朱焕章课题组在艾滋病基因治疗研究方面取得进展
发布时间:2016-03-25  阅读次数:2278

       日前,朱焕章课题组利用CRISPR/CAS9技术在体外靶向干预HIV潜伏又取得进展,为艾滋的功能治愈迈出了关键一步。该文发表在国际基因治疗领域著名杂志Mol Ther.(医学1区)在线发表(http://www.ncbi.nlm.nih.gov/pubmed/26775808),相关工作亦已申请了中国专利。
       目前,国际上已有近30种抗逆转录病毒药物应用于艾滋病的临床治疗,然而,现有这些药物只能抑制病毒复制,不能清除艾滋病病毒(HIV)感染, 其原因主要在于HIV可潜伏于少数免疫细胞,以此逃脱了机体免疫系统和抗病毒药物攻击。因此,研发能靶向干预HIV潜伏的新型治疗技术,已成为目前国际上艾滋病治愈研究领域发展趋势和研究的焦点。
基因编辑技术已有多个临床试验方案已被美国FDA批准应用到病人的抗肿瘤或艾滋病的基因治疗研究。该文研究者提出了利用CRISPR/Cas9技术靶向激活潜伏HIV前病毒的“引蛇出洞”的策略。研究人员设计并筛选出了能特异靶向多数HIV亚型基因保守区的dCas9-SunTag-VP64 系统,在多个HIV潜伏细胞系上证实了dCas9-SunTag-VP64 系统能特异靶向激活HIV-1前病毒,提示了该方法有可能成为一个可选择的抗HIV潜伏治疗手段。
       近几年来,朱焕章课题组在艾滋病基因治疗新型策略研究方面取得了阶段性的研究进展。2013年,首次提出并在体外证实利用基因编辑技术靶向切除整合HIV前病毒的“斩草除根”的AIDS/HIV治疗策略的可行性(Nucleic Acids Res,2013;41(16):7771-82);2014年,又获得了能特异靶向HIV调控区的锌指转录因子(ZFP)及转录样因子(TALE)融合基因,并在潜伏细胞模型上证实了这些融合蛋白可靶向激活潜伏HIV表达(Gene Therapy ,2014,21,90-95;AIDS Res Hum Retroviruses,2015,31:98-106)。


Specific Reactivation of Latent HIV-1 by dCas9-SunTag-VP64-mediated Guide RNA Targeting the HIV-1 Promoter
Abstract:HIV-1 escapes antiretroviral agents by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. Transcriptional activation is prerequisite for reactivation and the eradication of latent HIV-1 proviruses. dCas9-SunTag-VP64 transcriptional system has been reported that it can robustly activate the expression of an endogenous gene using a single guide RNA (sgRNA). Here, we systematically investigated the potential of dCas9-SunTag-VP64 with the designed sgRNAs for reactivating latent HIV-1. We found dCas9-SunTag-VP64 with sgRNA 4 or sgRNA 5 targeted from -164 to -146 or -124 to -106 bp upstream of the transcription start sites of HIV-1 could induce high expression of luciferase reporter gene after screening of sgRNAs targeting different regions of the HIV-1 promoter. Further, we confirmed that dCas9-SunTag-VP64 with sgRNA 4 or sgRNA 5 can effectively reactivate latent HIV-1 transcription in several latently infected human T-cell lines. Moreover, we confirmed that the reactivation of latent HIV-1 by dCas9-SunTag-VP64 with the designed sgRNA occurred through specific binding to the HIV-1 LTR promoter without genotoxicity and global T-cell activation. Taken together, our data demonstrated dCas9-SunTag-VP64 system can effectively and specifically reactivate latent HIV-1 transcription, suggesting that this strategy could offer a novel approach to anti-HIV-1 latency. (Mol Ther. 2016 Mar;24(3):508-21. doi: 10.1038/mt.2016.7. Epub 2016 Jan 18.)

 

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