Single cell RNA-seq and single molecule RNA-FISH approaches to study stochasticity of latent HIV-1 reactivation
单细胞 RNA-seq 和单分子 RNA-FISH 方法研究潜在 HIV-1 重新激活的随机性
基本信息
- 批准号:10082908
- 负责人:
- 金额:$ 25.15万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-05-20 至 2022-04-30
- 项目状态:已结题
- 来源:
- 关键词:AntibodiesBiological AssayCD4 Positive T LymphocytesCell Culture TechniquesCellsComputational algorithmDataDevelopmentFishesGene Expression RegulationGenesGeneticGenetic HeterogeneityGenetic TranscriptionGoalsHIVHIV-1ImageImmunofluorescence ImmunologicIndividualInfectionLeadLengthMethodologyMethodsMicroscopyModelingMolecularNoisePathway interactionsPatientsProceduresProteinsProvirusesQuantitative Reverse Transcriptase PCRRNARNA ProbesResolutionRestSamplingScanningShockSiteSlideSourceSpeedStimulusT-LymphocyteTechniquesTechnologyTranscriptVariantViralViral GenomeViral ProteinsVirus Integrationbasecell typecombatdifferential expressionepigenetic regulationin vivoinnovationintegration siteknock-downmultiple omicsnew therapeutic targetnovelnovel markernovel therapeuticsreactivation from latencysingle cell analysissingle moleculesingle-cell RNA sequencingtranscriptometranscriptome sequencingviral RNA
项目摘要
The persistent reservoirs are the ultimate hurdle to HIV-1 cure. ‘Shock and Kill’ strategy to eliminate HIV-
1 reservoirs requires reactivation of all latent proviruses with latency reversing agents (LRA), which is currently
not possible. Quantitation of patient derived latent reservoirs by QVOA (Quantitative Viral Outgrowth Assay)
indicated that only ~1/60th of full-length replication competent latent proviruses are activated by LRA. The reason
for this variability is not completely understood but has been attributed to differential epigenetic regulation of
provirus (due to different sites of proviral integration), differences in the viral genome (genetic variability),
differential expression of cellular factors or different transcriptional or post-transcriptional blocks in the patient-
derived latent cells. In the ExVivo and cell culture models of latency, where a single viral clone is used for infection
with no genetic heterogeneity, reactivation of only a small percentage of cells and different levels of expression
of viral RNA and proteins indicate cell-to-cell variation in reactivation. These studies also revealed that the
majority of latent cells are not activated by current approaches. Current strategies do not address cell-to-cell
variation in proviral reactivation and understanding this variability in vivo is essential to achieve either full
reactivation or full suppression of these reservoirs.
The goal of this application is to employ innovative single-cell and multi-omics platforms to investigate
mechanisms of HIV persistence at the single-cell level with greater precision and higher resolution than has been
achieved previously using traditional techniques. Stellaris-based quantitative single molecule RNA-FISH
(smRNA-FISH), that allows the greater resolution and precise quantitation of the number of RNA molecules
within single cells, have been used to study stochasticity in eukaryotic gene transcription. We have applied
smRNA-FISH combined with Immunofluorescence (IF) termed SMIRA (Single cell and single molecule IF and
RNA-FISH based Assay) to study HIV-1 reactivation in latency models and quantitated the single RNA molecules
using FISH-quant, a computational algorithm. In addition, we have combined these studies with a high speed
and high resolution scanning (HSHRS) microscopy to identify rare positive cell/s in a large pool of negative cells.
Using these methods, our goal is to quantitate the cell-to-cell variability in latent cells. Our hypothesis is that cell-
to-cell variation in HIV-1 reactivation is due to both ‘intrinsic’ and ‘extrinsic’ factors. By combining single cell and
single molecule approaches with single cell RNA-sequencing (scRNA-seq) methodologies we propose to identify
the determinants responsible for variation in the HIV-1 reactivation and extend these studies to identify these
markers in patient samples. In aim I, we will characterize and determine the genes and pathways involved
in the stochastic activation in HIV-1 latent cells using SMIRA, HSHRS and RNA-seq analysis. In aim II,
we will validate the genes and pathways identified to influence HIV-1 stochastic activation in models of
latency and in ART-suppressed patient samples. Our analyses will identify novel genes and pathways that
influence cell-to-cell variability in HIV-1 reactivation and ultimately will lead to the development of novel therapies
to combat HIV-1 latency.
持久的储存库是治愈HIV-1的最终障碍。消除艾滋病毒的“震惊和杀戮”战略
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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GANJAM V KALPANA其他文献
GANJAM V KALPANA的其他文献
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{{ truncateString('GANJAM V KALPANA', 18)}}的其他基金
RNA-mimicry to guide the intra-cellular targeting of host virus protein and viral RNA-protein interactions to inhibit HIV replication.
RNA模拟引导宿主病毒蛋白的细胞内靶向和病毒RNA-蛋白相互作用以抑制HIV复制。
- 批准号:
10554025 - 财政年份:2022
- 资助金额:
$ 25.15万 - 项目类别:
RNA-mimicry to guide the intra-cellular targeting of host virus protein and viral RNA-protein interactions to inhibit HIV replication.
RNA模拟引导宿主病毒蛋白的细胞内靶向和病毒RNA-蛋白相互作用以抑制HIV复制。
- 批准号:
10618961 - 财政年份:2022
- 资助金额:
$ 25.15万 - 项目类别:
Structure-based design of stapled peptides to target Gag-Pol and INI1 interaction to block assembly
基于结构的钉合肽设计,以靶向 Gag-Pol 和 INI1 相互作用来阻止组装
- 批准号:
10302316 - 财政年份:2020
- 资助金额:
$ 25.15万 - 项目类别:
Mechanism of HIV-1 Latency and Reactivation Kinetics Using Single Cell Analysis
使用单细胞分析研究 HIV-1 潜伏期和再激活动力学的机制
- 批准号:
9298589 - 财政年份:2016
- 资助金额:
$ 25.15万 - 项目类别:
Effect of drugs of abuse on CNS HIV-1 reservoirs and neuropathogenesis
滥用药物对 CNS HIV-1 储存库和神经发病机制的影响
- 批准号:
9532834 - 财政年份:2016
- 资助金额:
$ 25.15万 - 项目类别:
Effect of drugs of abuse on CNS HIV-1 reservoirs and neuropathogenesis
滥用药物对 CNS HIV-1 储存库和神经发病机制的影响
- 批准号:
9333307 - 财政年份:2016
- 资助金额:
$ 25.15万 - 项目类别:
Effect of drugs of abuse on CNS HIV-1 reservoirs and neuropathogenesis
滥用药物对 CNS HIV-1 储存库和神经发病机制的影响
- 批准号:
9977143 - 财政年份:2016
- 资助金额:
$ 25.15万 - 项目类别:
Effect of Drugs of Abuse on CNS HIV-1 Reservoirs and Neuropathogenesis
滥用药物对中枢神经系统 HIV-1 病毒库和神经发病机制的影响
- 批准号:
10419775 - 财政年份:2016
- 资助金额:
$ 25.15万 - 项目类别:
Mechanism of HIV-1 Latency and Reactivation Kinetics Using Single Cell Analysis
使用单细胞分析研究 HIV-1 潜伏期和再激活动力学的机制
- 批准号:
9207956 - 财政年份:2016
- 资助金额:
$ 25.15万 - 项目类别:
Effect of drugs of abuse on CNS HIV-1 reservoirs and neuropathogenesis
滥用药物对 CNS HIV-1 储存库和神经发病机制的影响
- 批准号:
9926378 - 财政年份:2016
- 资助金额:
$ 25.15万 - 项目类别:
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