Abe T, Suzuki S, Hatta T, Takai K, Yokota T, Takaku H
Department of Industrial Chemistry, Chiba Institute of Technology, Japan.
Antivir Chem Chemother. 1998 May;9(3):253-62. doi: 10.1177/095632029800900306.
We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza A virus replication in MDCK cells. Liposomally encapsulated and free antisense S-ODNs with four target sites (PB1, PB2, PA and NP genes) were tested for their abilities to inhibit virus-induced cytopathogenic effects in a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the site around the PB2 AUG initiation codon showed highly inhibitory effects. In contrast, the inhibitory effect of the liposomally encapsulated S-ODN targeted to PB1 was considerably decreased in comparison with that directed to the PB2 target site. The liposomally encapsulated antisense S-ODNs exhibited higher inhibitory activities than the free oligonucleotides, and showed sequence-specific inhibition, whereas free antisense S-ODNs were observed to inhibit viral adsorption to MDCK cells. Liposomal preparations of oligonucleotides facilitated their release from endocytic vesicles, and thus cytoplasmic and nuclear localization was observed. The activities of the antisense S-ODNs were effectively enhanced by using the liposomal carrier. Interestingly, the liposomally encapsulated FITC-S-ODN-PB2-as accumulated in the nuclear region of MDCK cells. However, weak fluorescence was observed within the endosomes and the cytoplasm of MDCK cells treated with the free antisense S-ODNs. The cationic lipid particles may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, appropriate for use in vitro or in vivo.
我们已经证明，硫代磷酸反义寡核苷酸（S-ODNs）可抑制甲型流感病毒在MDCK细胞中的复制。使用MDCK细胞，通过MTT试验检测了具有四个靶位点（PB1、PB2、PA和NP基因）的脂质体包裹型和游离反义S-ODNs抑制病毒诱导的细胞病变效应的能力。与PB2 AUG起始密码子周围位点互补的脂质体包裹型S-ODN显示出高度抑制作用。相比之下，脂质体包裹型靶向PB1的S-ODN的抑制作用与靶向PB2靶位点的相比显著降低。脂质体包裹型反义S-ODNs比游离寡核苷酸表现出更高的抑制活性，并表现出序列特异性抑制，而游离反义S-ODNs则被观察到可抑制病毒吸附到MDCK细胞上。寡核苷酸的脂质体制剂促进了它们从内吞小泡的释放，因此观察到了细胞质和细胞核定位。使用脂质体载体有效地增强了反义S-ODNs的活性。有趣的是，脂质体包裹的FITC-S-ODN-PB2-as积聚在MDCK细胞的核区域。然而，在用游离反义S-ODNs处理的MDCK细胞的内体和细胞质中观察到微弱的荧光。因此，阳离子脂质颗粒可能是一种潜在有用的寡核苷酸疗法和转基因递送载体，适用于体外或体内使用。
