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.     

Nonviral Gene Delivery: Principle, Limitations, and Recent Progress

Gene therapy is becoming a promising therapeutic modality for the treatment of genetic and acquired disorders. Nonviral approaches as alternative gene transfer vehicles to the popular viral vectors have received significant attention because of their favorable properties, including lack of immunogenicity, low toxicity, and potential for tissue specificity. Such approaches have been tested in preclinical studies and human clinical trials over the last decade. Although therapeutic benefit has been demonstrated in animal models, gene delivery efficiency of the nonviral approaches remains to be a key obstacle for clinical applications. This review focuses on existing and emerging concepts of chemical and physical methods for delivery of therapeutic nucleic acid molecules in vivo. The emphasis is placed on discussion about problems associated with current nonviral methods and recent efforts toward refinement of nonviral approaches.     

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 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.     

Innate immune response induced by gene delivery vectors

Gene therapy is a clinical strategy that has the potential to treat an array of genetic and nongenetic diseases. Vectors for gene transfer are the essential tools of gene therapy. For gene therapy to be successful, an appropriate amount of the therapeutic gene must be delivered into the target cells without substantial toxicity. A major limitation of the use of gene therapy vectors is the innate immune responses triggered by systemic administration of such vectors. It is essential to overcome vector-mediated innate immune responses, such as production of inflammatory cytokines, the maturation of antigen-presenting cells and tissue damage, because the induction of these responses not only shortens the period of gene expression but also leads to serious side effects. Viral vectors (for example, adenovirus (Ad) vectors) have been assumed to be more potent in inducing innate immune responses in spite of their high transduction efficiency since they contain pathogenic proteins. However, recent studies have demonstrated that not only viral vectors but also nonviral vectors, such as lipoplex (liposome/plasmid DNA complex), can induce innate immune responses. Indeed, nonviral vectors including lipoplex induce comparable or larger levels of innate immune response than viral vectors. In this review, we present an overview of the innate immune responses induced by Ad vector and lipoplex, which are used primarily for in vivo gene transfer.     

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.     

Polymeric vectors for ocular gene delivery

Gene therapy holds promise for the treatment of many inherited and acquired diseases of the eye. Successful ocular gene therapy interventions depend on efficient gene transfer to targeted cells with minimal toxicity. A major challenge is to overcome both intracellular and extracellular barriers associated with ocular gene delivery. Numerous viral and nonviral vectors were explored to improve transfection efficiency. Among nonviral delivery systems, polymeric vectors have gained significant attention in recent years owing to their nontoxic and non-immunogenic nature. Polyplexes or nanoparticles can be prepared by interaction of cationic polymers with DNA, which facilitate cellular uptake, endolysosomal escape and nuclear entry through active mechanisms. Chemical modification of these polymers allows for the generation of flexible delivery vectors with desirable properties. In this article several synthetic and natural polymeric systems utilized for ocular gene delivery are discussed.     

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.     

Role of calcium in gene delivery

The treatment of genetic diseases using therapeutic gene transfer is considered to be a significant development. This development has brought with it certain limitations, and the process of overcoming these barriers has seen a drastic change in gene delivery. Many metal ions such as Mg2+, Mn2+, Ba2+ and, most importantly, Ca2+ have been demonstrated to have significant roles in gene delivery. Recently, calcium phosphate alone, or in combination with viral and nonviral vectors, was found to exert a positive effect on gene transfer when incorporated in the colloidal particulate system, which is an advancing approach to gene delivery. This review elaborates on various successful methods of using calcium in gene delivery.     

Viral vectors for gene transfer: a review of their use in the treatment of human diseases

The efficient delivery of therapeutic genes and appropriate gene expression are the crucial issues for clinically relevant gene therapy. Viruses are naturally evolved vehicles which efficiently transfer their genes into host cells. This ability made them desirable for engineering virus vector systems for the delivery of therapeutic genes. The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. These are the major reasons why viral vectors derived from retroviruses, adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. Among these vector systems, retrovirus vectors represent the most prominent delivery system, since these vectors have high gene transfer efficiency and mediate high expression of therapeutic genes. Members of the DNA virus family such as adenovirus-, adeno-associated virus or herpesvirus have also become attractive for efficient gene delivery as reflected by the fast growing number of clinical trials using these vectors. The first clinical trials were designed to test the feasibility and safety of viral vectors. Numerous viral vector systems have been developed for ex vivo and in vivo applications. More recently, increasing efforts have been made to improve infectivity, viral targeting, cell type specific expression and the duration of expression. These features are essential for higher efficacy and safety of RNA- and DNA-virus vectors. From the beginning of development and utilisation of viral vectors it was apparent that they harbour risks such as toxicities, immunoresponses towards viral antigens or potential viral recombination, which limit their clinical use. However, many achievements have been made in vector safety, the retargeting of virus vectors and improving the expression properties by refining vector design and virus production. This review addresses important issues of the current status of viral vector design and discusses their key features as delivery systems in gene therapy of human inherited and acquired diseases at the level of laboratory developments and of clinical applications.     

Pharmaceutical emulsions: a new approach for gene therapy

The concept of gene therapy involves the experimental transfer of a therapeutic gene into an individual’s cells and tissues to replace an abnormal gene aiming to treat a disease, or to use the gene to treat a disease just like a medicine, improving the clinical status of a patient. The achievement of a foreigner nucleic acid into a population of cells requires its transfer to the target. Therefore, it is essential to create carriers (vectors) that transfer and protect the nucleic acid until it reaches the target. The obvious disadvantages of the use of viral vectors have directed the research for the development of a nonviral organized system such as emulsions. In fact, recently, there has been an increase of interest in its use in biotechnology as a nonviral vector for gene therapy. This review focuses on the progress of cationic emulsions and the improvement of the formulations, as a potential delivery system for gene therapy.     

Gene therapy of cancer

Gene therapy was initially thought of as a means to correct single gene defects in hereditary disease. Since then, cancer has become the most important indication for gene therapy in clinical trials. In the foreseeable future, the best way to achieve reasonable intratumoral concentrations of a transgene with available vectors will be direct intratumoral injection with or without the help of various techniques such as endoscopy or computed tomography guidance. At present, viral and nonviral methods of gene transfer are used either in vivo or ex vivo/in vitro. The most important viral vectors currently used in clinical trials are retroviruses, adenoviruses, adeno-associated viruses and herpes viruses. However, none of them satisfies all the criteria of an ideal gene therapeutic system, and vectors with only minimal residues of their parent viruses (gutless vectors) and completely synthetic viral vectors are gaining importance. Nonviral methods of gene therapy include liposomes, injection of vector-free (naked) DNA, protein-DNA complexes, delivery by gene gun, calcium-phosphate precipitation, electroporation and intracellular microinjection of DNA. The first clinical trial of human gene therapy was performed in 1990 and since then more than 5000 patients have been treated worldwide in over 400 clinical protocols. Side effects were rare and mostly mild in all of these studies and expression of the transgene was demonstrated in patients in vivo. Despite anecdotal reports of therapeutic responses in some patients, there is still no unequivocal proof of clinical efficacy of most approaches to gene therapy in cancer, primarily due to very low transduction and expression efficacy in vivo of available vectors. Strategies for gene therapy of cancer can be subdivided into four basic concepts: 1) strengthening of the immune response against a tumor, 2) repair of cell cycle defects caused by loss of tumor suppressor genes or inappropriate activation of oncogenes, 3) suicide gene strategies and 4) inhibition of tumor angiogenesis. Gene marker studies and gene protection of normal tissue are also discussed.     

Role of calcium in gene delivery

The treatment of genetic diseases using therapeutic gene transfer is considered to be a significant development. This development has brought with it certain limitations, and the process of overcoming these barriers has seen a drastic change in gene delivery. Many metal ions such as Mg²⁺, Mn²⁺, Ba²⁺ and, most importantly, Ca²⁺ have been demonstrated to have significant roles in gene delivery. Recently, calcium phosphate alone, or in combination with viral and nonviral vectors, was found to exert a positive effect on gene transfer when incorporated in the colloidal particulate system, which is an advancing approach to gene delivery. This review elaborates on various successful methods of using calcium in gene delivery.     

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.     

Electroporation for targeted gene transfer

The utilisation of nonviral gene delivery methods has been increasing steadily, however, a drawback has been the relative low efficiency of gene transfer with naked DNA compared with viral delivery methods. In vivo electroporation, which has previously been used clinically to deliver chemotherapeutic agents, also enhances the delivery of plasmid DNA and has been used to deliver plasmids to several tissue types, particularly muscle and tumour. Recently, a large number of preclinical studies for a variety of therapeutic modalities have demonstrated the potential of electrically mediated gene transfer. Although clinical trials using gene transfer with in vivo electroporation have not as yet been realised, the tremendous growth of this technology suggests that the first trials will soon be initiated.     

Nonviral approach for targeted nucleic acid delivery

Despite their relatively lower efficiency, nonviral approaches are emerging as safer alternatives in gene therapy to viral vectors. Delivery of nucleic acids to the target site is an important factor for effective gene expression (plasmid DNA) or knockdown (siRNA) with minimal side effects. Direct deposition at the target site by physical methods, including ultrasound, electroporation and gene gun, is one approach for local delivery. For less accessible sites, the development of carriers that can home into the target tissue is required. Cationic peptides, lipoplexes, polyplexes and nanoplexes have been used as carriers for delivery of nucleic acids. Targeting ligands, such as cell targeting peptides, have also been applied to decorate delivery vehicles in order to enhance their efficacy. This review focuses on delivery strategies and recent progress in non-viral carriers and their modifications to improve their performance in targeting and transfection.     

Current prospects for mRNA gene delivery

Replication-deficient viruses have been used most successfully in the field of gene therapy because of their high transfection efficiency. However, the risk of insertional mutagenesis and induction of unwanted immune responses remains still critical for their safe application. On the other hand, nonviral vectors have been intensively investigated for plasmid DNA (pDNA) delivery as a safer alternative although their gene transfer efficiency is still many folds lower than for viral vectors, which has been predominately attributed to the insufficient transport of pDNA into the nucleus. Instead of pDNA, messenger RNA (mRNA) has recently emerged as an attractive and promising alternative in the nonviral gene delivery field. This strategy combines several advantages compared to pDNA: (i) the nuclear membrane, which is a major obstacle for pDNA, can be avoided because mRNA exerts its function in the cytoplasm; (ii) the risk of insertional mutagenesis can be excluded; (iii) the determination and use of an efficient promoter is omitted; (iv) repeated application is possible; (v) mRNA is also effective in non-dividing cells, and (vi) vector-induced immunogenicity may be avoidable. In this review, we summarize recent improvements of mRNA gene delivery and discuss its opportunities for the usage in gene therapy.     

Glycosylated cationic liposomes for cell-selective gene delivery

Cationic liposomes have been considered as a potential nonviral vector for gene delivery because they possess low immunogenicity, unlike viral vectors. The gene transfer efficiency of cationic liposomes is lower than that ofviral vectors, but recent advances have shown that it is possible to enhance the gene expression levels of cationic liposomes. The main problem with cationic liposomes seems to be the lack of organ or cell selectivity because the lung has the highest level of gene expression after intravenous injection. Applying cell-specific targeting technology to liposomes would improve in vivo gene delivery and reduce any unexpected side effects. Both liver parenchymal and non-parenchymal cells exclusively express large numbers of high-affinity asialoglycoprotein and mannose receptors, respectively. Receptor-mediated gene delivery systems are able to introduce foreign DNA into specific cell types in vivo. However, we have confirmed that not only the nature of the ligands grafted to carriers but also the overall physicochemical properties of the complexes need to be optimized for effective cell-selective targeting of plasmid DNA. In this article, we attempt to evaluate a gene delivery system based on the physicochemical properties of plasmid DNA/glycosylated cationic complexes.     

Electroporation for targeted gene transfer

The utilisation of nonviral gene delivery methods has been increasing st-eadily, however, a drawback has been the relative low efficiency of gene transfer with naked DNA compared with viral delivery methods. Invivo electroporation, which has previously been used clinically to deliver chemotherapeutic agents, also enhances the delivery of plasmid DNA and has been used to deliver plasmids to several tissue types, particularly muscle and tumour. Recently, a large number of preclinical studies for a variety of therapeutic modalities have demonstrated the potential of electrically mediated gene transfer. Although clinical trials using gene transfer with invivo electroporation have not as yet been realised, the tremendous growth of this technology suggests that the first trials will soon be initiated.     

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.