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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.

Oligonucleotides are finding an extremely large number of applications in molecular biology and diagnostics, and might become a very selective class of drugs for the treatment of a vast palette of diseases. Oligonucleotides are polyanions that exert their specific activity following hybridization to a complementary sequence borne by another polyanionic nucleic acid. As drug candidates, they must also be capable of crossing the anionic cell membrane. Simple electrostatic considerations imply that hybridization energy and cell binding could benefit from the addition of cationic groups to the oligonucleotide structure. To reach this goal, many synthetic approaches for introducing ammonium or guanidinium residues into oligonucleotides have been explored: phosphate backbone replacement, ribose or nucleic base modification and end conjugation of a polycation. However, hybridization specificity, nucleic acid-processing enzyme activity and metabolite toxicity concerns all point to the diblock approach, in which the polycation is appended to an otherwise natural oligonucleotide, as the best solution. Unfortunately, stepwise automated synthesis of oligonucleotide–cationic peptide conjugates is not yet routine. Unfortunately, conjugation chemistry between preformed large blocks is not straightforward, especially in water, where “super” zwitterions raise intractable solubility, purification and characterization problems. Moreover, molecular biology and diagnostics applications require fast and straightforward synthesis of any given base sequence linked to any ACHTUNGTRENNUNGorganic cation length. With these considerations as guidelines, we have focused our efforts towards developing a high-yield automated synthesis of oligonucleotide–oligocation conjugates that makes exclusively use of classical phosphoramidite chemistry. In addition to the four A-, T-, Gand C-protected trityl phosphoramidite vials, a linear polyamine N-protected trityl phosphoramidite vial was plugged into the oligonucleotide synthetizer machine to allow online, computer-driven, synthesis (Scheme 1). Decamer oligonucleotides bearing a global charge of up to +9 were synthesized in good yield and characterized by ESMS and PAGE. Duplex melting temperature measurements showed that stability increases up to DTm=45 8C for the largest oligocation moiety. In order to avoid potential toxicity of metabolites, the cationic synthon was based on the largest naturally occurring polyamine, namely spermine. Spermine is present at millimolar concentration in cells, and end-alkylation seems harmless. Amine protection followed by a,w-bishydroxylalkylation led to a diol compatible with oligonucleotide synthesis (Scheme 2). Classical dimethoxytrityl (DMT)/phosphoramidite elongation chemistry was implemented together with base-labile trifluoroacetyl (TFA) protecting groups. The spermine synthon 1 was obtained in eight steps with an overall yield of 12%. A series of decamer oligonucleotides of identical sequences N10= CACCGTAGCG appended with increasing numbers of spermine residues S was synthesized on the micromolar scale on an Expedite 8909 oligonucleotide synthetizer. Individual coupling yields were determined by spectrophotometric determination of the total amount of DMT released during each step. For the nucleoside phosphoramidites, typical coupling yields over 97% were obtained under standard conditions, whereas coupling of 1 had to be optimized to finally reach 90– 96% routinely. The synthesis was stopped with the last DMT group remaining on the oligomer to facilitate separation from truncated sequences. Oligomer cleavage from the solid support and full deprotection were performed under standard aqueous ammonia conditions. Oligomers were purified by reversed-phase HPLC or by using a Poly-Pak II cartridge. DMT was removed by using either standard acetic acid conditions or 2% aqueous TFA to give 80–250 nmol decaoligonucleotide– spermine conjugates N10Sn with up to six spermines [a] B. Pons, Dr. M. Kotera, Dr. G. Zuber, Dr. J.-P. Behr Laboratoire de Chimie G!n!tique associ! au C.N.R.S. Universit! Louis Pasteur de Strasbourg, Facult! de Pharmacie B. P. 24, 67401 Illkirch (France) Fax: (+33)390-244-306 E-mail : kotera@bioorga.u-strasbg.fr behr@bioorga.u-strasbg.fr Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Scheme 1. Synthesis of oligonucleotide–oligospermine conjugates NmSn.

Human immunodeficiency virus (HIV) vaccines have the potential to improve antiretroviral drug treatment by inducing cytotoxic killing of HIV-infected cells. Prophylactic vaccines utilize new antigens to initiate immunity; however, in HIV-infected individuals the load of viral antigen is not the limiting factor for the restoration of immune responses. Here we describe a novel immunization strategy with DermaVir that improves viral antigen presentation using dendritic cells (DC). DermaVir contains a distinctive plasmid DNA expressing all HIV proteins except integrase to induce immune responses with broad specificity. The DNA is formulated to a mannosilated particle to target antigen-presenting cells and to protect the DNA from intracellular degradation. After topical application, DermaVir-transduced cells migrate from the skin to the draining lymph node and interdigitate as DermaVir-expressing, antigen-presenting DC. We compared the immunogenicity of topical and ex vivo DC-based DermaVir vaccinations in naïve rhesus macaques. Both vaccinations induced simian immunodeficiency virus-specific CD4 helper and CD8 memory T cells detected by an in vivo skin test and an in vitro intracellular cytokine-based assay. Topical DermaVir vaccination represents an improvement upon existing ex vivo DC-based immunization technologies and may provide a new therapeutic option for HIV-infected patients.

Polyethylenimine (PEI) has been described as one of the most efficient cationic polymers for in vitro gene delivery. Systemic delivery of PEI/DNA polyplexes leads to a lung-expression tropism. Selective in vivo gene transfer would require targeting and stealth particles. Here, we describe two strategies for chemically coupling polyethylene glycol (PEG) to PEI, to form protected ligand-bearing particles. Pre-grafted PEG-PEI polymers lost their DNA condensing property, hence their poor performances. Coupling PEG to pre-formed PEI/DNA particles led to the expected physical properties. However, low transfection efficacies raised the question of the fate of excess free polymer in solution. We have developed a straightforward a purification assay, which uses centrifugation-based ultrafiltration. Crude polyplexes were purified, with up to 60% of the initial PEI dose being removed. The resulting purified and unshielded PEI/DNA polyplexes are more efficient for transfection and less toxic to cells in culture than the crude ones. Moreover, the in vivo toxicity of the polyplexes was greatly reduced, without affecting their efficacy.

Background The major advantage in choosing non-viral vectors such as cationic polymers for in vitro and in vivo transfection is their higher biosafety than viral ones. Among the cationic polymers, polyethylenimines (PEIs) are promising molecules for gene delivery to a variety of cells. Efficient transfection of primary endothelial cells using PEIs could be regarded as an interesting strategy of treatment in some ischemic cardiovascular diseases. Methods Efficacies of a 22-kDa linear PEI (L-PEI) and its glucose-grafted derivative (L-PEI-Glc4) were compared for gene transfer into human umbilical vein endothelial cells (HUVEC) using the reporter gene luciferase. Cells were incubated for 2, 4 and 24 h with PEI/DNA complexes made in 150 mM sodium chloride (NaCl) or in 5% glucose solution. Luciferase activity was measured 24 h after the onset of transfection. The effects of low (2%) and high (30%) concentrations of serum on transfection efficacy were assessed as well. We then studied the intracellular fate of the PEI/DNA complexes labelled with the DNA intercalator YOYO-1 using flow cytometry analysis (FACS) and confocal microscopy. Results PEI/DNA complexes formed in NaCl led to a higher transfection efficacy than those made in glucose. The optimal formulation, depending on the incubation time and the presence of serum in the medium, was obtained using DNA complexed to L-PEI-Glc4 and incubated for 4 h with the cells. This condition led to 50% fluorescent cells after GFP transfection. A high serum concentration diminished the L-PEI associated toxicity but decreased L-PEI-Glc4 transfection efficiency. FACS analysis using both vectors showed that almost 90% of the cells had internalized the DNA complexes. Confocal microscopic observations showed a fast attachment of the complexes to the cell surface followed by inclusion into vesicles that migrated to the perinuclear region. Conclusions In this work, we defined the optimal conditions for gene delivery in HUVEC. These conditions were obtained when using derivatives L-PEI and L-PEI-Glc4 complexed with DNA in 150 mM NaCl and added to cells for 2 and 4 h, respectively. Cellular trafficking of the complexes suggested that cell entry was not a limiting factor for gene delivery using PEI. This study underlined the interest in PEIs as efficient vectors for gene transfer into human endothelial cells. Copyright © 2004 John Wiley & Sons, Ltd.

Nucleic acids delivery vectors have shown promising therapeutic potential in model systems. However, comparable clinical success is delayed essentially because of their poor biodistribution and of their ineffective intracellular trafficking. The size of condensed DNA particles is a key determinant for in vivo diffusion, as well as for gene delivery to the cell nucleus. Towards this goal, we have developed cationic thiol-detergents that individually compact plasmid DNA molecules into anionic particles. These particles are then 'stabilized' by air-induced dimerization of the detergent into a disulfide lipid on the template DNA. The particles all measure approximately 30 nm, which corresponds to the volume of a single molecule of plasmid DNA. The gel electrophoretic mobility of the anionic particles was found to be higher than that of the plasmid DNA itself. Similarly, particles formed with a 31-mer oligonucleotide measured 19 nm. Improved in vivo diffusion, as well as improved intracellular trafficking may be inferred from the faster migration of the complexes. Moreover, the size of the particles remains compatible with nuclear pore crossing. Finally, in an attempt to improve the biodistribution of these particles, we have coated the monomolecular particles with a poly(ethylene glycol) corona.

International audience; 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.

Conventional synthesis of heterobifunctional poly(ethylene glycol) derivatives, especially of medium size, is a rather tedious task. A straightforward solid-phase methodology has been developed that is illustrated here by the synthesis of alpha-pyridyldithio-omega-hydroxy-poly(ethylene glycol)600. This derivative was prepared from resin-bound PEG600 with a global yield of 65% for 6 individual steps, i.e., with an average yield of 93%/step. Intermediate purification steps simply consisted of resin washing. Progress of each reaction toward completion could conveniently be monitored by 13C NMR of the resin-bound PEG derivatives. This example highlights both the versatility and efficiency of combining polymer-supported synthesis with direct 13C-NMR characterization of the intermediate compounds.

International audience

International audience; The β-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.

International audience; 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.

Synthetic gene transfer vectors could be an attractive alternative to biological vehicles for gene therapy. In an effort to improve the previously developed lipopolyamine-mediated transfection technique, various amphiphilic DNA-binding molecules have been synthesized. Besides Transfectam, several lipospermines display very high gene delivery levels. The structure-activity relationship obtained points to the central role played by the polyamine headgroup in condensing the plasmid and binding it to the cell surface, provided the hydrophobic moiety is capable to generate nonmicellar mesomorphic structures. It also highlights other favorable (albeit more speculative) properties shared by protonable lipospermines as compared to quaternary ammonium-bearing lipids, such as their ability to act as a buffer and their strong affinity for chromatin. The former property may prevent the pH decrease along the degradative lysosomial pathway. The ability to bind to chromatin even in the presence of endogeneous polyamines should have two consequences: a nuclear tropism of the transfecting particles and plasmid uncoating in the nucleus by competitive dilution of the lipopolyamine into an ocean of DNA.