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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells
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Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells

机译：Ku70是必需基因，可调节人体细胞中rAAV介导的基因靶向频率

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摘要

Gene targeting has two important applications. One is the inacti-vation of genes ("knockouts"), and the second is the correction of a mutated allele back to wild-type ("gene therapy"). Central to these processes is the efficient introduction of the targeting DNA into the cells of interest. In humans, this targeting is often accomplished through the use of recombinant adeno-associated virus (rAAV). rAAV is presumed to use a pathway of DNA double-strand break (DSB) repair termed homologous recombination (HR) to mediate correct targeting; however, the specifics of this mechanism remain unknown. In this work, we attempted to generate Ku70-null human somatic cells by using a rAAV-based gene knockout strategy. Ku70 is the heterodimeric partner of Ku86, and together they constitute an end-binding activity that is required for a pathway [nonhomologous end joining (NHEJ)] of DSB repair that is believed to compete with HR. Our data demonstrated that Ku70 is an essential gene in human somatic cells. More importantly, however, in Ku70~(+/-) cells, the frequency of gene targeting was 5-to 10-fold higher than in wild-type cells. RNA interference and short-hairpinned RNA strategies to deplete Ku70 phenocopied these results in wild-type cells and greatly accentuated them in Ku70~(+/-) cell lines. Thus, Ku70 protein levels significantly influenced the frequency of rAAV-mediated gene targeting in human somatic cells. Our data suggest that gene-targeting frequencies can be significantly improved in human cells by impairing the NHEJ pathway, and we propose that Ku70 depletion can be used to facilitate both knockout and gene therapy approaches.
机译：基因靶向有两个重要的应用。第一个是基因的失活（“敲除”），第二个是将突变的等位基因校正回野生型（“基因疗法”）。这些过程的核心是将靶向DNA有效引入目标细胞。在人类中，这种靶向作用通常是通过使用重组腺相关病毒（rAAV）来实现的。据推测，rAAV使用称为同源重组（HR）的DNA双链断裂（DSB）修复途径来介导正确的靶向；但是，该机制的具体细节仍然未知。在这项工作中，我们试图通过使用基于rAAV的基因敲除策略来生成Ku70无人类体细胞。 Ku70是Ku86的异二聚体伴侣，它们共同构成了DSB修复途径（非同源末端连接（NHEJ））与HR竞争所必需的末端结合活性。我们的数据表明，Ku70是人类体细胞中必不可少的基因。然而，更重要的是，在Ku70〜（+/-）细胞中，基因靶向的频率比野生型细胞高5至10倍。 RNA干扰和短发的RNA策略消耗了Ku70表型，这些结果在野生型细胞中显着增强，并在Ku70〜（+/-）细胞系中大大增强。因此，Ku70蛋白水平显着影响人体细胞中rAAV介导的基因靶向的频率。我们的数据表明，通过破坏NHEJ途径，可以显着提高人类细胞中的基因靶向频率，并且我们建议使用Ku70耗竭法可以促进基因敲除和基因治疗方法的发展。

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