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Design of Potent Phosphorothioate Antisense Oligonucleotides Directed to Human Interleukin 10 Gene Product and Their Evaluation of Antisense Activity in U937 Cells

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Abstract

Purpose. The two objectives of this study were to design potent phosphorothioate antisense oligonucleotides (AS-S-oligos) directed against the human interleukin-10 (IL-10) gene product and to reveal the DNA sequence which best activates antisense effects.

Methods. The design of potent AS-S-oligo was performed by using melting temperature (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 folding 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 basis of having higher Tm values and favorable secondary structures of the IL-10 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 inhibitory effects on IL-10 production and this inhibition was dose- and time-dependent. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that the antisense effects of AS-S-oligos originated from a specific reduction 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-∝ (TNF-∝) production in the cells stimulated by lipopolysaccharide (LPS). Strong positive correlations between the inhibitory effect of AS-S-oligos on the IL-10 production and not only Tm values calculated by nearest neighbor method but also Tm values determined by absorbance versus temperature profiles were demonstrated except for AS25-S-oligo and AS1249-S-oligo.

Conclusions. These findings suggest AS367- and AS637-S-oligos powerfully inhibit IL-10 production in U937 cells via an antisense mechanism. In addition, it is suggested efficiency of AS-S-oligo directed against the sequence of the target gene product can be explained by these Tm values and the proposed secondary structures of the target gene product.

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Authors and Affiliations

  1. School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan

    Hidetoshi Arima, Mamiko Takahashi, Yukihiko Aramaki, Takatoshi Sakamoto & Seishi Tsuchiya

  2. Tsukuba Research Laboratories, Hisamitsu Pharmaceutical Co., Inc., 1-25-11 Kannondai, Tsukuba, Ibaraki, 305-0856, Japan

    Kazuhiro Yuda, Katsuhiko Akiyama & Takeshi Goto

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  1. Hidetoshi Arima
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  2. Mamiko Takahashi
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  3. Yukihiko Aramaki
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  4. Takatoshi Sakamoto
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  8. Seishi Tsuchiya
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Correspondence to Seishi Tsuchiya.

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Arima, H., Takahashi, M., Aramaki, Y. et al. Design of Potent Phosphorothioate Antisense Oligonucleotides Directed to Human Interleukin 10 Gene Product and Their Evaluation of Antisense Activity in U937 Cells. Pharm Res 16, 1163–1171 (1999). https://doi.org/10.1023/A:1018964625977

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