Use of ultrasound to enhance nonviral lung gene transfer in vivo

We have assessed if high-frequency ultrasound (US) can enhance nonviral gene transfer to the mouse lung. Cationic lipid GL67/pDNA, polyethylenimine (PEI)/pDNA and naked plasmid DNA (pDNA) were delivered via intranasal instillation, mixed with Optison microbubbles, and the animals were then exposed to 1 MHz US. Addition of Optison alone significantly reduced the transfection efficiency of all three gene transfer agents. US exposure did not increase GL67/pDNA or PEI/pDNA gene transfer compared to Optison-treated animals. However, it increased naked pDNA transfection efficiency by approximately 15-fold compared to Optison-treated animals, suggesting that despite ultrasound being attenuated by air in the lung, sufficient energy penetrates the tissue to increase gene transfer. US-induced lung haemorrhage, assessed histologically, increased with prolonged US exposure. The left lung was more affected than the right and this was mirrored by a lesser increase in naked pDNA gene transfer, in the left lung. The positive effect of US was dependent on Optison, as in its absence US did not increase naked pDNA transfection efficiency. We have thus established proof of principle that US can increase nonviral gene transfer, in the air-filled murine lung.     

Delivery and expression of pDNA embedded in collagen matrices.

Collagen matrices can be used as non-viral biocompatible gene carriers for localized implantable gene therapy. Collagen matrices embedding pDNA with enhanced binding through condensing agent linkage to the matrix or to the pDNA have been formulated, and characterized in various systems. pDNA and condensed pDNA were released intact from the matrices within 1-2 days. In vitro transfection with collagen matrices containing pDNA (luciferase encoding), pDNA in liposome (LIP), and pDNA with polyethylenimine (PEI) resulted in significantly higher expression levels in comparison to naked pDNA. pDNA-LIP matrices exhibited a dose response transfection of NIH 3T3, 293, MDA-MB-231 and smooth muscle cells (SMCs) in cell cultures. Subdermal implantations of collagen-polylysine-pDNA matrices in rats resulted in significantly higher gene expression levels in comparison to non-condensed pDNA matrices. Perivascular treatment with pDNA matrix and of naked pDNA solution in balloon-injured rat carotid arteries resulted in significant expression. In conclusion, a facile method for embedding cationic formulations of pDNA in collagen matrices was developed. These bioactive matrices seem to be suitable for tissue engineering and local gene therapy strategies.     

Sustained delivery and expression of DNA encapsulated in polymeric nanoparticles

Sustained release polymeric gene delivery systems offer increased resistance to nuclease degradation, increased amounts of plasmid DNA (pDNA) uptake, and the possibility of control in dosing and sustained duration of pDNA administration. Furthermore, such a system lacks the inherent problems associated with viral vectors. Biodegradable and biocompatible poly(DL-lactide-co-glycolide) polymer was used to enacapsulate pDNA (alkaline phosphatase, AP, a reporter gene) in submicron size particles. Gene expression mediated by the nanoparticles (NP) was evaluated in vitro and in vivo in comparison to cationic-liposome delivery. Nano size range (600 nm) pDNA-loaded in poly(DL-lactide-co-glycolide) polymer particles with high encapsulation efficiency (70%) were formulated, exhibiting sustained release of pDNA of over a month. The entrapped plasmid maintained its structural and functional integrity. In vitro transfection by pDNA-NP resulted in significantly higher expression levels in comparison to naked pDNA. Furthermore, AP levels increased when the transfection time was extended, indicating sustained activity of pDNA. However, gene expression was significantly lower in comparison with standard liposomal transfection. Seven days after i.m. injections in rats, naked pDNA and pDNA-NP were found to be significantly more potent (1-2 orders of magnitude) than liposomal pDNA. Plasmid DNA-NP treatment exhibited increased AP expression after 7 and 28 days indicating sustained activity of the NP.     

Water-soluble lipopolymer as an efficient carrier for gene delivery to myocardium

Water-soluble lipopolymer (WSLP), which consisted of polyethylenimine (PEI, 1800 Da) and cholesterol, was characterized as a gene carrier to smooth muscle cells and myocardium. Acid-base titration showed that WSLP had a proton-buffering effect. The size of WSLP/plasmid DNA (pDNA) complex was around 70 nm. WSLP/pDNA complex was transfected to A7R5 cells, a smooth muscle cell line. WSLP showed the highest transfection at a 40/1 N/P ratio. WSLP has higher transfection efficiency than PEI (1800 and 25 000 Da), SuperFect, and lipofectamine. In addition, WSLP has less cytotoxicity than PEI (25 000 Da), SuperFect, and lipofectamine. Since WSLP has cholesterol moiety, it may utilize cellular cholesterol uptake pathway, in which low-density lipoprotein (LDL) is involved. An inhibition study with free cholesterol or low-density lipoprotein (LDL) showed that transfection was inhibited by cholesterol or LDL, suggesting that WSLP/pDNA complex is transfected to the cells through the cholesterol uptake pathway. To evaluate the transfection efficiency to myocardium, WSLP/pDNA complex was injected into the rabbit myocardium. WSLP showed higher transfection than PEI and naked pDNA. WSLP expressed the transgene for more than 2 weeks. In conclusion, WSLP is an efficient carrier for local gene transfection to myocardium, and useful in in vivo gene therapy.     

Optimizing aerosol gene delivery and expression in the ovine lung.

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.     

A comparison of linear and branched polyethylenimine (PEI) with DCChol/DOPE liposomes for gene delivery to epithelial cells in vitro and in vivo

Polyethylenimine (PEI), a polycation with high ionic charge density, has recently been used as a gene therapy delivery agent. We have defined the optimal conditions for PEI-based transfection of airway epithelial cells in vitro and in vivo and used these conditions to restore Cl(-) channel activity in a CF mouse model. Three forms of PEI, a linear 22 kDa (ExGen 500) form and branched 25 or 50 kDa forms were evaluated. All forms of PEI significantly increased luciferase reporter gene expression compared to the liposome DCChol/DOPE in a human bronchial epithelial cell line (16HBE) irrespective of the extent of cell confluency. With subconfluent cells, gene expression was around 1000-, 200- and 25-fold higher than liposomes using linear 22, 25 and 50 kDa PEI, respectively. The transfection efficiency was reduced in confluent and polarized epithelial cells but linear 22 kDa PEI showed the smallest decrease and gave 8000-fold better transfection in polarized cells compared to liposomes. A comparison of linear 22 or 25 kDa PEI with DCChol/DOPE for airway delivery in vivo via intranasal instillation was also performed. Linear 22 kDa PEI gave significantly better luciferase reporter gene expression of 350-fold in the lung, 180-fold in the nose and 85-fold in the trachea compared to liposome. In contrast, the 25 kDa form of PEI was no better than DCChol/DOPE. Repeat dosing with linear 22 kDa PEI failed to give reporter gene delivery comparable to the initial dose. To establish that PEI can be used to deliver a physiologically relevent gene in vivo, we used it to restore Cl(-) secretion by CFTR gene delivery in the airways of a CF mouse model.     

Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein-Barr virus (EBV)-based plasmid vectors

Naked plasmid DNA (pDNA) injection could become an alternative procedure to viral and nonviral gene delivery systems. We have previously shown that Epstein-Barr virus (EBV)-based plasmid vectors containing the EBV nuclear antigen 1 (EBNA1) gene and the oriP sequence enable quite high and long-lasting expression in various in vitro and in vivo transfection systems. The EBV-based plasmids were intravenously injected into mice via their tail vein under high pressure. A large amount of the marker gene product was expressed in the liver; as much as 320 microg of luciferase was demonstrated per gram of liver at 8 to 24 h after a single injection with 10 microg of DNA. More than 70% of liver cells stained with X-gal when beta-gal gene was transferred. The expression level was significantly higher than that obtained by conventional pDNA lacking the EBNA1 gene and oriP. On day 35 after the transfection, the expression from the EBV-based plasmid was approximately 100-fold stronger than the conventional pDNA gene expression. Both the EBNA1 gene and oriP are a prerequisite for the augmentation of the transfection efficiency. These results suggest that the intravascular transfection with naked EBV-based plasmid may provide a quite efficient, simple and convenient means to transduce therapeutic genes in vivo into the liver.     

Evaluation of proinflammatory cytokine production induced by linear and branched polyethylenimine/plasmid DNA complexes in mice

The purpose of this study was to evaluate the cytokine response induced by linear and branched polyethylenimine (PEI)/plasmid DNA (pDNA) complex (polyplex) in relation to the ratio of PEI nitrogen and DNA phosphate (N/P ratio) of the polyplex, dose of pDNA, and structure and molecular weight of PEI, which are important for transfection efficacy of PEI polyplex. As a control, a N-[1-(2, 3-dioleyloxy) propyl]-n,n,n-trimethylammonium chloride/cholesterol liposome/pDNA complex (lipoplex) was selected for its high transfection efficacy in vivo. The concentration of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha were much lower after the administration of polyplex than lipoplex irrespective of the N/P ratio, dose of pDNA, or structure and molecular weight of PEI, although these factors affected the transfection efficacy in vivo. We demonstrated that the amount of activated nuclear factor-kappaB, which contributes substantially to the production of cytokines, was comparable with the control (no treatment) level, and significantly less than that obtained with lipoplex. Although the production of proinflammatory cytokines (TNF-alpha, interferon-gamma, and interleukin-12) was reduced on the administration of the linear PEI polyplex, serum alanine aminotransferase levels were significantly enhanced by pDNA in a dose-dependent manner, suggesting that such hepatic damage is not induced by proinflammatory cytokines.     

Controlled release of cell-permeable gene complex from poly(L-lactide) scaffold for enhanced stem cell tissue engineering

The use of tissue engineering to deliver genes to stem cells has been impeded by low transfection efficiency of the inserted gene and poor retention at the target site. Herein, we describe the use of non-viral gene transfer by cell-permeable peptide (CPP) to increase the transfection efficiency. The combination of this technique with the use of a controlled release concept using a poly (l-lactide) scaffold allowed for prolonged uptake in stem cells. High transfection efficiency was obtained using a human-derived arginine-rich peptide denoted as Hph-1 (YARVRRRGPRR). The formation of complex between pDNA and Hph-1 was monitored using gel retardation tests to measure size and zeta potential. Complex formation was further assessed using a DNase I protection assay. A sustained gene delivery system was developed using a fibrous 3-D scaffold coated with pDNA/Hph-1 complexes. Transfection efficiency and the mean fluorescence intensity of human adipose-derived stem cells (hASCs) on the sustained delivery scaffold were compared to those of cells transfected via bolus delivery. Plasmid DNA completely bound Hph-1 at a negative-to-positive (N/P) charge ratio of 10. After complex formation, Hph-1 appeared to effectively protect pDNA against DNase I attack and exhibited cytotoxicity markedly lower than that of the pDNA/PEI complex. Plasmid DNA/Hph-1 complexes were released from the scaffolds over 14 days and were successfully transfected hASCs seeded on the scaffolds. Flow cytometry revealed that the transfection efficiency in hASCs treated with pDNA/Hph-1 complex was approximately 5-fold higher than that in cells transfected using Lipofectamine. The sustained delivery system showed a significantly higher transfection efficiency and remained able to transfect cells for a longer period of time than bolus delivery. These results suggest that cell-scaffold-based tissue regeneration can be further improved by transduction concept using CPP and controlled release using polymeric scaffold.     

Combined delivery of dexamethasone and plasmid DNA in an animal model of LPS-induced acute lung injury

Dexamethasone was conjugated to low molecular weight polyethylenimine (2 kDa, PEI2k). Dexamethasone conjugated PEI2k (PEI2k-Dexa) was evaluated as a combined delivery carrier of dexamethasone and plasmid DNA (pDNA) in an animal model of lipopolysaccharide (LPS) induced acute lung injury (ALI). In vitro transfection of L2 lung epithelial cells, PEI2k-Dexa exhibited higher transfection efficiency than PEI2k or a simple mixture of PEI2k and dexamethasone. In addition, the PEI2k-Dexa/pβ-Luc complexes reduced the levels of pro-inflammatory cytokines in LPS activated Raw 264.7 macrophage cells. The anti-inflammatory effect of PEI2k-Dexa was higher than that of controls. The PEI2k-Dexa/pβ-Luc complexes were administered to mice via intratracheal injection. PEI2k-Dexa had higher pDNA delivery efficiency than PEI2k in the lung and decreased TNF-α and IL-6 in the lung homogenates and bronchoalveolar lavage (BAL) fluid compared with the controls. Furthermore, total protein and immunoglobulin M (IgM) concentrations in BAL fluid were reduced by the PEI2k-Dexa/pβ-Luc complexes. The intratracheal injection of the PEI2k-Dexa/pcDNA-EGFP complexes in the ALI model showed higher EGFP expression compared with PEI2k. Hematoxylin and eosin (H&E) staining showed that PEI2k-Dexa reduced inflammatory reaction in the lung. Therefore, PEI2k-Dexa may be useful for combination gene and drug therapy for ALI.     

Nonviral gene transfer into fetal mouse livers (a comparison between the cationic polymer PEI and naked DNA)

We investigated the efficacy and safety of the cationic polymer polyethylenimine (PEI) as a potential tool for intrauterine gene delivery into livers of fetal mice in the last trimester of pregnancy (E17.5). Using luciferase as a reporter gene, transferrin-conjugated and ligand-free PEI/DNA complexes (containing 3 microg DNA) with varying PEI-nitrogen/DNA-phosphate (N/P) ratios and different PEI forms, branched (800, 25 kDa) and linear (22 kDa), were compared with naked DNA. Transgene expression was measured 48 h after administration of PEI/DNA complexes or naked DNA. Highest luciferase activity (9.8 x 10(3) relative light units (RLU)/mg of tissue protein) was observed with ligand-free PEI22/DNA mixtures at N/P 6.0. In addition, this formulation was associated with very low toxicity as compared to the other PEI/DNA-injected groups. Using beta-galactosidase as a reporter gene, transfection of single, but also small, clusters of cells was demonstrated throughout the liver. Injection of 3 microg naked DNA resulted in an 11-fold lower transgene expression value (0.9 x 10(3) RLU/mg of tissue protein) as compared to PEI22/DNA complexes. However, the administration of higher concentrated naked DNA (9 microg) into fetal livers yielded expression levels of 3.2 x 10(4) RLU/mg of tissue protein, a more than three-fold increase compared to PEI22/DNA complexes. Furthermore, the gene transfer efficacy of concentrated naked DNA was approximately 40 times higher in fetuses than in adults (0.8 x 10(3) RLU/mg of tissue protein), indicating that fetal tissue is especially amenable to the uptake and expression of naked DNA.     

Synergistic effect of ultrasound and PEI on DNA transfection in vitro.

Ultrasound and poly(ethylenimine) (PEI) have each separately been shown to increase DNA transfection efficiency. This study tested the hypothesis that the combination of ultrasound and PEI can have a synergistic effect to increase DNA transfection. This in vitro study assessed transfection efficiency of two different DNA plasmids encoding green fluorescent protein and firefly luciferase in two different cell types, a primary culture of human aortic smooth muscle cells and an immortal line of human prostate cancer cells. We found that ultrasound sonication increased transfection up to 18-fold, DNA complexation with PEI increased transfection up to 90-fold, and the combination of ultrasound and PEI synergistically increased transfection up to 200-fold, which resulted in reporter gene expression by 34% of cells. Kinetic measurements found that the effects of ultrasound alone acted quickly, whereas increased transfection by PEI either alone or in combination with ultrasound strongly benefited from a 4-h incubation with the DNA plasmid after sonication. Although serum reduced absolute expression levels, it did not affect the relative increase in transfection when ultrasound was added to PEI enhancement. Flow cytometry measurements showed that sonication increased intracellular uptake of labeled DNA complexed to PEI by 55% relative to PEI complexation alone. Electrophoresis assay showed no damage to DNA or PEI-DNA complexes after sonication. Overall, these results suggest that the combination of ultrasound and PEI can have a synergistic effect to increase DNA transfection.     

Characterization and evaluation of the efficacy of cationic complex mediated plasmid DNA delivery in human embryonic palatal mesenchyme cells

The purpose of this study was to develop and test a non‐viral gene delivery system that can be employed to deliver genes of interest into a pre‐osteoblastic cell line. Human embryonic palatal mesenchymal (HEPM 1486) cells were transfected with vector‐plasmid DNA (pDNA) complexes. We explored calcium phosphate and polyethylenimine (PEI) as non‐viral vectors and compared their respective in vitro transfection efficacies. Plasmid DNA encoding luciferase protein (LUC) was complexed with PEI (with differing N:P ratios) and calcium phosphate (with differing Ca:P ratios), using established protocols. The complexes prepared were then characterized for size and surface charge, using a Malvern Zetasizer Nano‐ZS. The transfection efficiency and cytotoxicity of the prepared complexes were evaluated in HEPM cells. The PEI–pDNA complexes over the whole range of N:P ratios were found to be < 160 nm in size, while the calcium phosphate–pDNA complexes were relatively bigger. The PEI–pDNA complexes prepared at a N:P ratio of 10 were found to have maximum transfection efficiency at 4 h of treatment, with minimal cytotoxicity. The highest transfection efficiency obtained with calcium phosphate–pDNA complexes (Ca:P 200) was nearly 12‐fold lower than that obtained with PEI–pDNA complexes (N:P 10). Following this, transgene expression in the HEPM cells treated with complexes prepared at a N:P ratio of 10 was further examined, using pDNA coding for enhanced green fluorescent protein (EGFP‐N1) or therapeutically relevant platelet‐derived growth factor B (PDGF‐B). In conclusion, PEI was a more effective vector for delivering genes of interest to pre‐osteoblasts than calcium phosphate. Copyright © 2014 John Wiley & Sons, Ltd.     

Mobility and stability of gene complexes in biogels.

The tenacious secretions lining the conductive airways of cystic fibrosis (CF) patients may pose a significant barrier to successful gene therapy to the lung. In this work, we evaluated the diffusion of nanospheres and cationic DOTAP lipoplexes through CF sputum and the influence of CF mucus components on the physicochemical properties and gene expression of cationic DOTAP lipoplexes and neutral, pegylated GL67 lipoplexes. The number of particles transported through the sputum was extremely low (<0.5%) and strongly depending on the size of the particles, with almost no transport for the largest nanospheres (560 nm). For small particles (<150 nm), the low transport was primarily due to the long distance they have to travel through the sputum, while for larger particles also sterical obstruction was responsible for the low transport. Upon exposure of the cationic DOTAP lipoplexes to albumin, linear DNA or mucin (at concentration ratios expected to occur in vivo) a significant decrease in gene transfection activity was observed. This was primarily due to aggregation of the lipoplexes. However, exposure of pegylated GL67 lipoplexes to the same components did not affect their gene transfection activity. Indeed, it was determined that CF mucus components did not interact significantly with these neutral, pegylated GL67 lipoplexes.     

Pegylated GL67 lipoplexes retain their gene transfection activity after exposure to components of CF mucus

The highly viscous secretions lining the upper airways and bronchi of cystic fibrosis (CF) patients may pose a significant barrier to successful gene therapy of the lung. In this report we examined the influence of CF mucus components (albumin, DNA, mucin and phospholipids) on the gene transfection activity of cationic DOTAP-based lipoplexes and pegylated GL67-based lipoplexes which previously have been used in CF clinical studies. Upon exposure of the cationic DOTAP:DOPE lipoplexes to either albumin, linear DNA or mucin (at concentration ratios expected to occur in vivo) a significant decrease in gene transfection activity was observed. This was primarily due to aggregation of the lipoplexes. However, exposure of pegylated GL67 lipoplexes to the same components did not affect their gene transfection activity. Indeed, it was determined that CF mucus components did not interact significantly with these pegylated GL67 lipoplexes. These results suggest that charge shielding of cationic gene carriers with pEG may favor their physicochemical stability in CF mucus and thereby aid in preserving their transfection activity.     

PEGylated polyethylenimine for in vivo local gene delivery based on lipiodolized emulsion system.

Polyethylenimine (PEI) is one of the most efficient vectors for non-viral gene delivery, whereas its poor transfection activity, compared to viral vectors, and cytotoxicity need to be improved for in vivo applications. In this study, we prepared two PEI conjugates with 6 and 10 wt.% of poly(ethylene glycol) (PEG) grafts (referred to PEI-PEG-6 and PEI-PEG-10, respectively) in order to investigate the effects of PEGylation on cytotoxicity and transfection activity in vitro. In addition, their suitability as vectors for local gene delivery in vivo was assessed by injecting lipiodolized emulsions containing polymer/DNA complexes into the femoral artery of Sprague-Dawley (SD) rats, occluded by a surgical suture to block inflow of the blood to the leg. Both PEGylated PEIs showed significantly lower cytotoxicity and higher transfection activity in COS-1 cells than PEI taken as a control; in particular, PEI-PEG-10 produced the most promising results. The stable water-in-oil emulsion, composed of aqueous domains containing the complexes and lipiodol as an oil phase, was formed in the presence of a hydrogenated castor oil. From in vivo experiments, it was found that all the complexes, dispersed in the lipiodolized emulsion, delivered effectively gene to muscle, surrounding the injection site, rather than other organs such as liver, spleen, kidney, heart and lung. The in vivo transfection activity of PEI-PEG-10 was 3-folds higher in muscle than that of PEI. Based on these results, it can be concluded that PEGylated PEIs (based on the lipiodolized emulsion system) hold a promising potential for local gene delivery in vivo.     

Galactosylated chitosan-graft-polyethylenimine as a gene carrier for hepatocyte targeting

Chitosans have been proposed as alternative, biocompatible cationic polymers for nonviral gene delivery. However, the low transfection efficiency and low specificity of chitosan need to be addressed before clinical application. We prepared galactosylated chitosan-graft-polyethylenimine (GC-g-PEI) copolymer by an imine reaction between periodate-oxidized GC and low-molecular-weight PEI. The molecular weight and composition were characterized using gel permeation chromatography column with multi-angle laser scattering and (1)H nuclear magnetic resonance, respectively. The copolymer was complexed with plasmid DNA in various copolymer/DNA (N/P) charge ratios, and the complexes were characterized. GC-g-PEI showed good DNA-binding ability and superior protection of DNA from nuclease attack and had low cytotoxicity compared to PEI 25K. GC-g-PEI/DNA complexes showed higher transfection efficiency than PEI 25K in both HepG2 and HeLa cell lines. Transfection efficiency into HepG2, which has asialoglycoprotein receptors, was higher than that into HeLa, which does not. GC-g-PEI/DNA complexes also transfected liver cells in vivo after intraperitoneal (i.p.) administration more effectively than PEI 25K. These results suggest that GC-g-PEI can be used in gene therapy to improve transfection efficiency and hepatocyte specificity in vitro and in vivo.     

Pre-clinical evaluation of three non-viral gene transfer agents for cystic fibrosis after aerosol delivery to the ovine lung

We use both large and small animal models in our pre-clinical evaluation of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy. Here, we report the use of a large animal model to assess three non-viral GTAs: 25?kDa-branched polyethyleneimine (PEI), the cationic liposome (GL67A) and compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. GTAs complexed with plasmids expressing human cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA were administered to the sheep lung (n=8 per group) by aerosol. All GTAs gave evidence of gene transfer and expression 1 day after treatment. Vector-derived mRNA was expressed in lung tissues, including epithelial cell-enriched bronchial brushing samples, with median group values reaching 1-10% of endogenous CFTR mRNA levels. GL67A gave the highest levels of expression. Human CFTR protein was detected in small airway epithelial cells in some animals treated with GL67A (two out of eight) and PEI (one out of eight). Bronchoalveolar lavage neutrophilia, lung histology and elevated serum haptoglobin levels indicated that gene delivery was associated with mild local and systemic inflammation. Our conclusion was that GL67A was the best non-viral GTA currently available for aerosol delivery to the sheep lung, led to the selection of GL67A as our lead GTA for clinical trials in CF patients.     

Evaluation of polyether-polyethyleneimine graft copolymers as gene transfer agents

Cationic copolymers consisting of polycations linked to non-ionic polymers are evaluated as non-viral gene delivery systems. These copolymers are known to produce soluble complexes with DNA, but only a few studies have characterized the transfection activity of these complexes. This work reports the synthesis and characterization of a series of cationic copolymers obtained by grafting the polyethyleneimine (PEI) with non-ionic polyethers, poly (ethylene oxide) (PEO) or Pluronic 123 (P123). The PEO-PEI conjugates differ in the molecular mass of PEI (2 kDa and 25 kDa) and the degree of modification of PEI with PEO. All of these conjugates form complexes upon mixing with plasmids, which are stable in aqueous dispersion for several days. The sizes of the particles formed in these systems vary from 70 to 200 nm depending on the composition of the complex. However, transfection activity of these systems is much lower than that of PEI (25 kDa) or Superfect as assessed in in vitro transfection experiments utilizing a luciferase reporter expression in Cos-7 cells as a model system. In contrast, conjugate of P123 with PEI (2 kDa) mixed with free P123 (9:1(wt)) forms small and stable complexes with DNA (110 nm) that exhibit high transfection activity in vitro. Furthermore, gene expression is observed in spleen, heart, lungs and liver 24 h after i.v. injection of this complex in mice. Compared to 1,2-bis(oleoyloxy)-(trimethylammonio) propane:cholesterol (DOTAP:Chol) and PEI (25 kDa) transfection systems, the P123-PEI system reveals a more uniform distribution of gene expression between these organs, allowing a significant improvement of gene expression in liver.     

Chitosan as a nonviral gene delivery system. Structure-property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo

Chitosan is a natural cationic linear polymer that has recently emerged as an alternative nonviral gene delivery system. We have established the relationships between the structure and the properties of chitosan-pDNA polyplexes in vitro. Further, we have compared polyplexes of ultrapure chitosan (UPC) of preferred molecular structure with those of optimised polyethylenimine (PEI) polyplexes in vitro and after intratracheal administration to mice in vivo. Chitosans in which over two out of three monomer units carried a primary amino group formed stable colloidal polyplexes with pDNA. Optimized UPC and PEI polyplexes protected the pDNA from serum degradation to approximately the same degree, and they gave a comparable maximal transgene expression in 293 cells. In contrast to PEI, UPC was non toxic at escalating doses. After intratracheal administration, both polyplexes distributed to the mid-airways, where transgene expression was observed in virtually every epithelial cell, using a sensitive pLacZ reporter containing a translational enhancer element. However, the kinetics of gene expression differed - PEI polyplexes induced a more rapid onset of gene expression than UPC. This was attributed to a more rapid endosomal escape of the PEI polyplexes. Although this resulted in a more efficient gene expression with PEI polyplexes, UPC had an efficiency comparable to that of commonly used cationic lipids. In conclusion, this study provides insights into the use of chitosan as a gene delivery system. It emphasises that chitosan is a nontoxic alternative to other cationic polymers and it forms a platform for further studies of chitosan-based gene delivery systems.