Development of new DDS to individual organs by means of cell technology and its opplication to treatment of human diseases

我们开发了一种新的基因传递载体系统，称为hvj脂质体。在该传递系统中，DNA被包裹在带负电荷的脂质体中，脂质体与紫外线灭活的仙台病毒HVJ融合形成HVJ脂质体。目前，hvj脂质体作为一种高效的非病毒载体被广泛应用于将DNA、寡核苷酸和蛋白质引入各种动物器官。利用该载体系统，我们成功地预防球囊损伤大鼠动脉再狭窄，抑制肾小球硬化，保护移植器官，抑制播散性脑肿瘤。然而，在未来的基因治疗中，该系统还需要进一步改进，以获得更有效的体外和体内传递系统。采用旋流挤压法制备含有阳离子dc -胆固醇（DC-Chol）的hvj -阳离子脂质体后，fitc标记的反义寡核苷酸或核酶的递送量大大增加，荧光素酶基因的表达量也比含有磷脂酰丝氨酸（PS）的hvj -阳离子脂质体提高了约70倍。hvj - dc -胆脂质体对小鼠脑播散性胶质母细胞瘤的自杀性基因治疗非常有用。我们进一步优化了脂质体的脂质成分，开发了一种由鞘磷脂（Sph）、二油基磷脂酰乙醇胺（DOPE）、磷脂酰胆碱（PC）、胆固醇（Chol）和DC-Chol（摩尔比为1.7:1.7:1.7:4.0:1.0）组成的新型hvj脂质体，作为培养细胞中最有效的基因表达载体（比传统hvj脂质体高100-800倍）。然而，与传统的hvj脂质体相比，阳离子脂质体的使用反而降低了动物器官中的基因表达。然后，含有Sph、DOPE、PC、PS和Chol（摩尔比为1.3:1.3:1.0:5.0）模拟HIV包膜的hvj -脂质体（称为HVJ-AVE脂质体）使肝脏或肌肉中的基因表达比常规hvj -脂质体高5-10倍。因此，阳离子脂质体似乎在体外和体内基因表达中具有相互作用，我们的发现表明脂质体在体外和体内基因转移中的替代使用。少

We developed a novel gene delivery vector system called HVJ-liposome. In this delivery system, DNA was entrapped into negative-charge liposome and the liposome was fused with UV-inactivated HVJ,Sendai virus, to form HVJ-liposome. HVJ-liposome is now widely used as an efficient non-viral vector for introducing DNA,oligonucleotides and proteins into various animal organs. Using this vector system, we succeeded in prevention of restenosis of balloon injured areteries of rats, suppression of glomerulosclerosis, protection of transplanted organs, and inhibition of disseminated brain tumors. However, the system should be improved for more efficient delivery system both in vitro and in vivo toward future gene therapy.When HVJ-cationic liposome having cationic DC-cholesterol (DC-Chol) was prepared by vortexing and extrusion, the delivery of FITC-labeled antisense oligonucleotides or ribozymes was greatly enhanced, and luciferase gene expression was also increased about 70 times more than conve … More ntional HVJ-liposome containing phosphatidylserine (PS). The HVJ-DC-Chol-liposome was very useful for suicide gene therapy of disseminated glioblastoma in mouse brain. We further optimized lipid components of liposome and developed a novel HVJ-liposome consisting of sphingomyelin (Sph), dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine (PC), cholesterol (Chol) and DC-Chol (1.7 : 1.7 : 1.7 : 4.0 : 1.0 in molar ratio) as the most efficient vector for gene expression in culture cells (100-800 times higher than conventional HVJ-liposome). However, the use of cationic lipids rather reduced gene expression in animal organs, compared with conventional HVJ-liposome. Then, HVJ-liposome containing Sph, DOPE,PC,PS and Chol (1.3 : 1.3 : 1.0 : 5.0 in molar ratio) mimicking HIV envelope (called HVJ-AVE liposome) raised gene expression in either liver or muscle 5-10 times more than conventional HVJ-liposome. Thus, cationic lipids appeared to have reciprocal effects in gene expression in vitro and in vivo, and our finding indicates the alternative usage of liposomes for in vitro and in vivo gene transfer. Less