Keravala Annahita, Liu Dexi, Lechman Eric R, Wolfe Darren, Nash Joan A, Lampe David J, Robbins Paul D
Department of Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh, PA 15261, USA.
Hum Gene Ther. 2006 Oct;17(10):1006-18. doi: 10.1089/hum.2006.17.1006.
The use of nonviral delivery systems results in transient gene expression, in part because of the low efficiency of DNA integration. Previously, vectors based on transposon systems such as Sleeping Beauty have been shown to be able to increase stable transfection efficiencies in cell culture and in animal models. Himar1, a reconstructed active transposon belonging to the Tc1/mariner superfamily, also has been used as a vector for stable gene delivery, but the rate of transposition after transfection is low. In this paper, we evaluate the potential of the hyperactive Himar1 transposase C9, in combination with the Himar1 inverted repeat transposon, as a gene delivery vector. The C9 transposase is a hyperactive mutant of Himar1 with two amino acid substitutions, Q131R and E137K, that result in an increase in activity relative to wild type. Here we demonstrate that cotransfection of the C9 transposase with a Himar1-based vector increases the frequency of stable gene expression in human cells in a transposase concentration-dependent manner. In addition, we establish that C9 transposase mediates integration of the transgene in mammalian cells at a frequency similar to that of Sleeping Beauty under some of the conditions tested. Last, we show significantly higher levels of reporter gene expression in vivo in mouse liver and in synovium of rabbit knee joints after injection of the transposon plasmid expressing the transgene and the C9 transposase. These data suggest that vectors based on the Himar1 transposable element, in conjunction with the hyperactive mutant transposase C9, may be suitable vectors for gene therapy applications.
非病毒递送系统的使用会导致基因短暂表达，部分原因是DNA整合效率低。此前，基于转座子系统（如睡美人转座子）的载体已被证明能够提高细胞培养和动物模型中的稳定转染效率。Himar1是一种属于Tc1/水手超家族的重构活性转座子，也已被用作稳定基因递送的载体，但转染后的转座率较低。在本文中，我们评估了高活性Himar1转座酶C9与Himar1反向重复转座子结合作为基因递送载体的潜力。C9转座酶是Himar1的高活性突变体，有两个氨基酸替换，即Q131R和E137K，这导致其活性相对于野生型有所增加。在这里，我们证明C9转座酶与基于Himar1的载体共转染可在人细胞中以转座酶浓度依赖性方式提高稳定基因表达的频率。此外，我们确定在某些测试条件下，C9转座酶介导转基因在哺乳动物细胞中的整合频率与睡美人转座子相似。最后，我们显示在注射表达转基因的转座子质粒和C9转座酶后，小鼠肝脏和兔膝关节滑膜中的报告基因在体内有显著更高水平的表达。这些数据表明，基于Himar1转座元件并结合高活性突变转座酶C9的载体可能是适合基因治疗应用的载体。
