Coupling Epitranscriptomics to Molecular Disease Mechanisms and Nucleic Acid Therapeutics in Persistent Residual HIV Infection

项目摘要 人类免疫缺陷病毒（HIV）是一种高度可控的感染， 抗逆转录病毒疗法（cART）。然而，它还不能治愈，艾滋病毒感染者（PLWH）服用 cART患者的免疫健康状况欠佳，长期生活质量下降。的主要原因 持续的健康问题是持续的残余感染。持续感染PLWH的T细胞， cART仍然会产生病毒RNA和蛋白质，以及有缺陷的前病毒，这些病毒可以长期激活 免疫系统尽管无法检测到病毒载量。更好地了解生物学， HIV RNA代谢和基因表达在PLWH服用cART可以解锁额外的治疗 减少病毒基因产物和提高生活质量的策略。 细胞RNA和病毒RNA可以受到多种转录后化学修饰， 修改.这些修饰调节或微调分子相互作用，从而控制基因表达。 通过RNA加工、周转、定位或翻译来表达和发挥功能。共价碱 修饰及其对RNA调控的影响在广泛的水平上超出了四个典型的基础， 称为表转录组学。RNA修饰与RNA代谢的密切关系是 变得更受赞赏。然而，操纵这一过程用于治疗艾滋病毒和 接受cART的PLWH持续感染需要更深入地了解HIV表型组学。 在这里，我们提出了系统的表征和操作的HIV-1 RNA转录模型和 病人的T细胞在R61阶段，我们将利用三种强大的测序方法（总RNA-seq， 核糖体分析，和纳米孔直接RNA测序）来全面表征丰度， cART期间T细胞中病毒和宿主RNA的加工、翻译和修饰状态。这些 实验将包括从接受cART的PLWH中选择持续感染的T细胞。我们将瞄准 通过酶敲除和纳米孔测序的几种已知修饰。这些实验将 表征具有潜在意义的HIV-1 RNA化学修饰并识别那些具有高活性的修饰 可能影响T细胞中的HIV RNA和蛋白质载量。 在R33阶段，我们将使用化学修饰的寡核苷酸来改变高表达的修饰。 通过阻断或引导位点特异性修饰而引起兴趣。这将包括寡核苷酸， 阻止修改或引导新的修改，这两种治疗方式正在迅速成熟。 化学修饰的寡核苷酸将为表转录组操作提供概念验证 作为潜在的治疗方法，同时提供核酸治疗候选物。

PROJECT SUMMARY Human immunodeficiency virus (HIV) is a highly manageable infection when treated with combination anti-retroviral therapy (cART). However, it is not yet curable and people living with HIV (PLWH) taking cART suffer from suboptimal immune health and reduced quality of life long-term. A leading cause of ongoing health concerns is persistent, residual infection. Persistently infected T-cells of PLWH that take cART still produce viral RNA and proteins, as well as defective proviruses, that can chronically activate the immune system despite undetectable viral loads. A better understanding of the biology and metabolism of HIV RNA and gene expression in PLWH taking cART could unlock additional therapeutic strategies to reduce viral gene products and improve quality of life. Cellular RNA and viral RNA can be subjected to a variety of post-transcriptional chemical modifications. These modifications modulate or fine-tune molecular interactions and thereby control gene expression and function through RNA processing, turnover, localization, or translation. Covalent base modification and its effects on RNA regulation at a broad level beyond the four canonical bases is often termed epitranscriptomics. The intimate relationship between RNA modification and RNA metabolism is becoming better appreciated. However, manipulating this process for therapeutic treatment of HIV and persistent infection in PLWH taking cART requires a deeper understanding of HIV epitranscriptomics. Here we propose systematic characterization and manipulation of HIV-1 RNA transcripts in model and patient T-cells. In the R61 phase, we will utilize three powerful sequencing methodologies (total RNA-seq, ribosome profiling, and nanopore direct RNA sequencing) to globally characterize the abundance, processing, translation, and modification status of both viral and host RNA in T-cells during cART. These experiments will include selection of persistently infected T-cells from PLWH taking cART. We will target several known modifications via enzyme knockouts and nanopore sequencing. These experiments will characterize HIV-1 RNA chemical modifications of potential significance and identify those with high a likelihood of impacting HIV RNA and protein loads in T-cells. In the R33 phase, we will use chemically modified oligonucleotides to alter modifications of high interest by blocking or directing site-specific modification. This will include oligonucleotides that sterically block modification or guide new modification, both therapeutic modalities that are maturing rapidly. Chemically modified oligonucleotides will provide a proof-of-concept for epiptranscriptome manipulation as a potential therapeutic approach while offering nucleic acid therapeutic candidates.