Luo Phoebe, He Xianghui, Tsang Tom C, Harris David T
Gene Therapy Group, Department of Microbiology and Immunology, University of Arizona, Tucson, AZ 85721-0001, USA.
Int J Mol Med. 2004 Feb;13(2):319-25.
Safety and efficacy are both required for successful gene therapy. In this regard, our laboratory has created a novel expression system, pHi-Hot that combines inducible and amplifier strategies in one construct. In pHi-Hot, the first transcriptional unit contains an inducible heat shock protein (hsp70B) promoter controlling the expression of a transcriptional factor, Tat, which transactivates a second promoter, the HIV2 LTR, located downstream on the same construct. The second promoter drives the gene of interest. The magnitude of the amplified second gene expression can be regulated through manipulating the activity of the hsp promoter driving the Tat gene. Using the human interleukin-2 (IL-2) cytokine gene as the reporter gene, we demonstrated that moderate heat shock at 42 degrees C for 30 min, the pHi-Hot vector could achieve high gene expression levels while maintaining its inducibility. The induced IL-2 levels were 35- to 70-fold higher than achieved by using the hsp promoter alone, and 10- to 35-fold higher than achieved by using the CMV promoter. Using inducible and amplifier strategies, we can achieve high and controlled gene expression levels from a single construct. Finally, we discuss the advantages of using these strategies in developing new targeting and inducible vectors for genetic research and gene therapy.
成功的基因治疗需要安全性和有效性。在这方面，我们实验室创建了一种新型表达系统pHi-Hot，它在一个构建体中结合了诱导和放大策略。在pHi-Hot中，第一个转录单元包含一个诱导型热休克蛋白（hsp70B）启动子，控制转录因子Tat的表达，Tat可反式激活位于同一构建体下游的第二个启动子HIV2 LTR。第二个启动子驱动目的基因。通过操纵驱动Tat基因的hsp启动子的活性，可以调节扩增的第二个基因表达的幅度。以人白细胞介素-2（IL-2）细胞因子基因为报告基因，我们证明在42℃适度热休克30分钟时，pHi-Hot载体可以实现高基因表达水平，同时保持其诱导性。诱导的IL-2水平比单独使用hsp启动子高35至70倍，比使用CMV启动子高10至35倍。通过使用诱导和放大策略，我们可以从单个构建体实现高且可控的基因表达水平。最后，我们讨论了在开发用于基因研究和基因治疗的新型靶向和诱导载体中使用这些策略的优势。
