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

Abstract

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.

Fig. 1. The strategies for packaging recombinant adeno-associated virus (rAAV) expressing GSDMD^NT.

a Schematic of the strategy for the packaging of rAAV-GSDMD^NT (rAAV-P1) using the Sf9/rBac system and mammal-specific promoter mCBA. mCBA promoter can hardly drive gene expression in sf9 insect cells. b Fluorescence microscopy of Sf9 insect cells showing the expression of eGFP driven by different promoters at 48 h post transfection. Scale bars, 10 μm. c Images of the Sf9 insect cells infected with different baculoviruses. Arrows indicate pyroptotic cells. Scale bars, 20 μm. d The viral titer of different rAAVs packaged by the Sf9/rBac system. Data were expressed as mean ± s.e.m., from three independent replicates. Two-tailed unpaired t test with Welch’s correction was used for comparing the difference between two groups as indicated. e Schematic of the strategy using Cre/lox system to package rAAV-DIO-GSDMD^NT (rAAV-ef1α-DIO-GSDMD^NT). Coinfection of rAAV-Cre can revert the DIO-GSDMD^NT (rAAV-P2) to induce pyroptosis. f Images of HEK 293T cells transfected with pAAV-GSDMD^NT (pAAV-CMV-DIO-GSDMD^NT), pAAV-Cre, and pAAV-DIO-GSDMD^NT (pAAV-ef1α-DIO-GSDMD^NT), respectively. Arrows indicate pyroptotic cells. Scale bars, 20 μm. g Differential interference contrast (DIC) images of HEK 293T cells infected with rAAV-DIO-GSDMD^NT (rAAV-ef1α-DIO-GSDMD^NT), rAAV-P1, rAAV-P2, and PBS, respectively. Arrows indicate pyroptotic cells. Scale bars, 20 μm. All data are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 2. The pyroptosis induced by rAAV-P1 and rAAV-P2 in different cancer cell lines.

a–d Confocal images of the rAAV-treated HeLa (a), Hep3B (b), C6-luc (c), and 4T1-luc (d) cells. Scale bars, 20 μm. All the cells were added with propidium iodide and Annexin V-APC 15 min before imaging. e, f Comparison of LDH release-based cell death assay (e) and ATP cell viability assay (f) in HeLa, C6-luc, and 4T1-luc cells after treatment with rAAV-DIO-GSDMD^NT (rAAV-ef1α-DIO-GSDMD^NT), rAAV-P1, and rAAV-P2, respectively. All data are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 3. rAAV-P2 treatment prolongs the survival time of glioblastoma (GBM) rat model.

a Experimental timeline for rAAV-P2 treatment in GBM rat model. b Luciferase imaging of C6 tumors 20 days post implantation (n = 3 rats). c Corresponding quantification of luciferase expression in (b). Mean± s.e.m., n = 3 rats for each group, two-tailed unpaired t test with Welch’s correction. d Survival curves of C6-luc tumor-bearing rats treated with PBS, rAAV-DIO-GSDMD^NT (rAAV-ef1α-DIO-GSDMD^NT), and rAAV-P2, respectively, as indicated in (a). n = 7 rats for PBS and rAAV-DIO-GSDMD^NT (rAAV-ef1α-DIO-GSDMD^NT), six rats for rAAV-P2. Log-rank test was used for comparing two groups (PBS vs AAV-DIO-GSDMD^NT or PBS vs rAAV-P2). e H&E-stained coronal sections of C6-luc tumor-bearing rat brains treated as indicated, 35 days (three on the left) and 50 days (right) post-tumor cell implantation, respectively. Representative images of each group (n = 3 rats) are presented. Scale bar, 2000 μm. f Evans Blue staining of whole brains treated as indicated, 35 days (three on the left) and 50 days (right) post-tumor cell implantation, respectively. Representative images of each group (n = 3 rats) are presented. g Corresponding quantification of Evans Blue in (f). Data were expressed as mean ± s.e.m., n = 3 rats for each group. Two-tailed unpaired Student’s t test was used for comparing the difference between two groups. PBS vs rAAV-P2 (day 35), p = 0.0016; rAAV-DIO-GSDMD^NT vs rAAV-P2 (day 35), p = 0.0014; rAAV-P2 (day 50) vs rAAV-P2 (day 35), p = 0.0001. h Representative fluorescence images of CD3-BV421-stained of the C6-luc tumors following treatment as indicated. n = 3 rats for each group. Scale bar, 20 μm. All data are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 4. rAAV-P2 treatment activates antitumor immunity and inhibits the growth of triple-negative breast cancer (TNBC).

a Schematic of rAAV-P2 treatment on TNBC mouse model. b Tumor volume of an individual mouse. n = 7 mice per group. c Average tumor volume of mice as indicated. n = 7 mice per group. Data were expressed as mean ± s.e.m. Two-way ANOVA with the Geisser–Greenhouse correction was used for comparing two groups. d Photographs of the tumors 30 days post treatment with rAAV-P2 and rAAV-DIO-GSDMD^NT (rAAV-ef1α-DIO-GSDMD^NT), respectively. e Average tumor weight of mice as indicated. n = 7 mice per group. Data were expressed as mean ± s.e.m. Two-tailed unpaired Student’s t test was used for comparing two groups. f t-SNE plots of tumor-infiltrating single CD45⁺ immune cells of 4T1 tumors from mice treated with rAAV-DIO-GSDMD^NT (n = 2 mice) and rAAV-P2 (n = 2 mice), respectively. g The relative frequencies of different clusters. h Quantification of the tumor-infiltrating lymphocytes from TNBC mouse model. n = 4 mice for rAAV-DIO-GSDMD^NT (rAAV-ef1α-DIO-GSDMD^NT) and 5 mice for rAAV-P2. Data were expressed as mean ± s.e.m. Two-tailed unpaired t test with Welch’s correction was used for comparing two groups. i Condition-specific linkages between MDSC ligands and other lymphocyte cluster receptors in the rAAV-DIO-GSDMD^NT and rAAV-P2 treatment groups. j Diagram of the DEG interaction network of “the positive regulation of T cell proliferation” signaling pathway based on the STRING database. Data shown (a–e, h) are representative of two independent experiments. Source data are provided as a Source Data file.

Fig. 5. Anti-PD-L1 therapy boosts the effect of rAAV-P2 treatment on TNBC in the mouse model.

a Schematic of rAAV-P2 combined with anti-PD-L1 treatment on TNBC mouse model. b Luciferase imaging of 4T1-luc breast tumors 21 days post-tumor implantation. c Corresponding quantification of luciferase expression in (b). Mean ± s.e.m., from four independent replicates, one-tailed unpaired t test with Welch’s correction. d Tumor volume of sn individual mouse. n = 5 mice per group. e, g Average tumor volume (e) and weight (g) of mice as indicated. n = 5 mice per group. Data were expressed as mean ± s.e.m. Two-way ANOVA with the Geisser–Greenhouse correction was used for comparing the difference between two groups (e). Two-tailed unpaired t test with Welch’s correction was used for comparing the difference between two groups (g). f Photographs of representative tumors 29 days post treatment. h Propidium iodide staining of tumor cell pyroptosis induced by treatment as indicated. n = 3 mice for each group. Scale bar, 20 μm. All data are representative of two independent experiments. Source data are provided as a Source Data file.