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

用于人类基因治疗的病毒和非病毒载体的开发和进化。

• 批准号: 07557169
• 负责人: MATSUDA Ichiro
• 金额: $ 11.14万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557169/
• 关键词: gene therapy; vector liposome; virus vector; OTC deficiency; Wilson disease; ウイルソン病; ベクター; リポソームベクター

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.

1）非病毒基因转移脂质体的问题通常过于低效率，无法用作载体。我们开发了几个新的脂质体载体，这些载体在多层囊泡表面（TRX系列＃9,13,20）上进行了修饰，以获得较高的转染效率。为了评估TRX的效率，可在许多实验室中可用且受欢迎的TRX的效率被用作参考。在COS1，HEPG2（HepG2），以及来自LEC大鼠（Wilson病模型的动物模型）的培养细胞中测试了三种不同的TRX脂质体，这些ββ半乳糖苷酶，人OTC基因或Wilson疾病（WDEU）负责的Wilson病（WDEU）的负责人。 TRX载体（＃9,13和20）的转染效率均比Lipofectin和transfectin的转染效率高出5-10倍，而Farthermore则在TRX载体中有20％的血清，但在LipoFofectin中降低了Espession。当设计体外研究时，这是TRX载体的一个优点。携带人OTC基因和Wilson Gene的TRX载体分别通过尾静脉注射到OTC缺陷小鼠和LEC大鼠中。转基因的表达仅在库普弗细胞中发现，但在肝细胞中却没有。但是，当直接注射肝组织中的那些人时，即使在肝细胞中也观察到了一些表达。因此，需要对TRX表面表面进行更多修改，如果实现了TRX体外研究的可用性。2）2）病毒载体载体病毒矢量ADEX CAG HOTC，将人类OTC基因置于CAG启动子下的OTC启动子下，将其注入OTC不足小鼠，并在60天后将OTC活动的正常化为正常化。 3）尽管媒介的发展不足以申请人类治疗，但我们对基因转染的问题有了更多了解，基于该问题，应该继续进行进一步的研究。

项目成果

Hoshide,R.: "Prenatal monitoring in a family at high risk for ornithine transcarbamylase (OTC) deficiency ; a new mutation of an A-to-C transversion in position +4 of intron 1 of the OTC gene that is likely to abolish enzyme activity." Am J Med Genet. 64.

Hoshide,R.：“对鸟氨酸转氨甲酰酶 (OTC) 缺乏症高风险家庭进行产前监测；OTC 基因内含子 1 的位置 4 出现 A 到 C 转换的新突变，可能会消除酶活性

Shimadzu, M., et al.: "Ten novel mutations of ornithine transcarbamylase (OTC) deficiency" Human Mutation. (in press). (1997)

Shimadzu, M., et al.：“鸟氨酸转氨甲酰酶 (OTC) 缺乏症的十种新突变”人类突变。

Orimo H., et al.: "First trimester prenatal molecular diagnosis of infantile hypophosphatasia in a Japanese family" Prenatal Diag.16. 559-563 (1996)

Orimo H. 等人：“日本家庭中婴儿期低磷酸酯酶症的妊娠早期产前分子诊断”Prenatal Diag.16。

Palombo F.: "hMutsβ,a heterodimer of hMSH2 and hMSH3,binds to insertion/deletion loops in DNA." Current Biol.6. 1181-1184 (1996)

Palombo F.：“hMutsβ，hMSH2 和 hMSH3 的异二聚体，与 DNA 中的插入/删除环结合。”1181-1184 (1996)。

Uchino, T., et al.: "Three brothers with progressive hepatoc dysfucntion and severe hepatic steatosis due to a patent ductus venosus" Gastroenterology. 110. 1964-1966 (1996)

Uchino, T. 等人：“三兄弟因静脉导管未闭而患有进行性肝功能障碍和严重肝脂肪变性”胃肠病学。