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

将新半乳糖基白蛋白（GSA）活化后，与能与DNA形成静电复合物的树枝状分子（G4）反应，构建GSA-G4复合物，作为寡聚DNA（oliDNA）的载体。然后将GSA-G4与13 <111>In标记的olDNA混合，并将形成的GSA-G4/olDNA复合物静脉内施用到正常小鼠中。虽然GSA-G4/olDNA复合物显示出在肝脏中的积累，但它也显示出在肾、肺和脾中的高摄取，这可能是因为GSA-G4/olDNA复合物在血流中的不稳定性（即，在肾脏中积累的olDNA的脱离）并且还由于形成高分子量复合物及其聚集体，其被捕获在肺和脾中。一种高度糖基化的蛋白质，也已知其在肝脏中积累，被引入作为GSA的替代物。构建了两种Av-olDNA偶联物，并研究了它们的生物分布。首先将Av与生物素化的olDNA（bt-olDNA）混合以形成Av-bt-olDNA。其次，将Av与生物素化的G4（bt-G4）反应，形成Av-bt-G4，然后与olDNA混合，制备Av-bt-G4/olDNA复合物。在静脉内注射到正常小鼠中后，Av-bt-olDNA显示出特异性的和高的肝脏摄取的13 <111>In-标记的olDNA，而其他正常器官的摄取较低，导致非常高的肝脏与背景放射性比率。虽然Av-bt-G4/olDNA复合物显示相对高的肝摄取，但其在肺中显示极高的摄取。这可能是因为在肺内形成了大分子量的复合物及其聚集体。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复合物具有产生大聚集体的问题，并且需要改变给药途径，例如门静脉内注射。

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.