Dent C L, Lau G, Drake E A, Yoon A, Case C C, Gregory P D
Sangamo BioSciences Inc., Point Richmond Tech Center, Richmond, CA 94804, USA.
Gene Ther. 2007 Sep;14(18):1362-9. doi: 10.1038/sj.gt.3302985. Epub 2007 Jul 19.
Small-molecule-regulated gene expression offers the promise of titrating the dose and duration of action of DNA-based therapies. To this end, we show that engineered zinc-finger protein transcription factors (ZFP TFs) can be coupled with a drug-inducible regulatory domain to permit small-molecule control of endogenous gene transcription. We constructed a drug-responsive ZFP TF via the fusion of a ZFP DNA-binding domain (DBD) targeting the human VEGF-A gene and an effector domain containing a truncated progesterone receptor ligand-binding domain linked to the NFkappaB p65 activation domain. Introduction of this engineered ZFP TF into human or murine cells allowed expression of the chromosomal VEGF-A gene to be induced upon addition of mifepristone, a synthetic steroid analog. Mifepristone-dependent VEGF-A induction was rapid, dose-dependent and reversible. Moreover, stable lines expressing the drug-responsive ZFP TF could be maintained in a state of continuous induction for at least 30 days without loss of viability. Potent VEGF-A induction was demonstrated using different engineered ZFP DBDs, thus this approach may represent a general solution to small-molecule regulation of targeted endogenous genes.
小分子调控基因表达为滴定基于DNA的疗法的剂量和作用持续时间带来了希望。为此，我们表明，工程化锌指蛋白转录因子（ZFP TF）可以与药物诱导调控域偶联，以实现对内源基因转录的小分子控制。我们通过融合靶向人VEGF-A基因的ZFP DNA结合域（DBD）和包含与NFκB p65激活域相连的截短孕酮受体配体结合域的效应域，构建了一种药物响应性ZFP TF。将这种工程化ZFP TF导入人或鼠细胞后，加入米非司酮（一种合成类固醇类似物）可诱导染色体VEGF-A基因表达。米非司酮依赖性VEGF-A诱导迅速、呈剂量依赖性且可逆。此外，表达药物响应性ZFP TF的稳定细胞系可以在持续诱导状态下维持至少30天而不丧失活力。使用不同的工程化ZFP DBD证明了有效的VEGF-A诱导，因此这种方法可能代表了一种对靶向内源基因进行小分子调控的通用解决方案。
