Non-Invasive Gene Delivery to Brain Lymphatic System Using an Imaging Phased Array and Ultra-Short Focused Ultrasound Pulses

Neurodegenerative diseases such as Alzheimer’s and Parkinson’s are often characterized by abnormal depositions of waste products, which often occur due to ineffective metabolite clearance. Ependymal cells and the glymphatic system have been proposed as a potential contributor to the progression of these diseases, but effective and clinically translatable treatment methods targeting these cells have not yet been developed. Theranostic ultrasound-mediated blood-brain barrier opening (ThUS-BBBO) utilizes a repurposed imaging phased array to transiently open the blood-brain barrier (BBB) and has been used to increase adeno-associated virus delivery to the brain of mice. In this study, we employ ThUS-BBBO with different combinations of microbubbles and AAVs to assess the expression of the GFP transgene in periventricular areas. We hereby report ThUS-BBBO-enhanced AAV delivery with polydisperse and size-isolated microbubbles, with highest BBB opening volumes and transgene expression observed in mice injected microbubbles in the 3-5 μm range, and a positive correlation of BBB opening volume with cavitation dose across three microbubble groups. Additionally, we explore the role of AAV serotype and dose on the number and types of cells expressing GFP, with AAV9 transducing the largest number of cells in periventricular areas, including neurons and astrocytes, and achieving up to 30% increase in GFP expression with higher AAV doses. Our results lay the foundation for non-invasive gene delivery and therapy of the brain glymphatic system.