Nonviral gene therapy and its delivery systems

Nonviral gene therapy has significant clinical potential, yet its therapeutic utility has been hindered by low transfection efficiency due to a combination of extracellular and intracellular barriers. Recent developments in formulation and delivery methodology have allowed a number of advances toward high efficiency gene delivery to various cell types and organs. In particular, the extracellular and intracellular pharmacokinetics of plasmid DNA trafficking are better understood in a number of cell systems. Using cationic lipid or polymers (often with receptor targeting), more than 10(5) plasmids can be delivered to a single cell. Endosomolytic agents promote endosome disruption, and include: weak bases, proton-sponge polymers, fusogenic peptides, viral particles, and photosensitizing compounds. Both classical and nonclassical nuclear localization signal (NLS) peptides have also been tested for enhancement of the probability of nuclear import events, a major rate-limiting step in DNA delivery to nondividing cells. For example, the M9 sequence from heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) protein, a nonclassical NLS, has been found to increase gene expression level by more than 10 to 150-fold in a variety of cell types. This review will concentrate on the current understandings of the basic mechanisms of nonviral gene delivery and new approaches in the field.     

Polycation gene delivery systems: escape from endosomes to cytosol

Clinical success of gene therapy based on oligonucleotides (ODNs), ribozymes, RNA and DNA will be greatly dependent on the availability of effective delivery systems. Polycations have gained increasing attention as a non-viral gene delivery vector in the past decades. Significant progress has been made in understanding complex formation between polycations and nucleic acids, entry of the complex into the cells and subsequent entry into the nucleus. Sophisticated molecular architectures of cationic polymers have made the vectors more stable and less susceptible to binding by enzymes or proteins. Incorporation of specific ligands to polycations has resulted in more cell-specific uptake by receptor-mediated mechanisms. However, there are still other barriers limiting the transfection efficiency of polycation gene delivery systems. There is a consensus that polycation-DNA complexes (polyplexes) enter cells via the endocytotic pathway. It is not clearly understood, however, how the polyplexes escape (if they do) from endosomes, how DNA is released from the polyplexes or how the released DNA is expressed. The primary focus of this article is to review various polycation gene delivery systems, which are designed to translocate DNA from endosomes into cytosol. Many polycation gene delivery systems have tried to mimic the mechanisms that viruses use for the endosomal escape. Polycation gene delivery systems are usually coupled with synthetic amphipathic peptides mimicking viral fusogenic peptides, histidine-based gene delivery systems for pH-responsive endosomal escape, polycations with intrinsic endosomolytic activity by the proton sponge mechanism and polyanions to mimic the anionic amphiphilic peptides.     

Structure and design of polycationic carriers for gene delivery

The development of safe and effective gene delivery methods is a major challenge to enable gene therapy or DNA vaccines to become a reality. Currently there are two major approaches for delivery of genetic material, viral and non-viral. The majority of on-going clinical trials in gene therapy or DNA vaccines use retroviruses and adenoviruses for delivering genetic materials. Viral delivery systems are far more effective than non-viral delivery however there are concerns regarding toxicity, immunogenicity and possible integration of viral genetic material into the human genome. Given the negative charge of the phosphate backbone of DNA, polycationic molecules have been the major focus as carriers of DNA. There are several physiological barriers to overcome for effective systemic delivery of DNA. The ideal vector must be stable in the systemic circulation, escape the reticuloendothelial system, able to extravasate tissues, enter the target cell, escape lysosomal degradation and transport DNA to the nucleus to be transcribed. With increasing understanding of the physicochemical properties essential to overcome the various barriers, it is possible to apply rational design to the cationic carriers. A number of poly-amino acids, cationic block co-polymers, dendrimers and cyclodextrins have been rationally designed to optimize gene delivery. This review will discuss approaches that have been used to design various synthetic polycations with enhanced DNA condensing ability, serum stability and endosomolytic capability for efficient gene transfer in vitro and in vivo.     

Self-assembly of cyclodextrin complexes: aggregation of hydrocortisone/cyclodextrin complexes

Oral delivery of gene therapeutics would facilitate treatment of local intestinal disease, including colon cancer and inflammatory bowel disease, thus avoiding invasive surgery. The aims of this study were to investigate; if the orientation of the lipid tail on the cyclodextrin (CD) influenced the efficacy of a novel poly-6-cationic amphiphilic CD to transfect intestinal enterocytes; the endocytotic uptake pathway(s), and, the intracellular trafficking of the CD·DNA complexes. Inhibitors of clathrin- and caveolae-mediated endocytosis and macropinocytosis were used to determine the mechanism(s) of CD·DNA uptake by both undifferentiated and differentiated Caco-2 cells. Cell surface heparan sulphate proteoglycans were involved in the association of CD·DNA complexes with undifferentiated Caco-2 cells. Complexation of pDNA with CD facilitated significant levels of pDNA uptake and gene expression (comparable to PEI) in both undifferentiated and differentiated Caco-2 cells. Disruption of intracellular vesicular trafficking reduced transfection activity. CD was also capable of transfecting the more physiologically relevant differentiated Caco-2 model. Macropinocytosis was responsible for the uptake of CD·DNA transfection complexes by both undifferentiated and differentiated Caco-2 cells. The ability of this novel CD to transfect differentiated intestinal cells indicates the potential of this vector for oral gene delivery.     

Caveolae: from basic trafficking mechanisms to targeting transcytosis for tissue-specific drug and gene delivery in vivo

Continuous endothelium and epithelium create formidable barriers to endogenous molecules as well as targeted drug and gene therapies in vivo. Caveolae represent a possible vesicular trafficking pathway through cell barriers. Here we discuss recent discoveries regarding the basic function of caveolae in transport including transcellular trafficking, intracellular trafficking to distinct endosomes, and molecular mechanisms mediating their budding, docking and fusion (dynamin and SNARE machinery). New technologies to purify and map caveolae as well as generate new probes selectively targeting caveolae in vivo provide valuable tools not only for investigating caveolar endocytosis/transcytosis but also elucidating new potential applications for site-directed treatment of many diseases. Vascular targeting of the caveolar trafficking pathway may be a useful strategy for achieving tissue-specific pharmacodelivery that also overcomes key, normally restrictive cell barriers which greatly reduce the efficacy of many therapies in vivo.     

Non-viral vectors in cancer gene therapy: principles and progress

This review focuses on the use of synthetic (non-viral) delivery systems for cancer gene therapy. Therapeutic strategies such as gene replacement/mutation correction, immune modulation and molecular therapy/'suicide' gene therapy type approaches potentially offer unique and novel ways of fighting cancer, some of which have already shown promise in early clinical trials. However, the specific and efficient delivery of the genetic material to remote tumors/metastases remains a challenge, which is being addressed using a variety of viral and non-viral systems. Each of these disparate systems has distinct advantages and disadvantages, which need to be taken into account when a specific therapeutic gene is being used. The review concentrates on particulate gene delivery systems, which are formed through non-covalent complexation of cationic carrier molecules (e.g. lipids or polymers) and the negatively charged plasmid DNA. Such systems tend to be comparatively less efficient than viral systems, but have the inherent advantage of flexibility and safety. The DNA-carrier complex acts as a protective package, and needs to be inert and stable while in circulation. Once the remote site has been reached the complex needs to efficiently transfect the targeted (tumor) cells. In order to improve overall transfection specificity and efficiency it is necessary to optimize intracellular trafficking of the DNA complex as well as the performance after systemic administration. Common principles and specific advantages or disadvantages of the individual synthetic gene delivery systems are discussed, and their interaction with tumor-specific and generic biological barriers are examined in order to identify potential strategies to overcome them.     

Alternate routes for drug delivery to the cell interior: pathways to the Golgi apparatus and endoplasmic reticulum

The targeted delivery of drugs to the cell interior can be accomplished by taking advantage of the various receptor-mediated endocytic pathways operating in a particular cell. Among these pathways, the retrograde trafficking pathway from endosomes to the Golgi apparatus, and endoplasmic reticulum is of special importance since it provides a route to deliver drugs bypassing the acid pH, hydrolytic environment of the lysosome. The existence of pathways for drug or antigen delivery to the endoplasmic reticulum and Golgi apparatus has been to a large extent an outcome of research on the trafficking of A/B type-bacterial or plant toxins such as Shiga toxin within the cell. The targeting properties of these toxins reside in their B subunit. In this article we present an overview of the multiplicity of pathways to deliver drugs intracellularly. We highlight the retrograde trafficking pathway illustrated by Shiga toxin and Shiga-like toxin, and the potential role of the B subunit of these toxins as carriers of drugs, antigens and imaging agents.     

Effect of DC/mDC iontophoresis and terpenes on transdermal permeation of methotrexate: in vitro study

We have reported previously that a basic peptide, arginine peptide, can be used as an efficient system for delivery of foreign genes. In this work, to better understand the mechanism of arginine peptide-mediated gene delivery, we further evaluated the process of cellular uptake and nuclear localization of the peptide/DNA complex. To investigate the effect of cellular proteoglycans on arginine peptide/DNA complexes, interactions between polyanionic glycosaminoglycans (GAGs) and peptide/DNA complexes were examined by the ethidium bromide interaction assay. Sulfated GAGs were found to relax the complexed DNA at low peptide/DNA charge ratios. Condensed peptide/DNA complexes facilitate cellular uptake, but their mechanism of uptake is poorly understood. Studies of various endocytosis inhibitors suggested that the peptide/DNA complex internalization involved the caveolar-related endocytosis pathway. A critical step in the gene delivery is the cytosol-to-nucleus transport of exogenous DNA following initial complex uptake. Nuclear localization of peptide/DNA complex was confirmed by confocal laser scanning microscopic observation. Further, we show that transfections with peptides result in an early accumulation of plasmid DNA in the nucleus of growth-arrested cells, which suggest nuclear transport. To assess the potential for arginine peptide as an agent for therapeutic gene delivery, in vivo complexed DNA transduction studies were performed. Mice were injected subcutaneously with the reporter gene beta-galactosidase, resulting in high levels of gene expression in dermal tissue.     

Development of recombinant cationic polymers for gene therapy research

Cationic polymers created through recombinant DNA technology have the potential to fill a void in the area of gene delivery. The recombinant cationic polymers to be discussed here are amino acid based polymers synthesized in E. coli with the purpose to not only address the major barriers to efficient gene delivery but offer safety, biodegradability, targetability and cost-effectiveness. This review helps the readers to get a better understanding about the evolution of recombinant cationic polymers; and the potential advantages that they could offer over viral and synthetic non-viral vectors for gene delivery. It also discusses some of the major challenges that must be addressed in future studies to turn recombinant polymers into clinically effective gene delivery systems. Recent advances with the biopolymer design suggest that this emerging new class of gene delivery systems has the potential to address some of the major barriers to efficient, safe and cost-effective gene therapy.     

Intracellular routing of plasmid DNA during non-viral gene transfer

Gene transfer using non-viral vectors is a promising approach for the safe delivery of therapeutic DNA in genetic and acquired human diseases. Whereas the lack of specific immune response favors the use of plasmid-cationic polymer complexes, the limited efficacy and short duration of transgene expression impose major hurdles in the application of non-viral gene delivery techniques. Here, we review the major cellular, metabolic and physico-chemical impediments that non-viral vectors encounter before plasmid DNA enters the nucleus. Following endocytosis of DNA-polycation complexes, a large fraction of the DNA is targeted to the lysosomes. Since the cytosolic release of heterologous DNA is a prerequisite for nuclear translocation, entrapment and degradation of plasmid DNA in endo-lysosomes constitute one of the major impediments to efficient gene transfer. Plasmid DNA that escapes the endo-lysosomal compartment encounters the diffusional and metabolic barriers of the cytoplasm, reducing greatly the number of intact plasmids that reach the nucleosol. Nuclear translocation of DNA requires either the disassembly of the nuclear envelope or active nuclear transport via the nuclear pore complex. A better understanding of the cellular and molecular basis of non-viral vector trafficking from the extracellular compartment into the nucleus may provide strategies to overcome those obstacles that limit the efficiency of gene delivery.     

The role of the disulfide group in disulfide-based polymeric gene carriers

Background: An essential prerequisite for successful gene therapy is the development of safe and efficient gene delivery carriers. For this purpose, cationic polymers have been widely studied as non-viral carriers, but they generally suffer from low transfection efficiency and/or high cytotoxicity. To address these problems, disulfide-based cationic polymers have been designed as intelligent gene carriers that are capable of inducing highly efficient gene transfection with low cytotoxicity. Objective: The present review discusses the effects of the disulfide linker on the gene delivery properties of cationic polymers in relation to various gene delivery barriers. Methods: The literature regarding the gene delivery barriers encountered by polymeric gene delivery is reviewed and discussed in relation to the presence of the disulfide moiety in these gene carriers. Conclusions: The presence of disulfide linkages in cationic polymers can in many aspects favorably influence the gene delivery properties, such as increasing DNA binding ability, enabling de-shielding of ‘stealth’ (PEG) groups, fine-tuning of the buffer capacity for enhanced endosomal escape, improving carrier-unpacking and decreasing cytotoxicity. Therefore, disulfide-based cationic polymers are promising candidates for the next generation of non-viral carriers.     

Key issues in non-viral gene delivery

The future of non-viral gene therapy depends on a detailed understanding of the barriers to delivery of polynucleotides. These include physicomechanical barriers, which limit the design of delivery devices, physicochemical barriers that influence self-assembly of colloidal particulate formulations, and biological barriers that compromise delivery of the DNA to its target site. It is important that realistic delivery strategies are adopted for early clinical trials in non-viral gene therapy. In the longer term, it should be possible to improve the efficiency of gene delivery by learning from the attributes which viruses have evolved; attributes that enable translocation of viral components across biological membranes. Assembly of stable, organized virus-like particles will require a higher level of control than current practice. Here, we summarize present knowledge of the biodistribution and cellular interactions of gene delivery systems and consider how improvements in gene delivery will be accomplished in the future.     

Key issues in non-viral gene delivery

The future of non-viral gene therapy depends on a detailed understanding of the barriers to delivery of polynucleotides. These include physicomechanical barriers, which limit the design of delivery devices, physicochemical barriers that influence self-assembly of colloidal particulate formulations, and biological barriers that compromise delivery of the DNA to its target site. It is important that realistic delivery strategies are adopted for early clinical trials in non-viral gene therapy. In the longer term, it should be possible to improve the efficiency of gene delivery by learning from the attributes which viruses have evolved; attributes that enable translocation of viral components across biological membranes. Assembly of stable, organized virus-like particles will require a higher level of control than current practice. Here, we summarize present knowledge of the biodistribution and cellular interactions of gene delivery systems and consider how improvements in gene delivery will be accomplished in the future.