Mechanisms and Therapeutic Targeting of CD4 Down regulation in African Green Monkeys

项目摘要/摘要 鉴于完全抑制性抗逆转录病毒疗法(ART)的成功，努力已转移到 在过去的十年里，我们致力于旨在根除艾滋病毒体内的战略。这些策略中的绝大多数都在 VIVO依靠的是某种形式的“电击并杀死”方法。到目前为止，所有这些研究都未能 加速艾滋病毒储存库的腐烂。这些结果突显了改善当前经济状况的漫长道路 电击和杀戮方法以及探索替代艾滋病毒治疗途径的必要性。 在这种情况下，一种有希望的方法依赖于通过实验消融CCR5来呈现细胞 抵抗HIV-1感染。尽管存在持续的病毒血症，SIV的自然宿主仍可绕过疾病进展 已经进化到自然地调节HIV-1/SIV进入受体。非洲绿猴(AGM)，特别是后 胸腺下调CD4使其对SIV感染产生抵抗力，这一过程导致了轶事 明显治愈了SIV的AGM。尽管后果深远，但分子事件 管理这一过程是完全不确定的。我们之前的研究表明，CD4下调是 部分是由CD4启动子区域的DNA超甲基化介导的。在这项研究中，我们将检查总体 假设动眼小鼠中的CD4基因位点甲基化是独特调节的反式作用因子和 在渐进性宿主CD4T细胞中操纵这些反式作用可以诱导CD4不稳定和表型复制 AGM T细胞的抗病毒特性。我们发现了两个特殊的反式作用因子，它们在 失去CD4的AGM T细胞。在目标1中，我们将重点介绍Ten-11易位蛋白3(TET3)，这是一种DNA 在动植物生长因子中发现，当失去CD4时，去甲基酶下调。在目标2中，我们将重点介绍WNT信令 转录因子TCF-1，我们发现它在AGM CD4下调时唯一下调。两者都有 AIMS将遵循类似的工作流程，因果性地确定这些因素在CD4调节中的作用。由慢病毒感染 我们将确定过表达TET3或TCF-1是否能挽救AGM T细胞表面的CD4。 然后我们将使用CRISPR-Cas9基因编辑技术，询问在恒河猴中是否存在这些因子的遗传消融 或者人类细胞可以促进CD4的不稳定性，以及通过体外感染检测这是否会导致病毒耐药性。 这些目的是测试基于自然宿主与 SIV。拟议的实验将把这一模式扩展到进步的宿主，希望实现一个 “防弹”策略，使T细胞对所有HIV-1/SIV毒株具有抵抗力，而不考虑嗜性。如果成功，则 拟议的研究将为一项更全面的提案产生数据，该提案旨在对自体- 转移的、抗病毒的T细胞。

Project Summary/Abstract Given the success of fully-suppressive antiretroviral therapy (ART) regimens, efforts have shifted over the past decade to strategies aimed at eradicating the body of HIV. The vast majority of these strategies tested in vivo have relied on some form of the “shock and kill” approach. To date, all of these studies have failed to accelerate decay of the HIV reservoir. These outcomes highlight both the long road to refinement of current shock and kill approaches and the need to explore alternative HIV cure avenues. An approach with significant promise in this setting relies on experimentally ablating CCR5 to render cells resistant to HIV-1 infection. Natural hosts of SIV circumvent disease progression in spite of ongoing viremia and have evolved to naturally regulate HIV-1/SIV entry receptors. African green monkeys (AGMs), in particular, post- thymically down-regulate CD4 to become refractory to SIV infection, a process resulting in anecdotal instances of AGMs evidently curing themselves of SIV. Despite the profound consequences, the molecular events governing this process are entirely undefined. Our previous studies revealed that CD4 down-regulation is mediated in part by DNA hypermethylation of the CD4 promoter region. In this study we will examine the overall hypothesis that CD4 locus methylation in AGMs is the result of uniquely regulated trans-acting factors and manipulating these trans-acting in progressive host CD4 T cells can induce CD4 instability and phenocopy the virus-resistant qualities of AGM T cells. We identified two particular trans-acting factors uniquely regulated in AGM T cells that lose CD4. In aim 1 we will focus on the Ten-eleven translocation protein 3 (TET3), a DNA demethylase found to be down-regulated upon loss of CD4 in AGMs. In aim 2 we will focus on the wnt signaling transcription factor TCF-1, which we find to be uniquely down-regulated upon AGM CD4 down-regulation. Both aims will follow a similar workflow to causatively determine the role of these factors in CD4 regulation. By lentiviral transduction, we will determine if over-expression of TET3 or TCF-1 rescues CD4 on the surface of AGM T cells. We will then employ CRISPR-cas9 gene editing techniques, asking if genetic ablation of these factors in rhesus or human cells can promote CD4 instability, and whether this leads to virus resistance by in vitro infection assays. These aims test an overall model of CD4 gene promoter regulation based on natural host co-evolution with SIV. The proposed experiments will extend this model to progressive hosts in the hopes of implementing a “bulletproof” strategy to render T cells resistant to all HIV-1/SIV strains, regardless of tropism. If successful, the proposed studies will generate data for a more comprehensive proposal aimed at in vivo studies of autologously- transferred, virus-resistant T cells.