Genome Therapy of Myotonic Dystrophy Type 1 iPS Cells for Development of Autologous Stem Cell Therapy
Genome Therapy of Myotonic Dystrophy Type 1 iPS Cells for Development of Autologous Stem Cell Therapy
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DOI：
10.1038/mt.2016.97
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发表时间：
2016-08-01
影响因子：
12.4
通讯作者：
Xia, Guangbin
中科院分区：
文献类型：
--
作者：
Gao, Yuanzheng;Guo, Xiuming;Xia, Guangbin
肌强直性营养不良1型（DM1）是由肌强直性营养不良蛋白激酶（DMPK）基因3'-非翻译区（3' UTR）细胞嘧啶-胸腺嘧啶-鸟嘌呤（CTG）重复扩增引起的，目前尚无有效的治疗方法。本研究的目的是在人DM1诱导的多能干细胞（iPS）细胞中开发基因组疗法，以消除突变转录本并逆转表型，从而开发自体干细胞疗法。一般的方法包括在DMPK CTG重复序列的上游靶向插入polyA信号（PASs），这将导致转录的过早终止和毒性突变转录物的消除。通过位点特异性转录激活因子样效应核酸酶（TALEN）诱导的双链断裂引发的同源重组介导了PASs的插入。我们发现基因组处理的DM1 iPS细胞继续保持多能性。在线性分化的神经干细胞、心肌细胞和畸胎瘤组织中，PASs的插入导致突变转录物的消除和核RNA病灶的完全消失，以及异常剪接的逆转。综上所述，通过在扩增的DMPK CTG重复序列上游插入PASs进行基因组治疗，可以防止DM1 iPS细胞及其后代中毒性突变转录本的产生和表型逆转。这些经过基因处理的iPS细胞将在开发自体干细胞治疗DM1方面具有广泛的临床应用。
Myotonic dystrophy type 1 (DM1) is caused by expanded Cytosine-Thymine-Guanine (CTG) repeats in the 3'-untranslated region (3' UTR) of the Dystrophia myotonica protein kinase (DMPK) gene, for which there is no effective therapy. The objective of this study is to develop genome therapy in human DM1 induced pluripotent stem (iPS) cells to eliminate mutant transcripts and reverse the phenotypes for developing autologous stem cell therapy. The general approach involves targeted insertion of polyA signals (PASs) upstream of DMPK CTG repeats, which will lead to premature termination of transcription and elimination of toxic mutant transcripts. Insertion of PASs was mediated by homologous recombination triggered by site-specific transcription activator-like effector nuclease (TALEN)-induced double-strand break. We found genome-treated DM1 iPS cells continue to maintain pluripotency. The insertion of PASs led to elimination of mutant transcripts and complete disappearance of nuclear RNA foci and reversal of aberrant splicing in linear-differentiated neural stem cells, cardiomyocytes, and teratoma tissues. In conclusion, genome therapy by insertion of PASs upstream of the expanded DMPK CTG repeats prevented the production of toxic mutant transcripts and reversal of phenotypes in DM1 iPS cells and their progeny. These genetically-treated iPS cells will have broad clinical application in developing autologous stem cell therapy for DM1.