Short-hairpin RNAs (shRNA), targeting knockdown of specific genes, hold enormous promise for precision-based therapeutics to treat numerous neurodegenerative disorders. However, whether shRNA constructed molecules can modify neuronal circuits underlying certain behaviors has not been explored. We designed shRNA to knockdown the human HTR2A gene in vitro using iPSC-differentiated neurons. Multi-electrode array (MEA) results showed that the knockdown of the 5HT-2A mRNA and receptor protein led to a decrease in spontaneous electrical activity. In vivo, intranasal delivery of AAV9 vectors containing shRNA resulted in a decrease in anxiety-like behavior in mice and a significant improvement in memory in both mice (104%) and rats (92%) compared to vehicle-treated animals. Our demonstration of a non-invasive shRNA delivery platform that can bypass the blood-brain barrier has broad implications for treating numerous neurological mental disorders. Specifically, targeting the HTR2A gene presents a novel therapeutic approach for treating chronic anxiety and age-related cognitive decline.

The expanding field of precision gene editing using CRISPR/Cas9 has demonstrated its potential as a transformative technology in the treatment of various diseases. However, whether this genome-editing tool could be used to modify neural circuits in the central nervous system (CNS), which are implicated in complex behavioral traits, remains uncertain. In this study, we demonstrate the feasibility of noninvasive, intranasal delivery of adeno-associated virus serotype 9 (AAV9) vectors containing CRISPR/Cas9 cargo within the CNS resulting in modification of the HTR2A receptor gene. In vitro, exposure to primary mouse cortical neurons to AAV9 vectors targeting the HT2RA gene led to a concentration-dependent decrease in spontaneous electrical activity following multielectrode array (MEA) analysis. In vivo, at 5 weeks postintranasal delivery in mice, analysis of brain samples revealed single base pair deletions and nonsense mutations, leading to an 8.46-fold reduction in mRNA expression and a corresponding 68% decrease in the 5HT-2A receptor staining. Our findings also demonstrate a significant decrease in anxiety-like behavior in treated mice. This study constitutes the first successful demonstration of a noninvasive CRISPR/Cas9 delivery platform, capable of bypassing the blood-brain barrier and enabling modulation of neuronal 5HT-2A receptor pathways. The results of this study targeting the HTR2A gene provide a foundation for the development of innovative therapeutic strategies for a broad range of neurological disorders, including anxiety, depression, attentional deficits, and cognitive dysfunction.