Adeno-associated virus-mediated epithelial IL-33 silencing attenuates type 2 airway inflammation

BACKGROUND: While therapeutic strategies targeting the IL-33/ST2 axis are under active development, systemic blockade of IL-33 raises concerns because of its pleiotropic and protective functions in multiple organs. Therefore, strategies enabling cell- and tissue-specific control of IL-33 are needed. METHODS: We developed adeno-associated virus (AAV)-based gene silencing vectors encoding short hairpin RNA targeting IL-33. Cellular sources of IL-33 and AAV serotype tropism were characterized by immunostaining and flow cytometry. Therapeutic efficacy was evaluated in innate and adaptive models of type 2 airway inflammation in mice, as well as in human airway epithelial cell-ILC2 co-culture systems. RESULTS: In the mouse lungs, IL-33 was predominantly expressed in type II alveolar epithelial cells, and intratracheally administered AAV9 preferentially transduced these IL-33-expressing cells. AAV9 encoding shRNA targeting IL-33 efficiently transduced primary mouse type II alveolar epithelial cells and suppressed IL-33 expression in vitro. In vivo, AAV-mediated IL-33 silencing significantly attenuated type 2 airway inflammation in an Alternaria-induced innate immune model. In contrast, the suppressive effect was more modest in an OVA-induced adaptive immune model. In the human airway epithelium, IL-33 was primarily localized to basal cells, and primary human basal cells were efficiently transduced by AAV6. AAV6-mediated IL-33 knockdown in human basal cells significantly reduced IL-13 production by human ILC2s in a co-culture system. CONCLUSIONS: AAV-mediated epithelial cell-specific silencing of IL-33 effectively suppresses type 2 airway inflammation in mouse and human models. This strategy represents a qualitatively distinct approach to IL-33 modulation and provides a conceptual framework for tissue-targeted control of epithelial alarmins.