PEI-DNA Complexes Research Articles

Enhancement of plasmid DNA immunogenicity with linear polyethylenimine

The utility of plasmid DNA as an immunogen has been limited by its weak immunogenicity. In the present study, we evaluated the ability of a family of linear polyethylenimine (PEI) polymers, complexed to plasmid DNA, to augment DNA expression in vivo and to enhance antigen-specific adaptive immune responses. We showed that four of five structurally different PEIs that we evaluated increased in vivo DNA expression 20- to 400-fold, and enhanced DNA-induced epitope-specific CD8⁺ T-cell responses 10- to 25-fold in BALB/c and C57BL/6J mice respectively, when delivered intravenously. Functional studies of the PEI-DNA-induced CD8⁺ T-cell responses demonstrated that formulation of DNA with PEI was associated with increased numbers of cells secreting type I cytokines. In addition, PEI-DNA complexes improved antigen-specific T(H) 1-helper cell and humoral responses. Most importantly, the PEI-DNA complexes elicited memory cellular responses, capable of rapid expansion and accelerated clearance of a lethal dose of recombinant Listeria monocytogenes. Lastly, we identified physical properties of PEI-DNA complexes that are associated with enhanced DNA-elicited immunogenicity. These findings demonstrate that PEI polymers can play an important role in the development of DNA-based vaccines in the setting of infectious disease prevention and cancer therapy.

Role of non‐specific DNA in reducing coding DNA requirement for transient gene expression with CHO and HEK‐293E cells

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the TGE volumetric productivity has improved significantly over the past decade, the amount of plasmid DNA (pDNA) needed for transfection remains very high. Here, we examined the use of non-specific (filler) DNA to partially replace the transgene-bearing plasmid DNA (coding pDNA) in transfections of Chinese hamster ovary (CHO) and human embryo kidney (HEK-293E) cells. When the optimal amount of coding pDNA for either host was reduced by 67% and replaced with filler DNA, the recombinant protein yield decreased by only 25% relative to the yield in control transfections. Filler DNA did not affect the cellular uptake or intracellular stability of coding pDNA, but its presence lead to increases of the percentage of transfected cells and the steady-state level of transgene mRNA compared to control transfections. Studies of the physicochemical properties of DNA-polyethyleneimine (PEI) complexes with or without filler DNA did not reveal any differences in their size or surface charge. The results suggest that filler DNA allows the coding pDNA to be distributed over a greater number of DNA-PEI complexes, leading to a higher percentage of transfected cells. The co-assembly of filler DNA and coding pDNA within complexes may also allow the latter to be more efficiently utilized by the cell's transcription machinery, resulting in a higher level of transgene mRNA.

DNA Condensation by pH-Responsive Polycations

This work addresses the impact of pH variation on DNA-polyethylenimine (PEI) complex formation, in aqueous solution and at constant ionic strength. An initial potentiometric characterization of the acid-base behavior of PEI is carried out to measure the concentration of ionized species in the relevant systems. The characterization of the DNA-PEI complexes is performed by precipitation assays, agarose gel electrophoresis, photon correlation spectroscopy, and zeta potential analysis. It is observed that the variations on the electrophoretic mobility, size, and electrical properties of complexes display nonmonotonic, nontrivial trends with pH, if the same polycation/polyanion charge ratios are used for different values of pH. It is seen that both linear charge density and the relative number of chains of the condensing agent are important factors governing the condensation behavior. Complexes prepared at pH 4, for example, indicate strong binding and a large mean size, while those prepared at pH 8 are smaller, in a more uniform population. Finally, charge inversion was observed for all studied pH values (even below charge neutralization).

Formulation of a Peptide Nucleic Acid Based Nucleic Acid Delivery Construct

Gene delivery biomaterials need to be designed to efficiently achieve nuclear delivery of plasmid DNA. Polycations have been used to package DNA and other nucleic acids within submicrometer-sized particles, offering protection from shear-induced or enzymatic degradation. However, cytotoxicity issues coupled with limited in vivo transfection efficiencies minimize the effectiveness of this approach. In an effort to improve upon existing technologies aimed at delivering nucleic acids, an alternative approach to DNA packaging was explored. Peptide nucleic acids (PNAs) were used to directly functionalize DNA with poly(ethylene glycol) (PEG) chains that provide a steric layer and inhibit multimolecular aggregation during complexation. DNA prePEGylation by this strategy was predicted to enable the formation of more homogeneous and efficiently packaged polyplexes. In this work, DNA-PNA-peptide-PEG (DP3) conjugates were synthesized and self-assembled with 25 kDa poly(ethylenimine) (PEI). Complexes with small standard deviations and average diameters ranging 30-50 nm were created, with minimal dependence of complex size on N/P ratio (PEI amines to DNA phosphates). Furthermore, PEI-DNA interactions were altered by the derivatization strategy, resulting in tighter compaction of the PEI-DP3 complexes in comparison to PEI-DNA complexes. Transfection experiments in Chinese hamster ovary (CHO) cells revealed comparable transfection efficiencies but reduced cytotoxicities of the PEI-DP3 complexes relative to PEI-DNA complexes. The enhanced cellular activities of the PEI-DP3 complexes were maintained following the removal of free PEI from the PEI-DP3 formulations, whereas the cellular activity of the conventional PEI-DNA formulations was reduced by free PEI removal. These findings suggest that DNA prePEGylation by the PNA-based strategy might provide a way to circumvent cytotoxicity and formulation issues related to the use of PEI for in vivo gene delivery.

Development of a scalable process for high‐yield lentiviral vector production by transient transfection of HEK293 suspension cultures

Lentiviral vectors (LV) offer several advantages over other gene delivery vectors. Their potential for the integration and long-term expression of therapeutic genes renders them an interesting tool for gene and cell therapy interventions. However, large-scale LV production remains an important challenge for the translation of LV-based therapeutic strategies to the clinic. The development of robust processes for mass production of LV is needed. A suspension-grown HEK293 cell line was exploited for the production of green fluorescent protein-expressing LV by transient polyethylenimine (PEI)-based transfection with LV-encoding plasmid constructs. Using third-generation packaging plasmids (Gag/Pol, Rev), a vesicular stomatitis virus G envelope and a self-inactivating transfer vector, we employed strategies to increase volumetric and specific productivity. Functional LV titers were determined using a flow cytometry-based gene transfer assay. A combination of the most promising conditions (increase in cell density, medium selection, reduction of PEI-DNA complexes per cell, addition of sodium butyrate) resulted in significantly increased LV titers of more than 150-fold compared to non-optimized small-scale conditions, reaching infectious titers of approximately 10(8) transducing units/ml. These conditions are readily scalable and were validated in 3-liter scale perfusion cultures. Our process produces LV in suspension cultures and is consequently easily scalable, industrially viable and generated more than 10(11) total functional LV particles in a single bioreactor run. This process will allow the production of LV by transient transfection in sufficiently large quantities for phase I clinical trials at the 10-20-liter bioreactor scale.

Abstract B116: Adjuvant-mediated DNA immunotherapy for cancer

Abstract B116 Cancer immunotherapy invokes immunity against cancerous cells by stimulating the immune response from the host. In an attempt to investigate the effect of adjuvant-mediated therapy, polyethyleneimine (PEI), a nonviral polycationic gene delivery vector, was employed in this study as an adjuvant to investigate the immunostimulatory effect on the murine bone marrow-derived dendritic cells (BMDCs) and in the tumor-bearing animals. The physicochemical properties of the DNA-PEI complexes was characterized by the size and surface potential measurements. Murine bone-marrow-derived dendritic cells (BMDCs) at day 6 were treated with DNA-PEI complexes at various DNA/PEI ratios, followed by flow cytometric analysis of the expression of costimulatory molecules, including CD40, CD80, and CD86, at 24 h post-treatment. The production of the reactive oxygen species (ROS) after treatment with the PEI in the BMDCs was assessed with 2',7'-dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE), followed by flow cytometry. To determine the effect of adjuvant on immunotherapeutic response, approximately 2 x 105 B16F10 melanoma cells were inoculated s.c. on the back of C57BL/6. Control groups were injected with PBS. When the tumor sizes reached 100 mm3 on day 6 after inoculation of B16F10 cells, animals were treated with the PEI-DNA complexes, containing the IL2 or caspase (fusion of caspase-2 prodomain and amino-terminal truncated caspase-3) plasmid DNA. Tumor sizes were measured with a caliper, and the volumes were determined using the formula of (length x width2)/2. Each group contained 4 animals. Results obtained in this study demonstrated that treatment of BMDCs with PEI or LPS impaired the endocytic activity of BMDCs, and upregulated the expression of the costimulatory molecules, such as CD40 and CD86, suggesting the induction of maturation by PEI on the BMDCs. Treatment of BMDCs with the DNA-PEI complexes also lead to a concentration-dependent production of the reactive oxygen species (ROS), implicating the adjuvant function of PEI on immunostimulation. Injection of the animals with IL-2 or caspase plasmid DNA, complexed with PEI, was able to induce significant inhibition of the growth of tumors in the tumor-bearing animals. These results demonstrated the prevention and therapeutic effect of adjuvant-mediated DNA immunotherapy in the murine melanoma model. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B116.

Improvement in the suspension‐culture production of recombinant adeno‐associated virus–LacZ in HEK‐293 cells using polyethyleneimine–DNA complexes in combination with hypothermic treatment

rAAV (recombinant adeno-associated virus) has become a very useful gene-delivery vector for gene therapy. However, it is very difficult to generate rAAV using triple transfection on a commercial scale, owing to its low transfection efficiency. An optimal procedure for transfection in suspension-culture mode was developed for rAAV-LacZ production in suspension-cultured HEK-293 (human embryonic kidney-293) cells mediated by PEI (polyethyleneimine)-DNA complexes in combination with transient severe hypothermia at 4 degrees C for 1 h in the present study (LacZ is the product of the reporter gene lacZ, which codes for beta-D-galactosidase). It showed that the PEI/DNA ratio, cell density at the beginning of transfection and cell-cycle arrest in G2/M-phase were key factors affecting suspension-culture triple-transfection efficiency and rAAV-LacZ productivity. After incubation at 4 degrees C for 1 h and re-warming at 37 degrees C for 18 h, HEK-293 cells at 1x10(6) cells/ml were transfected with PEI-DNA complexes at a PEI/DNA ratio of 5:1 (w/w) with final concentrations of 30 mug/ml 25 kDa linear PEI and 6 mug/ml plasmid DNA in culture. After 6 h incubation for transfection, an equal volume of medium was added to the culture for additional 48 h growth until harvest. Finally, the high transfection efficiency of some 75% and rAAV-LacZ titre of (7.48+/-0.59)x10(11) physical particles or 1.86+/-0.96x10(10) infectious particles were achieved in 250 ml shake flasks with 60 ml working volume, indicating a promising application for scale-up.

Polyethyleneimine‐mediated gene delivery into rat pheochromocytoma PC‐12 cells

In this study, we examined the use of polyethyleneimine (PEI) as a non-viral gene carrier and lipofectamine(trade mark) 2000 as control for rat pheochromocytoma PC-12 cells. The complex formation of PEI and DNA or lipofectamine and DNA was characterized by gel electrophoresis and measurement of particle size and surface charge. A gradual increase in surface charge (from 0.7 to 43 mV) and a gradual decrease in particle size (from 900 to 130 nm) was observed in the PEI-DNA complex with higher PEI concentrations. The cytotoxicity of PC-12 cells for lipofectamine-DNA complex was similar to PEI-DNA complex at N:P charge ratios of 4 and 8. Transfection efficiency was 14% for lipofectamine and 15% for PEI. At low N:P ratio, DNA condenses poorly, so the particle size tends to be large and polydispersed, resulting in poor transfection efficiency. Meanwhile, a high N:P ratio results in high transfection efficiency and cytotoxicity. Transfected PC-12 cells showed the generation of neurites from transfected PC-12 cells in the presence of NGF, indicating the differentiation of PC-12 cells. NGF-differentiated PC-12 cells were transfected by PEI-DNA complex of N:P charge ratio 8. From real-time imaging for transfection, the enhanced green fluorescent protein (EGFP) started to localize in the nuclei of PC-12 cells at 5 h and localized in the cytoplasm from 15 h. Our study demonstrates that PEI or lipofectamine may be applied as an effective gene carrier for PC-12 cells.

Biochemical Investigation of Active Intracellular Transport of Polymeric Gene-Delivery Vectors

To design safe, efficient synthetic gene therapy vectors, it is desirable to understand the intracellular mechanisms that facilitate their delivery from the cell surface to the nucleus. Elements of the cytoskeleton and molecular motor proteins are known to play a pivotal role in most intracellular active transport processes. The actin depolymerizer cytochalasin D and microtubule effectors colchicine and paclitaxel were used to evaluate the function of these components of the cytoskeleton in the trafficking of polyethylenimine (PEI)-DNA complexes. In addition, ATPase inhibitors erythro-9[3-(2-hydroxynonyl)] adenine (EHNA), vanadate, adenylylimidodiphosphate (AMP-PNP), and rose bengal lactone (RBL), which have inhibitory activity against dynein and kinesin, were used to examine to the effects of these molecular motors on PEI-DNA delivery. Disruption of microfilaments decreased the delivery efficiency of PEI polyplexes 60-80%, though cytochalasin D did not significantly inhibit uptake. Depolymerization of microtubules by colchicine decreased transfection efficiency by 75%. Microtubule stabilization with paclitaxel, however, facilitated a 20-fold increase in gene expression. Treatment with EHNA and vanadate caused 50% and 80% decreases in transfection efficiency, respectively. Transfection efficiency was also decreased by RBL (80%) and AMP-PNP (98%). Our findings confirm the importance of microfilament- and microtubule-based active transport of PEI-DNA complexes. Further, the strong decrease in transfection efficiency caused by ATPase inhibitors that possess inhibitory activity against kinesin implies an unexpected role for these motors in gene delivery.

Transfection of HEK293-EBNA1 Cells in Suspension with Linear PEI for Production of Recombinant Proteins

INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a "must-have" technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, it is usually achieved in serum-containing medium under tightly controlled conditions that are difficult to achieve on a large scale. In contrast, polyethylenimine (PEI) is much easier to use: It binds to and precipitates DNA efficiently and the resulting DNA-PEI complexes are suitable for efficient transfection of mammalian cells. PEI has been used successfully on a large scale in serum-containing and serum-free cultures. In particular, the linear isoform of PEI is more effective for transfecting cells in suspension. This protocol describes the steps needed for successful transfection of HEK293 cells adapted to serum-supplemented or serum-free medium in suspension culture using linear PEI.

Transfection of Adherent HEK293-EBNA1 Cells in a Six-Well Plate with Branched PEI for Production of Recombinant Proteins

INTRODUCTIONFast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a "must-have" technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, polyethylenimine (PEI) is much easier to use: It binds to and precipitates DNA efficiently and the resulting DNA-PEI complexes are suitable for efficient transfection of mammalian cells. In particular, the branched isoform of PEI works well for adherent cells, as it promotes their attachment to the plastic surface. It is thus very useful in experiments requiring multiple medium exchanges or washing steps following transfection. Also, when used in conjunction with six-well CellBIND plates, branched PEI can be used to adhere transfected cells when establishing stable cell lines. This protocol describes the steps needed for successful transfection of HEK293 cells adapted to serum-supplemented or serum-free medium in adherent culture using branched PEI.

Lung-directed gene therapy in mice using the nonviral Sleeping Beauty transposon system

The Sleeping Beauty (SB) transposon is an integrative nonviral plasmid system. Here, we describe a protocol for SB-mediated transgene delivery using DNA/polyethyleneimine (PEI) complexes for long-term expression in mouse lungs. This protocol can be used for delivery of any plasmid-based vector system to mouse lungs, although long-term transgene expression will be obtained only when using the SB transposon or other integrating vector systems. The stages of this protocol are preparation of DNA-PEI complexes and injection of the complexes into the lateral tail vein of mice. We also provide protocols for assessing transgene expression using in vivo bioluminescence imaging and enzymatic assay of lung homogenates. The procedure can be completed within 24 h, starting from preparation of DNA-PEI complexes to analysis of transient transgene expression.

Phage phiC31 integrase‐mediated genomic integration and long‐term gene expression in the lung after nonviral gene delivery

Phage phiC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study investigated the activity of phiC31 integrase in murine lungs. Transfections in murine alveolar epithelial (MLE12) cells were performed with Lipofectamine 2000. For in vivo gene delivery, DNA was complexed with polyethylenimine (PEI) and PEI-DNA complexes were injected intravenously into mice. Expression of luciferase in mice was monitored by in vivo bioluminsecence imaging. Genomic integration and integration into a previously described 'hotspot' were confirmed by polymerase chain reaction (PCR). phiC31 integrase mediated intramolecular recombination between wild-type attB and attP sites in MLE12 cells. Long-term gene expression could be observed in MLE12 cells in the presence of integrase without any selection pressure. Long-term expression of luciferase after intravenous injection of PEI-DNA complexes could be observed only in the lungs of mice which were co-injected with the integrase-encoding plasmid. Increased amounts of integrase plasmid and administration of a second dose had no effect on the level of luciferase expression achieved with a single dose, which was three orders of magnitude lower than the values observed on 'day 1' post application. Genomic integration of the transgene in the mouse lungs was confirmed by PCR. Seven out of the fifteen treated mice showed integration at the mpsL1 site, a previously described 'hot spot' from liver. These results provide evidence for the activity of phiC31 integrase in lungs but also emphasize the need for optimization of the system to maintain long-term gene expression at high levels.

Trehalose Enhances Transgene Expression Mediated by DNA‐PEI Complexes

Using enhancers to improve the transfection efficiency of polyethylenimine (PEI) can circumvent the needs of chemical modifications as well as subsequent purification and characterization of the modified PEI. In this study, we found that incorporating trehalose into the transfection reagent could improve the transgene expression mediated by DNA-PEI complexes. Such enhancements were not observed when trehalose was replaced by other disaccharides. In an effort to explore the mechanisms, we examined how the timing of trehalose treatments and the durations of trehalose affected the percentages of cells expressing green fluorescent protein and the levels of intracellular ethidium monoazide labeled plasmid. Treatments with trehalose for 5-120 min prior to transfection could cause drops in transfection efficiency by 30-50%; such treatments, however, hardly affected the amounts of intracellular plasmid, indicating that the preexistence of intracellular trehalose could reduce transfection efficiency without lowering the endocytic activity. The transfection efficiency remained almost unchanged when the transfected cells were treated with trehalose after the removal of transfection reagents, indicating that trehalose had minimal effects on the machinery of protein synthesis. Despite the enhanced transgene expression, the presence of trehalose during transfection showed inhibitory effects on the internalization of DNA-PEI complexes. Additionally, the extent of enhancement in transgene expression strongly depended on the duration of trehalose. As the above observations suggested, only during the transfection process when complexes and trehalose coexisted, trehalose became an effective enhancer of transgene expression mediated by DNA-PEI complexes possibly by affecting the mechanisms of intracellular trafficking.

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 prostrate 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.

701. Gene Delivery of PEI-DNA Complexes to the Lungs of Mice by Inhalation Is Mouse Strain- Dependent

The cationic polymer polyethylenimine (PEI) has been widely used for efficient gene delivery to the lungs of mice via aerosol application. Various mouse strains have been used in these studies but direct comparisons between individual mouse strains have not been performed to date. However, due to the widespread use of mice as pre-clinical animal models of inherited and acquired diseases, investigation of interstrain variation of aerosol gene delivery could be of interest. PEI|[ndash]|based gene vectors were nebulized simultaneously to two commonly used mouse-strains, BALB/c and NMRI, and one transgenic heterozygous apoE-deficient mouse strain via an optimized whole body aerosol exposure device. Luciferase reporter gene expression measured in lung homogenates of BALB/c mice was significantly 3.2- and 3.8-fold higher than in NMRI and apoE- deficient mice, respectively. In order to assess dose-dependent effects due to different mouse strain-dependent deposition rates, radioactively-labeled plasmid DNA (I123) complexed with PEI was nebulized to the mice lungs. DNA deposition rates were not significantly different between each of the mouse-strains. These data suggest that the genetic background of mice could play an important role for nonviral aerosol gene delivery which should be considered in transgenic animal mouse models of inherited and acquired diseases for aerosol gene delivery studies.

Serum albumin enhances polyethylenimine‐mediated gene delivery to human respiratory epithelial cells

The interaction of polyethylenimine (PEI) polyplexes with proteins in cystic fibrosis (CF) airway secretions poses a significant hurdle to this nonviral delivery system. The aim of this study was to evaluate whether albumin may increase the efficiency of PEI complexes in mediating gene transfer into respiratory epithelial cells in the presence of CF mucus. PEI (25 kDa) was complexed to DNA in the presence of human serum albumin (HSA) and used to transfect confluent A549 and 9HTEo- cells. Alternatively, albumin was added to preformed PEI-DNA complexes. The cytotoxicity of complexes was analysed by the LDH (lactate dehydrogenase) assay. CF CFT1-C2 cells were allowed to polarise and were transfected either with luciferase- or CFTR-expressing plasmids. To evaluate the effect of CF respiratory secretions on transfection efficiency, confluent cells were transfected in the presence of sputum obtained from two CF patients. The ternary PEI-HSA complexes increased luciferase expression in confluent cultures in a dose-dependent fashion up to 100 times as compared to PEI-DNA. The number of GFP-expressing cells, as evaluated by epifluorescence, was augmented several-fold. When HSA was added to preformed PEI-DNA complexes, a further 5-10-fold increase in gene expression was observed. No significant cytotoxicity was observed with either PEI or PEI-HSA polyplexes. The ternary complexes determined detectable CFTR gene transfer and expression at the apical membrane in polarised CFT1-C2 cells, as evaluated by confocal microscopy. CF sputum inhibited PEI-mediated gene transfer by 7-186-fold. Although luciferase expression mediated by PEI-HSA was still inhibited by CF sputum, these levels were 18-83.8-fold higher than with PEI. Our results demonstrate that albumin increases PEI gene transfer efficiency in confluent and polarised respiratory epithelial cells and can allow CFTR gene expression in the appropriate cellular compartment. PEI-HSA complexes display a higher efficiency than PEI also in the presence of CF sputum, indicating that albumin-containing polyplexes may help overcome barriers imposed by CF airway secretions.

781. The Effect of Overexpression of Lysyl Oxidase on Dermal Wound Healing

Introduction: In this study, we aimed to increase crosslinking in extracellular matrix proteins through overexpression of lysyl oxidase (LO). Newly formed skin at a wound site never achieves the same mechanical strength as the original tissue and this condition is exacerbated in diabetic patients. It is therefore of interest to enhance mechanical strength of newly formed tissue in wounds. In this study, we have used a gene activated matrix (GAM) approach to locally deliver plasmid DNA complexed with polyethlyenimine (PEI) at the wound site. We demonstrated that PEI-DNA complexes were taken up and expressed by fibroblasts for at least 20 days. In vitro studies showed that fibroblast-seeded GAMs with LO transgene exhibited a 3-fold increase in mechanical strength as compared with a GFP-transgene control We applied this system to a wound healing model in vivo and showed that treatment with LO-producing GAMs led to significantly enhanced mechanical strength of newly formed skin at the wound site.

Mechanistic studies on aggregation of polyethylenimine‐DNA complexes and its prevention

Aggregation of polyethylenimine (PEI)-DNA complexes severely undermines their utility for gene delivery into mammalian cells. Herein we undertook to elucidate the mechanism of this deleterious phenomenon and to develop rational strategies for its prevention. The effect of temperature, surfactants, complex concentration, ionic strength, viscosity, and pH on the time course of this aggregation was systematically examined. The aggregation process was completely inhibited by 2.5% polyoxyethylene (100) stearate (POES) and to a lesser degree by other nonionic surfactants. Importantly, POES preserved the transfection efficiency of the complexes without inducing toxicity. The aggregation was also reduced by lowering the temperature and pH, diluting the complexes, and increasing the solution viscosity. It is concluded that PEI-DNA complexes aggregate primarily due to hydrophobic interactions, while electrostatic attractions play little role.

Enhanced gene transfer and cell death following p53 gene transfer using photochemical internalisation of glucosylated PEI-DNA complexes.

p53 is frequently mutated in many cancers including human head and neck squamous cell carcinoma and pancreatic cancer. In tumor models, wild-type (wt) p53 gene transfer induces apoptosis and tumor regression in vivo, justifying the extensive clinical investigation of p53 gene therapy. p53 nonviral-mediated gene transfer was achieved using glucosylated polyethylenimine (PEI) in conjunction with photochemical internalisation (PCI). Experimental conditions were optimised using the green fluorescent protein (GFP) as a reporter. p53 gene transfer was then evaluated using semi-quantitative RT-PCR in p53-deleted PANC3 and p53-mutated FaDu cell lines. Following gene transfer, induction of apoptosis was investigated using phosphatidylserine externalisation and nuclear fragmentation assays. Induction of long-term cell death was analysed using colony-forming assays. PCI was found to enhance GFP gene transfer after 48 h in both cell lines. Whether using glucosylated-PEI alone or associated with PCI, p53 gene transfer was achieved with subsequent recovery of p53 mRNA expression in PANC3 cells and a significant 4-fold increase in p53 mRNA expression in FaDu cells. PCI was found to further enhance p53 mRNA expression by 2.3-fold in PANC3 cells. Spontaneous induction of apoptosis following wt-p53 gene transfer was achieved in both cell lines. PCI was found to enhance apoptosis up to levels similar to those achieved with chemotherapy. As a consequence, long-term cell death was significantly enhanced after wt-p53 gene transfer when PCI was used in both cell lines, yielding up to 60% cell death. PCI increases glucosylated-PEI-mediated p53 gene transfer, apoptosis as well as cell death in mutant p53 human cancer cells.