Figure 7.

Dual-Targeting trans-Splicing RNA Triggers Enhanced Cell Death at 10-Fold Lower GCV Concentration

(A) Design of dual-targeting tsRNA for 3′ ER furnished each with two BDs (BD1 and BD2) specific for AFP and one of three alternative HCC biomarkers (HCCA2, CD24, or VEGF) (lower panel). All six combinations or orders of BDs were considered (upper panel). AFP BD 3mBDopen binds to AFP I5, and the additional BDs target I2 of the respective second target genes. Additional BDs were computationally selected to be unstructured binding target introns encompassing weak cis-splice partnering sites. (B) alamarBlue cell viability assay comparing the levels of death triggered by dual-targeting and the parental AFP-only targeting constructs in cells expressing endogenous AFP levels after 10 μM GCV treatment. Mean ± SEM (n = 3). Significance was tested using two-way ANOVA with Bonferroni post hoc test compared to mock. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also Figure S12.