Single Cell Transcriptomic and Epigenetic Analysis of CD4 T Cells Harboring Latent HIV during Antiretroviral Therapy

抗逆转录病毒治疗期间携带潜在 HIV 的 CD4 T 细胞的单细胞转录组学和表观遗传学分析

• 批准号: 10160363
• 负责人: Iain Clark
• 金额: $ 15.81万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10160363
• 关键词: ATAC-seq; Achievement; Address; Architecture; Biological Assay; Biological Markers; Blood; CD4 Positive T Lymphocytes; Cell Separation; Cell model; Cells; Cellular Structures; Chromatin; Chromatin Structure; Clinic; Clinical Data; Collaborations; Couples; DNA; Data; Data Set; Defective Viruses; Discrimination; Enrollment; Environment; Epigenetic Process; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Genome; Genomic DNA; Genomics; Goals; HIV; HIV Genome; HIV Seropositivity; Highly Active Antiretroviral Therapy; Immune system; Individual; Infection; Length; Life Cycle Stages; Link; Methodology; Methods; Microfluidics; Molecular; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Pharmacotherapy; Population; Process; Prospective Studies; Proviruses; RNA; RNA Sequences; Regulatory Element; Research; Sampling; Site; Sorting - Cell Movement; Source; System; Testing; Viral; Viral Genome; Viremia; Virion; Virus; Virus Diseases; Virus Integration; Work; antiretroviral therapy; base; deep sequencing; differential expression; drug development; genome-wide; in vivo; integration site; latent HIV reservoir; microfluidic technology; novel; prevent; programs; single-cell RNA sequencing; transcription factor; transcriptome; transcriptome sequencing; transcriptomics; viral rebound

PROJECT SUMMARY Human Immunodeficiency Virus (HIV) infection remains incurable, despite highly active antiretroviral therapy (ART) that effectively targets the viral life cycle. A small population of long-lived CD4 T cells harbors replication competent virus (the latent HIV reservoir) that rebounds when treatment is stopped. The latent reservoir is a major barrier to curing HIV but is poorly understood due to technical challenges that prevent its characterization. To address this, I developed an ultra-high throughput droplet microfluidic workflow called PCR activated cell sorting (PACS) that detects, sorts, and sequences cells containing a single copy of intracellular HIV genomic DNA. My preliminary work has demonstrated that PACS can 1) process millions of cells, 2) detect HIV DNA in single cells from ART-suppressed individuals, and 3) sort infected cells and determine their transcriptional differences by differential expression analysis. Based on these results, PACS provides a unique method to study HIV infected cells ex vivo. I hypothesize that there are specific genomic mechanisms in HIV infected cells that allow them to harbor replication competent provirus and evade the immune system. This proposal couples PACS to single cell RNA-seq and ATAC-seq in order to define what, if any, are the unique signatures of infected cells in vivo. Simultaneous sequencing of viral genomes and integration sites (in collaboration with Dr. Kearney) will allow discrimination of intact from defective virus and provide the unique opportunity to link RNA-seq and ATAC-seq datasets. This work will use samples from people on ART suppression enrolled in SCOPE, an ongoing prospective study with carefully curated clinical data, further increasing the translational value of this research. Understanding the cellular mechanisms that allow HIV to persist in vivo is critical for guiding the selection of latency reversing agents, and in the development of drugs that specifically target infected cells.

项目概要 尽管进行了高效的抗逆转录病毒治疗，人类免疫缺陷病毒（HIV）感染仍然无法治愈 （ART）有效地针对病毒生命周期。一小群长寿的 CD4 T 细胞具有复制能力 当治疗停止时，活性病毒（潜在的艾滋病毒储存库）会反弹。潜在储层是 治愈艾滋病毒的主要障碍，但由于技术挑战阻碍其表征，人们对此知之甚少。 为了解决这个问题，我开发了一种超高通量液滴微流体工作流程，称为 PCR 激活细胞 分选 (PACS)，用于检测、分选和测序含有细胞内 HIV 基因组单拷贝的细胞 脱氧核糖核酸。我的初步工作表明 PACS 可以 1) 处理数百万个细胞，2) 检测体内的 HIV DNA 来自 ART 抑制个体的单细胞，3) 对受感染的细胞进行分类并确定其转录 通过差异表达分析得出差异。基于这些结果，PACS 提供了一种独特的研究方法 HIV感染的细胞离体。 我假设 HIV 感染细胞中存在特定的基因组机制，使它们能够隐藏 具有复制能力的原病毒并逃避免疫系统。该提案将 PACS 与单细胞耦合 RNA-seq 和 ATAC-seq 旨在定义体内受感染细胞的独特特征（如果有）。 病毒基因组和整合位点的同步测序（与科尔尼博士合作）将允许 区分完整病毒和缺陷病毒，并提供连接 RNA-seq 和 ATAC-seq 的独特机会 数据集。这项工作将使用 SCOPE 中登记的 ART 抑制患者的样本，SCOPE 是一个正在进行的项目 精心策划的临床数据的前瞻性研究，进一步增加了这项研究的转化价值。 了解 HIV 在体内持续存在的细胞机制对于指导选择 潜伏期逆转剂，以及专门针对感染细胞的药物的开发。