Improving Viral Gene Delivery: Identifying and Preventing Central Nervous System Impacts of Adeno-Associated Viruses

Abstract

Adeno-associated viruses have revolutionized neuroscience research by enabling precise delivery of genetic cargo and facilitating targeted application of techniques such as optogenetics and chemogenetics. Clinically, AAV-mediated gene therapy has recently been approved by the FDA to treat nervous system disorders. However, recent evidence unexpectedly shows that AAVs can trigger innate immune responses through activation of Toll-like receptor 9 by unmethylated CpG motifs within the viral genome, leading to dendritic loss and synaptic weakening. As AAV-based therapies continue to gain clinical approval, further characterization of AAV-induced side effects — particularly behavioral outcomes — is crucial. To protect against these effects, strategies such as systemic TLR9 inhibitors have shown promise; however, challenges remain regarding immunosuppression, specificity, efficacy, and clinical applicability. This study aims to address these two major gaps in the field. To explore the link between AAV-induced functional and structural disruptions and behavioral outcomes, I investigated the behavioral consequences of AAV administration in the central nervous system, using a modified novel object recognition and whisker nuisance assay. To protect against AAV-induced circuit damage, I developed novel candidate oligodeoxynucleotide TLR9 inhibitors, and encoded one candidate TLR9 inhibitor directly in the AAV genome. This approach targets TLR9 inhibition to only those cells that are transduced with AAV, potentially improving efficacy and reducing immunosuppression compared to systemic TLR9 inhibitors. Together, these studies aim to improve the safety and efficacy of AAV-mediated gene delivery and to expand its therapeutic potential for treating neurological diseases.