title: Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold.
abstract: 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.
authors.affiliation: Laboratoire de Chimie Génétique associé au CNRS, Université Louis Pasteur, Faculté de Pharmacie, Illkirch, France.
authors.forename: O
authors.initials: O
authors.lastname: Boussif
authors.forename: M A
authors.initials: MA
authors.lastname: Zanta
authors.forename: J P
authors.initials: JP
authors.lastname: Behr
mesh_terms: D000818:Animals; D001769:Blood; D002412:Cations; D002478:Cells, Cultured; D002498:Centrifugation; D004247:DNA; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D008322:Mammals; D051379:Mice; D011094:Polyethyleneimine / Q000494:pharmacology*; D013096:Spermine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014162:Transfection
chemical_list: D002412:Cations; C061134:dioctadecylamidoglycylspermine; D013096:Spermine; D011094:Polyethyleneimine; D004247:DNA; D005998:Glycine