Development of liposomes for clinical research and application

我们的工作推动了阳离子脂质体在恶性肿瘤基因治疗中的应用。制备了多种阳离子脂质体，并进行了转导效率的评价。因此，我们证实了TMAG-脂质体和DDAB-脂质体是非常有用的外源基因转移。前者是阳离子脂质体，由N-（α-三甲基铵乙酰基）-双十二烷基-D-谷氨酸氯化物（TMAG）：二月桂酰磷脂酰胆碱（DLPC）：二油烯基磷脂酰乙醇胺（DOPE）以1：2：2的摩尔比组成。后者是以1：2：2或1：1：1的摩尔比作为二甲基-双十八烷基溴化铵（DDAB）：二月桂酰磷脂酰胆碱（DLPC）：二油酰磷脂酰乙醇胺（DOPE）组成的阳离子脂质体。我们还发现能够通过与单克隆抗体或去唾液酸糖蛋白缀合来赋予这些脂质体细胞或组织特异性。与此相反，我们也确认腺相关病毒（AAV）载体相关脂质体在体外基因转移到人脑胶质瘤细胞比含有质粒DNA的脂质体更有效，使用载体相关脂质体的转导效率增加超过10倍，相比单独的AAV的脂质体相比，超过6倍。从这些结果来看，AAV载体相关脂质体似乎是用于体内基因递送至恶性肿瘤的良好候选物。此外，研究了含有热休克蛋白启动子驱动的质粒的阳离子脂质体与小铁球磁铁矿的组合的有效性，并诱导了显著的抗肿瘤效果。另一方面，在名古屋大学医院建立了人基因治疗载体生产设施，并确认可以在该设施中制备用于临床研究的脂质体。

We have promoted the development of cationic liposomes for human gene therapy against malignant tumors. A lot of kinds of cationic liposomes were made and performed the evaluation of the transduction efficiency. As a result, we confirmed TMAG-liposomes and DDAB-liposomes were very useful to transfer the foreign genes to. The former is a cationic liposome composed in a molar ratio of 1 : 2 : 2 as N- (alpha-trimethylammonioacetyl)- didodecyl-D-glutamate chloride (TMAG) : dilauroyl phosphatidylcholine (DLPC) : diolenyl phosphatidyl ethanolamine (DOPE) . The latter is a cationic liposome composed in a molar ratio of 1 : 2 : 2 or 1 : 1 : 1 as dimethyl-dioctadecylammonium bromide (DDAB) : dilauroyl phosphatidylcholine (DLPC) : dioleoyl phosphatidylethanolamine (DOPE) . We also found to be able to give these liposomes cell - or tissue-specificity by conjugating with a monoclonal antibody or asialoglycoprotein. In contrast, we also confinned that adeno-associated virus (AAV) vector-associated liposomes were more effective for in vitro gene transfer to human glioma cells than are liposomes containing plasmid DNA.Using vector-associated liposomes increased transduction efficiency more than 1O-fold compared to liposomes containing plasmid DNA and more than 6-fold compared to AAV alone. From these results, AAV vector-associated liposomes appear to be good candidates for in vivo gene delivery to malignant tumors. Furthermore, we investigated the effectiveness of the combination of cationic liposomes containing the plasmids driven by heat shock protein promoter and small iron ball magnetite, and induced remarkable antitumor effect.On the other hand, we established human gene therapy vector producing facility in Nagoya University Hospital and confirmed that liposomes for clinical research could prepared in the facility.