Effects of linear polyethylenimine and polyethylenimine/DNA on lung function after airway instillation to rat lungs.

Lung transplantation is an acceptable treatment option for various end-stage pulmonary diseases, but long-term survival currently lags behind that after transplantation of other solid organs. We hypothesized that gene transfer to grafts before transplantation may be a useful method to deliver antioxidant and/or anti-inflammatory genes to modulate these processes. For this purpose, we assessed the efficiency of gene transfer and effects on lung function of the synthetic polycation, linear polyethylenimine (PEI), after airway instillation to the lungs of Fischer rats. Twenty-four hours after gene delivery, reporter gene activity in DNA/PEI treated rats was approximately 12-fold higher than that in rats treated with naked DNA, but by 72 hours there was no significant difference between groups and activity had decreased by at least 85%. Function of the transfected left lung was assessed by measuring arterial PaO(2) levels and was found to be significantly lower at 24 and 72 hours after gene transfer in the PEI/DNA group compared with the naked DNA group. The deterioration in lung function correlated with histological findings. Rats treated with PEI alone and sacrificed after 72 hours showed an impairment in lung function similar to that seen with PEI/DNA treatment. Our studies highlight the importance of assessing the functional capacity of a graft after gene transfer to determine suitability for subsequent transplantation.

Efficiency of non-viral gene delivery systems to rat lungs.

OBJECTIVE: Transient expression of therapeutic genes within lung allografts may modulate the pathological processes following allotransplantation. Whilst efficient gene transfer to lungs has been reported with viral vectors, their usefulness is limited on the grounds of safety. Since non-viral systems overcome many of these safety issues, our studies were designed to evaluate the efficiency of several non-viral gene delivery vectors for in vivo transfer of plasmid DNA to rat lungs via the airways. METHODS: Fischer rats (230--260 g) underwent a thoracotomy, right main bronchus occlusion and instillation of 300 microg naked or complexed DNA (pCIluci, luciferase gene/CMV promoter) to the left lung followed by ventilation for 10 min. Rats were divided into five treatment groups (n=5): (1) Glucose, (2) Naked DNA, (3) Linear polyethylenimine (PEI), (4) Branched PEI, (5) Lipid GL-67/DOPE and (6) DOTAP/cholesterol. Animals were sacrificed 24 h after gene delivery for measurement of reporter gene activity and gas exchange of the left lung. RESULTS: Linear PEI was the most efficient gene delivery vector and was significantly better than DOTAP/cholesterol (P=0.00002) and naked DNA (P=0.004). All gene delivery vectors impaired function of the transfected left lung compared with DNA alone. Of all the gene delivery vectors tested, lipid GL-67/DOPE exerted the least effect on lung function whilst DOTAP/cholesterol mediated the most adverse effect. CONCLUSION: Linear PEI was the most efficient vector for gene delivery to rat lungs in our experimental setting although it mediated a moderate impairment in lung function. Further studies are needed to evaluate whether this effect is transient.

Cationic lipid-mediated transfection of differentiated Caco-2 cells: a filter culture model of gene delivery to a polarized epithelium.

PURPOSE: The use of rapidly dividing in vitro cell culture systems to assess the efficiency of gene delivery is now recognised as a poor indicator of in vivo success. We investigated whether differentiated Caco-2 cell filter-cultures would make a more suitable model for studying gene transfer to an epithelium. METHODS: Caco-2 cells were cultured on semi-permeable membrane filters into differentiated polarised monolayers. Monolayer differentiation was assessed by monitoring the transport of taurocholic acid. Cells at different stages of differentiation were transfected with DNA/DOTAP lipoplexes and later analysed for reporter gene activity. The uptake of radiolabled DNA was also evaluated at various stages of differentiation. RESULTS: Caco-2 cultures developed a resistance to lipoplex-mediated transfection as early as three days, when some cells were still dividing and undifferentiated. As cultures matured, expression of reporter gene progressively decreased partly due to reduced internalisation of DNA. The resistance to transfection could be overcome in part by pre-treatment of monolayers with calcium chelating agents or surfactants. However, transgene expression in treated monolayers was still significantly lower than that in dividing cultures. CONCLUSIONS: Differentiated Caco-2 cells are a more appropriate model for gene-transfer studies to the intestinal epithelium because they demonstrate a resistance to transfection similar to that observed in vivo.

Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids.

DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.