Novel macrophage-tropic transmitted/founder SHIV model of CNS persistence to evaluate CRISPR/Cas9 gene editing

用于评估 CRISPR/Cas9 基因编辑的新型巨噬细胞嗜性传播/中枢神经系统持久性 SHIV 模型

基本信息

批准号：
10331568
负责人：
Katharine June Bar
金额：
$ 113.35万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-05 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10331568
关键词：
Aftercare Animal Model Antibodies Autologous Bar Codes Bioavailable Biodistribution Biological Biological Assay Brain Brain region CCR5 gene CD4 Positive T Lymphocytes CRISPR/Cas technology Cells Clinic Clinical Data Clonality Clustered Regularly Interspaced Short Palindromic Repeats Evolution Excision Genes Genetic Genome Guide RNA HIV HIV-1 Human Immunohistochemistry Infection Interruption Kinetics Lymphoid Tissue Macaca mulatta Measures Mediating Modeling Molecular Myelogenous Neuropathogenesis Pathogenesis Pattern Physiological Prevention Proviruses SIV Safety Source System Testing Time Tissues Tropism Variant Viral Viral Genome Viral reservoir Virus Virus Latency base cell type efficacy testing endonuclease experimental study gene delivery system gene therapy improved in vitro activity in vivo in vivo evaluation insight macrophage memory CD4 T lymphocyte monocyte nonhuman primate novel pre-clinical predictive modeling simian human immunodeficiency virus viral rebound virus host interaction

Aftercare Animal Model Antibodies Autologous Bar Codes Bioavailable Biodistribution Biological Biological Assay Brain Brain region CCR5 gene CD4 Positive T Lymphocytes CRISPR/Cas technology Cells Clinic Clinical Data Clonality Clustered Regularly Interspaced Short Palindromic Repeats Evolution Excision Genes Genetic Genome Guide RNA HIV HIV-1 Human Immunohistochemistry Infection Interruption Kinetics Lymphoid Tissue Macaca mulatta Measures Mediating Modeling Molecular Myelogenous Neuropathogenesis Pathogenesis Pattern Physiological Prevention Proviruses SIV Safety Source System Testing Time Tissues Tropism Variant Viral Viral Genome Viral reservoir Virus Virus Latency base cell type efficacy testing endonuclease experimental study gene delivery system gene therapy improved in vitro activity in vivo in vivo evaluation insight macrophage memory CD4 T lymphocyte monocyte nonhuman primate novel pre-clinical predictive modeling simian human immunodeficiency virus viral rebound virus host interaction

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项目摘要

Project Summary/Abstract. An eradicative HIV cure requires safe and effective clearance of replication competent virus from all reservoirs, including the brain. Characterization of the CNS reservoir and empiric testing of novel cure strategies require physiologically relevant animal models of HIV persistence in the brain. Here, we propose to integrate major advances from our groups in SHIV NHP models and AAV-delivered CRISPR/Cas9 editing to delineate key features of the CNS reservoir and determine the efficacy of CRISPR-based eradication in the brain. Transmitted/founder (TF) SHIVs, which encode minimally adapted TF HIV-1 Envs, represent a major advance in biologically relevant NHP models. TF SHIVs have demonstrated robust replication in rhesus macaques, with viral kinetics, cell tropism, and pathogenesis that mirror HIV-1 infection of humans. Further, they recently been shown to faithfully recapitulate virus – host interactions, persist through suppressive ART, and rebound with similar kinetics and clonality as HIV-1. Here, we will employ a novel, genetically barcoded TF SHIV model of CNS pathogenesis and persistence, based on TF SHIV.D.191859 (SHIV.D), which encodes a clade D TF HIV- 1 Env that is CCR5-tropic, efficiently replicates in CD4 T cells and monocyte-derived macrophages, and demonstrates consistent CNS replication, pathogenesis and persistence. Using this barcoded TF SHIV.D model CNS persistence, we will test a novel all-in-one AAV9-mediated CRISPR/Cas9 gene editing system. A recent first-in nonhuman-primate study of SIV-infected rhesus macaques demonstrated the tolerability and efficacy of this approach. The AAV9-CRISPR-Cas9 was broadly distributed across tissues, leading to cleavage and excision of the SIV genome and substantial reductions in the size of the proviral reservoir across tissues. Notably, the AAV was well distributed within CNS resulting in excision of provirus across brain regions. In this application, we will leverage advances in the macrophage-tropic barcoded SHIV.D model and AAV-delivered CRISPR/Cas9 editing to gain insight on CNS neuropathogenesis and persistence. Our hypothesis is that by (i) characterizing SHIV.D persistence in key CNS cells and tissues, (ii) optimizing AAV-delivered CRISPR-Cas9 approaches for SHIV.D persistence in the brain, and (iii) testing the effects of global and myeloid-targeting CRISPR approaches on CNS reservoir reduction in vivo, we will advance prospects for eradicating HIV from the brain. If this hypothesis is affirmed, the significance to HIV cure field would be substantial, since it would improve our understanding of the CNS reservoir, develop a robust model for HIV pathogenesis and persistence in the brain, and provide key pre-clinical data on the safety and efficacy of a promising CRISPR-based cure strategy.

项目摘要/摘要。根除的艾滋病毒治愈方法需要从所有储层中安全有效清除复制能力的病毒，包括大脑。中枢神经系统储层和新型治疗策略的经验测试的表征需要大脑中艾滋病毒持久性的生理相关动物模型。在这里，我们建议将专业整合我们小组的Shiv NHP模型和AAV交付的CRISPR/CAS9编辑的进步到Delineate Key 中枢神经系统储层的特征，并确定大脑基于CRISPR的消除的效率。传输/创始人（TF）Shivs编码最小化的TF HIV-1 Envs，代表了一个主要的进步在生物学相关的NHP模型中。 TF Shivs在恒河猕猴中表现出强大的复制病毒动力学，细胞感性和发病机理，反映了人类的HIV-1感染。此外，他们最近是忠实地概括病毒 - 寄主互动，通过抑制性艺术持续存在，并反弹类似的动力学和克隆性与HIV-1。在这里，我们将采用一种新颖的遗传性条形码TF SHIV模型 CNS发病机理和持久性，基于TF SHIV.D.D.191859（SHIV.D），它编码了进化枝D TF HIV- HIV- 1 env是CCR5循环，有效地在CD4 T细胞和单核细胞衍生的巨噬细胞中复制，以及表现出一致的中枢神经系统复制，发病机理和持久性。使用此条形码TF SHIV.D模型中枢神经系统的持久性，我们将测试一种新型的多合一AAV9介导的CRISPR/CAS9基因编辑系统。最近对SIV感染的恒河猕猴的首次非人类青春期研究证明了耐受性和效率这种方法。 AAV9-CRISPR-CAS9广泛分布在组织之间，导致裂解和 SIV基因组的切除和跨组织的前病毒作用者大小的大量减少。尤其， AAV在中枢神经系统中分布良好，从而使跨大脑区域的病毒感到满意。在此应用程序中我们将利用巨噬细胞的冰纹式SHIV.D模型和AAV交付的CRISPR/CAS9的进步编辑以了解有关CNS神经病发生和持久性的见解。我们的假设是（i）表征 SHIV.D在关键的中枢神经系统细胞和组织中的持久性，（ii）优化用于AAV的CRISPR-CAS9方法 Shiv.D在大脑中的持久性，以及（iii）测试全球和髓样靶向CRISPR方法的影响在CNS储层减少体内，我们将促进从大脑中消除HIV的前景。如果这个假设受到影响，对艾滋病毒治疗领域的意义将是重要的，因为这将改善我们的了解中枢神经系统储层，为大脑中的HIV发病机理和持久性开发一个可靠的模型，并提供有关承诺基于CRISPR的治疗策略的安全性和效率的关键临床前数据。