DEVELOPEMENT OF A NOVEL LIVER-SPECIFIC GENE TRANSFER METHOD USING HEPATIC-RECEPTOR IMAGING AGENT

新半乳糖白蛋白(GSA)被活化后与已知与DNA形成静电络合物的树状大分子(G4)反应，构建了GSA-G4化合物作为寡聚DNA(OlDNA)的载体进入肝脏。然后将GSA-G4与标记的olDNA混合，形成GSA-G4/olDNA复合体，静脉注射给正常小鼠。虽然GSA-G4/olDNA复合体在肝脏中有蓄积，但在肾、肺和脾中也表现出较高的摄取，这可能是由于GSA-G4/olDNA复合体在血液中的不稳定(即在肾脏中积累的olDNA的脱落)，也可能是因为形成了捕获在肺和脾中的高分子量复合体及其聚集体。然后，亲和素(Av)，一种高度糖化的蛋白质，也被认为积累在肝脏中，作为GSA的替代品。构建了两种Av-olDNA结合物，并研究了它们的生物分布。首先将Av与生物素标记的olDNA(bt-olDNA)混合，形成Av-bt-olDNA。其次，Av与生物素标记的G4(bt-G4)反应生成Av-bt-G4，再与olDNA混合形成Av-bt-G4/olDNA复合体。静脉注射给正常小鼠后，Av-bt-olDNA显示肝脏对标记的olDNA有很高的摄取率，而对其他正常器官的摄取率很低，导致肝脏与本底的放射性比值很高。虽然Av-bt-G4/olDNA复合体表现出较高的肝脏摄取，但在肺中表现出极高的摄取。这可能是由于大分子复合体及其聚集体的形成，它们被困在肺中。目前的研究表明，亲和素具有作为寡聚DNA载体进入肝脏的潜力。然而，Av-bt-G4/olDNA复合体存在形成大聚集体的问题，需要改变给药途径，如门静脉注射。

Neogalactosylalbumin (GSA) was activated and reacted with dendrimer (G4), which was known to make electrostatic complex with DNA, to construct GSA-G4 compound as a carrier of oligoDNA (olDNA) to the liver. GSA-G4 was then mixed with ^<111>In-labeled olDNA, and the formed GSA-G4/olDNA complex was intravenously administered into normal mice. Although GSA-G4/olDNA complex showed an accumulation to the liver, it also showed high uptake in the kidney, lung, and spleen, probably because of the instability of the GSA-G4/olDNA complex in the bloodstream (i.e. detachment of olDNA, which accumulated in the kidney) and also because of the formation high-molecular weight complexes and their aggregate, which were trapped in the lung and spleen.Then, avidin (Av), a highly glycosilated protein which is also known to accumulate in the liver, was introduced as an alternate to GSA. Two Av-olDNA conjugates were constructed and their biodistribution were studied. First Av was mixed with biotinylated olDNA (bt-olDNA) to form Av-bt-olDNA. Second, Av was reacted with biotinylated G4 (bt-G4) to form Av-bt-G4, and then mixed with olDNA to make Av-bt-G4/olDNA complex. After intravenous injection into normal mice, Av-bt-olDNA showed a specific and high hepatic uptake of ^<111>In-labeled olDNA with low uptake to other normal organs, resulting in very high liver-to-background radioactivity ratios. Although Av-bt-G4/olDNA complex showed relatively high hepatic uptake, it showed extremely high uptake in the lung. This may be because of the formation of large molecular weight complexes and their aggregates, which were trapped in the lung.Present investigations demonstrated that avidin had the potential as a carrier of oligoDNA to the liver. Av-bt-G4/olDNA complex, however, has a problem of making large aggregates and the change in administration route, such as intraportal injection, is necessary.