INTRACELLULAR-DISTRIBUTION AND MECHANISM OF DELIVERY OF OLIGONUCLEOTIDES MEDIATED BY CATIONIC LIPIDS

Purpose, To study the parameters influencing the intracellular traffic king of oligonucleotides delivered by cationic 1,2-dioleoyl-3-trimethy lammonium-propane (DOTAP) lipids and to elucidate the mechanism of upt ake. Methods. We have studied the intracellular localization of fluore scently labeled oligonucleotide (F-ODN) delivered by DOTAP using confo cal microscopy and measured inhibition of luciferase synthesis. The de livery mechanism of ODN/DOTAP complexes was investigated using inhibit ors of the endocytosis pathway. Results, F-ODN delivered by DOTAP lipo somes redistribute from punctate cytoplasmic regions into the nucleus. The nuclear uptake of F-ODN depends on: charge ratio (+/-), time of i ncubation, temperature and presence of serum. A positively charged com plex is required for enhanced uptake. The association of neutral lipid s with DOTAP reduced the optimum charge ratio without altering the del ivery efficiency. DOTAP lipids increased >100 fold the antisense activ ity of a specific anti-luciferase ODN. Inhibitors of the endocytosis p athway show that the majority of F-ODN are introduced through an endoc ytic pathway mainly involving uncoated vesicles. Nuclear accumulation of oligonucleotides can be decreased by inhibitors of actin microfilam ents, energy metabolism and proteins implicated in the fusion of endos omes. Nuclear uptake is independent of acidification of the endosomal vesicles and unaffected by inhibitors of microtubules. Conclusions, Ol igonucleotides are delivered by cationic lipids into the cytoplasm at an early stage of the endocytotic pathway which leads to a marked incr ease in antisense activity and oligonucleotide nuclear uptake.