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

ABSTRACT The effects of 15-mer phosphorothioate antisense oligodeoxynucleotides (aODNs) specifically designed against the RNAs of either of two closely related Na ϩ channel isoforms, hSkM1 or hH1, were tested in human myotubes. Fluorescence (3Ј-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 localization of ODNs. Intracellular fluorescence was stable for at least 3 days. At 5 M, the hH1-specific aODN was effective in suppressing ion channel function, but the hSkM1-specific aODN was not. Reverse transcription-polymerase chain reaction gave corresponding results on the mRNA level. However, in HEK-293 cells stably expressing hSkM1, the same hSkM1-specific aODN was able to reduce Na ϩ currents (2.4 Ϯ 0.5 nA, n ϭ 11; controls: 6.5 Ϯ 1.0 nA, n ϭ 14). We conclude that cellular uptake of aODNs and intracellular access to the RNA target are necessary, but not always sufficient conditions for an effective block of mRNA translation in intact cells. A modern strategy in pharmacology for interfering with receptor or channel proteins is to manipulate lifetime and translation efficacy of their coding mRNAs using antisense oligodeoxynucleotides (aODNs) For an effective inhibition of protein translation in vivo or in cultured cells, the aODNs have to be protected against extracellular degradation, they must also be able to pass through the lipophilic plasma membrane, and finally they must be made to escape intracellular degradation and sequestration into organelles Some of these tasks can now easily be solved, e.g., degradation is substantially slowed when phosphorothioate-protected ODNs are used instead of phosphodiester ODNs (Crooke, 1998). The accessibility of a specific target site in a given RNA can be tested in advance in vitro by means of binding assays or translation arrest assays (Schu and Brinkmeier, 1999). As for the specificity for closely related isoforms of voltage-gated Na ϩ channels, we have earlier developed highly specific 15-mer aODNs against the RNAs of the Na ϩ channels in human heart (hH1, Materials and Methods Cells and Application of Oligonucleotides. Human myotubes expressing both the tetrodotoxin (TTX)-sensitive skeletal musclespecific hSkM1 and the TTX-insensitive heart-specific hH1 Na ϩ channe