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Abstract：
X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene, resulting in absent or defective gp91phox protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation, we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients, which restored gp91phox expression and ROS production in iPSC-derived granulocytes. Alternate approaches for correcting the majority of X-CGD mutations were assessed, involving TALEN- or Cas9-mediated insertion of CYBB minigenes at exon 1 or 2 of the CYBB locus. Targeted insertion of an exon 1-13 minigene into CYBB exon 1 resulted in no detectable gp91phox expression or ROS activity in iPSC-derived granulocytes. In contrast, targeted insertion of an exon 2-13 minigene into exon 2 restored both gp91phox and ROS activity. This demonstrates the efficacy of two correction strategies: seamless repair of specific CYBB mutations by exon replacement or targeted insertion of an exon 2-13 minigene to CYBB exon 2 while retaining exon/intron 1. Furthermore, it highlights a key issue for targeted insertion strategies for expression from an endogenous promoter: retention of intronic elements can be necessary for expression.
摘要：
X连锁慢性肉芽肿病（X-CGD）是一种免疫缺陷，由吞噬细胞产生杀菌活性氧（ROS）的缺陷引起。引起突变发生在整个CYBB基因，导致gp91phox蛋白表达缺失或缺陷。为了纠正CYBB外显子5突变，同时保持正常的基因调控，我们利用TALEN或Cas9在患者的诱导多能干细胞（iPSCs）中进行外显子5替换，在iPSC衍生的粒细胞中恢复gp91phox表达和ROS产生。评估了纠正大多数X-CGD突变的替代方法，涉及TALEN-或Cas9介导的CYBB小基因在CYBB基因座外显子1或2的插入。将外显子1-13小基因靶向插入CYBB外显子1导致iPSC衍生的粒细胞中没有可检测的gp91phox表达或ROS活性。相比之下,将外显子2-13小基因靶向插入外显子2可以恢复gp91phox和ROS活性。这证明了两种校正策略的功效:通过外显子置换或外显子2-13小基因靶向插入CYBB外显子2同时保留外显子/内含子1来无缝修复特定CYBB突变。此外，它强调了从内源性启动子表达的靶向插入策略的关键问题:内含子元件的保留可能是表达所必需的。
