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.

项目总结 人类免疫缺陷病毒(HIV)持续在感染患者的长期潜伏宿主中，尽管 持续、长期使用高效抗逆转录病毒药物治疗。旨在治疗的疗法 通过使用潜伏期反转剂(LRA)重新激活和清除这些潜伏感染的细胞是已知的 随着“电击和杀戮”的临近，这些方法在很大程度上都失败了，因为 潜水池。而蜂窝环境和前病毒整合部位的差异被认为是原因 对于这种可变性，人们实际上对特定主机因素如何影响延迟和LRA知之甚少 在初级模型中的潜力。早期集成和延迟建立期间的事件最终是否会影响 延迟维护和重新激活？大部分潜在储集层被认为是由 静止的CD4+T细胞具有复制能力，但转录沉默的前病毒。这些细胞 传统上很难在体外产生，更难在基因上进行操作 用于研究特定的寄主因素。原代细胞潜伏期模型和基因组研究进展 然而，工程学第一次使这些研究变得可行。在这个提案中，我打算测试 假设HIV整合、前病毒沉默和潜伏期建立的早期事件有助于指示 潜伏期维护和重新激活潜力。为了验证这一假设，我将使用一个新的平台 在原代T细胞中编辑CRISPR/Cas9以消除整合位点偏好中涉及的两个宿主因素， LEDGF和CPSF6。这些细胞将被感染双荧光报告病毒以监测前病毒 由深度测序确定的沉默和整合位点图谱。这些单元格将返回到 静止状态，并使用针对不同功能通路的代表性LRA进行治疗以进行询问 潜伏期维持，并确定重新激活潜力是否与早期观察到的差异相关 每个遗传背景的感染(目标1)。相反，LRA最初设计为更改 延迟维护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，用于识别参与这些过程的新宿主因子。为此， 原代T细胞将用一组LRA进行预处理，感染双荧光报告病毒，以及 监测整合、前驱静默和潜伏期建立方面的差异(目标2)。正向同源 这些途径中的小分子和有针对性的基因敲除将被用于验证这些发现和 确认新的延迟主机因素的身份。总而言之，这些数据将是第一个分析 宿主因素、HIV整合、潜伏期建立和重新激活潜力之间的关系 初级T细胞。了解感染早期发生的宿主依赖事件如何与 慢性病的治疗效果将是新的个性化治疗方法发展的关键 不仅是艾滋病毒，而且是其他疾病状态的治疗和治愈战略。