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由一分点突变引起的遗传疾病的基因疗法。心胚层色素的突变（a）（XPA）已被很好地化。我们使用XPA作为模型，试图将基因插入染色体的特定位点。根据Kmiec，E。所描述的DNA/RNA嵌合寡核苷酸的校正点突变，我们构建了68个MER嵌合DNA/RNA寡核苷酸，该核苷酸含有内含子3和外显子4和外显子4的XPA基因边界的正常序列从G到C的转化发生在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，这些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，将细胞进行紫外线辐照。但是，没有出现殖民地。然后，我们尝试通过PCR检测XP2OSSV（XPA）细胞中点突变的校正，而无需紫外线辐射。 200000个细胞均未显示突变的校正。我们得出的结论是，嵌合寡核苷酸对于校正点突变的有效性不如先前的报告。接下来，我们尝试开发将转基因插入宿主染色体中的方法。其中一种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细胞中获得了大约30倍的新耐药稳定转化体，而没有转座酶。然后，我们使用HVJ-脂质体将新耐药基因与转座子/转座酶转移到小鼠肝脏中。 PCR在肝脏中检测到新耐药基因超过6周，而在没有转座酶的情况下，它在2周内消失了。该系统应更广泛地评估基因插入组织细胞，但对于长期基因表达和替代突变基因似乎是有希望的。