Eradication of latent SIV from the CNS

摘要。 中枢神经系统内的潜伏性HIV感染仍然是一个主要问题。精确识别潜伏感染细胞 类型，病毒再活化的机制，以及在CNS中消除HIV-1的方法， 多年来许多研究的主题。CRISPR/Cas9显示出在有效去除感染方面的前景。 艾德细胞从周边，但其渗透和影响，在大脑中还有待调查。凸 该方法的特征在于其能够编辑病毒基因组并将其永久性地插入到潜伏感染中， 通过去除病毒DNA的大片段而不需要潜伏的病毒激活， /reawakening。长期目标是为从宿主中根除艾滋病毒奠定基础，包括 CNS。本申请的目的是制定一项战略，并衡量有效的手段- 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。的 中心假设是从宿主细胞中去除整合DNA的关键片段会使病毒 复制能力，这将导致消除病毒蛋白质的生产，这有一个神经- 毒性作用，即达特、gp 120，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，脑中的神经胶质细胞活化（MacLean）， 感染的T细胞（Ling）和基于基因治疗的慢性疾病方法（邦内尔）。我们会委聘 CRISPR/Cas9基因编辑策略切除SIV-1的各个区域，包括SIV-1基因之间的序列。 5 '-LTR和gag，以及位于5'-和3 '-LTR之间的整个病毒编码序列。Ra- 一旦这个项目完成，将有可能从中枢神经系统中根除艾滋病病毒。 而不引起有害的神经炎症。具体目标1： 确定CNS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神经元。 中枢神经系统中病毒潜伏期的研究。我们假设这些细胞的潜伏感染（整合的DNA）导致 在不可逆的激活或其他表型变化，可以区分潜在感染的细胞在中枢神经系统。我们 这一目标的核心假设是CRISPR/Cas9基因编辑将减少甚至逆转这种激活 关于先天性免疫激活以及促炎细胞因子和病毒蛋白的表达。具体- 目的2：使用腺相关病毒递送载体将CRISPR/Cas9递送至CNS。我们的假设- 这里姐妹是，AAV将促进CRISPR/Cas9通过血脑屏障递送至潜伏感染者， SIV感染后的细胞，以及由此推断的HIV感染。我们的中心假设是， 较长的病毒序列将大大减少病毒储存库， 艾滋病毒感染者。

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.