decorated splenocytes effectively blocked alloreactive responses in naive and presensitized rodents and prevented the rejection of allogeneic pancreatic islets. This approach may serve as an alternative to gene transfer-based expression with broad research and therapeutic applications.     

Studies on neutral, cationic and biotinylated cationic microbubbles in enhancing ultrasound-mediated gene delivery in vitro and in vivo

Ultrasound-mediated gene transfer is emerging as a practical means of facilitating targeted gene expression and is significantly enhanced in the presence of exogenously added microbubbles. This study explores the influence of microbubble surface modifications on their interaction with plasmid DNA and target cells, and the functional consequences of those interactions in terms of ultrasound-mediated gene transfer. Polyethylene glycol-stabilized, lipid-shelled microbubbles with neutral (SDM201), cationic (SDM202) and biotinylated cationic (SDM302) surfaces were compared in terms of their abilities to interact with a luciferase-encoding reporter plasmid DNA and with target cells in vitro. The results demonstrate that the biotinylated cationic microbubble>cationic microbubble>neutral microbubble, in terms of their abilities to interact with target cells and to enhance ultrasound-mediated gene transfer, particularly at low microbubble concentration. The presence of a net positive charge on both cationic microbubbles promoted the formation of microbubble-nucleic acid complexes, although preformation of the complexes prior to addition to target cells inhibited the interaction between the microbubbles and target cells in vitro. The impact of these findings on potential in vitro or ex vivo therapeutic applications of microbubble-enhanced ultrasound-mediated gene transfer is discussed. All three microbubble preparations could be used to facilitate gene transfer in vivo and the potential advantages associated with the use of the cationic microbubbles for targeted gene delivery are discussed.     

Herpesvirus saimiri-based gene delivery vectors

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.     

Hyaluronan microspheres for sustained gene delivery and site-specific targeting

Hyaluronan is a naturally occurring polymer that has enjoyed wide successes in biomedical and cosmetic applications as coatings, matrices, and hydrogels. For controlled delivery applications, formulating native hyaluronan into microspheres could be advantageous but has been difficult to process unless organic solvents are used or hyaluronan has been modified by etherification. Therefore, we present a novel method of preparing hyaluronan microspheres using adipic dihydrazide mediated crosslinking chemistry. To evaluate their potential for medical applications, hyaluronan microspheres are incorporated with DNA for gene delivery or conjugated with an antigen for cell-specific targeting. The results show that our method, originally developed for preparing hyaluronan hydrogels, generates robust microspheres with a size distribution of 5-20mum. The release of the encapsulated plasmid DNA can be sustained for months and is capable of transfection in vitro and in vivo. Hyaluronan microspheres, conjugated with monoclonal antibodies to E- and P-selectin, demonstrate selective binding to cells expressing these receptors. In conclusion, we have developed a novel microsphere preparation using native hyaluronan that delivers DNA at a controlled rate and adaptable for site-specific targeting.     

Lipidots: competitive organic alternative to quantum dots for in vivo fluorescence imaging

The use of fluorescent nanostructures can bring several benefits on the signal to background ratio for in vitro microscopy, in vivo small animal imaging, and image-guided surgery. Fluorescent quantum dots (QDs) display outstanding optical properties, with high brightness and low photobleaching rate. However, because of their toxic element core composition and their potential long term retention in reticulo-endothelial organs such as liver, their in vivo human applications seem compromised. The development of new dye-loaded (DiO, DiI, DiD, DiR, and Indocyanine Green (ICG)) lipid nanoparticles for fluorescence imaging (lipidots) is described here. Lipidot optical properties quantitatively compete with those of commercial QDs (QTracker(?)705). Multichannel in vivo imaging of lymph nodes in mice is demonstrated for doses as low as 2 pmols of particles. Along with their optical properties, fluorescent lipidots display very low cytotoxicity (IC(50) > 75 nM), which make them suitable tools for in vitro, and especially in vivo, fluorescence imaging applications.     

Targeting specific neuronal populations using adeno- and lentiviral vectors: applications for imaging and studies of cell function

We employ viral vectors to address questions related to the function of specific types of neurones in the central control of blood pressure. Adenoviral vectors (AVVs) or lentiviral vectors (LVVs) can be used to visualize specifically living GABAergic or noradrenergic (NAergic) neurones or to interfere with intracellular signalling within these cell types. Here, we review recent in vitro, in situ and in vivo applications of these vectors in the rat brainstem as performed in our laboratories. In organotypic slice cultures prepared from defined cardiovascular brainstem areas, viral vectors were used to study the electrophysiological properties, intracellular signalling and gene expression in selected neuronal phenotypes. In vivo, vectors were microinjected into brainstem nuclei to inhibit specific aspects of cell signalling by expression of dominant negative proteins, for example. Outcomes for cardiovascular control were measured either acutely in situ or chronically in vivo with radio telemetry in freely moving rats. We showed that AVVs and LVVs have distinct properties that need to be considered prior to their application. For example, LVVs can be manufactured very quickly, have no immunogenicity and can be pseudotyped to display higher tropism for neurones than glia. However, comparatively lower production yields of LVVs may limit their use for some types of applications. In contrast, AVVs require a lengthy construction period, are easy to amplify to high yields at moderate cost but may trigger an immune response when used at high titres in vivo. These features make AVVs particularly suitable for in vitro applications. As the two vector types complement each other in several ways we generated a shuttle system that simplifies transfer of transgene cassettes between the backbones of AVVs and LVVs. Thus, AVVs and LVVs are powerful experimental tools that can be used in a variety of experimental designs in vivo, in situ and in vitro.     

Gemini surfactants: a new family of building blocks for non-viral gene delivery systems

Gemini surfactants provide a significant opportunity in the development of new non-viral delivery systems designed for gene therapy applications. This review summarizes the wide range of gemini surfactant structures that have been employed for DNA transfection in vitro. A general observation is that those structures capable of inducing a wide variety of polymorphic structures (lamellar, hexagonal, or cubic phases) demonstrate higher transfection efficiencies. Those compounds whose structures result in pH-dependent changes in aggregate structure similarly show higher levels of transfection. In vivo transfection using gemini surfactants has been demonstrated in only three cases, and in a recent study the transfection was linked to a specific therapeutic response.     

Construction and immunogenicity of a recombinant pseudotype baculovirus expressing the glycoprotein of rabies virus in mice

A pseudotype baculovirus with the glycoprotein of vesicular stomatitis virus (VSV-G) on the envelope was used as a vector for the construction of recombinant baculovirus expressing the G protein of rabies virus (RABV) under the cytomegalovirus (CMV) promoter. The generated recombinant baculovirus (BV-G) efficiently expressed the RABV G proteins in mammalian cells. Intramuscular vaccination with BV-G (10(9) PFU/mouse) induced the production of RABV G-specific neutralizing antibodies and strong T cell responses in mice. Our data clearly indicate that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop a new generation of vaccine against RABV infection.     

Receptor-mediated interleukin-2 gene transfer into human hepatoma cells.

Receptor-mediated gene delivery is an attractive method for gene transfer in vitro and shows promise for in vivo gene therapy applications. In the current study, we have selected the cytokine interleukin-2 (IL-2) gene to explore the feasibility of receptor-mediated gene transfer into human hepatocellular carcinoma HepG2 cells, using Epstein-Barr virus (EBV)-based vectors. We have developed a targeted DNA delivery system for the treatment of liver cancer by gene therapy. This system utilizes the hepatocyte-specific asialoglycoprotein receptor, which is uniquely expressed on liver cell membranes but not present on other cell types. Galactosylated histone, a ligand to the asialoglycoprotein receptors, was synthesized, and a new EBV-based expression vector bearing the human IL-2 cDNA was constructed and conjugated to the ligand through ionic interactions. The ligand/IL-2 DNA complex was able to bind specifically to cell-surface receptors on the target cell and, when incubated with HepG2 cells, resulted in elevated levels of IL-2 gene expression. These results indicate that therapeutic genes like IL-2 in ligand/DNA complex can be transferred into hepatoma cells via the hepatocyte receptor. This study constitutes an encouraging first step in the assessment of receptor-mediated gene transfer as a technique for gene therapy in liver cancer.