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J Virol:编辑HPV病毒基因 导致癌细胞自我毁灭

来源:生物谷 2014-08-13 08:59

2014年8月12日讯 /生物谷BIOON/--利用基因组编辑工具CRISPR,杜克大学研究人员们能够有选择性地破坏负责宫颈癌细胞生长和生存的两种乳头瘤病毒(HPV)基因,导致癌细胞的自我毁灭。

结果发表在病毒学Journal of Virology杂志上,新研究铺平了开发有针对性的抗病毒策略,来抵抗DNA病毒如B型肝炎病毒和单纯疱疹等的方法。

在这项研究中,研究人员利用CRISPR/Cas编辑病毒基因E6和E7,这两个基因是“原癌基因”,抑制宿主自身抵抗癌细胞的能力。具有讽刺意味的​​是,研究人员将此基因工程改造后的病毒感染宫颈癌细胞,以评估它是否能有效地破坏HPV感染和阻断癌细胞生长。研究结果发现癌细胞会立即停止生长。与此相反的是,对照病毒感染的宫颈癌细胞继续生长,未出现死亡现象。

然后,研究人员降低E6或E7水平,分析破坏癌细胞中E6或E7的后果。E6通常阻断p53蛋白,在这项研究中,E6使p53恢复其正常功能,能刺激肿瘤细胞的死亡。E7以类似方式工作,阻断另一种蛋白质--视网膜母细胞瘤或Rb,可触发癌细胞生长停滞和衰老。

当你关闭E6或E7,宿主的防御机制恢复很快。现在正在研究开发一种不同的病毒载体,在腺相关病毒的基础上,以递送CRISPR进入癌细胞。一旦研究人员实现了这一递送系统,他们将开始在动物模型测试中这种方法。(生物谷Bioon.com)

Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells using a bacterial CRISPR/Cas RNA-guided endonuclease

Edward M. Kennedya, Anand V. R. Kornepatia, Michael Goldsteinb, Hal P. Bogerda, Brigid C. Polinga, Adam W. Whisnanta, Michael B. Kastanb and Bryan R. Cullena#

High-risk human papillomaviruses (HPVs), including HPV-16 and HPV-18, are the causative agents of cervical carcinomas as well as being linked to several other tumors of the anogenital and oropharyngeal regions. The majority of HPV-induced tumors contain integrated copies of the normally episomal HPV genome that invariably retain intact forms of the two HPV oncogenes E6 and E7. E6 induces degradation of the cellular tumor suppressor p53, while E7 destabilizes the retinoblastoma (Rb) protein. Previous work has shown that loss of E6 function in cervical cancer cells induces p53 expression as well as downstream effectors that induce apoptosis and cell cycle arrest. Similarly, loss of E7 allows increased Rb expression, leading to cell cycle arrest and senescence.

Here, we demonstrate that expression of a bacterial Cas9 RNA-guided endonuclease, together with single guide RNAs (sgRNAs) specific for E6 or E7, is able to induce cleavage of the HPV genome, resulting in the introduction of inactivating deletion and insertion mutations into the E6 or E7 gene. This results in induction of p53 or Rb, leading to cell cycle arrest and eventual cell death. Both HPV-16 and HPV-18-transformed cells were found to be responsive to targeted HPV genome-specific DNA cleavage. These data provide proof of principle for the idea that vector-delivered Cas9/sgRNA combinations could represent effective treatment modalities for HPV-induced cancers.

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