Disabling the replication-competent HIV provirus in the reservoir by combined CRISPR-Cas action

总结。DNA可以成为CRISPR的靶标- CAS(CC)基因组编辑工具，该工具迅速 在抗艾滋病毒研究中实施1-8项。我们确认了 强有力的艾滋病毒抑制作用，但也表现出快速 病毒逃脱CC攻击9。视察 逃逸病毒表明NHEJ的作用 酶，修复dsDNA断裂和 中引入小的插入/删除(Indels) 进程。换句话说，我们描述了一种新的病毒 一条由细胞DNA促成的逃生路线 修复过程。这些结果得到了以下证实 其他实验室10-21。一种针对CC的组合攻击 保守序列触发HIV灭活 通过超突变，与 建议的切除路线(图1)。尽管前病毒 在我们的系统中可以检测到切除，它 以非常低的效率发生。相反，抄送 攻击产生了高度突变的前病毒 显然已经失去了所有的复制潜力。在其他 换句话说，连续的抄送行动可以在功能上 治愈感染艾滋病毒的细胞，让细胞拥有 灭活的艾滋病毒前病毒的墓地。这 令人惊讶的发现代表了艾滋病毒的一个主要标志 治愈研究。我们现在建议首先划定 潜在的机制和变量 确定CC攻击的结果(艾滋病毒切除 与超突变相比)。这将使我们能够 制定最佳治疗策略。与此同时，我们关注的是治愈的主要障碍： 将基因治疗药物输送到构成艾滋病毒储存库的细胞中。CC和交付工具 然后将在His小鼠体内进行体内治疗研究。

Summary. DNA can be targeted by the CRISPR- Cas (CC) genome editing tool, which was swiftly implemented in anti-HIV studies1-8. We confirmed potent HIV inhibition, but also demonstrated rapid virus escape from CC attack9. Inspection of the escape viruses indicated a role of the NHEJ enzyme, which repairs dsDNA breaks and introduces small insertions/deletions (indels) in the process. In other words, we described a novel viral escape route that is facilitated by a cellular DNA repair process. These results were confirmed by other labs10-21. A combinatorial CC attack against conserved sequences triggered HIV inactivation through hypermutation, quite different from the proposed excision route (Fig 1). Although provirus excision could be detected in our system, it occurred with a very low efficiency. Instead, CC attack generated hypermutated proviruses that apparently lost all replication potential. In other words, continuous CC action can functionally CURE HIV infected cells, leaving the cells with a graveyard of inactivated HIV proviruses. This surprise finding represents a major hallmark in HIV CURE studies. We now propose to first delineate the underlying mechanisms and variables that determine the outcome of CC attack (HIV excision versus hypermutation). This will allow us to formulate the best CURE strategy. At the same time we focus on the major obstacle towards a CURE: delivery of the gene therapeutics to cells that compose the HIV-reservoir. The CC and delivery tools will then be combined for in vivo CURE studies in the HIS mouse.