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.

Narrative Gene editing may offer a new therapeutic strategy to tackle many neurodegenerative disorders that remain untreatable. Current methodologies of delivering CRISPR-based gene editing elements to the brain are highly inefficient. We propose to develop a noninvasive, efficient approach to achieve gene editing in the brain using focused ultrasound technology.