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Single-Cell Transcriptomics of Non-Activated Latently Infected T cells Isolated from HIV+ Drug Users

从 HIV 吸毒者中分离出的非激活潜伏感染 T 细胞的单细胞转录组学

基本信息

批准号：
10548752
负责人：
Melanie Maria Ott
金额：
$ 94.18万
依托单位：
J. DAVID GLADSTONE INSTITUTES
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-03-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10548752
关键词：
Acquired Immunodeficiency Syndrome Address BCAR1 gene Biological Biological Markers Biology Brain CD4 Positive T Lymphocytes CRISPR/Cas technology Cell Line Cell Separation Cells Clustered Regularly Interspaced Short Palindromic Repeats Complex Computer Analysis Coupled DNA DNA Binding Data Data Set Detection Development Drug Exposure Drug usage Drug user Emerging Technologies Epidemic Epigenetic Process Failure Fluorescence Funding Gene Expression Gene Silencing Genetic Transcription Gold Guide RNA HIV HIV Infections HIV-1 Health Image Individual Infection Knowledge Label Life Mission Molecular Opioid Receptor Patients Phase Population Procedures Proteins Provirus Integration Proviruses Public Health Publishing RNA Regulation Reporter Research Research Support Rest Ribonucleoproteins Signal Transduction Sorting Stains Structure Surface System T memory cell T-Cell Activation T-Lymphocyte T-Lymphocyte Subsets Techniques Technology Testing United States National Institutes of Health Virus Virus Activation Visualization antiretroviral therapy biomarker development biomarker identification cell type computational platform drug of abuse fluorophore in vivo innovation insight latent HIV reservoir memory CD4 T lymphocyte nanoGold new technology new therapeutic target novel novel diagnostics opioid exposure opioid use opioid user particle receptor single cell analysis single-cell RNA sequencing specific biomarkers tool transcriptome transcriptome sequencing transcriptomics uptake

项目摘要

SINGLE-CELL TRANSCRIPTOMICS OF NON-ACTIVATED LATENTLY INFECTED T CELLS ISOLATED FROM HIV+ DRUG USERS Opioid use alters the epigenetic structure of the brain but its effects on CD4+ memory T cells, the main reservoir for latent HIV-1, remain unknown. The central hypothesis of this application is that the identity of memory T cells carrying latent HIV-1 is altered by opioid exposure, and thus, identifying specific biomarkers in patients with opioid use would be valuable. This hypothesis was formulated based on published results showing that opioid receptors are expressed on CD4+ T cells and signaling through these receptors modulates T-cell activation and differentiation. The central hypothesis will be tested in a two-pronged, highly milestone-driven approach. In the innovation phase (R61), our aims will optimize two necessary technologies: 1) Tracker-Cas9-Q, a new CRISPR- based in vivo DNA-labeling technique to visualize latently infected T cells, and 2) single-cell RNA sequencing and associated computational analysis for robust biomarker development. Aim 1: To label the HIV-1 proviral locus in intact cells by harnessing novel CRISPR-Cas protein technologies. Tracker-Cas9-Q is a new fluorescently labeled, but internally quenched, complex of catalytically inactive CRISPR/Cas9 and specific CRISPR guide RNAs that only fluoresces upon DNA binding (Murthy Lab). The underlying working hypothesis is that Tracker-Cas9-Q delivered within nanogold microparticles (CRISPR-Gold) allows efficient in vivo labeling and flow sorting of T cell lines containing latent HIV DNA. Aim 2: To establish single-cell RNA-Seq and computational biomarker identification in primary T cells ex vivo infected with dual-fluorescent HIV-1 with and without opioid exposure. The transcriptome of individual cells – alone or in complex cell populations – can now be analyzed at sufficient depth (Ott Lab) to allow reliable biomarker development in HIV-infected primary cell populations (Yosef Lab). Our working hypothesis is that individual latently infected primary T cells can be efficiently isolated and analyzed on a single-cell basis using RNA-Seq. In order to progress to the R33 phase, at the end of the R61 period we will have developed an HIV-specific DNA labeling system with efficient delivery (>50%) and sortable fluorescence intensities and established single-cell RNA-Seq and computational platforms for biomarker development. The R33 phase has one aim combining the experimental systems from Aims 1 and 2 in primary T cells isolated from aviremic HIV+ individuals under antiretroviral therapy. Aim 3: To characterize latently infected memory T cells at the single-cell level, isolated from HIV+ individuals with and without opioid use, using novel CRISPR-based labeling techniques. Our working hypothesis is that the newly developed Tracker-Cas9-Q and CRISPR-Gold technologies will efficiently label the latent provirus in patient-derived T cells, and combined with single-cell RNA-Seq and computational biomarker analysis, will yield fundamental new insight into the identity of latent reservoir cells in HIV+ individuals with and without opioid use. Our studies will contribute fundamentally new technologies and translatable biological knowledge to the study of HIV latency in opioid users.

hiv +药物分离的非活化潜伏感染t细胞的单细胞转录组学