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

用于精确切除潜伏 HIV-1 原病毒的下一代杂交核酸酶

• 批准号: 9010933
• 负责人: JEREMY LUBAN
• 金额: $ 91.43万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-15 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9010933
• 关键词: ATAC-seq; Architecture; Area; Beryllium; CD34 gene; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cell Line; Cells; Chromatin; DNA; Development; Disease; Drug Controls; Engineering; Excision; Gene Targeting; Generations; Genes; Genetic Transcription; Genome; Genomics; Goals; HIV; HIV Infections; HIV-1; Harvest; Health; Highly Active Antiretroviral Therapy; Hybrids; Individual; Infection; Interphase Cell; Life; Maps; Mediating; Memory; Methods; Molecular Conformation; Mus; Nucleosomes; Patients; Population; Positioning Attribute; Proviruses; Public Health; Research Personnel; Resolution; Resources; Rest; Site; Specificity; System; Technology; Transcription Coactivator; Viral; Viremia; base; chromosome conformation capture; clinical application; genome editing; humanized mouse; improved; in vitro Model; in vivo; integration site; medical specialties; memory CD4 T lymphocyte; mouse model; next generation; novel; nuclease; outcome forecast; tool; transcription activator-like effector nucleases

 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.

 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，即使多年来这些药物已经很好地控制了HIV-1病毒血症，终止HAART也会导致病毒反弹，很可能来自长期记忆的CD4+T细胞中的潜伏前病毒。大多数根除潜伏的HIV-1前病毒的努力都集中在重新激活前病毒转录以增强消除携带HIV-1前病毒的存储细胞，但这些努力在很大程度上是不成功的。因此，需要采用替代方法来消除前驱病毒。基因编辑工具的技术进步为直接灭活储存细胞中潜伏的HIV-1前病毒提供了一种潜在的方法。具体地说，Cas9/CRISPR可编程核酸酶系统是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系统的活性和精密度在临床应用中并不理想。我们开发了一种新的核酸酶结构，它结合了Cas9良好的切割活性和转录激活因子样效应域(TALE)的靶向性。这个Cas9系统极大地提高了DNA切割的活性和精确度。我们建议优化这个核酸酶平台，用于在记忆中的CD4+T细胞中灭活HIV-1前病毒，记忆CD4+T细胞是潜伏前病毒的主要细胞库之一。为了有效地中和前病毒，开发一个强大的核酸酶系统还需要一张前病毒在静止的水库细胞中的局部染色质景观的详细地图，以确定它特别容易受到攻击的区域。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，我们将使用基因组编辑技术来生成包含前病毒插入到基因组中特定位置和方向的CD34+细胞群体。一旦将CD34+细胞移植到NSG-BLT小鼠体内，这些CD34+细胞将产生具有统一的前病毒整合位点的静止的CD4+细胞群体，用于I-深度鉴定，这可能揭示储藏细胞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-Tell系统，以高效、准确地高精度灭活HIV-1前病毒。最初的优化将对基于Jurkat的细胞系进行，然后将转移到潜伏感染的中央记忆CD4+细胞的体外模型。在目标3中，这些优化的核酸酶将在HAART抑制的HIV-1感染人源化小鼠模型中的潜伏HIV前病毒上进行评估，并在接受HAART治疗的患者的静止CD4+细胞上进行评估。我们研究中经过验证的核酸酶将能够有效地灭活静止细胞中的前病毒，而不会损害宿主基因组。