抗ＨＳＶ活性を有するアンチセンス・オリゴＤＮＡの安定性およびウイルス感染細胞における動態の検討

Abstract

We have synthesized antisense phosphorothioate oligodeoxynucleotide (S-ODN) tageted 3' splice junction of immediate early (IE) pre-mRNA 4/5 in herpes simplex virus type 1 (HSV-1) and evaluated their antiviral activities as a novel anti-herpetic agent. The antisense S-ODN completely protected the permissive Vero cells from a cytopathic effect of HSV-1 at a lower concentration range of 0.78 to 1.56 microM in a serum-free assay system, whereas its sequence including mismatced bases of S-ODN and the sense sequence of S-ODN exhibited the protective effect at higher concentrations (=> 50 microM). The results indicated that the antisense S-ODNs targeted 3' splice junction inhibit viral growth in a sequence-specific manner. To get more informations for the usage as a therapeutic agent, the biological stability and the intracellular distribution of the antisense S-ODN were examined. In the nuclease degradation test, the antisense S-ODN had a half life of 5 to 7 hrs in culture medium containing 10% fetal calf serum at 37°C. The similar antisense phosphodiester (D) ODN was completely degraded within 60 mins. The S-ODN was shown to be considerablely more stable to nuclease degradation than D-ODN. In the same condition, the antiviral activity of the antisense S-ODN was 8 times less than that in the serum-free culture system. No antivial effect of the antisense D-ODN was detected at any concentration even in the serum-free culture system. In HSV-1 infected cells, a uniform distribution of S-ODN was observed both in nuclei and in cyto plasm at 12-hr incubation. On the other hand, in non-infected cells, S-ODN was localized into lysosome-like vesicles and such localization of S-ODN continued on and after 12-hr incubation. This indicates that a critical disadvantage of intracellular delivery to target site might be solved by viral infection. These findings suggest that the antisense S-ODN may be sufficiently worth developing as an antiherpetic therapeutic agent, and that some more devices for the improvement of nuclease-resistance and intracellular permeability could make the antisense S-ODN more effective against HSV-1 infection.