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感染需要生命期抗逆转录病毒治疗，因为其停止导致病毒迅速从HIV-1潜在的细胞/组织储存中反弹。新颖的射线座或永久沉默的方法迫切需要HIV-1预科病毒来实现艾滋病毒感染的“无菌”治疗基因组编辑已经开放了新的途径。在过去的几年中，我们和其他人使用了CAS9介导的基因组编辑，以激发体外，体内和体内的HIV-1病毒。临床前的挑战之一应用是如何有效，特别，安全地交付强大的基因组编辑机制到HIV-1 潜在感染的细胞。该提议的目的是为IN开发新型的合成纳米颗粒（NP） CAS9/SGRNA核糖核蛋白（RNP）的体内递送专门为CD4 T细胞，这是最重要的HIV-1 潜在的细胞晶状体。我们最近开发了一种新型的合成PEG-Morpholine共聚物（PEG-PMOR）在小鼠模型中用于体内药物输送的NP系统。我们的初步数据证明了可行性和该PEG-PMOR NP的效率在多个器官/组织中提供CAS9/SGRNA质粒或RNP，导致在体内消除HIV-1病毒DNA或宿主细胞基因。在此提案中，一种新颖的CD4特异性设计 Ankyrin重复蛋白（DARPIN）肽将显示在PEG-PMOR NP的表面上以实现在HIV-1感染的人源性小鼠模型中，将Cas9/sgrNA RNP靶向递送至人CD4 T细胞。在目的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对人类主要锻炼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，用于在体内递送Cas9/sgRNA RNP以消除HIV-1病毒DNA。在AIM III中，我们将评估 cas9/Quadruplex sgrna的CD4 T细胞靶向NP的组合治疗潜力 RNP（LTR1/GAGD+CCR5-A/B）在人源化小鼠模型中阻断或延迟潜在的HIV-1病毒反弹。这个高回报的提议着重于筛选新型CD4 T细胞特异性RNP基因组的递送经历HIV-1病毒和禁用HIV-1进入的CRECEPTOR CCR5基因的编辑。积极的结果将提供一种新颖的工具，将Cas9/sgrNA RNP传递到CD4 T细胞和/或其他储层细胞中，从而为开发治疗HIV-1的疗法提供了新的途径。