Eradication of latent SIV from the CNS

抽象的。 中枢神经系统内潜伏的艾滋病毒感染仍然是一个主要问题。准确识别潜伏感染细胞 病毒的类型、重新激活的机制以及在中枢神经系统中消除HIV-1的方法已被介绍 这是多年来许多研究的主题。CRISPR/Cas9在消除高效感染方面表现出了希望- 来自外周的ED细胞，但其对大脑的渗透和影响仍有待调查。突出之处 这种方法的特点在于它能够编辑病毒基因组，并在潜伏的时间内永久灭活它- 通过去除大片段的病毒DNA而不需要潜伏的病毒活性来感染细胞- 觉醒/再觉醒。长期目标是为从水库中根除艾滋病毒奠定基础，包括 中枢神经系统。本申请的目标是制定一项战略，并制定一种衡量有效- 使用CRISPR/Cas9从潜伏感染的细胞中去除病毒，而不杀死感染的细胞。这个 中心假设是，从宿主细胞中移除整合DNA的关键片段将使病毒 复制不能，这将导致病毒蛋白的产生，而病毒蛋白的产生具有神经- 毒性效应，即TAT、gp120，然后在脑环境中分泌。这是基于以前的工作 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的胶质细胞激活(MacLean)，经历了潜伏在- 感染T细胞(LING)和基于基因治疗的慢性病方法(Bunnell)。我们将聘用 CRISPR/Cas9基因编辑策略切除SIV-1的不同区域，包括 5‘-LtR和GAG，以及位于5’-和3‘-LtR之间的整个病毒编码序列。Ra- 这些研究的可能性是，一旦这个项目完成，就有可能从中枢神经系统中根除艾滋病毒 不会引起有害的神经炎。提出了两个具体目标：具体目标1： 确定中枢神经系统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反应物。 病毒潜伏在中枢神经系统。我们假设这些细胞的潜伏感染(整合的DNA)导致 在不可逆转的激活或其他表型变化中，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，以及致炎细胞因子和病毒蛋白的表达。指定- IC目标2：使用腺相关病毒递送载体将CRISPR/Cas9递送到CNS。我们的假说- 这里的SIS是AAV将促进CRISPR/Cas9通过血脑屏障传递到潜伏感染 SIV感染后的细胞，并由此推断，艾滋病毒感染。我们的中心假设是，移除 较长的病毒序列将大大减少病毒库，并减少中枢神经系统细胞的炎症状态 艾滋病病毒感染者。

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.