BIOAVAILABILITY OF ANTISENSE OLIGONUCLEOTIDES IN NEUROBLASTOMA-CELLS - COMPARISON OF EFFICACY AMONG DIFFERENT TYPES OF MOLECULES

To evaluate the real effectiveness of various chemical modifications i n enhancing the ability of antisense molecules to inhibit gene express ion, the toxicity, stability, uptake, and intracellular localization o f an identical sequence, synthetized either with a phosphodiester or a phosphorothioate backbone, with or without a cholesteryl moiety linke d to the 3'-end, were compared in three different human neuroblastoma cell lines. The toxicity, assessed by inhibition of cell viability, gr eatly depend on the presence of the lipid moiety and to a less extent on the cell line used. At high doses all the antisenses caused a necro tic lysis of plasma membranes. Typical features of apoptotic cell deat h were never observed. The presence of the lipid moiety enhanced the u ptake of antisense molecules while the phosphorothioate backbone, as e xpected, conferred higher stability. At late times, therefore, the com bination of lipid conjugation and phosphorothioate backbone seems to b e the most effective in obtaining a consistent antisense accumulation inside the cells. The presence of the cholesteryl moiety also caused a stronger association of the antisense to membraneous compartments, so that a quite different biodistribution occurred among the four antise nses tested. However, the actual amount of antisense molecules found i nside NE cells was low in all the conditions tested. Only following ce llular permeabilization a significant uptake was obtained, making the use of delivery system mandatory to achieve an efficient inhibition of highly expressed genes.