A multifunctional theranostic ultrasound platform for remote magnetogenetics and expanded blood-brain barrier opening.

Brain stimulation techniques are critical for unraveling the innerworkings of complex neuronal pathways governing both normal physiological function and pathologic states in neurological disorders. Focused ultrasound (FUS) is an emerging technique poised to significantly alter central nervous system (CNS) drug delivery and neuroscience research through non-invasive means. Magnetogenetics is a brain stimulation technique which may benefit from FUS technology in that alternating magnetic fields (AMF), like FUS, can pass through the skull without requiring surgery. Magnetogenetics involves the deposition of superparamagnetic iron-oxide nanoparticles (SPIONs) and overexpression of thermoreceptor transmembrane proteins (e.g. TRPV1 and TRPA1) in the brain. When an external AMF is applied, SPIONs generate local heating, which can activate thermoreceptors, depolarize the cell membrane and trigger action potentials in neurons. Monitoring neuronal activation by a magnetogenetics approach can be facilitated by the co-expression of genetically-encoded voltage indicators (GEVI), which enable fluorescence-based detection of membrane depolarization. However, traditional surgical methods used to introduce these components into the brain are invasive and highly focal, precluding investigation of brain-wide neuronal pathways. Here, we demonstrate that our recently developed, flexible configuration for FUS therapy and ultrasound imaging, called theranostic ultrasound (ThUS), can transiently open the blood-brain barrier (BBB) and facilitate the non-invasive delivery of SPION and viral vectors encoding thermoreceptors and GEVI, to enable remote magnetogenetic modulation. We also report significant advances in ThUS pulse sequence design, where we developed a novel multi-target opening volume expansion (MOVE) pulse sequence to maximize BBB opening volume within a single ThUS treatment. ThUS MOVE yielded increased gene delivery commensurate with the number of targeted focal zones and achieved brain-wide expression of GEVI. The results presented herein not only demonstrate the feasibility for ThUS to facilitate a non-invasive brain stimulation approach, but also showcase a method for eliciting larger volumes of BBB opening within a single sonication which could dramatically improve gene delivery procedures for both preclinical research and therapeutic purposes in the future.