Fig. 4: Sense-and-destroy against HSV-1 mediated by ALICE in mice.
From: Engineering antiviral immune-like systems for autonomous virus detection and inhibition in mice
a Schematic illustration of single- and dual-output ALICE systems for autonomous sense-and-destroy against HSV-1 in mice. Four types of cells, including (1) three sgRNAs (pYW102/pYW172/pYW188, respectively targeting US8/UL29/UL52)-transgenic HEKALICE-SEAP-Cas9 (ALICECas9), (2) pcDNA3.1-transgenic HEKALICE-Cas9-E317Ab (ALICEAb), (3) pYW102/pYW172/pYW188-transgenic HEKALICE-Cas9-E317Ab (ALICECas9+Ab), or (4) pcDNA3.1-transgenic HEK-293T cells (Control), were encapsulated into hydrogel-based scaffolds and transplanted into the abdomen of mice via intraperitoneal surgery. At 20 h post-transplantation of the hydrogel implants, HSV-1 (2 × 107 PFU) was intraperitoneally injected into each mouse. The antiviral effects of the single- and dual-output ALICE systems in mice were evaluated by detecting residual viral titers in indicated organs (liver, spleen, kidney) at 2, 4 and 6-days post HSV-1 injection (d–l). b Assay of HSV-1-inducible E317Ab expression and c western blot analysis of HSV-1-inducible Cas9 expression in hydrogel-scaffolds. Mice were transplanted with hydrogel-scaffolds containing pYW102/pYW172/pYW188-transgenic HEKALICE-Cas9-E317Ab cells, infected with HSV-1 (2 × 107 PFU, HSV-1 group) or uninfected (0, Vehicle group). The red arrowhead indicates the expected Cas9 band. d qPCR assay of HSV-1 UL23/US2 mRNA in mice. HSV-1 mRNA levels were performed in isolated liver/spleen/kidney using specific UL23/US2 primers listed in Supplementary Table 2 at 2 (d, e), 4 (g, h), and 6 (j, k) days post HSV-1 injection. White bars (Control), brown bars (ALICECas9), blue-gray bars (ALICEAb), red bars (ALICECas9+Ab) in (d–l). f Viral titers in mice. The mice were processed as described in (a). Virus in isolated tissues, such as liver, spleen, kidney, were titrated at 2 (f), 4 (i), and 6 (l) days post HSV-1 injection. The relative expression was calculated using the ΔΔct method based on the expression levels of viral genes UL23/US2 in organs of wild-type mice (WT). P values for all other group versus Control group in the same tissue. Data in d, e, g, h, j, k are normalized to wild-type mice (WT); Numbers 1–4 represents four indenpent mice in (c); Data in b, d–l are expressed as means ± SEM; n = 4 mice; P values were calculated by two-way ANOVA with Bonferroni’s post hoc test; n.s. not significant. Source data are provided as a Source Data file.
