Brain-Wide Genome Editing Enabled by Intravenously Administered Non-Viral Nanovectors As a Potential Therapy for Alzheimer’s Disease

项目摘要 目前，还没有有效的方法来减缓或预防阿尔茨海默病（AD）的进展。CRISPR 基因组编辑是一种革命性的、多功能的基因工程技术， 遗传性神经退行性疾病（NDD）的根本原因，如AD。然而，大脑基因的前景 治疗依赖于生物制剂向大脑的有效递送，这是非常具有挑战性的，因为血液- 脑屏障（BBB）。迄今为止，体内脑基因治疗主要是使用病毒载体实现的， 费力的定制和麻烦的安全配置文件。非病毒载体主要通过以下途径施用： 颅内给药是侵入性的，只能在小的局部脑中进行基因治疗 地区与其他NDD类似，AD影响多个大脑区域。因此，迫切需要开发高效的 能够绕过BBB的非病毒递送载体，用于安全和有效的全脑基因治疗。 本计画的主要目的为（1）设计谷胱甘肽（GSH）反应性矽奈米碳管（SNCs） 能够绕过BBB并将CRISPR基因组编辑器系统性地递送到整个大脑，以及（2） 评估优化SNC实现的全脑基因组编辑的治疗效果和生物安全性 使用新型淀粉样前体蛋白（APP）敲入AD小鼠模型和独特的 APP调节的基因靶点。独特的SNC拥有一长串理想的性能，包括多功能 有效载荷类型，用于配体缀合的多功能表面化学，高有效载荷装载含量和效率， 粒径小、体内稳定性好、生物相容性好、可规模化生产。我们的初步 数据显示静脉内施用的SNCs可以有效地递送mRNA、DNA、Cas9 mRNA/sgRNA 和Cas9/gRNA核糖核蛋白（RNP）对具有完整BBB的健康小鼠的全脑的影响。 我们的目标是进一步优化双重脑靶向配体的量（即，葡萄糖和狂犬病病毒 糖蛋白（RVG）肽）和用于两种类型的增强的全脑系统递送的SNC的剂量 CRISPR基因组编辑器（即，(1)Cas9 mRNA/sgRNA，和（2）具有神经元特异性人Cas9 mRNA/sgRNA的质粒DNA。 突触蛋白1（SYN 1）启动子并表达Cas9和sgRNA两者）。我们将进一步确定他们的治疗 使用新的APP敲入AD小鼠模型治疗AD的有效性和生物安全性，同时采用独特的 APP调节的基因靶点。SNC的基因编辑效率和生物安全性概况，以及 AD小鼠的病理和行为变化将通过各种技术来监测， 系列双光子断层全脑淀粉样蛋白成像平台。有了有希望的初步研究，最好的- 执行SNC将提交给NIA赞助的STOP-AD计划的临床前测试核心， 全面的临床前评价。该项目将为一种新的、安全的、非侵入性的、有效的 家族性AD的治疗方法。鉴于SNC的模块化和多功能性， 通过CRISPR系统对不同基因的测序，我们预计我们的SNC将适用于广泛的NDD。

Project Summary Currently, there is no effective way to slow down the progress of Alzheimer’s disease (AD) or prevent it. CRISPR genome editing is a revolutionary and versatile genetic engineering technique, making it possible to treat the root causes of genetic neurodegenerative diseases (NDDs) such as AD. However, the promise of brain gene therapy relies on the efficient delivery of biologics to the brain, which is extremely challenging due to the blood- brain barrier (BBB). To date, in vivo brain gene therapy has mostly been achieved using viral vectors that require laborious customization and have troublesome safety profiles. Non-viral vectors are largely administered via intracranial administration, which is invasive and can only enable gene therapy in a small and localized brain region. Similar to other NDDs, AD affects multiple brain regions. Thus, there is an urgent need to develop efficient non-viral delivery vehicles capable of bypassing the BBB for safe and efficient brain-wide gene therapy. The objectives of this project are (1) to engineer glutathione (GSH)-responsive silica nanocapsules (SNCs) capable of bypassing the BBB and delivering CRISPR genome editors to the whole brain systemically, and (2) to evaluate the therapeutic efficacy and biosafety of brain-wide genome editing enabled by the optimized SNC for the treatment of AD using a novel amyloid precursor protein (APP) knock-in AD mouse model and a unique gene target for APP modulation. The unique SNC possess a long list of desirable properties including versatile payload types, versatile surface chemistry for ligand conjugation, high payload loading content and efficiency, small particle sizes, excellent in vivo stability, good biocompatibility, and scalable production. Our preliminary data has shown that intravenously administered SNCs can efficiently deliver mRNA, DNA, Cas9 mRNA/sgRNA, and Cas9/gRNA ribonucleoprotein (RNP) to the whole brain of healthy mice with intact BBB. We aim to further optimize the amounts of the dual brain-targeting ligands (i.e., glucose and rabies virus glycoprotein (RVG) peptide) and dosages of the SNCs for enhanced brain-wide systemic delivery of two types of CRISPR genome editors (i.e., (1) Cas9 mRNA/sgRNA, and (2) plasmid DNA with a neuron-specific human synapsin 1 (SYN1) promoter and expressing both Cas9 and sgRNA). We will further determine their therapeutic efficacy and biosafety in treating AD using a novel APP knock-in AD mouse model while employing a unique gene target for APP modulation. The gene editing efficiency and biosafety profiles of the SNC, and the pathological and behavior changes of the AD mice will be monitored by various techniques including a novel serial two-photon tomography whole-brain amyloid imaging platform. With promising initial studies, the best- performing SNC will be submitted to the Preclinical Testing Core of the NIA-sponsored STOP-AD program for comprehensive preclinical evaluation. This project will pave the road for a new, safe, non-invasive and effective therapeutic approach for familial AD. Given the modularity and versatility of the SNCs, and ease of targeting different genes by the CRISPR system, we anticipate that our SNCs will be applicable for a wide range of NDDs.