ANTISENSE OLIGODEOXYNUCLEOTIDES TO THE CLONED DELTA-RECEPTOR DOR-1 - UPTAKE, STABILITY, AND REGULATION OF GENE-EXPRESSION

Phosphodiester antisense oligodeoxynucleotides (ODNs) directed against various domains of the cloned mouse delta receptor DOR-1 reduce delta -opioid receptor binding in vivo and in vitro. The present study exami nes the stability of an antisense ODN (275 nM) directed against the de lta-opioid receptor and its effect on DOR-1 mRNA in cultured neuroblas toma cells and in vivo. When added to NG108-15 cells, much of the anti sense ODN is degraded. However, >1% is intact, associated with cells, and stable for at least 72 h. Northern blot analysis demonstrates that treatment of NG108-15 cells with the antisense ODN reduces the levels of a species of DOR-1 mRNA by similar to 25%. Similarly, intrathecal administration of the antisense ODN results in the accumulation of int act ODN within the spinal cord, which is stable for at least 72 h, alt hough the levels of accumulation in vivo are lower than in vitro after either 4 or 72 h. Antisense ODN treatment lowers DOR-1 mRNA levels by similar to 25%. The loss of mRNA both in vivo and in vitro correspond s quite well to the decreases in receptor binding previously observed by our laboratory and is consistent with reduction of delta-opioid rec eptor protein in vitro as determined by western blot with a monoclonal antibody selective for the delta-opioid receptor. In conclusion, thes e studies indicate that a small, but significant, proportion of ODN is taken up by cells and remains intact for up to 72 h. This appears to be sufficient to down-regulate mRNA levels of delta-opioid receptors a nd their expression.