OPTIMIZED GALENICS IMPROVE IN-VITRO GENE-TRANSFER WITH CATIONIC MOLECULES UP TO 1000-FOLD

Reproducible and optimized complex formation between polyanionic DNA a nd a polycationic vector is a key aspect of nonviral gene transfer sys tems. To this end, several factors relevant to in vivo delivery have b een tested repeatedly on several cell types. Gene transfer with a lipo polyamine (transfectam) in the presence of serum was increased over 10 -fold by sequential addition of the lipid to DNA. Paradoxically, high complex concentrations (>200 mu g DNA/ml) led to large enhancements to o, which points to the fact that formation of productive complexes is a slow process. Each parameter more than compensates far the decreased efficiency generally observed with non viral vectors in serum. Transf ectam and PEI (polyethylenimine)-mediated transfection also improved a fter mild centrifugation of the complexes on to the cells. These indiv idual factors were shown to be essentially multiplicative, leading alt ogether to approximately a 1000-fold transfection increase with a luci ferase reporter gene. Finally, 25 cell lines and primary cells (includ ing fibroblasts, hepatocytes and endothelial cells) were successfully transfected over a five orders-of-magnitude efficiency range. From thi s large set of data, a general relation between the overall transfecti on level (as measured by luciferase reporter gene expression) and the fraction of transfected cells (histochemically stained for beta-galact osidase) could be inferred. Finally, transfectam and PEI displayed sim ilar trends over this large range of efficiencies, which reinforces th e hypothesis of a common transfection mechanism where the key endosome -releasing step occurs through a 'proton sponge' effect.