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

基本信息

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
项目类别：
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

项目摘要

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）：项目摘要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，即使这些药物多年来已很好地控制了 HIV-1 病毒血症，HAART 的终止很可能会导致病毒反弹。来自长寿命记忆 CD4+ T 细胞中的潜伏原病毒，大多数消除潜伏 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 的储存细胞。因此，基因编辑工具的技术进步为直接灭活储存细胞内潜伏的 HIV-1 原病毒提供了一种潜在的方法，但这些努力基本上都没有成功。系统是一种用于在基因组内产生靶向双链断裂的多功能平台，已被证明可以在细胞系中切除 HIV-1 原病毒。我们开发了一种新型核酸酶架构，将 Cas9 的有利切割活性与转录激活因子样效应器 (TALE) 结构域的靶向特异性结合起来，该 Cas9-TALE 系统显着提高了 DNA 切割的活性和精度。我们建议优化该核酸酶平台，以灭活记忆 CD4+ T 细胞中的 HIV-1 原病毒，这是潜在原病毒的主要细胞库之一。用于中和原病毒的核酸酶系统还需要静态储存细胞中原病毒局部染色质景观的详细图谱，以确定它特别容易受到攻击的区域。然而，获得所需的潜伏感染的原代细胞的数量。经过检查，在目标 1 中，我们将采用基因组编辑技术来生成在基因组内的特定位点和方向上包含原病毒插入的 CD34+ 细胞群。在 NSG-BLT 小鼠中，这些 CD34+ 细胞将生成具有统一原病毒整合位点的静息 CD4+ 细胞群，以进行深度表征，这可能揭示储库细胞中重要且意想不到的潜伏驱动因素。 TALE 系统能够以高精度高效、精确地灭活 HIV-1 原病毒。初步优化将在基于 Jurkat 的细胞系中进行，然后将转移到潜伏感染的体外模型。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对潜伏的 HIV 原病毒进行评估，并在接受 HAART 的患者的静息 CD4+ 细胞上进行评估。能够有效地从静止细胞中灭活原病毒，而不损害宿主基因组。