DNA Delivery Systems Research Articles

Poly(ethylene glycol)-Conjugated PAMAM Dendrimer for Biocompatible, High-Efficiency DNA Delivery

We demonstrate a simple and successful synthetic approach to devise a highly efficient DNA delivery system with low cytotoxicity and low cost. Polyamidoamine (PAMAM) dendrimer is a highly efficient DNA delivery agent, when compared to other chemical transfection reagents. Partially degraded, high-generation dendrimers offer even higher efficiency, presumably due to enhanced flexibility of the otherwise rigid dendrimer chains.1 We hypothesized that chemical modification of low generation dendrimer with biocompatible poly(ethylene glycol) (PEG) chains would create a conjugate of PAMAM core with flexible PEG chains, which mimics the fractured high-generation dendrimer and produces high transfection efficiency. Generation 5 PAMAM was modified with 3400 molecular weight PEG. The novel conjugate produced a 20-fold increase in transfection efficiency compared with partially degraded dendrimer controls. The cytotoxicity of PEGylated dendrimers was very low. This extremely efficient, highly biocompatible, low-cost DNA delivery system can be readily used in basic research laboratories and may find future clinical applications.

Block copolymer micelles for delivery of gene and related compounds

Block copolymers composed of a cationic segment and a hydrophilic segment spontaneously associate with polyanionic DNA to form block copolymer micelles. The distinct feature of the associate is that the core of the polyion complex between DNA and the polycation is coated by a layer of the hydrophilic polymer. The characteristic core-shell structure endows the associate with a high colloidal stability and reduced interaction with blood components. These desirable properties are the major advantages of the micellar DNA delivery system for in vivo application. In this article, the synthesis of block copolymers as well as graft copolymers utilized as DNA delivery systems are described. Particular emphasis is devoted to the association behavior and the physicochemical properties of polyion complex micelles entrapping DNA and related substances in relation to the biological aspects of the associates. Biodistribution and the factors that affect the intracellular fate of the micelles is also addressed based on recent studies in this field.

Analysis of gene function in bacterial pathogens by gambit

Publisher Summary This chapter describes practical guide to the use of the GAMBIT (genome analysis and mapping by in vitro transposition) approach for analysis of genetic function on a genomic scale or to study individual genomic regions of interest. As part of this discussion, versatile components of this method are described including transposase purification, in vitro transposon mutagenesis with mariner-derived transposons, and genetic footprinting. The chapter focuses on studies in naturally transformable bacteria; however, elements of the GAMBIT system have been adapted to genetic analysis of organisms. GAMBIT owes its versatility to its simplicity. The method creates a bank of transposon mutations spanning the entire genome or within user-defined subgenomic regions and uses an analytical PCR-based approach, genetic footprinting, to locate the positions of genes essential for growth or viability under any specific experimental condition. Transposition is conducted using purified components in vitro and therefore does not require complex genetic arrangements such as conditional replicons, organism-specific transposase expression systems, or “suicide” DNA delivery systems. The only requirement for conducting this procedure in any naturally transformable organism is the presence of a gene within the transposon conferring antibiotic resistance or other selectable traits.

Supramolecular assemblies of DNA delivery systems.

Gene delivery into cultured cells or in vivo is a promising approach to the treatment of diseases. Several gene delivery systems have been developed to promote gene expression either in vitro or in vivo. Concerns about viral-induced immune responses, the risk associated with replication-competent viruses, and production issues have stimulated efforts toward the development of alternative gene delivery systems such as cationic lipids and polymers. These positively charged molecules interact through electrostatic forces with DNA. This results in the formation of highly organized supramolecular structures where DNA molecules are condensed and protected against DNAses degradation. Association of DNA with cationic lipids under a micellar or liposomal form leads to lamellar organization with DNA molecules sandwiched between lipid bilayers. Although the lamellar phase is the common described structure, as evidenced by small-angle X-ray scattering and electron microscopy, monovalent cationic lipid combined with a hexagonal forming lipid resulted with DNA in an inverted hexagonal structure. Despite a lot of effort, the mechanism of gene transfer with cationic carrier is still ill-defined. Therefore, correlations need to be established between physicochemical properties of synthetic DNA delivery systems and in vitro and in vivo transfection efficiency.

Influence of Physicochemical Properties on Pharmacokinetics of Non-viral Vectors for Gene Delivery

The influence of physicochemical properties on the in vivo pharmacokinetics of gene delivery vectors after systemic administration is reviewed based on our studies. We have been studying the development of DNA delivery systems, such as plasmid DNA complexed with cationic polymers (polyplexes) and cationic liposomes (lipoplexes). Even if target-recognizable ligand is incorporated into the system, the overall physicochemical properties, notably size and charge, are predominant factors influencing in vivo disposition characteristics of the vector. Based on this consideration, liver cell-specific carrier systems via receptor-mediated endocytosis were successfully developed by optimizing physicochemical characteristics. In conclusion, rational design of gene delivery vectors requires an understanding of their pharmacokinetics in relation to the physicochemical properties. Optimization of the physicochemical properties is important for successful in vivo gene delivery by non-viral vectors.

Overcoming the Nuclear Barrier: Cell Cycle Independent Nonviral Gene Transfer with Linear Polyethylenimine or Electroporation

In many cases, nonviral particle-mediated gene delivery is highly dependent on the cell cycle status of transfected cells. Here we compare particle-mediated delivery with linear polyethylenimine (PEI) and physical transfer of DNA by electroporation with branched PEI and lipofection for their ability to transfect cells at different stages of the cell cycle. In contrast to other particle-mediated delivery methods (using Lipofectamine or branched PEI) linear PEI led to only small differences (within 1 log unit) in gene transfer between HeLa cells transfected in G1 and those in S/G2. Parallel transfections (lipofection or branched PEI) resulted in 2 to > 3 log-unit differ-ences in luciferase expression between cells transfected in G1 and S/G2. Gene transfer by elec-troporation also revealed hardly any cell cycle dependence and displayed completely different expression kinetics. Reporter gene expression is already very high 3 hours after electroporation with roughly the same level of reporter gene expression in all cell cycle phases. We suggest that DNA electroporation and DNA transfection with linear PEI particles have improved nuclear import characteristics relative to the other tested DNA delivery systems.

Extracellular Glycosaminoglycans Modify Cellular Trafficking of Lipoplexes and Polyplexes

It has been shown that extracellular glycosaminoglycans (GAGs) limit the gene transfer by cationic lipids and polymers. The purpose of this study was to clarify how interactions with anionic GAGs (hyaluronic acid and heparan sulfate) modify the cellular uptake and distribution of lipoplexes and polyplexes. Experiments on cellular DNA uptake and GFP reporter gene expression showed that decreased gene expression can rarely be explained by lower cellular uptake. In most cases, the cellular uptake is not changed by GAG binding to the lipoplexes or polyplexes. Reporter gene expression is decreased or blocked by heparan sulfate, but it is increased by hyaluronic acid; this suggests that intracellular factors are involved. Confocal microscopy experiments demonstrated that extracellular heparan sulfate and hyaluronic acid are taken into cells both with free and DNA-associated carriers. We conclude that extracellular GAGs may alter both the cellular uptake and the intracellular behavior of the DNA complexes.

Cationic bolasomes with delocalized charge centers as mitochondria-specific DNA delivery systems

Since their first discovery during the end of the 1980s, the number of diseases found to be associated with a defect in the mitochondrial genome has grown significantly. However, despite major advances in understanding mtDNA defects at the genetic and biochemical level, there is no satisfactory treatment available for the vast majority of patients. This is largely due to the fact that most of these patients have respiratory chain defects, i.e. defects that involve the final common pathway of oxidative metabolism, making it impossible to bypass the defect by giving alternative metabolic carriers of energy. These objective limitations of conventional biochemical treatment for patients with defects of mtDNA warrant the exploration of gene therapy approaches. However, mitochondrial gene therapy currently appears to be only theoretical and speculative. Any possibility for gene replacement is dependent on the use of a yet unavailable mitochondrial transfection vector. In this review we describe the current state of the development of mitochondrial DNA delivery systems. We also summarize our own efforts in exploring the properties of dequalinium, a cationic bolaamphiphile with delocalized charge centers, for the design of a vector suited for the transport of DNA to mitochondria in living cells.

A regulated, NFkappaB-assisted import of plasmid DNA into mammalian cell nuclei.

The success of synthetic DNA delivery systems in human gene therapy will be enhanced by increasing transfection efficiencies and by providing tighter control over targeting of the DNA into the nucleus. Here, we used DNA vectors that contain repetitive binding sites for the inducible transcription factor NFkappaB, which is transported into the nucleus by the nuclear import machinery. Nuclear entry of the modified vectors was augmented 12-fold and was associated with corresponding increase in gene expression. Depending on their position, the binding sites could also function as transcriptional enhancers, increasing gene expression levels up to an additional 19-fold. Notably, nuclear targeting of the DNA and transgene transcription could both be regulated by exogenous stimulators which modulate the intracellular distribution of NFkappaB. The approach provides a framework for the controlled targeting of constitutive or transcriptionally regulated synthetic vectors into mammalian cell nuclei.

Cationic microparticles are an effective delivery system for immune stimulatory cpG DNA.

Cationic microparticles are an effective delivery system for immune stimulatory cpG DNA.

Tuberculosis DNA vaccines.

DNA vaccines have been the subject of intense investigation for the past 10 y, during which time several tuberculosis (TB) DNA vaccines have been shown to confer protective immunity in animal models. So far, proof of principle for priming of immune responses by a naked DNA vaccine (malaria) has been demonstrated in humans, but potency remains a significant limitation. However, new DNA vaccine formulations and delivery systems are being developed with markedly improved potency in animal models. Therefore, there is a clear path to the human clinical testing of TB DNA vaccines.

Tuberculosis DNA Vaccines

DNA vaccines have been the subject of intense investigation for the past 10 y, during which time several tuberculosis (TB) DNA vaccines have been shown to confer protective immunity in animal models. So far, proof of principle for priming of immune responses by a naked DNA vaccine (malaria) has been demonstrated in humans, but potency remains a significant limitation. However, new DNA vaccine formulations and delivery systems are being developed with markedly improved potency in animal models. Therefore, there is a clear path to the human clinical testing of TB DNA vaccines.

Novel cationic amphiphilic 1,4-dihydropyridine derivatives for DNA delivery

In order to find new efficient and safe agents for gene delivery, we have designed and synthesized nine novel single- and double-charged amphiphiles on the base of 1,4-dihydropyridine (1,4-DHP) ring. Some biophysical properties of the amphiphilic dihydropyridines and their complexes with DNA were examined. We investigated the transfer of β-galactosidase gene into fibroblasts (CV1-P) and retinal pigment epithelial (D 4O7) cell lines in vitro. The structure–property relationships of the compounds were investigated in various ways. The net surface charges of 1,4-DHP liposomes were highly positive (25–49 mV). The double-charged compounds condensed DNA more efficiently than single-charged and the condensation increases with the increasing +/− charge ratio between the carrier and DNA. Double-charged compounds showed also buffering properties at endosomal pH and these compounds were more efficient in transfecting the cells, but transfection efficiency of amphiphiles was cell type-dependent. The length of alkyl chains in double-charged compounds affected the transfection efficacy. The most active amphiphile (compound VI) was double-charged and had two C12 alkyl chains. At optimal charge ratio (+/− 4), it was 2.5 times more effective than PEI 25 and 10 times better than DOTAP, known efficient polymeric and liposomal transfection agents. Formulation of amphiphiles with DOPE did not change their activities. Our data demonstrate some important effects of amphiphile structure on biophysics and activity. The data also suggest that cationic amphiphilic 1,4-DHP derivatives may find use as DNA delivery system.

Secretion of different listeriolysin cognates by recombinant attenuated Salmonella typhimurium: superior efficacy of haemolytic over non-haemolytic constructs after oral vaccination

Viable antigen (Ag) delivery systems expressing defined pathogen-derived proteins represent powerful candidates for future vaccination strategies. Here, recombinant (r) Salmonella typhimurium aroA strains secreting listeriolysin (Hly) of Listeria monocytogenes in haemolytic or non-haemolytic form were constructed to direct these carriers into cytosolic or phagosomal host cell compartments, respectively. Oral and intravenous (i.v.) vaccination of mice with either construct induced ‘transporter associated with antigen processing’-dependent protection against the intracellular bacterial pathogen L. monocytogenes. Comparison of oral immunization with both r Salmonella constructs revealed superior vaccine efficacy of the haemolytic r S. typhimurium Hlys construct as compared to the non-haemolytic r Salmonella Hlys 492 strain. In contrast, efficacy of i.v. vaccination with either r Salmonella strain did not significantly differ. Therefore, r Salmonella strains secreting biologically active Hly represent valuable delivery systems for heterologous rAg or DNA which should be exploited for future mucosal vaccination strategies.

A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro.

Diverse polycationic polymers have been used as nonviral transfection agents. Here we report the ability of colloidal silica particles with covalently attached cationic surface modifications to transfect plasmid DNA in vitro and make an attempt to describe the structure of the resulting transfection complexes. In analogy to the terms lipoplex and polyplex, we propose to describe the nanoparticle-DNA complexes by the term "nanoplex". Three batches, Si10E, Si100E, and Si26H, sized between 10 and 100 nm and with zeta potentials ranging from +7 to +31 mV at pH 7.4 were evaluated. The galactosidase expression plasmid DNA pCMVbeta was immobilized on the particle surface and efficiently transfected Cos-1 cells. The transfection activity was accompanied by very low cytotoxicity, with LD(50) values in the milligrams per milliliter range. The most active batch, Si26H, was produced by modification of commercially available silica particles with N-(6-aminohexyl)-3-aminopropyltrimethoxysilane, yielding spherical nanoparticles with a mean diameter of 26 nm and a zeta potential of +31 mV at pH 7.4. Complexes of Si26H and pCMVbeta plasmid DNA formed at w/w ratios of 10 were most effective in promoting transfection of Cos-1 cells in the absence of serum. At this ratio, >90% of the DNA was associated with the particles, yielding nanoplexes with a net negative surface charge. When the transfection medium was supplemented with 10% serum, maximum gene expression was observed at a w/w ratio of 30, at which the resulting particle-DNA complexes possessed a positive surface charge. Transfection was strongly increased in the presence of 100 microM chloroquine in the incubation medium and reached approximately 30% of the efficiency of a 60 kDa polyethylenimine. In contrast to polyethylenimine, no toxicity was observed at the concentrations required. Atomic force microscopy of Si26H-DNA complexes revealed a spaghetti-meatball-like structure. The surface of complexes prepared at a w/w ratio of 30 was dominated by particles half-spheres. Complex sizes correlated well with those determined previously by dynamic light scattering.

Antigen presentation and DNA vaccines.

There is reasonable evidence that both cross-priming and direct transfection of antigen-presenting cells (APCs) play a role in induction of immune responses by DNA vaccines. It is not known which mode is more important for priming cytotoxic T cell responses, but both are sufficient and neither alone is necessary. Hence, a rational strategy for increasing DNA vaccine potency would be to facilitate both pathways. With regard to cross-priming, a better understanding of the nature of the antigen transferred and the molecules/cells involved may suggest ways to design DNA vaccines to enhance this pathway. With respect to transfection of APCs, certain DNA formulations or delivery systems may be able to target APCs for increased DNA uptake. Other considerations include recruitment of APCs to the site of DNA injection and manipulation of these cells to ensure the proper activation state for priming immune responses. The burgeoning scientific literature in these areas indicates that much effort is currently being directed toward these goals.

In vivo correction of genetic defects of monocyte/macrophages using attenuated Salmonella as oral vectors for targeted gene delivery.

Macrophages are normal targets for Salmonella during natural infections, and it has been demonstrated that attenuated bacteria can deliver nucleic acid vaccine constructs. Therefore, we assessed if attenuated Salmonella can be used for the in vivo delivery of transgenes to their natural cellular target, in an attempt to correct genetic defects associated with monocytes/macrophages. This system would offer the distinct advantage of achieving a specific targeting of defective cells in a non-invasive form. Using a reporter gene, we demonstrated that attenuated Salmonella could be used as an effective in vitro delivery system to transfer genetic material into nondividing cells like murine macrophages. In vivo, the oral administration of attenuated Salmonella allows targeted delivery of transgenes to macrophages and subsequently expression of transgenes at a systemic level. IFNgamma-deficient mice (GKO) were thus selected as a model for the in vivo validation of the Salmonella-based delivery approach. Attenuated Salmonella, used as the carrier for a eukaryotic expression vector encoding the murine IFNgamma gene, was able to restore the production of this cytokine in GKO macrophages. Their oral administration to IFNgamma-deficient mice also re-established, in these immunocompromised animals, the natural resistance to bacterial infections. These results demonstrate, for the first time, that attenuated Salmonella can be successfully used in vivo as a DNA delivery system for the correction of a genetic defect associated with monocyte/macrophages.

Gene-based vaccines.

The development of vaccines is one of the best examples of the impact of biomedical advances upon human health. But, despite the successes, such as the elimination of smallpox from the planet, there remain a number of diseases for which we have no vaccines, and which infect and often kill millions of people each year (see Table 1). These diseases include those due to newly emerging pathogens such as HIV, as well as ancient scourges such as tuberculosis, where, although there is a vaccine, a better one is needed. A look at the new technologies being used to develop vaccines will be presented here.

Augmenting cancer vaccine strategies with dendritic cell boosts

Cationic microparticles for DNA adsorption were formulated by blending poly(lactide-co-glycolide) (PLGA) (50 : 50), with different cationic agents, either PEI 25 kDa (polyethylenimine) or CTAB (cetyl-trimethyl-ammonium-bromide). The aim was to create adjuvant delivery systems increasing the efficiency of DNA vaccines. Microparticles formulated with 10% PEI exhibited a highly positive ζ-potential, small particle sizes, in contrast to particles prepared with CTAB, which revealed highly aggregated structures in scanning electron micrographs. PEI 10% microparticles efficiently adsorbed DNA and protected DNA from enzymatic degradation.Microparticles with up to 10% PEI did not affect membrane integrity whereas CTAB particles showed higher LDH release. Transfection efficiencies were assessed using a luciferase reporter gene assay compared to naked DNA and PEI/DNA polyplexes. DNA adsorbed onto microspheres with 10% or 50% PEI generally had higher transfection efficiencies than CTAB but reached lower expression levels than PEI/DNA polyplexes alone. This documented the intact release of DNA. The mechanism of gene delivery to non-phagocytic cells was studied via covalent fluorescence labeling of both the DNA and PEI by confocal microscopy and suggested uptake of DNA.Immunization of mice was performed using plasmids encoding immunodominant antigens of Listeria monocytogenes adsorbed onto RG 502 H + PEI 10% microparticles. The efficiency was tested by intravenous challenge with an otherwise lethal dose of L. monocytogenes. PLGA + PEI microspheres can be used as adjuvant delivery systems for DNA but further optimization is necessary to exploit their full potential.

Development of DNA delivery system using Pseudomonas exotoxin A and a DNA binding region of human DNA topoisomerase I.

Gene therapy is defined as the delivery of a functional gene for expression in somatic tissues with the intent to cure a disease. Thus, highly efficient gene transfer is essential for gene therapy. Receptor-mediated gene delivery can offer high efficiency in gene transfer, but several technical difficulties need to be solved. In this study, we first examined the DNA binding regions of the human DNA topoisomerase I (Topo I), using agarose gel mobility shift assay, in order to identify sites of noncovalent binding of human DNA Topo I to plasmid DNA. We identified four DNA binding regions in human DNA Topo I. They resided in aa 51-200, 271-375, 422-596, and 651-696 of the human DNA Topo I. We then used one of the four regions as a DNA binding protein fragment in the construction of a DNA delivery vehicle. Based on the known functional property of each Pseudomonas exotoxin A (PE) domain and human DNA Topo I, we fused the receptor binding and membrane translocation domains of PE with a highly positively charged DNA binding region of the N-terminal 198 amino acid residues of human DNA Topo I. The resulting recombinant protein was examined for DNA binding in vitro and transfer efficiency in cultured cells. The results show that this DNA delivery protein is a general DNA delivery vehicle without DNA sequence, topology, and cell-type specificity. The DNA delivery protein could be used to target genes of interest into cells for genetic and biochemical studies. Therefore, this technique can potentially be applied to cancer gene therapy.