The Influence of Early Integration Events on HIV Latency and Reactivation Potential

早期整合事件对 HIV 潜伏期和再激活潜力的影响

• 批准号: 9750620
• 负责人: Judd F Hultquist
• 金额: $ 10.8万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-25 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750620
• 关键词: Ablation; Anti-Retroviral Agents; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cell Culture Techniques; Cell Line; Cell model; Cells; Chromatin; Chronic Disease; Complex; DNA; Data; Development; Disease; Effectiveness; Environment; Epigenetic Process; Event; Feasibility Studies; Flow Cytometry; Genes; Genetic; Genetic Epistasis; Genetic Transcription; Genome; Genome engineering; Goals; HIV; HIV Infections; HIV Long Terminal Repeat; Heterogeneity; Histone Deacetylase Inhibitor; Immune response; Immunologics; Infection; Integration Host Factors; Knock-out; Link; Location; Long Terminal Repeats; Maintenance; Modeling; Modification; Molecular; Molecular Probes; Monitor; Pathway interactions; Patients; Pattern; Penetrance; Pharmaceutical Preparations; Phenotype; Play; Predictive Factor; Probability; Process; Proviruses; Reporter; Role; Shock; Source; T-Lymphocyte; Testing; Therapeutic; Time; Treatment Efficacy; Virus; Virus Diseases; base; chromatin remodeling; curative treatments; deep sequencing; design; in vivo; inhibitor/antagonist; integration site; mouse model; novel; nucleocytoplasmic transport; personalized therapeutic; preference; promoter; reactivation from latency; small molecule; targeted agent; therapy design; tool; transcription factor; treatment strategy

PROJECT SUMMARY Human Immunodeficiency Virus (HIV) persists in long-lived, latent reservoirs in infected patients despite continuous, long-term treatment with highly active antiretroviral drugs. Curative therapies designed to reactivate and clear these latently infected cells through the use of latency reversing agents (LRAs) are known as “shock and kill” approaches, but these approaches have largely failed due to incomplete reactivation of the latent pool. While differences in cellular environment and proviral integration site are thought to be responsible for this variability, very little is actually known about how specific host factors can influence latency and LRA potential in primary models. Do events during early integration and latency establishment ultimately influence latency maintenance and reactivation? A majority of the latent reservoir is thought to be composed of quiescent CD4+ T cells harboring replication competent, but transcriptionally silent proviruses. These cells have traditionally been very difficult to generate ex vivo and have been even harder to manipulate genetically for the study of specific host factors. Recent advancements in primary cell latency models and genome engineering, however, have made these studies feasible for the first time. In this proposal, I intend to test the hypothesis that early events in HIV integration, proviral silencing, and latency establishment help dictate latency maintenance and reactivation potential. To test this hypothesis, I will use a novel platform for CRISPR/Cas9 editing in primary T cells to ablate two host factors involved in integration site preference, LEDGF and CPSF6. These cells will be infected with a dual fluorescent reporter virus to monitor proviral silencing and integration site profiles determined by deep sequencing. These cells will be returned to a quiescent state and treated with representative LRAs targeting distinct functional pathways to interrogate latency maintenance and determine if reactivation potential correlates with differences observed during early infection in each of genetic background (Aim 1). Conversely, LRAs that were originally designed to alter latency maintenance may alter events during early integration and latency establishment as well and therefore serve as molecular probes for identifying novel host factors involved in these processes. Towards this end, primary T cells will be pre-treated with a panel of LRAs, infected with a dual fluorescent reporter virus, and monitored for differences in integration, proviral silencing, and latency establishment (Aim 2). Orthologous small molecules in these pathways and targeted genetic knock-outs will be used to validate these findings and confirm the identity of novel latency host factors. Taken together, these data will be the first to analyze the relationship between host factors, HIV integration, latency establishment, and reactivation potential directly in primary T cells. Understanding how host-dependent events that occur early in infection are linked to therapeutic efficacy during chronic disease will be critical to the development of new personalized therapeutic strategies for the treatment and cure of not only HIV, but other disease states as well.

项目概要 尽管人类免疫缺陷病毒（HIV）在感染患者体内长期存在，但仍处于潜伏状态。 使用高活性抗逆转录病毒药物进行持续、长期治疗。 众所周知，通过使用潜伏期逆转剂（LRA）重新激活并清除这些潜伏感染的细胞 随着“休克和杀戮”的临近，但由于不完全重新激活这些方法，这些方法基本上都失败了 虽然细胞环境和原病毒整合位点的差异被认为是造成这种情况的原因。 对于这种可变性，实际上对于特定主机因素如何影响延迟和 LRA 知之甚少 早期集成和延迟建立期间的事件最终会影响主要模型中的潜力吗？ 潜伏期的维持和重新激活被认为是由大部分潜伏期组成的。 静止的 CD4+ T 细胞具有复制能力，但转录沉默的原病毒。 传统上很难在体外产生，并且更难以进行基因操纵 用于研究特定宿主因素的原代细胞潜伏期模型和基因组的最新进展。 然而，工程学首次使这些研究成为可能，在这个提案中，我打算测试一下。 假设 HIV 整合、前病毒沉默和潜伏期建立的早期事件有助于决定 为了测试这个假设，我将使用一个新的平台来测试延迟维持和重新激活的潜力。 原代 T 细胞中的 CRISPR/Cas9 编辑消除了参与整合位点偏好的两个宿主因子， LEDGF 和 CPSF6 将感染双荧光报告病毒以监测原病毒。 通过深度测序确定这些细胞的沉默和整合位点概况。 静止状态，并用代表性的 LRA 进行处理，针对不同的功能途径进行询问 潜伏期维持并确定再激活潜力是否与早期观察到的差异相关 每种遗传背景中的感染（目标 1），LRA 最初旨在改变。 延迟维护也可能会改变早期集成和延迟建立期间的事件，因此 作为分子探针来识别参与这些过程的新宿主因子。 原代 T 细胞将用一组 LRA 进行预处理，感染双荧光报告病毒，并且 监测整合、原病毒沉默和潜伏期建立方面的差异（目标 2）。 这些途径中的小分子和有针对性的基因敲除将用于验证这些发现和 综合起来，这些数据将首先分析新的延迟宿主因素的身份。 宿主因素、HIV 整合、潜伏期建立和再激活潜力之间的关系直接在 了解感染早期发生的宿主依赖性事件如何与原代 T 细胞相关。 慢性病期间的疗效将是开发新的个性化治疗的关键治疗方法 治疗和治愈艾滋病毒以及其他疾病的策略。