Gene editing of DNAH11 restores normal cilia motility in primary ciliary dyskinesia
Gene editing of DNAH11 restores normal cilia motility in primary ciliary dyskinesia
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DOI：
10.1136/jmedgenet-2015-103539
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发表时间：
2016-04-01
影响因子：
4
通讯作者：
Pistello, Mauro
中科院分区：
文献类型：
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作者：
Lai, Michele;Pifferi, Massimo;Pistello, Mauro
研究背景原发性纤毛运动障碍（PCD）是一种罕见的常染色体隐性遗传病，以纤毛运动功能障碍为特征。纤毛运动障碍导致粘膜纤毛清除不良，并导致肺功能受损和严重的呼吸道感染。PCD无特异性治疗。为了永久恢复基因功能并使纤毛运动正常化，我们使用基因编辑在缺陷细胞中用野生型序列替换突变的基因。方法靶基因是纤毛结构的重要组成部分动力蛋白重链11（DNAH11）。从两名具有DNAH11无义突变和改变的纤毛搏动和模式的PCD患者收集气道纤毛细胞。结果在含有DNAH11靶位点的上皮细胞系中，TALENs切割超过80%的突变DNAH11序列，并在约50%的细胞中用野生型序列取代突变序列。在PCD患者的气道纤毛细胞中，分别有33%和29%的细胞发生了纤毛跳动和模式的位点特异性重组和正常化，结论这项研究表明基因编辑可以在体外拯救纤毛跳动，为治疗PCD开辟了新的途径。
Background Primary ciliary dyskinesia (PCD) is a rare autosomal recessive genetic disorder characterised by dysfunction of motile cilia. Ciliary dysmotility causes poor mucociliary clearance and leads to impairment of pulmonary function and severe respiratory infections. PCD has no specific therapy. With the aim to permanently restore gene function and normalise ciliary motility, we used gene editing to replace mutated with wild-type sequence in defective cells.Methods The target gene was dynein heavy chain 11 (DNAH11), an essential component of ciliary structure. Airway ciliated cells were collected from two patients with PCD with DNAH11 nonsense mutations and altered ciliary beating and pattern. Repair of the genetic defect was performed ex vivo by site-specific recombination using transcription activator-like effector nucleases (TALENs).Results In an epithelial cell line engineered to contain the DNAH11 target site, TALENs cleaved over 80% of the mutated DNAH11 sequence and replaced the mutated sequence with wild-type sequence in about 50% of cells. In airway ciliated cells of patients with PCD, site-specific recombination and normalisation of ciliary beating and pattern occurred in 33% and 29% of cells, respectively.Conclusion This study demonstrates that gene editing can rescue ciliary beating ex vivo, opening up new avenues for treating PCD.