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.

项目总结