Directed gene therapy mediated by nucleases has become a new alternative to lead targeted integration of therapeutic genes in specific regions in the genome. In this work, we have compared the efficiency of two nuclease types, TALEN and meganucleases (MN), to introduce an EGFP reporter gene in a specific site in a safe harbor locus on chromosome 21 in an intergenic region, named here SH6. The efficiency of targeted integration mediated by SH6v5-MN and SH6-TALEN in HEK-293H cells was up to 16.3 and 15.0%. A stable expression was observed both in the pool of transfected cells and in established pseudoclones, with no detection of off-target integrations by Southern blot. In human hematopoietic stem and progenitor CD34⁺ cells, the nucleofection process preserved the viability and clonogenic capacity of nucleofected cells, reaching up to 3.1% of specific integration of the transgene in colony forming cells when the SH6-TALEN was used, although no expression of the transgene could be found in these cells. Our results show the possibility to specifically integrate genes at the SH6 locus in CD34⁺ progenitor cells, although further improvements in the efficacy of the procedure are required before this approach could be used for the gene editing of hematopoietic stem cells in patients with hematopoietic diseases.

由核酸酶介导的定向基因治疗已成为将治疗基因靶向整合到基因组特定区域的一种新选择。在这项工作中，我们比较了两种核酸酶类型，TALEN 和大范围核酸酶 (MN) 的效率，以在基因间区域的 21 号染色体安全港基因座的特定位点引入 EGFP 报告基因，此处命名为 SH6。SH6v5-MN和SH6-TALEN介导的靶向整合在HEK-293H细胞中的效率分别高达16.3%和15.0%。在转染细胞库和已建立的假克隆中均观察到稳定表达，Southern 印迹未检测到脱靶整合。在人类造血干细胞和祖细胞中 CD34 ⁺细胞，核转染过程保留了核转染细胞的活力和克隆能力，当使用 SH6-TALEN 时，高达 3.1% 的转基因在集落形成细胞中的特异性整合，尽管在这些细胞中没有发现转基因的表达. 我们的结果显示了在 CD34 ⁺祖细胞中的 SH6 基因座上特异性整合基因的可能性，尽管在这种方法可用于造血疾病患者的造血干细胞基因编辑之前，需要进一步提高该程序的功效。