1-2-dioleoyloxy-3-(trimethylammonium)propane and dioctadecylamidoglycylspermine

The aim of this study was to establish a nonviral method for gene delivery to the rat kidney. To this purpose, a panel of reagents was tested, including a monocationic lipid, DOTAP, a polycationic lipid, DOGS (or Transfectam), and three different forms of the cationic polymer polyethylenimine (PEI). A comparison among these compounds was performed in vivo, using luciferase as reporter gene. Complexes containing 10 microg of DNA were injected into the left renal artery of rats and allowed to remain in contact with the kidney for 10 min. Forty-eight hours later, luciferase expression levels in kidney extracts were measured. Kidneys injected with DNA complexed to the branched, 25-kD PEI polymer (PEI 25k) yielded activity levels significantly higher than control, sham-operated kidneys (2.7 x 10(4) vs. 5.2 x 10(3) RLU/kidney, respectively), whereas the other transfecting agents did not yield significant activity over controls. PEI 25k was therefore chosen for further optimization of transfection conditions. Dose-dependent luciferase expression was shown for 10, 50, and 100 microg of PEI-complexed DNA, reaching maximal levels of 2.4 x 10(5) RLU/kidney at 100 microg DNA. The optimal PEI nitrogen/DNA phosphate molar ratio was 10 equivalents. Luciferase activity peaked at 2 days, was still significantly higher than controls at 7 days, and was undetectable at 14 days post-injection. Using beta-galactosidase (beta-Gal) as a reporter, transgene expression was localized almost exclusively in proximal tubular cells.

Topics: Animals; beta-Galactosidase; Drug Carriers; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genes, Reporter; Glycine; Kidney; Kidney Tubules, Proximal; Luciferases; Male; Polyethyleneimine; Quaternary Ammonium Compounds; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Spermine; Tissue Distribution; Transfection; Transgenes

We have examined lipids as transfection agents to introduce recombinant plasmids into primary cultures of rat hippocampal neurons. By modifying the protocol for transfection mediated by the commercial reagent DOTAP, we were able to achieve a transfection efficiency of about 3%. Expression of various transfected gene products was sustained for several weeks in culture, the neurons developed normally and the transfected gene products were targeted to the appropriate subcellular compartment.

Topics: Animals; Cation Exchange Resins; Cells, Cultured; DNA; Drug Carriers; Fatty Acids, Monounsaturated; Fluorescent Antibody Technique; Fluorescent Dyes; Glycine; Hippocampus; Immunoglobulin kappa-Chains; Lipids; Mice; Neurons; Plasmids; Quaternary Ammonium Compounds; Rats; Recombinant Proteins; Spermine; Transfection

We have examined the complement-activating properties of synthetic cationic molecules and their complexes with DNA. Commonly used gene delivery vehicles include complexes of DNA with polylysine of various chain lengths, transferrin-polylysine, a fifth-generation poly(amidoamine) (PAMAM) dendrimer, poly(ethyleneimine), and several cationic lipids (DOTAP, DC-Chol/DOPE, DOGS/DOPE, and DOTMA/DOPE). These agents activate the complement system to varying extents. Strong complement activation is seen with long-chain polylysines, the dendrimer, poly(ethyleneimine), and DOGS (half-maximal at about 3 microM amine content in the assay used). Compared to these compounds, the other cationic lipids (in liposome formulations) are weak activators of the complement system (half-maximal approximately 50-100 microM positive charge in assay). Complement activation by polylysine is strongly dependent on the chain length. Short-chain oligolysines are comparable to cationic lipids in their activation of complement. Incubation of these compounds with DNA to form complexes reduces complement activation in virtually all cases. The degree of complement activation by DNA complexes is strongly dependent on the ratio of polycation and DNA (expressed as the charge ratio) for polylysine, dendrimer, poly(ethyleneimine), and DOGS. To a lesser degree, charge ratio also influences complement activation by monovalent cationic lipid-DNA complexes. For polylysine-DNA complexes, complement activation can be considerably reduced by modifying the surface of preformed DNA complexes with polyethyleneglycol (half-maximal approximately 20 microM amine content). The data suggests that, by appropriate formulation of DNA complexes, complement activation can be minimized or even avoided. These findings should facilitate the search for DNA complex formulations appropriate for reproducible intravenous gene delivery.

Topics: Animals; Cations; Complement Activation; DNA, Recombinant; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genetic Vectors; Glycine; Humans; Injections, Intravenous; Liposomes; Phosphatidylethanolamines; Phospholipids; Polylysine; Quaternary Ammonium Compounds; Sheep; Spermine

Direct in vivo transfection of tumor nodules in situ via liposome-DNA complexes has been employed as a strategy to accomplish antitumor immunization. To circumvent the potential safety hazards associated with systemic localization of delivered DNA, the utility of mRNA transcript-mediated gene delivery was explored. Capped, polyadenylated mRNA transcripts encoding the firefly luciferase and Escherichia coli lacZ reporter genes were derived by in vitro transcription. Transfection of the human breast cancer cell line MDA-MB-435 in vitro was accomplished employing cationic liposome-mRNA complexes. Evaluation of a panel of cationic liposome preparations demonstrated significant differences in the capacity of the various preparations to accomplish mRNA-mediated transfection. Quantitative evaluation of in vitro transfection demonstrated that target cells could be transfected at a high level of efficiency. The mRNA liposome-complexes were evaluated for in vivo transfection of tumor nodules in human xenografts in athymic nude mice. It could be demonstrated the liposome-mRNA complexes were comparable in efficacy to liposome-DNA complexes in accomplishing in situ tumor transfection. Thus, mRNA may be considered as an alternative to plasmid DNA as a gene transfer vector for genetic immunopotentiation applications.

Topics: beta-Galactosidase; Breast Neoplasms; Cation Exchange Resins; Cations; Drug Carriers; Fatty Acids, Monounsaturated; Genes, Reporter; Genetic Therapy; Glycine; Humans; Lipids; Liposomes; Luciferases; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; RNA, Messenger; Safety; Spermine; Transfection; Tumor Cells, Cultured

Topics: Base Sequence; Cations; Cell Line; Cell Membrane; Cell Nucleus; Chromatography, High Pressure Liquid; Cytoplasm; Drug Resistance, Multiple; Fatty Acids, Monounsaturated; Glycine; Humans; Lipids; Molecular Sequence Data; Oligonucleotides, Antisense; Quaternary Ammonium Compounds; Spermine

The power of antisense phosphodiester oligonucleotides (aODN) as regulatory molecules of gene expression is strongly limited by their low cellular uptake and very rapid nuclease-mediated degradation. This study deals with the effect of artificial cationic lipids on ODN cellular uptake and degradation in cell cultures and in human serum. At the ODN levels normally used in antisense-mediated gene regulation experiments, a cationic lipid, DOTAP, enhances the rate of ODN uptake more than 25 fold, but at lower ODN levels the effect of DOTAP is absent. These findings are consistent with a mechanism of ODN internalization by receptor-mediated saturable endocytosis that is bypassed by DOTAP. ODN degradation by nucleases is markedly prevented by DOTAP both in cultured cells and in human serum. Other cationic lipids, namely DOTMA and DOGS, exhibit very similar behaviour. The relatively slight cellular toxicity revealed by cationic lipids contribute to render these molecules very suitable for aODN vehiculation.

Topics: 3T3 Cells; Adenocarcinoma; Animals; Base Sequence; Biological Transport; Cell Line; Colonic Neoplasms; Drug Resistance; Fatty Acids, Monounsaturated; Glycine; Humans; Kinetics; Leukemia; Mice; Molecular Sequence Data; Oligonucleotides, Antisense; Quaternary Ammonium Compounds; Spermine; T-Lymphocytes; Time Factors; Tumor Cells, Cultured