转座酶
转座因子
诱导多能干细胞
睡美人转座系统
生物
整合酶
基因
转染
P元素
遗传学
基因组
细胞生物学
分子生物学
胚胎干细胞
作者
Irma Querques,Andreas Mades,Cecilia Zuliani,Csaba Miskey,Miriam Alb,Esther Grueso,Markus Machwirth,Tobias Rausch,Hermann Einsele,Zoltán Ivics,Michael Hudecek,O. Barábas
标识
DOI:10.1038/s41587-019-0291-z
摘要
The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells, but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk of genome instability, and by the inability to use the transposase protein directly. In this study, we used rational protein design based on the crystal structure of the hyperactive SB100X variant to create an SB transposase (high-solubility SB, hsSB) with enhanced solubility and stability. We demonstrate that hsSB can be delivered with transposon DNA to genetically modify cell lines and embryonic, hematopoietic and induced pluripotent stem cells (iPSCs), overcoming uncontrolled transposase activity. We used hsSB to generate chimeric antigen receptor (CAR) T cells, which exhibit potent antitumor activity in vitro and in xenograft mice. We found that hsSB spontaneously penetrates cells, enabling modification of iPSCs and generation of CAR T cells without the use of transfection reagents. Titration of hsSB to modulate genomic integration frequency achieved as few as two integrations per genome.
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