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

摘要 疱疹病毒是慢性病原体，终身感染;没有治愈方法。50年前， 对于第一种肿瘤病毒，疱疹病毒Epstein-Barr病毒（EBV），没有针对EBV的特异性抗病毒药物 在潜伏期，而且没有疫苗其他八个人中的大多数也是如此。 疱疹病毒该提案旨在开发新的工具来根除致癌γ疱疹病毒（gHV） 在癌细胞中。 我们建议调整我们现有的基于纳米颗粒的平台，以提供基于DNA的 CRISPR/Cas9系统，靶向基因组编辑的强大工具。我们将锁定并消除关键病毒 疱疹病毒基因组的基因和功能序列，以消除感染。 我们的纳米颗粒平台 利用碳纳米颗粒氧化石墨烯的显著多功能能力， 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） 颗粒可以用作多功能基因递送平台，以低细胞毒性和高细胞毒性递送DNA。 转染效率该项目的总体目标是设计一个基于O-GNR的平台， 将特异性核酸递送至靶细胞，以切割和裂解疱疹病毒基因组。 重要的是，我们将利用鼠gHV发病机制系统，使得我们可以快速地从细胞 基于培养的原理性实验证明，以公认的和 经验证的gHV感染动物模型。在目标1中，我们将设计O-GNR平台以将DNA递送到B 细胞.在目标2中，我们将确定驱动γ疱疹病毒基因组特异性编辑的指导RNA。 成功完成本提案的独立目标将使目标基因的整合 通过RNA指导的病毒基因组编辑递送至B细胞（Aim 1），以消除致癌基因 疱疹病毒从潜伏期水库（目的2）。第1阶段的体外验证将导致高度易处理的 在第2阶段期间在已建立的动物病原体系统中进行的体内研究。

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.