THE ROLE OF ENDOSOME DESTABILIZING ACTIVITY IN THE GENE-TRANSFER PROCESS MEDIATED BY CATIONIC LIPIDS

We used a P-32-labeled pCMV-CAT plasmid DNA to estimate the DNA uptake efficiency and unlabeled pCMV-CAT plasmid DNA to quantify the CAT act ivity after transfection of COS cells using each of the three followin g cationic compounds: [1] vectamidine (3-tetradecylamino-N-tert-butyl- N'-tetradecyl propionamidine, and previously described as diC14-amidin e [1], [2] lipofectin (a 1:1 mixture of N-(1-2,3-dioleyloxypropyl)-N,N ,N-triethylammonium (DOTMA) and dioleylphosphatid-ylethanolamine (DOPE )), and [3] DMRIE-C (a 1:1 mixture of ristyloxy)propyl]-N,N-dimethyl-N -(2-hydroxy-ethyl) ammonium bromide (DMRIE) and cholesterol), Surprisi ngly, a high CAT activity was observed with vectamidine although the D NA uptake efficiency was lower as compared to lipofectin and DMRIE-C, Transmission electron microscopy (TEM) revealed endocytosis as the maj or pathway of DNA-cationic lipid complex entry into COS cells for the three cationic lipids, However, the endosomal membrane in contact with complexes containing vectamidine or DMRIE-C often exhibited a disrupt ed morphology. This disruption of endosomes was much less frequently o bserved with the DNA-lipofectin complexes, This comparison of the thre e compounds demonstrate that efficient transfection mediated by cation ic lipids is not only correlated to their percentage of uptake but als o to their ability to destabilize and escape from endosomes. (C) 1997 Federation of European Biochemical Societies.