Herpes simplex virus/Sleeping Beauty vector-based embryonic gene transfer using the HSB5 mutant: loss of apparent transposition hyperactivity in vivo.
Herpes simplex virus/Sleeping Beauty vector-based embryonic gene transfer using the HSB5 mutant: loss of apparent transposition hyperactivity in vivo.
复制标题
使用HSB5突变体进行基于单纯疱疹病毒/睡美人载体的胚胎基因转移：体内明显转座过度活性的丧失。
DOI：
10.1089/hum.2010.062
复制
发表时间：
2010
影响因子：
4.2
通讯作者：
Bowers,WilliamJ
中科院分区：
文献类型：
--
作者：
deSilva,Suresh;Mastrangelo,MichaelA;LottaJr,LouisT;Burris,ClarkA;Izsvak,Zsuzsanna;Ivics,Zoltan;Bowers,WilliamJ
睡美人（SB）转座子系统已成功地用作非病毒和病毒载体平台上的基因传递工具。自其最初重建以来，已经产生了一系列的SB过度活跃的突变体。这些SB转座酶突变体体外活性的增强是否会转化为它们的体内设置，以及这种增加的整合效率是否会通过增加基因组插入突变的风险而最终损害转座子平台的安全性，这些问题仍然存在。在此，我们比较了单纯疱疹病毒（HSV）扩增子载体的“野生型”SB转座酶（SB10）和“过度活跃”SB突变体（HSB5）在胚胎期14.5天的C57BL/6小鼠体内与HSV -β地转座报告扩增子载体共传递的影响。SB10和HSB5转座对注射小鼠胚胎的生存能力和发育没有不同的影响。在出生后第21天，脑内β地理表达的定量显示，与注入sb10组相比，接受HSB5的小鼠仅表现出转基因表达的趋势增加，这一结果并不反映体外观察到的HSB5介导的转位的显着增强。这些发现表明，在体内应用高活性SB突变体，尽管对发育中的小鼠胚胎没有差异的遗传毒性，但在HSV/SB扩增子平台中传递时，并不一定比使用低活性SB提供显着的治疗优势。
TheSleeping Beauty(SB) transposon system has been successfully used as a gene delivery tool in nonviral and viral vector platforms. Since its initial reconstruction, a series of hyperactive mutants of SB have been generated. Questions remain as to whether the enhancedin vitroactivities of these SB transposase mutants translate to thein vivosetting, and whether such increased integration efficiencies will ultimately compromise the safety profile of the transposon platform by raising the risk of genomic insertional mutagenesis. Herein, we compared thein vivoimpact of a herpes simplex virus (HSV) amplicon-vectored “wild-type” SB transposase (SB10) and a “hyperactive” SB mutant (HSB5), codeliveredin uterowith the HSVT-βgeo transposable reporter amplicon vector to embryonic day 14.5 C57BL/6 mice. The SB10 and HSB5 transposases do not disparately affect the viability and development of injected mouse embryos. Quantitation of brain-resident βgeo expression on postnatal day 21 revealed that mice receiving HSB5 exhibited only a trending increase in transgene expression compared with the SB10-infused group, an outcome that did not mirror the marked enhancement of HSB5-mediated transposition observedin vitro.These findings indicate thatin vivoapplication of hyperactive SB mutants, although not differentially genotoxic to the developing mouse embryo, does not necessarily provide a significant therapeutic advantage over the employment of a lesser active SB when delivered in the context of the HSV/SB amplicon platform.