1] Interdisciplinary Graduate Program in Genetics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA [2] Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA [3] Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.
Mol Ther Nucleic Acids. 2012 Oct 16;1(10):e50. doi: 10.1038/mtna.2012.12.
Nonviral vector systems are used increasingly in gene targeting and gene transfer applications. The piggyBac transposon represents an alternative integrating vector for in vivo gene transfer. We hypothesized that this system could achieve persistent gene transfer to the liver when administered systemically. We report that a novel hyperactive transposase generated higher transposition efficiency than a codon-optimized transposase in a human liver cell line. Hyperactive transposase-mediated reporter gene expression persisted at levels twice that of codon-optimized transposase in the livers of mice for the 6-month study. Of note, expression persisted in mice following partial hepatectomy, consistent with expression from an integrated transgene. We also used the hyperactive transposase to deliver the human α(1)-antitrypsin gene and achieved stable expression in serum. To determine the integration pattern of insertions, we performed large-scale mapping in human cells and recovered 60,685 unique hyperactive transposase-mediated insertions. We found that a hyperactive piggyBac transposase conferred an altered pattern of integration from that of insect piggyBac transposase, with a decreased frequency of integration near transcription start sites than previously reported. Our results support that the piggyBac transposon combined with the hyperactive transposase is an efficient integrating vector system for in vitro and in vivo applications.Molecular Therapy - Nucleic Acids (2012) 1, e50; doi:10.1038/mtna.2012.12; published online 16 October 2012.
非病毒载体系统越来越多地用于基因靶向和基因转移应用。猪 bac 转座子代表了一种用于体内基因转移的替代整合载体。我们假设，当系统给药时，该系统可以实现对肝脏的持续基因转移。我们报告说，一种新型的超活性转座酶在人肝癌细胞系中比经过密码子优化的转座酶具有更高的转座效率。在为期 6 个月的研究中，超活性转座酶介导的报告基因表达在小鼠肝脏中的水平持续保持在密码子优化转座酶的两倍。值得注意的是，在部分肝切除术后，表达仍然持续存在，与整合转基因的表达一致。我们还使用超活性转座酶来递送人α(1)-抗胰蛋白酶基因，并在血清中实现稳定表达。为了确定插入的整合模式，我们在人细胞中进行了大规模作图，并回收了 60685 个独特的超活性转座酶介导的插入。我们发现，超活性猪 bac 转座酶赋予了不同于昆虫猪 bac 转座酶的整合模式，与先前报道的转录起始位点附近的整合频率降低。我们的结果支持猪 bac 转座子与超活性转座酶相结合是体外和体内应用的有效整合载体系统。分子治疗-核酸(2012)1, e50;doi:10.1038/mtna.2012.12;在线发表于 2012 年 10 月 16 日。
