Development of antisense oligonucleotide therapy for cancer.

反义寡核苷酸(ODN)基因治疗是一种合理的抑制靶基因的方法，但在传递性和特异性等方面仍存在一些问题。本研究以聚乙二醇(PLSP)或阳离子脂多胺(Transfettam)为载体，研究了针对c-raf m RNA的磷酸二酯(PO；核酸酶敏感)和硫代磷酸(PS；核酸酶抗性)反义ODN对胰腺癌(HS766T)和肝癌(SK-Hep)细胞的体内外抗增殖作用。RT-PCR检测P0和PS反义ODN均能降低c-raf基因的表达，但其抗增殖作用主要与反义机制无关。将SK-Hep细胞接种于裸鼠腹膜腔内建立腹膜移植瘤模型，肉眼观察移植瘤在肠系膜、肝门和肝脏的成瘤情况，包括腹水的发生情况。用ODN/Transfetam复合体(3亩)腹腔注射。GODN，每周2次，共6次)，接种SK-Hep细胞3周后，其体内抗肿瘤作用与体外实验基本一致。当使用6“杯ODN时，在接受PS反义复合体的小鼠身上观察到多个皮肤溃疡。我们的研究表明，P0反义ODN可能是一种有效的抗肿瘤药物，并用于安全的反义ODN治疗癌症。现在，我计划寻找新的靶基因和修饰载体，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.