Single Cell Transcriptomic and Epigenetic Analysis of CD4 T Cells Harboring Latent HIV during Antiretroviral Therapy

项目总结 尽管进行了高效的抗逆转录病毒治疗，但人类免疫缺陷病毒(HIV)感染仍无法治愈 (ART)有效地针对病毒生命周期。一小部分长寿的CD4T细胞存在复制 当治疗停止时，会反弹的有效病毒(潜伏的艾滋病毒蓄积者)。潜伏储集层是一种 这是治愈艾滋病毒的主要障碍，但由于技术挑战阻碍其定性，人们对此知之甚少。 为了解决这个问题，我开发了一种超高通量的液滴微流控工作流程，称为聚合酶链式反应激活细胞 分类(PAC)，检测、分类和排序包含单拷贝细胞内HIV基因组的细胞 DNA我的初步工作表明，PACS可以1)处理数百万个细胞，2)在 来自ART抑制个体的单个细胞，以及3)对感染细胞进行分类并确定它们的转录 差异表达分析。基于这些结果，PACS提供了一种独特的研究方法 体外感染艾滋病毒的细胞。 我推测，HIV感染细胞中存在特定的基因组机制，使它们能够容纳 复制能力强的前病毒并逃避免疫系统。该方案将PACS连接到单个细胞 RNA-seq和atac-seq，以便定义体内感染细胞的独特特征是什么(如果有的话)。 病毒基因组和整合位点的同步测序(与科尔尼博士合作)将使 区分完整病毒和缺陷病毒，并提供了将RNA-seq和atac-seq联系起来的独特机会 数据集。这项工作将使用在范围内登记的ART抑制的人的样本，正在进行的 经过精心挑选的临床数据的前瞻性研究，进一步增加了这项研究的翻译价值。 了解使艾滋病毒在体内持续存在的细胞机制对于指导选择 潜伏期反转剂，以及专门针对受感染细胞的药物的开发。

PROJECT SUMMARY Human Immunodeficiency Virus (HIV) infection remains incurable, despite highly active antiretroviral therapy (ART) that effectively targets the viral life cycle. A small population of long-lived CD4 T cells harbors replication competent virus (the latent HIV reservoir) that rebounds when treatment is stopped. The latent reservoir is a major barrier to curing HIV but is poorly understood due to technical challenges that prevent its characterization. To address this, I developed an ultra-high throughput droplet microfluidic workflow called PCR activated cell sorting (PACS) that detects, sorts, and sequences cells containing a single copy of intracellular HIV genomic DNA. My preliminary work has demonstrated that PACS can 1) process millions of cells, 2) detect HIV DNA in single cells from ART-suppressed individuals, and 3) sort infected cells and determine their transcriptional differences by differential expression analysis. Based on these results, PACS provides a unique method to study HIV infected cells ex vivo. I hypothesize that there are specific genomic mechanisms in HIV infected cells that allow them to harbor replication competent provirus and evade the immune system. This proposal couples PACS to single cell RNA-seq and ATAC-seq in order to define what, if any, are the unique signatures of infected cells in vivo. Simultaneous sequencing of viral genomes and integration sites (in collaboration with Dr. Kearney) will allow discrimination of intact from defective virus and provide the unique opportunity to link RNA-seq and ATAC-seq datasets. This work will use samples from people on ART suppression enrolled in SCOPE, an ongoing prospective study with carefully curated clinical data, further increasing the translational value of this research. Understanding the cellular mechanisms that allow HIV to persist in vivo is critical for guiding the selection of latency reversing agents, and in the development of drugs that specifically target infected cells.