Strategies to package recombinant Adeno-Associated Virus expressing the N-terminal gasdermin domain for tumor treatment

Pyroptosis induced by the N-terminal gasdermin domain (GSDM(NT)) holds great potential for anti-tumor therapy. However, due to the extreme cytoxicity of GSDM(NT), it is challenging to efficiently produce and deliver GSDM(NT) into tumor cells. Here, we report the development of two strategies to package recombinant adeno-associated virus (rAAV) expressing GSDM(NT): 1) drive the expression of GSDM(NT) by a mammal specific promoter and package the virus in Sf9 insect cells to avoid its expression; 2) co-infect rAAV-Cre to revert and express the double-floxed inverted GSDM(NT). We demonstrate that these rAAVs can induce pyroptosis and prolong survival in preclinical cancer models. The oncolytic-viruses induce pyroptosis and evoke a robust immune-response. In a glioblastoma model, rAAVs temporarily open the blood-brain barrier and recruit tumor infiltrating lymphocytes into the brain. The oncolytic effect is further improved in combination with anti-PD-L1. Together, our strategies efficiently produce and deliver GSDM(NT) into tumor cells and successfully induce pyroptosis, which can be exploited for anti-tumor therapy.