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

本研究的目的是发展基因治疗由一点突变引起的遗传性疾病。着色性干皮病组（A）（XPA）的突变已被很好地表征。以XPA为模型，我们尝试将基因插入到染色体的特定位点。根据Kmiec，E.，我们构建了含有人XPA基因内含子3和外显子4边界的正常序列的68 mer嵌合DNA/RNA寡核苷酸，因为在XP2OSSV（XPA）细胞中发生了从G到C的转换。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中的最有效载体。转移后，对细胞进行UV照射。但没有殖民地出现。然后，我们尝试在没有紫外线照射的情况下通过PCR检测XP2OSSV（XPA）细胞中点突变的校正。20万个细胞中没有一个显示出突变的纠正。我们的结论是，嵌合寡核苷酸是不那么有效的纠正点突变，因为在以前的报告。接下来，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细胞中获得的新抗性稳定转化体比没有转座酶的转化体效率高约30倍。然后，我们用HVJ-脂质体将带有转座子/转座酶的新耐药基因转移到小鼠肝脏。转座酶作用6周后，PCR检测到新耐药基因在肝脏中的表达，而不转座酶作用2周后，新耐药基因消失。该系统应更广泛地评估基因插入组织细胞，但它似乎是有前途的长期基因表达和突变基因的替代。

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.