Intracellular organelle-targeted non-viral gene delivery systems

Gene therapy is a rapidly growing approach for the treatment of various diseases. To achieve successful gene therapy, a gene delivery system is necessary to overcome several barriers in the extracellular and intracellular spaces. Polymers, peptides, liposomes and nanoparticles developed as gene carriers have achieved efficient cellular uptake of genes. Among these carriers, cationic polymers and peptides have been further developed as intracellular organelle-targeted delivery systems. The cytoplasm, nucleus and mitochondria have been considered primary targets for gene delivery using targeting moieties or environment-responsive materials. In this review, we explore recently developed non-viral gene carriers based on reducible systems specialized to target the cytoplasm, nucleus and mitochondria.     

Extracellular and intracellular barriers in non-viral gene delivery.

Complexes of DNA with cationic lipids and cationic polymers are frequently used for gene transfer. Extracellular interactions of the complexes with anionic glycosaminoglycans (GAGs) may interfere with gene transfer. Interactions of GAGs with carrier DNA complexes have been studied using tests for DNA relaxation (ethidium bromide intercalation), DNA release (electrophoresis), and transfection (pCMVbGal transfer into RAA smooth muscle cells). Several cationic lipid formulations (DOTAP, DOTAP/Chol, DOTAP/DOPE, DOTMA/DOPE, DOGS) and cationic polymers (fractured dendrimer, polyethylene imines 25 and 800 kDa, polylysines 20 and 200 kDa) were tested. Polycations condensed DNA more effectively than monovalent lipids. Hyaluronic acid did not release or relax DNA in any complex, but it inhibited transfection by some polyvalent systems (PEI, dendrimers, DOGS). Gene transfer by other carriers was not affected by hyaluronic acid. Sulfated GAGs (heparan sulfate, chondroitin sulfates B and C) completely blocked transfection, except in the case of liposomes with DOPE. Sulfated GAGs relaxed and released DNA from some complexes, but these events were not prerequisites for the inhibition of transfection. Furthermore, preliminary results suggest that cell surface GAGs, particularly heparan sulfate, inhibit gene transfer by cationic lipids and polymers.     

Uptake mechanisms of non-viral gene delivery

Non-viral gene delivery is currently a hot subject for its relative safety and simplicity of use; however, it is still far from being ideal enough to be clinically used for its comparatively lower efficiency than viral gene delivery. To improve the efficiency of non-viral gene delivery needs a comprehensive understanding of the uptake mechanisms. Macromolecules are internalized into cells by a variety of mechanisms, and their intracellular fates are usually relevant with the uptake pathways. The uptake pathways of non-viral gene complexes are usually determined by not only the gene/carrier interaction but also by the interaction between complexes and target cells. The best-characterized uptake pathway is the so-called clathrin-mediated endocytic pathway. However, there are numerous updates of knowledge about endocytic pathways and even non-endocytic pathways in recent years with the development of novel technologies for tracking and inhibiting. In this review, we will try to sort out our current understanding of the uptake mechanisms of non-viral gene delivery. In addition, factors for pathway selection are summarized in the third section. Finally, the useful inhibitors or tools for the study of these pathways will also be concluded in the last section.     

Histonefection: Novel and potent non-viral gene delivery.

Protein/peptide-mediated gene delivery has recently emerged as a powerful approach in non-viral gene transfer. In previous studies, we and other groups found that histones efficiently mediate gene transfer (histonefection). Histonefection has been demonstrated to be effective with various members of the histone family. The DNA binding domains and natural nuclear localisation signal sequences make histones excellent candidates for effective gene transfer. In addition, their positive charge promotes binding to anionic molecules and helps them to overcome the negative charge of cells that is an important barrier to cellular penetration. Histonefection appears to have particular promise in cancer gene transfer and therapy.     

Development of non-viral vectors for systemic gene delivery.

One of the major challenges for gene therapy is systemic delivery of a nucleic acid directly into an affected tissue. This requires developing a vehicle which is able to protect the nucleic acid from degradation, while delivering the gene of interest to the specific tissue and specific subcellular compartment. In this review, we summarize some of the recent advances in new non-viral delivery systems for systemic administration. Two types of gene delivery systems are described: (i) LPD1 (cationic liposome-entrapped, polycation-condensed DNA, type 1), and (ii) retention-time mediated naked DNA delivery. Hypothesized mechanisms for these systemic gene transfers are also discussed.     

Recent progress in gene delivery using non-viral transfer complexes.

The delivery of genetic material into cells is a field that is expanding very rapidly. Non-viral delivery methods, especially ones that focus on the use of chemical agents complexed with genetic material, are the focus of this mini-review. More-recent uses of known transfection agents such as poly(ethylenimine), poly(L-lysine), and various liposomes are discussed, and some novel approaches (both chemical and methodical) are reviewed as well. A very brief look at how non-viral gene delivery research is being aimed at the clinic is also included.     

Evolution of cross-linked non-viral gene delivery systems

Developing a non-viral gene delivery system that functions in vivo raises the challenge of finding solutions to efficiently deliver DNA to the cell surface that are also compatible with the efficient release of DNA into the cytosol. The stability, particle size and charge of DNA polyplexes and lipoplexes may be optimized to mediate efficient in vitro transfection only to find that different properties are necessary for successful in vivo transfection. Despite their versatility and improved safety, non-viral gene vectors still lack appreciable in vivo transfection efficiency compared to viral vectors. An emerging theme in recent studies is the use of cross-linking to achieve balance between the stability of polyplexes and lipoplexes in the blood and the controlled release of DNA in the cytosol. This review evaluates the evolution of cross-linking strategies aimed at transiently stabilizing non-viral gene delivery systems.     

Obstacles and advances in non-viral gene delivery

This review focuses on recent progress and novel strategies to improve the efficiency of in vivo non-viral gene delivery. Examples of the most promising attempts to overcome specific barriers are presented in fuller detail. Current research into several of the most difficult steps in the gene delivery pathway is discussed including particle stabilization, targeting, cytoplasmic entry and access to the nucleus. The impact of recent reports on our current understanding of the true limitations to in vivo delivery is also discussed. The importance of preclinical animal models for the development of clinical applications of gene therapy is noted.     

High-content screening of peptide-based non-viral gene delivery systems

High-content screening (HCS) uses high-capacity automated fluorescence imaging for the quantitative analysis of single cells and cell populations. Here, we developed an HCS assay for rapid screening of non-viral gene delivery systems as exemplified by the screening of a small library of peptide-based transfectants. These peptides were simultaneously screened for transfection efficiency, cytotoxicity, induction of cell permeability and the capacity to transfect non-dividing cells. We demonstrated that HCS is a valuable extension to the already existing screening methods for the in vitro evaluation of non-viral gene delivery systems with the added value that multiple parameters can be screened in parallel thereby obtaining more information from a single screening event, which will accelerate the development of novel gene delivery systems.     

Anti-GAD antibody targeted non-viral gene delivery to islet beta cells.

An islet cell targeting polymeric gene carrier was synthesized by conjugating anti-GAD Fab' fragment to PEI via PEG linker (PEI-PEG-Fab'). The Fab' fragment was prepared from a murine monoclonal antibody against glutamic acid decarboxylase (GAD), which has been identified as one of the major auto-antigens expressed in islet cells, and used as a targeting moiety for islet cell targeting. The electrophoretic migration of plasmid DNA (pCMVLuc)/PEI-PEG-Fab' complexes in agarose gel was completely retarded above the N/P ratio of 2. The complexes demonstrated a size of 100-275 nm with an almost neutral surface charge. Confocal microscopy revealed that the PEI-PEG-Fab' complexes showed much higher cellular binding and uptake efficiency compared to PEI-PEG complexes. The PEI-PEG-Fab' showed about 10-fold higher transfection efficiency (relative luciferase activity) than PEI-PEG in GAD-expressing mouse insulinoma cells (MIN6), however the transfection efficiency of PEI-PEG-Fab' reduced to that of PEI-PEG in GAD negative cells (293) and in the presence of competitive free Fab'. Considering the neutral surface charge of its complexes with DNA, and selectivity toward the islet cells expressing a specific antigen, the PEI-PEG-Fab' conjugate could be thought as a potential candidate of the systemic gene therapy for the treatment of type I diabetes.     

Aerosolized nanogram quantities of plasmid DNA mediate highly efficient gene delivery to mouse airway epithelium.

The lung is an important target of gene therapeutic interventions. In contrast to intratracheal instillation, inhalation would be the most practical route of administration in clinical applications. Here we show that aerosolized nanogram quantities of pDNA complexed to PEI (350 ng) yielded transfection levels 15-fold higher than a 140-fold higher dose (50 microg) of the same vector applied directly to the lungs of mice via intratracheal intubation. An important efficacy parameter is the osmolarity of the aerosol and not biophysical properties of the nebulized vector. Vectors formulated and nebulized in hypoosmotic distilled water yielded 57- and 185-fold higher expression levels than those in isotonic 5% glucose or Hepes-buffered saline, respectively. Pretreatment of mice with nebulized indomethacin, which prevents water-induced airway alteration, resulted in lower gene expression, whereas pretreatment with EGTA or polidocanol, which modulate tight-junction activity, had no effect. These results, together with histological analysis of regional lung deposition and gene expression, suggest that a temporary water-induced hypoosmotic shock permeabilizes the epithelium sufficiently to allow vector uptake. The so far observed inefficiency of nonviral gene delivery to the airways may be the result of an inappropriate method of vector administration.     

Trimethylated chitosans as non-viral gene delivery vectors: cytotoxicity and transfection efficiency.

Chitosans are linear polysaccharides of natural origin that show potential as carriers in drug and gene delivery. Introducing quaternisation on the chitosan backbone renders the polymer soluble over a wider pH range and confers controlled cationic character. This study aims to investigate the effect of increasing quaternisation and therefore, positive charge on cell viability and transfection. Oligomeric and polymeric chitosans were trimethylated, the toxicity and transfection efficiency of these derivatives were tested with respect to increasing degree of trimethylation. The cytoxicity of polymer and oligomer derivatives alone and of their complexes with plasmid DNA were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on COS-7 (monkey kidney fibroblasts) and MCF-7 (epithelial breast cancer) cells. Transfection efficiency was investigated using the pGL3 luciferase reporter gene on the same cell lines. Complexes were characterised for their stability by gel electrophoresis. Cytotoxicity results showed that all derivatives were significantly less toxic than linear polyethylenimine (PEI). A general trend of increasing toxicity with increasing degree of trimethylation was seen. However, higher toxicity was seen in polymeric chitosan derivatives over oligomeric chitosan derivatives at similar degrees of trimethylation. All derivatives complexed pGL3 luc plasmid DNA efficiently at 10:1 ratio and three (TMO44, TMC57 and TMC93) were able to transfect MCF-7 cells with greater efficiency than PEI; 16, 23 and 50-fold, respectively. TMC57, TMC93 and all TMOs gave appreciable transfection of COS-7 cells.     

Dry powder aerosols of polyethylenimine (PEI)-based gene vectors mediate efficient gene delivery to the lung

Aerosol gene delivery holds great therapeutical potential for many inherited and acquired pulmonary diseases. The physical instability of aqueous suspensions of non-viral vector complexes is a major limitation for their successful application. In this study, we investigated dry powder aerosols as novel gene vector formulations for gene transfer in vitro and murine lungs in vivo. Lyophilization was used to produce dry powder cakes followed by powderization to produce dry powder aerosols. Different sugars, namely lactose, sucrose and trehalose, were tested as lyoprotectants for gene delivery complexes consisting of branched polyethylenimine 25 kDa and plasmid DNA. Biophysical particle characterization demonstrated that lyophilization and powderization in the presence of lyoprotectants were well tolerated. In vitro transfection efficiency remained unaffected by the choice of lyoprotectant and subsequent lyophilization and/or powderization. In vivo screening of powderized samples, by applying the powder with an insufflator, resulted in highest gene expression with lactose as lyoprotectant. Delivering a plasmid coding for murine erythropoietin together with lactose as lyoprotectant resulted in increased blood hematocrit values post application thereby demonstrating the potential of dry powder aerosol as a promising method for pulmonary gene delivery.     

Development of a bio-electrospray system for cell and non-viral gene delivery

Bio-electrospray technology is a very attractive tool for preparing scaffolds and depositing desired solutions on various targets by electric force. In this study, we focused on the application of a bio-electrospray (BES) technique to spray cells on the target and to simultaneously deliver genetic constructs into the cells, called non-viral gene delivery-based bio-electrospray (NVG-BES). Using this method, we tried to harvest the electric charge produced during electrospray for the cellular internalization of cationic polymer/DNA nanoparticles as well as the delivery of living cells on the desired substrate. Furthermore, we optimized the voltage, culture medium and polymeric cationic charges for high transfection efficiency and cell viability during NVG-BES. As a result, the solutions used during the NVG-BES process played an important role in improving transfection efficiency. We determined that a voltage of 10 kV with PBS as the spraying solution showed high transfection efficiency, probably due to the facilitation of cationic polymer/DNA nanocomplexes in cellular internalization and their subsequent expression. In conclusion, NVG-BES, as a novel method, is expected to deliver genes to cells and simultaneously deliver transfected cells to any substrate or scaffold.

The NVG-BES system facilitated to introduce DNA to cells and delivered cells to a target simultaneously. In this method, a cationic polymer was used as non-viral carrier with electric force by bio-electrospray (BES) system to electrospray living cells onto a target.     

Synthesis and application of a non-viral gene delivery system for immunogene therapy of cancer.

The synthesis and gene delivery application of a novel lipopolymer, PEG-PEI-CHOL (PPC), is described. PPC is composed of a low molecular weight branched polyethylenimine (PEI) covalently linked with functional groups methoxypolyethyleneglycol (PEG) and cholesterol (CHOL). The potential utility of PPC as a gene delivery polymer was evaluated by showing its ability to form stable nanocomplexes with DNA, protect DNA from degradation by DNase and mediate gene transfer in vitro and in vivo in solid tumors. The ratio of PEG/PEI/CHOL and nitrogen to phosphate (Polymer/DNA) was optimized for physico-chemical properties and gene delivery efficiency of PPC/DNA complexes. The gene therapy application of the polymer was shown following administration of a murine IL-12 plasmid (pmIL-12) formulated with PPC into tumors in mice which resulted in significant inhibition of tumor growth. The inhibitory effects of pmIL-12/PPC were enhanced when combined with specific chemotherapeutic agents, demonstrating the potential usefulness of pIL-12/PPC as an adjuvant therapy for cancer treatment.     

Chitosan nanoparticles as non-viral gene delivery systems: Determination of loading efficiency

Chitosan has been studied for use in particle delivery systems for therapeutic purposes, since one of its most important applications is as a non-viral vector in gene therapy. Due to its positive charge, it is capable of forming DNA complexes (polyplexes) obtained through several methods and with the property of protecting nucleic acids. Two methods for obtaining the nanoparticles of chitosan-nucleic acids are reported in this study: simple complexation (of depolymerized chitosan or of different chitosan salts with plasmid) and ionic gelation (by adsorption of plasmid in the nanoparticles or by encapsulation of plasmid into nanoparticles). The determination of the loading efficiency of chitosan nanoparticles with the plasmid is carried out by electrophoretic mobility of the samples on agarose gel. Furthermore, the nanoparticles have been characterized according to their morphology, size and surface charge using AFM, TEM, laser diffraction and dynamic light scattering techniques. The polyplexes obtained have been found to be spherical and nanometric in size (between 100–230 nm) with a zeta potential between 37 and 48 mV. Positive results have been obtained by agarose gel electrophoresis for all studied cases: a concentration of between 20 and 30 μg/mL of chitosan salts is required while for the remaining chitosan samples studied, 100% loading efficiency does not occur until a concentration equal to 100 μg/mL (regardless of previous depolymerisation and the method performed). Chitosan–plasmid nanocapsules have been obtained at the polymer concentrations worked with (between 0.025 and 0.2%).     

Controllable delivery of non-viral DNA from porous scaffolds.

The inductive approach to tissue engineering combines three-dimensional porous scaffolds with drug delivery to direct the action of progenitor cells into a functional tissue. We present an approach to fabricate scaffolds capable of controlled, sustained delivery by the assembly and subsequent fusion of drug-loaded microspheres using a gas foaming/particulate leaching process. DNA-loaded microspheres were fabricated from the copolymers of lactide and glycolide (PLG) using a cryogenic double emulsion process. Microspheres were fabricated in four populations with mean diameters ranging from 12.3 microm to 92.5 microm. Scaffolds fabricated by fusion of these microspheres had an interconnected open pore structure, maintained DNA integrity, and exhibited sustained release for 21 days. Control over the release was obtained through manipulating the properties of the polymer, microspheres, and the foaming process. Decreasing the microsphere diameter or the molecular weight of the polymer used for microsphere fabrication led to increased rates of release from the porous scaffold. Additionally, increasing the pressure of CO(2) increased the DNA release rate. The ability to create porous polymer scaffolds capable of controlled release rates may provide a means to enhance and regulate gene transfer within a developing tissue, which will increase their utility in tissue engineering.     

Gene therapy progress and prospects: non-viral gene therapy by systemic delivery

Non-viral vectors continue to be an attractive alternative to viral vectors due to their safety, versatility and ease of preparation and scale-up. Over the past few years, investigators have been successful in developing gene carriers that can be targeted to the disease site. Several different delivery vectors for systemic use have been developed by different groups for plasmid DNA and oligonucleotide. Most of them are designed for targeted tumor therapy. The mechanism of inflammatory toxicity, the major toxicity of cationic lipoplex, has been studied and managed. In this review, we focus on the progress made over the last 2 years. We also discuss some future prospects for gene delivery.     

Histone-mediated transduction as an efficient means for gene delivery.

Gene delivery into the nucleus of eukaryotic cells is inefficient, largely because of the significant barriers within the target cell of the plasma membrane and nuclear envelope. Recently, a group of basic proteins, including the HIV-1 Tat protein and the four core histones, have been shown to enter cells through a novel energy- and receptor-independent manner. Here, we show that engineered histone H2B proteins are able to mediate the efficient delivery of either green fluorescent protein or DNA into HeLa cells through the process of "Histone-Mediated Transduction" (HMT), with further enhancement achieved by utilizing a dimer of histones H2B and H2A. Subsequent nuclear delivery was accelerated approximately two-fold by the addition of an optimized nuclear localization signal to histone H2B, thereby increasing the affinity of interaction with components of the cellular nuclear import machinery, resulting in increased expression of a reporter gene. Further, we demonstrate that the domains responsible for this histone transduction are located in the N-terminal tail and globular regions of histone H2B. HMT represents a new, efficient, and technically non-demanding means to deliver DNA to the nucleus of intact cells, including embryonic stem cells, which has important applications in gene therapy and cancer therapeutics.