Fig. 5: Long-term sense-and-destroy against HSV-1 mediated by ALICE_{Cas9+sgRNAs+Ab} in mice.

a Schematic illustration of ALICE_{Cas9+sgRNAs+Ab} system for autonomous sense-and-destroy against HSV-1 in mice. HEK-293T cells stably integrated with ALICE_{Cas9+sgRNAs+Ab} (HEK_{ALICE-Cas9-sgRNAs-E317Ab} cells) or pcDNA3.1-transgenic HEK-293T cells (Control) were encapsulated into hydrogel-based scaffolds and transplanted into mice abdomens via intraperitoneal surgery. At 28 d post-transplantation, HSV-1 (2 × 10⁷ PFU) was intraperitoneally injected into each mouse. The antiviral effects of the ALICE_{Cas9+sgRNAs+Ab} in mice was evaluated by detecting residual viral titers in the indicated organs (liver, spleen, kidney) at 2 days post HSV-1 infection. b Western blot analysis of HSV-1-inducible Cas9 expression in hydrogel-based scaffolds. The red arrowhead indicates the expected Cas9 band. Number 1–4 represents four independent samples. c Validation of HSV-1-inducible E317Ab expression in mice. The E317Ab level in the bloodstream from ALICE_{Cas9+sgRNAs+Ab}-treated mice, infected with or without HSV-1, was quantified at 30 days post-transplantation. d, e qPCR analysis of HSV-1 UL23/US2 mRNA levels in mice. f Viral titers in mice. Virus in isolated tissues (liver, spleen, kidney) from ALICE_{Cas9+sgRNAs+Ab}-treated mice, infected with or without HSV-1, were titrated at 30 days post-transplantation. g qPCR analysis of HSV-1 UL23/US2 mRNA in hydrogel implants at 30 days post-transplantation. h Cytokine levels in mice. Cytokines (IL-6, CCL5, CXCL10, TNF-α, and IFN-α) in the blood from HSV-1-infected mice implanted with or without ALICE_{Cas9+sgRNAs+Ab} were analyzed by flow cytometry at 30 days post-transplantation. i IgG expression in mice. Mice implanted with or without ALICE_{Cas9+sgRNAs+Ab} were infected with or without HSV-1. IgG levels in the bloods were analyzed using an IgG ELISA at 30 days post-transplantation. Data in d, e, and g are normalized to wild-type mice (WT); Data in c–i are expressed as means ± SEM; P values in c, h were calculated by two-tailed unpaired t-test; P values in d–f were calculated by two-way ANOVA with Bonferroni’s post hoc test; P values in i were calculated by one-way ANOVA followed by a Dunnett’s post hoc test; n = 4 mice in c–g, n = 4 or 5 mice in (h), n = 6 mice in (i). Source data are provided as a Source Data file.