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.

从 HIV+ 药物中分离出的未激活潜伏感染 T 细胞的单细胞转录组学 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 CD4+ 记忆 T 细胞（主要储存库）有影响 对于潜伏的 HIV-1，仍然未知。本申请的中心假设是记忆 T 细胞的身份 携带潜伏 HIV-1 的病毒会因暴露于阿片类药物而改变，因此可以识别患有潜伏性 HIV-1 的患者中的特定生物标志物 阿片类药物的使用将是有价值的。这一假设是根据已发表的结果制定的，该结果表明阿片类药物 受体在 CD4+ T 细胞上表达，通过这些受体的信号传导调节 T 细胞的激活和 差异化。中心假设将通过双管齐下、高度里程碑驱动的方法进行检验。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 标记技术来可视化潜伏感染的 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志物开发。目标 1：标记 HIV-1 前病毒 通过利用新型 CRISPR-Cas 蛋白质技术，在完整细胞中定位基因座。 Tracker-Cas9-Q 是一种新的 荧光标记但内部淬灭的催化失活 CRISPR/Cas9 和特异性复合物 CRISPR 引导 RNA 仅在 DNA 结合时发出荧光（Murthy 实验室）。基本的工作假设 纳米金微粒 (CRISPR-Gold) 内传递的 Tracker-Cas9-Q 可实现高效的体内标记 以及含有潜伏 HIV DNA 的 T 细胞系的流式分选。目标 2：建立单细胞 RNA-Seq 并 离体感染双荧光 HIV-1 的原代 T 细胞的计算生物标志物鉴定 没有阿片类药物暴露。单个细胞的转录组——单独的或复杂的细胞群——现在可以 进行足够深度的分析（Ott Lab），以便在感染 HIV 的原代细胞中开发可靠的生物标志物 人群（约瑟夫实验室）。我们的工作假设是，个体潜伏感染的原代 T 细胞可以 使用 RNA-Seq 在单细胞基础上进行有效分离和分析。为了进入R33阶段，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 (>50%) 和可分选的荧光强度以及已建立的单细胞 RNA-Seq 和计算平台 用于生物标志物开发。 R33 阶段的目标之一是结合目标 1 和 2 从接受抗逆转录病毒治疗的无病毒血症 HIV+ 个体中分离出的原代 T 细胞。目标 3：表征 单细胞水平上潜伏感染的记忆 T 细胞，从使用或不使用阿片类药物的 HIV+ 个体中分离出来 使用基于 CRISPR 的新型标记技术。我们的工作假设是新开发的 Tracker-Cas9-Q 和 CRISPR-Gold 技术将有效标记患者来源 T 细胞中的潜伏原病毒， 并与单细胞 RNA 测序和计算生物标志物分析相结合，将产生根本性的新见解 调查使用和不使用阿片类药物的 HIV+ 个体中潜在储存细胞的身份。我们的研究将做出贡献 根本性的新技术和可转化的生物学知识用于研究阿片类药物使用者的艾滋病毒潜伏期。