CRISPR-Cas9 Systems Delivered by Targeted Nanoparticles to Eradicate Herpesvirus Pathogens

摘要 疱疹病毒是一种慢性病原体，会终身感染；没有治愈方法。这一发现已有50年 在第一种肿瘤病毒，疱疹病毒爱泼斯坦-巴尔病毒(EBV)中，没有针对EBV的特异性抗病毒药物 在其潜伏期，没有疫苗。同样的道理也适用于其他八个人类中的大多数 疱疹病毒。这项提议旨在开发新的工具来根除致癌的伽马疱疹病毒(Ghv)。 在癌细胞中。 我们建议将我们现有的基于纳米颗粒的平台改造为基于DNA的 CRISPR/Cas9系统，靶向基因组编辑的强大工具。我们将瞄准并消灭关键病毒 疱疹病毒基因组的基因和功能序列，以根除感染。 我们的纳米颗粒平台 利用碳纳米颗粒氧化石墨烯的非凡多功能 纳米带(O-GNRs)。我们最近的体外实验结果表明，这些石墨烯(单层石墨) 颗粒可以作为一种多功能的基因传递平台，以低细胞毒性和高密度的方式传递DNA 转染率。该项目的总体目标是设计一个基于O-GNR的平台，它将 将特定的核酸输送到靶细胞，以切割和灭活疱疹病毒基因组。 重要的是，我们将利用小鼠的GHV致病系统，这样我们就可以快速地从细胞中移动 以培养为基础的原理验证实验以体内验证为基础的病毒根除在接受和 验证了GHV感染的动物模型。在目标1中，我们将设计O-GNR平台，将DNA运送到B 细胞。。在目标2中，我们将确定驱动伽马疱疹病毒基因组的特定编辑的引导RNA。 成功完成这项提案的独立目标将允许巩固目标基因 通过RNA引导的病毒基因组编辑将病毒传递到B细胞(目标1)，以推动消除致癌 潜伏库中的疱疹病毒(目标2)。第一阶段的体外验证将导致非常容易处理的 在第二阶段，在已建立的动物病原体系统中进行活体研究。

Abstract Herpesviruses are chronic pathogens that infect for life; there is no cure. 50 years since the discovery of the first tumor virus, the herpesvirus Epstein-Barr virus (EBV), there exist no specific antivirals for EBV during its latency phase and there are no vaccines. The same holds true for most of the other eight human herpesviruses. This proposal aims to develop novel tools to eradicate oncogenic gamma herpesviruses (gHV) in cancer cells. We propose to adapt our existing nanoparticle-based platform for the delivery of a DNA-based CRISPR/Cas9 system, a powerful tool for targeted genome editing. We will target and eliminate key viral genes and functional sequences of the herpesvirus genome to eradicate infection. Our nanoparticle platform harnesses the remarkable multifunctional capabilities of the carbon nanoparticle oxidized graphene nanoribbons (O-GNRs). Our recent in vitro results indicate that these graphene (single sheet of graphite) particles can serve as a versatile gene delivery platform to deliver DNA with low cytotoxicity and high transfection efficiency. The overall objective of this project is to engineer an O-GNR-based platform that will deliver specific nucleic acids to targeted cells in order to cleave and inactivate a herpesvirus genome. Importantly, we will utilize the murine gHV pathogenesis system, such that we can move quickly from cell culture-based proof of principle experiments to in vivo validation of virus eradication in an accepted and validated animal model of gHV infection . In Aim 1, we will engineer the O-GNR platform to deliver DNA to B cells. . In Aim 2, we will Identify the guideRNAs that drive specific editing of the gamma herpesvirus genome. Successful completion of the independent aims of this proposal will allow the consolidation of targeted gene delivery to B-cells (Aim 1) with RNA-guided editing of the virus genome to drive elimination of the oncogenic herpesvirus from the latency reservoir (Aim 2). The in vitro validation in phase 1 will lead to highly tractable in vivo studies in an established animal pathogen system during phase 2.