Fig. 5: Activity and functional performance of iCROP systems in vivo.

a Timeline for iCROP-fLuc study in vivo. Mice were hydrodynamically injected with the plasmid encoding iCROP-fLuc 24 h before rupintrivir administration. b Luminescence images were recorded at the indicated time points by the IVIS imaging system (n = 5 mice). c Quantification of the luminescence intensity of images in b. d Timeline for the in vivo study using iCROP-sr-IκBα to repress NF-κB signaling. The mice received the iCROP-sr-IκBα-encoding plasmid by hydrodynamic injection 24 h before the LPS treatment and rupintrivir administration. Blood samples were collected for cytokine analysis 6 h later. e Blood levels of TNFα, IL-1β and IL-6 6 h after the LPS treatment, analyzed by ELISA. Data are shown as mean ± SD, with individual data points (n = 6 mice per experimental group, n = 4 mice for wild-type group). f Timeline for the in vivo study using inducible iCROP-Cas9 for PCSK9 knockout. The DNA vectors expressing iCROP-Cas9 and sgRNA(mPCSK9) were delivered to mice 1 day before the first rupintrivir treatment, which was repeated for 3 consecutive days, before analysis. g Serum PCSK9 levels measured by ELISA (left) in wild-type mice, mice expressing only the iCROP-Cas9 system and mice expressing the iCROP-Cas9 system in combination with rupintrivir treatment. Results are shown as absolute PCSK9 values (left) and relative PCSK9 reduction compared to wild-type mice (right). Relative decrease was calculated by dividing the average PCSK9 levels of experimental groups by the average PCSK9 level of the wild-type group. h LDL-cholesterol levels in mouse serum, measured with an LDL-cholesterol assay kit 168 h after starting the rupintrivir dosing (left) and relative LDL-cholesterol reduction compared to wild-type mice (right). Relative decrease was calculated by dividing the average LDL-cholesterol levels of experimental groups by the average LDL-cholesterol level of the wild-type group. In panels g, h, data are shown as mean ± SD, with individual data points (n = 5–6 mice per group). Statistical significance was calculated by means of Welch’s two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.