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首页> 外文期刊>BioResearch open access. >Correction of the ΔF508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Gene by Zinc-Finger Nuclease Homology-Directed Repair
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Correction of the ΔF508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Gene by Zinc-Finger Nuclease Homology-Directed Repair

机译：通过锌指核酸酶同源性指导修复对囊性纤维化跨膜电导调节基因中ΔF508突变的校正

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摘要

The use of zinc-finger nucleases (ZFNs) to permanently and precisely modify the human genome offers a potential alternative to cDNA-based gene therapy. The ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is observed in ～70% of patients with cystic fibrosis (CF) and is a candidate for ZFN-mediated repair. Here, we report the modular design and synthesis of a pair of ZFNs that can create a double-stranded break (DSB) 203?bp upstream of the ΔF508 lesion, resulting in a nonhomologous end-joining (NHEJ) frequency of 7.8%. In spite of this relatively long distance between the DSB and the ΔF508 mutation, homology-directed repair (HDR) could be detected when using a DNA donor containing part of the wild-type (WT) CFTR . The ZFN target half-sites in CFTR are separated by a 4-bp spacer, but efficient cleavage of synthetic targets with either a 4- or 6-bp spacer was observed in vitro . These ZFNs may be suitable for a genome-editing strategy using a partial cDNA sequence-containing exons 10–24 of CFTR to restore CFTR function to cells containing not only the ΔF508 mutation but also potentially any mutation in or downstream of exon 10.
机译：使用锌指核酸酶（ZFN）永久且精确地修饰人类基因组为基于cDNA的基因治疗提供了潜在的替代方法。在约70％的囊性纤维化（CF）患者中观察到囊性纤维化跨膜电导调节剂（CFTR）基因中的ΔF508突变，它是ZFN介导的修复的候选者。在这里，我们报告了一对ZFN的模块化设计和合成，这些ZFN可以在ΔF508损伤上游产生一个203 bp的双链断裂（DSB），导致非同源末端连接（NHEJ）频率为7.8％。尽管DSB和ΔF508突变之间的距离相对较长，但当使用包含部分野生型（WT）CFTR的DNA供体时，仍可以检测到同源性定向修复（HDR）。 CFTR中ZFN靶的半位点被一个4 bp的间隔子隔开，但是在体外观察到了用4或6 bp的间隔子对合成靶的有效切割。这些ZFN可能适用于基因组编辑策略，即使用含有部分cDNA序列的CFTR外显子10-24恢复CFTR功能到不仅包含ΔF508突变而且还可能包含外显子10或下游任何突变的细胞。

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