UPTAKE OF ANTISENSE OLIGONUCLEOTIDES AND FUNCTIONAL BLOCK OF ACETYLCHOLINE-RECEPTOR SUBUNIT GENE-EXPRESSION IN PRIMARY EMBRYONIC NEURONS

Several recent studies have used antisense oligonucleotides in the ner vous system to probe the functional role of particular gene products. Since antisense oligonucleotide-mediated block of gene expression typi cally involves uptake of the oligonucleotides, we have characterized t he mechanism of this uptake into developing neurons from embryonic chi ckens. Antisense oligonucleotides (15 mers) added to the bathing media are taken up into the embryonic chicken sympathetic neurons maintaine d in vitro. A portion of the oligonucleotide uptake is temperature dep endent and saturates at extracellular oligonucleotide concentrations g reater-than-or-equal-to 20 muM. This temperature sensitive, saturable component is effectively competed by single nucleotides of ATP and AMP and is reminiscent of receptor-mediated endocytosis of oligonucleotid es described in non-neuronal cells. The efficiency of the oligonucleot ide uptake system is dependent on the developmental stage of the anima l but independent of the number of days that the neurons are maintaine d in vitro. Following the uptake of antisense oligonucleotides directe d against ion channel subunit genes expressed by these neurons (nicoti nic acetylcholine receptor subunit alpha3; nAChR alpha3), biophysical assays reveal that the functional expression of the target gene is lar gely blocked. Thus the number of wild type nAChR channels expressed is decreased by almost-equal-to 80%-90%. Furthermore, following antisens e deletion of alpha3, ''mutant'' nAChRs with distinct functional chara cteristics are expressed. In sum, these studies characterize the uptak e of antisense oligonucleotide and demonstrate the functional block of specific gene expression in primary developing neurons. In addition, the functional studies emphasize the need for sensitive and specific a ssay following antisense deletion, since other homologous gene product s may substitute for the targeted gene resulting in new phenotypes tha t are subtly different from wild type. (C) 1993 Wiley-Liss, Inc.