Targeting HIV-1 RNA modifications in latently infected CD4+ T cells for therapeutic development

项目摘要/摘要 尽管接受了高效的抗逆转录病毒疗法(ART)，但静息的CD4T细胞中潜伏的HIV-1储存库是 HIV-1感染的功能性治愈的主要障碍。我们这个R61/R33双阶段项目的总体目标是： 了解HIV-1感染者体内HIV-1 RNA转录后调控机制 正在进行抗逆转录病毒治疗(探索性R61期)，并开发一种新的治疗策略，以改变后的RNA。 转录修饰作为抑制HIV-1复制的潜在治疗平台(发展中 R33期)。 我们的多学科小组独一无二地准备解决在这笔资金中突出的几个关键问题 机会。我的实验室是独立发现N6-甲基腺苷(M6A)的三个小组之一。 HIV-1RNA的修饰在体外调节CD4T细胞中的病毒复制。使用CD4T细胞系和 健康供者的原代CD4T细胞，我们研究了m6A修饰的机制 调节HIV-1感染。我们还发现，HIV-1RNA的m6A修饰抑制了先天抗病毒免疫 健康供者原代巨噬细胞的反应。我们的体外研究表明，m6A修饰 HIV-1RNA在病毒复制和对病毒感染的先天免疫反应中起着关键作用。然而， HIV-1 RNA m6A修饰在调节HIV-1感染者体内病毒复制中的作用 仍然不为人知。我们的目标是填补这一重要的知识空白，并将这些发现转化为潜在的反 HIV-1治疗学。 我们假设HIV-1RNA的m6A修饰有助于在CD4T细胞中建立和维持病毒潜伏期 并避免接受抗逆转录病毒治疗的HIV-1感染者的先天抗病毒免疫反应。为了检验这一假说， 为了促进艾滋病毒-1治愈新战略的发展，我们分两个阶段设计了三个具体目标： (1)R61期(1-3年)：目的1.检测ART-T细胞亚群中HIV-1RNA的m6A谱。 治疗的患者；目的2.确定m6A通路中对HIV-1在初级患者中重新激活重要的细胞靶点 CD4T细胞；和(2)R33期(4-5年)：目的3.检测小分子抑制物的抗HIV-1作用 原代CD4T细胞中的M6A修饰。 总体影响：这些研究将揭示HIV-1RNA的m6A修饰如何调节ART中的病毒潜伏期- 治疗过的病人。R61阶段的研究将定义HIV-1持续存在的新机制，并确定 潜在的治疗靶点。R33阶段研究将开发一种针对m6A的策略来抑制HIV-1 在原代CD4T细胞中复制。

Project Summary/Abstract Despite highly effective anti-retroviral therapy (ART), the latent HIV-1 reservoir in resting CD4+ T cells is the major barrier to a functional cure of HIV-1 infection. Our overall objectives of this R61/R33 bi-phasic project are: to understand the mechanisms of post-transcriptional regulation of HIV-1 RNA in HIV-1-infected individuals who are on ART (Exploratory R61 phase), and to develop a novel therapeutic strategy to alter RNA post- transcriptional modifications as a potential therapeutic platform for inhibiting HIV-1 replication (Developmental R33 phase). Our multidisciplinary group is uniquely poised to address several key questions highlighted in this funding opportunity. My lab was among three groups that independently discovered that N6-methyladenosine (m6A) modifications of HIV-1 RNA modulate viral replication in CD4+ T cells in vitro. Using CD4+ T cell lines and primary CD4+ T cells from healthy donors, we investigated the mechanisms by which m6A modifications modulate HIV-1 infection. We also found that m6A modifications of HIV-1 RNA inhibit innate antiviral immune responses in primary macrophages from healthy donors. Our in vitro studies suggested that m6A modifications of HIV-1 RNA play a critical role in viral replication and innate immune responses to viral infection. However, the role of m6A modifications of HIV-1 RNA in regulating viral replication in HIV-1-infected individuals on ART remains unknown. We aim to fill this important knowledge gap and to translate the findings into potential anti- HIV-1 therapeutics. We hypothesize that m6A modifications of HIV-1 RNA help establish and maintain viral latency in CD4+ T cells and avoid innate antiviral immune responses in HIV-1 infected individuals on ART. To test this hypothesis and to facilitate the development of a novel strategy for HIV-1 cure, we designed three specific aims in two phases: (1) R61 phase (years 1-3): Aim 1. To determine m6A profile of HIV-1 RNA in subsets of CD4+ T cells from ART- treated patients; Aim 2. To identify cellular targets in the m6A pathway important for HIV-1 reactivation in primary CD4+ T cells; and (2) R33 phase (years 4-5): Aim 3. To examine anti-HIV-1 effects of small molecules inhibiting m6A modifications in primary CD4+ T cells. Overall Impact: These studies will reveal how m6A modifications of HIV-1 RNA regulate viral latency in ART- treated patients. The studies in the R61 phase will define new mechanisms of HIV-1 persistence and identify potential therapeutic targets. The R33 phase study will develop an m6A-specific strategy to inhibit HIV-1 replication in primary CD4+ T cells.