Highly efficient endogenous human gene correction using designed zinc-finger nucleases
Highly efficient endogenous human gene correction using designed zinc-finger nucleases
复制标题
DOI：
10.1038/nature03556
复制
发表时间：
2005-06-02
期刊：
影响因子：
64.8
通讯作者：
Holmes, MC
中科院分区：
文献类型：
--
作者：
Urnov, FD;Miller, JC;Holmes, MC
由于人类细胞中同源重组的低频率，体内的人类基因组的永久性修饰是不切实际的，这一事实使生物医学研究并朝着安全有效的基因治疗方向发展。在这里，我们报告了一种使用两个基本生物学过程的通用解决方案：C2H2锌指蛋白识别DNA以及DNA双链断裂的同源性修复。设计用于识别独特的染色体位点的锌指蛋白可以融合到核酸酶结构域，并且由此产生的锌指核酸酶引起的双链断裂可以通过刺激染色体和外鼻外DNA供体之间的同源重组来创造特定的序列改变。我们表明，IL2R伽马基因中针对X连锁严重的合并免疫缺陷（SCID）突变而设计的锌指核酸酶产生了18％以上基因改性的人类细胞而无需选择。值得注意的是，大约7％的细胞在两个X染色体上获得了所需的遗传修饰，细胞基因型在Messenger RNA和蛋白质水平上精确反射。我们观察到人类T细胞中的频率相当高，从而提高了基于锌指核酸酶治疗疾病的策略的可能性。
Permanent modification of the human genome in vivo is impractical owing to the low frequency of homologous recombination in human cells, a fact that hampers biomedical research and progress towards safe and effective gene therapy. Here we report a general solution using two fundamental biological processes: DNA recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA double-strand breaks. Zinc-finger proteins engineered to recognize a unique chromosomal site can be fused to a nuclease domain, and a double-strand break induced by the resulting zinc-finger nuclease can create specific sequence alterations by stimulating homologous recombination between the chromosome and an extrachromosomal DNA donor. We show that zinc-finger nucleases designed against an X-linked severe combined immune deficiency ( SCID) mutation in the IL2R gamma gene yielded more than 18% gene-modified human cells without selection. Remarkably, about 7% of the cells acquired the desired genetic modification on both X chromosomes, with cell genotype accurately reflected at the messenger RNA and protein levels. We observe comparably high frequencies in human T cells, raising the possibility of strategies based on zinc-finger nucleases for the treatment of disease.