基因工程化CRISPR/Csy4-新型抗HIV-1分子靶向药物的研发及机理研究

Highly active anti-retroviral therapy(HAART) function by inhibiting, rather than eliminating HIV-1 virus. As a result, they cannot completely cure the disease. The recent success in engineering CRISPR/Cas9 in gene targeting and employing substrate-linked protein evolution approach to redirect Cre DNA recombinase to recognize the HIV-1 DNA has highlighted the promise to cure AIDS. Our previous study has demonstrated that the CRISPR/Csy4 specifically recognizes a hairpin RNA that is highly homologous to the HIV-1 LTR-RNA. When incorporated the hairpin into the HIV-1 LTR, Csy4 destroys the virus at different steps of the infection, including post-transcription, viral packaging, and pro-virus DNA integration. In this study, we propose to employ a bacteria-based evolution approach to engineer the Csy4 protein, so that it can specifically recognize and cut the HIV-1 LTR RNA sequence. By examining the function of the engineered Csy4 and its underlying molecular mechanisms in HIV-1 cell models, we hope to develop a new generation of anti-HIV-1 drug, aiming to completely cure AIDS in near future.

高效抗反转录病毒治疗(highly active anti-retroviral therapy, HAART)能显著抑制HIV病毒，然而因其不能杀灭病毒而最终导致病毒耐药及疾病复发。高特异性CRISPR/Cas9基因编辑技术及识别HIV-LTR的Cre酶的基因工程改造成功，为根治HIV带来了希望。课题申请人前期研究发现：CRISPR/Csy4特异性识别的RNA发夹结构同HIV-1-LTR序列高度同源，基因工程改造的Csy4能从病毒转录、病毒包装及前病毒DNA整合等多个环节起抗HIV-1病毒作用。本课题拟在前期工作的基础上，结合基因工程技术对野生型Csy4进行优化改造，以筛选出能特异识别并酶切HIV-LTR的Csy4，并深入探讨其抗HIV-1作用的分子机理，研发出具有我国自主产权的新型抗HIV-1药物。

现有的抗HIV-1病毒药物因不能彻底清除病毒而需要终身服药。高特异性CRISPR/Cas9及识别HIV-LTR的Cre酶的基因工程改造成功为根治HIV带来了希望。课题申请人前期研究发现：CRISPR/ Csy4特异性识别的RNA发夹结构同HIV-1-LTR序列高度同源，并且计算机折叠模型显示，两者具有异常相似的二维结构。我们已经发表的结果证实：eCsy4能从HIV-1转录后、病毒包装及前病毒DNA整合等多个环节起抗HIV-1病毒作用。本课题拟在前期工作的基础上，结合基因工程技术对野生型Csy4进行优化改造，以筛选出能特异识别并酶切HIV-LTR的eCsy4，并深入探讨其抗HIV-1作用的分子机理，研发出具有我国自主产权的新型抗HIV-1药物?

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