AN ELECTRON-MICROSCOPY STUDY INTO THE MECHANISM OF GENE-TRANSFER WITHLIPOPOLYAMINES

Cationic amphiphiles have been shown to mediate gene transfer to eukar yotic cells, although the nature and fate of the lipid-DNA complexes i s still a matter of debate. Negative staining transmission electron mi croscopy (TEM) of the complexes in physiological medium, as well as th in-section TEM of transfected cells has been sued to visualize the par ticles and the possible pathways leading to transgene expression. Lipo polyamines form a network of tubular micelles into which plasmid DNA i s intertwined and condensed; the cationic particles contain hundreds o f plasmid molecules and are heterogeneous with respect to size (0.1-0. 5 mu) and shape. Adherent cells (293M, 3T3, MRC5, primary leptomeninge al cells) take them up readily within minutes by spontaneous endocytos is. Among suspension cells, lymphocytes only incidentally show cytopla smic inclusions and monocytes degrade the particles by phagocytosis. T he marked decrease in transfection efficiency generally observed betwe en adherent and nonadherent cells is thus due to reduced cell binding. This suggests that cationic particles bind to membrane components res ponsible for Ca2+-mediated cell anchoring to the extracellular matrix. Cation/anion-mediated endocytosis leads to endosomes that are entirel y filled with the particles. Consequently, two escape mechanisms may o perate; disruption of the lamellar envelope in close contact with tubu lar micelles, and endosome buffering by the lipopolyamine in response to proton entry, leading to osmotic swelling and endosome rupture. Eve n for moderately transfected MRC5 cells, 10(2)-10(3) particles are fou nd either free or in cytoplasmic vacuoles 24 h after transfection, hig hlighting a very inefficient nuclear translocation process. Such high numbers are also the clue to the small concentration window between tr ansfection and cytotoxicity that is often observed with nonviral vecto rs. Nuclear particle inclusions are sometimes seen, yet it is unclear whether plasmid uncoating (before expression) takes place by anion exc hange in the cytoplasm or in the nucleus. The still lower efficiency o f free plasmid translocation to the nucleus suggests an active role fo r the cationic lipid during this step. Although the last stages of the transfection mechanism remain unclear, the present work shows that th e major barrier which hampers in vitro gene delivery with cationic vec tors is nuclear translocation (and cell entry for nonadherent cells), providing precise targets for the design of improved nonviral vectors.