Design of potent phosphorothioate antisense oligonucleotides directed to human interleukin 10 gene product and their evaluation of antisense activityin U937 cells

Purpose. The two objectives of this study were to design potent phosphoroth ioate antisense oligonucleotides (AS-S-oligos) directed against the human i nterleukin-10 (IL-IO) gene product and to reveal the DNA sequence which bes t activates antisense effects. Methods. The design of potent AS-S-oligo was performed by using melting tem perature (Tm) value of a DNA/RNA hybrid calculated by the nearest neighbor method and a secondary structure of human IL-10 mRNA suggested by RNA foldi ng algorithms. U937 cells were used to estimate the antisense effect of the AS-S-oligos. Results. Of the eight candidates selected as potent AS-S-oligos on the basi s of having higher Tm values and favorable secondary structures of the IL-1 0 mRNA, AS-S-oligos directed against the translated (AS367-S-oligo) and 3'- untranslated (AS637-S-oligo) region of IL-10 mRNA showed the strongest inhi bitory effects on IL-10 production and this inhibition was dose- and time-d ependent. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that the antisense effects of AS-S-oligos originated from a specific reduc tion of target IL-10 mRNA by hybridization with AS367- and AS637-S-oligos. In addition, these AS-S-oligos did not affect human tumor necrosis factor-a lpha (TNF-alpha) production in the cells stimulated by lipopolysaccharide ( LPS). Strong positive correlations between the inhibitory effect of AS-S-ol igos on the IL-10 production and not only Tm values calculated by nearest n eighbor method but also Tm values determined by absorbance versus temperatu re profiles were demonstrated except for AS25-S-oligo and AS1249-S-oligo. Conclusions. These findings suggest AS367- and AS637-S-oligos powerfully in hibit IL-10 production in U937 cells via an antisense mechanism. In additio n, it is suggested efficiency of AS-S-oligo directed against the sequence o f the target gene product can be explained by these Tm values and the propo sed secondary structures of the target gene product.