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

项目摘要 尽管进行了高度有效的抗逆转录病毒治疗，但人类免疫缺陷病毒（艾滋病毒）感染仍然无法治愈 (ART)有效地针对病毒的生命周期。一小群长寿的CD 4 T细胞携带复制 当治疗停止时，有能力的病毒（潜伏的HIV宿主）反弹。潜在储层是一个 这是治疗艾滋病毒的主要障碍，但由于技术挑战阻碍了其特征化，人们对其了解甚少。 为了解决这个问题，我开发了一种超高通量液滴微流控工作流程，称为PCR激活细胞 检测、分类和测序含有单拷贝细胞内HIV基因组的细胞的PACS DNA.我的初步工作表明，PACS可以1）处理数百万个细胞，2）检测HIV DNA， 来自ART抑制个体的单个细胞，和3）分选感染的细胞并确定它们的转录 差异表达分析。基于这些结果，PACS提供了一种独特的方法来研究 离体HIV感染细胞。 我假设在HIV感染的细胞中有特定的基因组机制， 有复制能力的前病毒并逃避免疫系统。该提案将PACS与单细胞 RNA-seq和ATAC-seq，以确定体内感染细胞的独特特征（如果有的话）。 病毒基因组和整合位点的同步测序（与Kearney博士合作）将允许 区分完整病毒和缺陷病毒，并提供连接RNA-seq和ATAC-seq的独特机会 数据集。这项工作将使用来自SCOPE的ART抑制患者的样本，SCOPE正在进行 前瞻性研究与精心策划的临床数据，进一步增加了这项研究的转化价值。 了解允许HIV在体内持续存在的细胞机制对于指导选择治疗方案至关重要。 潜伏期逆转剂，以及开发专门针对感染细胞的药物。

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.