Department of Anatomy, John A Burns School of Medicine, Honolulu, HI 96822, USA.
Proc Natl Acad Sci U S A. 2010 May 4;107(18):8117-22. doi: 10.1073/pnas.1003674107. Epub 2010 Apr 19.
Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.
高效整合功能基因是成功进行基因传递（如细胞转染、动物转基因和基因治疗）的必要前提。基于病毒载体的基因传递策略目前是最有效的。然而，有限的载物能力、宿主免疫反应和插入突变的风险是限制因素，令人担忧。最近，有几个研究小组使用转座子为载体的方法将基因传递到各种细胞中。特别是 piggyBac（pB）转座酶因其分子修饰的灵活性、大容量载物能力和高转座活性，非常适合用于细胞转染和基因治疗方法。然而，关于转座酶基因插入宿主基因组的安全性考虑很少得到解决。在这里，我们报告了我们在工程无辅助 pB 质粒方面的结果。单质粒基因传递系统携带猪gyBac 转座酶（pBt）表达盒以及由末端重复元件序列侧翼的转座子货物。通过开发新的质粒，在质粒中转座子被切除后，pBt 基因失活，从而实现了无辅助结构的改进。因此，消除了转座后持续存在活性 pBt 基因可能产生的潜在负面影响。本文介绍的结果表明，我们的无辅助质粒是开发安全有效的基因传递方法的重要一步，在基因治疗和转基因方法中应该具有重要价值。
