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

基本信息

批准号：
10378164
负责人：
Joseph Christopher Mudd
金额：
$ 32.18万
依托单位：
TULANE UNIVERSITY OF LOUISIANA
依托单位国家：
美国
项目类别：
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/10378164
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

项目摘要

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.

项目概要/摘要鉴于完全抑制性抗逆转录病毒治疗 (ART) 方案的成功，工作重点已转向过去十年来，大多数旨在根除体内艾滋病毒的策略都经过了测试。迄今为止，所有这些研究都未能成功。这些结果凸显了改善当前病毒库的漫长道路。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，一种具有重大前景的方法依赖于通过实验消除 CCR5 来渲染细胞尽管病毒血症持续存在，但 SIV 的自然宿主仍能抵抗 HIV-1 感染，从而避免疾病进展。已经进化到能够自然调节 HIV-1/SIV 进入受体（AGM），特别是在感染后。胸腺下调 CD4 以抵抗 SIV 感染，这一过程导致了一些轶事实例尽管产生了深远的后果，但 AGM 显然可以治愈 SIV。我们之前的研究表明 CD4 下调是完全未定义的。部分由 CD4 启动子区域的 DNA 高甲基化介导。在本研究中，我们将检查整体。假设 AGM 中的 CD4 位点甲基化是独特调节的反式作用因子的结果，在进行性宿主 CD4 T 细胞中操纵这些反式作用可以诱导 CD4 不稳定并表现出表型我们确定了 AGM T 细胞的抗病毒特性，其中两种独特的反式作用因子受到调节。失去 CD4 的 AGM T 细胞在目标 1 中，我们将重点关注 10-11 易位蛋白 3 (TET3)，一种 DNA。发现 AGM 中 CD4 丢失后去甲基化酶下调。在目标 2 中，我们将重点关注 wnt 信号传导。转录因子 TCF-1，我们发现 AGM CD4 下调时其独特下调。目标将遵循类似的工作流程来因果性地确定这些因素在慢病毒调节中的作用。转导后，我们将确定 TET3 或 TCF-1 的过度表达是否可以拯救 AGM T 细胞表面的 CD4。然后我们将采用 CRISPR-cas9 基因编辑技术，探究恒河猴中这些因子的基因消除是否有效或人类细胞可以促进 CD4 不稳定性，以及通过体外感染测定这是否会导致病毒耐药性。这些目标测试基于自然宿主共同进化的 CD4 基因启动子调控的总体模型 SIV 提议的实验将把这个模型扩展到渐进的宿主，以期实现一个 “防弹”策略使 T 细胞对所有 HIV-1/SIV 毒株具有抵抗力，无论其趋向性如何。拟议的研究将为更全面的提案生成数据，该提案旨在自体体内研究转移的抗病毒 T 细胞。