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Abstract：
X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of the immune system. It is characterized by a defect in the production of reactive oxygen species (ROS) in phagocytic cells due to mutations in the NOX2 locus, which encodes gp91phox. Because the success of retroviral gene therapy for X-CGD has been hampered by insertional activation of proto-oncogenes, targeting the insertion of a gp91phox transgene into potential safe harbor sites, such as AAVS1, may represent a valid alternative. To conceptually evaluate this strategy, we generated X-CGD patient-derived induced pluripotent stem cells (iPSCs), which recapitulate the cellular disease phenotype upon granulocytic differentiation. We examined AAVS1-specific zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for their efficacy to target the insertion of a myelo-specific gp91phox cassette to AAVS1. Probably due to their lower cytotoxicity, TALENs were more efficient than ZFNs in generating correctly targeted iPSC colonies, but all corrected iPSC clones showed no signs of mutations at the top-ten predicted off-target sites of both nucleases. Upon differentiation of the corrected X-CGD iPSCs, gp91phox mRNA levels were highly up-regulated and the derived granulocytes exhibited restored ROS production that induced neutrophil extracellular trap (NET) formation. In conclusion, we demonstrate that TALEN-mediated integration of a myelo-specific gp91phox transgene into AAVS1 of patient-derived iPSCs represents a safe and efficient way to generate autologous, functionally corrected granulocytes.
摘要：
X连锁慢性肉芽肿病（X-CGD）是一种遗传性免疫系统疾病。它的特征是由于NOX2基因座的突变，吞噬细胞中活性氧（ROS）的产生存在缺陷，编码gp91phox。因为逆转录病毒基因治疗X-CGD的成功受到原癌基因插入激活的阻碍，靶向将gp91phox转基因插入潜在的安全港，例如AAVS1可以代表有效的替代方案。从概念上评估这个策略，我们产生了X-CGD患者来源的诱导多能干细胞（iPSCs），它概括了粒细胞分化后的细胞疾病表型。我们检查了AAVS1特异性锌指核酸酶（ZFN）和转录激活因子样效应核酸酶（TALEN）的功效，以靶向将骨髓特异性gp91phox盒插入AAVS1。可能是由于它们的细胞毒性较低，TALEN在产生正确靶向的iPSC菌落方面比ZFN更有效，但所有校正的iPSC克隆在两种核酸酶的前10个预测脱靶位点均未显示突变迹象.经校正的X-CGDiPSCs分化后，gp91phoxmRNA水平高度上调，衍生的粒细胞表现出恢复的ROS产生，从而诱导中性粒细胞胞外陷阱（NET）形成。总之,我们证明了TALEN介导的骨髓特异性gp91phox转基因整合到患者来源的iPSC的AAVS1中代表了一种安全有效的方法来产生自体,功能校正的粒细胞。
