Gene delivery systems: Bridging the gap between recombinant viruses and artificial vectors

Although most research in the field of somatic gene therapy has investigated the use of recombinant viruses for transferring genes into somatic target cells, various methods for nonviral gene delivery have also been proposed. Both types of gene delivery systems have advantages and drawbacks. Schematically, viral vectors are particularly efficient for gene delivery, whereas nonviral systems are free of the difficulties associated with the use of recombinant viruses but need to be further optimized to reach their full potential. In order to bridge the gap between viral vectors and synthetic reagents, we discuss here some specific features of the viral vector systems of today that could advantageously be taken into account for the design of improved nonviral gene delivery systems. Indeed, although nonviral systems differ fundamentally from viral systems, one possible approach towards enhanced artificial reagents aims at developing ‘artificial viruses' that mimic the highly efficient processes of viral infection.     

Recent development of nonviral gene delivery systems with virus-like structures and mechanisms

The concept of gene therapy includes not only the addition of normal genes to genetically deficient cells, but also the use of transgenes encoding several peptides that function to enhance the capacity of normal cells or to regulate cell differentiation. The application of gene therapy has been widely considered for various diseases, as well as for the field of tissue engineering. To overcome the problems with viral vectors, a broad range of nonviral systems for gene delivery have been developed, including systems composed of cationic lipids (lipoplexes) and cationic polymers (polyplexes). However, most of these systems are still much less efficient than viral vectors, especially for in vivo gene delivery. Paradoxically, to achieve a maximum transgene expression in the targeted cells, there is no question that natural viruses are the most effective nanocarriers. In this article, we highlight the approaches currently being taken to improve nonviral gene delivery systems so that they better replicate the typical structures and mechanisms of viruses, such as DNA (RNA) condensation in the core, surrounding structures with targeting molecules for specific receptors, as well as the toxic and immunogenic problems which should be avoided, with the ultimate goal of bringing these systems into a clinical setting.     

Gene-delivery systems using cationic polymers

Gene therapy will benefit a range of diseases from single-gene defects, to chronic diseases such as cancer, to vaccination. Initially, gene therapy used viral vectors, but the advantages of nonviral systems are now being fully appreciated. This review focuses on cationic polymers as a delivery system for DNA. The physicochemical characterization of DNA polycation complexes that condense and protect DNA from nuclease digestion are considered, together with further factors such as ligand targeting, endosomal escape, and nuclear localization. Where possible, the relative efficacy of different cationic polymer delivery systems is compared.     

Gene delivery for lung cancer using nonviral gene vectors

Multiple options for the treatment of lung cancer have often been described in the past, including surgery, chemotherapy and radiation, but the therapeutic effect is typically transient and mostly absent with advanced disease. New approaches to the treatment of lung cancer are urgently needed. Gene therapy has been widely proposed as a novel strategy to improve therapy. Although progress has been made using viral vectors, rapid advances in transfection technologies employing nonviral vectors, together with their relatively low toxicity, suggest that nonviral vectors may have significant potential for clinical applications. This paper briefly reviews general principles of gene delivery with emphasis on recent developments in the arena of lung cancer using nonviral vectors (naked DNA, polycationic polymers, cationic liposomes). Employing gene transfer techniques to achieve therapeutically useful levels of expression of therapeutic genes in the lung could provide a new strategy for treatment of lung cancer.     

Recent progress in polymeric gene delivery systems

Considerable progress in polymeric gene delivery systems has been made over the last several years. First generation polymers have been replaced by safer and more efficient carrier systems through molecular functionalization, improving polymer biocompatibility, biological stability, cell-specificity, and intracellular trafficking. Many new polymers have moved from in vitro characterization to preclinical validation in animal models of cancer, diabetes, and cardiovascular disorders. Although the transfection efficiency of most polymeric carriers is still significantly lower than that of viral vectors, their structural flexibility allows for continued improvement in polymer activity. Also, simple manufacturing and scale-up schemes and the low cost of manufacturing are likely to eventually compensate for the performance gap between viral and polymeric vectors and establish clinical recognition and commercialization of polymer-based gene therapy drugs.     

Development of gene delivery system using PLGA nanospheres

The development of nonviral vectors for the efficient and safe delivery to cells has long been awaited to facilitate gene therapy. Recently, many nonviral vectors modified with cationic lipids, cationic polymers, etc. have been reported. However, those nonviral vectors with cationic materials require improved stability, longer duration of gene expression, and reduced cytotoxicity. We successfully prepared mucoadhesive poly (lactide-co-glycolide) nanospheres (PLGA NS) by modifying the nanoparticulate surface with chitosan to improve mucosal peptide absorption after oral and pulmonary administration. Furthermore, we found that nucleic acid, which was not dispersed in the organic solvent, could be dispersed by forming a complex with cationic lipid. Using this phenomenon, polynucleic acids for gene therapy (plasmid DNA, antisense oligonucleotide, small interfering RNA, etc.) can be encapsulated into the matrix of the polymer particles with the emulsion solvent diffusion method. The advantages of this preparation method are its simple process and avoidance of an ultrasonication process for submicronization of particles. The resultant nanospheres show better cellular uptake and different gene therapeutic effects compared with conventional vectors due to their improved adherence to cells and sustained release of polynucleic acid in the cells. In conclusion, chitosan-coated PLGA NS can possibly be applied in nonviral vectors for gene therapy.     

Emerging vectors and targeting methods for nonviral gene therapy

Until recently, nonviral vectors were outside the mainstream of gene transfer technology. Recent problems in clinical trials using viral vectors renewed interest in these methods. The clinical usefulness of nonviral methods is still hindered by their relatively low gene delivery/transgene expression efficiencies. Vectors must navigate a series of obstacles before the therapeutic gene can be expressed. This review considers these barriers and the properties of components of nonviral vectors that are essential for nucleic acid transfer. Although developments of new physical methods (hydrodynamic delivery, ultrasound, electroporation) have made a significant impact on gene transfer efficiency, various chemical carriers (lipids and polymers) have been shown to achieve high-level gene delivery and functional expression. Success of nonviral gene targeting will depend not only on the efficacy, but also safety of this methodology, and this aspect is also discussed. Understanding problems associated with nonviral targeting can also help in designing better viral vectors. In fact, interplay between viral and nonviral technologies should lead to a continued refinement of both methodologies.     

Emerging vectors and targeting methods for nonviral gene therapy

Until recently, nonviral vectors were outside the mainstream of gene transfer technology. Recent problems in clinical trials using viral vectors renewed interest in these methods. The clinical usefulness of nonviral methods is still hindered by their relatively low gene delivery/transgene expression efficiencies. Vectors must navigate a series of obstacles before the therapeutic gene can be expressed. This review considers these barriers and the properties of components of nonviral vectors that are essential for nucleic acid transfer. Although developments of new physical methods (hydrodynamic delivery, ultrasound, electroporation) have made a significant impact on gene transfer efficiency, various chemical carriers (lipids and polymers) have been shown to achieve high-level gene delivery and functional expression. Success of nonviral gene targeting will depend not only on the efficacy, but also safety of this methodology, and this aspect is also discussed. Understanding problems associated with nonviral targeting can also help in designing better viral vectors. In fact, interplay between viral and nonviral technologies should lead to a continued refinement of both methodologies.     

Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery

The successful delivery of therapeutic genes to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful gene therapy. Nonviral gene delivery is currently a subject of increasing attention because of its relative safety and simplicity of use; however, its use is still far from being ideal because of its comparatively low efficiency. Most of the currently available nonviral gene vectors rely on two main components, cationic lipids and cationic polymers, and a variety of functional devices can be added to further optimize the systems. The design of these functional devices depends mainly on our understanding of the mechanisms involved in the cellular uptake and intracellular disposition of the therapeutic genes as well as their carriers. Macromolecules are internalized into cells by a variety of mechanisms, and their intracellular fate is usually linked to the entry mechanism. Therefore, the successful design of a nonviral gene delivery system requires a deep understanding of gene/carrier interactions as well as the mechanisms involved in the interaction of the systems with the target cells. In this article, we review the different uptake pathways that are involved in nonviral gene delivery from a gene delivery point of view. In addition, available knowledge concerning cellular entry and the intracellular trafficking of cationic lipid-DNA complexes (lipoplexes) and cationic polymer-DNA complexes (polyplexes) is summarized.     

Decationized polyplexes for gene delivery

Gene therapy has received much attention in the field of drug delivery. Synthetic, nonviral gene delivery systems have gained increasing attention as vectors for gene therapy mainly due to a favorable immunogenicity profile and ease of manufacturing as compared to viral vectors. The great majority of these formulations are based on polycationic structures, due to their ability to interact with negatively charged nucleic acids to spontaneously form nanoparticles. In recent years, several polycationic systems have demonstrated high transfection in vitro. However, progress toward clinical applications has been slow, mainly because the cationic nature of these systems leads to intolerable toxicity levels, inappropriate biodistribution and unsatisfactory efficiency in vivo, particularly after systemic administration. Decationized polyplexes are a new class of gene delivery systems that have been developed as an alternative for conventional polycation-based systems. The major innovation introduced by decationized polyplexes is that these systems are based on neutral polymers, without any detrimental effect on the physicochemical stability or encapsulation ability, due to the transient presence of cationic charge and disulfide cross-links between the polymer chains by which the nucleic acids are physically entrapped in the particles. This editorial summarizes the most important features of decationized polyplexes and discusses potential implications for the development of new safe and efficient gene delivery systems.     

Nonviral delivery of genetic medicine for therapeutic angiogenesis

Genetic medicines that induce angiogenesis represent a promising strategy for the treatment of ischemic diseases. Many types of nonviral delivery systems have been tested as therapeutic angiogenesis agents. However, their delivery efficiency, and consequently therapeutic efficacy, remains to be further improved, as few of these technologies are being used in clinical applications. This article reviews the diverse nonviral gene delivery approaches that have been applied to the field of therapeutic angiogenesis, including plasmids, cationic polymers/lipids, scaffolds, and stem cells. This article also reviews clinical trials employing nonviral gene therapy and discusses the limitations of current technologies. Finally, this article proposes a future strategy to efficiently develop delivery vehicles that might be feasible for clinically relevant nonviral gene therapy, such as high-throughput screening of combinatorial libraries of biomaterials.     

Efficient, specific and targeted delivery of genes to the lung

Many inherited and acquired pulmonary disorders without satisfactory therapies may be amenable to gene therapy. Despite numerous advances, efficient delivery and expression of the therapeutic transgene at physiological levels for phenotypic correction of disease has proved elusive. This article focuses on various strategies aimed at achieving targeted delivery to the lungs. Both physical methods and biological targeting have been successfully applied in various gene delivery systems. Targeting of different cell types has been achieved by pseudotyping of viral vectors with capsids from different serotypes and modification of nonviral vectors with targeting ligands. Both classes of vectors are discussed with respect to their gene delivery and expression efficiencies, longevity of expression and immunogenicity. Moreover, gene therapy clinical trials for different lung diseases are discussed.     

Advances in polymeric and inorganic vectors for nonviral nucleic acid delivery

Nonviral systems for nucleic acid delivery offer a host of potential advantages compared with viruses, including reduced toxicity and immunogenicity, increased ease of production and less stringent vector size limitations, but remain far less efficient than their viral counterparts. In this article we review recent advances in the delivery of nucleic acids using polymeric and inorganic vectors. We discuss the wide range of materials being designed and evaluated for these purposes while considering the physical requirements and barriers to entry that these agents face and reviewing recent novel approaches towards improving delivery with respect to each of these barriers. Furthermore, we provide a brief overview of past and ongoing nonviral gene therapy clinical trials. We conclude with a discussion of multifunctional nucleic acid carriers and future directions.     

Tumor-selective targeted delivery of genes and antisense oligodeoxyribonucleotides via the folate receptor

Gene therapy is a promising approach for the treatment of cancer. The main obstacle for the clinical application of cancer gene therapy is the lack of gene transfer vectors that are safe, efficacious, and tumor-selective. In recent years, targeted gene delivery through cellular receptors, using either viral or nonviral vectors, is emerging as a novel approach to enhance the efficacy of tumor-selective gene delivery. The folate receptor (FR), which is absent in most normal tissues and elevated in over 90% of ovarian carcinomas and at a high frequency in other human malignancies, is an attractive tumor-selective target. FR-targeted vectors include folate-derivatized adenoviruses, cationic polymers, cationic liposomes, and pH-sensitive liposomes. In addition, FR-targeted liposomes have been evaluated for the targeted delivery of antisense oligodeoxyribonucleotides (ODNs). These vectors have invariably shown impressive FR-selectivity in cell culture assays and, in addition, shown promising tumor-specific gene transfer activity in several in vivo models. There are important theoretical advantages for FR-targeted vectors over traditional non-targeted vectors in therapeutic gene and oligodeoxyribonucleotides delivery in vivo to cancer cells. Further preclinical characterization of these vectors is, therefore, warranted to determine their potential utility in cancer gene therapy.     

Gene vectors for cytokine expression in vivo

The understanding of cytokine networks and the exploitation of these networks for the treatment of immune and inflammatory diseases as well as cancer depend on in vivo delivery of cytokines. Due to instability of recombinant cytokine proteins, investigators have employed cytokine-encoding gene therapy vectors to induce high levels of cytokine expression in vivo. Numerous gene therapy vectors have been developed recently which are suitable for this purpose. Recent advances in the design of adenovirus, adeno-associated virus, poxvirus, retrovirus, lentivirus, and nonviral vectors are described here. Properties of the various vector systems which determine their usefulness for cytokine gene delivery are compared. The implementations of cytokine-encoding gene therapy vectors for analyzing immune responses and for the therapy of inflammatory disorders, immune disease, infections and cancer are reviewed.     

Peptide-guided gene delivery

Although currently less efficient than their viral counterparts, nonviral vectors are under intense investigation as a safer alternative for gene therapy. For successful delivery, the nonviral vector must be able to overcome many barriers to protect DNA and specifically deliver it for efficient gene expression in target cells. The use of peptides as gene delivery vectors is advantageous over other nonviral agents in that they are able to achieve all of these goals. This review will focus on the application of peptides to mediate nonviral gene delivery. By examining the literature over the past 20 years, it becomes clear that no other class of biomolecules are simultaneously capable of DNA condensation, blocking metabolism, endosomal escape, nuclear localization, and receptor targeting. Based on virtually limitless diversity of peptide sequence and function information from nature, it is increasingly clear that peptide-guided gene delivery is still in its infancy.     

Polymeric nanoparticles for gene delivery

Since the evolution of the concept of gene therapy, delivering therapeutic genes to the diseased cells has been a major challenge. Although viral vectors have been shown to be efficient in delivering genes, the issue of their safety is still to be solved. Meanwhile, the field of developing nonviral expression vectors has seen considerable progress. As compared with viruses, these are relatively safe but are confronted with the problem of poor transfection efficiency. With the growing understanding of the biology of gene transfection, and the continued efforts at enhancing the efficiency of nonviral expression vectors, it could soon become a preferred option for human gene therapy. In this review, the potential of polymeric nanoparticles as a gene expression vector is discussed. Furthermore, the importance of understanding the pathophysiology of disease conditions in developing gene expression vectors is discussed in Section 6.     

Mesenchymal stem cells as a novel carrier for targeted delivery of gene in cancer therapy based on nonviral transfection

The success of gene therapy relies largely on an effective targeted gene delivery system. Till recently, more and more targeted delivery carriers, such as liposome, nanoparticles, microbubbles, etc., have been developed. However, the clinical applications of these systems were limited for their several disadvantages. Therefore, design and development of novel drug/gene delivery vehicles became a hot topic. Cell-based delivery systems are emerging as an alternative for the targeted delivery system as we described previously. Mesenchymal stem cells (MSCs) are an attractive cell therapy carrier for the delivery of therapeutic agents into tumor sites mainly for their tumor-targeting capacities. In the present study, a nonviral vector, PEI(600)-Cyd, prepared by linking low molecular weight polyethylenimine (PEI) and β-cyclodextrin (β-CD), was used to introduce the therapeutical gene, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), to MSCs. Meanwhile, the characterization, transfection efficiency, cytotoxicity, cellular internalization, and its mechanism of this nonviral vector were evaluated. The in vitro expression of TRAIL from MSCs-TRAIL was demonstrated by both enzyme-linked immunosorbent assay and Western blot analysis. The lung tumor homing ability of MSCs was further confirmed by the in vitro and in vivo model. Moreover, the therapeutic effects as well as the safety of MSCs-TRAIL on lung metastases bearing C57BL/6 mice and normal C57BL/6 mice were also demonstrated. Our results supported both the effectiveness of nonviral vectors in transferring the therapeutic gene to MSCs and the feasibility of using MSCs as a targeted gene delivery carrier, indicating that MSCs could be a promising tumor target delivery vehicle in cancer gene therapy based on nonviral gene recombination.     

Current status of polymeric gene delivery systems

Gene therapy provides great opportunities for treating diseases from genetic disorders, infections and cancer. To achieve successful gene therapy, development of proper gene delivery systems could be one of the most important factors. Several non-viral gene transfer methods have been developed to overcome the safety problems of their viral counterpart. Polymer-based non-viral gene carriers have been used due to their merits in safety including the avoidance of potential immunogenecity and toxicity, the possibility of repeated administration, and the ease of the establishment of good manufacturing practice (GMP). A wide range of polymeric vectors have been utilized to deliver therapeutic genes in vivo. The modification of polymeric vectors has also shown successful improvements in achieving target-specific delivery and in promoting intracellular gene transfer efficiency. Various systemic and cellular barriers, including serum proteins in blood stream, cell membrane, endosomal compartment and nuclear membrane, were successfully circumvented by designing polymer carriers having a smart molecular structure. This review explores the recent development of polymeric gene carriers and presents the future directions for the application of the polymer-based gene delivery systems in gene therapy.     

Octaarginine-modified liposomes: enhanced cellular uptake and controlled intracellular trafficking

Gene therapy is a promising new approach for treating a variety of genetic and acquired diseases. While viral vectors are highly efficient for gene therapy, their use is associated with high toxicity and immunogenicity. Synthetic or nonviral vectors are attractive alternatives to viral vectors because of their low immunogenicity and low acute toxicity. The main disadvantage of the nonviral vectors is the low transfection efficiency compared to viral vectors. Novel functional devices to enhance the transfection activities of nonviral vectors are needed. In this review, we discuss the modification of liposomal drug carriers with a novel functional device, the octaarginine (R8) peptide, for drug and gene delivery. Decoration of liposomes with R8 enhanced their cellular uptake. In addition, by optimizing the density of the peptide as well as its topology, the liposomes could be internalized via clathrin-independent pathways, which improved the intracellular trafficking through avoiding lysosomal degradation. A special emphasis is given to the need for optimizing the conditions of using the peptide to not only enhance the cellular uptake but also to improve the intracellular trafficking of its cargos. In addition, the use of R8-modified liposomes and nano-particles in gene delivery is discussed.