Enabling Nanoplatforms for Targeted in vivo Delivery of CRISPR/Cas9 Ribonucleoproteins in the Brain

项目总结： CRISPR/Cas9是一种革命性的、多功能的基因组编辑技术，具有广泛的实用价值。体内基因组 编辑预计将成为针对各种主要健康威胁的下一波治疗浪潮，包括Neurode- 生殖性疾病。然而，为了安全，迫切需要开发高效的、非病毒的递送工具。 和有效的在体CRISPR基因组编辑。此外，将CRISPR基因组编辑机器交付给 由于其致密的结构和血脑屏障(BBB)，脑/神经元是一个主要障碍。 该项目目标是设计一种多功能的、新颖的、非病毒的Cas9-gRNA核糖核蛋白家族 (RNP)递送纳米胶囊(NCS)，可在神经元中稳健而安全地产生靶向基因编辑 大脑。我们设想，我们强大而通用的RNP交付纳米平台将使创新 治疗毁灭性的神经退行性疾病。为了实现这一目标，我们将评估我们 在演示中，通过靶向与阿尔茨海默病相关的淀粉样前体蛋白(APP)的方法 (AD)在健康的小鼠和猴子模型中。 在我们的初步研究中，我们开发了具有高RNP负载量(68wt.%)的聚乙二醇化NC， 多种表面化学，超小尺寸(dh~13 nm)，可控的化学计量比，出色的生物兼容性，以及 体外和体内基因组编辑效率高。在UG3目标1中，我们将进一步优化NC的设计 用于大脑/神经元目标基因组编辑。特别是，我们将研究混合动力的协同效应。 靶向配体，包括(1)静脉注射葡萄糖+RVG多肽。注射以加强交叉 血脑屏障和神经元特异性编辑，以及(2)CPP+RVG多肽脑内注射增强摄取 以及神经元特有的基因组编辑。不同类型/数量的靶向配体对细胞的影响 NCS在两个Neuro2a中的摄取、生物相容性、基因组编辑效率和功能后果 并将对原代神经元细胞进行研究。在《UG3目标2》中，我们将评估大脑/神经元靶向 静脉注射的特异性、基因组编辑效率、潜在的免疫反应和全身毒性。或 健康小鼠脑内给药NCS与不同靶向配体结合。在UH3目标1中，我们 将开发建立和合成工艺，以扩大NCS的生产。在UH3目标2中，我们将进一步 健康恒河猴脑/神经元靶向神经干细胞的基因组编辑效率和生物相容性评价 猕猴。 我们独特设计的NCS有望实现高大脑积累、高渗透深度和高 神经元特有的基因组编辑效率得益于它们所需的特性。考虑到模块化和易用性 通过CRISPR系统靶向不同的基因，我们预计得到的NCS将在广泛的 一系列人类疾病，包括衰弱的神经退行性疾病，目前尚无治愈方法。

Project Summary: CRISPR/Cas9 is a revolutionary and versatile genome editing technique with wide-ranging utility. In vivo genome editing is anticipated to be the next wave of therapeutics for various major health threats, including neurode- generative diseases. However, there is an urgent need to develop efficient, non-viral delivery vehicles for safe and efficient in vivo CRISPR genome editing. Furthermore, delivering CRISPR genome editing machinery to the brain/neuron represents a major hurdle due to its dense structure and the blood–brain barrier (BBB). The objective of this project is to engineer a family of versatile, novel, non-viral Cas9-gRNA ribonucleoprotein (RNP) delivery nanocapsules (NCs) that can robustly and safely generate targeted gene edits in neurons within the brain. We envision that our robust and universal RNP delivery nanoplatforms will enable innovative treatments for devastating neurodegenerative diseases. Towards this goal, we will evaluate the feasibility of our approach, in a demonstration, by targeting the amyloid precursor protein (APP) – relevant to Alzheimer's disease (AD) in healthy mice and monkey models. During our preliminary studies, we developed a PEGylated NC with a high RNP loading content (68 wt.%), versatile surface chemistry, ultrasmall size (dH~13 nm), controllable stoichiometry, excellent biocompatibility, and high genome editing efficiency in vitro and in vivo. In UG3 Aim 1, we will further optimize the design of the NC for brain/neuron-targeted genome editing. In particular, we will investigate the synergistic effects of hybrid targeting ligands, including (1) glucose+RVG peptide for intravenous (i.v.) injection to enhance the crossing of the BBB and neuron-specific editing, and (2) CPP+RVG peptide for intracerebral injection to enhance uptake and neuron-specific genome editing. The effects of different types/amounts of targeting ligands on the cellular uptake, biocompatibility, genome editing efficiency, and functional consequences of the NCs in both Neuro2a and primary neuron cells will be investigated. In UG3 Aim 2, we will evaluate the brain/neuron targeting specificity, genome editing efficiency, and potential immune response and systemic toxicity of the i.v. or intracerebrally administered NCs conjugated with various targeting ligands in healthy mice. In UH3 Aim 1, we will develop the set up and synthesis process to scale up the production of NCs. In UH3 Aim 2, we will further evaluate the genome editing efficiency and biocompatibility of the brain/neuron-targeted NCs in healthy rhesus macaques. Our uniquely designed NCs are expected to achieve high brain accumulation, high penetration depth, and high neuron-specific genome editing efficiency due to their desirable characteristics. Given the modularity and ease of targeting different genes by the CRISPR system, we anticipate that the resulting NCs will be useful for a wide range of human diseases, including debilitating neurodegenerative diseases for which there are no cures.