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.

项目摘要 人类免疫缺陷病毒（HIV）持续存在于感染患者目的地的长寿命潜在储层中 连续长期用高度活性抗逆转录病毒药物治疗。旨在的治疗疗法 通过使用潜伏期逆转剂（LRA）重新激活并清除这些潜在感染的细胞 作为“震惊和杀戮”的方法，但是由于不完整的重新激活，这些方法在很大程度上失败了 潜在游泳池。虽然人们认为细胞环境和前病毒整合位点的差异是负责任的 对于这种可变性，实际上几乎没有了解特定宿主因素如何影响潜伏期和LRA 在主要模型中的潜力。在早期整合和潜伏期期间做事件最终会影响 延迟维护和重新激活？人们认为大多数潜在储层由 静止的CD4+ T细胞具有具有胜任的复制能力，但在转录中无声的遗嘱病毒。这些细胞 传统上很难产生离体，甚至更难操纵很难 用于研究特定宿主因素。原代细胞潜伏模型和基因组的最新进展 但是，工程学使这些研究首次可行。在此提案中，我打算测试 假设艾滋病毒整合，前病毒沉默和潜伏期的早期事件有助于决定 延迟维持和重新激活潜力。为了检验这一假设，我将使用一个新颖的平台进行 原代T细胞中的CRISPR/CAS9编辑以消除涉及集成站点偏好涉及的两个宿主因素， LEDGF和CPSF6。这些细胞将被双荧光报告病毒感染以监测提供商 通过深度测序确定的沉默和集成位点曲线。这些单元将返回到 静态状态并用代表性的LRA对靶向不同的功能途径进行质疑 延迟维持并确定重新激活电位是否与早期观察到的差异相关 在每个遗传背景中的感染（AIM 1）。相反，最初旨在改变的LRA 延迟维护可能会在早期整合和延迟建立期间改变事件，因此 作为识别这些过程中涉及的新宿主因素的分子问题。在这一目标中， 原代T细胞将用一组LRA进行预处理，并感染了双荧光记者病毒，并且 监视整合，临时沉默和潜伏期的差异（AIM 2）。直系同源 这些途径中的小分子和靶向遗传敲除将用于验证这些发现，并且 确认新型延迟宿主因素的身份。综上所述，这些数据将是第一个分析 直接在 原代T细胞。了解感染早期发生的宿主依赖性事件与 慢性病期间的治疗效率对于开发新的个性化疗法至关重要 治疗和治愈不仅艾滋病毒的策略，还可以治愈其他疾病。