Development of antisense oligonucleotide therapy for cancer.

这种以反义寡脱氧核苷酸（ODN）基因治疗为目的的合理抑制靶基因的方法是非常有吸引力的，但在递送和特异性方面还存在一些问题。本研究以聚乙二醇（PLSP）或阳离子脂多胺（Transfectam）偶联聚赖氨酸/丝氨酸共聚物为载体，研究了针对c-raf mRNA的磷酸二酯（PO；核酸酶敏感）和磷酸硫酸（PS；核酸酶抗性）反义ODN对胰腺癌（HS766T）和肝癌（SK-HEP）细胞体外和体内的抗增殖作用。PO反义ODN与PLSP （2 μ m ODN）或Transfectam （0.5 μ m ODN）复合物的抗增殖作用均大于PS DON。RT-PCR检测的c-raf mRNA水平均被P0和PS反义ODN降低，但其抗增殖作用主要与反义机制无关。将SK-HEP细胞接种于裸鼠腹腔制作腹膜播散模型，宏观观察肠系膜、肝门、肝脏的肿瘤形成情况，包括细胞的发生情况。腹腔注射ODN/Transfectam复合物(3 μ。g ODN；接种SK-HEP细胞3周后，体内各复合物的抗肿瘤作用与体外实验基本一致。当使用6”杯的ODN时，在接受PS反义复合物的小鼠身上观察到多发皮肤溃疡。我们的研究表明，P0反义ODN可能是一种有效的抗肿瘤药物，可以安全地用于反义ODN治疗癌症。现在，我正计划寻找新的靶基因，并修改载体，试图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.