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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.

这项研究的目的是开发针对单点突变引起的遗传性疾病的基因疗法。着色性干皮病组 (A) (XPA) 的突变已得到很好的表征。使用 XPA 作为模型，我们尝试将基因插入染色体的特定位点。根据Kmiec，E.描述的使用DNA/RNA嵌合寡核苷酸对点突变的校正，我们构建了包含人类XPA基因的内含子3和外显子4边界的正常序列的68聚体嵌合DNA/RNA寡核苷酸，因为从G到C的转变发生在XP2OSSV（XPA）细胞中。使用 HVJ 脂质体将嵌合寡核苷酸转移到 XP2OSSV (XPA) 细胞中，HVJ 脂质体已被评估为将寡核苷酸引入细胞的最有效载体。转移后，对细胞进行紫外线照射。然而，没有出现集落。然后，我们尝试通过无需紫外线照射的PCR来检测XP2OSSV（XPA）细胞中点突变的校正。 200000个细胞中没有一个显示出突变的纠正。我们得出的结论是，嵌合寡核苷酸对于点突变的校正并不像以前的报道那样有效。接下来，我们尝试开发增加转基因插入宿主染色体的方法。其中一项策略是使用源自鱼类的转座子/转座酶系统。使用该系统，在培养的 HeLa 细胞中获得新抗性稳定转化体的效率比没有转座酶的细胞高约 30 倍。然后，我们使用HVJ-脂质体将新抗性基因与转座子/转座酶一起转移至小鼠肝脏。通过PCR在肝脏中检测到新抗性基因超过6周，而在没有转座酶的情况下，新抗性基因在2周内消失。该系统应该针对基因插入组织细胞进行更广泛的评估，但它似乎对于长期基因表达和突变基因的替换很有希望。