CD4 T cell-targeted nanoparticle in vivo delivery of CRISPR/Cas9 genome editors for HIV cure

CD4 T 细胞靶向纳米颗粒体内递送 CRISPR/Cas9 基因组编辑器以治疗 HIV

基本信息

批准号：
10591410
负责人：
Wenhui Hu
金额：
$ 32.61万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-08 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10591410
关键词：
Ankyrin Repeat Anti-Retroviral Agents Biodistribution Blood Circulation CCR5 gene CD4 Positive T Lymphocytes CRISPR/Cas technology Cells Charge Clinic DNA Data Development Devices Drug Delivery Systems Drug Kinetics Excision Genes Genome HIV HIV Infections HIV therapy HIV-1 HIV/AIDS Human Immune response Immunologic Stimulation In Vitro Infection Knock-out Life Mediating Micelles Mission Mus Organ Outcome Peptides Pharmaceutical Preparations Plasmids Polymers Production Proteins Proviruses Public Health RNA Research Ribonucleoproteins Ribonucleotides Serum Proteins Small Interfering RNA Specificity Sterility Surface System T-Lymphocyte Technology Testing Therapeutic Tissues Toxic effect Transgenic Mice United States National Institutes of Health Vaccines Viral Virus antiretroviral therapy biomaterial compatibility biophysical properties cell suicide clinical application copolymer design genome editing high reward humanized mouse improved in vivo latent HIV reservoir morpholine mouse model nanoparticle nanoparticle delivery novel novel strategies novel therapeutics pre-clinical response screening self assembly side effect success suicidal morbidity targeted delivery therapeutic development tool vector viral rebound

项目摘要

SUMMARY HIV-1 infection requires a life-term antiretroviral treatment because its cessation leads to rapid viral rebound from the HIV-1 latent cellular/tissue reservoir. Novel approaches to eradicate or permanently silence HIV-1 proviruses are urgently needed to achieve a “sterile” cure of HIV infection, for which CRIPSR/Cas9 genome editing has opened a new avenue. In the past years, we and others have utilized Cas9-mediated genome editing to excise HIV-1 provirus in vitro, ex vivo and in vivo. One of challenges before clinical application is how to deliver effectively, specifically and safely the powerful genome editing machinery to HIV-1 latently infected cells. The objective of this proposal is to develop novel synthetic nanoparticle (NP) for the in vivo delivery of Cas9/sgRNA ribonucleoprotein (RNP) specifically to CD4 T cells, the most important HIV-1 latent cellular reservoir. We have recently developed a novel synthetic PEG-Morpholine copolymer (PEG-pMor) NP system for in vivo drug delivery in mouse model. Our preliminary data demonstrated the feasibility and efficiency of this PEG-pMor NP to deliver Cas9/sgRNA plasmid or RNP in multiple organs/tissues resulting in eradication of HIV-1 proviral DNA or host cellular genes in vivo. In this proposal, a novel CD4-specific designed ankyrin repeat protein (DARPin) peptide will be displayed on the surface of the PEG-pMor NP to achieve targeted delivery of Cas9/sgRNA RNP to human CD4 T cells in HIV-1-infected humanized mouse models. In Aim I, we will develop and characterize CD4 T cell-targeting NP both in vitro and in vivo and determine the efficiency of CD4-specific DARPin-mediated Cas9/duplex sgRNA RNP to excise CCR5 gene in human primary T cells (in vitro) and humanized mouse model (in vivo). In Aim II, we will determine the efficiency of CD4 T cell- targeting NP for in vivo delivery of Cas9/sgRNA RNP to excise HIV-1 proviral DNA. In Aim III, we will assess the combinatory therapeutic potential of CD4 T cell-targeting NP in vivo delivery of Cas9/quadruplex sgRNA RNP (LTR1/GagD+CCR5-A/B) in blocking or delaying latent HIV-1 viral rebound in humanized mouse model. This high-reward proposal focuses on the screening of novel CD4 T cell-specific delivery of RNP genome editors to excise HIV-1 provirus and disable HIV-1 entry coreceptor CCR5 gene. The positive outcome will offer a novel tool to deliver Cas9/sgRNA RNP to CD4 T cells and/or other reservoir cells in vivo, and thus provide new avenues for the development of therapeutics to cure HIV-1.

概括 HIV-1 感染需要终生抗逆转录病毒治疗，因为停止治疗会导致病毒快速传播根除或永久沉默 HIV-1 潜伏细胞/组织库的反弹。迫切需要 HIV-1 原病毒来实现 HIV 感染的“无菌”治疗，为此 CRIPSR/Cas9 在过去的几年里，我们和其他人已经利用了 Cas9 介导的基因组编辑开辟了一条新途径。基因组编辑在体外、离体和体内切除 HIV-1 原病毒是临床面临的挑战之一。应用程序是如何有效地、特异地、安全地向 HIV-1 提供强大的基因组编辑机制该提案的目的是开发新型合成纳米颗粒（NP）。将 Cas9/sgRNA 核糖核蛋白 (RNP) 体内特异性递送至 CD4 T 细胞（最重要的 HIV-1）我们最近开发了一种新型合成 PEG-吗啉共聚物 (PEG-pMor)。我们的初步数据证明了用于小鼠模型体内药物递送的 NP 系统的可行性和有效性。该 PEG-pMor NP 在多个器官/组织中递送 Cas9/sgRNA 质粒或 RNP 的效率在此提议中，设计了一种新型的CD4特异性的体内消灭HIV-1前病毒DNA或宿主细胞基因的方法。锚蛋白重复蛋白（DARPin）肽将展示在PEG-pMor NP的表面以实现在 HIV-1 感染的人源化小鼠模型中，将 Cas9/sgRNA RNP 靶向递送至人类 CD4 T 细胞。目标 I，我们将在体外和体内开发和表征 CD4 T 细胞靶向 NP，并确定 CD4特异性DARPin介导的Cas9/双链sgRNA RNP在人原代细胞中切除CCR5基因的效率 T 细胞（体外）和人源化小鼠模型（体内）在 Aim II 中，我们将确定 CD4 T 细胞的效率。靶向 NP 体内递送 Cas9/sgRNA RNP 以切除 HIV-1 前病毒 DNA 在目标 III 中，我们将评估。 CD4 T 细胞靶向 NP 体内 Cas9/四联体 sgRNA 的联合治疗潜力 RNP (LTR1/GagD+CCR5-A/B) 在人源化小鼠模型中阻断或延迟潜在 HIV-1 病毒反弹。这项高回报提案的重点是筛选新型 CD4 T 细胞特异性递送 RNP 基因组编辑者切除HIV-1原病毒并禁用HIV-1进入辅助受体CCR5基因将获得积极结果。提供了一种将 Cas9/sgRNA RNP 递送至体内 CD4 T 细胞和/或其他储存细胞的新工具，从而为开发治愈 HIV-1 的疗法提供新途径。