Attenuating AAV-Triggered Innate Immunity in the Adult Mouse Nervous System via cGAS-STING Pathway Inhibition

While adeno-associated virus (AAV)-mediated gene delivery has emerged as a promising therapeutic modality for neurological disorders, dose-dependent immune responses remain a critical barrier to clinical translation. Here we identify the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway as a key mediator of innate immune activation following intracranial AAV administration. Through comparative analyses in genetic and pharmacological intervention models, we demonstrate that STING signaling mediates key neuroinflammatory sequelae including glia reactivation, cytotoxic T cell infiltration, and neuronal injury. Mechanistically, microglia serve as the predominant sentinels detecting AAV immunogenicity via cGAS-STING activation. Therapeutic inhibition of this pathway by either microglia depletion or antagonism of STING by small molecules significantly mitigates high-dose AAV9-induced neurotoxicity while enhancing transgene delivery efficacy. Our work delineates a unified mechanistic framework linking AAV-triggered DNA sensing to neuroinflammatory pathology, and provides two clinically actionable approaches to decouple therapeutic gene delivery from detrimental immune activation in nervous system targeted gene therapy. Overall design: step1:In this study, we first used bulk-RNA sequencing technology to investigate the early changes in gene expression profiles of AAV-injected brain tissue with different dose(contributor:Yaowei Guo). Bulk RNA sequencing analysis showed that in the brains administered with 1010 GC AAV9, classical innate immune responses were significantly upregulated, including TLRs and NOD-like receptors (NLRs). In addition, high dose AAV administration robustly upregulates gene expression that involved in the cGAS-STING pathway. Using Sting knockout transgenic mice, we further confirmed that the cGAS-STING signaling pathway plays a crucial role in mediating inflammation, immune responses, and neuronal injury in the adult mouse brain following high-dose AAV9 intracranial injection. Further studies have found that both reactive microglia and astrocytes are involved in the activation of the cGAS-STING pathway. step2:Microglia depletion significantly inhibited cGAS-STING pathway activation and subsequent inflammation and T cell infiltration. H-151 is a novel STING inhibitor that effectively suppressed inflammation, reactive glial cells, T cell infiltration and neuronal damage, while also significantly increasing the expression of transgene in AAV vectors. Altogether, our findings suggest that inhibiting the cGAS-STING pathway may improve the safety and efficacy of AAV gene therapy in the nervous system.(contributor:Yaowei Guo)