Highly active anti-retroviral therapy (HAART) cannot clear the infected cells harboring HIV-1 proviral DNA from the HIV-1 infected patients. We previously demonstrated that Zinc-finger nucleases (ZFNs) could specifically and efficiently excise HIV-1 proviral DNA from the latently infected human T cells by targeting the long terminal repeats (LTRs), a novel and alternative antiretroviral strategy for eradicating HIV-1 infection. To prevent the unwanted off-target effects from constantly expressed ZFNs, in this study, we engineered the expression of ZFNs under the control of HIV-1 LTR, by which ZFNs expression can be activated by the HIV-1 Tat protein. Our results show that functional expression of ZFNs induced by Tat excised the integrated proviral DNA of HIV-NL4-3-eGFP in approximate 30% population of HIV-1 infected cells. Meanwhile, the results from HIV-1 infected human primary T cells and latently infected T cells treated with the regulated ZFNs further validated proviral DNA could be excised. Taken together, positively regulated expression of ZFNs in the presence of HIV-1 Tat may provide a safer and novel implementation of the genome-editing technology for eradicating the HIV-1 proviral DNA from the infected host cells.

高效抗逆转录病毒疗法（HAART）无法从感染HIV-1的患者中清除带有HIV-1前病毒DNA的感染细胞。我们先前证明，锌指核酸酶（ZFN）可以通过靶向长末端重复序列（LTR），从潜在感染的人类T细胞中特异性和有效地切除HIV-1前病毒DNA，这是一种新的替代性抗逆转录病毒策略，可消除HIV-1感染。为了防止持续表达的ZFN产生不必要的脱靶效应，在这项研究中，我们设计了HIV-1 LTR控制下ZFNs的表达，通过该表达，HIV-1 Tat蛋白可以激活ZFNs的表达。我们的结果表明，由Tat诱导的ZFNs的功能表达在大约30％的HIV-1感染细胞中切除了HIV-NL4-3-eGFP的整合前病毒DNA。同时，从HIV-1感染的人原代T细胞和用调节的ZFNs处理的潜伏感染的T细胞得到的结果可以进一步切除，以进一步验证原病毒DNA。综上所述，在存在HIV-1 Tat的情况下ZFNs的正向表达可能会为从受感染宿主细胞中消除HIV-1前病毒DNA的基因组编辑技术提供更安全，新颖的实施方法。