A highly soluble Sleeping Beauty transposase improves control of gene insertion
A highly soluble Sleeping Beauty transposase improves control of gene insertion
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DOI：
10.1038/s41587-019-0291-z
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发表时间：
2019-12-01
影响因子：
46.9
通讯作者：
Barabas, Orsolya
中科院分区：
文献类型：
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作者：
Querques, Irma;Mades, Andreas;Barabas, Orsolya
睡美人（SB）转座子系统是哺乳动物细胞中有效的非病毒基因转移工具，但其广泛使用受到来自DNA载体的不受控制的转座酶基因活性的阻碍，从而造成基因组不稳定的风险，并且不能直接使用转座酶蛋白。在这项研究中，我们使用合理的蛋白质设计的基础上的晶体结构的超活性SB100X变体，以创建一个SB转座酶（高溶解度SB，hsSB）具有增强的溶解性和稳定性。我们证明了hsSB可以与转座子DNA一起递送以遗传修饰细胞系和胚胎、造血和诱导多能干细胞（iPSC），克服不受控制的转座酶活性。我们使用hsSB产生嵌合抗原受体（CAR）T细胞，其在体外和异种移植小鼠中表现出有效的抗肿瘤活性。我们发现hsSB自发地穿透细胞，使得能够在不使用转染试剂的情况下修饰iPSC和产生CAR T细胞。滴定hsSB以调节基因组整合频率实现了每个基因组少至两个整合。
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.