Sorting and Sequencing Latent Reservoirs in HIV+ Opioid Users

HIV阿片类药物使用者中潜在储库的分类和测序

基本信息

批准号：
10789790
负责人：
Adam R. Abate
金额：
$ 163.09万
依托单位：
J. DAVID GLADSTONE INSTITUTES
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-15 至 2028-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10789790
关键词：
Acquired Immunodeficiency Syndrome Adherence Affect Award Binding Biochemical Biological Assay Biology Brain CD4 Positive T Lymphocytes CRISPR-mediated transcriptional activation Caring Cell Surface Proteins Cell Survival Cells Characteristics Chemistry Clinical Clustered Regularly Interspaced Short Palindromic Repeats DNA DNA Fragmentation DNA analysis Data Defective Viruses Detection Development Drug usage Drug user Engineering Equipment Failure Funding G Protein-Coupled Receptor Signaling Gene Expression Gene Expression Profile Generations Genes Genomics Gifts Goals HIV HIV Genome Heroin Heroin Users Infection Intake Investigation Knowledge Lymphoid Lymphoid Cell Maintenance Measurement Medical Methods Microfluidics Microglia Mission Molecular Morphine Myelogenous Myeloid Cells Nature Opioid Participant Pathway interactions Persons Process Property Protein Analysis Proteins Proteomics Proviruses Public Health Publishing RNA Sequences RNA analysis Research Research Support Role Sorting Spleen System T-Lymphocyte Technology Testing Tissues United States National Institutes of Health Viral reservoir Vulnerable Populations antiretroviral therapy beneficiary clinical care cohort cold temperature differential expression experimental study fluorescence activated cell sorter device genome sequencing induced pluripotent stem cell insight integration site medical vulnerability memory CD4 T lymphocyte multimodality multiple omics new technology opioid use opioid user personalized approach prevent protein expression proteogenomics proteomic signature social vulnerability substance use transcriptional reprogramming transcriptomics translational approach

项目摘要

ABSTRACT Opioid users are prime beneficiaries of cure strategies for HIV due to their medical and social vulnerabilities and low adherence to antiretroviral therapies, but the nature of the latent reservoir in people using morphine or heroin is unknown. The central hypothesis of this multi-PI proposal is that HIV DNA+ cells from opioid users show unique transcriptional and proteomic signatures that provide fundamental insight into initiation, establishment and maintenance of the latent reservoir in drug users. This hypothesis is based on our recent data in Nature characterizing HIV DNA+ CD4+ T cells without prior activation using a new sorting and sequencing strategy called FIND-Seq. We further show that specific silencing and cell survival pathways are altered in latently infected cells and identify 55 differentially expressed genes (DEGs) implicated in the underlying biology of HIV latency. Here we propose to extend these studies to tissue reservoirs from opioid users, test the functional relevance of DEGs on latency biology, and develop important genomic, proteomic and biochemical advancements of FIND-Seq. The central hypothesis will be tested in four specific aims: 1) Define the cellular mechanisms of HIV persistence and their modulation by morphine in HIV-infected T cells and microglia from Last Gift participants. We will use FIND-seq to sort and RNA sequence HIV+ cells from the spleen, gut, and brain of Last Gift participants with and without morphine to elucidate the role of opioids on latently infected T cells and microglia across different tissues. 2) Determine how DEGs and opioids regulate HIV latency in T cells and microglia. We will perform ex vivo CRISPR activation/interference experiments with isolated CD4+ T cells and microglia derived from induced pluripotent stem cells to determine the functional relevance of identified DEGs for latency establishment with and without morphine or heroin. 3) Perform a proteogenomic analysis of HIV+CD4+ T cells from the spleen of Last Gift participants. We will use DAb-seq, which allows high-coverage genome sequencing and simultaneous analysis of cell-surface protein expression on CD4+ T cells from spleens from Last Gift participants with or without morphine to determine the intactness of the provirus, its integration site, and changes in protein expression of latently infected cells induced by opioid use. 4) Simultaneously sequence the RNA, protein, and full HIV genome from HIV DNA+ cells. We will develop FIND-Seq into a multimodal assay that sorts HIV DNA+ cells on a flow cytometer and simultaneously performs genomic, transcriptomic, and proteomic analysis on recovered cells. We have assembled a highly complementary team of world experts in the biology and advanced investigation of HIV latency with cutting-edge microfluidics, CRISPR and multi-omics sequencing technologies combined with leading experts in clinical care of people living with HIV and opioid use from the Last Gift Cohort and the Johns Hopkins HIV Clinical Cohort. We expect to gain paradigm-shifting insight into the “underlying molecular mechanisms by which HIV latency is initiated, established, and maintained in the CNS, lymphoid and myeloid tissues and how substance use might influence these processes” and are fully aligned with this RFA.

抽象的阿片类药物使用者是治愈艾滋病毒策略的主要受益人，因为其医疗和社会脆弱性和对抗逆转录病毒疗法的依从性低，但是使用吗啡或海洛因的人的潜在水库的性质是未知的。该多PI提案的中心假设是来自阿片类药物使用者的HIV DNA+细胞显示独特转录和蛋白质组学签名，可为主动性，建立和在吸毒者中维护潜在水库。该假设基于我们自然界的最新数据表征HIV DNA+ CD4+ T细胞，而没有事先激活的新分类和测序策略称为 find-seq。我们进一步表明，在潜在感染的细胞中，特定的沉默和细胞存活途径改变了并确定在HIV潜伏期基本生物学中实现的55个不同表达的基因（DEG）。这里我们建议将这些研究扩展到阿片类药物使用者的组织库，测试DEG的功能相关性关于潜伏期生物学，并发展出重要的基因组，蛋白质组学和生化的进步。中心假设将以四个特定目的进行检验：1）定义HIV持久性的细胞机制以及它们在HIV感染的T细胞和上次礼物参与者的小胶质细胞中对吗啡的调节。我们将使用从最后的礼物参与者和没有吗啡可以阐明阿片类药物对不同组织的潜在感染T细胞和小胶质细胞的作用。 2）确定DEGS和阿片类药物如何调节T细胞和小胶质细胞中的HIV潜伏期。我们将执行Ex Vivo CRISPR激活/干扰实验，该实验是通过诱导的分离的CD4+ T细胞和小胶质细胞进行的多能干细胞确定已确定的DEG与与和没有吗啡或海洛因。 3）对最后的脾脏进行HIV+ CD4+ T细胞的蛋白质分析礼物参与者。我们将使用dab-seq，它允许高覆盖的基因组测序和简单性分析来自或没有或没有的最后礼物参与者的脾脏CD4+ T细胞上的细胞表面蛋白表达吗啡以确定病毒的完整性，其整合位点以及蛋白质表达的变化通过使用阿片类药物诱导的潜在感染细胞。 4）同时对RNA，蛋白质和全HIV基因组进行序列来自HIV DNA+细胞。我们将将发现seq开发到多模式的测定中，该测定法将HIV DNA+细胞分类为细胞仪并简单地对回收细胞进行基因组，转录组和蛋白质组学分析。我们已经组建了一支高度完整的世界专家团队，从事艾滋病毒的生物学和高级调查具有尖端微流体，CRISPR和多摩学测序技术的潜伏期最后礼物队列和约翰群岛的艾滋病毒和阿片类药物使用的人的临床护理领先专家霍普金斯艾滋病毒临床队列。我们期望获得“基础分子”的范式转移见解在中枢神经系统，淋巴机和髓样中启动，确定和维持艾滋病毒潜伏期的机制组织以及物质使用如何影响这些过程”，并与此RFA完全排列。