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:
- 金额:$ 94.18万
- 依托单位:
- 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:
- 关键词:Acquired Immunodeficiency SyndromeAddressBCAR1 geneBiologicalBiological MarkersBiologyBrainCD4 Positive T LymphocytesCRISPR/Cas technologyCell LineCell SeparationCellsClustered Regularly Interspaced Short Palindromic RepeatsComplexComputer AnalysisCoupledDNADNA BindingDataData SetDetectionDevelopmentDrug ExposureDrug usageDrug userEmerging TechnologiesEpidemicEpigenetic ProcessFailureFluorescenceFundingGene ExpressionGene SilencingGenetic TranscriptionGoldGuide RNAHIVHIV InfectionsHIV-1HealthImageIndividualInfectionKnowledgeLabelLifeMissionMolecularOpioid ReceptorPatientsPhasePopulationProceduresProteinsProvirus IntegrationProvirusesPublic HealthPublishingRNARegulationReporterResearchResearch SupportRestRibonucleoproteinsSignal TransductionSortingStainsStructureSurfaceSystemT memory cellT-Cell ActivationT-LymphocyteT-Lymphocyte SubsetsTechniquesTechnologyTestingUnited States National Institutes of HealthVirusVirus ActivationVisualizationantiretroviral therapybiomarker developmentbiomarker identificationcell typecomputational platformdrug of abusefluorophorein vivoinnovationinsightlatent HIV reservoirmemory CD4 T lymphocytenanoGoldnew technologynew therapeutic targetnovelnovel diagnosticsopioid exposureopioid useopioid userparticlereceptorsingle cell analysissingle-cell RNA sequencingspecific biomarkerstooltranscriptometranscriptome sequencingtranscriptomicsuptake
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
(>;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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Melanie Maria Ott其他文献
Melanie Maria Ott的其他文献
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{{ truncateString('Melanie Maria Ott', 18)}}的其他基金
Modeling intestinal dysfunction in HIV infection with organoid technology
利用类器官技术模拟 HIV 感染的肠道功能障碍
- 批准号:
10542390 - 财政年份:2020
- 资助金额:
$ 94.18万 - 项目类别:
Modeling intestinal dysfunction in HIV infection with organoid technology
利用类器官技术模拟 HIV 感染的肠道功能障碍
- 批准号:
9894660 - 财政年份:2020
- 资助金额:
$ 94.18万 - 项目类别:
Modeling intestinal dysfunction in HIV infection with organoid technology
利用类器官技术模拟 HIV 感染的肠道功能障碍
- 批准号:
10083740 - 财政年份:2020
- 资助金额:
$ 94.18万 - 项目类别:
Modeling intestinal dysfunction in HIV infection with organoid technology
利用类器官技术模拟 HIV 感染的肠道功能障碍
- 批准号:
10322720 - 财政年份:2020
- 资助金额:
$ 94.18万 - 项目类别:
Exploring HIV-associated Neurocognitive Disorder (HAND) and HIV Latency at the Single Cell Level in Cerebral Organoids
在脑类器官的单细胞水平上探索 HIV 相关神经认知障碍 (HAND) 和 HIV 潜伏期
- 批准号:
10466829 - 财政年份:2019
- 资助金额:
$ 94.18万 - 项目类别:
Harnessing the RNA-Binding Properties of Cas13a for HIV-1 Self-Testing
利用 Cas13a 的 RNA 结合特性进行 HIV-1 自检
- 批准号:
10423661 - 财政年份:2019
- 资助金额:
$ 94.18万 - 项目类别:
Harnessing the RNA-Binding Properties of Cas13a for HIV-1 Self-Testing
利用 Cas13a 的 RNA 结合特性进行 HIV-1 自检
- 批准号:
10456229 - 财政年份:2019
- 资助金额:
$ 94.18万 - 项目类别:
Exploring HIV-associated Neurocognitive Disorder (HAND) and HIV Latency at the Single Cell Level in Cerebral Organoids
在脑类器官的单细胞水平上探索 HIV 相关神经认知障碍 (HAND) 和 HIV 潜伏期
- 批准号:
10678898 - 财政年份:2019
- 资助金额:
$ 94.18万 - 项目类别:
PROJECT 2: Determine clinically relevant host-viral dependency networks for respiratory infections including SARS-CoV-2
项目 2:确定呼吸道感染(包括 SARS-CoV-2)的临床相关宿主病毒依赖性网络
- 批准号:
10550002 - 财政年份:2018
- 资助金额:
$ 94.18万 - 项目类别:
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