Development and evoluation of viral and non-viral vectors for human gene therapy.

1)非病毒基因转移脂质体作为载体的效率普遍较低。为了获得更高的转基因转染效率，我们开发了几种新的脂质体载体，并将其修饰在多层囊泡（TRX系列#9,13,20）表面。为了评价TRX的效率，我们以Lipofectin和Transfectin作为参考，这两种药物在很多实验室都很流行。在培养细胞Cos1、HepG2和建立的LEC大鼠肝细胞（威尔逊病动物模型）中，对CAG启动子下含有-半乳糖苷酶、人OTC基因或威尔逊病负责基因（Wdeu）的三种不同TRX脂质体进行了检测。TRX载体（9号、13号和20号）的转染效率均比Lipofectin和transferectin高5-10倍，且在20%的血清存在下，TRX载体的表达增加，而Lipofectin的表达降低。这是TRX载体在设计体外研究时的一个优势。将携带人OTC基因和Wilson基因的TRX载体分别经尾静脉注射到OTC缺失小鼠和LEC大鼠体内。该基因仅在库普弗细胞中表达，而在肝细胞中不表达。而直接注射肝组织时，肝细胞中也有表达。因此，需要对TRX的表面进行更多的修饰，如果能够实现，TRX在体外的可用性研究将会有很大的进展。2)病毒载体将CAG启动子下携带人OTC基因的腺病毒载体Adex CAG hOTC注射到OTC缺失小鼠体内，注射后60 d OTC活性明显恢复正常。目前正在研究携带相同基因的AAV载体。3)虽然载体的发展还不足以应用于人类治疗，但我们对基因转染的问题有了更多的了解，在此基础上还需要进一步的研究。

1) non-viral gene transferThe issue of liposome is generally of too low efficiency to be used as vector. We developed several new liposome vectors, which were modified on surface of multilameral vesicle (TRX series #9,13,20) in order to obtain higher transfection efficiency of transgenes. For evaluating efficiency of the TRX,Lipofectin and Transfectin, which are commercialty avalable and popular in many laboratories, were used as references. Three different TRX liposomes, which havor beta galactosidase, human OTC gene or the responsible gene for Wilson disease (Wdeu) under CAG promotor, were tested in cultured cells including Cos1, HepG2, and established liver cells from LEC rat (animal model of Wilson disease). Transfection efficiency of TRX vectors (#9,13 and 20) was all 5-10 times higher than those of Lipofectin and Transfectin, and farthermore, the espession was increased in the presence of 20% of serum in TRX vectors, but was reduced in Lipofectin. This is an advantage of TRX vector when in vitro study is designed.TRX vector harboring human OTC gene and Wilson gene was injected into OTC deficient mice and LEC rat through tail vein, respectively. Expression of the transgene was found only in kupffer cells but not in hepatocyte. However, when injected those in liver tissue directly, some expression was observed even in hepatocyte. Thus, some more modification of surface of TRX are necessary, and, if achieved, availability of TRX in vitro study will be much progressed.2) Virus vectorAdenovirus vector Adex CAG hOTC,which harbor human OTC gene under CAG promoter was injected into OTC deficient mice, and normalization of OTC activity was evident for 60 days after the injection. AAV vector harboring the same gene is now studying.3) Though the development of vector is not enough to apply for human therapy, we learned more about the issue for transfection of gene, based on which further study should be continued.