Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold

Reproducible and optimized complex formation between polyanionic DNA and a polycationic vector is a key aspect of nonviral gene transfer systems. To this end, several factors relevant to in vivo delivery have been tested repeatedly on several cell types. Gene transfer with a lipopolyamine (transfectam) in the presence of serum was increased over 10-fold by sequential addition of the lipid to DNA. Paradoxically, high complex concentrations (> 200 micrograms DNA/ml) led to large enhancements too, which points to the fact that formation of productive complexes is a slow process. Each parameter, more than compensates for the decreased efficiency generally observed with nonviral vectors in serum. Transfectam and PEI (polyethylenimine)-mediated transfection also improved after mild centrifugation of the complexes on to the cells. These individual factors were shown to be essentially multiplicative, leading altogether to approximately a 1000-fold transfection increase with a luciferase reporter gene. Finally, 25 cell lines and primary cells (including fibroblasts, hepatocytes and endothelial cells) were successfully transfected over a five orders-of-magnitude efficiency range, From this large set of data, a general relation between the overall transfection level (as measured by luciferase reporter gene expression) and the fraction of transfected cells (histochemically stained for beta-galactosidase) could be inferred. Finally, transfectam and PEI displayed similar trends over this large range of efficiencies, which reinforces the hypothesis of a common transfection mechanism where the key endosome-releasing stop occurs through a "proton sponge' effect.     

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.     

Efficiency of cationic lipid-mediated transfection of polarized and differentiated airway epithelial cells in vitro and in vivo

Systematic analysis of a large number of different cationic lipids has led to the identification of novel structures (GL-67) and formulations of cationic lipid:plasmid DNA (pDNA) complexes that facilitate high levels of gene expression in lungs of mice. However, despite significant improvement in gene transfer activity, we show here that the efficiency of GL-67-mediated gene transduction of intact airway epithelia is still relatively low. Administration of GL-67:pCF1-CFTR (encoding the cystic fibrosis transmembrane conductance regulator) complexes into the nasal epithelium of cystic fibrosis (CF) transgenic mice resulted only in marginal correction of the ion transport defects. Measurements of nasal potential differences (PD) showed no correction of the sodium (Na+) transport defect, and only partial restitution of the chloride (Cl-) transport defect was achieved in a small proportion of the animals after perfusion of the nasal epithelium with the complexes. Furthermore, in contrast to results obtained following instillation of GL-67:pDNA complexes into the lungs of mice, perfusion of GL-67:pDNA into the nasal epithelium resulted only in a moderate enhancement of gene transduction activity relative to that attained with naked pDNA alone. To determine the basis for this low efficiency of transfection, a series of studies was conducted to identify some of the barriers governing cationic lipid-mediated gene transfer to the airway epithelium. We show here that the transfection activity of GL-67 was affected by the polarization, differentiation, and proliferative state of the cells. Diminished transfection activity was observed with nonmitotic, highly polarized and differentiated airway epithelial cells. This observed reduction in gene expression with nonmitotic cells was determined to be due in part to inefficient nuclear translocation of the pDNA from the cytoplasm. Together these data indicate that much improvement in the ability of cationic lipids to transfect polarized and differentiated airway epithelial cells is a necessary prerequisite for effective cationic lipid-mediated gene therapy of airway diseases such as CF.     

2-(Dimethylamino)ethyl methacrylate based (co)polymers as gene transfer agents

Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) is a water-soluble cationic polymer, which is able to bind to DNA by electrostatic interactions. At a polymer/plasmid ratio above 2 (w/w) positively charged complexes were formed with a size around 0.2 microm. The transfection efficiency of polymer/plasmid complexes was evaluated in cell culture (COS-7 and OVCAR-3 cells) using a pCMV-lacZ plasmid, encoding for beta-galactosidase, as a reporter gene. The optimal transfection efficiency was found at a PDMAEMA/plasmid ratio of 3-5 (w/w). Under these conditions 3-6% of the cells were actually transfected. Like other cationic polymers, PDMAEMA is slightly cytotoxic. This activity was partially masked by complexing the polymer with DNA. A pronounced effect of the molecular weight of the polymer on the transfection efficiency was observed. An increasing molecular weight resulted in an increasing number of transfected cells. Dynamic light scattering experiments showed that high molecular weight polymers (Mw>300 kDa) were able to condense DNA effectively (particle size 0.15-0.20 microm). In contrast, when plasmid was incubated with low molecular weight PDMAEMA, large complexes were formed (size 0.5-1.0 microm). Copolymers of DMAEMA with methyl methacrylate (MMA), ethoxytriethylene glycol methacrylate (triEGMA) or N-vinyl-pyrrolidone (NVP) also acted as transfection agents. A copolymer with 20 mol % of MMA showed a reduced transfection efficiency and a substantial increased cytotoxicity compared with a homopolymer of the same molecular weight. A copolymer with triEGMA (48 mol %) showed both a reduced transfection efficiency and a reduced cytotoxicity, whereas a copolymer with NVP (54 mol %) showed an increased transfection efficiency and a decreased cytotoxicity as compared to a DMAEMA homopolymer.     

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Efficient transfection conditions for a number of human, rat and rabbit primary cells and established lines of vascular origin have been determined using a complex of a commercially available cationic lipid transfection agent (Tfx-50) and luciferase reporter plasmid constructs. The optimised conditions have also been successfully applied to rabbit carotid arteries in vivo and a series of human arteries in vitro. The most critical factors influencing the efficiency of gene transfection with this protocol are: DNA concentration; ratio of lipid reagent to DNA; transfection time and the presence or absence of serum. Immunohistochemical analysis shows that a high percentage of cells (approximately 30-80% dependent on lineage) were transfected under optimal conditions with minimal toxicity effects. Similar analyses performed on undamaged rabbit carotid vessels transfected in vivo and human arteries transfected in vitro show high-efficiency transfer and strong expression of the luciferase vector as demonstrated by reporter gene expression. The optimisation of gene transfer into vascular cells with this cationic lipid complex will be valuable for molecular studies of genes implicated in cardiovascular diseases and as a possible method of gene delivery with therapeutic intent.     

Biodistribution and gene expression of lipid/plasmid complexes after systemic administration

The objectives of this study were to investigate the influence of physicochemical properties of lipid/plasmid complexes on in vivo gene transfer and biodistribution characteristics. Formulations based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and novel biodegradable cationic lipids, such as ethyl dioleoyl phosphatidylcholine (EDOPC), ethyl palmitoyl myristyl phosphatidylcholine (EPMPC), myristyl myristoyl carnitine ester (MMCE), and oleyl oleoyl L-carnitine ester (DOLCE), were assessed for gene expression after tail vein injection of lipid/plasmid complexes in mice. Gene expression was influenced by cationic lipid structure, cationic lipid-to-colipid molar ratios, plasmid-to-lipid charge ratios, and precondensation liposome size. Detectable levels of human growth hormone (hGH) in serum, human factor IX (hFIX) in plasma, and chloramphenicol acetyltransferase (CAT) in the lung and liver were observed with positively charged lipid/plasmid complexes prepared from 400-nm extruded liposomes with a cationic lipid-to-colipid ratio of 4:1 (mol/mol). Intravenous administration of lipid/CAT plasmid complexes resulted in distribution of plasmid DNA mainly to the lung at 15 min after injection. Plasmid DNA accumulation in the liver increased with time up to 24 hr postinjection. There was a 10-fold decrease in the amount of plasmid DNA in the lung at 15 min after injection, when the lipid/plasmid complex charge ratio was decreased from 3:1 to 0.5:1 (+/-). Bright fluorescent aggregates were evident in in vivo-transfected lung with the positively charged pCMV-CAT/DOLCE:dioleyl phosphatidylethanolamine (DOPE) (1:1, mol/mol) complexes, while more discrete punctate fluorescence was observed with a 4:1 molar ratio of cationic lipid:colipid formulations. Preinjection of polyanions such as plasmid, dextran sulfate, polycytidic acid, and polyinosinic acid decreased hGH expression, whereas the preinjection of both positively charged and neutral liposomes had no effect on hGH serum levels. Of the cationic lipids tested, DOLCE was found to be the most effective potentially biodegradable cationic lipid. A correlation between gene expression and cationic lipid:colipid ratios and lipid-to-plasmid charge ratio was also observed for DOTMA- and DOLCE-based formulations.     

Plasmid DNA is protected against ultrasonic cavitation-induced damage when complexed to cationic liposomes

Cationic liposomes bound to plasmid DNA are currently used for in vitro and in vivo gene therapy applications, but such complexes readily form large, heterogeneous aggregates that are not appropriate for pharmaceutical development. More importantly, size heterogeneity makes studies focused on optimizing gene transfer to cells difficult to conduct or understand. For this reason we have evaluated the effect of microprobe sonication on these complexes in an effort to achieve process-controlled size homogeneity. Complexes were prepared using a 7.2 kb reporter plasmid and the following liposomal lipid combinations: DDAB/DOPE (50:50 mol %), DDAB/DOPE/PEG-PE (50:45:5 mol %), DDAB/EPC (50:50 mol %), DDAB/EPC/PEG-PE (50:45:5, 50:40:10, 50:35:15 mol %), DODAC/DOPE (50:50 mol %), and DODAC/EPC (50:50 mol %) (DDAB, dimethyldioctadecylammonium bromide; DOPE, dioleoylphosphatidylethanolamine; PEG-PE, monomethoxypolyethylene glycol2000 succinate- distearoylphosphatidylethanolamine; EPC, egg phosphatidylcholine; DODAC, dioleoyldimethylammonium chloride). The influence of complex composition and lipid:DNA ratio was evaluated. Particle size was determined before and after complexation and again after sonication using the quasi-elastic light scattering technique. DNA integrity was assessed via agarose gel electrophoresis. Finally, gene transfection was evaluated using CHO cells that were transfected in vitro with sonicated and unsonicated complexes. It is established in this study that size reduction can occur, but this is dependent on cationic and neutral lipid composition and, in some cases, lipid:DNA ratio. Surprisingly, the process of sonication leaves a significant percentage of the plasmid DNA intact and capable of in vitro transfection. This study shows that plasmid DNA can be protected from damage due to sonication by liposome complex formation. This may indicate that more common pharmaceutical methods for size reduction which subject particles to mechanical stress may be applicable in preparation of liposome/DNA formulations for in vivo application.     

Gene delivery by negatively charged ternary complexes of DNA, cationic liposomes and transferrin or fusigenic peptides

Potential problems with the use of viral vectors for gene therapy necessitate the development of efficient nonviral vectors. The association of transferrin, or the pH-sensitive peptide GALA, with cationic liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane and its equimolar mixture with dioleoylphosphatidylethanolamine, under conditions where the liposome/DNA complex is negatively charged, drastically increased luciferase expression from pCMVluc. The percentage of cells transfected, measured by beta-galactosidase expression, was also increased by about 10-fold. The zeta potential of the ternary complexes was lower than that of the liposome/DNA complexes. Transfection activity of positively charged complexes was also enhanced by association with transferrin, GALA or the influenza hemagglutinin N terminal peptide HA-2, but to a smaller extent compared with the negatively charged complexes. The enhancement of gene delivery by transferrin or GALA was not affected significantly by the presence of serum and did not cause significant cytotoxicity. Our results indicate that negatively charged ternary complexes of cationic liposomes, DNA and transferrin, or fusigenic peptides, can facilitate efficient transfection of cultured cells, and that they may alleviate the drawbacks of the use of highly positively charged complexes for gene delivery in vivo.     

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We have investigated the morphology and transfection activity of cationic liposome-DNA complexes (CLDC) under conditions relevant to both in vivo and in vitro studies. Moreover we have attempted to establish structure-function relationships relevant for high transfection activities under both conditions. CLDC were composed of dimethyldioctadecylammonium bromide with either 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol (Chol) interacting either with pre-condensed DNA or with uncondensed plasmid DNA. Furthermore for steric stabilization 1% poly(ethylene glycol)-phospholipid conjugate was added to CLDC containing Chol and plasmid DNA. The in vivo studies were carried out in mice following i.v. injection, and the in vitro studies were performed on SK-BR-3 human breast cancer cells in the presence of media with serum. The morphology of the CLDC, monitored by freeze-fracture electron microscopy, was investigated after mixing with mouse serum or the medium where the cells were kept. The substitution of DOPE with Chol, and the addition of N-[omega-methoxypoly(oxyethylene)-alpha-oxycarbonyl-DSPE+ ++ are producing CLDC which are stabilized with respect to time and serum, and are relatively small (100-300 nm). These stabilized complexes show high expression of a marker gene in mouse lungs reaching expression values up to 10 ng luciferase per mg tissue protein, but relatively low expression in SK-BR-3 cells in vitro. Additionally, some of the complexes containing pre-condensed DNA look like 'map-pin' structures showing heads of the size of liposomes and short, stiff and tapering tails. The in vivo transfection activity of these preparations is highest. Similar complexes containing DOPE rather than Chol as helper lipid precipitate in the presence of serum and especially of cell medium and convert into hexagonal lipid (HII) phase. Such complexes, despite their high transfection activity in vitro, show very little transfection activity in vivo. These comparisons may help us to understand the fundamental difference between in vitro and in vivo activity of CLDC: high in vitro transfection activity seems to be associated with hexagonal lipid precipitates whereas high in vivo activity seems to be related with small, stabilized complexes, which in our case also exhibit some protrusions (map-pin structures).     

Cationic lipid-mediated transfer of the hIL-10 gene prolongs survival of allogeneic hepatocytes in Nagase analbuminemic rats

Gene transfer techniques can be used as a drug delivery system to achieve local immunosuppression. We performed a series of experiments to identify the cationic lipid that most efficiently transfects isolated, cultured, rat hepatocytes; to optimize conditions for efficient transfection; to determine the duration of gene expression in vitro; and finally, to determine the survival of allogeneic hepatocytes transplanted into Nagase rats. Our results suggest that DOTAP is the best cationic lipid for transfection of cultured rat hepatocytes. In addition, the following conditions appear to optimize transfection efficiency: a DNA:DOTAP ratio of 1:6; a 24 exposure time of the hepatocytes to the DNA-DOTAP complex; a DNA dose of 4 microg/35 mm culture plate seeded with 2.5x10(5) rat hepatocytes. When transfected as described above, cultured hepatocytes expressed the hIL-10 gene for approximately 14 days. Accordingly, Nagase rats transplanted with 4x10(7) DOTAP-hIL-10 transfected, allogeneic hepatocytes had an abrupt rise in serum albumin levels that peaked within 7 days of the transplant, decreased abruptly after 15 days, and approached baseline by day 40. In contrast, control animals had a smaller albumin peak that returned to baseline within 10 days (P<0.01). In all animals, serum hIL-10 levels were undetectable when tested. We conclude that DOTAP is the best cationic lipid for transfection of cultured rat hepatocytes. Furthermore, hIL-10 transfected hepatocytes have a prolonged survival in an allogeneic host which is probably limited by loss of gene expression. Further studies using other vectors capable of prolonged gene expression will help determine if indefinite hIL-10 gene expression leads to indefinite graft survival.     

Surfactant inhibits cationic liposome-mediated gene transfer

We have demonstrated that tracheal insufflation of recombinant plasmid DNA results in transfection of rat lungs to the same extent as insufflation of plasmid-cationic liposome complex. To understand this observation better, we investigated the in vitro gene transfer of plasmid DNA in the presence and absence of cationic liposome and the effect of surfactant on gene transfer. The chloramphenicol acetyltransferase (CAT) expression plasmids pBL-CAT and pSV-CAT were studied in three cell types: rat fetal lung fibroblast (RFL-6), calf pulmonary artery endothelial cell (CPAE), and rat type II alveolar epithelial cell (type II AE). Three cationic liposomes were tested: DDAB (dimethyl-dioctadecyl ammonium bromide)-liposome, DOTAP (dioleoyltrimethyl ammonium propane)-liposome, and lipofectin. The results revealed that (i) plasmid DNA alone caused a dose-dependent, low-level transfection, most efficiently in RFL-6 followed by CPAE and type II AE, (ii) DDAB-liposome markedly enhanced gene transfer, most efficiently in RFL-6 followed by CPAE and type II AE, (iii) Survanta, a naturally derived surfactant preparation, and Exosurf, a synthetic surfactant, while having no effect on in vitro gene transfer by plasmid DNA alone, markedly inhibited cationic liposome-mediated gene transfer, (iv) dipalmitoyl phosphatidylcholine was responsible for the inhibitory effect of Exosurf, and (v) inhibition of cationic liposome-mediated gene transfer by Exosurf was not due to inhibition of plasmid DNA-cationic liposome complex uptake or interference with the promoter and enhancer. The observed inhibition of cationic liposome-mediated gene transfer by surfactant may in part explain our previous observation that tracheal insufflation of plasmid DNA and plasmid-cationic liposome complex results in equal lung gene transfer.     

Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability

PURPOSE: Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery. With the aim of developing this system, various biophysical characteristics of chitosan-condensed DNA complexes were measured, and transfections were performed. METHODS: Transmission electronic microscopy (TEM) visualizations, sedimentation experiments, dynamic light scattering (DLS), and zeta potential measurements were realized. Transfections were made by using the luciferase reporter gene. RESULTS: In defined conditions, plasmid DNA formulated with chitosan produced homogenous populations of complexes which were stable and had a diameter of approximately 50-100 nm. Discrete particles of nicely condensed DNA had a donut, rod, or even pretzel shape. Chitosan/DNA complexes efficiently transfected HeLa cells, independently of the presence of 10% serum, and did not require an added endosomolytic agent. In addition, gene expression gradually increased over time. from 24 to 96 hours, whereas in the same conditions the efficacy of polyethylenimine-mediated transfection dropped by two orders of magnitude. At 96 hours, chitosan was found to be 10 times more efficient than PEI. However, chitosan-mediated transfection depended on the cell type. This dependency is discussed here. CONCLUSIONS: Chitosan presents some characteristics favorable for gene delivery, such as the ability to condense DNA and form small discrete particles in defined conditions.     

Monomolecular collapse of plasmid DNA into stable virus-like particles

Cationic lipids are being widely used for cell transfection in vitro. The lipid/DNA complexes, however, tend to aggregate into large and polydisperse particle mixtures; this hampers their use in vivo. Cationic detergents, on the contrary, do not mediate cell transfection per se, yet are capable of condensing individual DNA molecules into discrete entities. We have taken (only) the interesting features of both types of amphiphiles for the two-step formation of stable core particles reminiscent of viruses. Individual anionic plasmid molecules were cooperatively collapsed with a carefully tailored cationic cysteine-based detergent. The resulting 23-nm particles were then simply "frozen" by spontaneous aerobic dimerization of the cysteine-detergent into a cystine-lipid on the template DNA. The population of spherical particles is monodisperse and stable over days, in physiological conditions. Together with a negative surface potential, these properties should ensure good tissue dissemination and escape from the blood stream after i.v. injection.     

Effects of membrane-active agents in gene delivery

A large variety of membrane-modifying agents have been used for the enhancement of DNA(lipo)polycation complex based gene transfer. The magnitude of improvement depends on the nature of the membrane-modifying agent and the (poly)cationic carrier. Within the lipid-free polymer-based systems (polyfection), ligand-polylysine mediated gene transfer can be improved up to more than 1000-fold by pH-specific endosomolytic peptides, glycerol, bacterial proteins or adenovirus particles. Ligand-polyethylenimine or dendrimer-based systems with per se higher efficiency are only slightly (about ten-fold) enhanced by endosomolytic agents. Membrane-active agents show only minor effects when applied to cationic lipid-based gene transfer (lipofection) with DNA complexes formed under optimized conditions using an three- to four-fold excess of positive charges. Less positively charged lipofection complexes can be strongly improved by the addition of membrane-active peptides.     

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Nebulisation is currently the most acceptable and practical delivery system for repeated applications of gene therapy to the lower airways of cystic fibrosis (CF) patients. We have assessed whether this route of administration offers other benefits with regard to respiratory gene transfer. A standard jet nebuliser (Acorn System 22, Medicaid) was used to transfer the reporter gene beta-galactosidase complexed with the cationic liposome DC-Chol/DOPE to three epithelial cell lines in vitro, two non-CF and one CF, using a novel collection system. In all three cell lines, nebulisation resulted in significantly (P < 0.05) improved transfection efficiency compared with instillation. At a constant DNA: liposome ratio of 1:5 (wt:wt), transfection efficiency was inversely related to increasing concentrations of DNA-liposomes before nebulisation. This effect was not related to the amount of DNA delivered and measurements of both zeta potential and mean aerodynamic particle size before and after nebulisation did not show concentration-related differences. The increased transfection efficiency did not relate either to the physical consequences of the nebulisation processes nor the effects of nebulisation on the complexes before instillation. Significantly increased transfection efficiency was seen following nebulisation with 95% O2/5% CO2 in comparison with 21% O2/78% N2 (air); this did not relate to changes in either the pH or temperature of the solution bathing the cells. The data confirm that nebulisation is appropriate for gene delivery to the lower airways in clinical practice and points to factors that may optimise gene transfer efficiency.     

Direct gene transfer to the respiratory tract of mice with pure plasmid and lipid-formulated DNA

Direct gene transfer into the respiratory system could be carried out for either therapeutic or immunization purposes. Here we demonstrate that cells in the lung can take up and express plasmid DNA encoding a luciferase reporter gene whether it is administered in naked form or formulated with cationic liposomes. Depending on the lipid used, the transfection efficiency with liposome-formulated DNA may be higher, the same as, or less than that with pure plasmid DNA. Tetramethyltetraalkylspermine analogs with alkyl groups of 16 or 18 carbons and DMRIE/cholesterol formulations proved particularly effective. Similar results for reporter gene expression in the lung were obtained whether the DNA (naked or lipid formulated) was administered by indirect, noninvasive intranasal delivery (inhaled or instilled) or by invasive, direct intratracheal delivery (injected or via a cannula). Reporter gene expression peaks around 4 days, then falls off dramatically by 9 days. The dose-response is linear, at least up to 100 microg plasmid DNA, suggesting better transfection efficiencies might be realized if there was not a volume limitation. For a given dose of DNA, the best results are obtained when the DNA is mixed with the minimum amount of lipid that can complex it completely. These results are discussed in the context of direct gene transfer for either gene therapy or delivery of a mucosal DNA vaccine.     

Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells

The beta-galactosidase reporter gene, either free or complexed with various cationic vectors, was microinjected into mammalian cells. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus. Polyethylenimine and to a lesser extent polylysine, but not cationic lipids, enhance transgene expression when complexes are injected into the cytoplasm. This latter effect was independent of the polymer vector/cDNA ionic charge ratio, suggesting that nucleic acid compaction rather than surface charge was critical for efficient nuclear trafficking. Cell division was not required for nuclear entry. Finally, comparative transfection and microinjection experiments with various cell lines confirm that barriers to gene transfer vary with cell type. We conclude that polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and that transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.     

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The factors controlling the transfection efficiency of cationic lipid carrier systems following intravenous administration are poorly understood. Using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) combined with Tween 80 as a carrier system and cDNA of luciferase or beta-galactosidase gene as a reporter, we investigated the importance of DOTMA to DNA ratio and the ratio of DOTMA to Tween 80 in the lipid formulation in determining the site and level of transgene expression following intravenous administration. The data show that all of the internal organs, including lung, liver, spleen, heart and kidneys, expressed the transgene upon systemic administration into animals with 25 micrograms of plasmid DNA when complexed with DOTMA-Tween 80 lipid formulation. The transfection efficiency was dependent on both DOTMA to DNA, and DOTMA to Tween 80 ratios. Among the organs examined, the lung appeared to be more transfectable than other organs. A better transfection activity was obtained with higher DOTMA to DNA and DOTMA to Tween 80 ratios. Time-response curve shows that gene expression was transient with a maximal level between 10 and 24 h after injection. Results from tissue distribution studies with 125I-labeled plasmid DNA and Southern analysis suggest that the transient expression is the result of the loss of transgene from the transfected cells. These results suggest that cationic lipid-based delivery systems can be efficient for gene delivery if the composition of the DNA-lipid complexes is properly controlled.     

Inhibitor and temperature effect on catalase in the liver of adult diploid and haploid Rana rugosa

Cellular uptake and gene expression of plasmid DNA and its cationic liposome complexes were studied using primary cultures of bovine brain microvessel endothelial cells (BMEC) developed as an in vitro model of the blood-brain barrier. An avid association of naked plasmid DNA with the BMEC monolayer was observed at 37 degreesC, which is comparable to that of the DNA/liposome complex. The cellular association significantly decreased at low temperature (4 degreesC). The binding at 4 degreesC was saturable and significantly inhibited by polyanions involving polyinosinic acid and dextran sulfate, typical ligands for the macrophage scavenger receptors, but not by polycytidylic acid or in the presence of EDTA. Unexpectedly, a significant gene expression in the BMEC was obtained by transfection with naked plasmid DNA although the expression level was lower than that obtained by plasmid DNA/cationic liposome complex. Taken together, cultured capillary endothelial cells derived from the brain are able to take up naked plasmid DNA via a scavenger receptor like-mediated mechanism for polyanions and gene expression in the cells takes place.     

Stability of peptide-condensed plasmid DNA formulations

Low molecular weight homogeneous peptides were used to form peptide/DNA condensates. A peptide possessing 18 lysines was found to protect plasmid DNA from serum endonuclease and sonicative-induced degradation whereas a shorter peptide possessing 8 lysines dissociated in 0.1 M sodium chloride and failed to protect DNA from enzymatic degradation. Peptide-condensed DNA showed no change in the ratio of supercoiled to circular DNA following 100 W sonication for up to 60 s and was able to transfect HepG2 cells with equivalent efficiency as untreated condensed plasmid DNA. Alternatively, uncondensed plasmid DNA was rapidly fragmented by sonication and serum endonucleases and resulted in negligible gene expression following condensation with peptide. Cationic lipid/DNA complexes were only partially effective at stabilizing DNA in serum compared to the complete stabilization afforded by peptide/DNA condensation. These results indicate that the stabilization afforded by condensation with a peptide protects DNA during formulation and preserves its structure in serum. These functions are important to achieve optimal gene expression from a nonviral gene delivery system.