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Basic study of correction of mutated gene in xeroderma pigmentosum group A

着色性干皮病A组突变基因纠正的基础研究

基本信息

批准号：
10470505
负责人：
KANEDA Yasufumi
金额：
$ 8.26万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

The objective of this study is the development of gene therapeutics for genetic disorders caused by one-point mutation. Mutations of xeroderma pigmentosum group (A) (XPA) have been well chracterized. Using XPA as a model, we attempted to insert genes to specific sites of chromosomes. According to the correction of a point mutation using DNA/RNA chimeric oligonucleotides described by Kmiec, E., we constructed 68 mer chimeric DNA/RNA oligonucleotides containing normal sequence of the boundary of the intron 3 and exon 4 of the human XPA gene because the conversion from G to C occurs in XP2OSSV (XPA) cells. The chimeric oligonucleotides were transferred to XP2OSSV (XPA) cells using HVJ-liposomes which have been evalutated as the most efficient vehicle for introducing oligonucleotides into cells. After transfer, cells were subjected to UV-irradiation. However, no colonies appeared. Then, we tried to detect the correction of the point mutation in XP2OSSV (XPA) cells by PCR without UV-irradiation. None of 200000 cells showed the correction of the mutation. We concluded that the chimeric oligonucleotides were not so effective for the correction of point mutation as in the previous reports. Next, we attempted to develop the methods for increasing the insertion of a transgene into host chromosomes. One of the strategies was the use of transposon/transposase system derived from fish. Using this system, neo-resistant stable transformants were obtained approximately 30 fold more efficiently in cultured HeLa cells than that without the transposase. Then, we transferred neo-resistant gene with the transposon/transposase to mouse liver using HVJ-liposomes. Neo-resistant gene was detected in the liver by PCR for more than 6 weeks whereas it disappeared in 2 weeks without the transposase. This system should be more extensively evaluated for gene insertion into tissue cells, but it seems to be promising for long-term gene expression and the replacement of mutant genes.

这项研究的目的是发展基因疗法来治疗由一点突变引起的遗传疾病。着色性干皮病(A)群(XPA)的突变已被很好地描述。以XPA为模型，我们尝试将基因插入到染色体的特定位置。根据Kmiec，E.所描述的DNA/RNA嵌合寡核苷酸对点突变的纠正作用，我们构建了68个含有人类XPA基因内含子3和外显子4边界正常序列的DNA/RNA嵌合寡核苷酸，因为XP2OSSV(XPA)细胞存在G到C的转换。利用HVJ脂质体将嵌合寡核苷酸转移到XP2OSSV(XPA)细胞中，该脂质体被认为是将寡核苷酸导入细胞的最有效载体。转移后的细胞接受紫外线照射。然而，没有出现殖民地。然后，我们尝试在没有紫外线照射的情况下，用聚合酶链式反应检测XP2OSSV(XPA)细胞中点突变的校正情况。在200000个细胞中，没有一个细胞表现出突变的纠正。我们的结论是，嵌合寡核苷酸在纠正点突变方面并不像以前的报道那样有效。下一步，我们试图开发增加转基因插入宿主染色体的方法。其中一个策略是使用来自FISH的转座子/转座酶系统。使用该系统，在培养的HeLa细胞中获得了抗NEO的稳定转化子，效率大约是没有转座酶的30倍。然后，利用hvj-脂质体将转座子/转座酶介导的neo抗性基因转入小鼠肝脏。在6周以上的肝脏中检测到neo抗性基因，而在没有转座酶的情况下，该基因在2周内消失。这个系统在基因插入组织细胞方面应该得到更广泛的评估，但它似乎在基因长期表达和突变基因的替换方面很有前途。