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

总结 HIV-1感染需要终身抗逆转录病毒治疗，因为它的停止会导致快速的病毒感染。 从HIV-1潜伏细胞/组织库反弹。消除或永久沉默的新方法 迫切需要HIV-1前病毒来实现HIV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，我们和其他人利用Cas9介导的 基因组编辑以在体外、离体和体内切除HIV-1前病毒。临床前的挑战之一 应用是如何有效地，特异性地和安全地将强大的基因组编辑机制传递给HIV-1 潜伏感染细胞该提案的目的是开发用于纳米粒子的新型合成纳米粒子（NP）。 Cas9/sgRNA核糖核蛋白（RNP）特异性地体内递送至CD 4 T细胞，最重要的HIV-1 潜在细胞库我们最近开发了一种新的合成聚乙二醇-吗啉共聚物（PEG-pMor） 用于小鼠模型中体内药物递送的NP系统。我们的初步数据证明了可行性， 该PEG-pMor NP在多个器官/组织中递送Cas9/sgRNA质粒或RNP的效率， 在体内根除HIV-1前病毒DNA或宿主细胞基因。在这项提议中，一种新的CD 4特异性设计， 锚蛋白重复蛋白（DARPin）肽将展示在PEG-pMor NP的表面上，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CD 4 T细胞。在 目的一，我们将在体外和体内开发和表征靶向CD 4 T细胞的NP，并确定其生物学活性。 CD 4特异性DARPin介导的Cas9/双链体sgRNA RNP切除人原发性肝癌中CCR 5基因的效率 T细胞（体外）和人源化小鼠模型（体内）。在目标II中，我们将确定CD 4 T细胞的效率- 靶向NP用于体内递送Cas9/sgRNA RNP以切除HIV-I前病毒DNA。在目标III中，我们将评估 靶向CD 4 T细胞的NP体内递送Cas9/四链体sgRNA的组合治疗潜力 RNP（LTR 1/GagD+ CCR 5-A/B）在人源化小鼠模型中阻断或延迟潜伏的HIV-1病毒反弹。 这个高回报的建议集中在筛选新的CD 4 T细胞特异性递送RNP基因组 编辑器切除HIV-1前病毒并使HIV-1进入辅助受体CCR 5基因失活。积极的结果将 提供了一种新的工具来将Cas9/sgRNA RNP递送到体内的CD 4 T细胞和/或其他储库细胞，因此 为开发治疗HIV-1的药物提供了新的途径。

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.