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和多功能的基因工程技术，使治疗 阿尔茨海默病等遗传性神经退行性疾病(NDDS)的根本原因。然而，大脑基因的前景 治疗依赖于将生物制剂有效地输送到大脑，这是极其具有挑战性的，因为血液- 脑屏障(BBB)。到目前为止，活体脑基因治疗大多是通过使用需要 繁琐的定制和麻烦的安全配置文件。非病毒载体主要通过 颅内给药，这是侵入性的，只能在小而局部的大脑中进行基因治疗 区域。与其他NDD类似，AD会影响多个大脑区域。因此，迫切需要发展高效的 非病毒载体能够绕过血脑屏障，进行安全有效的全脑基因治疗。 该项目的目标是(1)设计谷胱甘肽(GSH)响应型二氧化硅纳米胶囊(SNCs) 能够绕过血脑屏障，将CRISPR基因组编辑系统地传递到整个大脑，以及(2) 评价优化后的SNC实现全脑基因组编辑的疗效和生物安全性 使用一种新的淀粉样前体蛋白(APP)敲入AD小鼠模型和一种独特的 APP调控的基因靶点。独特的SNC具有一长串所需的特性，包括 有效负载类型，用于配基连接的多种表面化学，高有效负载含量和效率， 颗粒小，体内稳定性好，生物相容性好，生产可扩展。我们的预赛 有数据表明，静脉注射SNCs可以有效地递送mRNA、DNA、Cas9 mRNA/sgRNA， 和Cas9/gRNA核糖核蛋白(RNP)给血脑屏障完整的健康小鼠全脑。 我们的目标是进一步优化双重脑靶向配体(即葡萄糖和狂犬病病毒)的数量 糖蛋白(RVG)和SNCs的剂量促进两种类型的全脑全身给药 CRISPR基因组编辑(即(1)Cas9 mRNA/sgRNA，和(2)带有神经元特异性人的质粒DNA 突触素1(Synapsin 1，SYN1)启动子，同时表达Cas9和sgRNA)。我们将进一步确定他们的治疗方法 使用一种新的APP敲入AD小鼠模型治疗AD的有效性和生物安全性 APP调控的基因靶点。SNC的基因编辑效率和生物安全概况，以及 将通过包括一种新的技术在内的各种技术来监测AD小鼠的病理和行为变化 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 全面的临床前评估。该项目将为新的、安全、非侵入性和有效的 家族性AD的治疗方法。考虑到SNC的模块化和多功能性，以及目标确定的简便性 由于CRISPR系统提供了不同的基因，我们预计我们的SNCs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.