Cellular uptake and efficacy of antisense oligonucleotides against RNAs oftwo Na+ channel isoforms

The effects of 15-mer phosphorothioate antisense oligodeoxynucleotides (aOD Ns) specifically designed against the RNAs of either of two closely related Na+ channel isoforms, hSkM1 or hH1, were tested in human myotubes. Fluores cence (3'-fluorescein isothiocyanate) labeling showed that mere incubation of cultures with aODNs did not result in aODN uptake, but liposome-mediated transfer was successful and resulted in cytoplasmic and nuclear localizati on of ODNs. Intracellular fluorescence was stable for at least 3 days. At 5 mu M, the hH1-specific aODN was effective in suppressing ion channel funct ion, but the hSkM1-specific aODN was not. Reverse transcription-polymerase chain reaction gave corresponding results on the mRNA level. However, in HE K-293 cells stably expressing hSkM1, the same hSkM1-specific aODN was able to reduce Na+ currents (2.4 +/- 0.5 nA, n = 11; controls: 6.5 +/- 1.0 nA, n = 14). We conclude that cellular uptake of aODNs and intracellular access to the RNA target are necessary, but not always sufficient conditions for a n effective block of mRNA translation in intact cells.