Hyperactive piggyBac Gene Transfer in Human Cells and In Vivo
Hyperactive piggyBac Gene Transfer in Human Cells and In Vivo
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DOI：
10.1089/hum.2011.138
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发表时间：
2012-03-01
影响因子：
4.2
通讯作者：
Wilson, Matthew H.
中科院分区：
文献类型：
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作者：
Doherty, Joseph E.;Huye, Leslie E.;Wilson, Matthew H.
我们的特点是最近开发的高度活跃的piggyBac（PB）转座酶[含有7个突变（7 PB）]的基因转移在人体细胞在体外和小鼠体内的体细胞。尽管蛋白质水平表达与天然pB相似，但7 pB显著增加了HEK 293和HeLa培养的人细胞中新霉素抗性盒转座子的基因转移效率。天然pB和SB 100 X，睡美人转座子系统中最活跃的转座酶，在培养的人类细胞系中表现出相似的转座效率。与piggyBac和SB 100 X相比，当离体递送至原代人T细胞时，7 pB使基因递送增加2至3倍。过度活跃的7 pB转座酶的活性不受增加的24-kDa的N-末端标签的影响，而SB 100 X表现出50%的减少转座。在小鼠体内，使用有限剂量的转座酶DNA与荧光素酶报告转座子组合的流体动力学尾静脉注射，将超活性7 pB与天然pB和SB 100 X进行比较。我们跟踪转基因表达长达6个月，并观察到与注射天然pB或SB 100 X的小鼠相比，注射密码子优化版本的7 pB的小鼠的长期基因表达增加了约10倍。我们的结论是，过度活跃的piggyBac元件可以增加在人类细胞和体内的基因转移，并应能够改善基因传递使用piggyBac转座子系统在各种细胞和基因治疗应用。
We characterized a recently developed hyperactive piggyBac (pB) transposase enzyme [containing seven mutations (7pB)] for gene transfer in human cells in vitro and to somatic cells in mice in vivo. Despite a protein level expression similar to that of native pB, 7pB significantly increased the gene transfer efficiency of a neomycin resistance cassette transposon in both HEK293 and HeLa cultured human cells. Native pB and SB100X, the most active transposase of the Sleeping Beauty transposon system, exhibited similar transposition efficiency in cultured human cell lines. When delivered to primary human T cells ex vivo, 7pB increased gene delivery two- to threefold compared with piggyBac and SB100X. The activity of hyperactive 7pB transposase was not affected by the addition of a 24-kDa N-terminal tag, whereas SB100X manifested a 50% reduction in transposition. Hyperactive 7pB was compared with native pB and SB100X in vivo in mice using hydrodynamic tail-vein injection of a limiting dose of transposase DNA combined with luciferase reporter transposons. We followed transgene expression for up to 6 months and observed approximately 10-fold greater long-term gene expression in mice injected with a codon-optimized version of 7pB compared with mice injected with native pB or SB100X. We conclude that hyperactive piggyBac elements can increase gene transfer in human cells and in vivo and should enable improved gene delivery using the piggyBac transposon system in a variety of cell and gene-therapy applications.