Targeting latent HIV Astroglial Reservoirs without Reactivation

靶向潜在的 HIV 星形胶质细胞库而不重新激活

基本信息

批准号：
9350661
负责人：
Anuja Ghorpade
金额：
$ 21.9万
依托单位：
UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-05-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9350661
关键词：
Address Astrocytes Biological Markers Biological Models Blood - brain barrier anatomy Brain Cell Line Cells Chronic Color Comorbidity Cytomegalovirus DNA Data Disease Excision Failure Fluorescent Dyes Future Gene Expression Genes Goals Guide RNA HIV HIV Infections HIV Seropositivity HIV therapy HIV-1 HIV-associated neurocognitive disorder Homeostasis Human Individual Infection Inflammation Investigation Ligands Mediating Modeling Monitor Nerve Degeneration Neuroglia Neurologic Neurons Patients Peripheral Blood Mononuclear Cell Physiological Population Proviruses Recovery Reporter Research Personnel Sorting - Cell Movement System TNF gene Techniques Therapeutic Time Tissues Vesicular stomatitis Indiana virus Viral Viral Genes Viral Proteins Viral reservoir Virion Virus Virus Diseases Virus Latency Work antiretroviral therapy astrogliosis base cytokine digital extracellular improved insight latent infection nanoparticle neuroAIDS neuroinflammation novel novel therapeutics prevent promoter receptor response restoration scale up synaptic function targeted biomarker therapeutic gene tool

项目摘要

Project Summary/Abstract Despite the advent of successful antiretroviral therapy (ART), the quest for a human immunodeficiency virus (HIV)-1 cure continues. In order to achieve total eradication, we must successfully resolve persistent viral reservoirs in various tissue compartments. HIV-associated neurocognitive disorders (HAND) remain a significant societal burden in post-ART era. In response to the RFA-MH-17-100, we propose to uncover strategies to address astrocytes as a major CNS reservoir for HIV-1. Our preliminary data show HIV-1 latency can be recapitulated in cultured human astrocytes. The lack of powerful experimental tools to evaluate latency versus reactivation has in part, been responsible for the lack of information in viral latency in the CNS. We propose to employ the powerful dual color HIV-1 reporter virus that harbors CMV-driven mCherry (red, R) and HIV-1 LTR-driven GFP (green, G). Preliminary studies using primary human astrocytes infected with RG-HIV-1 VSV-pseudotyped virions are shown. All astrocytes infected with RG- HIV-1 express the CMV-driven mCherry (red), whereas only those that have an active HIV-1 LTR will express GFP (green) and are positive for HIV-1 p24. Red-alone astrocytes (mCherry+/GFP-) represent latently infected cells amenable to easy monitoring, sorting, manipulation and mechanistic investigations. We have established this model system using primary human astrocytes, which presents a simple, convenient and powerful tool for these works. We propose that latently infected astrocytes express identifiable biomarkers, even in the absence of reactivation. And that guide RNA/Cas9 editing can excise or silence proviral gene expression to prevent reactivation and improve astrocyte physiological function. To mitigate the conundrum of distinguishing latently infected astrocytes from those that have active HIV-1 LTR function, our first goal is to identify targetable biomarkers for HIV-1 latency in human astrocytes (Aim 1). Second, we will target latently infected astrocytes without reactivation using guide RNA and Cas9 gene editing techniques to excise proviral components, rendering it silent and incapable of reactivation (Aim 2). A successful integration of Aims 1 and 2 will lead to novel therapies to cure and eradicate HIV-1 CNS reservoirs. In summary, these investigations responding to RFA-MH-17-100 will provide novel and timely insight into delineating approaches to identify and target astrocyte viral reservoirs, key CNS barriers to HIV-1/HAND cure, and eliminate them without viral reactivation.

项目摘要/摘要尽管成功的抗逆转录病毒疗法（ART）出现了，但寻求人类免疫缺陷病毒（HIV）-1治疗继续。为了完全消除，我们必须成功解决持续的病毒各种组织室中的储层。艾滋病毒相关的神经认知障碍（手）仍然是艺术后时代的重大社会负担。为了响应RFA-MH-17-100，我们提议发现作为HIV-1的主要中枢神经系统水库，解决星形胶质细胞的策略。我们的初步数据表明，可以在培养的人类星形胶质细胞中概括HIV-1潜伏期。缺乏评估潜伏期与重新激活的强大实验工具部分原因是缺乏中枢神经系统中病毒潜伏期的信息。我们建议采用强大的双色HIV-1记者病毒 Harbors CMV驱动的MCHERRY（RED，R）和HIV-1 LTR驱动的GFP（Green，G）。使用主要的研究显示了感染了RG-HIV-1 VSV-PESUDYTY型病毒体的人类星形胶质细胞。所有感染RG-的星形胶质细胞 HIV-1表达CMV驱动的MCHERRY（红色），而只有活性HIV-1 LTR的麦克场会表达 GFP（绿色），对HIV-1 P24呈阳性。红色的星形胶质细胞（MCHERRY+/GFP-）代表被延迟感染细胞可轻松监测，分类，操纵和机械研究。我们已经建立了该模型系统使用主要的人类星形胶质细胞，该系统呈现出一种简单，方便且功能强大的工具这些作品。我们提出，即使在不存在的情况下重新激活。并且该指导RNA/CAS9编辑可以切除或沉默前病毒基因表达到防止重新激活并改善星形胶质细胞生理功能。减轻将潜在感染的星形胶质细胞与具有活性HIV-1 LTR功能的星形胶质细胞区分开来，我们的第一个目标是确定人类星形细胞中HIV-1潜伏期的目标生物标志物（AIM 1）。其次，我们将潜在目标使用指南RNA和CAS9基因编辑技术进行消除病毒的感染星形胶质细胞无重新激活组件，使其保持沉默且无能力激活（AIM 2）。目标1和2的成功整合将导致新颖的疗法治愈和消除HIV-1 CNS储层。总之，这些调查响应RFA-MH-17-100将提供新颖而及时的见解，以识别和靶向星形胶质细胞病毒储存库，HIV-1/手动治疗的关键CNS障碍，并在没有病毒的情况下消除它们重新激活。