In situ genetic correction of F8 intron 22 inversion in hemophilia A patient-specific iPSCs.
In situ genetic correction of F8 intron 22 inversion in hemophilia A patient-specific iPSCs.
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A 型血友病患者特异性 iPSC 中 F8 内含子 22 倒位的原位遗传校正
DOI：
10.1038/srep18865
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发表时间：
2016-01-08
影响因子：
4.6
通讯作者：
Liang D
中科院分区：
文献类型：
--
作者：
Wu Y;Hu Z;Li Z;Pang J;Feng M;Hu X;Wang X;Lin-Peng S;Liu B;Chen F;Wu L;Liang D
近一半的严重血友病A（HA）病例是由F8内含子22倒位（Inv 22）引起的。这个0.6-Mb倒位将186-kb F8分裂成具有相反转录方向的两个部分。反向5′部分（141 kb）保留了由内在F8启动子驱动的前22个外显子，由于缺少外显子23-26的最后627 bp编码序列，导致F8转录物截短。在这里，我们描述了在患者特异性诱导多能干细胞（iPSC）中Inv 22的原位遗传校正。通过使用TALENs，627 bp序列加上polyA信号通过同源重组（HR）精确靶向外显子22和内含子22的连接处，具有62.5%和52.9%的高靶向效率。在使用Cre-LoxP系统去除药物选择盒后，基因校正的iPSC保留了正常的核型。重要的是，F8转录和FVIII分泌在HA基因治疗的候选细胞类型中得到拯救，包括内皮细胞（EC）和源自基因校正的iPSC的间充质干细胞（MSC）。这是第一个报告的一个有效的原位遗传校正的大倒位突变，使用的策略，靶向基因添加。
Nearly half of severe Hemophilia A (HA) cases are caused by F8 intron 22 inversion (Inv22). This 0.6-Mb inversion splits the 186-kb F8 into two parts with opposite transcription directions. The inverted 5′ part (141 kb) preserves the first 22 exons that are driven by the intrinsic F8 promoter, leading to a truncated F8 transcript due to the lack of the last 627 bp coding sequence of exons 23–26. Here we describe an in situ genetic correction of Inv22 in patient-specific induced pluripotent stem cells (iPSCs). By using TALENs, the 627 bp sequence plus a polyA signal was precisely targeted at the junction of exon 22 and intron 22 via homologous recombination (HR) with high targeting efficiencies of 62.5% and 52.9%. The gene-corrected iPSCs retained a normal karyotype following removal of drug selection cassette using a Cre-LoxP system. Importantly, both F8 transcription and FVIII secretion were rescued in the candidate cell types for HA gene therapy including endothelial cells (ECs) and mesenchymal stem cells (MSCs) derived from the gene-corrected iPSCs. This is the first report of an efficient in situ genetic correction of the large inversion mutation using a strategy of targeted gene addition.