Next generation hybrid nucleases for precise excision of latent HIV-1 provirus

 描述（由申请人提供）： 高效抗逆转录病毒疗法（HAART）极大地改变了HIV-1感染者的预后。然而，即使HIV-1病毒血症已经被这些药物很好地控制了多年，HAART的终止也会导致病毒反弹，最有可能来自长寿记忆CD 4 + T细胞中的潜伏前病毒。大多数消除潜伏的HIV-1前病毒的努力都集中在重新激活前病毒转录以加强消除携带HIV-1前病毒的储库细胞，但这些努力在很大程度上是不成功的。因此，需要用于前病毒消除的替代方法。 基因编辑工具的技术进步提供了一种潜在的方法，可以直接灭活储存细胞内潜伏的HIV-1前病毒。具体而言，Cas9/CRISPR可编程核酸酶系统，一种用于在基因组内产生靶向双链断裂的通用平台，已被证明可以切除细胞系中的HIV-1前病毒。然而，Cas9/CRISPR系统的活性和精确度对于临床应用而言是次优的。我们已经开发了一种新的核酸酶结构，其将Cas9的有利切割活性与转录激活因子样效应物（TALE）结构域的靶向特异性相结合。这种Cas9-TALE系统显著提高了DNA切割的活性和精度。我们建议优化这种核酸酶平台，用于记忆性CD 4 + T细胞中HIV-1前病毒的灭活，记忆性CD 4 + T细胞是潜伏前病毒的主要细胞库之一。 开发一个强大的核酸酶系统，有效中和原病毒，也需要一个详细的地图，局部染色质景观的原病毒在静止的水库细胞，以确定在哪些领域，它是特别容易受到攻击。然而，获得这些分析所需的潜伏感染原代细胞的数量是不可行的。因此，在目标1中，我们将采用基因组编辑技术来产生在基因组内的特定位点和方向含有前病毒插入的CD 34+细胞群体。一旦移植到NSG-BLT小鼠中，这些CD 34+细胞将产生具有用于i-深度表征的均匀前病毒整合位点的静息CD 4+细胞群，这可能揭示储库细胞中潜伏期的重要和意想不到的驱动因素。 在目标2中，我们将优化Cas9-TALE系统，以高精度高效精确地表达HIV-1前病毒。将在基于Jurkat的细胞系中进行初始优化，然后将转移到潜伏感染的中央记忆CD 4+细胞的体外模型。在目标3中，将在HAART抑制的人源化HIV-1感染小鼠模型中的潜伏HIV前病毒上以及在来自经历HAART的患者的静息CD 4+细胞上评价这些优化的核酸酶。我们的研究验证的核酸酶将能够有效地从静止细胞中去除前病毒，而不会损害宿主基因组。

 DESCRIPTION (provided by applicant): Project Summary Highly active antiretroviral therapy (HAART) has dramatically changed the prognosis for individuals infected with HIV-1. Yet, even when HIV-1 viremia has been well controlled by these drugs for years, termination of HAART results in viral rebound, most likely coming from latent provirus in long-lived memory CD4+ T cells. Most efforts to eradicate latent HIV-1 proviruses have focused on reactivation of proviral transcription to potentiate the elimination of reservoir cells harboring HIV-1 provirus, but these efforts have largely been unsuccessful. Alternative approaches for proviral elimination are therefore needed. Technological advances in gene editing tools provide a potential method for direct inactivation of latent HIV-1 provirus within reservoir cells. Specifically, the Cas9/CRISPR programmable nuclease system, a versatile platform for the generation of targeted double-strand breaks within the genome, has been shown to excise HIV-1 provirus in cell lines. However, the activity and precision of the Cas9/CRISPR system is suboptimal for clinical application. We have developed a novel nuclease architecture that combines the favorable cleavage activity of Cas9 with the targeting specificity of Transcription Activator-Like Effector (TALE) domains. This Cas9-TALE system dramatically improves the activity and precision of DNA cleavage. We propose to optimize this nuclease platform for the inactivation of HIV-1 provirus in memory CD4+ T cells, one of the primary cellular reservoirs of latent provirus. Development of a robust nuclease system for the efficient neutralization of provirus will also require a detailed map of the local chromatin landscape of the provirus in quiescent reservoir cells to identify areas at which it is particularly vulnerable to attack. However, obtaining the quantities of latently-infected primary cells needed for these analyses is not feasible. Consequently, in Aim 1 we will employ genome-editing technologies to generate populations of CD34+ cells containing a proviral insertion at a specific site and orientation within the genome. Once engrafted in NSG-BLT mice, these CD34+ cells will generate populations of resting CD4+ cells with uniform proviral integration sites for i-depth characterization, which may reveal important and unexpected drivers of latency in reservoir cells. In Aim 2, we will optimize the Cas9-TALE system to efficiently and precisely inactivate HIV-1 provirus with a high degree of precision. Initial optimization will be performed i Jurkat-based cell lines and then will move to an in vitro model of latently-infected central memory CD4+ cells. In Aim 3, these optimized nucleases will be evaluated on latent HIV provirus in a HAART-suppressed humanized mouse model of HIV-1 infection, and on resting CD4+ cells from patients undergoing HAART. Validated nucleases from our studies will be able to inactivate provirus efficiently from quiescent cells without compromising the host genome.