Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene.
Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene.
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DOI：
10.1038/nbt.3843
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发表时间：
2017-06
影响因子：
46.9
通讯作者：
Luo JH
中科院分区：
文献类型：
--
作者：
Chen ZH;Yu YP;Zuo ZH;Nelson JB;Michalopoulos GK;Monga S;Liu S;Tseng G;Luo JH
特异性靶向肿瘤细胞中的基因组重排和突变仍然是癌症治疗中难以实现的目标。在这里，我们使用基于Cas9的基因组编辑将前体药物转化酶“单纯疱疹病毒1型胸苷激酶”（HSV 1-tk）的基因引入携带基因组重排产生的独特序列的癌细胞的基因组中。具体而言，我们在体外和小鼠异种移植物中靶向人前列腺癌或肝细胞癌细胞中TMEM 135-CCDC 67和MAN 2A 1-FER融合的断点。我们设计了一种腺病毒以递送切口酶Cas 9D 10A和靶向断点序列的gRNA，并且另一种腺病毒以递送EGFP-HSV 1-tk构建体，所述构建体侧翼为与断点周围的序列同源的序列。两种病毒感染导致EGFP-tk和更昔洛韦介导的细胞凋亡的断点依赖性表达。当用腺病毒和更昔洛韦治疗小鼠异种移植物时，所有动物显示肿瘤负荷减少，在观察期内没有死亡。我们的研究结果表明，Cas9介导的自杀基因插入可能是一种可行的基因型特异性癌症治疗方法。
Specifically targeting genomic rearrangements and mutations in tumor cells has remained an elusive goal in cancer therapy. Here, we use Cas9-based genome editing to introduce the gene for the pro-drug converting enzyme ‘herpes simplex virus type 1 thymidine kinase’ (HSV1-tk) into the genome of cancer cells that carry unique sequences resulting from genome rearrangements. Specifically, we targeted the breakpoints of TMEM135-CCDC67 and MAN2A1-FER fusions in human prostate cancer or hepatocellular carcinoma cells in vitro and in mouse xenografts. We designed one adenovirus to deliver the nickase Cas9D10A and gRNAs targeting the breakpoint sequences and another to deliver an EGFP-HSV1-tk construct flanked by sequences homologous to those surrounding the breakpoint. Infection with both viruses resulted in breakpoint-dependent expression of EGFP-tk and ganciclovir-mediated apoptosis. When mouse xenografts were treated with adenoviruses and ganciclovir, all animals showed reduction of tumor burden with no mortality over the observation period. Our results suggest that Cas9-mediated suicide gene insertion might be a viable genotype-specific therapy for cancer.