德国欧洲分子生物学实验室Orsolya Barabas与维尔茨堡大学Michael Hudecek等研究人员开发出一种高溶解度的睡美人（SB）转座酶系统，其可提高基因插入的效率。2019年11月4日，《自然—生物技术》杂志在线发表了该研究成果。

基于过度活跃的SB100X变体的晶体结构，研究人员使用了合理的蛋白质设计来创建具有增强的溶解性和稳定性的SB转座酶（高溶解度SB，hsSB）。研究人员证明，hsSB可以与转座子DNA一起传递，从而遗传修饰细胞系和胚胎、造血和诱导多能干细胞（iPSCs），克服了不受控制的转座酶活性。研究人员使用hsSB产生了嵌合抗原受体（CAR）T细胞，在体外和异种移植小鼠中均显示出强大的抗肿瘤活性。研究人员发现hsSB可自发穿透细胞，从而无需使用转染试剂即可修饰iPSC和生成CAR T细胞。每个基因组仅需两次整合，hsSB即可以调节基因组整合频率。

据悉，SB转座子系统是哺乳动物细胞中一种有效的非病毒基因转移工具，但其使用受到DNA载体转座酶基因活性不受控制、基因组不稳定的风险以及无法直接使用转座酶蛋白等因素的困扰。

附：英文原文

Title: A highly soluble Sleeping Beauty transposase improves control of gene insertion

Author: Irma Querques, Andreas Mades, Cecilia Zuliani, Csaba Miskey, Miriam Alb, Esther Grueso, Markus Machwirth, Tobias Rausch, Hermann Einsele, Zoltn Ivics, Michael Hudecek, Orsolya Barabas

Issue&Volume: 2019-11-04

Abstract: 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.

DOI: 10.1038/s41587-019-0291-z

Source: https://www.nature.com/articles/s41587-019-0291-z