Le Thi Thanh Huong, Tran Ngoc Tung, Dao Thi Mai Lan, Nguyen Dinh Dung, Do Huy Duong, Ha Thi Lien, Kühn Ralf, Nguyen Thanh Liem, Rajewsky Klaus, Chu Van Trung
Department of Gene Technology, Vinmec Research Institute of Stem Cell and Gene Technology, Hanoi, Vietnam.
Immune Regulation and Cancer, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.
Front Genet. 2019 Jul 2;10:625. doi: 10.3389/fgene.2019.00625. eCollection 2019.
Patients with Rett syndrome (RTT) have severe mental and physical disabilities. The majority of RTT patients carry a heterozygous mutation in methyl-CpG binding protein 2 (MECP2), an X-linked gene encoding an epigenetic factor crucial for normal nerve cell function. No curative therapy for RTT syndrome exists, and cellular mechanisms are incompletely understood. Here, we developed a CRISPR/Cas9-mediated system that targets and corrects the disease relevant regions of the MECP2 exon 4 coding sequence. We achieved homologous recombination (HR) efficiencies of 20% to 30% in human cell lines and iPSCs. Furthermore, we successfully introduced a MECP2 mutation into the MECP2 gene in human induced pluripotent stem cells (iPSCs). Consequently, using CRISPR/Cas9, we were able to repair such mutations with high efficiency in human mutant iPSCs. In summary, we provide a new strategy for MECP2 gene targeting that can be potentially translated into gene therapy or for iPSCs-based disease modeling of RTT syndrome.
雷特综合征（RTT）患者存在严重的智力和身体残疾。大多数RTT患者在甲基化CpG结合蛋白2（MECP2）中携带杂合突变，MECP2是一个X连锁基因，编码对正常神经细胞功能至关重要的表观遗传因子。目前尚无针对RTT综合征的治愈性疗法，其细胞机制也尚未完全明确。在此，我们开发了一种CRISPR/Cas9介导的系统，该系统靶向并校正MECP2外显子4编码序列的疾病相关区域。我们在人类细胞系和诱导多能干细胞（iPSC）中实现了同源重组（HR）效率达到20%至30%。此外，我们成功地在人类诱导多能干细胞（iPSC）的MECP2基因中引入了MECP2突变。因此，利用CRISPR/Cas9，我们能够在人类突变iPSC中高效修复此类突变。总之，我们提供了一种靶向MECP2基因的新策略，该策略有可能转化为基因治疗或用于基于iPSC的RTT综合征疾病建模。
