Focused Ultrasound-mediated Delivery of Gene-editing Elements to the Brain for Neurodegenerative Disorders

聚焦超声介导的基因编辑元件脑内注射治疗神经退行性疾病 摘要 CRISPR技术作为精确基因编辑的变革性工具包，可能会解决许多顽固的神经退行性变 疾病。然而，将基于CRISPR的基因编辑元件传递到大脑的效率非常低。我们之前 率先使用聚焦超声(FUS)技术实现非侵入性、脑部特异性递送。我们 证明了FUS与单分散、充气的微泡相结合可以提供治疗有效载荷， 包括小分子、蛋白质和腺相关病毒(AAV)，通过静脉注射到大脑的特定区域 在啮齿动物和非人灵长类动物(NHP)模型中注射。此外，我们还重新设计了多路复用系统 目的：提高非病毒CRISPR介导的体内外基因编辑效率和特异性。有了那些 初步研究，我们建议将组织特异性(FUS)和细胞特异性(AAV和非病毒)的优点结合起来 多重载体)递送系统，使CRISPR能够递送到大脑及其疾病相关细胞类型。整体而言 该项目的目标是开发一种非侵入性的、FUS介导的技术来传递AAV载体和非病毒载体 将CRISPR元件携带到大脑的复合体，并评估其对两种主要神经退行性疾病的疗效， 阿尔茨海默氏症和帕金森氏症。我们建议在UG3发展阶段追求三个具体目标和一个 UH3演示阶段的第四个目标：(1)使用定义的微泡成分、声学成分优化FUS系统 参数并优化携带CRISPR基因敲除、抑制以及激活元件的AAV载体以 在小鼠脑内实现高效的基因编辑效率；(2)开发高效的CRISPR递送非病毒多聚体系统 通过FUS技术进入脑内的瞬时表达谱；(3)评价治疗效果和 使用优化的AAV和非病毒CRISPR递送系统对阿尔茨海默氏症和帕金森氏病模型的安全性； (4)在NHP模型中对优化后的配送系统的配送效率和安全性进行评价。作为当前CRISPR 脑内注射仅依赖于颅内注射，拟议的项目将是第一个研究开发 在大脑中实现基因编辑的非侵入性、高效的方法。该项目的成功将刺激新的战略 治疗神经退行性疾病的方法仍然具有挑战性，甚至无法治愈。

Focused Ultrasound-mediated Delivery of Gene-editing Elements to the Brain for Neurodegenerative Disorders Abstract CRISPR technology as a transformative toolkit for precise gene editing may tackle many intractable neurodegenerative diseases. However, delivery of CRISPR-based gene editing elements to the brain is highly inefficient. We previously pioneered the use of focused ultrasound (FUS) technology to achieve noninvasive, brain-specific delivery. We demonstrated that FUS in conjunction with monodispersed, gas-filled microbubbles could deliver therapeutic payloads, including small molecules, proteins and adeno-associated virus (AAV), to a specific region of the brain through intravenous injection in both rodent and non-human primate (NHP) models. Furthermore, we have also redesigned polyplex systems to improve the nonviral CRISPR-mediated gene editing efficiency and specificity both in vitro and in vivo. With those preliminary studies, we propose to integrate the merits of tissue-specific (FUS) and cell-specific (AAV and non-viral polyplex vectors) delivery systems to enable CRISPR delivery to the brain and its disease-related cell types. The overall objective of this project is to develop a non-invasive, FUS-mediated technology for delivering AAV vectors and non-viral polyplexes carrying CRISPR elements to the brain and to evaluate the efficacy on two major neurodegenerative disorders, Alzheimer’s and Parkinson’s diseases. We propose to pursue three specific aims in the UG3 developmental phase and a fourth aim in the UH3 demonstration phase: (1) Optimize the FUS system with defined microbubble composition, acoustic parameters and optimize the AAV vector carrying the CRISPR knockout, suppression as well as activation elements to achieve high gene editing efficacy in mouse brain; (2) Develop an efficient CRISPR delivery non-viral polyplex system with a transient expression profile for delivery into the brain via FUS technology; (3) Evaluate the therapeutic efficacy and the safety on both Alzheimer’s and Parkinson’s disease models with the optimized AAV and non-viral CRISPR delivery systems; and (4) Evaluate the delivery efficiency and safety of the optimized delivery system in the NHP model. As current CRISPR delivery to the brain relies only on intracranial injection, the proposed project will be the first study developing a noninvasive, efficient approach to achieve gene editing in the brain. Success of this project will stimulate new strategic approaches of tacking neurodegenerative disorders that remain challenging or even untreatable.