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

非洲绿猴 CD4 下调的机制和治疗目标

• 批准号: 10254703
• 负责人: Joseph Christopher Mudd
• 金额: $ 28.95万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10254703
• 关键词: Ablation; Acquired Immunodeficiency Syndrome; Acute; Address; African Green Monkey; Appearance; Autologous; Awareness; Biological Assay; CCR5 gene; CD4 Positive T Lymphocytes; CD4 gene; CRISPR/Cas technology; Cell Count; Cell division; Cell physiology; Cells; Complex; Cytomegalovirus; Cytosol; DNA; DNA Methylation; DNA Modification Methylases; Data; Development; Disease; Disease Progression; Down-Regulation; Elements; Epigenetic Process; Equilibrium; Event; Evolution; Gene Expression Regulation; Gene Silencing; Gene set enrichment analysis; Genes; Genetic; Genetic Transcription; Goals; HIV; HIV-1; Human; Hypermethylation; In Vitro; Infection; Inherited; Link; Mediating; Methylation; Modeling; Molecular; Outcome; Pathway interactions; Peptides; Phenocopy; Population; Process; Promoter Regions; Protein translocation; Proteins; Recrudescences; Refractory; Regimen; Regulation; Repression; Resistance; Rhesus; Role; SIV; Shock; Sum; Surface; T-Lymphocyte; TCF Transcription Factor; Techniques; Testing; Text; Thymus Gland; Trans-Activators; Transcriptional Regulation; Transduction Gene; Tropism; Viral; Viremia; Virus; WNT Signaling Pathway; antiretroviral therapy; base; beta catenin; experimental study; functional outcomes; gene repression; genetic manipulation; genome editing; in vivo; in vivo evaluation; methylation pattern; overexpression; promoter; receptor; success; therapeutic target; viral resistance; virology

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.

项目总结/摘要 鉴于完全抑制性抗逆转录病毒治疗（ART）方案的成功， 在过去十年中，艾滋病毒感染者的身体被消灭。这些策略中的绝大多数都在 vivo依赖于某种形式的“休克和杀死”方法。迄今为止，所有这些研究都未能 加速HIV病毒库的衰变。这些结果既突出了完善当前 休克和杀死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，一种具有重要前景的方法依赖于实验性地消融CCR 5以使细胞 抗HIV-1感染。SIV的天然宿主尽管存在持续的病毒血症， 已经进化到自然调节HIV-1/SIV进入受体。非洲绿色猴（AGM），特别是， 胸腺下调CD 4，使其对SIV感染不敏感，这一过程导致了一些轶事 年度股东大会显然治愈了SIV。尽管有着深远的影响， 这一过程是完全不确定的。我们以前的研究表明，CD 4下调是 部分由CD 4启动子区的DNA超甲基化介导。在这项研究中，我们将研究整体 假设AGM中的CD 4基因座甲基化是独特调节的反式作用因子的结果， 在进行性宿主CD 4 T细胞中操纵这些反式作用可以诱导CD 4不稳定性和表型， AGM T细胞的抗病毒特性。我们确定了两种特殊的反式作用因子， 失去CD 4的AGM T细胞。在目标1中，我们将关注10 - 11易位蛋白3（TET 3），一种DNA 在AGM中，发现脱甲基酶在CD 4丧失后下调。在aim 2中，我们将重点关注wnt信号 转录因子TCF-1，我们发现其在AGM CD 4下调时独特地下调。两 目标将遵循类似的工作流程，以确定这些因素在CD 4调节中的作用。通过慢病毒 通过转导，我们将确定TET 3或TCF-1的过表达是否拯救AGM T细胞表面上的CD 4。 然后，我们将采用CRISPR-cas9基因编辑技术，询问恒河猴中这些因子的基因切除是否 或人细胞可以促进CD 4不稳定性，以及这是否通过体外感染测定导致病毒抗性。 这些目标测试了基于自然宿主共进化的CD 4基因启动子调控的整体模型， SIV所提出的实验将把这个模型扩展到渐进式主机，希望实现一个 “防弹”策略，使T细胞对所有HIV-1/SIV毒株都具有抗性，无论其嗜性如何。如果成功，则 拟议的研究将为更全面的提案提供数据，该提案旨在进行自体- 转移的抗病毒T细胞