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Development of Gene Targeting Technique Using Radiolabeled Antisense DNA

使用放射性标记反义 DNA 开发基因打靶技术

基本信息

批准号：
10470194
负责人：
TONAMI Norihisa
金额：
$ 3.39万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

Using radiolabeled antisense oligonucletides (DNA), lesions of amplified mRNA can be imaged with gamma cameras. For this approach, the stable radiometal chelates, which are appropriate for oligonucletide, is required. We have developed the in vivo model system where nude mice xenografted tumor cells express P-glycoprotein (P-gp). To image multidrug resistant tumors caused by P-gp, we designed the 15mer of antisense DNA sequence for the mdr1 gene coding P-gp. The 5'-end of the oligonucletide was modified with the thiol group and the maleimido-C6-benzyl-EDTA chelate was reacted. The final product was identified as the objective compound by ODS chromatography. Further investigation was warranted. The small molecules like oligonucleotides tend to be cleared very rapidly from the blood and therefore, the absolute tumor uptake of this kind of the tracer is limited. We have tried to increase the tumor uptake of radiolabeled antibodies by modifying the delivery system to the tumor tissue. The combination usage of angiotensin-II, continuously infused at 2μg/kg/min, and a kininase inhibitor, enalapril maleate, 30 μg increased the mouse blood pressure from 95/61 to 153/67. And the tumor uptake was also increased by the factor of 1.62 with little change in normal organ distribution. The autoradiography showed that more homogeneous distribution of the radiolabeled antibody in the tumor because of recanalization of vascular beds and increased vascular permeability. In conclusions, enhanced tumor uptake was achieved by manipulating hemodynamics and vascular permeability of the tumor tissue and this technique can be applied for smaller molecule as the oligonucleotides.

使用放射性标记的反义寡核苷酸（DNA），扩增的mRNA的病变可以用γ照相机成像。对于这种方法，需要适合于寡核苷酸的稳定的放射性金属螯合物。我们已经建立了裸鼠移植瘤细胞表达P-糖蛋白（P-gp）的体内模型系统。为了研究P-糖蛋白（P-gp）引起的多药耐药肿瘤，我们设计了一段15聚体的反义DNA序列。寡核苷酸的5 '端用巯基修饰，并与马来酰亚胺-C6-苄基-EDTA螯合物反应。最终产物经ODS色谱鉴定为目标化合物。需要进一步调查。像寡核苷酸这样的小分子往往会非常迅速地从血液中清除，因此，这种示踪剂的绝对肿瘤摄取是有限的。我们已经尝试通过修改肿瘤组织的递送系统来增加放射性标记抗体的肿瘤摄取。以2μg/kg/min持续输注血管紧张素II和30 μg激酶抑制剂马来酸依那普利的联合使用使小鼠血压从95/61升高至153/67。肿瘤摄取也增加了1.62倍，而正常器官分布变化不大。放射自显影显示，由于血管床的再通和血管通透性的增加，放射性标记抗体在肿瘤中的分布更加均匀。总之，通过操纵肿瘤组织的血流动力学和血管通透性来实现增强的肿瘤摄取，并且该技术可以应用于更小分子的寡核苷酸。