State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, China.
Gene Ther. 2020 Jun;27(6):266-280. doi: 10.1038/s41434-020-0128-x. Epub 2020 Feb 7.
The transcription factor NF-κB is an attractive target for cancer therapy due to its over-activation in all tumours; however, NF-κB inhibitors developed in the past decades rarely became drugs due to undesirable side effects. In this study, we developed a gene therapy strategy named NF-κB-activated gene expression (Nage), which could induce the death of cancer cells in vitro and in vivo by utilising NF-κB over-activity in cancer cells, but had no effects on normal cells. Nage was consisted of an NF-κB-specific promoter formed by fusing an NF-κB decoy sequence with a minimal promoter, which could be bound by the intracellular over-activated NF-κB and thus activated the expression of downstream effector genes in an NF-κB-specific manner. In this study, we first confirmed the cancer cell-specific over-activation of NF-κB and then tested the cancer cell specificity of the Nage vector by expressing the reporter gene ZsGreen in various in vitro cultivated cells. We next demonstrated that the Nage vector could be used to express CRISPR/Cas9 protein only in cancer cells. The Cas9 protein was then guided by a sgRNA targeting telomeric DNA and induced cancer cell death. The Nage vector expressing Cas9/sgRNA could be packaged into adeno-associated virus (AAV) and intravenously injected to inhibit tumour growth in mice without visible side effects and toxicity.
转录因子 NF-κB 在所有肿瘤中过度激活，因此成为癌症治疗的一个有吸引力的靶点；然而，过去几十年来开发的 NF-κB 抑制剂由于不良反应而很少成为药物。在这项研究中，我们开发了一种名为 NF-κB 激活基因表达（Nage）的基因治疗策略，该策略利用癌细胞中 NF-κB 的过度活性，在体外和体内诱导癌细胞死亡，但对正常细胞没有影响。Nage 由一个 NF-κB 特异性启动子组成，该启动子由 NF-κB 诱饵序列与最小启动子融合而成，可以被细胞内过度激活的 NF-κB 结合，从而以 NF-κB 特异性方式激活下游效应基因的表达。在这项研究中，我们首先证实了 NF-κB 在癌细胞中的特异性过度激活，然后通过在各种体外培养的细胞中表达报告基因 ZsGreen 来测试 Nage 载体的癌细胞特异性。接下来，我们证明了 Nage 载体只能在癌细胞中表达 CRISPR/Cas9 蛋白。Cas9 蛋白随后被靶向端粒 DNA 的 sgRNA 引导，诱导癌细胞死亡。表达 Cas9/sgRNA 的 Nage 载体可以被包装成腺相关病毒（AAV），并静脉注射到小鼠体内，抑制肿瘤生长，没有明显的副作用和毒性。
