Targeting latent HIV Astroglial Reservoirs without Reactivation

项目总结/摘要 尽管成功的抗逆转录病毒疗法（ART）的出现，寻求人类免疫缺陷病毒（HIV） 病毒（HIV）-1治疗仍在继续。为了实现彻底根除，我们必须成功地解决持续存在的病毒 各种组织隔室中的储库。HIV相关的神经认知障碍（HAND）仍然是一个 后ART时代的重大社会负担。针对RFA-MH-17-100，我们建议 将星形胶质细胞作为HIV-1的主要中枢神经系统储存库。 我们的初步数据显示，HIV-1潜伏期可以在培养的人星形胶质细胞中重现。缺乏 强大的实验工具来评估延迟与重新激活，部分原因是缺乏 病毒在中枢神经系统潜伏期的信息。我们建议使用强大的双色HIV-1报告病毒， 含有CMV驱动的mCherry（红色，R）和HIV-1 LTR驱动的GFP（绿色，G）。初步研究使用主要 显示了用RG-HIV-1 VSV-假型病毒体感染的人星形胶质细胞。所有感染RG-的星形胶质细胞 HIV-1表达CMV驱动的mCherry（红色），而只有那些具有活性HIV-1 LTR的人才会表达 GFP（绿色）和HIV-1 p24阳性。单独的红色星形胶质细胞（mCherry+/GFP-）代表潜伏感染 细胞易于监测、分选、操作和机械研究。我们建立 该模型系统使用原代人星形胶质细胞，这为以下方面提供了简单、方便和强大的工具： 这些作品。 我们认为，潜伏感染的星形胶质细胞表达可识别的生物标志物，即使在缺乏 重新激活。指导RNA/Cas9编辑可以切除或沉默前病毒基因表达， 防止再活化和改善星形胶质细胞生理功能。为了缓解 区分潜伏感染的星形胶质细胞与具有活跃的HIV-1 LTR功能的星形胶质细胞，我们的第一个目标是 鉴定人类星形胶质细胞中HIV-1潜伏期的靶向生物标志物（目标1）。第二，我们将瞄准潜在目标 使用指导RNA和Cas9基因编辑技术切除前病毒， 组件，使其沉默和无法重新激活（目标2）。目标1和目标2的成功融合 将导致治疗和根除HIV-1 CNS储库的新疗法。总之，这些调查 响应RFA-MH-17-100将提供新的和及时的见解，描绘方法，以确定和 靶向星形胶质细胞病毒储库，HIV-1/HAND治疗的关键CNS屏障，并在没有病毒的情况下消除它们 重新激活

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.