锌指蛋白核酸内切酶在HIV感染细胞及潜伏细胞上介导整合HIV前病毒DNA有效切除

目前，艾滋病不能完全被治愈的原因不仅与其"治标不治本"的治疗方法有关，而且也与HIV前病毒整合后潜伏有关。针对这一关键科学问题，本申请项目提出了锌指蛋白核酸内切酶（ZFN）介导整合HIV前病毒切除的"斩草除根"的策略；拟以已经建立的HIV感染细胞或潜伏感染细胞为靶细胞，以在HIV-1各亚型中高度保守的LTR序列为特异靶点，设计并筛选出了一对靶向切除率较高的ZFN-LTR；将ZFN-LTR及其对照载体分别转入靶细胞，检测分析靶细胞前病毒基因缺失的效率，利用DNA序列分析方法进一步证实HIV前病毒基因的缺失，探讨DNA修复的分子机制；利用定量PCR及ELISA方法，检测分析其抗病毒活性；检测分析细胞毒性和基因毒性，评价ZFN-LTR的 安全性。通过上述研究，旨在获得具有靶向性和有效性于一体的介导HIV前病毒基因缺失的ZFN-LTR，探索出有效清除整合病毒的新方法，为HIV彻底治愈开辟新途径。

HIV-infected individuals currently cannot be completely cured because existing antiviral therapy cannot specifically delete the integrated HIV proviral DNA and the latently infected cells establish a stable reservoir which cannot be easily eradicated. To solve this key scientific challenge, we propose here in this project a new strategy that can treat the disease at the root cause by using specially designed zinc-finger nucleases (ZFN) to remove integrated HIV provirus, flanked by long terminal repeats (LTRs). Using an LTR sequence highly conserved in all HIV-1 subtypes, we commercially designed and screened out one ZFN pair, named ZFN-LTR, which can specifically target HIV-1 provirus LTR with high efficiency in yeast MEL-1 reporter assay . Transfecting ZFN-LTR and control plasmids into the HIV-1 infected or latently infected cell models we've established, we will detect the effectiveness of ZFN-LTR by flow cytometry after treat the cells with ZFN-LTR; and we will further verify the removal of integrated HIV proviral DNA by sequence analysis and explore the mechanism of DNA repair. To address the antiretroviral activity of ZFN-LTR, we will measure the copy number of HIV-1-encoded gag gene by quantitative real-time PCR and the expression level of P24 protein by ELISA. On the other hand, we will analyze the cytotoxicity and genotoxicity of ZFN-LTR to assay its safety. Through the above research, this project aim is to get one ZFN pair that can specifically and effectively mediate HIV proviral gene deletion, exploring a new method for effective removal of the integrated virus, and opening a new avenue to the complete cure for HIV.