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递送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以解决许多棘手的神经退行性疾病 疾病然而，将基于CRISPR的基因编辑元件递送到大脑是非常低效的。我们之前 率先使用聚焦超声（FUS）技术实现非侵入性、脑特异性递送。我们 证明了FUS与单分散的充气微泡结合可以递送治疗有效载荷， 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，我们还重新设计了复合系统 以提高非病毒CRISPR介导的基因编辑效率和特异性在体外和体内。与 初步研究，我们建议整合组织特异性（FUS）和细胞特异性（AAV和非病毒）的优点， 多聚物载体）递送系统，以使CRISPR能够递送到大脑及其疾病相关的细胞类型。整体 本项目的目的是开发一种非侵入性的，FUS介导的技术，用于递送AAV载体和非病毒载体。 将CRISPR元件携带到大脑的复合物，并评估对两种主要神经退行性疾病的功效， 阿尔茨海默氏症和帕金森氏症。我们建议在UG 3发展阶段追求三个具体目标， UH 3演示阶段的第四个目标：（1）优化FUS系统，具有定义的微泡成分，声学 参数，并优化携带CRISPR敲除、抑制以及激活元件的AAV载体， 在小鼠脑中实现高基因编辑功效;（2）开发有效的CRISPR递送非病毒复合物系统， 通过FUS技术递送到脑中的瞬时表达谱;（3）评估治疗功效和治疗效果。 使用优化的AAV和非病毒CRISPR递送系统对阿尔茨海默病和帕金森病模型的安全性; （4）评价NHP模型中优化后的给药系统的给药效率和安全性。作为目前的CRISPR 输送到大脑只依赖于颅内注射，拟议的项目将是第一个研究开发一个 非侵入性的，有效的方法来实现大脑中的基因编辑。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，甚至无法治疗。

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.