Development of Gene Targeting Technique Using Radiolabeled Antisense DNA

利用放射性标记的反义寡核苷酸（DNA），可以用伽马照相机对扩增mRNA的病变进行成像。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。我们建立了裸鼠异种移植肿瘤细胞表达p糖蛋白（P-gp）的体内模型系统。为了成像P-gp引起的多药耐药肿瘤，我们设计了编码P-gp的mdr1基因的15mer反义DNA序列。用巯基修饰寡核苷酸的5′端，反应马来酰氨基- c6 -苄基- edta螯合物。最终产物经ODS色谱鉴定为目的化合物。有必要进一步调查。像寡核苷酸这样的小分子往往会很快从血液中清除，因此，肿瘤对这种示踪剂的绝对摄取是有限的。我们试图通过改变肿瘤组织的递送系统来增加肿瘤对放射性标记抗体的吸收。血管紧张素- ii以2μg/kg/min持续输注，与肌动酶抑制剂马来酸依那普利30 μg联合使用，使小鼠血压由95/61提高到153/67。在正常器官分布变化不大的情况下，肿瘤摄取增加了1.62倍。放射自显像显示，由于血管床再通和血管通透性增加，放射标记抗体在肿瘤中的分布更为均匀。综上所述，通过控制肿瘤组织的血流动力学和血管通透性可以增强肿瘤的摄取，该技术可以应用于小分子的寡核苷酸。

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.