权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Eradication of latent SIV from the CNS

根除中枢神经系统中潜在的 SIV

基本信息

批准号：
9473820
负责人：
Binhua Julie Ling
金额：
$ 78.75万
依托单位：
TULANE UNIVERSITY OF LOUISIANA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-05-01 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9473820
关键词：
Animals Astrocytes Blood - brain barrier anatomy Brain Brain region CD8B1 gene CRISPR/Cas technology Cells Chinese People Chronic Disease Code Confocal Microscopy DNA Dependovirus Environment Excision Fingerprint Functional disorder Genes Goals Gold HIV HIV Envelope Protein gp120 HIV Infections HIV-1 HIV-associated neurocognitive disorder Histology Human Immunology Individual Infection Inflammation Inflammatory Intermediate Filaments Latent Virus Lead Macaca Macaca mulatta Measures Methods Microglia Modeling Molecular Molecular Genetics Molecular Virology Monkeys Neuroglia Neurosciences Penetration Pharmacology Phenotype Primates Production Quantitative Reverse Transcriptase PCR Regimen Research Resolution SIV Shock Stretching System T-Lymphocyte Therapeutic Viral Viral Genome Viral Proteins Viral reservoir Virus Virus Latency Virus Replication Work adeno-associated viral vector antiretroviral therapy base cell type cellular transduction cytokine cytotoxic effectiveness measure experience gene therapy genetic approach glial activation immune activation immunological status improved in vivo latent infection latent virus activation macrophage neuroAIDS neuroimmunology neuroinflammation neurotoxic nonhuman primate novel reactivation from latency vector viral DNA virology

项目摘要

ABSTRACT. Latent HIV infection within the CNS remains a major problem. Precisely identifying the latently infected cell types, the mechanisms of viral reactivation, and the approach for elimination of HIV-1 in the CNS have been the topics of much research over the years. CRISPR/Cas9 has shown promise in removing productively infect- ed cells from the periphery, but its penetration to and impact in the brain has yet to be investigated. The salient feature of this method lies in its ability to edit the viral genome and permanently inactivate it in the latently in- fected cells by removing a large fragment of viral DNA with no requirement for latent virus activa- tion/reawakening. The Long-Term Goal is to establish a basis for eradication of HIV from reservoirs, including the CNS. The Objective of this Application is to develop a strategy for, and a means of measuring effective- ness of, removing virus from latently infected cells using CRISPR/Cas9, without killing the infected cell. The Central Hypothesis is that removal of key stretches of integrated DNA from the host cell would render the virus replication incompetent, and that this would result in elimination of viral protein production, which has a neuro- toxic effect, i.e. Tat, gp120 and then secretion in the brain environment. This is based this on previous work from the interdisciplinary team of neuroimmunology (Khalili & MacLean), HIV-1 molecular geneticist (Khalili), glia activation in brains in the absence of viral replication (MacLean), experience with reactivation of latently in- fected T cells (Ling), and gene therapy based approaches to chronic diseases (Bunnell). We will employ CRISPR/Cas9 gene editing strategy to excise various regions of SIV-1 including the sequences between the 5'-LTR and gag, as well as the entire viral coding sequence located between the 5'- and 3'-LTRs. The Ra- tionale for these studies, is that once this project is completed, it will be possible to eradicate HIV from CNS reservoirs without causing deleterious neuroinflammation. Two Specific Aims are proposed: Specific Aim 1: Determine the phenotype of latently-infected cells in the CNS. Microglia and astrocytes are the major res- ervoirs for viral latency in the CNS. We hypothesize that latent infection (integrated DNA) of these cells results in irreversible activation or other phenotypic changes that can distinguish latently infected cells in the CNS. Our central hypothesis for this aim is that CRISPR/Cas9 gene editing will reduce or even reverse this activation with regard to innate immune activation, and expression of proinflammtory cytokines and viral proteins. Specif- ic Aim 2: Deliver CRISPR/Cas9 to the CNS using an adeno-associated viral delivery vector. Our hypothe- sis here is that AAV will facilitate delivery of CRISPR/Cas9 through the blood-brain barrier to latently infected cells following SIV infection, and by inference, HIV infection. Our central hypothesis is that the removal of longer sequences of virus will substantially reduce viral reservoirs, and the inflammatory profile of CNS cells in HIV infected individuals.

抽象的。中枢神经系统内的潜在艾滋病毒感染仍然是一个主要问题。精确识别潜伏感染细胞类型、病毒再激活机制以及消除中枢神经系统中 HIV-1 的方法已被研究多年来许多研究的主题。 CRISPR/Cas9 在有效消除感染方面显示出前景—— 外周细胞，但其对大脑的渗透和影响还有待研究。突出的这种方法的特点在于它能够编辑病毒基因组并使其在潜伏期永久失活。通过去除病毒 DNA 的大片段来感染细胞，无需潜伏病毒激活化/苏醒。长期目标是为根除病毒储存者奠定基础，包括中枢神经系统。该应用程序的目的是制定一种策略和衡量有效的方法使用 CRISPR/Cas9 从潜伏感染的细胞中去除病毒，而不杀死受感染的细胞。这中心假设是，从宿主细胞中去除整合 DNA 的关键片段将使病毒复制能力不足，这将导致病毒蛋白产生的消除，这种蛋白具有神经- 毒性作用，即Tat、gp120然后分泌到脑环境中。这是基于之前的工作来自神经免疫学跨学科团队 (Khalili & MacLean)、HIV-1 分子遗传学家 (Khalili)、在没有病毒复制的情况下大脑中神经胶质细胞的激活（MacLean），重新激活潜伏的神经胶质细胞的经验受感染的 T 细胞 (Ling) 和基于基因治疗的慢性疾病方法 (Bunnell)。我们将聘用 CRISPR/Cas9 基因编辑策略切除 SIV-1 的各个区域，包括 SIV-1 之间的序列 5'-LTR 和 gag，以及位于 5'- 和 3'-LTR 之间的整个病毒编码序列。拉- 这些研究的要点是，一旦该项目完成，就有可能根除中枢神经系统中的艾滋病毒不会引起有害的神经炎症。提出了两个具体目标：具体目标 1：确定中枢神经系统中潜伏感染细胞的表型。小胶质细胞和星形胶质细胞是主要的研究对象中枢神经系统病毒潜伏期。我们假设这些细胞的潜伏感染（整合 DNA）导致不可逆的激活或其他表型变化可以区分中枢神经系统中潜伏感染的细胞。我们的这一目标的中心假设是 CRISPR/Cas9 基因编辑将减少甚至逆转这种激活关于先天免疫激活以及促炎细胞因子和病毒蛋白的表达。具体- ic 目标 2：使用腺相关病毒递送载体将 CRISPR/Cas9 递送至 CNS。我们的假设—— 这里的观点是，AAV 将促进 CRISPR/Cas9 通过血脑屏障传递给潜伏感染者 SIV 感染后的细胞，并由此推断 HIV 感染。我们的中心假设是去除更长的病毒序列将大大减少病毒库，并且中枢神经系统细胞的炎症特征 HIV感染者。