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，并对其转导效率进行了评价。结果表明，tmag -脂质体和ddab -脂质体对外源基因的转移非常有用。前者是一种阳离子脂质体，其摩尔比为1:2:2:N- （α -三甲氨酰乙酰）-二十二烷基- d -谷氨酸氯（TMAG）：二酰磷脂酰胆碱（DLPC）：二烯酰磷脂酰乙醇胺（DOPE）。后者是一种阳离子脂质体，其摩尔比为1:2:2或1:1:1，为二甲基二十八烷基溴化铵（DDAB）：二酰磷脂酰胆碱（DLPC）：二酰磷脂酰乙醇胺（DOPE）。我们还发现，通过与单克隆抗体或asialal糖蛋白偶联，能够使这些脂质体具有细胞或组织特异性。相比之下，我们还发现腺相关病毒（AAV）载体相关脂质体比含有质粒DNA的脂质体更有效地将体外基因转移到人胶质瘤细胞。与含有质粒DNA的脂质体相比，使用载体相关脂质体可将转导效率提高10倍以上，与单独使用AAV相比可提高6倍以上。从这些结果来看，AAV载体相关脂质体似乎是恶性肿瘤体内基因传递的良好候选者。此外，我们还研究了含热休克蛋白启动子驱动质粒的阳离子脂质体与小铁球磁铁矿结合的有效性，并诱导了显著的抗肿瘤作用。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设施，并确定该设施可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.