Gene editing strategies to target HIV for elimination in periphery and brain

 描述（由申请人提供）：尽管联合抗逆转录病毒治疗（cART）有效，但在感染的早期和/或晚期阶段，HIV-1进入大脑导致绝大多数个体形成病毒储库。在这方面，HIV-1前病毒DNA整合到脑血管周围巨噬细胞、小神经胶质细胞、星形胶质细胞和可能的脑内皮细胞的染色体中导致在HIV疾病过程中在这些重要的细胞区室以及外周循环和可能的其他组织中的静息记忆T细胞和单核细胞-巨噬细胞谱系的细胞内建立病毒潜伏/持续。病毒基因在外周和病毒脑渗透后的激活和表达程度可能取决于在给定储库中的治疗功效、病毒准种的多样性、宿主免疫激活谱和许多合并症因素，如药物滥用、衰老和其他慢性感染或癌症。在不同程度上，这些因子整体参与调节全长和截短的病毒RNA、毒性病毒蛋白（达特、Nef、Vpr和gp 120）以及脑内外感染性病毒的产生。因此，迫切需要新的策略来从潜伏/持续感染的细胞中消除所有形式的整合前病毒，从而防止感染性产生以及神经毒性病毒蛋白的产生，所述神经毒性病毒蛋白也可以由目前可用的治疗策略未消除的缺陷基因组产生。为此，一个经验丰富的研究小组已经组建，在病毒多样性和HIV-1基因组的分子结构方面具有互补的专业知识（B。Wigdahl，Drexel大学），病毒潜伏期（J. Karn，Case Western Reserve大学）和基因切除技术（K. Khalili，坦普尔大学）来检验CRISPR/Cas9基因编辑平台可以被定制以开发精确指导的基因编辑策略的假设，以从潜伏感染的静息记忆CD 4 + T细胞库和大脑的储存细胞中消除HIV-1。为了解决这一假设，提出了三个具体目标。在目标1中，来自对HIV-1感染患者进行的病毒遗传研究的序列和生物信息学信息将用于设计gRNA以精确地指导HIV-1切除过程（Drexel University）。在目标2中，我们将开发和测试体外HIV-1特异性基因编辑系统（坦普尔大学），结合HIV-1潜伏期分子生物学方面的专业知识（凯斯西储大学），这将在目标3中对HIV-1感染样本进行体外实验达到高潮。这些研究将为未来的体内动物研究奠定基础，以验证临床应用的方法。总体目标是开发一种稳健的实验方法，可以通过各种基因递送平台（如纳米分子、慢病毒或干/祖细胞工程）来治疗艾滋病。

 DESCRIPTION (provided by applicant): The entry of HIV-1 into the brain during the early and/or late stages of infection leads to the development of a viral reservoir in the vast majority f individuals despite effective combination antiretroviral therapy (cART). In this regard, HIV-1 proviral DNA integration into the chromosomes of brain perivascular macrophages, microglial cells, astrocytes, and perhaps brain endothelial cells leads to the establishment of viral latency/persistence within these important cellular compartments as well as resting memory T cells and cells of the monocyte-macrophage lineage in the peripheral circulation and likely other tissues during the course of HIV disease. The extent of viral gene activation and expression in the periphery and following viral brain penetration may be dependent on therapeutic efficacy in a given reservoir, the diversity of the viral quasispecies, host immune activation profiles, and a number of comorbidity factors such as substance abuse, aging, and other chronic infections or cancers. To varying degrees, these factors are integrally involved in modulating the production of full-length and truncated viral RNA, toxic viral proteins (Tat, Nef, Vpr, and gp120), and infectious virus within and outside the brain. Consequently, there is a critical need for new strategies to eliminate all forms of integrated provirus from latently/persistently infected cells thereby preventing infectious production as well as the production of neurotoxic viral proteins that could also be produced from defective genomes not eliminated by currently available therapeutic strategies. To this end, a team experienced investigators has been assembled with complementary expertise in viral diversity and molecular architecture of the HIV-1 genome (B. Wigdahl, Drexel University), viral latency (J. Karn, Case Western Reserve University), and gene excision technology (K. Khalili, Temple University) to examine the Hypothesis that the CRISPR/Cas9 gene editing platform can be tailored to develop precision-guided gene editing strategies to eliminate HIV-1 from the latently infected resting memory CD4+ T-cell reservoir and reservoir cells with the brain. To address this hypothesis, three specific aims are proposed. In Aim 1 sequence and bioinformatic information from viral genetic studies performed with HIV-1-infected patients will be utilized to design gRNAs to precisely guide the HIV-1 excision process (Drexel University). In Aim 2 we will develop and test in vitro HIV-1- specific gene editing systems (Temple University), combined with expertise in the molecular biology of HIV-1 latency (Case Western Reserve University), which will culminate in ex vivo experimentation in Aim 3 on HIV-1- infected samples. These studies will set the stage for future in vivo animal studies for validating the approach toward clinical application. The overarching goal is to develop a robust experimental procedure which can be employed through various gene delivery platforms, such as nanomolecules, lentivirus, or stem/progenitor cell engineering, to provide a cure for AIDS.