Development of antisense oligonucleotide therapy for cancer.

反义寡脱氧核苷酸（ODN）基因治疗是一种理想的靶基因抑制方法，但在靶向和特异性方面仍存在一些问题。在这项研究中，磷酸二酯的抗增殖作用（PO ;核酸酶敏感）和硫代磷酸酯（PS：应用多聚核苷酸技术检测c-raf基因反义寡核苷酸对胰腺癌细胞株HS 766 T和肝癌细胞株SK-HEP的作用与聚乙二醇（PLSP）或阳离子脂多胺缀合的（赖氨酸/丝氨酸）共聚物PO反义ODN与PLSP（2 μ M ODN）或Transfectam（0.5 μ M ODN）联合应用的抗增殖作用均大于PS DON。P0和PS反义ODN均能降低c-raf mRNA水平，但其抗增殖作用与反义机制无关。将SK-HEP细胞接种于裸鼠腹腔内建立腹膜播散模型，肉眼观察肠系膜、肝门和肝脏的肿瘤形成情况，包括腹水的发生情况。腹膜内给予ODN/Transfectam复合物（3 μ g. g臭氧消耗物质;每周两次，共6次）接种SK-HEP细胞3周后，各复合物的体内抗肿瘤效果与体外实验中观察到的效果大致相同。当使用6 μ g ODN时，在接受PS反义复合物的小鼠上观察到多个皮肤溃疡。我们的研究表明P0反义ODN是一种有效的抗肿瘤药物，可安全地用于肿瘤的反义ODN治疗，目前，我正计划寻找新的靶基因，并对载体进行修饰，以提高对癌细胞或组织的特异性。

Such rational approach to suppress target genes as antisense oligodeoxynucleotide (ODN) gene therapy is very attractive, but still has some problems concerning delivery and specificity. In this study, antiproliferative effects of phosphodiester (PO ; nuclease-sensitive) and phosphorothioate (PS ; nuclease-resistant) antisense ODN targeted against c-raf mRNA on pancreatic cancer (HS766T) and hepatoma (SK-HEP) cells in vitro and in vivo were assessed using poly (lysine/serine) copolymers conjugated with polyethylene glycol (PLSP) or cationic lipopolyamines (Transfectam) as carriers.The antiproliferative effect of the PO antisense ODN was greater than that of the PS DON, either compelxed with PLSP (2 muM ODN) or the Transfectam (0.5 muM ODN). The c-raf mRNA levels assessed by RT-PCR were reduced by both P0 and PS antisense ODN, but the antiproliferative effects were mainly unrelated to antisense mechanisms. SK-HEP cells were inoculated into the peritoneal cavity of nude mice to make a peritoneal dissemination model, and tumor formation on the mesentery, liver hilus and liver were macroscopically assessed, including the occurrence of acites. With the intraperitoneal administration of the ODN/Transfectam complexes (3 mu. g ODN ; twice a week 6 times in total) 3 weeks after the inoculation of SK-HEP cells, the antitumor effects of the respective complexes in vivo were much the same as those seen in the in-vitro experiment. When 6 "mug of ODN was used, multiple skin ulcers were observed on the mice that received the PS antisense complexes. Our study suggests that P0 antisense ODN might be potent as an antitumor agent and used in safe for the antisense ODN therapy for cancer.Now, I am planning to search for new target genes and to modify carriers in an attempt to improve the specificity for cancer cells or tissue.