Development of liposomes for clinical research and application

我们推动了用于人类恶性肿瘤基因治疗的阳离子脂质体的开发。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载体。前者为阳离子脂质体，摩尔比为：N-(α-三甲基氨乙酰)-D-十二烷基谷氨酸氯(TMAG)：二月桂酰磷脂酰胆碱(DLPC)：双烯基磷脂酰乙醇胺(DOPE)。后者为阳离子脂质体，摩尔比为1：2：2或1：1：1，组成为二甲基二十八烷基溴化铵(DDAB)：二月桂酰磷脂酰胆碱(DLPC)：二油酰磷脂酰乙醇胺(DOPE)。我们还发现，通过与单抗或去唾液酸糖蛋白结合，能够赋予这些脂质体细胞或组织特异性。相反，我们还限制腺相关病毒(AAV)载体相关的脂质体在体外对人胶质瘤细胞的基因转移比含质粒DNA的脂质体更有效。与含有质粒DNA的脂质体相比，载体相关脂质体的转导效率提高了10倍以上，与AAV单独使用相比提高了6倍以上。从这些结果来看，AAV载体相关的脂质体似乎是很好的体内基因输送到恶性肿瘤的候选者。此外，我们还考察了热休克蛋白启动子驱动的阳离子脂质体与小铁球磁铁复合后的抗肿瘤效果。另一方面，我们在名古屋大学医院建立了人类基因治疗载体生产装置，证实在该装置中可以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.