腺相关病毒介导锌指核酸酶体外切除HIV-1基因组研究  

Study on HIV-1 Genome Excision by ZFN Delivered via AAV in vitro

作　　者：梁智铭 杨辛毅 王亚楠 王菁 沈晓婷 潘浛雨 杨金龙 陆盼盼 赵琳 梁玥 朱焕章[1] LIANG Zhiming;YANG Xinyi;WANG Ya nan;WANG Jing;SHEN Xiaoting;PAN Hanyu;YANG Jinlong;LU Panpan;ZHAO Lin;LIANG Yue;ZHU Huanzhang(School of Life Sciences,Fudan University,Shanghai 200433,China)

机构地区：[1]复旦大学生命科学学院,上海200433

出　　处：《复旦学报（自然科学版）》2023年第6期753-764,共12页Journal of Fudan University：Natural Science

摘　　要：人类免疫缺陷病毒1型(Human Immunodeficiency Virus type 1,HIV-1)能够整合到宿主细胞基因组并通过基因沉默的方式逃避高效抗逆转录病毒疗法(Highly Active Antietroviral Therapy,HAART),而高效特异性的基因编辑工具的出现,有望成功切除HIV-1前病毒基因组。重组腺相关病毒(AAV)是基因治疗的重要载体系统,为了获得能够高效感染T细胞的基因编辑工具递送系统,本研究通过比较过表达增强绿色荧光蛋白的血清型2、血清型6、血清型DJ的重组腺相关病毒,筛选出T细胞系的偏好血清型为6型。通过比较CMV启动子、SFFV启动子,筛选出T细胞系的偏好启动子为SFFV启动子。最后,我们通过制备过表达靶向HIV-1长末端重复序列(Long Terminal Region,LTR)的锌指核酸酶(Zinc Finger Nuclease,ZFN)的AAV6递送载体系统,感染模拟HIV-1阳性表达的细胞系Ya,T7E1检测证实HIV-1前病毒基因组被特异性切除,流式细胞仪检测证实切除效率约为17.1%,测序分析进一步证实靶向HIV-1 LTR的ZFN能介导从细胞基因组中切除HIV-1前病毒基因组。实验证明,6型重组腺相关病毒递送靶向HIV-1 LTR的ZFN能有效切除HIV-1前病毒基因组,为该系统未来的体内应用奠定了基础。Acquired Immune Deficiency Syndrome(AIDS)is a serious infectious disease caused by human immunodeficiency virus(HIV-1)infection.HIV-1 can latently integrate into the host cell genome to escape the highly active antiretroviral therapy.Through gene editing tools,the excision of the HIV-1 proviral genome holds promise for a cure for AIDS.In this study,by comparing the recombinant adeno-associated viruses of serotype 2,serotype 6,and serotype DJ overexpressing the enhanced green fluorescent protein,the preferred serotype of lymphoid T cell line was screened:serotype 6.By comparing the CMV promoter and SFFV promoter,the preferred promoter for T cell lines was screened:SFFV promoter.The recombinant adeno-associated virus of serotype 6 overexpressing the zinc finger nuclease(ZFN)targeting HIV-1 long terminal repeat(LTR)was prepared and used to transfect the HIV-1 positive-infected cell line Ya.That the HIV-1 proviral genome was excised was confirmed by T7E1 assay and flow cytometry confirmed that the excision efficiency was about 17.1%.The sequencing analysis further confirmed that the ZFN targeting HIV-1 LTR mediated the excision of the HIV-1 proviral genome in the cell genome.Experiments have shown that ZFN targeting HIV-1 LTR delivered by recombinant adeno-associated virus can effectively excise the HIV-1 provirus genome,and extended application of ZFN to cure AIDS.

关 键 词：基因编辑工具 人类免疫缺陷病毒1型 获得性免疫缺陷综合征 锌指核酸酶 腺相关病毒 

分 类 号：Q3[生物学—遗传学]