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

项目概要/摘要 尽管有高效的抗逆转录病毒疗法（ART），潜伏在静息CD 4 + T细胞中的HIV-1储存库是HIV-1的主要来源。 HIV-1感染功能性治愈的主要障碍。我们的R61/R33双相项目的总体目标是： 了解HIV-1感染者HIV-1 RNA转录后调控的机制， 处于ART（探索性R61阶段），并开发一种新的治疗策略来改变RNA后， 转录修饰作为抑制HIV-1复制的潜在治疗平台（开发 R33相）。 我们的多学科小组是唯一准备解决几个关键问题，强调在这一资金 机会我的实验室是三个独立发现N6-甲基腺苷（m6 A） HIV-1 RNA的修饰在体外调节CD 4 + T细胞中的病毒复制。使用CD 4 + T细胞系和 我们从健康供体的原代CD 4 + T细胞中，研究了m6 A修饰 调节HIV-1感染。我们还发现HIV-1 RNA的m6 A修饰抑制了先天性抗病毒免疫， 来自健康供体的原代巨噬细胞的反应。我们的体外研究表明，m6 A修饰 HIV-1 RNA的合成在病毒复制和对病毒感染的先天免疫应答中起关键作用。但 HIV-1 RNA的m6 A修饰在接受ART的HIV-1感染者中调节病毒复制的作用 仍然未知。我们的目标是填补这一重要的知识空白，并将研究结果转化为潜在的反 HIV-1治疗。 我们假设HIV-1 RNA的m6 A修饰有助于在CD 4 + T细胞中建立和维持病毒潜伏期 并避免接受ART的HIV-1感染者的先天性抗病毒免疫应答。为了验证这一假设， 为了促进HIV-1治疗新策略的发展，我们分两个阶段设计了三个具体目标： (1)R61阶段（1-3年）：目标1。为了确定来自ART的CD 4 + T细胞亚群中HIV-1 RNA的m6 A谱， 治疗患者;目标2.确定m6 A通路中对HIV-1再活化重要的细胞靶点， CD 4 + T细胞;和（2）R33期（4-5岁）：目标3。为了检测抑制HIV-1的小分子的抗HIV-1作用， 原代CD 4 + T细胞中的m6 A修饰。 总体影响：这些研究将揭示HIV-1 RNA的m6 A修饰如何调节ART中的病毒潜伏期。 治疗的病人。R61阶段的研究将确定HIV-1持续存在的新机制， 潜在的治疗目标。R33期研究将开发一种m6 A特异性策略来抑制HIV-1 在原代CD 4 + T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.