Department of Biological Sciences, National University of Singapore, 117543 Singapore, Department of Surgery, Program of Innovative Cancer Therapeutics, First Affiliated Hospital of Zhejiang University College of Medicine, 310009 Hangzhou, China and Institute of Bioengineering and Nanotechnology, 138669 Singapore.
Nucleic Acids Res. 2013 Oct;41(19):e180. doi: 10.1093/nar/gkt721. Epub 2013 Aug 13.
Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells.
在人类基因组中，基因整合的安全性和可靠性仍然是干细胞基因治疗的挑战。在这里，我们报告了一种用于 AAVS1 基因座导向同源重组的杆状病毒转录激活因子样效应物核酸酶系统，用于人类诱导多能干细胞（iPSC）。该病毒系统在优化后的人 U87 细胞中，在整合 Neo-eGFP 盒时提供了 95.21%的靶向整合效率，并能够介导长达 13.5kb 的 DNA 插入物的整合。在 iPSC 中，实现了靶向整合和持续的转基因表达，而不会影响基因组稳定性。修饰后的 iPSC 继续表达干细胞多能性标志物，并保持在衍生的胚状体中分化为三个生殖谱系的能力。在 iPSC 中使用杆状病毒-Cre/LoxP 系统，可以将 AAVS1 基因座上的 Neo-eGFP 盒替换为 Hygro-mCherry 盒，证明了盒交换的可行性。此外，通过测量 γ-H2AX 表达水平评估，在用表达转录激活因子样效应物核酸酶的中等剂量杆状病毒载体转导后，未检测到与染色体双链断裂相关的基因组毒性。鉴于高靶向整合效率、转基因交换的灵活性和低基因组毒性，我们的杆状病毒转导方法为人类多能干细胞的精确遗传操作提供了巨大的潜力和有吸引力的选择。
