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

• 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
• 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

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+药物未激活潜伏感染T细胞的单细胞转录 用户 阿片类药物的使用改变了大脑的表观遗传结构，但它对主要储存库CD4+记忆T细胞的影响 至于潜伏的HIV-1，仍不得而知。这一应用的中心假设是记忆T细胞的身份 携带潜伏的HIV-1病毒会因阿片类药物暴露而改变，因此，在患有阿片类药物的患者中识别特定的生物标志物 阿片类药物的使用将是有价值的。这一假说是基于已发表的结果提出的，这些结果表明阿片类药物 受体在CD4+T细胞上表达，通过这些受体信号调节T细胞的激活和 差异化。核心假设将以一种双管齐下、高度里程碑驱动的方式进行检验。在 创新阶段(R61)，我们的目标将优化两项必要的技术：1)Tracker-Cas9-Q，一种新的CRISPR- 基于体内DNA标记技术以可视化潜伏感染的T细胞，以及2)单细胞RNA测序 以及相关的计算分析，以实现稳健的生物标记物开发。目标1：给HIV-1前病毒贴上标签 利用新的CRISPR-Cas蛋白质技术在完整细胞中定位。Tracker-Cas9-Q是一款新的 荧光标记的，但内部猝灭的，催化不活性的CRISPR/Cas9和特定的 CRISPR指导仅在DNA结合时发出荧光的RNA(Murthy Lab)。潜在的工作假说 在纳米金微粒(CRISPR-Gold)内传递的Tracker-Cas9-Q允许有效的活体标记 以及含有潜伏的HIV DNA的T细胞系的流动分选。目的2：建立单细胞RNA-Seq和 双荧光HIV-1体外感染原代T细胞的计算机生物标志物鉴定 不接触阿片类药物。单个细胞的转录组--单独的或复杂的细胞群体--现在可以 在足够深的地方进行分析(Ott Lab)，以便在感染艾滋病毒的原代细胞中进行可靠的生物标志物开发 人口(约瑟夫实验室)。我们的工作假设是，个体潜伏感染的初级T细胞可以是 使用RNA-Seq在单细胞基础上有效地分离和分析。为了进入R33阶段，在 在R61期结束时，我们将开发出一种高效交付的HIV特异性DNA标记系统 (&gt；50%)和可分类的荧光强度，并建立了单细胞RNA-Seq和计算平台 用于生物标记物的开发。R33阶段有一个目标，组合了AIMS 1和AIMS的实验系统 2从接受抗逆转录病毒治疗的无核型HIV+患者分离的原代T细胞中。目标3：刻画 在单细胞水平潜伏感染的记忆T细胞，从使用和不使用阿片类药物的HIV+患者中分离出来 使用，使用基于CRISPR的新标签技术。我们的工作假设是，新开发的 Tracker-Cas9-Q和CRISPR-Gold技术将有效地标记患者来源T细胞中的潜伏前病毒， 并与单细胞RNA-Seq和计算生物标志物分析相结合，将产生根本性的新见解 在使用和不使用阿片类药物的HIV+患者中，潜伏的储藏细胞的身份。我们的研究将有助于 从根本上讲，新技术和可翻译的生物学知识有助于研究阿片类药物使用者的艾滋病毒潜伏期。