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

项目摘要： CRISPR/Cas9是一种革命性的多功能基因组编辑技术，具有广泛的实用性。体内基因组 编辑预计将成为各种主要健康威胁的下一波治疗方法，包括神经退行性疾病， 生殖疾病然而，迫切需要开发有效的、非病毒的递送载体，以安全地递送药物。 和高效的体内CRISPR基因组编辑。此外，将CRISPR基因组编辑机器交付给 脑/神经元由于其致密结构和血脑屏障（BBB）而代表主要障碍。 该项目的目标是设计一个多功能的、新型的、非病毒的Cas9-gRNA核糖核蛋白家族。 (RNP)递送纳米胶囊（NC），可以在神经元中稳健且安全地产生靶向基因编辑， 大脑我们设想，我们强大的和通用的RNP交付纳米平台将使创新 神经退行性疾病的治疗方法为达致这个目标，我们会评估 在一个演示中，通过靶向与阿尔茨海默病相关的淀粉样前体蛋白（APP）， (AD)在健康的小鼠和猴子模型中。 在我们的初步研究期间，我们开发了具有高RNP负载含量（68重量%）的PEG化NC， 多功能表面化学、超小尺寸（dH~13 nm）、可控化学计量、优异的生物相容性，以及 体外和体内基因组编辑效率高。在UG 3 Aim 1中，我们将进一步优化NC的设计 用于大脑/神经元靶向基因组编辑。特别地，我们将研究混合物的协同效应。 靶向配体，包括（1）葡萄糖+RVG肽，用于静脉内（i. v.）注，以提高过境 BBB和神经元特异性编辑，以及（2）CPP+RVG肽用于脑内注射以增强摄取 和神经元特异性基因组编辑。不同类型/量的靶向配体对细胞凋亡的影响 神经细胞的摄取、生物相容性、基因组编辑效率和神经细胞在Neuro 2a 和初级神经元细胞进行研究。在UG 3目标2中，我们将评估大脑/神经元靶向 特异性、基因组编辑效率和潜在的免疫应答和静脉内或静脉内注射的全身毒性。 在健康小鼠中脑内施用与各种靶向配体缀合的NC。在UH 3目标1中，我们 将开发设置和合成工艺，以扩大NC的生产。在UH 3目标2中，我们将进一步 评估健康恒河猴脑/神经元靶向NC的基因组编辑效率和生物相容性 猕猴 我们独特设计的NC有望实现高脑积累、高穿透深度、高 神经元特异性基因组编辑效率，这是由于它们的理想特性。考虑到模块化和易用性 通过CRISPR系统靶向不同基因，我们预计所得的NC将用于广泛的研究。 一系列人类疾病，包括无法治愈的衰弱性神经退行性疾病。

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.