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

摘要 HIV-1感染需要终生抗逆转录病毒治疗，因为它的停止会导致快速病毒 从HIV-1潜伏的细胞/组织储存库反弹。根除或永久沉默的新方法 迫切需要艾滋病毒-1前体来实现艾滋病毒感染的“无菌”治愈，为此，CRIPSR/Cas9 基因组编辑开辟了一条新的道路。在过去的几年里，我们和其他人使用了Cas9中介的 在体外、体外和体内进行基因组编辑以切除HIV-1前病毒。临床面临的挑战之一 应用是如何有效、具体和安全地将强大的基因组编辑机器传递给HIV-1 潜伏感染的细胞。本研究的目的是开发新型的合成纳米粒子(NP)。 体内特异性地将Cas9/sgRNA核糖核蛋白(RNP)传递给HIV-1最重要的CD4T细胞 潜在的细胞储集层。我们最近开发了一种新型的合成聚乙二醇-吗啉共聚物(聚乙二醇聚吗啉)。 用于小鼠体内给药的纳米粒系统。我们的初步数据证明了该方法的可行性和 此PEG-pMor NP在多个器官/组织中传递Cas9/sgRNA质粒或RNP的效率导致 体内根除HIV-1前病毒DNA或宿主细胞基因。在这项建议中，一种新颖的针对CD4的设计 将Ankyrin Repeat Protein(DARPin)肽展示在聚乙二醇单核苷酸的表面，实现 在HIV-1感染的人源化小鼠模型中靶向人CD4T细胞的Cas9/sgRNA RNP。在……里面 目的一、我们将在体外和体内建立和鉴定以CD4T细胞为靶点的NP，并确定 CD4DARPin介导的Cas9/双链sgRNA RNP对人原代CCR5基因的切割效率 T细胞(体外)和人源化小鼠模型(体内)。在AIM II中，我们将测定CD4T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AIM III中，我们将评估 CD4T细胞靶向NP体内递送Cas9/四链sgRNA的联合治疗作用 RNP(LTR1/GagD CCR5-A/B)阻断或延缓HIV-1病毒在人源化小鼠模型中的潜伏反弹 这项高回报的建议侧重于筛选新型的RNP基因组的CD4T细胞特异性递送 编辑切除HIV-1前病毒并禁用HIV-1进入共受体CCR5基因。积极的结果将是 提供了一种新的工具来将Cas9/sgRNA RNP运送到体内的CD4T细胞和/或其他存储细胞，从而 为治疗HIV-1病毒的疗法的发展提供新的途径。

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.