Intraperitoneal linear polyethylenimine (L-PEI)-mediated gene delivery to ovarian carcinoma nodes in mice.

Linear polyethylenimine (L-PEI) is an efficient transfection agent for ovarian carcinoma cells in vitro and ex vivo. In the present work, we go a step further and evaluate the efficacy of L-PEI in human ovarian tumor nodes developed in mice. PEI/DNA complexes were administered intraperitoneally instead of intravenously to avoid sequestering of complexes in the lung and liver and to allow transfection of nonvascularized tumor nodes. Plasmid biodistribution was studied by PCR and gene expression was characterized using complementary luciferase and beta-galactosidase assays. Intraperitoneal (i.p.) injection of L-PEI/DNA complexes allowed the straightforward distribution of plasmid in the whole peritoneal cavity. Gene expression occurred in many organs, but tumor nodes appeared as preferential sites for transgene expression. The i.p. delivery route allowed repeated injections and administration of large amounts of DNA (up to 400 mug) without signs of toxicity, even for doses well beyond the intravenous lethal dose. Transgene expression was dose-dependent and transient. However, multiple injections allowed its persistence to increase. These results provide encouraging elements towards the development of PEI-based gene therapy protocols for the treatment of advanced stage ovarian carcinoma.

Polyethylenimine shows properties of interest for cystic fibrosis gene therapy.

Before being considered for a cystic fibrosis (CF) gene therapy trial, any gene delivery agent must be able to show that it produces low levels of toxicity as well as being able to protect the DNA from nuclease degradation. Here we show that complexes of linear polyethylenimine (L-PEI) and DNA can repeatedly be administered to animals (up to 21 consecutive days) without eliciting an immune response against PEI/DNA particles or inducing toxic side effects due to accumulation of PEI in the lungs. However, the host response to the exogenous protein resulted in some decrease of expression. PEI-mediated transfection was unaffected by treatment of the complexes with DNase (frequently used to reduce the viscosity of lung secretions in CF patients). Taken together, these properties make L-PEI a valuable vector for gene therapy of CF.

Gene transfer into intact vertebrate embryos.

Intact chick embryos at 40 h incubation were transfected in vivo with chimeric vectors expressing chloramphenicol acetyl transferase (CAT) under different promoter sequences. The cationic lipid, dioctadecylamidoglycyl spermine (DOGS) used as the transfecting agent had no noticeable toxic effects on embryonic development. CAT activity was monitored 48 h post-transfection on homogenates of embryos dissected free of all annexes. Of the various constructs tested, those containing the AP-1 response element linked to CAT (TRE-tk-CAT) gave high expression and consistent enzyme responses within groups. Co-transfection experiments in which embryos were exposed simultaneously to a CAT vector containing the cAMP response element and to a vector expressing the catalytic subunit of protein kinase A showed that the promoters of the introduced genes can be regulated by their respective transacting factors. This method may therefore represent a general tool for introducing genes into intact vertebrate embryos at precise developmental times.

Lipospermine-based gene transfer into the newborn mouse brain is optimized by a low lipospermine/DNA charge ratio.

Nonviral, plasmid-based gene transfer into somatic tissues offers the prospect of various simple and safe therapeutic possibilities as well as applications in fundamental research. Although cationic lipids display efficient transfection activities in many in vitro systems, only low success rates using these vectors in vivo have been reported. We succeeded in defining conditions providing high levels of in vivo transfection in the brains of newborn mice. Our hypothesis was that conditions favorable for in vitro transfection (highly positively charged particles) were unlikely to be appropriate for in vivo conditions. When using the cationic lipid dioctadecylamido glycylspermine (Transfectam, DOGS) with a cytomegalovirus (CMV)-luciferase reporter gene, the best levels of transfection were obtained when using a low ratio of positive charges (supplied by the DOGS) to negative charges (carried by the DNA). Moreover, addition of the neutral lipid dioleoylphosphatidyl ethanolamine (DOPE) significantly enhanced transfection. Expression of the transgene diminished over time, independently of lipopolysaccharide content of the plasmid preparation used. This suggests that either a mitotic population of cells was preferentially transfected, or that promoter silencing was occurring. Histological examination of the spatial distribution of a beta-galactosidase-expressing transgene showed numerous groups of transfected cells both within the striatal parenchyma and in the paraventricular area. Thus, DNA-lipid complexes bearing overall charges close to neutrality open promising possibilities for modulating gene expression in the developing central nervous system and for therapy in the brain.

A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine.

Nonviral gene transfer into the central nervous system (CNS) offers the prospect of providing safe therapies for neurological disorders and manipulating gene expression for studying neuronal function. However, results reported so far have been disappointing. We show that the cationic polymer polyethylenimine (PEI) provides unprecedentedly high levels of transgene expression in the mature mouse brain. Three different preparations of PEI (25-, 50-, and 800-kD) were compared for their transfection efficiencies in the brains of adult mice. The highest levels of transfection were obtained with the 25-kD polymer. With this preparation, DNA/PEI complexes bearing mean ionic charge ratios closest to neutrality gave the best results. Under such conditions, and using a cytomegalovirus (CMV)-luciferase construction, we obtained up to 0.4 10(6) RLU/microgram DNA (equivalent to 0.4 ng of luciferase), which is close to the values obtained using PEI to transfect neuronal cultures and the more easily transfected newborn mouse brain (10(6) RLU/microgram DNA). Widespread expression (over 6 mm3) of marker (luciferase) or functional genes (bcl2) was obtained in neurons and glia after injection into the cerebral cortex, hippocampus, and hypothalamus. Transgene expression was found more than 3 months post-injection in cortical neurons. No morbidity was observed with any of the preparations used. Thus, PEI, a low-toxicity vector, appears to have potential for fundamental research and genetic therapy of the brain.

In vivo delivery to tumors of DNA complexed with linear polyethylenimine.

Synthetic gene delivery vectors have shown promise in several organs, including brain and lung. Tumor cell targeting, however, is still hindered by their low efficacy. A linear polyethylenimine (L-PEI, Exgen 500) was found to be effective in vivo. Our first attempts to use L-PEI for intratumoral gene delivery were not successful, presumably because of poor diffusion of the complexes within the tumor mass after injection with a syringe. Here we show that L-PEI-mediated transfection can be strongly enhanced when the complexes are delivered slowly into a solid tumor mass, using a micropump. Furthermore, L-PE/DNA complexes actively transfect pseudocystic tumor cells when injected into the cyst cavity. In both cases L-PEI induced a significant and long-lasting (> or =15 days) expression of the reporter gene. Finally, even though systemic delivery of L-PEI/DNA complexes leads to high levels of expression in the lung, this method is not adapted for transfection of subcutaneous tumors implanted in the thigh nor for transfection of lung metastases. Altogether, these results show that L-PEI has promising features for transfection of tumor cells, provided that the mode of delivery is adapted.

Towards synthetic viruses.

Gene delivery is too complex to be performed with a single carrier molecule. Synthetic multicomponent vectors are being designed that mimic key properties of viruses. Some solutions, such as diverting cell-anchoring molecules or the endogenous nuclear import machinery from their normal function, are directly copied from bacteria and viruses. Some other solutions are original ones: monomolecular genome condensation via detergent dimerization or endosome disruption by the proton sponge effect are not exploited by natural cell invaders. All these components, however, still have to be assembled into a unique supramolecular system, an 'artificial virus'.

High-efficiency transfection of primary human keratinocytes with positively charged lipopolyamine:DNA complexes.

The ability to introduce DNA into mammalian cells has provided a powerful means to examine the regulation of gene expression and the function of gene products. However, the most commonly used techniques for DNA transfection are not always suitable for primary cells. Primary human keratinocytes are particularly stringent in their growth requirements and are also very refractory to transfection, rendering transient gene expression studies difficult. We have investigated the ability of several polycationic lipids to promote DNA uptake into human epidermal keratinocytes, as monitored with the bacterial beta-galactosidase reporter gene. We report that the cationic lipopolyamine dipalmitoyl phosphatidylethanolamine spermine as well as another procedure using Polybrene can achieve a 20% to 30% transfection efficiency, superior to any other agent tested on these cells. Gene transfer was accomplished by a 3-h exposure of monolayer cells to DNA complexes formed with either reagent by simple mixing in a serum-free medium, followed by a brief osmotic shock with glycerol. Neither DNA carrier showed any toxicity at the effective concentrations nor interfered with cell attachment, growth or differentiation. The use of a fully biodegradable lipopolyamine as DNA carrier should make it possible to extend this transfection method to gene transfer for in vivo therapeutic applications.

Ovarian carcinoma cells are effectively transfected by polyethylenimine (PEI) derivatives.

As a prerequisite to nonviral gene therapy approaches of ovarian carcinoma, we evaluated the possibility of transfecting established tumor cell lines (SKOV3, IGROV1) as well as primary mesothelial and tumor cells by various polyethylenimine (PEI) derivatives. Several PEI-based vectors were able to effectively transfect these cells, as shown by high luciferase expression levels (10(8) to 10(9) relative light units per milligram of cell protein) that corresponded with 25-50% of green fluorescent protein-positive cells after 24 hours. However, unpredictable differences were observed among the vectors and cell types that a posteriori justified the screening procedure. We also showed that cells that were not transfected after the first experiment remained transfectable in a subsequent transfection experiment to a level similar to that of the initial population. This experiment does not support the emergence of a transfection-resistant cell population and opens the door to multiple therapeutic gene deliveries. Although efficacy and cell targeting still remain to be improved, PEI derivatives appear to be promising molecules for the development of nonviral gene therapy of ovarian carcinoma.

Improvement of nonviral p53 gene transfer in human carcinoma cells using glucosylated polyethylenimine derivatives.

Polyethylenimine (PEI) derivatives are potent polycationic nonviral vectors for gene transfer. The gene transfer efficiency of glucosylated and galactosylated PEI derivatives was optimized using green fluorescent protein gene as reporter gene in FaDu and PANC3 human carcinoma cell lines. Glucosylated or galactosylated PEI derivatives were found to be slightly less cytotoxic than unsubstituted PEI. Gene transfer efficiency was found to be related to DNA/cell number ratio and optimal gene transfer efficiency was achieved at 4 microg DNA/10(5) cells. PEI-DNA complexes were found to enter cells rapidly and were detected into cytoplasmic vesicles 2 hours post-transfection. Green fluorescent protein gene expression was detected 4-6 hours after transfection and reached maximal value 24 hours post-transfection. The results achieved demonstrated that glucosylated PEI yield higher and longer gene transfer efficiency than unsubstituted PEI. Using glucosylated PEI allowed to achieve significant gene transfer in more than 10% of the total cell population for more than 4 days. These data were then applied to p53 gene transfer in PANC3 cells bearing p53 gene deletion and consequently unable to initiate apoptosis. Using glucosylated PEI, p53 gene transfer was successfully achieved with subsequent recovery of p53 mRNA expression and transient P53 protein expression. P53 protein functionality was further demonstrated because transfected cells underwent apoptosis.

Temporal and spatial expression of lipospermine-compacted genes transferred into chick embryos in vivo.

We have optimized a lipospermine-based transfection method for introducing genes into intact vertebrate embryos in vivo. The method employs small amounts of the cationic lipid Transfectam (DOGS), in a concentrated (40 mM) ethanolic solution, to compact and to transfer exogenous genes into chick embryos during the early stages of development (< 36 h of incubation). Plasmid vectors containing the reporter gene luciferase were used to follow the time course of expression. Luciferase activity was detected as early as 12 h post-transfection and was highest at this time. Enzyme activity then decreased over the next two days and was usually undetectable by 72-h post-transfection. To follow the spatial expression of the exogenous genes, a Rous sarcoma virus (RSV)-beta-galactosidase vector was used. When the transfection complex was applied externally around the developing embryo, the main site of expression was the cardiac tissue. Expression could be targeted to the nervous system by micro-injecting the DNA/DOGS (DNA/dioctadecylamidoglycylspermine) complex into the developing brain. The results show that reporter genes can be efficiently expressed in both the developing central nervous system and heart. This raises the possibility that lipospermines can be used to transfer functional genes into embryos during defined periods of development and also to deliver genes in other species and in other in vivo contexts.

Gene transfer optimization with lipospermine-coated DNA.

Designed synthetic DNA carriers represent an attractive alternative to the widely used calcium phosphate gene transfer technique. In this context, we developed a class of nucleic acid binding lipids, the lipopolyamines, which spontaneously condense DNA on a cationic lipid layer. The resulting nucleolipidic particles transfect most animal cells efficiently. However, compaction depends on many experimental factors, some of which have been varied here to give optimal transfection efficiency. When plasmid condensation by the lipospermine is performed in the absence of competing polyions or serum proteins, or when the gene of interest is diluted into carrier DNA, transfection efficiency is increased by 2-3 orders of magnitude. With these improvements, chloramphenicol acetyl transferase activity resulting from transfection of as little as 25 ng could easily be detected by a nonradioactive ELISA test.

Lipospermine-mediated gene transfer technique into murine cultured cortical cells.

In order to transfer exogenous DNA into embryonic cortical cells, we have chosen a transfection technique using a synthetic lipospermine (dipalmitoylphosphatidylethanolamylspermine, DPPES) which complexes DNA molecules and allows their penetration into the intracellular compartment. The procedure was optimized after testing several parameters: DPPES/DNA ratio, incubation time, kinetics of transgene expression, and growth medium. The protocol was achieved by following the expression of the E. coli LacZ reporter gene under the control of the cytomegalovirus promoter. The lipopolyamine-mediated transfection is efficient for terminally differentiated cells, since we routinely obtained transfection efficiencies of 30% for neurons.

A photoaffinity probe for the polyamine site regulating the NMDA receptor.

Polyamines produce on the NMDA-receptor channel activity a regulatory effect subsequent to their binding to specific sites on the receptor-complex. The photoactivatable polyamine derivative L-azidophenylspermine shows properties which suggest its potential as a photoaffinity probe to investigate the nature and topology of these sites. In the dark, its effect on the binding of tritiated N-(1-[thienyl]cyclohexyl)piperidine ([3H]TCP) to synaptosomal plasma membranes is similar to that of diaminodecane. Arcaine antagonizes both the effects of L-azidophenylspermine and diaminodecane on [3H]TCP binding. L-Azidophenylspermine competes in post-synaptic densities with tritiated spermidine for a unique binding site with an EC50 similar to that of spermine. Upon irradiation, L-azidophenyl-spermine incorporates into this material with a high efficiency to a level consistent with both the Bmax for tritiated spermidine and the estimated density of NMDA receptors in this fraction.

DNA condensation by an oxidizable cationic detergent. Interactions with lipid vesicles.

Cationic amphiphile-mediated delivery of plasmid DNA is the non-viral gene transfer method most often used. In the present work, we considered a new cysteine-detergent, ornithinyl-cysteinyl-tetradecylamide (C(14)-CO), able to convert itself, via oxidative dimerization, into a cationic cystine-lipid. By using fluorescence techniques, we first characterized the structure of complexes of plasmid DNA with C(14)-CO molecules either kept as monomers, or oxidized into dimers. Both forms are able to condense DNA, with the formation of hydrophobic micelle-like domains along the DNA chain. Domains with a larger molecular order were obtained with dimeric C(14)-CO/DNA complexes. In a second step, the interactions of these complexes with lipid vesicles considered as membrane models were investigated. In the presence of vesicles, we observed a decondensation of the DNA involved in complexes obtained with C(14)-CO monomers. With anionic vesicles, the DNA is released into the bulk solution, while with neutral vesicles, it remains bound to the vesicles via electrostatic interactions with inserted C(14)-CO molecules. In sharp contrast, the complexes with C(14)-CO dimers are unaffected by the addition of either neutral or anionic vesicles and show no interaction with them. These results may partly explain the low transfection efficiency of these complexes at the +/-charge ratios used in this study.

Recognition of DNA by strand invasion with oligonucleotide-spermine conjugates.

Modified oligonucleotides bearing spermine groups (ODN-sper) with increased binding affinity to DNA have been synthesized. The ability of these ODN-sper to bind within superhelical double-stranded DNA by strand invasion has been studied. The uptake by a supercoiled plasmid was 3 fold higher for the ODN-sper than for the unmodified oligonucleotides.

[Gene transfer with amino lipids and amino polymers]. / Transfert de gènes par l'intermédiaire de lipides et de polymères aminés.

Several non-permanent polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyethylenimines, are efficient transfection agents per se, i.e. without the addition of lysosomotropic bases, or cell targeting or membrane disruption agents. These vectors have been shown to deliver genes as well as oligonucleotides both in vitro and in vivo. Our hypothesis is that their efficiency relies on extensive endosome swelling and rupture that provides an escape mechanism for the polycation/DNA particles.